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The incidence of group B Streptococcus (GBS) neonatal disease continues to be a significant cause of concern worldwide. Strains belonging to clonal complex 17 (CC17) are the most frequently responsible for GBS infections in neonates, especially among late-onset disease cases. Therefore, we undertook the largest genomic study of GBS CC17 strains to date to decipher the genetic bases of their remarkable colonization and infection ability. We show that crucial functions involved in different steps of the colonization or infection process of GBS are distinctly mutated during the adaptation of CC17 to the human host. In particular, our results implicate the CovRS two-component regulator of virulence in the differentiation between carriage- and disease-associated isolates. Not only does this work raise important implications for the ongoing development of a vaccine against GBS but might also drive the discovery of key functions for GBS adaptation and pathogenesis that have been overlooked until now., Group B Streptococcus (GBS) is a commensal of the gastrointestinal and genitourinary tracts, while a prevailing cause of neonatal disease worldwide. Of the various clonal complexes (CCs), CC17 is overrepresented in GBS-infected newborns for reasons that are still largely unknown. Here, we report a comprehensive genomic analysis of 626 CC17 isolates collected worldwide, identifying the genetic traits behind their successful adaptation to humans and the underlying differences between carriage and clinical strains. Comparative analysis with 923 GBS genomes belonging to CC1, CC19, and CC23 revealed that the evolution of CC17 is distinct from that of other human-adapted lineages and recurrently targets functions related to nucleotide and amino acid metabolism, cell adhesion, regulation, and immune evasion. We show that the most distinctive features of disease-specific CC17 isolates were frequent mutations in the virulence-associated CovS and Stk1 kinases, underscoring the crucial role of the entire CovRS regulatory pathway in modulating the pathogenicity of GBS. Importantly, parallel and convergent evolution of major components of the bacterial cell envelope, such as the capsule biosynthesis operon, the pilus, and Rib, reflects adaptation to host immune pressures and should be taken into account in the ongoing development of a GBS vaccine. The presence of recurrent targets of evolution not previously implicated in virulence also opens the way for uncovering new functions involved in host colonization and GBS pathogenesis. IMPORTANCE The incidence of group B Streptococcus (GBS) neonatal disease continues to be a significant cause of concern worldwide. Strains belonging to clonal complex 17 (CC17) are the most frequently responsible for GBS infections in neonates, especially among late-onset disease cases. Therefore, we undertook the largest genomic study of GBS CC17 strains to date to decipher the genetic bases of their remarkable colonization and infection ability. We show that crucial functions involved in different steps of the colonization or infection process of GBS are distinctly mutated during the adaptation of CC17 to the human host. In particular, our results implicate the CovRS two-component regulator of virulence in the differentiation between carriage- and disease-associated isolates. Not only does this work raise important implications for the ongoing development of a vaccine against GBS but might also drive the discovery of key functions for GBS adaptation and pathogenesis that have been overlooked until now. Author Video: An author video summary of this article is available.

The heptavalent pneumococcal conjugate vaccine (PCV7) was introduced in the United States (US) in 2000 and has significantly reduced invasive pneumococcal disease; however, the incidence of nonvaccine serotype invasive disease, particularly due to serotype 19A, has increased. The serotype 19A increase can be explained in part by expansion of a genotype that has been circulating in the US prior to vaccine implementation (and other countries since at least 1990), but also by the emergence of a novel “vaccine escape recombinant” pneumococcal strain. This strain has a genotype that previously was only associated with vaccine serotype 4, but now expresses a nonvaccine serotype 19A capsule. Based on prior evidence for capsular switching by recombination at the capsular locus, the genetic event that resulted in this novel serotype/genotype combination might be identifiable from the DNA sequence of individual pneumococcal strains. Therefore, the aim of this study was to characterise the putative recombinational event(s) at the capsular locus that resulted in the change from a vaccine to a nonvaccine capsular type. Sequencing the capsular locus flanking regions of 51 vaccine escape (progeny), recipient, and putative donor pneumococci revealed a 39 kb recombinational fragment, which included the capsular locus, flanking regions, and two adjacent penicillin-binding proteins, and thus resulted in a capsular switch and penicillin nonsusceptibility in a single genetic event. Since 2003, 37 such vaccine escape strains have been detected, some of which had evolved further. Furthermore, two new types of serotype 19A vaccine escape strains emerged in 2005. To our knowledge, this is the first time a single recombinational event has been documented in vivo that resulted in both a change of serotype and penicillin nonsusceptibility. Vaccine escape by genetic recombination at the capsular locus has the potential to reduce PCV7 effectiveness in the longer term., Author Summary The 7-valent pneumococcal conjugate vaccine is a remarkable public health success story. It has significantly reduced invasive pneumococcal disease in the United States not only by protecting vaccinated children, but also by protecting unvaccinated older children and adults by herd immunity. However, there was always a concern that use of a limited-valency vaccine would result in an increase in disease due to nonvaccine serotypes, and this has now occurred in the US. The predominant nonvaccine serotype causing invasive disease is 19A, and this increase is partially explained by “vaccine escape” pneumococci, strains that have exchanged the genes that encode a vaccine serotype 4 capsule for genes that encode a nonvaccine serotype 19A capsule. These strains are then able to escape vaccine-induced immunity. Characterisation of the genetic event that resulted in these vaccine escape strains was the focus of our study and the results were surprising. The results of this study have important relevance to the long-term effectiveness of the current vaccine and to the development of future pneumococcal vaccines.

Genetic makeup of the host plays a significant role in the course and outcome of infection. Inbred strains of mice display a wide range of sensitivities to Listeria monocytogenes infection and thus serve as a good model for analysis of the effect of genetic polymorphism. The outcome of L. monocytogenes infection in mice is influenced by the ability of this bacterium to induce expression of interferon beta mRNA, encoded in mouse by the Ifnb1 (interferon beta 1, fibroblast) gene. Mouse strains that lack components of the IFNβ signaling pathway are substantially more resistant to infection. We found that macrophages from the ByJ substrain of the common C57BL/6 inbred strain of mice are impaired in their ability to induce Ifnb1 expression in response to bacterial and viral infections. We mapped the locus that controls differential expression of Ifnb1 to a region on Chromosome 7 that includes interferon regulatory factor 3 (Irf3), which encodes a transcription factor responsible for early induction of Ifnb1 expression. In C57BL/6ByJ mice, Irf3 mRNA was inefficiently spliced, with a significant proportion of the transcripts retaining intron 5. Analysis of the Irf3 locus identified a single base-pair polymorphism and revealed that intron 5 of Irf3 is spliced by the atypical U12-type spliceosome. We found that the polymorphism disrupts a U12-type branchpoint and has a profound effect on the efficiency of splicing of Irf3. We demonstrate that a naturally occurring change in the splicing control element has a dramatic effect on the resistance to L. monocytogenes infection. Thus, the C57BL/6ByJ mouse strain serves as an example of how a mammalian host can counter bacterial virulence strategies by introducing subtle alteration of noncoding sequences., Author Summary Specific variances in an individual's DNA, known as genetic polymorphisms, can play a significant role in determining susceptibility to an infectious disease. To identify the genetic polymorphisms that are associated with resistance to the common human bacterial pathogen L. monocytogenes, we have carried out a series of genetic and molecular biology experiments using closely related strains of mice that are differentially susceptible to Listeria infection. Through this analysis, we have identified a spontaneous mutation in an intron of the Irf3 gene, which encodes a key transcription factor involved in innate immunity. This single nucleotide change affects the efficiency with which Irf3 mRNA is spliced, thus limiting the ability of bacteria to induce interferon beta expression in order to suppress innate immune defense. By analyzing this mutation, we found that processing of mouse Irf3 mRNA relies on an atypical U12 splicing mechanism that has been suggested to be a rate-limiting step in gene expression. Our findings not only provide an additional example of an important role of noncoding polymorphisms in control of gene function, but also demonstrate how such polymorphisms can fine tune innate immune response.

Shigella infection, the cause of bacillary dysentery, induces caspase-1 activation and cell death in macrophages, but the precise mechanisms of this activation remain poorly understood. We demonstrate here that caspase-1 activation and IL-1β processing induced by Shigella are mediated through Ipaf, a cytosolic pattern-recognition receptor of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, and the adaptor protein apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC). We also show that Ipaf was critical for pyroptosis, a specialized form of caspase-1-dependent cell death induced in macrophages by bacterial infection, whereas ASC was dispensable. Unlike that observed in Salmonella and Legionella, caspase-1 activation induced by Shigella infection was independent of flagellin. Notably, infection of macrophages with Shigella induced autophagy, which was dramatically increased by the absence of caspase-1 or Ipaf, but not ASC. Autophagy induced by Shigella required an intact bacterial type III secretion system but not VirG protein, a bacterial factor required for autophagy in epithelial-infected cells. Treatment of macrophages with 3-methyladenine, an inhibitor of autophagy, enhanced pyroptosis induced by Shigella infection, suggesting that autophagy protects infected macrophages from pyroptosis. Thus, Ipaf plays a critical role in caspase-1 activation induced by Shigella independently of flagellin. Furthermore, the absence of Ipaf or caspase-1, but not ASC, regulates pyroptosis and the induction of autophagy in Shigella-infected macrophages, providing a novel function for NLR proteins in bacterial–host interactions., Author Summary Shigella are bacterial pathogens that are the cause of bacillary dysentery known as shigellosis. A crucial aspect of the propensity of Shigella to cause diseases lies in its ability to invade the cytoplasm of epithelial cells as well as macrophages. The bacterial invasion of macrophages induces pyroptosis, the proinflammatory cell death associated with caspase-1 activation. Activated caspase-1 then cleaves and activates prointerleukin (proIL)-1β and proIL-18, which are proinflammatory cytokines involved in host inflammatory responses. However, the precise mechanisms of caspase-1 activation induced by Shigella infection remain poorly understood. Ipaf, a cytosolic pattern-recognition receptor of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, is a crucial host factor that activates caspase-1 through the sensing of flagellin produced by some bacteria, such as Salmonella or Legionella. We discovered that Ipaf and the adaptor protein ASC are required for caspase-1 activation induced by non-flagellated Shigella infection. Thus, Ipaf and ASC mediate caspase-1 activation by sensing an unknown bacterial factor, but not flagellin. Autophagy, a cellular system for eliminating intracellular pathogens, was dramatically enhanced in Shigella-infected macrophages by the absence of caspase-1 or Ipaf, but not ASC. The inhibition of autophagy promoted Shigella-induced cell death, suggesting that autophagy protects infected macrophages from pyroptosis. This study provides evidence that in Shigella-infected macrophages, autophagy is inhibited by Ipaf and caspase-1, but positively regulated by ASC, providing a novel function for NLR proteins in bacterial–host interactions.

Mitochondria are eukaryotic organelles that originated from the endosymbiosis of an alpha-proteobacterium. To gain insight into the evolution of the mitochondrial proteome as it proceeded through the transition from a free-living cell to a specialized organelle, we compared a reconstructed ancestral proteome of the mitochondrion with the proteomes of alpha-proteobacteria as well as with the mitochondrial proteomes in yeast and man. Overall, there has been a large turnover of the mitochondrial proteome during the evolution of mitochondria. Early in the evolution of the mitochondrion, proteins involved in cell envelope synthesis have virtually disappeared, whereas proteins involved in replication, transcription, cell division, transport, regulation, and signal transduction have been replaced by eukaryotic proteins. More than half of what remains from the mitochondrial ancestor in modern mitochondria corresponds to translation, including post-translational modifications, and to metabolic pathways that are directly, or indirectly, involved in energy conversion. Altogether, the results indicate that the eukaryotic host has hijacked the proto-mitochondrion, taking control of its protein synthesis and metabolism., Author Summary Mitochondria are compartments from the eukaryotic cell that originated from the endosymbiosys of an alpha-proteobacterium. The bacterial-like metabolism of this early endosymbiont was thought to differ substantially from that of modern mitochondria, but so far we do not know the details of this bacterium-to-organelle transformation. To address this issue, we used an evolutionary approach to find genes derived from the ancestor of mitochondria. By identifying eukaryotic genes that are closely related to alpha-proteobacterial ones, we reconstructed a set of genes derived from the mitochondrial ancestor. We used that set to infer the ancestral mitochondrial metabolism, and subsequently compared it with those of modern mitochondria, as reconstructed from proteomics data from yeast and human. This allowed us to trace the metabolic evolution of mitochondria. What we found is that there has been a large turnover of the protein content of mitochondria, which has affected some pathways more than others. Pathways for protein synthesis and those involved in energy conversion have been preferentially retained in the mitochondrion, whereas those involved in replication, transcription, cell division, transport, regulation, and signal transduction have been replaced by eukaryotic proteins. Our findings show how the eukaryotic host has taken control of the endosymbiont, effectively hijacking those pathways that it could use.

Background Only a fraction of the microbial species used for anaerobic digestion in biogas production plants are methanogenic archaea. We have analyzed the taxonomic profiles of eubacteria and archaea, a set of chemical key parameters, and biogas production in samples from nine production plants in seven facilities in Thuringia, Germany, including co-digesters, leach-bed, and sewage sludge treatment plants. Reactors were sampled twice, at a 1-week interval, and three biological replicates were taken in each case. Results A complex taxonomic composition was found for both eubacteria and archaea, both of which strongly correlated with digester type. Plant-degrading Firmicutes as well as Bacteroidetes dominated eubacteria profiles in high biogas-producing co-digesters; whereas Bacteroidetes and Spirochaetes were the major phyla in leach-bed and sewage sludge digesters. Methanoculleus was the dominant archaea genus in co-digesters, whereas Methanosarcina and Methanosaeta were the most abundant methanogens in leachate from leach-bed and sewage sludge digesters, respectively. Conclusions This is one of the most comprehensive characterizations of the microbial communities of biogas-producing facilities. Bacterial profiles exhibited very low variation within replicates, including those of semi-solid samples; and, in general, low variation in time. However, facility type correlated closely with the bacterial profile: each of the three reactor types exhibited a characteristic eubacteria and archaea profile. Digesters operated with solid feedstock, and high biogas production correlated with abundance of plant degraders (Firmicutes) and biofilm-forming methanogens (Methanoculleus spp.). By contrast, low biogas-producing sewage sludge treatment digesters correlated with high titers of volatile fatty acid-adapted Methanosaeta spp. Electronic supplementary material The online version of this article (doi:10.1186/s13068-015-0271-6) contains supplementary material, which is available to authorized users.

