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Streptococcus pyogenes is a Gram-positive human pathogen that is recognized by yet unknown pattern recognition receptors (PRRs). Engagement of these receptor molecules during infection with S. pyogenes, a largely extracellular bacterium with limited capacity for intracellular survival, causes innate immune cells to produce inflammatory mediators such as TNF, but also type I interferon (IFN). Here we show that signaling elicited by type I IFNs is required for successful defense of mice against lethal subcutaneous cellulitis caused by S. pyogenes. Type I IFN signaling was accompanied with reduced neutrophil recruitment to the site of infection. Mechanistic analysis revealed that macrophages and conventional dendritic cells (cDCs) employ different signaling pathways leading to IFN-beta production. Macrophages required IRF3, STING, TBK1 and partially MyD88, whereas in cDCs the IFN-beta production was fully dependent on IRF5 and MyD88. Furthermore, IFN-beta production by macrophages was dependent on the endosomal delivery of streptococcal DNA, while in cDCs streptococcal RNA was identified as the IFN-beta inducer. Despite a role of MyD88 in both cell types, the known IFN-inducing TLRs were individually not required for generation of the IFN-beta response. These results demonstrate that the innate immune system employs several strategies to efficiently recognize S. pyogenes, a pathogenic bacterium that succeeded in avoiding recognition by the standard arsenal of TLRs., Author Summary Streptococcus pyogenes is an important human pathogen that causes a broad range of diseases. The bacterium colonizes the throat and the skin where it can evoke usually mild illness such as strep throat or scarlet fever. Systemic infections with S. pyogenes are less frequent but can develop into life-threatening diseases such as necrotizing fasciitis and streptococcal toxic shock syndrome. The immune system launches a usually successful response that is initiated by a so far not understood recognition of this pathogen by the cells of the innate immune system. These cells produce upon infection a variety of cytokines that orchestrate a full blown protective response. Among these cytokines, type I interferons play a critical role as demonstrated by our study. We further show that IFN-beta, the key type I interferon, is produced only after macrophages and dendritic cells have taken up the pathogen and liberated the bacterial nucleic acids for recognition in the intracellular vesicles. Importantly, macrophages and dendritic cells recognize different nucleic acids and employ different signaling pathways to respond. Our data suggest that the innate immune system employs several strategies to efficiently recognize S. pyogenes, a pathogenic bacterium that succeeded in avoiding recognition by the standard recognition mechanisms.

In Borrelia burgdorferi (Bb), the Lyme disease spirochete, the alternative σ factor σ54 (RpoN) directly activates transcription of another alternative σ factor, σS (RpoS) which, in turn, controls the expression of virulence-associated membrane lipoproteins. As is customary in σ54-dependent gene control, a putative NtrC-like enhancer-binding protein, Rrp2, is required to activate the RpoN-RpoS pathway. However, recently it was found that rpoS transcription in Bb also requires another regulator, BosR, which was previously designated as a Fur or PerR homolog. Given this unexpected requirement for a second activator to promote σ54-dependent gene transcription, and the fact that regulatory mechanisms among similar species of pathogenic bacteria can be strain-specific, we sought to confirm the regulatory role of BosR in a second virulent strain (strain 297) of Bb. Indeed, BosR displayed the same influence over lipoprotein expression and mammalian infectivity for strain Bb 297 that were previously noted for Bb strain B31. We subsequently found that recombinant BosR (rBosR) bound to the rpoS gene at three distinct sites, and that binding occurred despite the absence of consensus Fur or Per boxes. This led to the identification of a novel direct repeat sequence (TAAATTAAAT) critical for rBosR binding in vitro. Mutations in the repeat sequence markedly inhibited or abolished rBosR binding. Taken together, our studies provide new mechanistic insights into how BosR likely acts directly on rpoS as a positive transcriptional activator. Additional novelty is engendered by the facts that, although BosR is a Fur or PerR homolog and it contains zinc (like Fur and PerR), it has other unique features that clearly set it apart from these other regulators. Our findings also have broader implications regarding a previously unappreciated layer of control that can be involved in σ54–dependent gene regulation in bacteria., Author Summary Lyme disease, caused by the bacterium Borrelia burgdorferi (Bb), remains the most common arthropod-borne illness in the United States. A critical strategy for Bb to maintain its presence in nature is adaptation to its diverse tick and mammalian (mouse) hosts. To accomplish this, Bb encodes a potential gene regulator, BB0647 (BosR). Herein, we confirmed that BosR is essential for Bb to establish mammalian infection. We then found that purified recombinant BosR bound to the promoter DNA (regulatory region) of rpoS, suggesting that BosR directly controls the expression of the rpoS gene. This study has revealed a new mechanism of bacterial gene control. The discovery that BosR governs Bb's virulence may lead to new strategies to interrupt the bacterium's complex life cycle.

The F region downstream of the mecI gene in the SCCmec element in hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) contains two bidirectionally overlapping open reading frames (ORFs), the fudoh ORF and the psm-mec ORF. The psm-mec ORF encodes a cytolysin, phenol-soluble modulin (PSM)-mec. Transformation of the F region into the Newman strain, which is a methicillin-sensitive S. aureus (MSSA) strain, or into the MW2 (USA400) and FRP3757 (USA300) strains, which are community-acquired MRSA (CA-MRSA) strains that lack the F region, attenuated their virulence in a mouse systemic infection model. Introducing the F region to these strains suppressed colony-spreading activity and PSMα production, and promoted biofilm formation. By producing mutations into the psm-mec ORF, we revealed that (i) both the transcription and translation products of the psm-mec ORF suppressed colony-spreading activity and promoted biofilm formation; and (ii) the transcription product of the psm-mec ORF, but not its translation product, decreased PSMα production. These findings suggest that both the psm-mec transcript, acting as a regulatory RNA, and the PSM-mec protein encoded by the gene on the mobile genetic element SCCmec regulate the virulence of Staphylococcus aureus., Author Summary Methicillin-resistant Staphylococcus aureus (MRSA) causes opportunistic infectious diseases in immunocompromised patients in hospitals, which are difficult to cure with antibiotics. These MRSA are called hospital-associated MRSA (HA-MRSA). In contrast, other MRSA strains, which are called community-acquired MRSA (CA-MRSA), have high virulence and cause serious infections in previously healthy individuals. Why the CA-MRSA phenotype has high virulence is not well understood. MRSA becomes resistant to antibiotics by acquiring a foreign DNA called SCCmec. The SCCmec region contains several genes, including the mecA gene, which confers resistance against methicillin. We focused our attention on the structural difference of the SCCmec region between HA-MRSA and CA-MRSA. The SCCmec region of CA-MRSA lacks the F region, which suppresses HA-MRSA virulence. In this study, we revealed that both the transcription and translation products of a cytolysin gene, psm-mec, which is encoded in the F region, suppress MRSA virulence. This finding contributes to our understanding of the high virulence of CA-MRSA at the molecular level and will help to establish new strategies to combat infectious diseases due to CA-MRSA.

Non-typeable Haemophilus influenzae (NTHi), a common commensal of the human pharynx, is also an opportunistic pathogen if it becomes established in the lower respiratory tract (LRT). In comparison to colonizing isolates from the upper airway, LRT isolates, especially those associated with exacerbations of chronic obstructive pulmonary disease, have increased resistance to the complement- and antibody-dependent, bactericidal effect of serum. To define the molecular basis of this resistance, mutants constructed in a serum resistant strain using the mariner transposon were screened for loss of survival in normal human serum. The loci required for serum resistance contribute to the structure of the exposed surface of the bacterial outer membrane. These included loci involved in biosynthesis of the oligosaccharide component of lipooligosaccharide (LOS), and vacJ, which functions with an ABC transporter encoded by yrb genes in retrograde trafficking of phospholipids from the outer to inner leaflet of the cell envelope. Mutations in vacJ and yrb genes reduced the stability of the outer membrane and were associated with increased cell surface hyrophobicity and phospholipid content. Loss of serum resistance in vacJ and yrb mutants correlated with increased binding of natural immunoglobulin M in serum as well as anti-oligosaccharide mAbs. Expression of vacJ and the yrb genes was positively correlated with serum resistance among clinical isolates. Our findings suggest that NTHi adapts to inflammation encountered during infection of the LRT by modulation of its outer leaflet through increased expression of vacJ and yrb genes to minimize recognition by bactericidal anti-oligosaccharide antibodies., Author Summary Haemophilus influenzae generally colonizes the human upper respiratory tract. When isolated from the lower respiratory tract, this opportunistic pathogen is associated with inflammatory conditions such as pneumonia and exacerbations of chronic obstructive pulmonary disease (COPD). Here we show that one of the adaptations made by H. influenzae isolated from the lower respiratory tract is increased resistance to the bactericidal effect of antibody and complement. To define the mechanism for increased resistance, mutants were screened to identify the complete set of genes required to inhibit killing by antibody and complement. These included multiple genes that all contribute to biosynthesis of the organism's surface oligosaccharide (lipooligosaccharide), which is targeted by bactericidal antibody. Our results also revealed a novel function for additional genes that maintain the lipid asymmetry of the surface membrane and thereby limit recognition of the pathogen by anti-oligosaccharide antibodies.

Identifying the major routes of disease transmission and reservoirs of infection are needed to increase our understanding of disease dynamics and improve disease control. Despite this, transmission events are rarely observed directly. Here we had the unique opportunity to study natural transmission of Bordetella bronchiseptica – a directly transmitted respiratory pathogen with a wide mammalian host range, including sporadic infection of humans – within a commercial rabbitry to evaluate the relative effects of sex and age on the transmission dynamics therein. We did this by developing an a priori set of hypotheses outlining how natural B. bronchiseptica infections may be transmitted between rabbits. We discriminated between these hypotheses by using force-of-infection estimates coupled with random effects binomial regression analysis of B. bronchiseptica age-prevalence data from within our rabbit population. Force-of-infection analysis allowed us to quantify the apparent prevalence of B. bronchiseptica while correcting for age structure. To determine whether transmission is largely within social groups (in this case litter), or from an external group, we used random-effect binomial regression to evaluate the importance of social mixing in disease spread. Between these two approaches our results support young weanlings – as opposed to, for example, breeder or maternal cohorts – as the age cohort primarily responsible for B. bronchiseptica transmission. Thus age-prevalence data, which is relatively easy to gather in clinical or agricultural settings, can be used to evaluate contact patterns and infer the likely age-cohort responsible for transmission of directly transmitted infections. These insights shed light on the dynamics of disease spread and allow an assessment to be made of the best methods for effective long-term disease control., Author Summary A lack of understanding regarding determinants of infectious disease transmission has hindered improved disease control efforts. Here we had the unique opportunity to study the natural transmission of the respiratory pathogen Bordetella bronchiseptica within a commercial rabbitry. B. bronchiseptica is a directly transmitted gram-negative bacterium belonging to the genus Bordetella, which also comprises B. pertussis and B. parapertussis, the etiological agents of whooping cough in humans. In this study we estimated the importance of rabbit sex, age and social group on disease spread. To do this we first outlined a set of hypotheses about how natural B. bronchiseptica infections may be transmitted between rabbits. We then discriminated between these hypotheses by estimating the rate at which susceptible individuals acquire infection (or force-of-infection) using B. bronchiseptica age-prevalence data. The importance of social structure in disease spread was then evaluated using random-effect binomial regression. Our results support young weanlings as the age cohort primarily responsible for B. bronchiseptica transmission and demonstrate that easy to collect age-prevalence data can be used to infer the likely age-cohort responsible for disease transmission. Such insights shed light on the dynamics of disease spread and allow an assessment to be made of the best methods for effective disease control.

Pathogen-associated secretion systems translocate numerous effector proteins into eukaryotic host cells to coordinate cellular processes important for infection. Spatiotemporal regulation is therefore important for modulating distinct activities of effectors at different stages of infection. Here we provide the first evidence of “metaeffector,” a designation for an effector protein that regulates the function of another effector within the host cell. Legionella LubX protein functions as an E3 ubiquitin ligase that hijacks the host proteasome to specifically target the bacterial effector protein SidH for degradation. Delayed delivery of LubX to the host cytoplasm leads to the shutdown of SidH within the host cells at later stages of infection. This demonstrates a sophisticated level of coevolution between eukaryotic cells and L. pneumophila involving an effector that functions as a key regulator to temporally coordinate the function of a cognate effector protein., Author Summary Many bacterial pathogens encode a large array of “effector proteins” that are essential for successful infection. By definition, effector proteins are synthesized in bacteria and transported from bacteria into host cells. Within host cells, effector proteins directly interact with host factors in order to modulate their functions. Effector expression, translocation or activity within host cells must be precisely regulated over infection stages. Here we demonstrate the first example of an effector protein which targets and regulates another effector within host cells: Legionella effector protein LubX targets another effector protein SidH to proteasome-mediated protein degradation in the host cells. Expression and delivery of these effector proteins are differentially regulated, which results in LubX-dependent SidH shutdown at late stages of infection. We propose the designation “metaeffector” for this class of bacterial effector protein: an effector that targets and regulates another effector within host cells. Future studies may reveal that metaeffectors which play critical roles in coordinating the functional expression of other effectors spatiotemporally are prevalent among bacterial pathogens.

