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Nitric oxide (NO˙) is a radical molecule produced by mammalian phagocytic cells as part of the innate immune response to bacterial pathogens. It exerts its antimicrobial activity in part by impairing the function of metalloproteins, particularly those containing iron and zinc cofactors. The pathogenic Gram-negative bacterium Salmonella enterica serovar typhimurium undergoes dynamic changes in its cellular content of the four most common metal cofactors following exposure to NO˙ stress. Zinc, iron and magnesium all decrease in response to NO˙ while cellular manganese increases significantly. Manganese acquisition is driven primarily by increased expression of the mntH and sitABCD transporters following derepression of MntR and Fur. ZupT also contributes to manganese acquisition in response to nitrosative stress. S. Typhimurium mutants lacking manganese importers are more sensitive to NO˙, indicating that manganese is important for resistance to nitrosative stress.

A variety of eubacteria, plants and protozoa can modify membrane lipids by cyclopropanation, which is reported to modulate membrane permeability and fluidity. The ability to cyclopropanate membrane lipids has been associated with resistance to oxidative stress in Mycobacterium tuberculosis, organic solvent stress in Escherichia coli, and acid stress in E. coli and Salmonella. In bacteria, the cfa gene encoding cyclopropane fatty acid (CFA) synthase is induced during the stationary phase of growth. In the present study we constructed a cfa mutant of Salmonella enterica serovar Typhimurium 14028s (S. Typhimurium) and determined the contribution of CFA-modified lipids to stress resistance and virulence in mice. Cyclopropane fatty acid content was quantified in wild-type and cfa mutant S. Typhimurium. CFA levels in a cfa mutant were greatly reduced compared to wild-type, indicating that CFA synthase is the major enzyme responsible for cyclopropane modification of lipids in Salmonella. S. Typhimurium cfa mutants were more sensitive to extreme acid pH, the protonophore CCCP, and hydrogen peroxide, compared to wild-type. In addition, cfa mutants exhibited reduced viability in murine macrophages and could be rescued by addition of the NADPH phagocyte oxidase inhibitor diphenyleneiodonium (DPI) chloride. S. Typhimurium lacking cfa was also attenuated for virulence in mice. These observations indicate that CFA modification of lipids makes an important contribution to Salmonella virulence.

A variety of eubacteria, plants, and protozoa can modify membrane lipids by cyclopropanation, which is reported to modulate membrane permeability and fluidity. The ability to cyclopropanate membrane lipids has been associated with resistance to oxidative stress in Mycobacterium tuberculosis, organic solvent stress in Escherichia coli, and acid stress in E. coli and Salmonella. In bacteria, the cfa gene encoding cyclopropane fatty acid (CFA) synthase is induced during the stationary phase of growth. In the present study, we constructed a cfa mutant of Salmonella enterica serovar Typhimurium 14028s (S. Typhimurium) and determined the contribution of CFA-modified lipids to stress resistance and virulence in mice. Cyclopropane fatty acid content was quantified in wild-type and cfa mutant S. Typhimurium. CFA levels in the cfa mutant were greatly reduced compared to CFA levels in the wild type, indicating that CFA synthase is the major enzyme responsible for cyclopropane modification of lipids in Salmonella. S. Typhimurium cfa mutants were more sensitive to extreme acid pH, the protonophore CCCP, and hydrogen peroxide compared to the wild type. In addition, cfa mutants exhibited reduced viability in murine macrophages and could be rescued by the addition of the NADPH phagocyte oxidase inhibitor diphenyleneiodonium (DPI) chloride. S. Typhimurium lacking cfa was also attenuated for virulence in mice. These observations indicate that CFA modification of lipids makes an important contribution to Salmonella virulence.

Bacteria primarily evolve via horizontal gene transfer, acquiring new traits such as virulence and antibiotic resistance in single transfer events. However, newly acquired genes must be integrated into existing regulatory networks to allow appropriate expression in new hosts. This is accommodated in part by the opposing mechanisms of xenogeneic silencing and countersilencing. An understanding of these mechanisms is necessary to understand the relationship between gene regulation and bacterial evolution. Here we examine the functional evolution of an important lineage of countersilencers belonging to the ancient MarR family of classical transcriptional repressors. We show that although members of the SlyA lineage retain some ancestral features associated with the MarR family, their cis-regulatory sequences have evolved significantly to support their new function. Understanding the mechanistic requirements for countersilencing is critical to understanding the pathoadaptation of emerging pathogens and also has practical applications in synthetic biology., Gene duplication and subsequent evolutionary divergence have allowed conserved proteins to develop unique roles. The MarR family of transcription factors (TFs) has undergone extensive duplication and diversification in bacteria, where they act as environmentally responsive repressors of genes encoding efflux pumps that confer resistance to xenobiotics, including many antimicrobial agents. We have performed structural, functional, and genetic analyses of representative members of the SlyA/RovA lineage of MarR TFs, which retain some ancestral functions, including repression of their own expression and that of divergently transcribed multidrug efflux pumps, as well as allosteric inhibition by aromatic carboxylate compounds. However, SlyA and RovA have acquired the ability to countersilence horizontally acquired genes, which has greatly facilitated the evolution of Enterobacteriaceae by horizontal gene transfer. SlyA/RovA TFs in different species have independently evolved novel regulatory circuits to provide the enhanced levels of expression required for their new role. Moreover, in contrast to MarR, SlyA is not responsive to copper. These observations demonstrate the ability of TFs to acquire new functions as a result of evolutionary divergence of both cis-regulatory sequences and in trans interactions with modulatory ligands.

Nitric oxide (NO·) produced by mammalian cells exerts antimicrobial actions that result primarily from the modification of protein thiols (S-nitrosylation) and metal centers. A comprehensive approach was used to identify novel targets of NO· in Salmonella enterica serovar Typhimurium (S. Typhimurium). Newly identified targets include zinc metalloproteins required for DNA replication and repair (DnaG, PriA, and TopA), protein synthesis (AlaS and RpmE), and various metabolic activities (ClpX, GloB, MetE, PepA, and QueC). The cytotoxic actions of free zinc are mitigated by the ZntA and ZitB zinc efflux transporters, which are required for S. Typhimurium resistance to zinc overload and nitrosative stress in vitro. Zinc efflux also ameliorates NO·-dependent zinc mobilization following internalization by activated macrophages and is required for virulence in NO·-producing mice, demonstrating that host-derived NO· causes zinc stress in intracellular bacteria., IMPORTANCE Nitric oxide (NO·) is produced by macrophages in response to inflammatory stimuli and restricts the growth of intracellular bacteria. Mechanisms of NO·-dependent antimicrobial actions are incompletely understood. Here, we show that zinc metalloproteins are important targets of NO· in Salmonella, including the DNA replication proteins DnaG and PriA, which were hypothesized to be NO· targets in earlier studies. Like iron, zinc is a cofactor for several essential proteins but is toxic at elevated concentrations. This study demonstrates that NO· mobilizes free zinc in Salmonella and that specific efflux transporters ameliorate the cytotoxic effects of free zinc during infection.