Trophic relationships, such as those between predator and prey or between pathogen and host, are key interactions linking species in ecological food webs. The structure of these links and their strengths have major consequences for the dynamics and stability of food webs. The existence and strength of particular trophic links has often been assessed using observational data on changes in species abundance through time. Here we show that very strong links can be completely missed by these kinds of analyses when changes in population abundance are accompanied by contemporaneous rapid evolution in the prey or host species. Experimental observations, in rotifer-alga and phage-bacteria chemostats, show that the predator or pathogen can exhibit large-amplitude cycles while the abundance of the prey or host remains essentially constant. We know that the species are tightly linked in these experimental microcosms, but without this knowledge, we would infer from observed patterns in abundance that the species are weakly or not at all linked. Mathematical modeling shows that this kind of cryptic dynamics occurs when there is rapid prey or host evolution for traits conferring defense against attack, and the cost of defense (in terms of tradeoffs with other fitness components) is low. Several predictions of the theory that we developed to explain the rotifer-alga experiments are confirmed in the phage-bacteria experiments, where bacterial evolution could be tracked. Modeling suggests that rapid evolution may also confound experimental approaches to measuring interaction strength, but it identifies certain experimental designs as being more robust against potential confounding by rapid evolution., Author Summary The presence and strength of interactions between species has frequently been inferred from observational data on changes in species abundance. For example, correlated cycles in potential predator and prey species may be interpreted as evidence that the species interact, while the absence of such coupled oscillations might be interpreted as evidence for lack of interaction. Here we show that prey abundance can be decoupled from changes in predator abundance when there is genetic variability in the prey for antipredator defense traits, allowing rapid evolutionary changes in prey defense levels. It then appears that the two species are not interacting, when in fact they are. We deduce this from studies of two laboratory microcosm systems, one with algae consumed by rotifers and the other with bacteria attacked by phage. In each, when the prey vary genetically for defense traits and undefended genotypes are superior competitors, defended and undefended prey frequencies evolve in a cyclical way that is almost exactly counterbalancing, so that total prey density remains nearly constant. We show mathematically that these “cryptic cycles” occur whenever conditions are right for predator-prey cycles, when prey vary genetically for defense traits, and when prey defense against predation is effective but inexpensive to produce. Under these conditions, observations of predator and prey population dynamics cannot be trusted to be informative about the strength or even the existence of interspecific trophic links., Contemporary rapid evolution in prey and pathogen species masks strong tropic interactions with predators and hosts. These "cryptic dynamics" reveal a need for a new approach to measuring interaction strengths in food webs.

Symbiosis is a ubiquitous phenomenon generating biological complexity, affecting adaptation, and expanding ecological capabilities. However, symbionts, which can be subject to genetic limitations such as clonality and genomic degradation, also impose constraints on hosts. A model of obligate symbiosis is that between aphids and the bacterium Buchnera aphidicola, which supplies essential nutrients. We report a mutation in Buchnera of the aphid Acyrthosiphon pisum that recurs in laboratory lines and occurs in field populations. This single nucleotide deletion affects a homopolymeric run within the heat-shock transcriptional promoter for ibpA, encoding a small heat-shock protein. This Buchnera mutation virtually eliminates the transcriptional response of ibpA to heat stress and lowers its expression even at cool or moderate temperatures. Furthermore, this symbiont mutation dramatically affects host fitness in a manner dependent on thermal environment. Following a short heat exposure as juveniles, aphids bearing short-allele symbionts produced few or no progeny and contained almost no Buchnera, in contrast to aphids bearing symbionts without the deletion. Conversely, under constant cool conditions, aphids containing symbionts with the short allele reproduced earlier and maintained higher reproductive rates. The short allele has appreciable frequencies in field populations (up to 20%), further supporting the view that lowering of ibpA expression improves host fitness under some conditions. This recurring Buchnera mutation governs thermal tolerance of aphid hosts. Other cases in which symbiont microevolution has a major effect on host ecological tolerance are likely to be widespread because of the high mutation rates of symbiotic bacteria and their crucial roles in host metabolism and development., Author Summary Aphids are sap-feeding insects that depend on obligate bacterial symbionts of the genus Buchnera for biosynthesis of needed nutrients. Studying Buchnera gene expression in pea aphids, we identified a recurring mutation (a single-base deletion) in the transcriptional promoter of the small heat-shock protein, ibpA. This mutation arose and was fixed twice in sublines derived from a single female aphid in the lab and kept at constant 20 °C. Experiments using aphid lines that differed only in the presence of this Buchnera mutation revealed that it eliminates the ibpA transcriptional response to heat shock and affects ibpA expression at low temperatures. In aphids containing Buchnera with the mutation, a short heat treatment as juveniles leads to elimination of most or all symbionts and to reproductive failure; the same treatment has little effect on aphids containing Buchnera without the mutation. Conversely, at constant lower temperatures, aphids with Buchnera bearing this mutation enjoy a reproductive advantage. Pea aphid populations are polymorphic for the Buchnera mutation, suggesting that it is maintained at substantial frequencies by selection. This study indicates that mutations in obligate symbionts can have major consequences for host fitness and geographic distributions., A recurring single-nucleotide deletion in a heat-shock transcriptional promoter in the bacterialBuchnera symbiont of the pea aphid(Acyrthosiphon pisum) dramatically affects host fitness in a thermal-dependent manner.

In phylogenetics, the unrooted model of phylogeny and the strict molecular clock model are two extremes of a continuum. Despite their dominance in phylogenetic inference, it is evident that both are biologically unrealistic and that the real evolutionary process lies between these two extremes. Fortunately, intermediate models employing relaxed molecular clocks have been described. These models open the gate to a new field of “relaxed phylogenetics.” Here we introduce a new approach to performing relaxed phylogenetic analysis. We describe how it can be used to estimate phylogenies and divergence times in the face of uncertainty in evolutionary rates and calibration times. Our approach also provides a means for measuring the clocklikeness of datasets and comparing this measure between different genes and phylogenies. We find no significant rate autocorrelation among branches in three large datasets, suggesting that autocorrelated models are not necessarily suitable for these data. In addition, we place these datasets on the continuum of clocklikeness between a strict molecular clock and the alternative unrooted extreme. Finally, we present analyses of 102 bacterial, 106 yeast, 61 plant, 99 metazoan, and 500 primate alignments. From these we conclude that our method is phylogenetically more accurate and precise than the traditional unrooted model while adding the ability to infer a timescale to evolution., This new method can simultaneously infer phylogeny and estimate the molecular clock. The authors run their method on several large alignments to show its phylogenetic accuracy and ability to infer a timescale to evolution.

The extent and nature of epistatic interactions between mutations are issues of fundamental importance in evolutionary biology. However, they are difficult to study and their influence on adaptation remains poorly understood. Here, we use a systems-level approach to examine epistatic interactions that arose during the evolution of Escherichia coli in a defined environment. We used expression arrays to compare the effect on global patterns of gene expression of deleting a central regulatory gene, crp. Effects were measured in two lineages that had independently evolved for 20,000 generations and in their common ancestor. We found that deleting crp had a much more dramatic effect on the expression profile of the two evolved lines than on the ancestor. Because the sequence of the crp gene was unchanged during evolution, these differences indicate epistatic interactions between crp and mutations at other loci that accumulated during evolution. Moreover, a striking degree of parallelism was observed between the two independently evolved lines; 115 genes that were not crp-dependent in the ancestor became dependent on crp in both evolved lines. An analysis of changes in crp dependence of well-characterized regulons identified a number of regulatory genes as candidates for harboring beneficial mutations that could account for these parallel expression changes. Mutations within three of these genes have previously been found and shown to contribute to fitness. Overall, these findings indicate that epistasis has been important in the adaptive evolution of these lines, and they provide new insight into the types of genetic changes through which epistasis can evolve. More generally, we demonstrate that expression profiles can be profitably used to investigate epistatic interactions., Author Summary The effect of a genetic mutation can depend on the genotype of the organism in which it occurs. For example, a mutation that is beneficial in one genetic background might be neutral or even deleterious in another. The interactions between genes that cause this dependence—known as epistasis—play an important role in many evolutionary theories. However, they are difficult to study and remain poorly understood. We used a novel approach to examine the evolution of interactions arising between a key regulatory gene, crp, and mutations that occurred during the adaptation of a bacterium, Escherichia coli, to a laboratory environment. To do this, we measured the effect of deleting crp on the expression of all genes in the organism, providing a sensitive measure to identify new interactions involving this gene. We found that deleting crp had a dramatic and parallel effect on gene expression in two independently evolved populations, but much less effect in their ancestor. An analysis of these changes identified a number of regulatory genes as candidates for harboring beneficial mutations that could account for the parallel changes. These findings indicate that epistasis has played an important role in the evolution of these populations, and they provide insight into the types of genetic changes through which epistasis can evolve.

Interleukin (IL)-10 increases host susceptibility to microorganisms and is involved in intracellular persistence of bacterial pathogens. IL-10 is associated with chronic Q fever, an infectious disease due to the intracellular bacterium Coxiella burnetii. Nevertheless, accurate animal models of chronic C. burnetii infection are lacking. Transgenic mice constitutively expressing IL-10 in macrophages were infected with C. burnetti by intraperitoneal and intratracheal routes and infection was analyzed through real-time PCR and antibody production. Transgenic mice exhibited sustained tissue infection and strong antibody response in contrast to wild-type mice; thus, bacterial persistence was IL-10-dependent as in chronic Q fever. The number of granulomas was low in spleen and liver of transgenic mice infected through the intraperitoneal route, as in patients with chronic Q fever. Macrophages from transgenic mice were unable to kill C. burnetii. C. burnetii–stimulated macrophages were characterized by non-microbicidal transcriptional program consisting of increased expression of arginase-1, mannose receptor, and Ym1/2, in contrast to wild-type macrophages in which expression of inducible NO synthase and inflammatory cytokines was increased. In vivo results emphasized macrophage data. In spleen and liver of transgenic mice infected with C. burnetii by the intraperitoneal route, the expression of arginase-1 was increased while microbicidal pathway consisting of IL-12p40, IL-23p19, and inducible NO synthase was depressed. The overexpression of IL-10 in macrophages prevents anti-infectious competence of host, including the ability to mount granulomatous response and microbicidal pathway in tissues. To our knowledge, this is the first efficient model for chronic Q fever pathogenesis., Author Summary The interaction between immune system and invading bacteria is sufficient to eradicate microorganisms in the majority of bacterial infections, but the suppression of the microbicidal response leads to reactivation or chronic evolution of infections and to bacterial persistence. Coxiella burnetii, an obligate intracellular bacterium, is responsible for Q fever. This infectious disease is characterized by a primary infection that may become chronic as endocarditis in patients with valvular damage and immunocompromised patients. Clinical and in vitro studies have suggested a role for interleukin-10 in the chronic evolution of Q fever. However, an efficient mouse model for chronic Q fever pathogenesis, which could serve as a platform for anti–C. burnetii drug or immunotherapy development, is lacking. Here we use transgenic mice with constitutive overexpression of interleukin-10 in the macrophage lineage to study C. burnetii infection. We report an efficient mouse model for chronic Q fever pathogenesis, which associates high levels of specific antibodies, sustained tissue infection, and reduced granuloma formation, as in human Q fever. We also find an anti-inflammatory transcriptional program and altered expression of chemokines in infected tissues.

Previous model-based analysis of the metabolic network of Geobacter sulfurreducens suggested the existence of several redundant pathways. Here, we identified eight sets of redundant pathways that included redundancy for the assimilation of acetate, and for the conversion of pyruvate into acetyl-CoA. These equivalent pathways and two other sub-optimal pathways were studied using 5 single-gene deletion mutants in those pathways for the evaluation of the predictive capacity of the model. The growth phenotypes of these mutants were studied under 12 different conditions of electron donor and acceptor availability. The comparison of the model predictions with the resulting experimental phenotypes indicated that pyruvate ferredoxin oxidoreductase is the only activity able to convert pyruvate into acetyl-CoA. However, the results and the modeling showed that the two acetate activation pathways present are not only active, but needed due to the additional role of the acetyl-CoA transferase in the TCA cycle, probably reflecting the adaptation of these bacteria to acetate utilization. In other cases, the data reconciliation suggested additional capacity constraints that were confirmed with biochemical assays. The results demonstrate the need to experimentally verify the activity of key enzymes when developing in silico models of microbial physiology based on sequence-based reconstruction of metabolic networks., Author Summary Geobacter sulfurreducens is a member of the Geobacteraceae family of micro-organisms that breathe metals and have a unique mode of metabolism. Stimulation of the activity of this species in the environment has been shown to result in the removal of radioactive contaminants in groundwater. Similarly, the respiration of these micro-organisms also has been linked to electricity generation in a microbial fuel cell. Both the rate of electricity generation and the efficiency of ground water clean-up can be enhanced through the improved understanding of the growth and metabolism of Geobacteraceae. In order to better understand the growth and metabolism of this organism, we had constructed a large-scale mathematical model of the metabolic network of this organism. Using this model, we identified reaction alternates that sustain metabolism in the event of gene deletions. We then experimentally confirmed the role of these metabolic reactions through gene deletion mutants and biochemical assays and improved the predictive ability of the mathematical model. Such an integrated computational and experimental approach can be used to study the activity and function of metabolic network in a rapid manner for other poorly characterized organisms of environmental relevance.