The “enhanced intracellular survival” (eis) gene of Mycobacterium tuberculosis (Mtb) is involved in the intracellular survival of M. smegmatis. However, its exact effects on host cell function remain elusive. We herein report that Mtb Eis plays essential roles in modulating macrophage autophagy, inflammatory responses, and cell death via a reactive oxygen species (ROS)-dependent pathway. Macrophages infected with an Mtb eis-deletion mutant H37Rv (Mtb-Δeis) displayed markedly increased accumulation of massive autophagic vacuoles and formation of autophagosomes in vitro and in vivo. Infection of macrophages with Mtb-Δeis increased the production of tumor necrosis factor-α and interleukin-6 over the levels produced by infection with wild-type or complemented strains. Elevated ROS generation in macrophages infected with Mtb-Δeis (for which NADPH oxidase and mitochondria were largely responsible) rendered the cells highly sensitive to autophagy activation and cytokine production. Despite considerable activation of autophagy and proinflammatory responses, macrophages infected with Mtb-Δeis underwent caspase-independent cell death. This cell death was significantly inhibited by blockade of autophagy and c-Jun N-terminal kinase-ROS signaling, suggesting that excessive autophagy and oxidative stress are detrimental to cell survival. Finally, artificial over-expression of Eis or pretreatment with recombinant Eis abrogated production of both ROS and proinflammatory cytokines, which depends on the N-acetyltransferase domain of the Eis protein. Collectively, these data indicate that Mtb Eis suppresses host innate immune defenses by modulating autophagy, inflammation, and cell death in a redox-dependent manner., Author Summary Tuberculosis is a global health problem: at least one-third of the world's population is infected with Mycobacterium tuberculosis (Mtb). Mtb is a successful pathogen that enhances its own intracellular survival by arresting phagolysosomal fusion. Recently, autophagy has emerged as a host defense strategy against Mtb infection, through stimulation of the fusion of phagosomes and lysosomes. However, excessive and uncontrolled autophagic activity can be detrimental to host cells and can result in their death. The Mtb “enhanced intracellular survival” (eis) gene has been implicated in the intracellular survival of M. smegmatis. However, its exact role and how it regulates host innate immune responses have not been fully explained. Here, we provide evidence that Eis suppresses macrophage autophagy, inflammation, and cell death through the inhibition of reactive oxygen species (ROS) generation. Although it has previously been demonstrated that autophagy is a key host defense response to mycobacterial infections, our data indicate that excessive autophagy, and the resulting cell death, do not significantly affect host defense responses to mycobacteria. Additionally, our data reveal that Eis's ability to regulate ROS generation and proinflammatory responses depends on its N-acetyltransferase domain. These results underscore a previously unrecognized role of Eis in modulating host inflammatory responses, oxidative stress, and cell survival/death during mycobacterial infection.

M. tuberculosis (MTB) species-specific antigenic determinants of the human T cell response are important for immunodiagnosis and vaccination. As hypoxia is a stimulus in chronic tuberculosis infection, we analyzed transcriptional profiles of MTB subject to 168 hours of hypoxia to test the hypothesis that upregulation by hypoxia might result in gene products being recognized as antigens. We identified upregulation of two region of difference (RD) 11 (Rv2658C and Rv2659c), and one RD2 (Rv1986) absent from commonly used BCG strains. In MTB infected persons, the IL-2 ELISpot response to Rv1986 peptides was several times greater than the corresponding IFN-γ response to the reference immunodominant ESAT-6 or CFP-10 antigens. The IL-2 response was confined to two epitopic regions containing residues 61–80 and 161–180. The biggest population of IL-2 secreting T cells was single cytokine positive central memory T cells. The IL-2 response to live MTB bacilli lacking Rv1986 was significantly lower than the response to wild type or mutant complemented with Rv1986. In addition, the IL-2 response to Rv1986 was significantly lower in HIV-TB co-infected persons than in HIV uninfected persons, and significantly increased during antiretroviral therapy. These findings demonstrate that Rv1986 is an immunodominant target of memory T cells and is therefore of relevance when considering the partial efficacy of currently used BCG vaccines and provide evidence for a clinical trial comparing BCG strains., Author Summary Mycobacterium tuberculosis (the cause of tuberculosis) can persist for many years in humans without causing disease but has the potential to reactivate. One of the conditions the bacterium must survive in these circumstances is hypoxia. In order to do so, the bacterium uses a characteristic set of genes that help alter its metabolism. It follows that the products of such genes may encode protein antigens that can be recognized by the immune response. We therefore analyzed gene response patterns of tuberculosis subject to prolonged hypoxia as a guide to the discovery of new antigens that might be useful as vaccines or diagnostic agents. Amongst the genes most strongly increased by low oxygen levels, one was identified (known as Rv1986) that is missing from most strains of the tuberculosis vaccine Mycobacterium bovis BCG. When we analyzed human immune responses to this protein in tuberculosis infected people our experiments showed it was particularly well recognized by cells that produce a chemical messenger (cytokine) called interleukin-2. Interleukin-2 is important for long-term immunological memory. The BCG vaccine is only partially effective and our experiments therefore suggest one of the reasons could be that an important immunological target is missing from many strains. Further evaluation of BCG strains in which Rv1986 is present or absent is therefore warranted in the hope that this might improve the efficacy of existing or new tuberculosis vaccines.

Ticks are distributed worldwide and affect human and animal health by transmitting diverse infectious agents. Effective vaccines against most tick-borne pathogens are not currently available. In this study, we characterized a tick histamine release factor (tHRF) from Ixodes scapularis and addressed the vaccine potential of this antigen in the context of tick engorgement and B. burgdorferi transmission. Results from western blotting and quantitative Reverse Transcription-PCR showed that tHRF is secreted in tick saliva, and upregulated in Borrelia burgdorferi-infected ticks. Further, the expression of tHRF was coincident with the rapid feeding phase of the tick, suggesting a role for tHRF in tick engorgement and concomitantly, for efficient B. burgdorferi transmission. Silencing tHRF by RNA interference (RNAi) significantly impaired tick feeding and decreased B. burgdorferi burden in mice. Interfering with tHRF by actively immunizing mice with recombinant tHRF, or passively transferring tHRF antiserum, also markedly reduced the efficiency of tick feeding and B. burgdorferi burden in mice. Recombinant tHRF was able to bind to host basophils and stimulate histamine release. Therefore, we speculate that tHRF might function in vivo to modulate vascular permeability and increase blood flow to the tick bite-site, facilitating tick engorgement. These findings suggest that blocking tHRF might offer a viable strategy to complement ongoing efforts to develop vaccines to block tick feeding and transmission of tick-borne pathogens., Author Summary Ticks are distributed worldwide and affect human and animal health by transmitting diverse infectious agents. Safe and effective vaccines against most tick-borne pathogens are not currently available. Typical vaccines target microbes directly, using extracts of the organism, or recombinant antigens as the immunogen; the transmission of tick-borne pathogens can also theoretically be prevented by interfering with the ability of ticks to feed on a mammalian host. In this study, we have characterized a putative histamine release factor (tHRF) from I. scapularis ticks, the predominant vector of B. burgdorferi, the agent of Lyme disease in North America. Our results suggested that tHRF is presented in tick saliva and critical for tick feeding; blocking tHRF markedly reduced the efficiency of tick feeding, and reduced the B. burgdorferi burden in mice. This finding provides novel insights into the molecular mechanisms of tick feeding and provides a potential vaccine target to block tick feeding and pathogen transmission.

Murine models of urinary tract infection (UTI) have provided substantial data identifying uropathogenic E. coli (UPEC) virulence factors and assessing their expression in vivo. However, it is unclear how gene expression in these animal models compares to UPEC gene expression during UTI in humans. To address this, we used a UPEC strain CFT073-specific microarray to measure global gene expression in eight E. coli isolates monitored directly from the urine of eight women presenting at a clinic with bacteriuria. The resulting gene expression profiles were compared to those of the same E. coli isolates cultured statically to exponential phase in pooled, sterilized human urine ex vivo. Known fitness factors, including iron acquisition and peptide transport systems, were highly expressed during human UTI and support a model in which UPEC replicates rapidly in vivo. While these findings were often consistent with previous data obtained from the murine UTI model, host-specific differences were observed. Most strikingly, expression of type 1 fimbrial genes, which are among the most highly expressed genes during murine experimental UTI and encode an essential virulence factor for this experimental model, was undetectable in six of the eight E. coli strains from women with UTI. Despite the lack of type 1 fimbrial expression in the urine samples, these E. coli isolates were generally capable of expressing type 1 fimbriae in vitro and highly upregulated fimA upon experimental murine infection. The findings presented here provide insight into the metabolic and pathogenic profile of UPEC in urine from women with UTI and represent the first transcriptome analysis for any pathogenic E. coli during a naturally occurring infection in humans., Author Summary Animal models of infection have been used extensively to study how bacteria and other pathogens cause disease. These models provide valuable information and have led to the development of numerous vaccines and antimicrobial therapies. However, it is important to recognize how these animal models compare to human infection and to understand how bacteria cause disease in humans. This study measured gene expression in E. coli, a major cause of urinary tract infection, immediately after collection from the urine of women with bladder infection symptoms. The data showed that E. coli gene expression in the urine from women with urinary tract infection was very often similar to what had been observed in a mouse model, but these studies also identified several potentially important differences, including a bacterial surface structure that is necessary for infection in mice but not detected in most E. coli in human urine. Although more precise measurements are still needed, these findings contribute to our understanding of bacterial infection in humans and will help in the development of vaccines and treatments for urinary tract infection.

Neonatal meningitis due to Escherichia coli K1 is a serious illness with unchanged morbidity and mortality rates for the last few decades. The lack of a comprehensive understanding of the mechanisms involved in the development of meningitis contributes to this poor outcome. Here, we demonstrate that depletion of macrophages in newborn mice renders the animals resistant to E. coli K1 induced meningitis. The entry of E. coli K1 into macrophages requires the interaction of outer membrane protein A (OmpA) of E. coli K1 with the alpha chain of Fcγ receptor I (FcγRIa, CD64) for which IgG opsonization is not necessary. Overexpression of full-length but not C-terminal truncated FcγRIa in COS-1 cells permits E. coli K1 to enter the cells. Moreover, OmpA binding to FcγRIa prevents the recruitment of the γ-chain and induces a different pattern of tyrosine phosphorylation of macrophage proteins compared to IgG2a induced phosphorylation. Of note, FcγRIa−/− mice are resistant to E. coli infection due to accelerated clearance of bacteria from circulation, which in turn was the result of increased expression of CR3 on macrophages. Reintroduction of human FcγRIa in mouse FcγRIa−/− macrophages in vitro increased bacterial survival by suppressing the expression of CR3. Adoptive transfer of wild type macrophages into FcγRIa−/− mice restored susceptibility to E. coli infection. Together, these results show that the interaction of FcγRI alpha chain with OmpA plays a key role in the development of neonatal meningitis by E. coli K1., Author Summary Escherichia coli K1 is the most common cause of meningitis in premature infants; the mortality rate of this disease ranges from 5% to 30%. A better understanding of the pathogenesis of E. coli K1 meningitis is needed to develop new preventative strategies. We have shown that outer membrane protein A (OmpA) of E. coli K1, independent of antibody opsonization, is critical for bacterial entrance and survival within macrophages. Using a newborn mouse model, we found that depletion of macrophages renders the animals resistant to E. coli K1 induced meningitis. OmpA binds to α-chain of Fcγ-receptor I (FcγRIa) in macrophages, but does not induce expected gamma chain association and signaling. FcγRIa knockout mice are resistant to E. coli K1 infection because their macrophages express more CR3 and are thus able to kill bacteria with greater efficiency, preventing the development of high-grade bacteremia, a pre-requisite for the onset of meningitis. These novel observations demonstrate that inhibiting OmpA binding to FcγRIa is a promising therapeutic target for treatment or prevention of neonatal meningitis.

Chemokines have been found to exert direct, defensin-like antimicrobial activity in vitro, suggesting that, in addition to orchestrating cellular accumulation and activation, chemokines may contribute directly to the innate host response against infection. No observations have been made, however, demonstrating direct chemokine-mediated promotion of host defense in vivo. Here, we show that the murine interferon-inducible CXC chemokines CXCL9, CXCL10, and CXCL11 each exert direct antimicrobial effects in vitro against Bacillus anthracis Sterne strain spores and bacilli including disruptions in spore germination and marked reductions in spore and bacilli viability as assessed using CFU determination and a fluorometric assay of metabolic activity. Similar chemokine-mediated antimicrobial activity was also observed against fully virulent Ames strain spores and encapsulated bacilli. Moreover, antibody-mediated neutralization of these CXC chemokines in vivo was found to significantly increase host susceptibility to pulmonary B. anthracis infection in a murine model of inhalational anthrax with disease progression characterized by systemic bacterial dissemination, toxemia, and host death. Neutralization of the shared chemokine receptor CXCR3, responsible for mediating cellular recruitment in response to CXCL9, CXCL10, and CXCL11, was not found to increase host susceptibility to inhalational anthrax. Taken together, our data demonstrate a novel, receptor-independent antimicrobial role for the interferon-inducible CXC chemokines in pulmonary innate immunity in vivo. These data also support an immunomodulatory approach for effectively treating and/or preventing pulmonary B. anthracis infection, as well as infections caused by pathogenic and potentially, multi-drug resistant bacteria including other spore-forming organisms., Author Summary Innate immunity is critical to host defense and plays a central role in protecting the lungs from respiratory pathogens. Among the mediators important in the innate host response to pulmonary infection are chemokines, proteins originally described for their ability to regulate immune cell trafficking during an inflammatory response. More recently, chemokines have been found to exert direct antimicrobial activity against a broad range of bacteria and fungi in vitro. While these observations suggest chemokines may contribute to host defense by killing microorganisms at local sites of infection through activities not associated with cellular chemokine receptors, the biological relevance of direct chemokine-mediated antimicrobial activity in vivo has not been established. Here we show that the murine chemokines CXCL9, CXCL10, and CXCL11 exert direct antimicrobial effects against B. anthracis in vitro and that neutralization of these CXC chemokines, but not their shared receptor CXCR3, increases host susceptibility to pulmonary B. anthracis infection in vivo. These data provide unique insight into the host mediators important in host-pathogen interaction and pathogenesis of disease and support the emerging concept that host chemokines mediate efficient, pleiotropic roles that include receptor-independent promotion of host defense in vivo.