Integration of horizontally acquired genes into transcriptional networks is essential for the regulated expression of virulence in bacterial pathogens. In Salmonella enterica, expression of such genes is repressed by the nucleoid-associated protein H-NS, which recognizes and binds to AT-rich DNA. H-NS-mediated silencing must be countered by other DNA-binding proteins to allow expression under appropriate conditions. Some genes that can be transcribed by RNA polymerase (RNAP) associated with the alternative sigma factor σ(S) or the housekeeping sigma factor σ(70) in vitro appear to be preferentially transcribed by σ(S) in the presence of H-NS, suggesting that σ(S) may act as a counter-silencer. To determine whether σ(S) directly counters H-NS-mediated silencing and whether co-regulation by H-NS accounts for the σ(S) selectivity of certain promoters, we examined the csgBA operon, which is required for curli fimbriae expression, and is known to be regulated by both H-NS and σ(S). Using genetics and in vitro biochemical analyses, we found that σ(S) is not directly required for csgBA transcription, but rather up-regulates csgBA via an indirect upstream mechanism. Instead, the biofilm master regulator CsgD directly counter-silences the csgBA promoter by altering the DNA-protein complex structure to disrupt H-NS-mediated silencing in addition to directing the binding of RNAP.

Summary Staphylococcus aureus is a commensal bacterium that can asymptomatically colonize its host but also causes invasive infections. Quorum sensing regulates S. aureus virulence and the transition from a commensal to a pathogenic organism. However, little is known about how host innate immunity affects interbacterial communication. We show that nitric oxide suppresses staphylococcal virulence by targeting the Agr quorum sensing system. Nitric oxide-mediated inhibition occurs through direct modification of cysteine residues C55, C123, and C199 of the AgrA transcription factor. Cysteine modification decreases AgrA promoter occupancy as well as transcription of the agr operon and quorum sensing-activated toxin genes. In a staphylococcal pneumonia model, mice lacking inducible nitric oxide synthase develop more severe disease with heightened mortality and proinflammatory cytokine responses. In addition, staphylococcal α-toxin production increases in the absence of nitric oxide or nitric oxide-sensitive AgrA cysteine residues. Our findings demonstrate an anti-virulence mechanism for nitric oxide in innate immunity.

The Rcs phosphorelay and Psp (phage shock protein) systems are envelope stress responses that are highly conserved in gammaproteobacteria. The Rcs regulon was found to be strongly induced during metal deprivation of Salmonella enterica serovar Typhimurium lacking the Psp response. Nineteen genes activated by the RcsA-RcsB response regulator make up an operon responsible for the production of colanic acid capsular polysaccharide, which promotes biofilm development. Despite more than half a century of research, the physiological function of colanic acid has remained elusive. Here we show that Rcs-dependent colanic acid production maintains the transmembrane electrical potential and proton motive force in cooperation with the Psp response. Production of negatively charged exopolysaccharide covalently bound to the outer membrane may enhance the surface potential by increasing the local proton concentration. This provides a unifying mechanism to account for diverse Rcs/colanic acid-related phenotypes, including susceptibility to membrane-damaging agents and biofilm formation. IMPORTANCE Colanic acid is a negatively charged polysaccharide capsule produced by Escherichia coli , Salmonella , and other gammaproteobacteria. Research conducted over the 50 years since the discovery of colanic acid suggests that this exopolysaccharide plays an important role for bacteria living in biofilms. However, a precise physiological role for colanic acid has not been defined. In this study, we provide evidence that colanic acid maintains the transmembrane potential and proton motive force during envelope stress. This work provides a new and fundamental insight into bacterial physiology.

Salmonella enterica serovar Typhimurium ( S. Typhimurium) causes systemic inflammatory disease in mice by colonizing cells of the mononuclear leukocyte lineage. Mouse strains resistant to S. Typhimurium, including Sv129S6, have an intact Nramp1 ( Slc11a1) allele and survive acute infection, whereas C57/BL6 mice, homozygous for a mutant Nramp1 allele, Nramp1G169D, develop lethal infections. Restoration of Nramp1 (C57/BL6 Nramp1G169) reestablishes resistance to S. Typhimurium; mice survive at least 3 to 4 weeks postinfection. Since many transgenic mouse strains are on a C57/BL6 genetic background, C57/BL6 Nramp1G169 mice provide a model to examine host genetic determinants of resistance to infection. To further evaluate host immune response to S. Typhimurium, we performed comparative analyses of Sv129S6 and C57/BL6 Nramp1G169 mice 3 weeks following oral S. Typhimurium infection. C57/BL6 Nramp1G169mice developed more severe inflammatory disease with splenic bacterial counts 1000-fold higher than Sv129S6 mice and relatively greater splenomegaly and blood neutrophil and monocyte counts. Infected C57/BL6 Nramp1G169 mice developed higher proinflammatory serum cytokine and chemokine responses (interferon-γ, tumor necrosis factor–α, interleukin [IL]–1β, and IL-2 and monocyte chemotactic protein–1 and chemokine [C-X-C motif] ligand 1, respectively) and marked decreases in anti-inflammatory serum cytokine concentrations (IL-10, IL-4) compared with Sv129S6 mice postinfection. Splenic dendritic cells and macrophages in infected compared with control mice increased to a greater extent in C57/BL6 Nramp1G169mice than in Sv129S6 mice. Overall, data show that despite the Nramp1 gene present in both strains, C57/BL6 Nramp1G169mice develop more severe, Th1-skewed, acute inflammatory responses to S. Typhimurium infection compared with Sv129S6 mice. Both strains are suitable model systems for studying inflammation in the context of adaptive immunity.