Proliferation of bacterial pathogens in blood represents one of the most dangerous stages of infection. Growth in blood serum depends on the ability of a pathogen to adjust metabolism to match the availability of nutrients. Although certain nutrients are scarce in blood and need to be de novo synthesized by proliferating bacteria, it is unclear which metabolic pathways are critical for bacterial growth in blood. In this study, we identified metabolic functions that are essential specifically for bacterial growth in the bloodstream. We used two principally different but complementing techniques to comprehensively identify genes that are required for the growth of Escherichia coli in human serum. A microarray-based and a dye-based mutant screening approach were independently used to screen a library of 3,985 single-gene deletion mutants in all non-essential genes of E. coli (Keio collection). A majority of the mutants identified consistently by both approaches carried a deletion of a gene involved in either the purine or pyrimidine nucleotide biosynthetic pathway and showed a 20- to 1,000-fold drop in viable cell counts as compared to wild-type E. coli after 24 h of growth in human serum. This suggests that the scarcity of nucleotide precursors, but not other nutrients, is the key limitation for bacterial growth in serum. Inactivation of nucleotide biosynthesis genes in another Gram-negative pathogen, Salmonella enterica, and in the Gram-positive pathogen Bacillus anthracis, prevented their growth in human serum. The growth of the mutants could be rescued by genetic complementation or by addition of appropriate nucleotide bases to human serum. Furthermore, the virulence of the B. anthracis purE mutant, defective in purine biosynthesis, was dramatically attenuated in a murine model of bacteremia. Our data indicate that de novo nucleotide biosynthesis represents the single most critical metabolic function for bacterial growth in blood and reveal the corresponding enzymes as putative antibiotic targets for the treatment of bloodstream infections., Author Summary Bacterial growth in the bloodstream is a common manifestation of a number of bacterial infections. When growing in blood, bacteria not only have to evade the host's immune response, but also adjust their metabolism to suit availability of nutrients. Although the concentrations of various metabolites in human blood are known, it is difficult to predict which nutrients are abundant and which are scarce. To proliferate in human blood, bacteria need to synthesize metabolites that are present in the limiting concentrations. For that, they need to produce specific enzymes that are, thus, critical for the bacterial growth in the bloodstream. We carried out a comprehensive, genome-wide search for Escherichia coli genes that are essential for growth in human serum. We found that inactivation of nucleotide biosynthesis genes leads to a significant growth defect in human serum not only for E. coli but also for two other pathogens, Salmonella Typhimurium and Bacillus anthracis. The results of this study demonstrate that the limiting amounts of the nucleotide bases in human serum force invading pathogens to rely on de novo nucleotide biosynthesis. Hence, our findings reveal nucleotide biosynthesis enzymes as a possible target for the treatment of bloodstream infections.

The control of Mycobacterium tuberculosis (Mtb) infection is heavily dependent on the adaptive Th1 cellular immune response. Paradoxically, optimal priming of the Th1 response requires activation of priming dendritic cells with Th1 cytokine IFN-γ. At present, the innate cellular mechanisms required for the generation of an optimal Th1 T cell response remain poorly characterized. We hypothesized that innate Mtb-reactive T cells provide an early source of IFN-γ to fully activate Mtb-exposed dendritic cells. Here, we report the identification of a novel population of Mtb-reactive CD4− αβTCR+ innate thymocytes. These cells are present at high frequencies, respond to Mtb-infected cells by producing IFN-γ directly ex vivo, and display characteristics of effector memory T cells. This novel innate population of Mtb-reactive T cells will drive further investigation into the role of these cells in the containment of Mtb following infectious exposure. Furthermore, this is the first demonstration of a human innate pathogen-specific αβTCR+ T cell and is likely to inspire further investigation into innate T cells recognizing other important human pathogens., Author Summary Mycobacterium tuberculosis (Mtb) infects about one-third of the world's population. Most people who are exposed remain healthy, but control of this intracellular bacterium requires a robust cellular immune response. Production of the pro-inflammatory cytokine IFN-γ from cells in the adaptive immune response is critically important in the immune control of Mtb. However, this cytokine is also essential in initiating an optimal adaptive immune response. We hypothesized that innate cells could provide an early source of IFN-γ to aid in generation of an optimal adaptive immune response. We looked for IFN-γ producing cells in human neonates that were unlikely to have been previously exposed to either Mtb or other environmental mycobacteria. Here, we report the identification of a novel T cell population from the thymus that produces IFN-γ in response to Mtb-infected cells. Mtb-reactive thymocytes are present at high frequencies, are present in nearly all newborns tested, and display characteristics of T cells normally associated with a memory response. This novel innate population of Mtb-reactive cells will drive further investigation into the role of these cells in the containment of Mtb following infectious exposure and is likely to inspire further investigation into innate T cells recognizing other important human pathogens.

To what extent can modes of gene regulation be explained by systems-level properties of metabolic networks? Prior studies on co-regulation of metabolic genes have mainly focused on graph-theoretical features of metabolic networks and demonstrated a decreasing level of co-expression with increasing network distance, a naïve, but widely used, topological index. Others have suggested that static graph representations can poorly capture dynamic functional associations, e.g., in the form of dependence of metabolic fluxes across genes in the network. Here, we systematically tested the relative importance of metabolic flux coupling and network position on gene co-regulation, using a genome-scale metabolic model of Escherichia coli. After validating the computational method with empirical data on flux correlations, we confirm that genes coupled by their enzymatic fluxes not only show similar expression patterns, but also share transcriptional regulators and frequently reside in the same operon. In contrast, we demonstrate that network distance per se has relatively minor influence on gene co-regulation. Moreover, the type of flux coupling can explain refined properties of the regulatory network that are ignored by simple graph-theoretical indices. Our results underline the importance of studying functional states of cellular networks to define physiologically relevant associations between genes and should stimulate future developments of novel functional genomic tools., Author Summary Why do certain genes in a biological network show tight transcriptional co-regulation while others are more or less independently regulated? Prior studies showed that the degree of co-regulation between enzymatic genes decreases with their distance in the metabolic network. However, there are fundamental reasons to suspect that network distance is an incomplete descriptor of functional coherence (hence gene co-regulation), and other, biochemically more relevant measures, have been proposed to capture the functional dependencies between enzymes. We systematically examine whether flux coupling, a biochemically sound and computationally tractable measure of functional interaction between reactions, can better explain gene co-regulation than network distance in the metabolisms of Escherichia coli and Saccharomyces cerevisiae. After validating the flux coupling method using published experimental data on in vivo flux correlations (i.e., coherence of reaction usage), we demonstrate that it not only outperforms metabolic network distance in relation to in vivo flux correlations, but also in explaining transcriptional co-regulation and operonic organization. Future functional genomics studies could benefit from the concept of flux coupling by using it as a basis to test the reliability of computationally predicted functional associations.

How the innate immune system tailors specific responses to diverse microbial infections is not well understood. Cells use a limited number of host receptors and signaling pathways to both discriminate among extracellular and intracellular microbes, and also to generate responses commensurate to each threat. Here, we have addressed these questions by using DNA microarrays to monitor the macrophage transcriptional response to the intracellular bacterial pathogen Listeria monocytogenes. By utilizing combinations of host and bacterial mutants, we have defined the host transcriptional responses to vacuolar and cytosolic bacteria. These compartment-specific host responses induced significantly different sets of target genes, despite activating similar transcription factors. Vacuolar signaling was entirely MyD88-dependent, and induced the transcription of pro-inflammatory cytokines. The IRF3-dependent cytosolic response induced a distinct set of target genes, including IFNβ. Many of these cytosolic response genes were induced by secreted cytokines, so we further identified those host genes induced independent of secondary signaling. The host response to cytosolic bacteria was reconstituted by the cytosolic delivery of L. monocytogenes genomic DNA, but we observed an amplification of this response by NOD2 signaling in response to MDP. Correspondingly, the induction of IFNβ was reduced in nod2 −/− macrophages during infection with either L. monocytogenes or Mycobacterium tuberculosis. Combinatorial control of IFNβ induction by recognition of both DNA and MDP may highlight a mechanism by which the innate immune system integrates the responses to multiple ligands presented in the cytosol by intracellular pathogens., Author Summary Macrophages are critical cells of the innate immune system, contributing to immediate and robust defense against microbial infections. Macrophages detect pathogens using host receptors located on the cell surface, in phagosomal vacuoles, and in the cytosol. While fundamental to innate immunity, it is not clear if these different receptors merely provide redundant mechanisms for sensing microbial infection, or if instead they induce distinct gene expression programs that may allow for threat-specific host responses. We addressed this question by dissecting the macrophage transcriptional responses to the model intracellular bacterial pathogen Listeria monocytogenes. Using genetic and genomic approaches, we found that the macrophage response to L. monocytogenes trapped in phagosomal compartments was distinct and separable from the response to live bacteria replicating in the host cytosol. The macrophage response to cytosolic bacteria was recapitulated by bacterial nucleic acid and cell wall fragments, and induced surprisingly few primary response genes. These findings highlight a mechanism by which the innate immune system may specifically sense intracellular bacteria, as the macrophage response to Mycobacterium tuberculosis was similarly regulated.

Yersinia pseudotuberculosis binds to β1 integrin receptors, and uses the type III secretion proteins YopB and YopD to introduce pores and to translocate Yop effectors directly into host cells. Y. pseudotuberculosis lacking effectors that inhibit Rho GTPases, YopE and YopT, have high pore forming activity. Here, we present evidence that Y. pseudotuberculosis selectively modulates Rho activity to induce cellular changes that control pore formation and effector translocation. Inhibition of actin polymerization decreased pore formation and YopE translocation in HeLa cells infected with Y. pseudotuberculosis. Inactivation of Rho, Rac, and Cdc42 by treatment with Clostridium difficile toxin B inhibited pore formation and YopE translocation in infected HeLa cells. Expression of a dominant negative form of Rac did not reduce the uptake of membrane impermeable dyes in HeLa cells infected with a pore forming strain YopEHJT−. Similarly, the Rac inhibitor NSC23766 did not decrease pore formation or translocation, although it efficiently hindered Rac-dependent bacterial uptake. In contrast, C. botulinum C3 potently reduced pore formation and translocation, implicating Rho A, B, and/or C in the control of the Yop delivery. An invasin mutant (Y. pseudotuberculosis invD911E) that binds to β1 integrins, but inefficiently transduces signals through the receptors, was defective for YopE translocation. Interfering with the β1 integrin signaling pathway, by inhibiting Src kinase activity, negatively affected YopE translocation. Additionally, Y. pseudotuberculosis infection activated Rho by a mechanism that was dependent on YopB and on high affinity bacteria interaction with β1 integrin receptors. We propose that Rho activation, mediated by signals triggered by the YopB/YopD translocon and from engagement of β1 integrin receptors, stimulates actin polymerization and activates the translocation process, and that once the Yops are translocated, the action of YopE or YopT terminate delivery of Yops and prevents pore formation., Author Summary The type III secretion system (TTSS) is essential for the virulence of a number of Gram-negative human pathogens of enormous clinical significance. The molecular mechanisms by which TTSS effector proteins are translocated into the host cell are not well understood. The work presented here proposes a new model in which the enteropathogen Yersinia pseudotuberculosis manipulates the host cell machinery to control effector translocation. This involves activation of the host cell Rho GTPase by the cooperative action of adhesin-mediated high affinity binding to specific cell receptor molecules known as β1 integrins, and interaction of components of the TTSS with the host cell membrane. This molecular mechanism of controlling TTSS may not be restricted to Y. pseudotuberculosis and might take place during infection of host cells with other pathogens that encode homologues of Yersinia TTSS proteins. Our findings provide a good starting point to study the molecular nature of the complex interaction between bacterial pathogens bearing TTSSs and the host cell. Importantly, components that act by modulating the TTSS are potential targets for novel antimicrobials.

The type III secretion systems (TTSS) encoded in Salmonella pathogenicity island-1 and -2 (SPI-1 and -2) are virulence factors required for specific phases of Salmonella infection in animal hosts. However, the host cell types targeted by the TTSS have not been determined. To investigate this, we have constructed translational fusions between the ß-lactamase reporter and a broad array of TTSS effectors secreted via SPI-1, SPI-2, or both. Secretion of the fusion protein to a host cell was determined by cleavage of a specific fluorescent substrate. In cultured cells, secretion of all six effectors could be observed. However, two to four days following i.p. infection of mice, only effectors secreted by SPI-2 were detected in spleen cells. The cells targeted were identified via staining with nine different cell surface markers followed by FACS analysis as well as by conventional cytological methods. The targeted cells include B and T lymphocytes, neutrophils, monocytes, and dendritic cells, but not mature macrophages. To further investigate replication in these various cell types, Salmonella derivatives were constructed that express a red fluorescent protein. Bacteria could be seen in each of the cell types above; however, most viable bacteria were present in neutrophils. We find that Salmonella is capable of targeting most phagocytic and non-phagocytic cells in the spleen but has a surprisingly high preference for neutrophils. These findings suggest that Salmonella specifically target splenic neutrophils presumably to attenuate their microbicidal functions, thereby promoting intracellular survival and replication in the mouse., Author Summary Bacteria of the Salmonella genus are important human pathogens and a leading cause of food-borne illness. Salmonella species' ability to cause disease relies on the activities of two sophisticated molecular syringes that allow the bacteria to pump proteins into cells that they infect. The activities of these syringes have been studied extensively in cells grown under laboratory conditions and shown to be essential for the infectious process in animal models. However, the specific cells within infected organs that are targeted by these syringes have not been identified. In this work we describe the specific spleen cells targeted by Salmonella in the mouse. We find that Salmonella is capable of targeting most cell types using their molecular syringes. Quite surprisingly, we find that Salmonella mostly targets neutrophils, a cell type not thought to be associated with live Salmonella in host tissues. These findings challenge our current views of Salmonella infection and may lead to new insight for treating the disease.