The host response to mycobacterial infection depends on host and pathogen genetic factors. Recent studies in human populations suggest a strain specific genetic control of tuberculosis. To test for mycobacterial-strain specific genetic control of susceptibility to infection under highly controlled experimental conditions, we performed a comparative genetic analysis using the A/J- and C57BL/6J-derived recombinant congenic (RC) mouse panel infected with the Russia and Pasteur strains of Mycobacterium bovis Bacille Calmette Guérin (BCG). Bacillary counts in the lung and spleen at weeks 1 and 6 post infection were used as a measure of susceptibility. By performing genome-wide linkage analyses of loci that impact on tissue-specific bacillary burden, we were able to show the importance of correcting for strain background effects in the RC panel. When linkage analysis was adjusted on strain background, we detected a single locus on chromosome 11 that impacted on pulmonary counts of BCG Russia but not Pasteur. The same locus also controlled the splenic counts of BCG Russia but not Pasteur. By contrast, a locus on chromosome 1 which was indistinguishable from Nramp1 impacted on splenic bacillary counts of both BCG Russia and Pasteur. Additionally, dependent upon BCG strain, tissue and time post infection, we detected 9 distinct loci associated with bacillary counts. Hence, the ensemble of genetic loci impacting on BCG infection revealed a highly dynamic picture of genetic control that reflected both the course of infection and the infecting strain. This high degree of adaptation of host genetics to strain-specific pathogenesis is expected to provide a suitable framework for the selection of specific host-mycobacteria combinations during co-evolution of mycobacteria with humans., Author Summary Susceptibility to mycobacterial infection results from a complex interaction between host and bacterial genetic factors. To examine the effect of host and pathogen genetic variability on the control of mycobacterial infection, we infected a panel of genetically related recombinant congenic (RC) mouse strains with two closely related strains of Mycobacterium bovis BCG. Bacterial counts of BCG Russia and BCG Pasteur were determined in the lung and spleen at 1 and 6 weeks following infection and used for genetic analysis. A novel analytical approach was developed to perform genome-wide linkage analyses using the RC strains. Comparative linkage analysis using this model identified a strong genetic effect on chromosome 1 controlling counts of BCG Pasteur at 1 week and of BCG Russia at 1 week and 6 weeks in the spleen. A locus impacting on late BCG Russia counts in the lung and spleen was identified on chromosome 11. Nine additional loci were shown to control bacterial counts in a tissue-, time-, and BCG strain-specific manner. Our findings suggest that the host genetic control of mycobacterial infection is highly dynamic and adapted to the stage of pathogenesis and to the infecting strain. Such a high degree of genetic plasticity in the host-pathogen interplay is expected to favour evolutionary co-adaptation in mycobacterial disease.

If insufficiently treated, Lyme borreliosis can evolve into an inflammatory disorder affecting skin, joints, and the CNS. Early innate immunity may determine host responses targeting infection. Thus, we sought to characterize the immediate cytokine storm associated with exposure of PBMC to moderate levels of live Borrelia burgdorferi. Since Th17 cytokines are connected to host defense against extracellular bacteria, we focused on interleukin (IL)-17 and IL-22. Here, we report that, despite induction of inflammatory cytokines including IL-23, IL-17 remained barely detectable in response to B. burgdorferi. In contrast, T cell-dependent expression of IL-22 became evident within 10 h of exposure to the spirochetes. This dichotomy was unrelated to interferon-γ but to a large part dependent on caspase-1 and IL-1 bioactivity derived from monocytes. In fact, IL-1β as a single stimulus induced IL-22 but not IL-17. Neutrophils display antibacterial activity against B. burgdorferi, particularly when opsonized by antibodies. Since neutrophilic inflammation, indicative of IL-17 bioactivity, is scarcely observed in Erythema migrans, a manifestation of skin inflammation after infection, protective and antibacterial properties of IL-22 may close this gap and serve essential functions in the initial phase of spirochete infection., Author Summary Lyme borreliosis displays multifaceted clinical manifestations caused by the Borrelia burgdorferi sensu lato complex. If insufficiently treated, infection may proceed to inflammatory complications of chronic infection. Th17-like cytokines, foremost IL-17 and IL-22, are crucial for host defense against extracellular bacteria. IL-17/IL-22 secretion by human leukocytes exposed to live Borreliae has not been analyzed. Here we report that B. burgdorferi-activated PBMC lack immediate IL-17 expression despite being highly activated and robust T cell-dependent production of IL-22 that to a large part is mediated by monocyte-derived IL-1. Early innate immunity may shape dermal infection, thus likely affecting bacterial dissemination. Specifically, insufficient neutrophil recruitment/function, supposedly due to insufficient early IL-17 production along with a lack of opsonizing antibodies, may favor the spread of B. burgdorferi. Indeed, neutrophilic inflammation, indicative of IL-17 bioactivity, is scarcely observed in Erythema migrans, a manifestation of skin inflammation after infection. Production of IL-22 may fill this gap. Current knowledge on the role of IL-22 in epithelial biology in fact supports the hypothesis that IL-22 may serve as protection, particularly under conditions of inadequate neutrophil-driven host defense as seen early in B. burgdorferi infection.

A fundamental, but unanswered question in host-pathogen interactions is the timing, localization and population distribution of virulence gene expression during infection. Here, microarray and in situ single cell expression methods were used to study Vibrio cholerae growth and virulence gene expression during infection of the rabbit ligated ileal loop model of cholera. Genes encoding the toxin-coregulated pilus (TCP) and cholera toxin (CT) were powerfully expressed early in the infectious process in bacteria adjacent to epithelial surfaces. Increased growth was found to co-localize with virulence gene expression. Significant heterogeneity in the expression of tcpA, the repeating subunit of TCP, was observed late in the infectious process. The expression of tcpA, studied in single cells in a homogeneous medium, demonstrated unimodal induction of tcpA after addition of bicarbonate, a chemical inducer of virulence gene expression. Striking bifurcation of the population occurred during entry into stationary phase: one subpopulation continued to express tcpA, whereas the expression declined in the other subpopulation. ctxA, encoding the A subunit of CT, and toxT, encoding the proximal master regulator of virulence gene expression also exhibited the bifurcation phenotype. The bifurcation phenotype was found to be reversible, epigenetic and to persist after removal of bicarbonate, features consistent with bistable switches. The bistable switch requires the positive-feedback circuit controlling ToxT expression and formation of the CRP-cAMP complex during entry into stationary phase. Key features of this bistable switch also were demonstrated in vivo, where striking heterogeneity in tcpA expression was observed in luminal fluid in later stages of the infection. When this fluid was diluted into artificial seawater, bacterial aggregates continued to express tcpA for prolonged periods of time. The bistable control of virulence gene expression points to a mechanism that could generate a subpopulation of V. cholerae that continues to produce TCP and CT in the rice water stools of cholera patients., Author Summary Most pathogenic microorganisms infect in a stepwise manner: colonization of host surfaces is followed by invasion and injury of host tissues and, late in the infectious process, dissemination to other hosts occurs. During its residence in the host, the pathogen produces essential virulence determinants and often replicates rapidly, leading to a vast expansion of its biomass. Although this scenario is well established also for Vibrio cholerae, the cause of a potentially fatal diarrheal illness, it has not previously been possible to identify precisely when or where virulence determinants are produced in the intestine. We addressed this question by investigating the expression of virulence genes by individual V. cholerae during infection of the small intestine. Virulence genes were found to be powerfully expressed early in the infectious process by bacteria in close proximity to epithelial surfaces. Increased replication rates were also localized to epithelial surfaces. During later stages of the infection, the population of V. cholerae bifurcates into two fractions: one subpopulation continues to express virulence genes, whereas these genes are silenced in the other subpopulation. The genetic program controlling the continued production of virulence genes may mediate the persistence of a hyper-infectious subpopulation of bacteria in the stools of cholera patients.

Toll-like receptor signaling requires functional Toll/interleukin-1 (IL-1) receptor (TIR) domains to activate innate immunity. By producing TIR homologous proteins, microbes inhibit host response induction and improve their own survival. The TIR homologous protein TcpC was recently identified as a virulence factor in uropathogenic Escherichia coli (E. coli), suppressing innate immunity by binding to MyD88. This study examined how the host MyD88 genotype modifies the in vivo effects of TcpC and whether additional, TIR-domain containing proteins might be targeted by TcpC. In wild type mice (wt), TcpC enhanced bacterial virulence, increased acute mortality, bacterial persistence and tissue damage after infection with E. coli CFT073 (TcpC+), compared to a ΔTcpC deletion mutant. These effects were attenuated in Myd88−/− and Tlr4−/− mice. Transcriptomic analysis confirmed that TcpC inhibits MYD88 dependent gene expression in CFT073 infected human uroepithelial cells but in addition the inhibitory effect included targets in the TRIF and IL-6/IL-1 signaling pathways, where MYD88 dependent and independent signaling may converge. The effects of TcpC on bacterial persistence were attenuated in Trif −/− or Il-1β −/− mice and innate immune responses to ΔTcpC were increased, confirming that Trif and Il-1β dependent targets might be involved in vivo, in addition to Myd88. Furthermore, soluble TcpC inhibited Myd88 and Trif dependent TLR signaling in murine macrophages. Our results suggest that TcpC may promote UTI-associated pathology broadly, through inhibition of TIR domain signaling and downstream pathways. Dysregulation of the host response by microbial TcpC thus appears to impair the protective effects of innate immunity, while promoting inflammation and tissue damage., Author Summary The clinical manifestations of infection range from beneficial, asymptomatic states to life threatening disease, depending on the arsenal of virulence factors carried by the bacteria and the host immune defence repertoire. Pathogenic bacteria have evolved many sophisticated ways of avoiding the host defence and especially the immune response to infection. In this study, we present a very interesting case where bacteria actively inhibit the immune response by producing a host defence like protein, TcpC, which acts by promoting bacterial survival and corrupting the tissue response to infection such that the tissues are damaged rather than protected. The importance of TcpC is demonstrated in a mouse model of urinary tract infection (UTI) and in isolated human and murine kidney cells. The results suggest that TcpC expressing bacteria cause death, inflammation and tissue damage in normal hosts by creating a dysfunctional innate immune response and that partial inhibition of adaptor proteins turns the normally protective defense into lethal inflammation, followed by kidney tissue damage. In human cells, TcpC was a broad innate immune inhibitor, acting via the MYD88, TRIF and IL-1/IL-6 pathways. Our report increases the understanding of how TcpC and microbial proteins with similar targets succeed in shifting the balance in favor of the pathogen, thus promoting disease. These data are fundamentally important in showing pathways for host defense that can be fine tuned by a bacterial virulence factor in order to paradoxically promote bacterial replication thereby illustrating the host response as a generator of pathology.

The cycle inhibiting factors (Cif), produced by pathogenic bacteria isolated from vertebrates and invertebrates, belong to a family of molecules called cyclomodulins that interfere with the eukaryotic cell cycle. Cif blocks the cell cycle at both the G1/S and G2/M transitions by inducing the stabilization of cyclin-dependent kinase inhibitors p21waf1 and p27kip1. Using yeast two-hybrid screens, we identified the ubiquitin-like protein NEDD8 as a target of Cif. Cif co-compartmentalized with NEDD8 in the host cell nucleus and induced accumulation of NEDD8-conjugated cullins. This accumulation occurred early after cell infection and correlated with that of p21 and p27. Co-immunoprecipitation revealed that Cif interacted with cullin-RING ubiquitin ligase complexes (CRLs) through binding with the neddylated forms of cullins 1, 2, 3, 4A and 4B subunits of CRL. Using an in vitro ubiquitylation assay, we demonstrate that Cif directly inhibits the neddylated CUL1-associated ubiquitin ligase activity. Consistent with this inhibition and the interaction of Cif with several neddylated cullins, we further observed that Cif modulates the cellular half-lives of various CRL targets, which might contribute to the pathogenic potential of diverse bacteria., Author Summary Among the arsenal of virulence factors used by bacterial pathogens to infect and manipulate their hosts, cyclomodulins are a growing family of bacterial toxins that interfere with the eukaryotic cell-cycle. Cif is one of these cyclomodulins produced by both mammalian and invertebrate pathogenic bacteria. Cif blocks the host cell cycle by inducing the accumulation of two regulators of cell cycle progression: the cyclin-dependent kinase inhibitors p21 and p27. To decipher the mode of action of Cif, we performed yeast two-hybrid screenings. We show that Cif binds to NEDD8 and induce accumulation of neddylated cullins early after infection. Cullins are scaffold components of cullin-RING ubiquitin ligases (CRLs), which ubiquitinate proteins and target them for degradation by the 26S proteasome. We demonstrate that Cif directly inhibits the ubiquitin ligase activity of these CRLs and consequently the targeting of p21 and p27 for ubiquitin-dependent degradation. Targeting at NEDD8 represents a novel strategy for modulation of host cell functions by bacterial pathogens. By inhibiting the most prominent class of ubiquitin-ligases, Cif controls the stability of a cohort of key regulators and impinge on not only cell cycle progression but also on many cellular and biological processes such as immunity, development, transcription, and cell signaling.

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen chronically infecting the lungs of patients with chronic obstructive pulmonary disease (COPD), pneumonia, cystic fibrosis (CF), and bronchiectasis. Cif (PA2934), a bacterial toxin secreted in outer membrane vesicles (OMV) by P. aeruginosa, reduces CFTR-mediated chloride secretion by human airway epithelial cells, a key driving force for mucociliary clearance. The aim of this study was to investigate the mechanism whereby Cif reduces CFTR-mediated chloride secretion. Cif redirected endocytosed CFTR from recycling endosomes to lysosomes by stabilizing an inhibitory effect of G3BP1 on the deubiquitinating enzyme (DUB), USP10, thereby reducing USP10-mediated deubiquitination of CFTR and increasing the degradation of CFTR in lysosomes. This is the first example of a bacterial toxin that regulates the activity of a host DUB. These data suggest that the ability of P. aeruginosa to chronically infect the lungs of patients with COPD, pneumonia, CF, and bronchiectasis is due in part to the secretion of OMV containing Cif, which inhibits CFTR-mediated chloride secretion and thereby reduces the mucociliary clearance of pathogens., National Institutes of Health (U.S.) (NIH grants R01-HL074175), National Institutes of Health (U.S.) (R01-DK/HL-45881), National Institutes of Health (U.S.) (5P20RR018787-08), National Institutes of Health (U.S.) (R01-AI083256), Cystic Fibrosis Foundation (CF RDP), Cystic Fibrosis Foundation (BOMBER08F0)

Bacillus anthracis infects hosts as a spore, germinates, and disseminates in its vegetative form. Production of anthrax lethal and edema toxins following bacterial outgrowth results in host death. Macrophages of inbred mouse strains are either sensitive or resistant to lethal toxin depending on whether they express the lethal toxin responsive or non-responsive alleles of the inflammasome sensor Nlrp1b (Nlrp1bS/S or Nlrp1bR/R, respectively). In this study, Nlrp1b was shown to affect mouse susceptibility to infection. Inbred and congenic mice harboring macrophage-sensitizing Nlrp1bS/S alleles (which allow activation of caspase-1 and IL-1β release in response to anthrax lethal toxin challenge) effectively controlled bacterial growth and dissemination when compared to mice having Nlrp1bR/R alleles (which cannot activate caspase-1 in response to toxin). Nlrp1bS-mediated resistance to infection was not dependent on the route of infection and was observed when bacteria were introduced by either subcutaneous or intravenous routes. Resistance did not occur through alterations in spore germination, as vegetative bacteria were also killed in Nlrp1bS/S mice. Resistance to infection required the actions of both caspase-1 and IL-1β as Nlrp1bS/S mice deleted of caspase-1 or the IL-1 receptor, or treated with the Il-1 receptor antagonist anakinra, were sensitized to infection. Comparison of circulating neutrophil levels and IL-1β responses in Nlrp1bS/S,Nlrp1bR/ R and IL-1 receptor knockout mice implicated Nlrp1b and IL-1 signaling in control of neutrophil responses to anthrax infection. Neutrophil depletion experiments verified the importance of this cell type in resistance to B. anthracis infection. These data confirm an inverse relationship between murine macrophage sensitivity to lethal toxin and mouse susceptibility to spore infection, and establish roles for Nlrp1bS, caspase-1, and IL-1β in countering anthrax infection., Author Summary In this study, we show that anthrax lethal toxin activation of Nlrp1b in toxin-sensitive mouse macrophages imparts resistance to infection. Inbred and congenic mice harboring macrophage-sensitizing Nlrp1b alleles control bacterial growth and dissemination independent of infection route or effects on germination efficiency. Knockout mice demonstrate that resistance imparted by Nlrp1b requires caspase-1 activity and IL-1 signaling. Mice in which lethal toxin activates the Nlrp1b inflammasome show an IL-1β response and increased neutrophil recruitment leading to increased resistance to infection. Neutrophil depletion experiments verify the importance of this cell type in resistance to B. anthracis infection. These data confirm an inverse relationship between murine macrophage sensitivity to lethal toxin and mouse susceptibility to spore infection and demonstrate that the activation of the inflammasome in response to anthrax infection in mice is a protective event that occurs through IL-1β induction of neutrophil recruitment.