Nitric oxide (NO•) is a ubiquitous molecular mediator in biology. Many signalling actions of NO• generated by mammalian NO• synthase (NOS) result from targeting of the haem moiety of soluble guanylate cyclase. Some pathogenic and environmental bacteria also produce a NOS that is evolutionary related to the mammalian enzymes, but a bacterial haem-containing receptor for endogenous enzymatically generated NO• has not been identified previously. Here, we show that NOS of the human pathogen Staphylococcus aureus, in concert with an NO•-metabolizing flavohaemoprotein, regulates electron transfer by targeting haem-containing cytochrome oxidases under microaerobic conditions to maintain membrane bioenergetics. This process is essential for staphylococcal nasal colonization and resistance to the membrane-targeting antibiotic daptomycin and demonstrates the conservation of NOS-derived NO•-haem receptor signalling between bacteria and mammals.

The four-cysteine zinc finger motif of the bacterial RNA polymerase regulator DksA is essential for protein structure, canonical control of the stringent response to nutritional limitation, and thiol-based sensing of oxidative and nitrosative stress. This interdependent relationship has limited our understanding of DksA-mediated functions in bacterial pathogenesis. Here, we have addressed this challenge by complementing ΔdksA Salmonella with Pseudomonas aeruginosa dksA paralogues that encode proteins differing in cysteine and zinc content. We find that four-cysteine, zinc-bound (C4) and two-cysteine, zinc-free (C2) DksA proteins are able to mediate appropriate stringent control in Salmonella and that thiol-based sensing of reactive species is conserved among C2 and C4 orthologues. However, variations in cysteine and zinc content determine the threshold at which individual DksA proteins sense and respond to reactive species. In particular, zinc acts as an antioxidant, dampening cysteine reactivity and raising the threshold of posttranslational thiol modification with reactive species. Consequently, C2 DksA triggers transcriptional responses in Salmonella at levels of oxidative or nitrosative stress normally tolerated by Salmonella expressing C4 orthologues. Inappropriate transcriptional regulation by C2 DksA increases the susceptibility of Salmonella to the antimicrobial effects of hydrogen peroxide and nitric oxide, and attenuates virulence in macrophages and mice. Our findings suggest that the redox-active sensory function of DksA proteins is finely tuned to optimize bacterial fitness according to the levels of oxidative and nitrosative stress encountered by bacterial species in their natural and host environments., IMPORTANCE In order to cause disease, pathogenic bacteria must rapidly sense and respond to antimicrobial pressures encountered within the host. Prominent among these stresses, and of particular relevance to intracellular pathogens such as Salmonella, are nutritional restriction and the enzymatic generation of reactive oxygen and nitrogen species. The conserved transcriptional regulator DksA controls adaptive responses to nutritional limitation, as well as to oxidative and nitrosative stress. Here, we demonstrate that each of these functions contributes to bacterial pathogenesis. Our observations highlight the importance of metabolic adaptation in bacterial pathogenesis and show the mechanism by which DksA orthologues are optimized to sense the levels of oxidative and nitrosative stress encountered in their natural habitats. An improved understanding of the conserved processes used by bacteria to sense, respond to, and limit host defense will inform the development of novel strategies to treat infections caused by pathogenic, potentially multidrug-resistant bacteria.

Nontyphoidal Salmonella enterica serovar Typhimurium is a frequent cause of bloodstream infections in children and HIV-infected adults in sub-Saharan Africa. Most isolates from African patients with bacteremia belong to a single sequence type, ST313, which is genetically distinct from gastroenteritis-associated ST19 strains, such as 14028s and SL1344. Some studies suggest that the rapid spread of ST313 across sub-Saharan Africa has been facilitated by anthroponotic (person-to-person) transmission, eliminating the need for Salmonella survival outside the host. While these studies have not ruled out zoonotic or other means of transmission, the anthroponotic hypothesis is supported by evidence of extensive genomic decay, a hallmark of host adaptation, in the sequenced ST313 strain D23580. We have identified and demonstrated 2 loss-of-function mutations in D23580, not present in the ST19 strain 14028s, that impair multicellular stress resistance associated with survival outside the host. These mutations result in inactivation of the KatE stationary-phase catalase that protects high-density bacterial communities from oxidative stress and the BcsG cellulose biosynthetic enzyme required for the RDAR (red, dry, and rough) colonial phenotype. However, we found that like 14028s, D23580 is able to elicit an acute inflammatory response and cause enteritis in mice and rhesus macaque monkeys. Collectively, these observations suggest that African S. Typhimurium ST313 strain D23580 is becoming adapted to an anthroponotic mode of transmission while retaining the ability to infect and cause enteritis in multiple host species., IMPORTANCE The last 3 decades have witnessed an epidemic of invasive nontyphoidal Salmonella infections in sub-Saharan Africa. Genomic analysis and clinical observations suggest that the Salmonella strains responsible for these infections are evolving to become more typhoid-like with regard to patterns of transmission and virulence. This study shows that a prototypical African nontyphoidal Salmonella strain has lost traits required for environmental stress resistance, consistent with an adaptation to a human-to-human mode of transmission. However, in contrast to predictions, the strain remains capable of causing acute inflammation in the mammalian intestine. This suggests that the systemic clinical presentation of invasive nontyphoidal Salmonella infections in Africa reflects the immune status of infected hosts rather than intrinsic differences in the virulence of African Salmonella strains. Our study provides important new insights into the evolution of host adaptation in bacterial pathogens.

SummaryHost nitric oxide (NO⋅) production is important for controlling intracellular bacterial pathogens, including Salmonella enterica serovar Typhimurium, but the underlying mechanisms are incompletely understood. S. Typhmurium 14028s is prototrophic for all amino acids but cannot synthesize methionine (M) or lysine (K) during nitrosative stress. Here, we show that NO⋅-induced MK auxotrophy results from reduced succinyl-CoA availability as a consequence of NO⋅ targeting of lipoamide-dependent lipoamide dehydrogenase (LpdA) activity. LpdA is an essential component of the pyruvate and α-ketoglutarate dehydrogenase complexes. Additional effects of NO⋅ on gene regulation prevent compensatory pathways of succinyl-CoA production. Microarray analysis indicates that over 50% of the transcriptional response of S. Typhimurium to nitrosative stress is attributable to LpdA inhibition. Bacterial methionine transport is essential for virulence in NO⋅-producing mice, demonstrating that NO⋅-induced MK auxotrophy occurs in vivo. These observations underscore the importance of metabolic targets for antimicrobial actions of NO⋅.