Wolbachia are among the most widespread intracellular bacteria, carried by thousands of metazoan species. The success of Wolbachia is due to efficient vertical transmission by the host maternal germline. Some Wolbachia strains concentrate at the posterior of host oocytes, which promotes Wolbachia incorporation into posterior germ cells during embryogenesis. The molecular basis for this localization strategy is unknown. Here we report that the wMel Wolbachia strain relies upon a two-step mechanism for its posterior localization in oogenesis. The microtubule motor protein kinesin-1 transports wMel toward the oocyte posterior, then pole plasm mediates wMel anchorage to the posterior cortex. Trans-infection tests demonstrate that factors intrinsic to Wolbachia are responsible for directing posterior Wolbachia localization in oogenesis. These findings indicate that Wolbachia can direct the cellular machintery of host oocytes to promote germline-based bacterial transmission. This study also suggests parallels between Wolbachia localization mechanisms and those used by other intracellular pathogens., Author Summary This study focuses on Wolbachia, a genus of intracellular bacteria carried by insect and nematode host species. It was recently shown that Wolbachia carried into the human body by the host nematode Onchocerca volvulus trigger an immune response that leads to African river blindness. Findings like these raise fundamental questions of how Wolbachia interact with host cells to perpetuate Wolbachia infection. Distinct from many pathogenic bacteria, Wolbachia are transmitted throughout host populations primarily from females to their offspring, similar to mitochondrial inheritance. The molecular basis for this transmission strategy is unclear. Here we show that Wolbachia transmission is aided by a complex mechanism in egg development. Our study suggests that Wolbachia are transported inside the egg as cargo of molecular motors that walk along microtubule filaments. This directs Wolbachia to the posterior of maturing eggs, thus placing Wolbachia at the site where reproductive cells form during embryogenesis and ensuring Wolbachia integration into those cells. Furthermore, both factors intrinsic to Wolbachia and host molecules specifying reproductive cell fates are necessary to maximize posterior concentration of Wolbachia in the egg. This suggests that Wolbachia manipulate conserved cellular machinery in egg development to direct their transmission to the next host generation.

Gene duplication with subsequent interaction divergence is one of the primary driving forces in the evolution of genetic systems. Yet little is known about the precise mechanisms and the role of duplication divergence in the evolution of protein networks from the prokaryote and eukaryote domains. We developed a novel, model-based approach for Bayesian inference on biological network data that centres on approximate Bayesian computation, or likelihood-free inference. Instead of computing the intractable likelihood of the protein network topology, our method summarizes key features of the network and, based on these, uses a MCMC algorithm to approximate the posterior distribution of the model parameters. This allowed us to reliably fit a flexible mixture model that captures hallmarks of evolution by gene duplication and subfunctionalization to protein interaction network data of Helicobacter pylori and Plasmodium falciparum. The 80% credible intervals for the duplication–divergence component are [0.64, 0.98] for H. pylori and [0.87, 0.99] for P. falciparum. The remaining parameter estimates are not inconsistent with sequence data. An extensive sensitivity analysis showed that incompleteness of PIN data does not largely affect the analysis of models of protein network evolution, and that the degree sequence alone barely captures the evolutionary footprints of protein networks relative to other statistics. Our likelihood-free inference approach enables a fully Bayesian analysis of a complex and highly stochastic system that is otherwise intractable at present. Modelling the evolutionary history of PIN data, it transpires that only the simultaneous analysis of several global aspects of protein networks enables credible and consistent inference to be made from available datasets. Our results indicate that gene duplication has played a larger part in the network evolution of the eukaryote than in the prokaryote, and suggests that single gene duplications with immediate divergence alone may explain more than 60% of biological network data in both domains., PLoS Computational Biology, 3 (11), ISSN:1553-734X, ISSN:1553-7358

Infection of macrophages by Yersinia species results in YopJ-dependent apoptosis, and naïve macrophages are highly susceptible to this form of cell death. Previous studies have demonstrated that macrophages activated with lipopolysaccharide (LPS) prior to infection are resistant to YopJ-dependent cell death; we found this simultaneously renders macrophages susceptible to killing by YopJ− Yersinia pseudotuberculosis (Yptb). YopJ− Yptb-induced macrophage death was dependent on caspase-1 activation, resulting in rapid permeability to small molecules, followed by membrane breakdown and DNA damage, and accompanied by cleavage and release of proinflammatory interleukin-18. Induction of caspase-1-dependent death, or pyroptosis, required the bacterial type III translocon but none of its known translocated proteins. Wild-type Yptb infection also triggered pyroptosis: YopJ-dependent activation of proapoptotic caspase-3 was significantly delayed in activated macrophages and resulted in caspase-1-dependent pyroptosis. The transition to susceptibility was not limited to LPS activation; it was also seen in macrophages activated with other Toll-like receptor (TLR) ligands and intact nonviable bacteria. Yptb infection triggered macrophage activation and activation of caspase-1 in vivo. Y. pestis infection of activated macrophages also stimulated caspase-1 activation. These results indicate that host signaling triggered by TLR and other activating ligands during the course of Yersinia infection redirects both the mechanism of host cell death and the downstream consequences of death by shifting from noninflammatory apoptosis to inflammatory pyroptosis., Author Summary Pathogenic Yersinia are bacteria capable of interacting with host immune cells and inhibiting their function. Macrophages are potent antimicrobial immune cells that eliminate invading microbes, and represent a major target for Yersinia during infection. Yersinia triggers death of resting macrophages by apoptosis, a process thought to be advantageous for Yersinia growth during early stages of infection because important host cells are eliminated without perturbing the surrounding tissue. However, activated macrophages with enhanced antimicrobial activity play a crucial role in controlling Yersinia infection. To elucidate the mechanisms involved in successful defense against infection, the authors investigated the response of activated macrophages to Yersinia, which revealed induction of a proinflammatory cell death pathway termed pyroptosis. Unlike apoptosis, pyroptosis unleashes inflammatory mediators capable of enhancing immune responses and clearing bacteria. Macrophage activation and pyroptosis was observed in infected host tissue. Thus, regulating the mechanism of cell death is important for effective responses to Yersinia infection: activated macrophages resisting apoptosis are redirected to utilize pyroptosis, an inflammatory process facilitating host resistance.

Serratia marcescens is an entomopathogenic bacterium that opportunistically infects a wide range of hosts, including humans. In a model of septic injury, if directly introduced into the body cavity of Drosophila, this pathogen is insensitive to the host's systemic immune response and kills flies in a day. We find that S. marcescens resistance to the Drosophila immune deficiency (imd)-mediated humoral response requires the bacterial lipopolysaccharide O-antigen. If ingested by Drosophila, bacteria cross the gut and penetrate the body cavity. During this passage, the bacteria can be observed within the cells of the intestinal epithelium. In such an oral infection model, the flies succumb to infection only after 6 days. We demonstrate that two complementary host defense mechanisms act together against such food-borne infection: an antimicrobial response in the intestine that is regulated by the imd pathway and phagocytosis by hemocytes of bacteria that have escaped into the hemolymph. Interestingly, bacteria present in the hemolymph elicit a systemic immune response only when phagocytosis is blocked. Our observations support a model wherein peptidoglycan fragments released during bacterial growth activate the imd pathway and do not back a proposed role for phagocytosis in the immune activation of the fat body. Thanks to the genetic tools available in both host and pathogen, the molecular dissection of the interactions between S. marcescens and Drosophila will provide a useful paradigm for deciphering intestinal pathogenesis., Author Summary The gut is a crucial interface of the host with its environment and represents an important portal of entry for pathogens. Here, we have developed a novel model of intestinal infections in the genetic model organism Drosophila melanogaster using the potent entomopathogen bacterium Serratia marcescens. In contrast to other enteropathogens, this bacterium traverses the intestinal epithelium despite a local immune response and gains access to the body cavity of the fruit fly. The cellular arm of innate immunity controls its proliferation in the hemocoele. Interestingly, ingested bacteria that have moved to the hemolymph compartment are not detected by the humoral immune system of the fly unless phagocytosis is blocked. In a septic injury model, S. marcescens kills its host in a day. In contrast, the flies succumb slowly to an intestinal infection, even though the bacterium is present in the hemolymph. We surmise that the bacterium expresses distinct virulence programs according to the mode of infection. Thanks to the genetic tools available in both host and pathogen, the molecular dissection of the interactions between S. marcescens and Drosophila will provide a useful paradigm to decipher intestinal pathogenesis.

Chlamydia spp. are intracellular obligate bacterial pathogens that infect a wide range of host cells. Here, we show that C. caviae enters, replicates, and performs a complete developmental cycle in Drosophila SL2 cells. Using this model system, we have performed a genome-wide RNA interference screen and identified 54 factors that, when depleted, inhibit C. caviae infection. By testing the effect of each candidate's knock down on L. monocytogenes infection, we have identified 31 candidates presumably specific of C. caviae infection. We found factors expected to have an effect on Chlamydia infection, such as heparansulfate glycosaminoglycans and actin and microtubule remodeling factors. We also identified factors that were not previously described as involved in Chlamydia infection. For instance, we identified members of the Tim-Tom complex, a multiprotein complex involved in the recognition and import of nuclear-encoded proteins to the mitochondria, as required for C. caviae infection of Drosophila cells. Finally, we confirmed that depletion of either Tom40 or Tom22 also reduced C. caviae infection in mammalian cells. However, C. trachomatis infection was not affected, suggesting that the mechanism involved is C. caviae specific., Author Summary Chlamydia spp. are intracellular bacterial pathogens that infect a wide range of hosts and cause various diseases, including preventable blindness in developing countries, sexually transmitted disease, and pneumonia. Chlamydia spp. are able to establish their replication niche inside the host cell, residing in a membrane-bound compartment that serves as a protector shield against immune surveillance and antimicrobial agents but also acts as a “filter” to exchange factors with the host cell. Despite the primary importance of Chlamydia for human health, little is known about the mechanisms underlying the infection process. The study of Chlamydia pathogenesis is challenging because Chlamydia spp. are not amenable to genetic manipulation and it is difficult to conduct extensive genetic approaches in the mammalian host. To circumvent these difficulties, we have used Drosophila cells to model Chlamydia infection. We conducted a genome-wide RNA interference screen and identified host factors that, when depleted, reduce Chlamydia infection. Validating our approach, we further showed that the identified factors were also required for infection in mammalian cells. This work will help us better understand the complex interaction between Chlamydia and its host and potentially identify novel targets for therapeutic treatment.

Analysis of mycobacterial strains that have lost their ability to cause disease is a powerful approach to identify yet unknown virulence determinants and pathways involved in tuberculosis pathogenesis. Two of the most widely used attenuated strains in the history of tuberculosis research are Mycobacterium bovis BCG (BCG) and Mycobacterium tuberculosis H37Ra (H37Ra), which both lost their virulence during in vitro serial passage. Whereas the attenuation of BCG is due mainly to loss of the ESAT-6 secretion system, ESX-1, the reason why H37Ra is attenuated remained unknown. However, here we show that a point mutation (S219L) in the predicted DNA binding region of the regulator PhoP is involved in the attenuation of H37Ra via a mechanism that impacts on the secretion of the major T cell antigen ESAT-6. Only H37Ra “knock-ins” that carried an integrated cosmid with the wild-type phoP gene from M. tuberculosis H37Rv showed changes in colony morphology, increased virulence, ESAT-6 secretion, and induction of specific T cell responses, whereas other H37Ra constructs did not. This finding established a link between the PhoP regulator and ESAT-6 secretion that opens exciting new perspectives for elucidating virulence regulation in M. tuberculosis., Author Summary Mycobacterium tuberculosis, the causative agent of human tuberculosis is an extremely successful human pathogen in spite of its lack of classical virulence factors, such as toxins. The pathogenesis of this bacterium is closely linked with its ability to circumvent destruction by the host cell, which depends on a large variety of mycobacterial lipids and secreted proteins. Genome comparison of fully virulent strains with closely related, but attenuated strains that have lost their ability to cause disease is a powerful approach to identify factors contributing to mycobacterial virulence. In this study we have compared the virulent paradigm strain of tuberculosis research M. tuberculosis H37Rv, with the widely used, attenuated laboratory variant, H37Ra. This analysis, combined with gene complementation studies, has identified a mutation in the two-component system regulator PhoP that impacts on the secretion and T cell recognition of the 6-kD early-secreted antigenic target ESAT-6. In previous studies, both PhoP and the ESAT-6 secretion system ESX-1 have been identified independently as major virulence determinants of M. tuberculosis, so the finding that their action is interconnected opens exciting new insights and perspectives into the virulence regulation of tubercle bacilli.

For over 30 years a phospholipase C enzyme called alpha-toxin was thought to be the key virulence factor in necrotic enteritis caused by Clostridium perfringens. However, using a gene knockout mutant we have recently shown that alpha-toxin is not essential for pathogenesis. We have now discovered a key virulence determinant. A novel toxin (NetB) was identified in a C. perfringens strain isolated from a chicken suffering from necrotic enteritis (NE). The toxin displayed limited amino acid sequence similarity to several pore forming toxins including beta-toxin from C. perfringens (38% identity) and alpha-toxin from Staphylococcus aureus (31% identity). NetB was only identified in C. perfringens type A strains isolated from chickens suffering NE. Both purified native NetB and recombinant NetB displayed cytotoxic activity against the chicken leghorn male hepatoma cell line LMH; inducing cell rounding and lysis. To determine the role of NetB in NE a netB mutant of a virulent C. perfringens chicken isolate was constructed by homologous recombination, and its virulence assessed in a chicken disease model. The netB mutant was unable to cause disease whereas the wild-type parent strain and the netB mutant complemented with a wild-type netB gene caused significant levels of NE. These data show unequivocally that in this isolate a functional NetB toxin is critical for the ability of C. perfringens to cause NE in chickens. This novel toxin is the first definitive virulence factor to be identified in avian C. perfringens strains capable of causing NE. Furthermore, the netB mutant is the first rationally attenuated strain obtained in an NE-causing isolate of C. perfringens; as such it has considerable vaccine potential., Author Summary Clostridium perfringens can cause gas gangrene and food poisoning in humans and causes several enterotoxemic diseases in animals including avian necrotic enteritis. This disease affects all chicken producing countries worldwide and is a considerable burden on the commercial chicken production industry. Until recently alpha-toxin was thought to be the major virulence factor involved in necrotic enteritis. However, by using an alpha-toxin null mutant it has been demonstrated that this toxin is not essential for disease. This paper details the identification and characterisation of a novel toxin, NetB, and provides evidence that the protein is an essential factor in causing necrotic enteritis in chickens. NetB has limited protein sequence identity to the beta-toxin of C. perfringens, which causes mucosal necrosis of the small intestine in humans and animals. We demonstrate that NetB null mutants can no longer cause disease in chickens, whereas both the wild-type and mutant complemented with a wild-type netB gene caused significant levels of necrotic enteritis. The identification of this important toxin advances our understanding of the pathogenesis of the disease and opens significant opportunities for the development of novel vaccines against necrotic enteritis in poultry.