Pseudomonas aeruginosa is a nearly ubiquitous human pathogen, and infections can be lethal to patients with impaired respiratory and immune systems. Prior studies have established that strong loss-of-function mutations in the egl-9 gene protect the nematode C. elegans from P. aeruginosa PAO1 fast killing. EGL-9 inhibits the HIF-1 transcription factor via two pathways. First, EGL-9 is the enzyme that targets HIF-1 for oxygen-dependent degradation via the VHL-1 E3 ligase. Second, EGL-9 inhibits HIF-1-mediated gene expression through a VHL-1-independent mechanism. Here, we show that a loss-of-function mutation in hif-1 suppresses P. aeruginosa PAO1 resistance in egl-9 mutants. Importantly, we find stabilization of HIF-1 protein is not sufficient to protect C. elegans from P. aeruginosa PAO1 fast killing. However, mutations that inhibit both EGL-9 pathways result in higher levels of HIF-1 activity and confer resistance to the pathogen. Using forward genetic screens, we identify additional mutations that confer resistance to P. aeruginosa. In genetic backgrounds that stabilize C. elegans HIF-1 protein, loss-of-function mutations in swan-1 increase the expression of hypoxia response genes and protect C. elegans from P. aeruginosa fast killing. SWAN-1 is an evolutionarily conserved WD-repeat protein belonging to the AN11 family. Yeast two-hybrid and co-immunoprecipitation assays show that EGL-9 forms a complex with SWAN-1. Additionally, we present genetic evidence that the DYRK kinase MBK-1 acts downstream of SWAN-1 to promote HIF-1-mediated transcription and to increase resistance to P. aeruginosa. These data support a model in which SWAN-1, MBK-1 and EGL-9 regulate HIF-1 transcriptional activity and modulate resistance to P. aeruginosa PAO1 fast killing., Author Summary Pseudomonas aeruginosa is a common bacterial pathogen that can infect a wide range of animals. In some conditions, P. aeruginosa produces cyanide, a toxin that limits cellular capacity to metabolize oxygen and produce energy. The nematode Caenorhabditis elegans is a powerful genetic model system for understanding the mechanisms of stress response and pathogen resistance. Here, we show that HIF-1, a DNA-binding transcription factor that mediates cellular responses to low oxygen, can protect C. elegans from P. aeruginosa fast killing. Additionally, we identify swan-1 as a gene that functions to inhibit HIF-1 activity and suppress P. aeruginosa resistance. The SWAN-1 protein binds directly to the oxygen-sensing EGL-9 enzyme that controls HIF-1 stability and activity. This study advances understanding of HIF-1 regulatory networks, defines connections between hypoxia response and P. aeruginosa fast killing, and provides new insights into mechanisms by which animals can resist this bacterial pathogen.

Previous work revealed that conditional depletion of the core proteasome subunits PrcB and PrcA impaired growth of Mycobacterium tuberculosis in vitro and in mouse lungs, caused hypersusceptibility to nitric oxide (NO) and impaired persistence of the bacilli during chronic mouse infections. Here, we show that genetic deletion of prcBA led to similar phenotypes. Surprisingly, however, an active site mutant proteasome complemented the in vitro and in vivo growth defects of the prcBA knockout (ΔprcBA) as well as its NO hypersensitivity. In contrast, long-term survival of M. tuberculosis in stationary phase and during starvation in vitro and in the chronic phase of mouse infection required a proteolytically active proteasome. Inhibition of inducible nitric oxide synthase did not rescue survival of ΔprcBA, revealing a function beyond NO defense, by which the proteasome contributes to M. tuberculosis fitness during chronic mouse infections. These findings suggest that proteasomal proteolysis facilitates mycobacterial persistence, that M. tuberculosis faces starvation during chronic mouse infections and that the proteasome serves a proteolysis-independent function., Author Summary The eukaryotic proteasome is ubiquitous and essential for many basic cellular processes. In contrast to most bacteria, which do not express a proteasome, Mycobacterium tuberculosis encodes a proteasome predicted to be essential or required for optimal growth of the pathogen. Genetic silencing of the proteasome core genes further suggested that the M. tuberculosis proteasome plays an important role in defense against nitric oxide and in persistence of the pathogen during chronic mouse infections. In this manuscript we generated a genetic deletion mutant of the proteasome core genes proving that the 20S proteasome is not essential for growth of M. tuberculosis. We complemented the proteasome knockout with a proteolytically active and a mutated, proteolysis defective proteasome. This revealed that proteasomal proteolysis is dispensable for in vitro and in vivo growth and nitric oxide resistance of M. tuberculosis and suggests that the proteasome core serves a proteolysis-independent function. In contrast, long-term survival of the pathogen in vitro and in the chronic phase of mouse infection required a proteolytically active proteasome. We further provide evidence that nitric oxide is not responsible for killing of the proteasome knockout during chronic mouse infections. Thus, proteasomal proteolysis facilitates mycobacterial persistence independently of defense against nitric oxide. We propose that the failure to survive starvation contributes to the impaired persistence of M. tuberculosis lacking a proteolytically active proteasome during chronic infections.

Upon infection of mammalian cells, enterohemorrhagic E. coli (EHEC) O157:H7 utilizes a type III secretion system to translocate the effectors Tir and EspFU (aka TccP) that trigger the formation of F-actin-rich ‘pedestals’ beneath bound bacteria. EspFU is localized to the plasma membrane by Tir and binds the nucleation-promoting factor N-WASP, which in turn activates the Arp2/3 actin assembly complex. Although N-WASP has been shown to be required for EHEC pedestal formation, the precise steps in the process that it influences have not been determined. We found that N-WASP and actin assembly promote EHEC-mediated translocation of Tir and EspFU into mammalian host cells. When we utilized the related pathogen enteropathogenic E. coli to enhance type III translocation of EHEC Tir and EspFU, we found surprisingly that actin pedestals were generated on N-WASP-deficient cells. Similar to pedestal formation on wild type cells, Tir and EspFU were the only bacterial effectors required for pedestal formation, and the EspFU sequences required to interact with N-WASP were found to also be essential to stimulate this alternate actin assembly pathway. In the absence of N-WASP, the Arp2/3 complex was both recruited to sites of bacterial attachment and required for actin assembly. Our results indicate that actin assembly facilitates type III translocation, and reveal that EspFU, presumably by recruiting an alternate host factor that can signal to the Arp2/3 complex, exhibits remarkable versatility in its strategies for stimulating actin polymerization., Author Summary The food-borne pathogen enterohemorrhagic E. coli (EHEC) O157:H7 can cause severe diarrhoea and life-threatening systemic illnesses. During infection, EHEC attaches to cells lining the human intestine and injects Tir and EspFU, two bacterial molecules that alter the host cell actin cytoskeleton and stimulate the formation of “pedestals” just beneath bound bacteria. Pedestal formation promotes colonization during the later stages of infection. N-WASP, a host protein known to regulate actin assembly in mammalian cells, was previously shown to be manipulated by Tir and EspFU to stimulate actin assembly, and to be required for EHEC to generate actin pedestals. Surprisingly, we show here that N-WASP promotes the efficient delivery of Tir and EspFU into mammalian cells, and that when we utilized a related E. coli to enhance type III delivery of Tir and EspFU, actin pedestals assembled even in its absence. Thus, EHEC stimulates at least two pathways of actin assembly to generate pedestals, one mediated by N-WASP and one by an unidentified alternate factor. This flexibility likely reflects an important function of pedestal formation by EHEC, and study of the underlying mechanisms may provide new insights into the pathogenesis of infection as well as the regulation of the actin cytoskeleton of mammalian cells.

The bacterial pathogen Staphylococcus aureus seeds abscesses in host tissues to replicate at the center of these lesions, protected from host immune cells via a pseudocapsule. Using histochemical staining, we identified prothrombin and fibrin within abscesses and pseudocapsules. S. aureus secretes two clotting factors, coagulase (Coa) and von Willebrand factor binding protein (vWbp). We report here that Coa and vWbp together are required for the formation of abscesses. Coa and vWbp promote the non-proteolytic activation of prothrombin and cleavage of fibrinogen, reactions that are inhibited with specific antibody against each of these molecules. Coa and vWbp specific antibodies confer protection against abscess formation and S. aureus lethal bacteremia, suggesting that coagulases function as protective antigens for a staphylococcal vaccine., Author Summary Clinical isolates of the human pathogen Staphylococcus aureus secrete coagulase (Coa), a polypeptide that binds to and activates prothrombin, thereby converting fibrinogen to fibrin and promoting clotting of plasma or blood. Another secreted coagulase, designated von-Willebrand factor binding protein (vWbp), catalyzes a similar reaction. Staphylococcal binding to fibrinogen or fibrin is an important attribute of disease pathogenesis, which leads to the formation of abscesses and bacterial persistence in host tissues. We report here that Coa and vWbp are essential for S. aureus strain Newman abscess formation and persistence in host tissues. Antibodies directed against Coa or vWbp prevent coagulase binding to prothrombin or fibrinogen and confer protection against challenge with S. aureus Newman or the methicillin-resistant S. aureus isolate USA300 LAC in mouse models of abscess formation or lethal sepsis. These results suggest that coagulases may be used as vaccine antigens to elicit antibodies that protect humans against S. aureus infections.

Bacterial growth in multicellular communities, or biofilms, offers many potential advantages over single-cell growth, including resistance to antimicrobial factors. Here we describe the interaction between the biofilm-promoting components curli fimbriae and cellulose of uropathogenic E. coli and the endogenous antimicrobial defense in the urinary tract. We also demonstrate the impact of this interplay on the pathogenesis of urinary tract infections. Our results suggest that curli and cellulose exhibit differential and complementary functions. Both of these biofilm components were expressed by a high proportion of clinical E. coli isolates. Curli promoted adherence to epithelial cells and resistance against the human antimicrobial peptide LL-37, but also increased the induction of the proinflammatory cytokine IL-8. Cellulose production, on the other hand, reduced immune induction and hence delayed bacterial elimination from the kidneys. Interestingly, LL-37 inhibited curli formation by preventing the polymerization of the major curli subunit, CsgA. Thus, even relatively low concentrations of LL-37 inhibited curli-mediated biofilm formation in vitro. Taken together, our data demonstrate that biofilm components are involved in the pathogenesis of urinary tract infections by E. coli and can be a target of local immune defense mechanisms., Author Summary Most infections of the urinary tract are caused by uropathogenic E. coli. On abiotic surfaces, these bacteria are able to form biofilms, which protect them from various adverse environmental conditions. In this study, we sought to investigate whether two E. coli biofilm components, curli fimbriae and cellulose, provide a similar protection against innate immune defense mechanisms of the urinary tract. We put special emphasis on the interaction with the human antimicrobial peptide LL-37, which plays a crucial role in the protection against uropathogenic E. coli. We demonstrate that curli expression specifically reduces bacterial sensitivity to LL-37 by binding the peptide before reaching the bacterial cell membrane and exhibiting its bactericidal activity. A more general protection is mediated by cellulose, possibly by hiding immunogenic surface structures of the bacterium. In addition to providing protection, curli are also targeted by the immune system. The formation of new curli fibers is inhibited in the presence of LL-37. Moreover, curliated bacteria show higher immunogenicity than their non-curliated counterparts. Cellulose expression, on the other hand, appears to impair initial host colonization. In conclusion, our findings demonstrate an example of the tight interplay between bacterial virulence factors and the host immune defense.

Large bacterial protein toxins autotranslocate functional effector domains to the eukaryotic cell cytosol, resulting in alterations to cellular functions that ultimately benefit the infecting pathogen. Among these toxins, the clostridial glucosylating toxins (CGTs) produced by Gram-positive bacteria and the multifunctional-autoprocessing RTX (MARTX) toxins of Gram-negative bacteria have distinct mechanisms for effector translocation, but a shared mechanism of post-translocation autoprocessing that releases these functional domains from the large holotoxins. These toxins carry an embedded cysteine protease domain (CPD) that is activated for autoprocessing by binding inositol hexakisphosphate (InsP(6)), a molecule found exclusively in eukaryotic cells. Thus, InsP(6)-induced autoprocessing represents a unique mechanism for toxin effector delivery specifically within the target cell. This review summarizes recent studies of the structural and molecular events for activation of autoprocessing for both CGT and MARTX toxins, demonstrating both similar and potentially distinct aspects of autoprocessing among the toxins that utilize this method of activation and effector delivery.