Salmonella enterica serovar Typhi, the cause of typhoid fever, is host-adapted to humans and unable to cause disease in mice. Here, we show that S. Typhi can replicate in vivo in nonobese diabetic (NOD)- scid IL2rγ null mice engrafted with human hematopoietic stem cells (hu-SRC-SCID mice) to cause a lethal infection with pathological and inflammatory cytokine responses resembling human typhoid. In contrast, S. Typhi does not exhibit net replication or cause illness in nonengrafted or immunocompetent control animals. Screening of transposon pools in hu-SRC-SCID mice revealed both known and previously unknown Salmonella virulence determinants, including Salmonella Pathogenicity Islands 1, 2, 3, 4, and 6. Our observations indicate that the presence of human immune cells allows the in vivo replication of S. Typhi in mice. The hu-SRC-SCID mouse provides an unprecedented opportunity to gain insights into S. Typhi pathogenesis and devise strategies for the prevention of typhoid fever.

FimH is an adhesive subunit of type 1 fimbriae expressed by different enterobacterial species. The enteric bacterium Klebsiella pneumoniae is an environmental organism that is also a frequent cause of sepsis, urinary tract infection (UTI), and liver abscess. Type 1 fimbriae have been shown to be critical for the ability of K. pneumoniae to cause UTI in a murine model. We show here that the K. pneumoniae fimH gene is found in 90% of strains from various environmental and clinical sources. The fimH alleles exhibit relatively low nucleotide and structural diversity but are prone to frequent horizontal-transfer events between different bacterial clones. Addition of the fimH locus to multiple-locus sequence typing significantly improved the resolution of the clonal structure of pathogenic strains, including the K1 encapsulated liver isolates. In addition, the K. pneumoniae FimH protein is targeted by adaptive point mutations, though not to the same extent as FimH from uropathogenic Escherichia coli or TonB from the same K. pneumoniae strains. Such adaptive mutations include a single amino acid deletion from the signal peptide that might affect the length of the fimbrial rod by affecting FimH translocation into the periplasm. Another FimH mutation (S62A) occurred in the course of endemic circulation of a nosocomial uropathogenic clone of K. pneumoniae. This mutation is identical to one found in a highly virulent uropathogenic strain of E. coli , suggesting that the FimH mutations are pathoadaptive in nature. Considering the abundance of type 1 fimbriae in Enterobacteriaceae , our present finding that fimH genes are subject to adaptive microevolution substantiates the importance of type 1 fimbria-mediated adhesion in K. pneumoniae .

CorA is the primary Mg2+channel inSalmonella entericaserovar Typhimurium. AcorAmutant is attenuated in mice and defective for invasion of and replication within epithelial cells. Microarray studies show that several virulence effectors are repressed in acorAmutant strain, which ultimately manifests itself as a decrease in virulence.

Salmonella enterica serovar Typhimurium harbors five pathogenicity islands (SPI) required for infection in vertebrate hosts. Although the role of SPI1 in promoting epithelial invasion and proinflammatory cell death has been amply documented, SPI4 has only more recently been implicated in Salmonella virulence. SPI4 is a 24-kb pathogenicity island containing six open reading frames, siiA to siiF . Secretion of the 595-kDa SiiE protein requires a type I secretory system encoded by siiC , siiD , and siiF . An operon polarity suppressor ( ops ) sequence within the 5′ untranslated region upstream of siiA is required for optimal SPI4 expression and predicted to bind the antiterminator RfaH. SiiE concentrations are decreased in a SPI1 mutant strain, suggesting that SPI1 and SPI4 may have common regulatory inputs. SPI1 gene expression is positively regulated by the transcriptional activators HilA, HilC, and HilD, encoded within SPI1, and negatively regulated by the regulators HilE and PhoP. Here, we show that mutations in hilA , hilC , or hilD similarly reduce expression of siiE , and mutations in hilE or phoP enhance siiE expression. Individual overexpression of HilA, HilC, or HilD in the absence of SPI1 cannot activate siiE expression, suggesting that these transcriptional regulators act in concert or in combination with additional SPI1-encoded regulatory loci to activate SPI4. HilA is no longer required for siiE expression in an hns mutant strain, suggesting that HilA promotes SPI4 expression by antagonizing the global transcriptional silencer H-NS. Coordinate regulation suggests that SPI1 and SPI4 play complementary roles in the interaction of S. enterica serovar Typhimurium with the host intestinal mucosa.

Lateral gene transfer has played a prominent role in bacterial evolution, but the mechanisms allowing bacteria to tolerate the acquisition of foreign DNA have been incompletely defined. Recent studies show that H-NS, an abundant nucleoid-associated protein in enteric bacteria and related species, can recognize and selectively silence the expression of foreign DNA with higher adenine and thymine content relative to the resident genome, a property that has made this molecule an almost universal regulator of virulence determinants in enteric bacteria. These and other recent findings challenge the ideas that curvature is the primary determinant recognized by H-NS and that activation of H-NS-silenced genes in response to environmental conditions occurs through a change in the structure of H-NS itself. Derepression of H-NS-silenced genes can occur at specific promoters by several mechanisms including competition with sequence-specific DNA-binding proteins, thereby enabling the regulated expression of foreign genes. The possibility that microorganisms maintain and exploit their characteristic genomic GC ratios for the purpose of self/non-self-discrimination is discussed.

Salmonella enterica serovar Typhimurium must successfully transition the broad fluctuations in oxygen concentrations encountered in the host. In Escherichia coli , FNR is one of the main regulatory proteins involved in O 2 sensing. To assess the role of FNR in serovar Typhimurium, we constructed an isogenic fnr mutant in the virulent wild-type strain (ATCC 14028s) and compared their transcriptional profiles and pathogenicities in mice. Here, we report that, under anaerobic conditions, 311 genes (6.80% of the genome) are regulated directly or indirectly by FNR; of these, 87 genes (28%) are poorly characterized. Regulation by FNR in serovar Typhimurium is similar to, but distinct from, that in E. coli. Thus, genes/operons involved in aerobic metabolism, NO· detoxification, flagellar biosynthesis, motility, chemotaxis, and anaerobic carbon utilization are regulated by FNR in a fashion similar to that in E. coli . However, genes/operons existing in E. coli but regulated by FNR only in serovar Typhimurium include those coding for ethanolamine utilization, a universal stress protein, a ferritin-like protein, and a phosphotransacetylase. Interestingly, Salmonella -specific genes/operons regulated by FNR include numerous virulence genes within Salmonella pathogenicity island 1 (SPI-1), newly identified flagellar genes ( mcpAC , cheV ), and the virulence operon ( srfABC ). Furthermore, the role of FNR as a positive regulator of motility, flagellar biosynthesis, and pathogenesis was confirmed by showing that the mutant is nonmotile, lacks flagella, is attenuated in mice, and does not survive inside macrophages. The inability of the mutant to survive inside macrophages is likely due to its sensitivity to the reactive oxygen species generated by NADPH phagocyte oxidase.