Transcription factors (TFs) form large paralogous gene families and have complex evolutionary histories. Here, we ask whether putative orthologs of TFs, from bidirectional best BLAST hits (BBHs), are evolutionary orthologs with conserved functions. We show that BBHs of TFs from distantly related bacteria are usually not evolutionary orthologs. Furthermore, the false orthologs usually respond to different signals and regulate distinct pathways, while the few BBHs that are evolutionary orthologs do have conserved functions. To test the conservation of regulatory interactions, we analyze expression patterns. We find that regulatory relationships between TFs and their regulated genes are usually not conserved for BBHs in Escherichia coli K12 and Bacillus subtilis. Even in the much more closely related bacteria Vibrio cholerae and Shewanella oneidensis MR-1, predicting regulation from E. coli BBHs has high error rates. Using gene–regulon correlations, we identify genes whose expression pattern differs between E. coli and S. oneidensis. Using literature searches and sequence analysis, we show that these changes in expression patterns reflect changes in gene regulation, even for evolutionary orthologs. We conclude that the evolution of bacterial regulation should be analyzed with phylogenetic trees, rather than BBHs, and that bacterial regulatory networks evolve more rapidly than previously thought., Author Summary Living organisms use transcription factors (TFs) to control the production of proteins. For example, the bacterium E. coli contains a TF that prevents it from making enzymes that degrade lactose when lactose is absent. Bacterial genomes encode a huge diversity of TFs, and except in a few well-studied organisms, the function of these TFs is not known. To predict the function of a TF, biologists often search for a similar TF, from another organism, that has been characterized. It is generally believed that orthologous TFs—TFs that are derived from the organisms' common ancestor—will have conserved functions. The authors show that a commonly used method to identify orthologous TFs gives misleading results when applied to distantly related bacteria: the “orthologous” TFs are evolutionarily distant, they sense different signals, and they regulate different pathways. Biologists often predict, more specifically, that orthologous TFs will regulate orthologous genes. However, the authors show that even in more closely related bacteria, where the orthologous TFs do have conserved functions, these specific predictions are often incorrect. It seems that gene regulation in bacteria evolves rapidly, and it will be difficult to predict regulation in diverse bacteria from our knowledge of a few well-studied bacteria.

Marine sediments are frequently covered by mats of the filamentous Beggiatoa and other large nitrate-storing bacteria that oxidize hydrogen sulfide using either oxygen or nitrate, which they store in intracellular vacuoles. Despite their conspicuous metabolic properties and their biogeochemical importance, little is known about their genetic repertoire because of the lack of pure cultures. Here, we present a unique approach to access the genome of single filaments of Beggiatoa by combining whole genome amplification, pyrosequencing, and optical genome mapping. Sequence assemblies were incomplete and yielded average contig sizes of approximately 1 kb. Pathways for sulfur oxidation, nitrate and oxygen respiration, and CO2 fixation confirm the chemolithoautotrophic physiology of Beggiatoa. In addition, Beggiatoa potentially utilize inorganic sulfur compounds and dimethyl sulfoxide as electron acceptors. We propose a mechanism of vacuolar nitrate accumulation that is linked to proton translocation by vacuolar-type ATPases. Comparative genomics indicates substantial horizontal gene transfer of storage, metabolic, and gliding capabilities between Beggiatoa and cyanobacteria. These capabilities enable Beggiatoa to overcome non-overlapping availabilities of electron donors and acceptors while gliding between oxic and sulfidic zones. The first look into the genome of these filamentous sulfur-oxidizing bacteria substantially deepens the understanding of their evolution and their contribution to sulfur and nitrogen cycling in marine sediments., Author Summary In 1888 Winogradsky proposed the concept of chemolithotrophy—growth using inorganic compounds as an energy source—after studying the sulfur bacterium Beggiatoa. These filamentous bacteria and related organisms inhabit the surface of marine and freshwater sediments, where they oxidize hydrogen sulfide using either oxygen or nitrate. In particular, conspicuously large marine representatives accumulate nitrate in vacuoles to survive anoxia, a unique feature among prokaryotes. Since nitrate-storing Beggiatoa are not available in pure culture, we amplified and sequenced the genomic DNA of single multicellular filaments. We comprehensively tested the incomplete sequence assemblies for foreign DNA. We show that the Beggiatoa genome encodes the pathways of chemolithoautotrophy but also appears to support the use of alternative electron donors and acceptors. We propose that vacuolar-type ATPases generate an electrochemical gradient to drive nitrate transport over the vacuole membrane, a mechanism similar to eukaryotic solute accumulation. Intriguingly, we found evidence for substantial gene exchange between Beggiatoa and cyanobacteria. In both phyla, hemagglutinins are possibly involved in filament formation. The breadth of storage and metabolic capabilities encoded in its genome enables Beggiatoa to act as a “rechargeable battery,” which glides between oxic and sulfidic zones to overcome non-overlapping availabilities of electron donors and acceptors., Examining the genome of single filaments of Beggiatoa deepens our understanding of their contribution to sulfur and nitrogen cycling in marine sediments.

An increasing number of small RNAs (sRNAs) have been shown to regulate critical pathways in prokaryotes and eukaryotes. In bacteria, regulation by trans-encoded sRNAs is predominantly found in the coordination of intricate stress responses. The mechanisms by which sRNAs modulate expression of its targets are diverse. In common to most is the possibility that interference with the translation of mRNA targets may also alter the abundance of functional sRNAs. Aiming to understand the unique role played by sRNAs in gene regulation, we studied examples from two distinct classes of bacterial sRNAs in Escherichia coli using a quantitative approach combining experiment and theory. Our results demonstrate that sRNA provides a novel mode of gene regulation, with characteristics distinct from those of protein-mediated gene regulation. These include a threshold-linear response with a tunable threshold, a robust noise resistance characteristic, and a built-in capability for hierarchical cross-talk. Knowledge of these special features of sRNA-mediated regulation may be crucial toward understanding the subtle functions that sRNAs can play in coordinating various stress-relief pathways. Our results may also help guide the design of synthetic genetic circuits that have properties difficult to attain with protein regulators alone., Author Summary The activation of stress response programs, while crucial for the survival of a bacterial cell under stressful conditions, is costly in terms of energy and substrates and risky to the normal functions of the cell. Stress response is therefore tightly regulated. A recently discovered layer of regulation involves small RNA molecules, which bind the mRNA transcripts of their targets, inhibit their translation, and promote their cleavage. To understand the role that small RNA plays in regulation, we have studied the quantitative aspects of small RNA regulation by integrating mathematical modeling and quantitative experiments in Escherichia coli. We have demonstrated that small RNAs can tightly repress their target genes when their synthesis rate is smaller than some threshold, but have little or no effect when the synthesis rate is much larger than that threshold. Importantly, the threshold level is set by the synthesis rate of the small RNA itself and can be dynamically tuned. The effect of biochemical properties—such as the binding affinity of the two RNA molecules, which can only be altered on evolutionary time scales—is limited to setting a hierarchical order among different targets of a small RNA, facilitating in principle a global coordination of stress response., In bacteria, small RNAs can regulate the expression of genes at the translational level. The many advantages of this type of control include a tuneable threshold response and resistance to biochemical noise.

Comparing gene expression profiles over many different conditions has led to insights that were not obvious from single experiments. In the same way, comparing patterns of natural selection across a set of ecologically distinct species may extend what can be learned from individual genome-wide surveys. Toward this end, we show how variation in protein evolutionary rates, after correcting for genome-wide effects such as mutation rate and demographic factors, can be used to estimate the level and types of natural selection acting on genes across different species. We identify unusually rapidly and slowly evolving genes, relative to empirically derived genome-wide and gene family-specific background rates for 744 core protein families in 30 γ-proteobacterial species. We describe the pattern of fast or slow evolution across species as the “selective signature” of a gene. Selective signatures represent a profile of selection across species that is predictive of gene function: pairs of genes with correlated selective signatures are more likely to share the same cellular function, and genes in the same pathway can evolve in concert. For example, glycolysis and phenylalanine metabolism genes evolve rapidly in Idiomarina loihiensis, mirroring an ecological shift in carbon source from sugars to amino acids. In a broader context, our results suggest that the genomic landscape is organized into functional modules even at the level of natural selection, and thus it may be easier than expected to understand the complex evolutionary pressures on a cell., Author Summary Natural selection promotes the survival of the fittest individuals within a species. Over many generations, this may result in the maintenance of ancestral traits (conservation through purifying selection), or the emergence of newly beneficial traits (adaptation through positive selection). At the genetic level, long-term purifying or positive selection can cause genes to evolve more slowly, or more rapidly, providing a way to identify these evolutionary forces. While some genes are subject to consistent purifying or positive selection in most species, other genes show unexpected levels of selection in a particular species or group of species—a pattern we refer to as the “selective signature” of the gene. In this work, we demonstrate that these patterns of natural selection can be mined for information about gene function and species ecology. In the future, this method could be applied to any set of related species with fully sequenced genomes to better understand the genetic basis of ecological divergence.

While pleiotropic adaptive mutations are thought to be central for evolution, little is known on the downstream molecular effects allowing adaptation to complex ecologically relevant environments. Here we show that Escherichia coli MG1655 adapts rapidly to the intestine of germ-free mice by single point mutations in EnvZ/OmpR two-component signal transduction system, which controls more than 100 genes. The selective advantage conferred by the mutations that modulate EnvZ/OmpR activities was the result of their independent and additive effects on flagellin expression and permeability. These results obtained in vivo thus suggest that global regulators may have evolved to coordinate activities that need to be fine-tuned simultaneously during adaptation to complex environments and that mutations in such regulators permit adjustment of the boundaries of physiological adaptation when switching between two very distinct environments., Author Summary The mammalian intestine is a privileged physiological site to study how coevolution between hosts and the trillions of bacteria present in the microbiota has shaped the genome of each partner and promoted the development of mutualistic interactions. Herein we have used germ-free mice, a simplified albeit ecologically relevant system, to analyse intestinal adaptation of a model bacterial strain, Escherichia coli MG1655. Our results show that single point mutations in the ompB master regulator confer a striking selective adaptive advantage. OmpB comprises EnvZ, a transmembrane sensor with a dual kinase/phosphatase activity, and OmpR, a transcription factor controlling more than 100 target genes. In response to environmental changes, EnvZ modulates the phosphorylation and thereby the transcriptional activity of OmpR. We further show that the selective advantage conferred by OmpB mutations is related to their additive and independent effects on genes regulating permeability and flagellin expression, two major set of genes controlled by OmpR. These results suggest that global regulators may have evolved to coordinate physiological activities necessary for adaptation to complex environments and that mutations offer a complementary genetic mechanism to adjust the scale of the physiological regulation controlled by these regulators in distinct environments.

Specialized secretion systems of pathogenic bacteria commonly transport multiple effectors that act in concert to control and exploit the host cell as a replication-permissive niche. Both the Mycobacterium marinum and the Mycobacterium tuberculosis genomes contain an extended region of difference 1 (extRD1) locus that encodes one such pathway, the early secretory antigenic target 6 (ESAT-6) system 1 (ESX-1) secretion apparatus. ESX-1 is required for virulence and for secretion of the proteins ESAT-6, culture filtrate protein 10 (CFP-10), and EspA. Here, we show that both Rv3881c and its M. marinum homolog, Mh3881c, are secreted proteins, and disruption of RD1 in either organism blocks secretion. We have renamed the Rv3881c/Mh3881c gene espB for ESX-1 substrate protein B. Secretion of M. marinum EspB (EspBM) requires both the Mh3879c and Mh3871 genes within RD1, while CFP-10 secretion is not affected by disruption of Mh3879c. In contrast, disruption of Mh3866 or Mh3867 within the extRD1 locus prevents CFP-10 secretion without effect on EspBM. Mutants that fail to secrete only EspBM or only CFP-10 are less attenuated in macrophages than mutants failing to secrete both substrates. EspBM physically interacts with Mh3879c; the M. tuberculosis homolog, EspBT, physically interacts with Rv3879c; and mutants of EspBM that fail to bind Mh3879c fail to be secreted. We also found interaction between Rv3879c and Rv3871, a component of the ESX-1 machine, suggesting a mechanism for the secretion of EspB. The results establish EspB as a substrate of ESX-1 that is required for virulence and growth in macrophages and suggests that the contribution of ESX-1 to virulence may arise from the secretion of multiple independent substrates., Author Summary A major mechanism used by pathogenic bacteria for disabling host defenses is secretion of virulence proteins. These effectors are often transported by specialized secretion machines. One such pathway, present in Mycobacterium and other Gram-positive genera, is ESX-1 (early secretory antigenic target 6 system 1). Although ESX-1 is required for multiple phenotypes related to the pathogenesis of infection, only three substrates of the secretion machine have been identified to date, and the mechanism by which these substrates are exported is not understood. In our efforts to understand this virulence-related secretion mechanism, we identified a novel substrate and found that its delivery to the ESX-1 machine requires different protein interactions than previously identified substrates. Finally, we present data that the various ESX-1 substrates contribute additively to virulence. These data are incorporated into a model of ESX-1 function.