The genetically tractable model host Caenorhabditis elegans provides a valuable tool to dissect host-microbe interactions in vivo. Pseudomonas aeruginosa and Staphylococcus aureus utilize virulence factors involved in human disease to infect and kill C. elegans. Despite much progress, virtually nothing is known regarding the cytopathology of infection and the proximate causes of nematode death. Using light and electron microscopy, we found that P. aeruginosa infection entails intestinal distention, accumulation of an unidentified extracellular matrix and P. aeruginosa-synthesized outer membrane vesicles in the gut lumen and on the apical surface of intestinal cells, the appearance of abnormal autophagosomes inside intestinal cells, and P. aeruginosa intracellular invasion of C. elegans. Importantly, heat-killed P. aeruginosa fails to elicit a significant host response, suggesting that the C. elegans response to P. aeruginosa is activated either by heat-labile signals or pathogen-induced damage. In contrast, S. aureus infection causes enterocyte effacement, intestinal epithelium destruction, and complete degradation of internal organs. S. aureus activates a strong transcriptional response in C. elegans intestinal epithelial cells, which aids host survival during infection and shares elements with human innate responses. The C. elegans genes induced in response to S. aureus are mostly distinct from those induced by P. aeruginosa. In contrast to P. aeruginosa, heat-killed S. aureus activates a similar response as live S. aureus, which appears to be independent of the single C. elegans Toll-Like Receptor (TLR) protein. These data suggest that the host response to S. aureus is possibly mediated by pathogen-associated molecular patterns (PAMPs). Because our data suggest that neither the P. aeruginosa nor the S. aureus–triggered response requires canonical TLR signaling, they imply the existence of unidentified mechanisms for pathogen detection in C. elegans, with potentially conserved roles also in mammals., Author Summary Pseudomonas aeruginosa and Staphylococcus aureus are bacteria that can form part of the human microbiota, but can also cause severe disease. Despite their great clinical importance, little is known about their interactions with the human host before disease onset, particularly regarding the molecules that host cells use to prevent and combat infection. The invertebrate Caenorhabditis elegans is a powerful model host to study host-pathogen interactions and, because of its simplicity and highly-developed experimental methods, can illuminate fundamental mechanisms of bacterial pathogenesis. We used C. elegans to understand the cellular events that occur during early stages of P. aeruginosa and S. aureus infection. We found that P. aeruginosa slowly colonizes the intestine, producing virulence-related membrane vesicles, and causing accumulation of electron-dense biofilm-like material on intestinal cells and abnormalities in them. In contrast, S. aureus colonizes rapidly, disrupting microvilli and lysing host cells. We found that these different strategies result in different host gene expression responses. Focusing on S. aureus infection, the C. elegans response is mainly microbicidal and detoxifying, aiding host survival and bearing similarities with the human response. Our study provides new insights into mechanisms that P. aeruginosa and S. aureus use to cause disease, and into C. elegans defenses, with potential implications for human immunity and disease.

Leprosy is an infectious disease caused by the obligate intracellular pathogen Mycobacterium leprae and remains endemic in many parts of the world. Despite several major studies on susceptibility to leprosy, few genomic loci have been replicated independently. We have conducted an association analysis of more than 1,500 individuals from different case-control and family studies, and observed consistent associations between genetic variants in both TLR1 and the HLA-DRB1/DQA1 regions with susceptibility to leprosy (TLR1 I602S, case-control P = 5.7×10−8, OR = 0.31, 95% CI = 0.20–0.48, and HLA-DQA1 rs1071630, case-control P = 4.9×10−14, OR = 0.43, 95% CI = 0.35–0.54). The effect sizes of these associations suggest that TLR1 and HLA-DRB1/DQA1 are major susceptibility genes in susceptibility to leprosy. Further population differentiation analysis shows that the TLR1 locus is extremely differentiated. The protective dysfunctional 602S allele is rare in Africa but expands to become the dominant allele among individuals of European descent. This supports the hypothesis that this locus may be under selection from mycobacteria or other pathogens that are recognized by TLR1 and its co-receptors. These observations provide insight into the long standing host-pathogen relationship between human and mycobacteria and highlight the key role of the TLR pathway in infectious diseases., Author Summary Mycobacterium leprae is an obligate intracellular pathogen that causes leprosy, a disease that shares a long history with the human population but which remains endemic in many parts of the world. Despite the fact that the genome of M. leprae has been sequenced, our understanding of its pathogenesis and interaction with the human host is limited, in part due to the inability to culture the bacterium in vitro. In this gene-centric microarray study, we have genotyped SNPs in over 2,000 genes and identified TLR1 and HLA-DRB1/DQA1 as major leprosy susceptibility genes. Studying the geographical distribution of this hypo-functional TLR1 variant demonstrated extreme population differentiation at this locus. These results suggest that leprosy may have contributed to the evolution of this genomic region, and provide insight into the long history of the host-pathogen relationship between humans and M. leprae.

Disruption of p53/Puma-mediated apoptosis protects against lethality due to DNA damage. Here we demonstrate the unexpected requirement of the pro-apoptotic p53-target gene Puma to mount a successful innate immune response to bacterial sepsis. Puma−/− mice rapidly died when challenged with bacteria. While the immune response in Puma−/− mice was unchanged in cell migration, phagocytosis and bacterial killing, sites of infection accumulated large abscesses and sepsis was progressive. Blocking p53/Puma-induced apoptosis during infection caused resistance to ROS-induced cell death in the CD49d+ neutrophil subpopulation, resulting in insufficient immune resolution. This study identifies a biological role for p53/Puma apoptosis in optimizing neutrophil lifespan so as to ensure the proper clearance of bacteria and exposes a counter-balance between the innate immune response to infection and survival from DNA damage., Author Summary Protection against fulminant, lethal bacterial sepsis requires a robust and rapid response from the neutrophil subclass of white blood cells. However, prolonged survival of these activated cells leads to organ damage. The cellular processes determining the optimal life span of neutrophils are not understood. The p53/Puma pathway drives programmed cell death by initiating apoptosis. This response is beneficial in preventing cellular transformation by eliminating abnormal cells that have sustained DNA damage. Here we demonstrate an opposing effect of the pathway in the context of a successful immune response to bacterial sepsis. Puma−/− mice rapidly died when challenged with bacteria and sites of infection accumulated large abscesses. Blocking p53/Puma-induced apoptosis during infection prolonged the life of the CD49d+ neutrophil subpopulation, resulting in insufficient immune resolution. This study expands the biological role of p53/Puma-induced apoptosis to the proper clearance of bacteria by neutrophils and exposes a counter-balance between the innate immune response to infection and survival from DNA damage.

Although there is tremendous interest in understanding the evolutionary roles of horizontal gene transfer (HGT) processes that occur during chronic polyclonal infections, to date there have been few studies that directly address this topic. We have characterized multiple HGT events that most likely occurred during polyclonal infection among nasopharyngeal strains of Streptococcus pneumoniae recovered from a child suffering from chronic upper respiratory and middle-ear infections. Whole genome sequencing and comparative genomics were performed on six isolates collected during symptomatic episodes over a period of seven months. From these comparisons we determined that five of the isolates were genetically highly similar and likely represented a dominant lineage. We analyzed all genic and allelic differences among all six isolates and found that all differences tended to occur within contiguous genomic blocks, suggestive of strain evolution by homologous recombination. From these analyses we identified three strains (two of which were recovered on two different occasions) that appear to have been derived sequentially, one from the next, each by multiple recombination events. We also identified a fourth strain that contains many of the genomic segments that differentiate the three highly related strains from one another, and have hypothesized that this fourth strain may have served as a donor multiple times in the evolution of the dominant strain line. The variations among the parent, daughter, and grand-daughter recombinant strains collectively cover greater than seven percent of the genome and are grouped into 23 chromosomal clusters. While capturing in vivo HGT, these data support the distributed genome hypothesis and suggest that a single competence event in pneumococci can result in the replacement of DNA at multiple non-adjacent loci., Author Summary Bacterial infections have long been studied using Koch's postulates wherein the paradigm is that a single clone leads to a given infection. Over the past decade, it has become clear that chronic bacterial infections often do not fit this paradigm. Instead these are associated with the presence of multiple strains or species (polyclonal) of bacteria that are organized into highly structured communities, termed biofilms, which can persist in the body and are recalcitrant to antibiotic treatment. In addition, there is extensive evidence that bacteria can incorporate genes from neighboring bacteria into their own genomes. This process can produce new strains and is known as horizontal gene transfer. In this study, we investigated for the first time, the tempo and relevance of gene transfer among bacterial strains of Streptococcus pneumoniae during a naturally occurring chronic childhood infection. We identified extensive gene transfer among multiple infecting strains, by sequencing of isolates recovered sequentially over a seven-month period. This gene transfer may serve as a counterpoint to the host's adaptive immune response and help explain the phenomenon of bacterial persistence, since, as occurs with some chronic viral and parasitic infections, the immune system may become overwhelmed by a set of related strains.

Entry of Staphylococcus aureus into the bloodstream can lead to metastatic abscess formation and infective endocarditis. Crucial to the development of both these conditions is the interaction of S. aureus with endothelial cells. In vivo and in vitro studies have shown that the staphylococcal invasin FnBPA triggers bacterial invasion of endothelial cells via a process that involves fibronectin (Fn) bridging to α5β1 integrins. The Fn-binding region of FnBPA usually contains 11 non-identical repeats (FnBRs) with differing affinities for Fn, which facilitate the binding of multiple Fn molecules and may promote integrin clustering. We thus hypothesized that multiple repeats are necessary to trigger the invasion of endothelial cells by S. aureus. To test this we constructed variants of fnbA containing various combinations of FnBRs. In vitro assays revealed that endothelial cell invasion can be facilitated by a single high-affinity, but not low-affinity FnBR. Studies using a nisin-inducible system that controlled surface expression of FnBPA revealed that variants encoding fewer FnBRs required higher levels of surface expression to mediate invasion. High expression levels of FnBPA bearing a single low affinity FnBR bound Fn but did not invade, suggesting that FnBPA affinity for Fn is crucial for triggering internalization. In addition, multiple FnBRs increased the speed of internalization, as did higher expression levels of FnBPA, without altering the uptake mechanism. The relevance of these findings to pathogenesis was demonstrated using a murine sepsis model, which showed that multiple FnBRs were required for virulence. In conclusion, multiple FnBRs within FnBPA facilitate efficient Fn adhesion, trigger rapid bacterial uptake and are required for pathogenesis., Author Summary Staphylococcus aureus is a frequent cause of bacteremia and sepsis. Adhesion to and invasion of endothelial cells lining blood vessels by S. aureus can lead to colonization of the heart valves and/or dissemination into surrounding tissues and the establishment of secondary (metastatic) infections. Uptake by endothelial cells is triggered by the interaction of fibronectin-binding protein A (FnBPA) with host cell receptors called integrins via a fibronectin (Fn) bridge. FnBPA contains 11 non-identical repeats (FnBRs) that mediate binding to Fn. Previous work has shown that partial deletions in the FnBR region do not significantly affect binding to Fn or invasion of host cells in vitro. It was therefore unclear why FnBPA contains so many repeats. This work demonstrates that the number of repeats affects the amount of surface-exposed FnBPA required to trigger invasion, as well as affecting the speed of uptake. As such, the presence of multiple repeats within FnBPA facilitates adhesion and invasion even at low surface expression levels. Experiments using a murine model of sepsis demonstrate that these differences have a significant impact on virulence; S. aureus expressing FnBPA variants with no or few FnBRs are significantly less virulent compared to FnBPA with the full complement of repeats.

Arginine is a crucial amino acid that serves to modulate the cellular immune response during infection. Arginine is also a common substrate for both inducible nitric oxide synthase (iNOS) and arginase. The generation of nitric oxide from arginine is responsible for efficient immune response and cytotoxicity of host cells to kill the invading pathogens. On the other hand, the conversion of arginine to ornithine and urea via the arginase pathway can support the growth of bacterial and parasitic pathogens. The competition between iNOS and arginase for arginine can thus contribute to the outcome of several parasitic and bacterial infections. There are two isoforms of vertebrate arginase, both of which catalyze the conversion of arginine to ornithine and urea, but they differ with regard to tissue distribution and subcellular localization. In the case of infection with Mycobacterium, Leishmania, Trypanosoma, Helicobacter, Schistosoma, and Salmonella spp., arginase isoforms have been shown to modulate the pathology of infection by various means. Despite the existence of a considerable body of evidence about mammalian arginine metabolism and its role in immunology, the critical choice to divert the host arginine pool by pathogenic organisms as a survival strategy is still a mystery in infection biology.

Intestinal epithelial cells (IECs) compose the first barrier against microorganisms in the gastrointestinal tract. Although the NF-κB pathway in IECs was recently shown to be essential for epithelial integrity and intestinal immune homeostasis, the roles of other inflammatory signaling pathways in immune responses in IECs are still largely unknown. Here we show that p38α in IECs is critical for chemokine expression, subsequent immune cell recruitment into the intestinal mucosa, and clearance of the infected pathogen. Mice with p38α deletion in IECs suffer from a sustained bacterial burden after inoculation with Citrobacter rodentium. These animals are normal in epithelial integrity and immune cell function, but fail to recruit CD4+ T cells into colonic mucosal lesions. The expression of chemokines in IECs is impaired, which appears to be responsible for the impaired T cell recruitment. Thus, p38α in IECs contributes to the host immune responses against enteric bacteria by the recruitment of immune cells., Author Summary The cellular responses of intestinal epithelial cells (IECs) to microorganisms in the gastrointestinal tract are mediated by activation of a number of intracellular signaling pathways. It was shown that the NF-κB pathway in IECs is essential for epithelial integrity and intestinal immune homeostasis, and here we show that p38α-mediated signaling in IECs is not important for epithelial integrity and immune cell function, but is critical for the clearance of the infected pathogen. p38α in IECs is essential for pathogen-induced chemokine expression in IECs and for subsequent immune cell recruitment into the intestinal mucosa, which leads to the clearance of the infectious pathogen. Our results indicate that different intracellular signaling pathways in IECs mediate distinct cellular responses to microorganisms in the gastrointestinal tract, and this information should be taken into consideration in the development of pathway-targeted therapeutic interventions for gastrointestinal infection.