Summary Analysis of the transcriptome of slyA mutant Salmo- nella enterica serovar Typhimurium revealed that many SlyA-dependent genes, including pagC , pagD , ugtL , mig-14 , virK , phoN , pgtE , pipB2 , sopD2 , pagJ and pagK , are also controlled by the PhoP/PhoQ regulatory system. Many SlyA- and PhoP/PhoQ-co- regulated genes have functions associated with the bacterial envelope, and some have been directly implicated in virulence and resistance to antimicro- bial peptides. Purified His-tagged SlyA binds to the pagC and mig-14 promoters in regions homologous to a previously proposed 'SlyA-box'. The pagC pro- moter lacks a consensus PhoP binding site and does not bind PhoP in vitro , suggesting that the effect of PhoP on pagC transcription is indirect. Stimulation of pagC expression by PhoP requires SlyA. Levels of SlyA protein and mRNA are not significantly changed under low-magnesium PhoP-inducing conditions in which pagC expression is profoundly elevated, how- ever, indicating that the PhoP/PhoQ system does not activate pagC expression by altering SlyA protein concentration. Models are proposed in which PhoP may control SlyA activity via a soluble ligand or SlyA may function as an anti-repressor to allow PhoP acti- vation. The absence of almost all SlyA-activated genes from the Escherichia coli K12 genome sug- gests that the functional linkage between the SlyA and PhoP/PhoQ regulatory systems arose as Salmo- nella evolved its distinctive pathogenic lifestyle.

Phagocytic cells inhibit the growth of intracellular pathogens by producing nitric oxide (NO). NO causes cell filamentation, induction of the SOS response, and DNA replication arrest in the Gram-negative bacterium Salmonella enterica . NO also induces double-stranded chromosomal breaks in replication-arrested Salmonella lacking a functional RecBCD exonuclease. This DNA damage depends on actions of additional DNA repair proteins, the RecG helicase, and RuvC endonuclease. Introduction of a recG mutation restores both resistance to NO and the ability of an attenuated recBC mutant Salmonella strain to cause lethal infection in mice, demonstrating that bacterial DNA replication is inhibited during host–pathogen interactions. Inhibition of DNA replication during nitrosative stress is invariably accompanied by zinc mobilization, implicating DNA-binding zinc metalloproteins as critical targets of NO-related antimicrobial activity.

Salmonella enterica serovar Arizona ( S. enterica subspecies IIIa) is a common Salmonella isolate from reptiles and can cause serious systemic disease in humans. The spv virulence locus, found on large plasmids in Salmonella subspecies I serovars associated with severe infections, was confirmed to be located on the chromosome of serovar Arizona. Sequence analysis revealed that the serovar Arizona spv locus contains homologues of spvRABC but lacks the spvD gene and contains a frameshift in spvA , resulting in a different C terminus. The SpvR protein functions as a transcriptional activator for the spvA promoter, and SpvB and SpvC are highly conserved. The analysis supports the proposal that the chromosomal spv sequence more closely corresponds to the ancestral locus acquired during evolution of S. enterica , with plasmid acquisition of spv genes in the subspecies I strains involving addition of spvD and polymorphisms in spvA .

Bacteria must contend with conditions of nutrient limitation in all natural environments. Complex programmes of gene expression, controlled in part by the alternative sigma factors sigmaS (sigma38, RpoS) and sigmaH (sigma32, RpoH), allow a number of bacterial species to survive conditions of partial or complete starvation. We show here that the alternative sigma factor sigmaE (sigma24, RpoE) also facilitates the survival of Salmonella typhimurium under conditions of nutrient deprivation. Expression of the sigmaE regulon is strongly induced upon entry of Salmonella into stationary phase. A Salmonella mutant lacking sigmaE has reduced survival during stationary phase as well as increased susceptibility to oxidative stress. A Salmonella strain lacking both sigmaE and sigmaS is non-viable after just 24 h in stationary phase, but survival of these mutants is completely preserved under anaerobic stationary-phase conditions, suggesting that oxidative injury is one of the major mechanisms of reduced microbial viability during periods of nutrient deprivation. Moreover, the attenuated virulence of sigmaE-deficient Salmonella for mice can be largely restored by genetic abrogation of the host phagocyte respiratory burst, suggesting that the sigmaE regulon plays an important antioxidant role during Salmonella infection of mammalian hosts.

The pathogenesis of serious systemic Salmonella infections is characterized by survival and proliferation of bacteria inside macrophages. Infection of human monocyte-derived macrophages in vitro with S. typhimurium or S. dublin produces cytopathology characterized by detachment of cells that contain large numbers of proliferating bacteria. This cytopathology is dependent on the expression of the bacterial spv genes, a virulence locus previously shown to markedly enhance the ability of Salmonella to produce systemic disease. After 24 h of infection, macrophage cultures contain two populations of bacteria: (i) proliferating organisms present in a detached cell fraction; and (ii) a static bacterial population in macrophages remaining attached to the culture well. Mutations in either the essential transcriptional activator SpvR or the key SpvB protein markedly reduce the cytopathic effect of Salmonella infection. The spv-dependent cytopathology in macrophages exhibits characteristics of apoptosis, with release of nucleosomes into the cytoplasm, nuclear condensation and DNA fragmentation. The current findings suggest that the mechanism of the spv effect is through induction of increased cytopathology in host macrophages.

Mutation of slyA , which reduces Salmonella typhimurium virulence in mice, caused only minor attenuation of S. typhimurium virulence in orally inoculated calves. This correlated with modest reductions in intestinal invasion and enteropathogenic responses in bovine ligated ileal loops. slyA appears to regulate virulence genes involved in systemic, but not enteric, salmonellosis.