Airway infection by the Gram-positive pathogen Streptococcus pneumoniae (Sp) leads to recruitment of neutrophils but limited bacterial killing by these cells. Co-colonization by Sp and a Gram-negative species, Haemophilus influenzae (Hi), provides sufficient stimulus to induce neutrophil and complement-mediated clearance of Sp from the mucosal surface in a murine model. Products from Hi, but not Sp, also promote killing of Sp by ex vivo neutrophil-enriched peritoneal exudate cells. Here we identify the stimulus from Hi as its peptidoglycan. Enhancement of opsonophagocytic killing was facilitated by signaling through nucleotide-binding oligomerization domain-1 (Nod1), which is involved in recognition of γ-D-glutamyl-meso-diaminopimelic acid (meso-DAP) contained in cell walls of Hi but not Sp. Neutrophils from mice treated with Hi or compounds containing meso-DAP, including synthetic peptidoglycan fragments, showed increased Sp killing in a Nod1-dependent manner. Moreover, Nod1−/− mice showed reduced Hi-induced clearance of Sp during co-colonization. These observations offer insight into mechanisms of microbial competition and demonstrate the importance of Nod1 in neutrophil-mediated clearance of bacteria in vivo., Author Summary Pathogens are generally studied in the laboratory one species at a time. Most exist, however, in complex environments where they must adapt not only to their host but also to other members of the microbial flora. Using a mouse model of co-colonization, we have shown that one bacterial species (Haemophilus influenzae) can take advantage of the innate immune response of its host to outcompete and eliminate another species (Streptococcus pneumoniae) that resides in the same microenvironment of the upper respiratory tract. The molecular mechanism for this effect involves recognition of a cell wall fragment found on H. influenzae, but not on S. pneumoniae. The response to this immunostimulatory fragment requires Nod1, a host molecule that transmits inflammatory signals in response to specific peptides of the bacterial cell wall. This Nod1-mediated inflammatory stimulation triggers an increase in the ability of a type of white blood cell (neutrophil) to engulf and then kill S. pneumoniae, effectively removing it from its niche on the mucosal surface of the host airway. Our study, therefore, provides a demonstration of the importance of Nod1 in neutrophil-mediated clearance of bacterial infection. In addition, we have described a mechanism for interspecies competition between microbes that occurs through selective stimulation of host innate immune responses.

Filtration usually eliminates water-living bacteria. Here, we report on the complete genome sequence of Minibacterium massiliensis, a β-proteobacteria that was recovered from 0.22-μm filtered water used for patients in the hospital. The unexpectedly large 4,110,251-nucleotide genome sequence of M. massiliensis was determined using the traditional shotgun sequencing approach. Bioinformatic analyses shows that the M. massiliensis genome sequence illustrates characteristic features of water-living bacteria, including overrepresentation of genes encoding transporters and transcription regulators. Phylogenomic analysis based on the gene content of available bacterial genome sequences displays a congruent evolution of water-living bacteria from various taxonomic origins, principally for genes involved in energy production and conversion, cell division, chromosome partitioning, and lipid metabolism. This phylogenomic clustering partially results from lateral gene transfer, which appears to be more frequent in water than in other environments. The M. massiliensis genome analyses strongly suggest that water-living bacteria are a common source for genes involved in heavy-metal resistance, antibiotics resistance, and virulence factors., Author Summary Microorganisms are ubiquitous, found in environments including humans and animals, air, soil, and water, even in extreme conditions. Indeed, we isolated an emerging small bacterium M. massiliensis in hemodialysis water despite microbiological control by filtration and chemicals. Its very small size allowed this bacterium to pass through filters. Decoding of its genome revealed the presence of numerous so-called heavy-metal resistance genes encoding protection against chemicals. The genome also encodes virulence factors and antibiotic resistances. Study of M. massiliensis gene content revealed that it shares many genes with other bacteria in its β-proteobacteria family, but also with many other water-living bacteria from other families. Comparison of the M. massiliensis genome with other completely sequenced genomes indicated that a high fraction of genes (17%) had closest neighbors in water-living bacteria from other families. Such lateral gene transfer was further generalized to all water-living bacteria, which mutualize a higher fraction of their genome than bacteria living in other environments. Water is a privileged ecosystem for the exchange of bacterial genes and the emergence of new combinations of virulence and resistance. As new technologies increase the contact of humans with water, its use for medical and recreational usages has to be thoroughly controlled.

Geographic partitioning is postulated to foster divergence of Helicobacter pylori populations as an adaptive response to local differences in predominant host physiology. H. pylori's ability to establish persistent infection despite host inflammatory responses likely involves active management of host defenses using bacterial proteins that may themselves be targets for adaptive evolution. Sequenced H. pylori genomes encode a family of eight or nine secreted proteins containing repeat motifs that are characteristic of the eukaryotic Sel1 regulatory protein, whereas the related Campylobacter and Wolinella genomes each contain only one or two such “Sel1-like repeat” (SLR) genes (“slr genes”). Signatures of positive selection (ratio of nonsynonymous to synonymous mutations, dN/dS = ω > 1) were evident in the evolutionary history of H. pylori slr gene family expansion. Sequence analysis of six of these slr genes (hp0160, hp0211, hp0235, hp0519, hp0628, and hp1117) from representative East Asian, European, and African H. pylori strains revealed that all but hp0628 had undergone positive selection, with different amino acids often selected in different regions. Most striking was a divergence of Japanese and Korean alleles of hp0519, with Japanese alleles having undergone particularly strong positive selection (ωJ > 25), whereas alleles of other genes from these populations were intermingled. Homology-based structural modeling localized most residues under positive selection to SLR protein surfaces. Rapid evolution of certain slr genes in specific H. pylori lineages suggests a model of adaptive change driven by selection for fine-tuning of host responses, and facilitated by geographic isolation. Characterization of such local adaptations should help elucidate how H. pylori manages persistent infection, and potentially lead to interventions tailored to diverse human populations., Author Summary Helicobacter pylori is a genetically diverse bacterial species that infects billions of people worldwide, typically for decades. Long-term infection is a major risk factor for stomach ulcers and cancer, although most infections are benign, and the risks of various disease outcomes vary markedly among human populations. Analyses of housekeeping genes, whose encoded proteins perform normal cellular metabolic functions, had established that H. pylori strains from different geographic regions differed in their DNA sequences. Here, we analyzed the H. pylori slr multigene family that encodes up to nine secreted proteins (called SLR proteins) quite similar to the human protein Sel1. We showed that most members of the H. pylori slr gene family evolved significantly more rapidly than normal housekeeping genes. Different amino acids were selected in different H. pylori lineages, often on exposed surfaces of SLR proteins where they were potentially positioned to interact with host components. We propose that these amino acid differences affect the SLR protein function, likely contributing to H. pylori's adaptation to local differences in human stomach physiology. Further characterization of H. pylori proteins with lineage-specific differences in amino acids should improve understanding of geographic differences in H. pylori–host interactions and human disease, and of the interplay between different evolutionary forces in natural populations of any species.

Salmonella enterica subspecies can establish persistent, systemic infections in mammals, including human typhoid fever. Persistent S. enterica disease is characterized by an initial acute infection that develops into an asymptomatic chronic infection. During both the acute and persistent stages, the bacteria generally reside within professional phagocytes, usually macrophages. It is unclear how salmonellae can survive within macrophages, cells that evolved, in part, to destroy pathogens. Evidence is presented that during the establishment of persistent murine infection, macrophages that contain S. enterica serotype Typhimurium are hemophagocytic. Hemophagocytic macrophages are characterized by the ingestion of non-apoptotic cells of the hematopoietic lineage and are a clinical marker of typhoid fever as well as certain other infectious and genetic diseases. Cell culture assays were developed to evaluate bacterial survival in hemophagocytic macrophages. S. Typhimurium preferentially replicated in macrophages that pre-phagocytosed viable cells, but the bacteria were killed in macrophages that pre-phagocytosed beads or dead cells. These data suggest that during persistent infection hemophagocytic macrophages may provide S. Typhimurium with a survival niche., Author Summary Microbes that establish persistent infections present serious problems for world health but are not well understood. The bacteria Salmonella enterica cause asymptomatic chronic infection in humans. Carriers shed the bacteria into the environment, leading to periodic acute typhoid fever epidemics. Antibiotics are effective at treating typhoid fever, but Salmonellae strains resistant to multiple antibiotics have caused recent epidemics. New therapeutic strategies are needed and may develop from a molecular understanding of how the bacteria avoid killing by our immune systems. During acute and chronic infection, Salmonellae reside within macrophages, a kind of white blood cell type that normally destroys bacteria. Evidence is presented that during the establishment of chronic infection of mice, the bacteria can live within a special kind of macrophage. Hemophagocytic macrophages are macrophages that have ingested white and red blood cells. They are a clinical marker of typhoid fever and many other kinds of microbial infections. Cell culture assays showed that Salmonellae preferentially survive in hemophagocytic macrophages. These data suggest that hemophagocytic macrophages may provide S. Typhimurium with a survival niche during chronic infection. Moreover, a natural mouse model and a cell culture assay now exist for studying the medically important phenomenon of hemophagocytosis.

Infectious diseases result in millions of deaths each year. Mechanisms of infection have been studied in detail for many pathogens. However, many questions are relatively unexplored. What are the properties of human proteins that interact with pathogens? Do pathogens interact with certain functional classes of human proteins? Which infection mechanisms and pathways are commonly triggered by multiple pathogens? In this paper, to our knowledge, we provide the first study of the landscape of human proteins interacting with pathogens. We integrate human–pathogen protein–protein interactions (PPIs) for 190 pathogen strains from seven public databases. Nearly all of the 10,477 human-pathogen PPIs are for viral systems (98.3%), with the majority belonging to the human–HIV system (77.9%). We find that both viral and bacterial pathogens tend to interact with hubs (proteins with many interacting partners) and bottlenecks (proteins that are central to many paths in the network) in the human PPI network. We construct separate sets of human proteins interacting with bacterial pathogens, viral pathogens, and those interacting with multiple bacteria and with multiple viruses. Gene Ontology functions enriched in these sets reveal a number of processes, such as cell cycle regulation, nuclear transport, and immune response that participate in interactions with different pathogens. Our results provide the first global view of strategies used by pathogens to subvert human cellular processes and infect human cells. Supplementary data accompanying this paper is available at http://staff.vbi.vt.edu/dyermd/publications/dyer2008a.html., Author Summary Many pathogens, such as viruses and bacteria, cause disease in humans. Pathogen infections result in illness and death for millions of people each year. Pathogens communicate with human cells through physical interactions with various human proteins on the surface of the cell and within the interior of the cell. These interactions allow the pathogen to enter the host cell, manipulate important cellular processes, multiply, and invade other cells. In this paper, we compare interactions between human and pathogen proteins from 190 different pathogens to provide important insights into strategies used by pathogens to infect human cells. We show that both viral and bacterial proteins interact with human proteins that themselves interact with many human proteins or with human proteins that lie on many communication channels between other human proteins. Pathogens may have evolved to interact with these human proteins since they may control critical human cellular process. We also demonstrate that many viruses share common infection strategies, e.g., lengthening particular stages of the cell cycle, controlling programmed cell death, and interacting with the nuclear membrane to transfer viral genetic material into and out of the nucleus. Such studies may help us better understand the process of infection and identify better strategies to prevent or cure infection.

Campylobacter jejuni is one of the major causes of infectious diarrhea world-wide, although relatively little is know about its mechanisms of pathogenicity. This bacterium can gain entry into intestinal epithelial cells, which is thought to be important for its ability to persistently infect and cause disease. We found that C. jejuni is able to survive within intestinal epithelial cells. However, recovery of intracellular bacteria required pre-culturing under oxygen-limiting conditions, suggesting that C. jejuni undergoes significant physiological changes within the intracellular environment. We also found that in epithelial cells the C. jejuni–containing vacuole deviates from the canonical endocytic pathway immediately after a unique caveolae-dependent entry pathway, thus avoiding delivery into lysosomes. In contrast, in macrophages, C. jejuni is delivered to lysosomes and consequently is rapidly killed. Taken together, these studies indicate that C. jejuni has evolved specific adaptations to survive within host cells., Author Summary Campylobacter jejuni is one of the most common causes of food-borne illness in the United States and a major cause of diarrheal disease throughout the world. After infection through the oral route, this bacterium invades the cells of the intestinal epithelium, a property that is important for its ability to cause disease. Usually, bacteria and other material entering the cell move to compartments called lysosomes, where an acidic mix of enzymes breaks it down. This study shows that C. jejuni can survive within intestinal epithelial cells by avoiding delivery to lysosomes. In contrast, in macrophages, which are specialized cells with the capacity to engulf and kill bacteria, C. jejuni cannot avoid delivery into lysosomes and consequently is rapidly killed. These studies help explain an important virulence attribute of C. jejuni.