The host vasculature is believed to constitute the principal route of dissemination of Neisseria meningitidis (Nm) throughout the body, resulting in septicaemia and meningitis in susceptible humans. In vitro, the Nm outer membrane protein Opc can enhance cellular entry and exit, utilising serum factors to anchor to endothelial integrins; but the mechanisms of binding to serum factors are poorly characterised. This study demonstrates that Nm Opc expressed in acapsulate as well as capsulate bacteria can increase human brain endothelial cell line (HBMEC) adhesion and entry by first binding to serum vitronectin and, to a lesser extent, fibronectin. This study also demonstrates that Opc binds preferentially to the activated form of human vitronectin, but not to native vitronectin unless the latter is treated to relax its closed conformation. The direct binding of vitronectin occurs at its Connecting Region (CR) requiring sulphated tyrosines Y56 and Y59. Accordingly, Opc/vitronectin interaction could be inhibited with a conformation-dependent monoclonal antibody 8E6 that targets the sulphotyrosines, and with synthetic sulphated (but not phosphorylated or unmodified) peptides spanning the vitronectin residues 43–68. Most importantly, the 26-mer sulphated peptide bearing the cell-binding domain 45RGD47 was sufficient for efficient meningococcal invasion of HBMECs. To our knowledge, this is the first study describing the binding of a bacterial adhesin to sulphated tyrosines of the host receptor. Our data also show that a single region of Opc is likely to interact with the sulphated regions of both vitronectin and of heparin. As such, in the absence of heparin, Opc-expressing Nm interact directly at the CR but when precoated with heparin, they bind via heparin to the heparin-binding domain of the activated vitronectin, although with a lower affinity than at the CR. Such redundancy suggests the importance of Opc/vitronectin interaction in meningococcal pathogenesis and may enable the bacterium to harness the benefits of the physiological processes in which the host effector molecule participates., Author Summary Neisseria meningitidis is a human pathogen that can cross the natural cellular barriers to reach the blood and the brain, causing septicaemia and meningitis. One of its surface molecules, Opc, has the capacity to attach to human cells lining the blood vessels. In vitro, the bacterium can do this by coating itself with the human serum factor vitronectin; and, by mimicking as vitronectin, it can bind to human cellular vitronectin-binding proteins (receptors). In this study, we have investigated the structural features of vitronectin that N. meningitidis recognises; such knowledge could help develop future strategies to control bacterial spread. We describe two different aspects of bacterial binding to vitronectin and demonstrate that one of these is a novel method, which occurs directly through the sulphated tyrosines of vitronectin available only when the molecule presents itself in an unfolded form. Such unfolded or activated vitronectin levels may be elevated during bacterial presence in the blood. Thus our observations imply that when bacteria appear in the blood, their presence may help to generate the form of vitronectin that they require for binding to and invading the cells that line human blood vessels to spread throughout body tissues including the brain.

The Haemophilus influenzae HMW1 adhesin is a high-molecular weight protein that is secreted by the bacterial two-partner secretion pathway and mediates adherence to respiratory epithelium, an essential early step in the pathogenesis of H. influenzae disease. In recent work, we discovered that HMW1 is a glycoprotein and undergoes N-linked glycosylation at multiple asparagine residues with simple hexose units rather than N-acetylated hexose units, revealing an unusual N-glycosidic linkage and suggesting a new glycosyltransferase activity. Glycosylation protects HMW1 against premature degradation during the process of secretion and facilitates HMW1 tethering to the bacterial surface, a prerequisite for HMW1-mediated adherence. In the current study, we establish that the enzyme responsible for glycosylation of HMW1 is a protein called HMW1C, which is encoded by the hmw1 gene cluster and shares homology with a group of bacterial proteins that are generally associated with two-partner secretion systems. In addition, we demonstrate that HMW1C is capable of transferring glucose and galactose to HMW1 and is also able to generate hexose-hexose bonds. Our results define a new family of bacterial glycosyltransferases., Author Summary Decoration of proteins with carbohydrates has an important impact on protein function throughout biology and has been recognized increasingly in pathogenic bacteria. Haemophilus influenzae is a common cause of both bacterial respiratory tract disease and bacterial invasive disease and initiates infection by colonizing the upper respiratory tract. The Haemophilus HMW1 adhesin is a large protein that resides on the bacterial surface and mediates bacterial attachment to respiratory epithelial cells, an essential step in the process of colonization. In recent work, we discovered that HMW1 is decorated at multiple sites with short carbohydrate units that serve to prevent degradation and to stabilize association with the bacterial surface. In the current study we identify the enzyme responsible for adding carbohydrate units at specific sites of HMW1. In addition, we demonstrate that this enzyme is capable of creating both carbohydrate-protein and carbohydrate-carbohydrate bonds. The amino acid sequence of this enzyme is similar to the sequences of proteins in several other bacteria, suggesting a new family of bacterial enzymes capable of creating carbohydrate-protein and carbohydrate-carbohydrate bonds.

Hypervirulent strains of Clostridium difficile have emerged over the past decade, increasing the morbidity and mortality of patients infected by this opportunistic pathogen. Recent work suggested the major C. difficile virulence factor, TcdB, from hypervirulent strains (TcdBHV) was more cytotoxic in vitro than TcdB from historical strains (TcdBHIST). The current study investigated the in vivo impact of altered TcdB tropism, and the underlying mechanism responsible for the differences in activity between the two forms of this toxin. A combination of protein sequence analyses, in vivo studies using a Danio rerio model system, and cell entry combined with fluorescence assays were used to define the critical differences between TcdBHV and TcdBHIST. Sequence analysis found that TcdB was the most variable protein expressed from the pathogenicity locus of C. difficile. In line with these sequence differences, the in vivo effects of TcdBHV were found to be substantially broader and more pronounced than those caused by TcdBHIST. The increased toxicity of TcdBHV was related to the toxin's ability to enter cells more rapidly and at an earlier stage in endocytosis than TcdBHIST. The underlying biochemical mechanism for more rapid cell entry was identified in experiments demonstrating that TcdBHV undergoes acid-induced conformational changes at a pH much higher than that of TcdBHIST. Such pH-related conformational changes are known to be the inciting step in membrane insertion and translocation for TcdB. These data provide insight into a critical change in TcdB activity that contributes to the emerging hypervirulence of C. difficile., Author Summary Clostridium difficile is a spore-forming bacterium that contaminates hospitals and infects patients undergoing antibiotic therapy. C. difficile is now the leading cause of hospital-acquired diarrhea in developed countries. Most concerning has been the recent increase in mortality of C. difficile patients due to the emergence of a hypervirulent strain of this pathogen. Results from the current study suggest this change in disease severity may be due to new strains producing a variant form of C. difficile's major virulence factor, TcdB. The findings indicate TcdB from hypervirulent strains targets a much broader range of cells in vivo and is able to translocate into target cells more quickly than TcdB from historical strains of C. difficile. The more rapid cell entry by TcdB from hypervirulent C. difficile appears to be due to the toxin's capacity to undergo conformational changes necessary for membrane translocation at a higher pH than TcdB from historical strains. To date, very little has been learned about the underlying reasons for the increased virulence of emerging C. difficile strains. These findings provide insight into this problem and suggest variations in TcdB activity could be an important contributing factor to the hypervirulence of emerging strains of C. difficile.

While a primary genital tract infection with C. trachomatis stimulates partial-protection against re-infection, it may also result in severe inflammation and tissue destruction. Here we have dissected whether functional compartments exist in the genital tract that restrict Th1-mediated protective immunity. Apart from the Th1-subset, little is known about the role of other CD4+ T cell subsets in response to a genital tract chlamydial infection. Therefore, we investigated CD4+ T cell subset differentiation in the genital tract using RT-PCR for expression of critical transcription factors and cytokines in the upper (UGT) and lower genital tract (LGT) of female C57BL/6 mice in response to C. trachomatis serovar D infection. We found that the Th1 subset dominated the UGT, as IFN-γ and T-bet mRNA expression were high, while GATA-3 was low following genital infection with C. trachomatis serovar D. By contrast, IL-10 and GATA-3 mRNA dominated the LGT, suggesting the presence of Th2 cells. These functional compartments also attracted regulatory T cells (Tregs) differently as increased FoxP3 mRNA expression was seen primarily in the UGT. Although IL-17A mRNA was somewhat up-regulated in the LGT, no significant change in RORγ-t mRNA expression was observed, suggesting no involvement of Th17 cells. The dichotomy between the LGT and UGT was maintained during infection by IL-10 because in IL-10-deficient mice the distinction between the two compartments was completely lost and a dramatic shift to the predominance of Th1 cells in the LGT occurred. Unexpectedly, the major source of IL-10 was CD11c+ CD11b+ DC, probably creating an anti-inflammatory privileged site in the LGT., Author Summary The immune response to the genital tract pathogen C. trachomatis can result in a number of pathological outcomes including tubal scarring and consequently, infertility. CD4+ T helper 1 (Th1) cells are critical for host protection against infection, but may also contribute to immunopathology. Apart from the Th1 cells, little is known about the role of other CD4+ T cell subsets in response to a genital tract chlamydial infection. By tracking the development of T helper cells in the genital tract using RT-PCR for distinct transcription factors associated with these subsets, we found vastly different immune responses in the upper genital tract (UGT) compared to the lower genital tract (LGT) of female mice during infection. The LGT was dominated by anti-inflammatory IL-10 production from dendritic cells (DC) and the non-protective Th2 subset. In contrast, the upper genital tract was populated by protective-Th1 cells. In the absence of IL-10, though, the LGT and UGT were both dominated by Th1 cells, arguing that DC-derived IL-10 secures an anti-inflammatory privileged site in the LGT. These findings provide a break-through in our understanding of functional compartments in the genital tract immune system with potentially strong impact on vaccine development.

The binding of bacteria to human platelets is a likely central mechanism in the pathogenesis of infective endocarditis. We have previously found that platelet binding by Streptococcus mitis SF100 is mediated by surface components encoded by a lysogenic bacteriophage, SM1. We now demonstrate that SM1-encoded lysin contributes to platelet binding via its direct interaction with fibrinogen. Far Western blotting of platelets revealed that fibrinogen was the major membrane-associated protein bound by lysin. Analysis of lysin binding with purified fibrinogen in vitro confirmed that these proteins could bind directly, and that this interaction was both saturable and inhibitable. Lysin bound both the Aα and Bβ chains of fibrinogen, but not the γ subunit. Binding of lysin to the Bβ chain was further localized to a region within the fibrinogen D fragment. Disruption of the SF100 lysin gene resulted in an 83±3.1% reduction (mean ± SD) in binding to immobilized fibrinogen by this mutant strain (PS1006). Preincubation of this isogenic mutant with purified lysin restored fibrinogen binding to wild type levels. When tested in a co-infection model of endocarditis, loss of lysin expression resulted in a significant reduction in virulence, as measured by achievable bacterial densities (CFU/g) within vegetations, kidneys, and spleens. These results indicate that bacteriophage-encoded lysin is a multifunctional protein, representing a new class of fibrinogen-binding proteins. Lysin appears to be cell wall-associated through its interaction with choline. Once on the bacterial surface, lysin can bind fibrinogen directly, which appears to be an important interaction for the pathogenesis of endocarditis., Author Summary The binding of bacteria to human platelets is thought to be a central event in the development of endocarditis (a life-threatening cardiovascular infection). We have previously found that platelet binding by Streptococcus mitis is mediated by surface components encoded by a bacteriophage contained within the host bacterium. We now show that lysin (an enzyme of bacteriophage origin) contributes to platelet binding via its direct interaction with fibrinogen on the platelet surface. Lysin bound to purified fibrinogen in vitro, and this interaction specifically involved the Aα and Bβ chains of fibrinogen. Binding of lysin to the Bβ chain was further localized to a region within the fibrinogen D fragment. Disruption of the gene encoding lysin gene resulted in a significant reduction in binding to fibrinogen by S. mitis, as well as a major reduction in virulence, as measured by a rat model of endocarditis. These results indicate that lysin is a multifunctional protein, representing a new class of fibrinogen-binding molecules. Lysin is localized to the bacterial surface via its interaction with cell wall choline, where it then can bind fibrinogen directly. Cell surface lysin apparently also contributes to the development of endovascular infections via its previously unrecognized fibrinogen binding activity.

Many bacterial pathogens produce extracellular proteases that degrade the extracellular matrix of the host and therefore are involved in disease pathogenesis. Dichelobacter nodosus is the causative agent of ovine footrot, a highly contagious disease that is characterized by the separation of the hoof from the underlying tissue. D. nodosus secretes three subtilisin-like proteases whose analysis forms the basis of diagnostic tests that differentiate between virulent and benign strains and have been postulated to play a role in virulence. We have constructed protease mutants of D. nodosus; their analysis in a sheep virulence model revealed that one of these enzymes, AprV2, was required for virulence. These studies challenge the previous hypothesis that the elastase activity of AprV2 is important for disease progression, since aprV2 mutants were virulent when complemented with aprB2, which encodes a variant that has impaired elastase activity. We have determined the crystal structures of both AprV2 and AprB2 and characterized the biological activity of these enzymes. These data reveal that an unusual extended disulphide-tethered loop functions as an exosite, mediating effective enzyme-substrate interactions. The disulphide bond and Tyr92, which was located at the exposed end of the loop, were functionally important. Bioinformatic analyses suggested that other pathogenic bacteria may have proteases that utilize a similar mechanism. In conclusion, we have used an integrated multidisciplinary combination of bacterial genetics, whole animal virulence trials in the original host, biochemical studies, and comprehensive analysis of crystal structures to provide the first definitive evidence that the extracellular secreted proteases produced by D. nodosus are required for virulence and to elucidate the molecular mechanism by which these proteases bind to their natural substrates. We postulate that this exosite mechanism may be used by proteases produced by other bacterial pathogens of both humans and animals., Author Summary Extracellular proteases are produced by many bacterial pathogens and are commonly involved in the degradation of the host extracellular matrix, facilitating invasion and colonization. One such pathogen is Dichelobacter nodosus, the causative agent of ovine footrot, a disease of major economic significance to the international sheep industry. D. nodosus secretes a number of serine proteases, which are thought to cause the tissue damage associated with virulent footrot. Our study showed that a D. nodosus mutant lacking one of the three secreted proteases, AprV2, failed to cause virulent disease in sheep. We used x-ray crystallography to solve the structure of AprV2 and the closely related protease AprB2. Our structures revealed an unusual extended disulphide-tethered loop that is located next to, but does not form part of, the primary substrate binding site. Through targeted mutagenesis studies we were able to show that this loop functions as an exosite, mediating effective enzyme-substrate interactions. Bioinformatic analyses suggest that subtilases from other pathogenic bacteria may contain this loop and may therefore utilize a similar mechanism. Our multidisciplinary research approach has provided a comprehensive understanding of the functional role of extracellular proteases in the pathogenesis of ovine footrot.