Escherichia coli K-12 carries the cryptic hemolysin gene sheA which is under the control of positive and negative transcriptional regulators. The objectives of the present study were to further analyze the regulation of the sheA gene in E. coli, to compare the sheA genes from E. coli K-12 and a pathogenic E. coli strain, and to characterize the SheA hemolytic activity. Northern blot analysis demonstrated that the transcriptional regulator SlyA activates the E. coli K-12 sheA gene. The main transcriptional start site of the sheA gene was 56 nucleotides upstream from the start codon as determined by primer extension analysis. The sheA genes from E. coli K-12 and a pathogenic E. coli strain were identical. SheA hemolytic activity was cell associated and Ca2+ independent.

Horizontal gene transfer plays a major role in microbial evolution. However, newly acquired sequences can decrease fitness unless integrated into preexisting regulatory networks. We found that the histone-like nucleoid structuring protein (H-NS) selectively silences horizontally acquired genes by targeting sequences with GC content lower than the resident genome. Mutations in hns are lethal in Salmonella unless accompanied by compensatory mutations in other regulatory loci. Thus, H-NS provides a previously unrecognized mechanism of bacterial defense against foreign DNA, enabling the acquisition of DNA from exogenous sources while avoiding detrimental consequences from unregulated expression of newly acquired genes. Characteristic GC/AT ratios of bacterial genomes may facilitate discrimination between a cell's own DNA and foreign DNA.

Appropriate regulation of genes enables Salmonella typhimurium to adapt to the intracellular environment of the host. The Salmonella slyA gene is in a family of transcriptional regulators that may play an important role in this adaptation. We have previously shown that slyA mutant Salmonella strains are profoundly attenuated for virulence and do not survive in macrophages. In this study, we demonstrate that the expression of multiple Salmonella proteins is regulated by SlyA during stationary phase and during infection of macrophages. Both of these conditions also induced the expression of a slyA::lacZ transcriptional fusion. Expression of the slyA::lacZ transcriptional fusion increased 15-fold in stationary phase and was not dependent on the stationary-phase sigma factor, RpoS. slyA mutant Salmonella strains were sensitive to oxidative products of the respiratory burst, including hydrogen peroxide and the products of the redox cycling compound paraquat, but not to nitric oxide donors. These results suggest that the SlyA regulon is activated during infection of the host and is required for resistance to toxic oxidative products of the reticuloendothelial system.

Survival of Salmonella typhimurium within macrophages is associated with virulence. Most data on the fate of Salmonella during infection of macrophages are derived from viable counts of intracellular bacteria. These counts are a result of a combination of bacterial death and growth within the intracellular population but may not reflect the true levels of either macrophage killing of Salmonella or bacterial growth inside cells. In this study, two independent methods have been used to obtain a more accurate measurement of absolute levels of both death and growth of Salmonella inside macrophages. A purine auxotroph (purD) was used to measure Salmonella death in the absence of bacterial growth and then bacterial growth was measured by supplementing the purD cultures with adenosine. Numbers of dead and live Salmonella were also quantitated using the BacLight staining system, which distinguishes dead from live bacteria. Both methods demonstrate that killing of Salmonella by macrophages is considerably greater than detected using traditional cell counts and that bacterial inactivation occurs throughout the infection period. Salmonella was inactivated at a similar rate in both J774 macrophages (most permissive macrophages) and peritoneal exuadate macrophages (least permissive macrophages), suggesting that the major difference between these cells is the ability to limit bacterial growth. These studies also demonstrate that growth of Salmonella within murine macrophages occurs simultaneously with significant amounts of bacterial death. Identifying the factors responsible for shifting the interaction between macrophages and bacteria toward conditions that favor bacterial growth will be critical to understanding Salmonella virulence.Key words: Salmonella, macrophage, purD, purine auxotroph.

Isocitrate lyase is required for fatty acid utilization via the glyoxylate shunt. Although isocitrate lyase is essential for Salmonella persistence during chronic infection, it is dispensable for acute lethal infection in mice. Substrate availability in the phagosome appears to evolve over time, with increasing fatty acid dependence during chronic infection.

SummaryHost inflammation alters the availability of nutrients such as iron to limit microbial growth. However, Salmonella enterica serovar Typhimurium thrives in the inflamed gut by scavenging for iron with siderophores. By administering Escherichia coli strain Nissle 1917, which assimilates iron by similar mechanisms, we show that this nonpathogenic bacterium can outcompete and reduce S. Typhimurium colonization in mouse models of acute colitis and chronic persistent infection. This probiotic activity depends on E. coli Nissle iron acquisition, given that mutants deficient in iron uptake colonize the intestine but do not reduce S. Typhimurium colonization. Additionally, the ability of E. coli Nissle to overcome iron restriction by the host protein lipocalin 2, which counteracts some siderophores, is essential, given that S. Typhimurium is unaffected by E. coli Nissle in lipocalin 2-deficient mice. Thus, iron availability impacts S. Typhimurium growth, and E. coli Nissle reduces S. Typhimurium intestinal colonization by competing for this limiting nutrient.

Adaptation to the intracellular environment of host cells is crucial for the pathogenesis of Salmonella infections. The alternative sigma factor RpoS is a global regulator of gene expression during starvation and stress conditions and is required for virulence in Salmonella spp. We have used lacZ reporter fusions to rpoS and rpoS-dependent genes to study rpoS regulation after entry of Salmonella typhimurium into macrophages and epithelial cells. The results demonstrate that expression of an rpoS::lacZ translational fusion increases rapidly in S. typhimurium after phagocytosis. Activity of RpoS also increases after bacterial entry into both macrophages and epithelial cells, as demonstrated by the induction of the rpoS-regulated genes katE and spvB. A control rpoS-independent promoter for neomycin resistance does not show significant induction after cell entry. These results demonstrate that the regulatory system mediated by RpoS in S. typhimurium is activated by the intracellular environment of eukaryotic cells.