Bacteria–host interactions are dynamic processes, and understanding transcriptional responses that directly or indirectly regulate the expression of genes involved in initial infection stages would illuminate the molecular events that result in host colonization. We used oligonucleotide microarrays to monitor (in vitro) differential gene expression in group A streptococci during pharyngeal cell adherence, the first overt infection stage. We present neighbor clustering, a new computational method for further analyzing bacterial microarray data that combines two informative characteristics of bacterial genes that share common function or regulation: (1) similar gene expression profiles (i.e., co-expression); and (2) physical proximity of genes on the chromosome. This method identifies statistically significant clusters of co-expressed gene neighbors that potentially share common function or regulation by coupling statistically analyzed gene expression profiles with the chromosomal position of genes. We applied this method to our own data and to those of others, and we show that it identified a greater number of differentially expressed genes, facilitating the reconstruction of more multimeric proteins and complete metabolic pathways than would have been possible without its application. We assessed the biological significance of two identified genes by assaying deletion mutants for adherence in vitro and show that neighbor clustering indeed provides biologically relevant data. Neighbor clustering provides a more comprehensive view of the molecular responses of streptococci during pharyngeal cell adherence., Author Summary Microarray technology is commonly used to reveal genome-wide transcriptional changes in bacterial pathogens during interactions with the host. Clustering algorithms, which group genes with similar expression patterns, facilitate microarray data organization and are based on assumptions that co-expressed genes share common function or regulation; however, clustering solely by co-expression may not reveal all of the information contained in bacterial array data. We introduce neighbor clustering, a new tool for analyzing bacterial gene expression profiles, which distinguishes itself from other programs by incorporating details unique to the architecture of bacterial chromosomes into the analysis. Neighbor clustering combines two informative characteristics of bacterial genes that share common function or regulation—(1) similar expression profiles and (2) physical proximity on the chromosome—and extracts statistically significant clusters of gene neighbors that are potentially related by function or regulation. We present the analysis of microarray data from group A streptococci during adherence to human pharyngeal cells, the first overt infection step. We show that neighbor clustering identifies more differentially expressed genes than rigorous statistical analyses alone, and can provide functional clues about unknown genes. We extended the analysis to include a previously published streptococcal array study to demonstrate the applicability of the method.

Correlated changes of nucleic or amino acids have provided strong information about the structures and interactions of molecules. Despite the rich literature in coevolutionary sequence analysis, previous methods often have to trade off between generality, simplicity, phylogenetic information, and specific knowledge about interactions. Furthermore, despite the evidence of coevolution in selected protein families, a comprehensive screening of coevolution among all protein domains is still lacking. We propose an augmented continuous-time Markov process model for sequence coevolution. The model can handle different types of interactions, incorporate phylogenetic information and sequence substitution, has only one extra free parameter, and requires no knowledge about interaction rules. We employ this model to large-scale screenings on the entire protein domain database (Pfam). Strikingly, with 0.1 trillion tests executed, the majority of the inferred coevolving protein domains are functionally related, and the coevolving amino acid residues are spatially coupled. Moreover, many of the coevolving positions are located at functionally important sites of proteins/protein complexes, such as the subunit linkers of superoxide dismutase, the tRNA binding sites of ribosomes, the DNA binding region of RNA polymerase, and the active and ligand binding sites of various enzymes. The results suggest sequence coevolution manifests structural and functional constraints of proteins. The intricate relations between sequence coevolution and various selective constraints are worth pursuing at a deeper level., Author Summary The sequences of different components within and across genes often undergo coordinated changes in order to maintain the structures or functions of the genes. Identifying the coordinated changes—the “coevolution”—of those components in the context of evolution is important in predicting the structures, interactions, and functions of genes. The authors incur a large-scale screening on all the known protein sequences and build a compendium about the coevolving relations of all protein domains—subunits of proteins. The majority of the coevolving protein domains either belongs to the same proteins, appears in the same protein complexes, or shares the same functional annotations. Furthermore, coevolving positions in the same proteins or protein complexes are spatially coupled, as they tend to be closer than random positions in the 3-D structures of the proteins/protein complexes. More strikingly, many coevolving positions are located at functionally important sites of the molecules. The results provide useful insights about the relations between sequence evolution and protein structures and functions.

We have developed a powerful experimental framework that combines competitive selection and microarray-based genetic footprinting to comprehensively reveal the genetic basis of bacterial behaviors. Application of this method to Escherichia coli motility identifies 95% of the known flagellar and chemotaxis genes, and reveals three dozen novel loci that, to varying degrees and through diverse mechanisms, affect motility. To probe the network context in which these genes function, we developed a method that uncovers genome-wide epistatic interactions through comprehensive analyses of double-mutant phenotypes. This allows us to place the novel genes within the context of signaling and regulatory networks, including the Rcs phosphorelay pathway and the cyclic di-GMP second-messenger system. This unifying framework enables sensitive and comprehensive genetic characterization of complex behaviors across the microbial biosphere., Author Summary Bacteria thrive in a limitless range of extreme environments, accompanied by exotic metabolisms and sophisticated behaviors. However, our modern molecular understanding of bacteria comes from studies of a limited range of phenotypes in a handful of model organisms such as E. coli and Bacillus subtilis. With the availability of thousands of sequenced bacterial genomes, there is now an urgent need for methods that rapidly and comprehensively reveal the genetic basis of phenotypes across the microbial biosphere. To this end, we have developed a genome-wide experimental framework that quantifies the degree to which every gene in the genome contributes to a phenotype of interest, and reveals the organization of genes within regulatory networks and signaling pathways. We show here that the application of this methodology to E. coli swimming and surface motility reveals essentially all the previously known components of flagellar-mediated chemotaxis on the time scale of weeks. Remarkably, we also identify three dozen additional novel loci that operate through diverse mechanisms to affect a behavior that was assumed to be completely characterized. The speed, ease, and broad applicability of this framework should greatly accelerate the global analysis of a wide range of uncharacterized bacterial behaviors.

The chemotaxis system in the bacterium Escherichia coli is remarkably sensitive to small relative changes in the concentrations of multiple chemical signals over a broad range of ambient concentrations. Interactions among receptors are crucial to this sensitivity as is precise adaptation, the return of chemoreceptor activity to prestimulus levels in a constant chemoeffector environment. Precise adaptation relies on methylation and demethylation of chemoreceptors by the enzymes CheR and CheB, respectively. Experiments indicate that when transiently bound to one receptor, these enzymes act on small assistance neighborhoods (AN) of five to seven receptor homodimers. In this paper, we model a strongly coupled complex of receptors including dynamic CheR and CheB acting on ANs. The model yields sensitive response and precise adaptation over several orders of magnitude of attractant concentrations and accounts for different responses to aspartate and serine. Within the model, we explore how the precision of adaptation is limited by small AN size as well as by CheR and CheB kinetics (including dwell times, saturation, and kinetic differences among modification sites) and how these kinetics contribute to noise in complex activity. The robustness of our dynamic model for precise adaptation is demonstrated by randomly varying biochemical parameters., Author Summary Bacteria swim in relatively straight lines and change directions through tumbling. In the process of chemotaxis, a network of receptors and other proteins controls the tumbling frequency to direct an otherwise random walk toward nutrients and away from repellents. Receptor clustering and adaptation to persistent stimuli through covalent modification allow chemotaxis to be sensitive over a large range of ambient concentrations. The individual components of the chemotaxis network are well characterized, and signaling measurements by fluorescence microscopy quantify the network's response, making the system well suited for modeling and analysis. In this paper, we expand upon a previous model based on experiments indicating that the covalent modifications required for adaptation occur through the action of enzymes on groups of neighboring receptors, referred to as assistance neighborhoods. Simulations show that our proposed molecular model of a strongly coupled complex of receptors produces accurate responses to different stimuli and is robust to parameter variation. Within this model, the correct adaptation response is limited by small assistance-neighborhood size as well as enzyme kinetics. We also explore how these kinetics contribute to noise in the chemotactic response.

The obligate intracellular, gram-negative bacterium Chlamydophila pneumoniae (Cpn) has impact as a human pathogen. Little is known about changes in the Cpn transcriptome during its biphasic developmental cycle (the acute infection) and persistence. The latter stage has been linked to chronic diseases. To analyze Cpn CWL029 gene expression, we designed a pathogen-specific oligo microarray and optimized the extraction method for pathogen RNA. Throughout the acute infection, ratio expression profiles for each gene were generated using 48 h post infection as a reference. Based on these profiles, significantly expressed genes were separated into 12 expression clusters using self-organizing map clustering and manual sorting into the “early”, “mid”, “late”, and “tardy” cluster classes. The latter two were differentiated because the “tardy” class showed steadily increasing expression at the end of the cycle. The transcriptome of the Cpn elementary body (EB) and published EB proteomics data were compared to the cluster profile of the acute infection. We found an intriguing association between “late” genes and genes coding for EB proteins, whereas “tardy” genes were mainly associated with genes coding for EB mRNA. It has been published that iron depletion leads to Cpn persistence. We compared the gene expression profiles during iron depletion–mediated persistence with the expression clusters of the acute infection. This led to the finding that establishment of iron depletion–mediated persistence is more likely a mid-cycle arrest in development rather than a completely distinct gene expression pattern. Here, we describe the Cpn transcriptome during the acute infection, differentiating “late” genes, which correlate to EB proteins, and “tardy” genes, which lead to EB mRNA. Expression profiles during iron mediated–persistence led us to propose the hypothesis that the transcriptomic “clock” is arrested during acute mid-cycle., Author Summary Chlamydophila (Chlamydia) pneumoniae (Cpn) accounts for approximately one-tenth of the cases of community-acquired pneumonia worldwide, and persistent Cpn infections are thought to be associated with a variety of chronic diseases. Little is known about Cpn transcriptome changes during its biphasic developmental cycle (the acute infection) and persistence stages. Iron limitation, among several other treatments, has recently been shown to lead to persistent Cpn infection. How this pathogen reacts to iron-limiting host defense mechanisms is of great interest, as iron is an important factor affecting virulence. This article reports on the Cpn transcriptome during the developmental cycle and iron depletion–mediated persistence and reveals that genes coding for proteins of the infectious particle (the elementary body [EB]) were expressed constantly at the end of the cycle. In contrast, genes contributing to EB mRNA but not to EB protein showed an increasing expression at the end of the cycle. This suggested that most EB proteins are made in mid-cycle, and the redifferentiation process is initiated only by a limited number of genes. During iron depletion–mediated persistence, the Cpn transcriptome was altered in such a way that an arrest in Cpn gene expression can be proposed.

The role of stochasticity on gene expression is widely discussed. Both potential advantages and disadvantages have been revealed. In some systems, noise in gene expression has been quantified, in among others the lac operon of Escherichia coli. Whether stochastic gene expression in this system is detrimental or beneficial for the cells is, however, still unclear. We are interested in the effects of stochasticity from an evolutionary point of view. We study this question in the lac operon, taking a computational approach: using a detailed, quantitative, spatial model, we evolve through a mutation–selection process the shape of the promoter function and therewith the effective amount of stochasticity. We find that noise values for lactose, the natural inducer, are much lower than for artificial, nonmetabolizable inducers, because these artificial inducers experience a stronger positive feedback. In the evolved promoter functions, noise due to stochasticity in gene expression, when induced by lactose, only plays a very minor role in short-term physiological adaptation, because other sources of population heterogeneity dominate. Finally, promoter functions evolved in the stochastic model evolve to higher repressed transcription rates than those evolved in a deterministic version of the model. This causes these promoter functions to experience less stochasticity in gene expression. We show that a high repression rate and hence high stochasticity increases the delay in lactose uptake in a variable environment. We conclude that the lac operon evolved such that the impact of stochastic gene expression is minor in its natural environment, but happens to respond with much stronger stochasticity when confronted with artificial inducers. In this particular system, we have shown that stochasticity is detrimental. Moreover, we demonstrate that in silico evolution in a quantitative model, by mutating the parameters of interest, is a promising way to unravel the functional properties of biological systems., Author Summary Gene expression is a process that is inherently stochastic because of the low number of molecules that are involved. In recent years it has become possible to measure the amount of stochasticity in gene expression, which has inspired a debate about the importance of stochasticity in gene expression. Little attention, however, has been paid to stochasticity in gene expression from an evolutionary perspective. We studied the evolutionary consequences of stochastic gene expression in one of the best-known systems of genetic regulation, the lac operon of E. coli, which regulates lactose uptake and metabolism. We used a computational approach, in which we let cells evolve their lac operon promoter function in a fluctuating, spatially explicit, environment. Cells can in this way adapt to the environment, but also change the amount of stochasticity in gene expression. We find that cells evolve their repressed transcription rates to higher values in a stochastic model than in a deterministic model. Higher repressed transcription rates means less stochasticity, and, hence, these cells appear to avoid stochastic gene expression in this particular system. We find that this can be explained by the fact that stochastic gene expression causes a larger delay in lactose uptake, compared with deterministic gene expression.

Bacillus anthracis causes three forms of anthrax: inhalational, gastrointestinal, and cutaneous. Anthrax is characterized by both toxemia, which is caused by secretion of immunomodulating toxins (lethal toxin and edema toxin), and septicemia, which is associated with bacterial encapsulation. Here we report that, contrary to the current view of B. anthracis pathogenesis, B. anthracis spores germinate and establish infections at the initial site of inoculation in both inhalational and cutaneous infections without needing to be transported to draining lymph nodes, and that inhaled spores establish initial infection in nasal-associated lymphoid tissues. Furthermore, we found that Peyer's patches in the mouse intestine are the primary site of bacterial growth after intragastric inoculation, thus establishing an animal model of gastrointestinal anthrax. All routes of infection progressed to the draining lymph nodes, spleen, lungs, and ultimately the blood. These discoveries were made possible through the development of a novel dynamic mouse model of B. anthracis infection using bioluminescent non-toxinogenic capsulated bacteria that can be visualized within the mouse in real-time, and demonstrate the value of in vivo imaging in the analysis of B. anthracis infection. Our data imply that previously unrecognized portals of bacterial entry demand more intensive investigation, and will significantly transform the current perception of inhalational, gastrointestinal, and cutaneous B. anthracis pathogenesis., Author Summary Anthrax is caused by Bacillus anthracis, a bacterial pathogen that forms spores, dormant bacteria that are highly resistant to destruction. Infections initiate from the introduction of spores into airways or damaged skin, or from the consumption of contaminated food. Within the host, spores germinate, then bacteria secrete toxins that cripple the immune response and sheath themselves in a capsule that prevents them from being phagocytosed. We strove to determine in real space and time where and when spores introduced by these three routes of infection germinate and how bacteria subsequently disseminate in a mouse model. This was achieved through the development of light-emitting B. anthracis that could be tracked inside a living mouse. Contrary to current models, our studies indicated that spores germinated in situ in the skin, the intestines, and the nasal passages without needing to be transported to lymph nodes. Furthermore, bacteria disseminate from initial sites of infection in a similar fashion, first to the draining lymph nodes, then the spleen, and finally the lungs and blood. These findings imply that spore interactions with local sites of entry are critical in the development of systemic disease and that disruption of these interactions may offer new methods of anthrax prevention.