Streptococcal toxic shock syndrome (STSS) is a severe invasive infection characterized by the sudden onset of shock and multiorgan failure; it has a high mortality rate. Although a number of studies have attempted to determine the crucial factors behind the onset of STSS, the responsible genes in group A Streptococcus have not been clarified. We previously reported that mutations of csrS/csrR genes, a two-component negative regulator system for multiple virulence genes of Streptococcus pyogenes, are found among the isolates from STSS patients. In the present study, mutations of another negative regulator, rgg, were also found in clinical isolates of STSS patients. The rgg mutants from STSS clinical isolates enhanced lethality and impaired various organs in the mouse models, similar to the csrS mutants, and precluded their being killed by human neutrophils, mainly due to an overproduction of SLO. When we assessed the mutation frequency of csrS, csrR, and rgg genes among S. pyogenes isolates from STSS (164 isolates) and non-invasive infections (59 isolates), 57.3% of the STSS isolates had mutations of one or more genes among three genes, while isolates from patients with non-invasive disease had significantly fewer mutations in these genes (1.7%). The results of the present study suggest that mutations in the negative regulators csrS/csrR and rgg of S. pyogenes are crucial factors in the pathogenesis of STSS, as they lead to the overproduction of multiple virulence factors., Author Summary Group A streptococcus (GAS) causes life-threatening severe invasive diseases, including necrotizing fasciitis and streptococcal toxic shock-like syndrome. Although many studies have attempted to determine factors that are crucial for the onset of streptococcal toxic shock syndrome (STSS), bacterial factors responsible for it have not been clarified. By comparing genome sequences of clinical GAS isolates from STSS with those of non-invasive infections, we showed that mutations of negative regulator genes (csrS, csrR, rgg) were detected at a high frequency of more than 50% in STSS isolates, but at a low frequency of less than 2% in non-invasive isolates. These mutations of negative regulators were found in various emm-genotyped STSS isolates but not in a particular emm genotype. These mutants enhanced virulence in mouse models. Such results indicated that mutations of bacterial negative regulators are crucial for the pathogenesis of STSS due to the overproduction of multiple virulence factors under the de-repressed conditions.

Legionella pneumophila is an intracellular pathogen responsible for Legionnaires' disease. This bacterium uses the Dot/Icm type IV secretion system to inject a large number of bacterial proteins into host cells to facilitate the biogenesis of a phagosome permissive for its intracellular growth. Like many highly adapted intravacuolar pathogens, L. pneumophila is able to maintain a neutral pH in the lumen of its phagosome, particularly in the early phase of infection. However, in all cases, the molecular mechanisms underlying this observation remain unknown. In this report, we describe the identification and characterization of a Legionella protein termed SidK that specifically targets host v-ATPase, the multi-subunit machinery primarily responsible for organelle acidification in eukaryotic cells. Our results indicate that after being injected into infected cells by the Dot/Icm secretion system, SidK interacts with VatA, a key component of the proton pump. Such binding leads to the inhibition of ATP hydrolysis and proton translocation. When delivered into macrophages, SidK inhibits vacuole acidification and impairs the ability of the cells to digest non-pathogenic E. coli. We also show that a domain located in the N-terminal portion of SidK is responsible for its interactions with VatA. Furthermore, expression of sidK is highly induced when bacteria begin to enter new growth cycle, correlating well with the potential temporal requirement of its activity during infection. Our results indicate that direct targeting of v-ATPase by secreted proteins constitutes a virulence strategy for L. pneumophila, a vacuolar pathogen of macrophages and amoebae., Author Summary One hallmark of the lysosome is a low luminal pH that is important for its maturation as well as the activity of many hydrolyzing enzymes responsible for efficient digestion of phagocytosed contents. To survive and replicate in phagocytes, successful intracellular pathogens have evolved various mechanisms to circumvent the challenges posed by lysosomal killing. One salient feature associated with infection of the intracellular bacterial pathogen Legionella pneumophila is the maintenance of a neutral pH of the Legionella containing vacuoles (LCVs) that supports its intracellular growth in the early phase of infection, while the nonpathogenic mutants are believed to be immediately trafficked to an acidic compartment. In eukaryotic cells, organelle acidification is mediated by the vacuolar H+-ATPase that translocates protons into target compartments in a process energized by ATP hydrolysis. The recent discovery of the association of v-ATPase with LCVs points to the necessity for active modulation of v-ATPase activity by the bacterium. By screening L. pneumophila proteins that cause a yeast phenotype similar to its v-ATPase mutants, we have identified a substrate of the L. pneumophila Dot/Icm type IV secretion system that specifically inhibits the activity of the proton transporter. This protein, termed SidK, inhibits the activity of v-ATPase by directly interacting with the VatA subunit that is responsible for hydrolyzing ATP. Moreover, macrophages harboring SidK display defects in phagosomal acidification and lysosomal killing of non-pathogenic bacteria. We also found that expression of sidK is highly induced right after stationary bacteria are diluted into fresh medium, suggesting that SidK plays an important role in the early phase of infection. Our results reveal a mechanism by which an intravacuolar pathogen engages the v-ATPase protein and inhibits its activity, rather than actively avoiding its association with the pathogen's vacuolar membrane.

Lipopolysaccharide (LPS) is a major component on the surface of Gram negative bacteria and is composed of lipid A-core and the O antigen polysaccharide. O polysaccharides of the gastric pathogen Helicobacter pylori contain Lewis antigens, mimicking glycan structures produced by human cells. The interaction of Lewis antigens with human dendritic cells induces a modulation of the immune response, contributing to the H. pylori virulence. The amount and position of Lewis antigens in the LPS varies among H. pylori isolates, indicating an adaptation to the host. In contrast to most bacteria, the genes for H. pylori O antigen biosynthesis are spread throughout the chromosome, which likely contributed to the fact that the LPS assembly pathway remained uncharacterized. In this study, two enzymes typically involved in LPS biosynthesis were found encoded in the H. pylori genome; the initiating glycosyltransferase WecA, and the O antigen ligase WaaL. Fluorescence microscopy and analysis of LPS from H. pylori mutants revealed that WecA and WaaL are involved in LPS production. Activity of WecA was additionally demonstrated with complementation experiments in Escherichia coli. WaaL ligase activity was shown in vitro. Analysis of the H. pylori genome failed to detect a flippase typically involved in O antigen synthesis. Instead, we identified a homolog of a flippase involved in protein N-glycosylation in other bacteria, although this pathway is not present in H. pylori. This flippase named Wzk was essential for O antigen display in H. pylori and was able to transport various glycans in E. coli. Whereas the O antigen mutants showed normal swimming motility and injection of the toxin CagA into host cells, the uptake of DNA seemed to be affected. We conclude that H. pylori uses a novel LPS biosynthetic pathway, evolutionarily connected to bacterial protein N-glycosylation., Author Summary Bacterial surfaces are decorated with glycans. The human stomach pathogen Helicobacter pylori exposes lipopolysaccharide (LPS) containing Lewis antigens that mimic human glycan structures. H. pylori alters its Lewis antigen display in adaptation to the individual host. Lewis antigens can interact with human dendritic cells, thereby inducing a suppression of the immune response and facilitating a chronic H. pylori infection. Whereas three general LPS biosynthesis pathways are known, the route of LPS assembly in H. pylori remained to be elucidated. We identified and characterized two components of the H. pylori LPS pathway, WecA and WaaL, which demonstrated that, as in other bacteria, the glycan is initially assembled onto a polyprenoid lipid carrier. This intermediate then has to cross a membrane barrier, requiring specialized translocases. H. pylori does not employ a translocase from common LPS pathways. We show that instead H. pylori uses a translocase named Wzk, which is involved in protein N-glycosylation in other bacteria. Wzk was able to translocate various glycan structures. The identification of Wzk as the H. pylori translocase involved in LPS biosynthesis indicates an evolutionary connection between LPS and glycoprotein biosynthesis pathways.

The major autolysins (Atl) of Staphylococcus epidermidis and S. aureus play an important role in cell separation, and their mutants are also attenuated in virulence. Therefore, autolysins represent a promising target for the development of new types of antibiotics. Here, we report the high-resolution structure of the catalytically active amidase domain AmiE (amidase S. epidermidis) from the major autolysin of S. epidermidis. This is the first protein structure with an amidase-like fold from a bacterium with a gram-positive cell wall architecture. AmiE adopts a globular fold, with several α-helices surrounding a central β-sheet. Sequence comparison reveals a cluster of conserved amino acids that define a putative binding site with a buried zinc ion. Mutations of key residues in the putative active site result in loss of activity, enabling us to propose a catalytic mechanism. We also identified and synthesized muramyltripeptide, the minimal peptidoglycan fragment that can be used as a substrate by the enzyme. Molecular docking and digestion assays with muramyltripeptide derivatives allow us to identify key determinants of ligand binding. This results in a plausible model of interaction of this ligand not only for AmiE, but also for other PGN-hydrolases that share the same fold. As AmiE active-site mutations also show a severe growth defect, our findings provide an excellent platform for the design of specific inhibitors that target staphylococcal cell separation and can thereby prevent growth of this pathogen., Author Summary Although Staphylococci are common habitants of the human skin and the respiratory tract, a number of highly pathogenic strains are a major cause of hospital-associated infections and can be life threatening especially in immunocompromised patients. Moreover, an increasing number of strains has acquired resistance against commonly used antibiotics, which makes treatment of infections a challenge. Therefore, antibacterial drugs that act on new targets are needed to counteract the further spread of multiresistent staphylococci. The autolysins, which are cell wall associated enzymes that are essential for cell proliferation, represent one promising such new target. We used x-ray crystallography to solve the structure of a catalytically active region of the autolysin of Staphylococcus epidermidis, AmiE, at atomic resolution. Our studies reveal a defined binding groove for a specific cell wall component on the protein surface. Using in silico calculations in combination with biochemical studies, we are able to identify key motifs that are required for the recognition of the cell wall by autolysins. Our data further indicate that, besides these core motifs, species-specific alterations of bacterial cell walls are responsible for the unambiguous identification of ligands. Knowledge of the interactions in an enzyme-substrate complex, as well as information about the mechanism of catalysis, are prerequisites for the successful development of antimicrobial drugs. Our results therefore provide a platform from which a new class of inhibitors can be launched.

Many Gram-negative bacteria colonize and exploit host niches using a protein apparatus called a type III secretion system (T3SS) that translocates bacterial effector proteins into host cells where their functions are essential for pathogenesis. A suite of T3SS-associated chaperone proteins bind cargo in the bacterial cytosol, establishing protein interaction networks needed for effector translocation into host cells. In Salmonella enterica serovar Typhimurium, a T3SS encoded in a large genomic island (SPI-2) is required for intracellular infection, but the chaperone complement required for effector translocation by this system is not known. Using a reverse genetics approach, we identified a multi-cargo secretion chaperone that is functionally integrated with the SPI-2-encoded T3SS and required for systemic infection in mice. Crystallographic analysis of SrcA at a resolution of 2.5 Å revealed a dimer similar to the CesT chaperone from enteropathogenic E. coli but lacking a 17-amino acid extension at the carboxyl terminus. Further biochemical and quantitative proteomics data revealed three protein interactions with SrcA, including two effector cargos (SseL and PipB2) and the type III-associated ATPase, SsaN, that increases the efficiency of effector translocation. Using competitive infections in mice we show that SrcA increases bacterial fitness during host infection, highlighting the in vivo importance of effector chaperones for the SPI-2 T3SS., Author Summary Systemic typhoid fever caused by Salmonella enterica serovar Typhi leads to high mortality in the developing world and can be linked with chronic, persistent infections in survivors. To cause disease, Salmonella uses a specialized secretion device called a type III secretion system to disarm cells of the immune system and replicate within them. The assembly and function of this secretion system requires a set of chaperone proteins to direct the process, but the chaperone proteins themselves have remained elusive. Here, we found a new chaperone protein, called SrcA, which is required for proper function of the type III secretion system. Using a bacterial mutant lacking the srcA gene, we found that this chaperone was needed for Salmonella to compete against wild type cells during systemic disease because it controls secretion of at least 2 key proteins involved in immune escape and cell-to-cell transmission. This chaperone is present in all types of virulent Salmonella, but not in Salmonella that don't cause human infections, providing new insights into the pathogenic nature of this organism.

Differentiation, distribution and immune regulation of human IL-22-producing T cells in infections remain unknown. Here, we demonstrated in a nonhuman primate model that M. tuberculosis infection resulted in apparent increases in numbers of T cells capable of producing IL-22 de novo without in vitro Ag stimulation, and drove distribution of these cells more dramatically in lungs than in blood and lymphoid tissues. Consistently, IL-22-producing T cells were visualized in situ in lung tuberculosis (TB) granulomas by confocal microscopy and immunohistochemistry, indicating that mature IL-22-producing T cells were present in TB granuloma. Surprisingly, phosphoantigen HMBPP activation of Vγ2Vδ2 T cells down-regulated the capability of T cells to produce IL-22 de novo in lymphocytes from blood, lung/BAL fluid, spleen and lymph node. Up-regulation of IFNγ-producing Vγ2Vδ2 T effector cells after HMBPP stimulation coincided with the down-regulated capacity of these T cells to produce IL-22 de novo. Importantly, anti-IFNγ neutralizing Ab treatment reversed the HMBPP-mediated down-regulation effect on IL-22-producing T cells, suggesting that Vγ2Vδ2 T-cell-driven IFNγ-networking function was the mechanism underlying the HMBPP-mediated down-regulation of the capability of T cells to produce IL-22. These novel findings raise the possibility to ultimately investigate the function of IL-22 producing T cells and to target Vγ2Vδ2 T cells for balancing potentially hyper-activating IL-22-producing T cells in severe TB., Author Summary TB remains one of the leading causes of morbidity and mortality among infectious diseases worldwide. Protective immunity against TB in humans remains poorly characterized, although the importance of CD4 T cells is well described in HIV-related TB. Similarly, little is known about the role of newly-defined T helper 17 (Th17) cells producing pro-inflammatory cytokine IL-17 or IL-22 during M. tuberculosis infection of humans. Understanding immune response, kinetics, distribution and regulation of Th17 cells will help to elucidate immune function of these cells in TB, and to explore Th17-targeted immunotherapeutics. We therefore employed the nonhuman primate TB model to study Th17 cells and their interaction with Vγ2Vδ2 T cells, a major human γδ T-cell subset having regulatory potential. We demonstrated that M. tuberculosis infection drove induction and distribution of IL-22-producing T cells in blood, airway, lung, lymph node and spleen. IL-22-producing T cells were most frequent in lungs, and involved in TB granuloma formation. We further showed that activation of Vγ2Vδ2 T cells could remarkably down-regulate IL-22-producing T cells in blood and tissue lymphocytes, and that such a down-regulation was mediated by an IFNγ-associated cytokine network. Our findings provide new information regarding kinetics, distribution and immune regulation of IL-22-producing T cells in TB.