The plasmid virulence genes spvABCD of Salmonella spp. are regulated by SpvR and the stationary-phase sigma factor RpoS. The transcription of spv genes is induced during the post-exponential phase of bacterial growth in vitro. We sought to investigate the relationship between growth phase and RpoS in spv regulation. rpoS insertion mutations were constructed in S. dublin Lane and plasmid-cured LD842 strains, and the mutants were found to be attenuated for virulence and deficient in spv gene expression. We utilized the plasmid pBAD::rpoS to express rpoS independent of the growth phase under the control of the arabinose-inducible araBAD promoter. SpvA expression was induced within 2 h after the addition of 0.1% arabinose, even when bacteria were actively growing. This suggested that the level of RpoS, instead of the growth phase itself, controls induction of the spv genes. However, RpoS did not activate transcription of spvA in the absence of SpvR protein. Using a constitutive tet promoter to express spvR, we found that the spvA gene can be partially expressed in the rpoS mutant, suggesting that RpoS is required for SpvR synthesis. We confirmed that spvR is poorly expressed in the absence of RpoS. With an intact rpoS gene, spvR expression is not dependent on an intact spvR gene but is enhanced by spvR supplied in trans. We propose a model for Salmonella spv gene regulation in which both RpoS and SpvR are required for maximal expression at the spvR and spvA promoters.

Virulence genes in the genus Salmonella are regulated by growth phase and by environmental signals, which allows a sequential program of expression during infection. Conditions that promote the expression of loci required in systemic infection, including the plasmid-encoded spv genes, are the opposite of the factors that induce genes involved in the invasion of epithelial cells in the gastrointestinal tract.

The Salmonella plasmid-borne spvR gene encodes a 33-kDa regulatory protein that activates transcription of the spvABCD operon during the stationary phase of bacterial growth. We used gel mobility shift assays to demonstrate that SpvR recognizes a specific target DNA sequence within a 318-bp EcoRI-Apa1 fragment upstream of spvA. The addition of unlabeled target DNA to the radioactive labeled DNA-SpvR complex resulted in competitive inhibition of band retardation confirming the specificity of SpvR binding. Introduction of target DNA on a high copy number plasmid into wild-type Salmonella dublin Lane resulted in a substantial decrease of SpvB synthesis, confirming the binding properties of this DNA segment in vivo. Three SpvR mutants were constructed and were shown to abolish the positive regulatory function of SpvR: By site-specific mutagenesis of spvR, three single amino acids within the putative SpvR N-terminal α-helix domains were substituted by prolines. This resulted in loss of binding to the spvA promotor sequence and in loss of activation of the spvABCD genes. This study demonstrates that the regulatory function of SpvR is mediated by specific binding to the promotor region of the spvABCD operon.

Nramp1 confers resistance of macrophages to Salmonella Typhimurium via NF-κB-dependent induction of the siderophore capturing peptide lipocalin-2. The expression of the cation transporter Nramp1 (Slc11a1) in late phagolysosomes confers resistance to infection with several intracellular pathogens, such as Salmonella enterica, in mice. The antimicrobial actions of Nramp1 are attributable, in part, to modulation of macrophage immune function and cellular iron metabolism—the latter affecting the availability of the essential nutrient iron for intraphagosomal bacteria. Here, we provide novel evidence that Nramp1 functionality increases the expression of the peptide Lcn2, which exerts its antimicrobial activity by scavenging iron-loaded bacterial siderophores and mediating iron efflux from macrophages. With the use of macrophage cell lines expressing functional or nonfunctional Nramp1, we found significantly elevated Lcn2 mRNA and protein levels in Nramp1-expressing cells. These resulted from Nramp1-mediated alterations in the production of ROS, which stimulated NF-κB activity and subsequently, Lcn2 transcription. We observed that increased Lcn2 levels in primary Nramp1-positive macrophages resulted in a significant suppression of S. enterica serovar typhimurium growth. Stimulation of Lcn2 expression is a novel mechanism by which Nramp1 confers resistance against infection with the intracellular bacterium S. typhimurium.

Whereas the majority of pathogenic Salmonella serovars are capable of infecting many different animal species, typically producing a self-limited gastroenteritis, serovars with narrow host-specificity exhibit increased virulence and their infections frequently result in fatal systemic diseases. In our study, a genetic and functional analysis of the mannose-specific type 1 fimbrial adhesin FimH from a variety of serovars of Salmonella enterica revealed that specific mutant variants of FimH are common in host-adapted (systemically invasive) serovars. We have found that while the low-binding shear-dependent phenotype of the adhesin is preserved in broad host-range (usually systemically non-invasive) Salmonella, the majority of host-adapted serovars express FimH variants with one of two alternative phenotypes: a significantly increased binding to mannose (as in S. Typhi, S. Paratyphi C, S. Dublin and some isolates of S. Choleraesuis), or complete loss of the mannose-binding activity (as in S. Paratyphi B, S. Choleraesuis and S. Gallinarum). The functional diversification of FimH in host-adapted Salmonella results from recently acquired structural mutations. Many of the mutations are of a convergent nature indicative of strong positive selection. The high-binding phenotype of FimH that leads to increased bacterial adhesiveness to and invasiveness of epithelial cells and macrophages usually precedes acquisition of the non-binding phenotype. Collectively these observations suggest that activation or inactivation of mannose-specific adhesive properties in different systemically invasive serovars of Salmonella reflects their dynamic trajectories of adaptation to a life style in specific hosts. In conclusion, our study demonstrates that point mutations are the target of positive selection and, in addition to horizontal gene transfer and genome degradation events, can contribute to the differential pathoadaptive evolution of Salmonella., Author Summary The process of Salmonella host-adaptation is traditionally considered to involve acquisition of novel genetic elements encoding specific virulence factors or loss of genes representing liability for the more pathogenic strains. Here, by analysis of the mannose-sensitive fimbrial adhesin FimH, we demonstrate that in addition to horizontal gene transfer and genome degradation, single amino acid replacement plays an important role in the differential adaptive evolution of Salmonella. We show that acquisition of specific structural mutations in FimH variants of host-adapted (systemically invasive) serovars results in either significantly enhanced or, alternatively, completely inactivated mannose-binding, whereas systemically non-invasive serovars retain a primordial relatively low-binding (shear-dependent) phenotype. A phylogenetic analysis indicates that these mutations are commonly of a convergent nature and occur under strong positive selection illustrating the role of point amino acid changes in convergent evolution of host-adapted Salmonella. Although we show increased bacterial adhesiveness and cell-invasiveness of the high-binding mutants, the physiologic role of the non-binding mutations in FimH remains to be determined.