The MglA protein is the only known regulator of virulence gene expression in Francisella tularensis, yet it is unclear how it functions. F. tularensis also contains an MglA-like protein called SspA. Here, we show that MglA and SspA cooperate with one another to control virulence gene expression in F. tularensis. Using a directed proteomic approach, we show that both MglA and SspA associate with RNA polymerase (RNAP) in F. tularensis, and that SspA is required for MglA to associate with RNAP. Furthermore, bacterial two-hybrid and biochemical assays indicate that MglA and SspA interact with one another directly. Finally, through genome-wide expression analyses, we demonstrate that MglA and SspA regulate the same set of genes. Our results suggest that a complex involving both MglA and SspA associates with RNAP to positively control virulence gene expression in F. tularensis. The F. tularensis genome is unusual in that it contains two genes encoding different α subunits of RNAP, and we show here that these two α subunits are incorporated into RNAP. Thus, as well as identifying SspA as a second critical regulator of virulence gene expression in F. tularensis, our findings provide a framework for understanding the mechanistic basis for virulence gene control in a bacterium whose transcription apparatus is unique., Author Summary The Gram-negative bacterium Francisella tularensis is an intracellular pathogen and the causative agent of tularemia. In F. tularensis, the MglA protein is the only known regulator of virulence genes that are important for intracellular survival, yet it is not known how MglA functions. F. tularensis also contains an MglA-like protein called SspA whose function is not known. In this study, we show that both MglA and SspA associate with RNA polymerase (RNAP) and positively regulate virulence gene expression in F. tularensis. Our study provides evidence that MglA and SspA interact with one another directly and that the association of MglA with RNAP is dependent on the presence of SspA. We also show that, unlike RNAP from any other bacterium, RNAP from F. tularensis contains two distinct α subunits. Given the fundamental roles the α subunit plays in transcription regulation, this may have far-reaching implications for how gene expression is controlled in F. tularensis. Our study therefore uncovers a new critical regulator of virulence gene expression in F. tularensis (SspA), provides mechanistic insight into how MglA and SspA cooperate to control virulence gene expression, and reveals that the F. tularensis transcription machinery has an unusual composition.

Multi-drug tolerance is a key phenotypic property that complicates the sterilization of mammals infected with Mycobacterium tuberculosis. Previous studies have established that iniBAC, an operon that confers multi-drug tolerance to M. bovis BCG through an associated pump-like activity, is induced by the antibiotics isoniazid (INH) and ethambutol (EMB). An improved understanding of the functional role of antibiotic-induced genes and the regulation of drug tolerance may be gained by studying the factors that regulate antibiotic-mediated gene expression. An M. smegmatis strain containing a lacZ gene fused to the promoter of M. tuberculosis iniBAC (PiniBAC) was subjected to transposon mutagenesis. Mutants with constitutive expression and increased EMB-mediated induction of PiniBAC::lacZ mapped to the lsr2 gene (MSMEG6065), a small basic protein of unknown function that is highly conserved among mycobacteria. These mutants had a marked change in colony morphology and generated a new polar lipid. Complementation with multi-copy M. tuberculosis lsr2 (Rv3597c) returned PiniBAC expression to baseline, reversed the observed morphological and lipid changes, and repressed PiniBAC induction by EMB to below that of the control M. smegmatis strain. Microarray analysis of an lsr2 knockout confirmed upregulation of M. smegmatis iniA and demonstrated upregulation of genes involved in cell wall and metabolic functions. Fully 121 of 584 genes induced by EMB treatment in wild-type M. smegmatis were upregulated (“hyperinduced”) to even higher levels by EMB in the M. smegmatis lsr2 knockout. The most highly upregulated genes and gene clusters had adenine-thymine (AT)–rich 5-prime untranslated regions. In M. tuberculosis, overexpression of lsr2 repressed INH-mediated induction of all three iniBAC genes, as well as another annotated pump, efpA. The low molecular weight and basic properties of Lsr2 (pI 10.69) suggested that it was a histone-like protein, although it did not exhibit sequence homology with other proteins in this class. Consistent with other histone-like proteins, Lsr2 bound DNA with a preference for circular DNA, forming large oligomers, inhibited DNase I activity, and introduced a modest degree of supercoiling into relaxed plasmids. Lsr2 also inhibited in vitro transcription and topoisomerase I activity. Lsr2 represents a novel class of histone-like proteins that inhibit a wide variety of DNA-interacting enzymes. Lsr2 appears to regulate several important pathways in mycobacteria by preferentially binding to AT-rich sequences, including genes induced by antibiotics and those associated with inducible multi-drug tolerance. An improved understanding of the role of lsr2 may provide important insights into the mechanisms of action of antibiotics and the way that mycobacteria adapt to stresses such as antibiotic treatment., Author Summary Understanding the cellular processes stimulated when Mycobacterium tuberculosis is treated with antibiotics may provide clues as to why months of therapy and use of several drugs simultaneously are required to prevent antibiotic resistance. Antibiotic treatment “turns on” or induces certain M. tuberculosis genes. These genes are of special interest because they appear to help M. tuberculosis survive the stress of antibiotic treatment. Our study of the regulation of antibiotic-induced genes, including iniBAC, in two mycobacterial species revealed that a small protein called Lsr2 controls iniBAC and other antibiotic-induced genes, especially ones related to the cell wall. Lsr2 binds to DNA in a relatively non-specific manner and appears to inhibit certain enzymes that must interact with DNA as part of their function. These properties differentiate Lsr2 from classical regulators of gene expression that bind to specific DNA sequences, and suggest that Lsr2 is a novel histone-like protein. These proteins regulate genes by changing the way DNA is shaped, and, indeed, we found that Lsr2 can change the shape of DNA by introducing a small number of coils into its structure. Our results suggest that Lsr2 is a major regulator of antibiotic-induced responses in mycobacteria.

Wolbachia are maternally inherited bacteria that commonly spread through host populations by causing cytoplasmic incompatibility, often expressed as reduced egg hatch when uninfected females mate with infected males. Infected females are frequently less fecund as a consequence of Wolbachia infection. However, theory predicts that because of maternal transmission, these “parasites” will tend to evolve towards a more mutualistic association with their hosts. Drosophila simulans in California provided the classic case of a Wolbachia infection spreading in nature. Cytoplasmic incompatibility allowed the infection to spread through individual populations within a few years and from southern to northern California (more than 700 km) within a decade, despite reducing the fecundity of infected females by 15%–20% under laboratory conditions. Here we show that the Wolbachia in California D. simulans have changed over the last 20 y so that infected females now exhibit an average 10% fecundity advantage over uninfected females in the laboratory. Our data suggest smaller but qualitatively similar changes in relative fecundity in nature and demonstrate that fecundity-increasing Wolbachia variants are currently polymorphic in natural populations., Author Summary Wolbachia are endosymbiotic bacteria that live inside the cells of their invertebrate hosts. They are transmitted directly from mother to offspring, and spread through populations by manipulating the reproduction of their hosts. The most common reproductive manipulation responsible for the spread of these bacteria, called “cytoplasmic incompatibility,” arises when infected males mate with uninfected females, resulting in fewer offspring than normal. There are fitness costs for the hosts associated with Wolbachia infections, most commonly involving a reduction in egg production. Theory predicts that this detrimental effect of Wolbachia on its host should result in selection for the bacteria to evolve a more benign lifestyle, changing the bacterium from being parasitic to more mutualistic. We document such a shift in a Wolbachia infection of fruit flies (Drosophila simulans) from California. The shift occurred extremely rapidly, over 20 years. Consequently, Wolbachia-infected hosts now have higher rates of egg production than their uninfected counterparts. Changes in the genome of Wolbachia seem to be responsible for this, rather than changes in the host genome. Our study reveals that bacteria and their hosts represent components of a dynamic interacting system that can evolve rapidly over time., A parasitic endosymbiont,Wolbachia, has rapidly evolved towards a more mutualistic lifestyle with itsDrosophila host in under 20 years, suggesting a dynamic interaction between parasitic and mutualistic lifestyles.

A description of many biological processes requires knowledge of the 3-D structure of proteins and, in particular, the defined active site responsible for biological function. Many proteins, the genes of which have been identified as the result of human genome sequencing, and which were synthesized experimentally, await identification of their biological activity. Currently used methods do not always yield satisfactory results, and new algorithms need to be developed to recognize the localization of active sites in proteins. This paper describes a computational model that can be used to identify potential areas that are able to interact with other molecules (ligands, substrates, inhibitors, etc.). The model for active site recognition is based on the analysis of hydrophobicity distribution in protein molecules. It is shown, based on the analyses of proteins with known biological activity and of proteins of unknown function, that the region of significantly irregular hydrophobicity distribution in proteins appears to be function related., Author Summary We present here a method of defining functional site recognition in proteins. The active site (enzymatic cavity or ligand-binding site) is localized on the basis of hydrophobicity deficiency, which is understood as the difference between empirical (dependent on amino acid positions) and idealized (3-D Gauss function, or Fuzzy Oil Drop model) distribution of hydrophobicity. It is assumed that the localization of amino acids representing a high difference of hydrophobic density reveals the functional site. The analysis of the structure of 33 proteins of known biological activity and of 33 proteins of unknown function (with comparable polypeptide chain lengths) seems to verify the applicability of the method to binding cavity localization. The comparative analysis with other methods oriented on biological function is also presented. The validation of predictability accuracy is shown with respect to the enzyme classes.

P pili are important adhesive fibres involved in kidney infection by uropathogenic Escherichia coli strains. P pili are assembled by the conserved chaperone–usher pathway, which involves the PapD chaperone and the PapC usher. During pilus assembly, subunits are incorporated into the growing fiber via the donor–strand exchange (DSE) mechanism, whereby the chaperone's G1 β-strand that complements the incomplete immunoglobulin-fold of each subunit is displaced by the N-terminal extension (Nte) of an incoming subunit. P pili comprise a helical rod, a tip fibrillum, and an adhesin at the distal end. PapA is the rod subunit and is assembled into a superhelical right-handed structure. Here, we have solved the structure of a ternary complex of PapD bound to PapA through donor–strand complementation, itself bound to another PapA subunit through DSE. This structure provides insight into the structural basis of the DSE reaction involving this important pilus subunit. Using gel filtration chromatography and electron microscopy on a number of PapA Nte mutants, we establish that PapA differs in its mode of assembly compared with other Pap subunits, involving a much larger Nte that encompasses not only the DSE region of the Nte but also the region N-terminal to it., Author Summary Bacterial adhesion to a host is a crucial step that determines the onset of bacterial infection. It is mediated through recognition of a receptor on the host cell surface by a protein called an adhesin displayed on the surface of the bacterium. Many adhesins are displayed at the tip of specialized organelles called pili, some of which are assembled by the ubiquitous chaperone–usher pathway. In this pathway, each pilus subunit is assisted in folding by a chaperone. The resulting chaperone–subunit complex is targeted to a pore located in the outer membrane, called the usher, that serves as assembly platform. There, pilus subunits dissociate from the chaperone and polymerize, resulting in a surface organelle, the pilus, that protrudes out of the usher. Here, we have determined the structure of the major subunit of the P pilus, PapA. The P pilus, produced in uropathogenic Escherichia coli, displays the adhesin PapG responsible for targeting the bacterium to the kidney epithelium. We have determined the structure of PapA either bound to its cognate chaperone, PapD, or bound to another PapA subunit. These structures provide a view of PapA before and after its assembly in the pilus and shed light on the mechanism of PapA assembly.

The vertebrate gut harbors a vast community of bacterial mutualists, the composition of which is modulated by the host immune system. Many gastrointestinal (GI) diseases are expected to be associated with disruptions of host-bacterial interactions, but relatively few comprehensive studies have been reported. We have used the rhesus macaque model to investigate forces shaping GI bacterial communities. We used DNA bar coding and pyrosequencing to characterize 141,000 sequences of 16S rRNA genes obtained from 100 uncultured GI bacterial samples, allowing quantitative analysis of community composition in health and disease. Microbial communities of macaques were distinct from those of mice and humans in both abundance and types of taxa present. The macaque communities differed among samples from intestinal mucosa, colonic contents, and stool, paralleling studies of humans. Communities also differed among animals, over time within individual animals, and between males and females. To investigate changes associated with disease, samples of colonic contents taken at necropsy were compared between healthy animals and animals with colitis and undergoing antibiotic therapy. Communities from diseased and healthy animals also differed significantly in composition. This work provides comprehensive data and improved methods for studying the role of commensal microbiota in macaque models of GI diseases and provides a model for the large-scale screening of the human gut microbiome., Author Summary Bacterial mutualists within the gastrointestinal tract aid digestion, promote development of the gut immune system, and provide competitive barriers to pathogen invasion. The host, in return, provides bacteria with safe housing and food during lean times. The composition of the gut microbiota is controlled in part by the host immune system. In a variety of disease states, immune function can be altered, and gut morbidity is often associated, leading to the hypothesis that alterations in the GI microbiota may contribute to disease. In this study, the gut microbiota was characterized in 100 samples from rhesus macaques using pyrosequencing, which allowed 141,000 sequences from 16S rRNA genes to be generated and analyzed. Healthy animals were compared to animals with gut disorders, induced, for example by advanced simian AIDS. Many factors contributed to changes in the microbiota, including the sex of the animal of origin. Animals with chronic colitis showed differences in composition of the GI microbiota compared to healthy animals, providing an association between altered microbiota and disease.