Anthrax lethal toxin (LT) is a bipartite protease-containing toxin and a key virulence determinant of Bacillus anthracis. In mice, LT causes the rapid lysis of macrophages isolated from certain inbred strains, but the correlation between murine macrophage sensitivity and mouse strain susceptibility to toxin challenge is poor. In rats, LT induces a rapid death in as little as 37 minutes through unknown mechanisms. We used a recombinant inbred (RI) rat panel of 19 strains generated from LT-sensitive and LT-resistant progenitors to map LT sensitivity in rats to a locus on chromosome 10 that includes the inflammasome NOD-like receptor (NLR) sensor, Nlrp1. This gene is the closest rat homolog of mouse Nlrp1b, which was previously shown to control murine macrophage sensitivity to LT. An absolute correlation between in vitro macrophage sensitivity to LT-induced lysis and animal susceptibility to the toxin was found for the 19 RI strains and 12 additional rat strains. Sequencing Nlrp1 from these strains identified five polymorphic alleles. Polymorphisms within the N-terminal 100 amino acids of the Nlrp1 protein were perfectly correlated with LT sensitivity. These data suggest that toxin-mediated lethality in rats as well as macrophage sensitivity in this animal model are controlled by a single locus on chromosome 10 that is likely to be the inflammasome NLR sensor, Nlrp1., Author Summary Inflammasomes are multiprotein cytoplasmic complexes that respond to a variety of danger signals by activating the host innate immune response. The sensor components of these complexes are NLR (NOD-like receptor) proteins. In this report, a recombinant inbred rat strain collection was used to genetically map anthrax lethal toxin (LT) susceptibility to a limited region of chromosome 10 containing one such sensor, Nlrp1. Similar to its mouse ortholog, Nlrp1b, which controls murine macrophage sensitivity to this toxin, the locus containing rat Nlrp1 was shown to control macrophage sensitivity to anthrax LT. However, unlike the situation in mice, where multiple genetic loci influence animal susceptibility to LT, the single chromosome 10 locus alone appears to control the rapid anthrax LT-induced death, which can occur in as little as 37 minutes. Sequencing of Nlrp1 from 12 rat strains identified polymorphisms which correlated perfectly with animal sensitivity to toxin. These polymorphisms were within the N-terminal 100-amino acid portion of Nlrp1, in an area of unknown function, which suggests that the N-terminus of rodent Nlrp1 could be an important functional domain.

Staphylococcus aureus frequently invades the human bloodstream, leading to life threatening bacteremia and often secondary foci of infection. Failure of antibiotic therapy to eradicate infection is frequently described; in some cases associated with altered S. aureus antimicrobial resistance or the small colony variant (SCV) phenotype. Newer antimicrobials, such as linezolid, remain the last available therapy for some patients with multi-resistant S. aureus infections. Using comparative and functional genomics we investigated the molecular determinants of resistance and SCV formation in sequential S. aureus isolates from a patient who had a persistent and recurrent S. aureus infection, after failed therapy with multiple antimicrobials, including linezolid. Two point mutations in key staphylococcal genes dramatically affected clinical behaviour of the bacterium, altering virulence and antimicrobial resistance. Most strikingly, a single nucleotide substitution in relA (SACOL1689) reduced RelA hydrolase activity and caused accumulation of the intracellular signalling molecule guanosine 3′, 5′-bis(diphosphate) (ppGpp) and permanent activation of the stringent response, which has not previously been reported in S. aureus. Using the clinical isolate and a defined mutant with an identical relA mutation, we demonstrate for the first time the impact of an active stringent response in S. aureus, which was associated with reduced growth, and attenuated virulence in the Galleria mellonella model. In addition, a mutation in rlmN (SACOL1230), encoding a ribosomal methyltransferase that methylates 23S rRNA at position A2503, caused a reduction in linezolid susceptibility. These results reinforce the exquisite adaptability of S. aureus and show how subtle molecular changes cause major alterations in bacterial behaviour, as well as highlighting potential weaknesses of current antibiotic treatment regimens., Author Summary The treatment of serious infections caused by Staphylococcus aureus is complicated by the development of antibiotic resistance, and in some cases the appearance of more persistent bacteria that have a reduced growth rate resulting in small colony variants (SCV). Here we have shown using whole genome sequencing and gene replacement experiments on sequential S. aureus isolates obtained from a patient with a serious bloodstream infection, how S. aureus evolved into a multi-antibiotic resistant, persistent and almost untreatable SCV. Specifically we show that a minor DNA change in a S. aureus gene encoding an enzyme called RelA causes an accumulation of a small signalling molecule called (p)ppGpp, which in turn leads to persistent activation of the important bacterial stress response known as the stringent response. This is the first report of the involvement of the stringent response in S. aureus SCV formation and its association with persistent infection. Additionally, we have uncovered a novel mechanism of resistance to the new antimicrobial linezolid, caused by a mutation in a gene encoding a 23S rRNA methyltransferase. This study highlights the exquisite adaptability of this important pathogen in the face of antimicrobial treatment.

NleG homologues constitute the largest family of type 3 effectors delivered by pathogenic E. coli, with fourteen members in the enterohaemorrhagic (EHEC) O157:H7 strain alone. Identified recently as part of the non-LEE-encoded (Nle) effector set, this family remained uncharacterised and shared no sequence homology to other proteins including those of known function. The C-terminal domain of NleG2-3 (residues 90 to 191) is the most conserved region in NleG proteins and was solved by NMR. Structural analysis of this structure revealed the presence of a RING finger/U-box motif. Functional assays demonstrated that NleG2-3 as well as NleG5-1, NleG6-2 and NleG9′ family members exhibited a strong autoubiquitination activity in vitro; a characteristic usually expressed by eukaryotic ubiquitin E3 ligases. When screened for activity against a panel of 30 human E2 enzymes, the NleG2-3 and NleG5-1 homologues showed an identical profile with only UBE2E2, UBE2E3 and UBE2D2 enzymes supporting NleG activity. Fluorescence polarization analysis yielded a binding affinity constant of 56±2 µM for the UBE2D2/NleG5-1 interaction, a value comparable with previous studies on E2/E3 affinities. The UBE2D2 interaction interface on NleG2-3 defined by NMR chemical shift perturbation and mutagenesis was shown to be generally similar to that characterised for human RING finger ubiquitin ligases. The alanine substitutions of UBE2D2 residues Arg5 and Lys63, critical for activation of eukaryotic E3 ligases, also significantly decreased both NleG binding and autoubiquitination activity. These results demonstrate that bacteria-encoded NleG effectors are E3 ubiquitin ligases analogous to RING finger and U-box enzymes in eukaryotes., Author Summary Many bacterial pathogens utilize a multiprotein ‘‘injection needle’’ termed the type III secretion system to deliver a set of proteins called effectors into the host cell. These effectors then manipulate host signalling pathways to the advantage of the pathogen, often mimicking eukaryote-specific activities. We present a study of an uncharacterised family of effectors called NleG, secreted primarily by enterohaemorrhagic E. coli (EHEC) O157:H7, a causative agent of human gastroenteritis. We solved the solution structure of a conserved C-terminal region of an NleG family member by NMR. Structural analysis demonstrated that the NleG structure is similar to the RING finger/U-box domain found primarily in eukaryotic ubiquitin ligases. The activity of these domains in eukaryotes is an essential part of the ubiquitination signalling system. Due to its central role in cell metabolism and the host immune response, the ubiquitination system has emerged as a primary target for bacterial effectors. Our biochemical analysis demonstrated that NleG proteins selectively interact with human E2 ubiquitin conjugating enzymes and exhibit in vitro activity typical of eukaryotic E3 ligases. Our data reveal that NleG effectors structurally and functionally mimic host U-box/RING E3 ubiquitin ligases. Future research will focus on determining targets of NleG ubiquitin ligase activity and the role in E. coli pathogenesis.

Many organisms respond to DNA damage by inducing expression of DNA repair genes. We find that the human stomach pathogen Helicobacter pylori instead induces transcription and translation of natural competence genes, thus increasing transformation frequency. Transcription of a lysozyme-like protein that promotes DNA donation from intact cells is also induced. Exogenous DNA modulates the DNA damage response, as both recA and the ability to take up DNA are required for full induction of the response. This feedback loop is active during stomach colonization, indicating a role in the pathogenesis of the bacterium. As patients can be infected with multiple genetically distinct clones of H. pylori, DNA damage induced genetic exchange may facilitate spread of antibiotic resistance and selection of fitter variants through re-assortment of preexisting alleles in this important human pathogen., Author Summary All organisms have genetic programs to respond to stressful conditions. The human stomach pathogen, Helicobacter pylori, survives on the surface of the stomach lining for the lifetime of its host and causes a chronic inflammatory response. In this niche, H. pylori is likely exposed to constant DNA damage and requires DNA repair systems to survive in the host. Many bacteria encode a genetic program for a coordinated response to DNA damage called the SOS response, which typically includes transcriptional induction of DNA repair systems and mutagenic DNA polymerases and a temporary halt to cell division. This study demonstrates that H. pylori has a distinct DNA damage response: instead of activating DNA repair systems, it induces both DNA uptake machinery and an enzyme that liberates DNA from neighboring cells. This capacity for genetic exchange enhances recombination of exogenous DNA into the genome, thus contributing to both the high genetic diversity observed between H. pylori clinical isolates and the spread of antibiotic resistance.

Since the concentration of free iron in the human host is low, efficient iron-acquisition mechanisms constitute important virulence factors for pathogenic bacteria. In Gram-negative bacteria, TonB-dependent outer membrane receptors are implicated in iron acquisition. It is far less clear how other metals that are also scarce in the human host are transported across the bacterial outer membrane. With the aim of identifying novel vaccine candidates, we characterized in this study a hitherto unknown receptor in Neisseria meningitidis. We demonstrate that this receptor, designated ZnuD, is produced under zinc limitation and that it is involved in the uptake of zinc. Upon immunization of mice, it was capable of inducing bactericidal antibodies and we could detect ZnuD-specific antibodies in human convalescent patient sera. ZnuD is highly conserved among N. meningitidis isolates and homologues of the protein are found in many other Gram-negative pathogens, particularly in those residing in the respiratory tract. We conclude that ZnuD constitutes a promising candidate for the development of a vaccine against meningococcal disease for which no effective universal vaccine is available. Furthermore, the results suggest that receptor-mediated zinc uptake represents a novel virulence mechanism that is particularly important for bacterial survival in the respiratory tract., Author Summary The outer membrane protects Gram-negative bacteria against harmful compounds from the environment. Nutrients usually pass this barrier by passive diffusion via pore-forming proteins. However, nutrients that are scarce in the environment are taken up via an active, receptor-mediated process. The vast majority of Gram-negative bacterial receptors described to date are involved in iron acquisition. Since free iron is scarce in the human host, these receptors constitute important virulence factors. In a search for putative vaccine components, we have characterized here a new receptor of Neisseria meningitidis, a resident of the nasopharynx that occasionally causes sepsis and meningitis. We show that expression of this receptor is induced under zinc limitation and that the protein is involved in the uptake of zinc. Homologues of this protein are present in many other Gram-negative pathogens, particularly in those residing in the respiratory tract, suggesting that receptor-mediated zinc acquisition is important for bacteria residing in this niche. We also found that the protein is highly conserved among N. meningitidis isolates and that it induces bactericidal antibodies upon immunization of mice. Therefore, the protein appears an excellent candidate for the development of a vaccine against N. meningitidis, for which no universal vaccine is available yet.

Sequence analysis of the genome of the strict intracellular pathogen Chlamydia trachomatis revealed the presence of a SET domain containing protein, proteins that primarily function as histone methyltransferases. In these studies, we demonstrated secretion of this protein via a type III secretion mechanism. During infection, the protein is translocated to the host cell nucleus and associates with chromatin. We therefore named the protein nuclear effector (NUE). Expression of NUE in mammalian cells by transfection reconstituted nuclear targeting and chromatin association. In vitro methylation assays confirmed NUE is a histone methyltransferase that targets histones H2B, H3 and H4 and itself (automethylation). Mutants deficient in automethylation demonstrated diminished activity towards histones suggesting automethylation functions to enhance enzymatic activity. Thus, NUE is secreted by Chlamydia, translocates to the host cell nucleus and has enzymatic activity towards eukaryotic substrates. This work is the first description of a bacterial effector that directly targets mammalian histones., Author Summary Chlamydia trachomatis is a particularly prevalent human pathogen responsible for loss of eyesight through trachoma and is the most common sexually transmitted disease of bacterial origin. Unlike most other bacterial pathogens, chlamydiae survive only within another cell and thus must develop sophisticated mechanisms to subvert immune clearance by their host. To this end, the bacteria secrete proteins into the cells they occupy which disrupt normal cellular function. In this work, we identify one such protein, NUE, which is injected into the nucleus of human cells during infection. Sequence analysis of NUE revealed the presence of a SET domain which suggests its involvement in chromatin remodeling. Indeed, we found the protein associated with chromatin both during infection and when transfected into human cells. Importantly, we demonstrate NUE is an active histone methyltransferase that targets host cell histones but does not modify bacterial histone-like proteins. This is the first bacterial protein identified which is able to penetrate the nucleus and directly modify mammalian histones.