The starvation stress response of Salmonella typhimurium encompasses the genetic and physiologic changes that occur when this bacterium is starved for an essential nutrient such as phosphate (P), carbon (C), or nitrogen (N). The responses to the limitation of each of these nutrients involve both unique and overlapping sets of proteins important for starvation survival and virulence. The role of the alternative sigma factor RpoS in the regulation of the starvation survival loci, stiA, stiB, and stiC, has been characterized. RpoS (sigma S) was found to be required for the P, C, and N starvation induction of stiA and stiC. In contrast, RpoS was found to be required for the negative regulation of stiB during P and C starvation-induced stationary phase but not during logarithmic phase. This role was independent of the relA gene (previously found to be needed for stiB induction). The role of RpoS alone and in combination with one or more sti mutations in the starvation survival of the organism was also investigated. The results clearly demonstrate that RpoS is an integral component of the complex interconnected regulatory systems involved in S. typhimurium's response to nutrient deprivation. However, differential responses of various sti genes indicate that additional signals and regulatory proteins are also involved.

Background Escherichia coli serogroup O157:H7 has emerged as an important zoonotic bacterial pathogen, causing a range of symptoms from self-limiting bloody diarrhea to severe hemorrhagic colitis and hemolytic-uremic syndrome in humans. Beef and dairy cattle are considered the most important animal reservoirs for this pathogen. One of the important virulence characteristics of E. coli O157:H7 is the eaeA gene encoding the 97 kDa surface protein intimin. Intimin is required for attachment and effacement during the interaction of enterohemorrhagic E. coli with human and bovine neonatal enterocytes. The present study was undertaken to test the hypothesis that an adaptive mucosal immune response directed against intimin will reduce or prevent enteric colonization and fecal shedding of E. coli O157:H7 in cattle. Results Cattle were orally inoculated with either milk (control), milk with live attenuated Salmonella enterica serovar Dublin (vector), or milk with live attenuated recombinant S. Dublin expressing intimin (vaccinated) on days 0, 14 and 28. On day 98, all calves were challenged orally with E. coli O157:H7 to evaluate whether vaccination with the recombinant S. Dublin expressing intimin would reduce the level of E. coli O157:H7 fecal shedding. During the first 28 days, vaccinated calves shed both the vector strain and the intimin-expressing S. Dublin strain at a similar level. The vector strain was shed for a significantly longer period as compared to the level of recombinant vaccine strain. Calves that received the intimin-expressed vaccine ceased shedding S. Dublin from day 28 to day 63. All calves were challenged with E. coli O157:H7 on day 98 to determine the effect on fecal shedding of E. coli O157:H7. The amount of E. coli O157:H7 in feces was measured for 30 days post-challenge. We observed a transient clearance of E. coli O157:H7 from the feces in the vaccinated calves. The magnitude of fecal E. coli O157:H7 shedding did not correlate with the presence of intimin-specific fecal IgA. Conclusion Oral vaccination with live attenuated recombinant S. Dublin expressing intimin reduced enteric colonization and fecal shedding of E. coli O157:H7. However, the transient clearance of E. coli O157:H7 was not associated with an enhanced IgA-mediated mucosal immune response.

Serpula (Treponema) hyodysenteriae, the etiologic agent of swine dysentery, produces a hemolysin which is thought to be an important factor in the pathogenesis of the disease. We report the cloning, sequencing, and expression of a hemolysin gene (tly) from S. hyodysenteriae B204. A pUC19 gene bank of strain B204 was constructed in the Escherichia coli K-12 strain DH5 alpha, and hemolytic recombinants were identified by plating the library on blood agar plates. From the hemolytic recombinants, a 1.5-kb DNA fragment could be isolated that contained information necessary for the production of a hemolysin/cytotoxin in E. coli. Nucleotide sequence determination of this 1.5-kb fragment showed that it contained an open reading frame capable of encoding a 26.9-kDa protein. The recombinant hemolysin was easily released from E. coli by osmotic shock. As with the native hemolysin, the recombinant hemolysin is EDTA insensitive, thermolabile, and cytotoxic for several eukaryotic cell lines. Southern blot hybridization showed that the cloned S. hyodysenteriae hemolysin gene tly is present in all pathogenic strains of S. hyodysenteriae tested and absent in the nonpathogenic, weakly hemolytic spirochete S. innocens.

Intracellular pathogens must withstand nitric oxide (NO·) generated by host phagocytes. Salmonella enterica serovar Typhimurium interferes with intracellular trafficking of inducible nitric oxide synthase (iNOS) and possesses multiple systems to detoxify NO·. Consequently, the level of NO· stress encountered by S. Typhimurium during infection in vivo has been unknown. The Base Excision Repair (BER) system recognizes and repairs damaged DNA bases including cytosine and guanine residues modified by reactive nitrogen species. Apurinic/apyrimidinic (AP) sites generated by BER glycosylases require subsequent processing by AP endonucleases. S. Typhimurium xth nfo mutants lacking AP endonuclease activity exhibit increased NO· sensitivity resulting from chromosomal fragmentation at unprocessed AP sites. BER mutant strains were thus used to probe the nature and extent of nitrosative damage sustained by intracellular bacteria during infection. Here we show that an xth nfo S. Typhimurium mutant is attenuated for virulence in C3H/HeN mice, and virulence can be completely restored by the iNOS inhibitor L-NIL. Inactivation of the ung or fpg glycosylase genes partially restores virulence to xth nfo mutant S. Typhimurium, demonstrating that NO· fluxes in vivo are sufficient to modify cytosine and guanine bases, respectively. Mutants lacking ung or fpg exhibit NO·–dependent hypermutability during infection, underscoring the importance of BER in protecting Salmonella from the genotoxic effects of host NO·. These observations demonstrate that host-derived NO· damages Salmonella DNA in vivo, and the BER system is required to maintain bacterial genomic integrity., Author Summary The host innate immune system represents the first line of defense against invading microorganisms. An integral part of this response involves the production of nitric oxide (NO·), a potent antimicrobial effector. Bacterial pathogens have evolved a variety of systems to counteract the inhibitory effects of host NO·. Here we describe the role of a complex DNA repair pathway in the bacterium Salmonella Typhimurium that minimizes the mutagenic nature of host NO·. We have determined the mutation rate of bacteria during a model infection. Our results reveal that the Salmonella Base Excision Repair system (BER), comprised of DNA glycosylases and AP endonucleases, is able to eliminate excess mutations that accumulate in NO·–exposed cells during their interaction with the host. Thus, during Salmonella infections, the BER system protects the bacterium against potentially detrimental DNA damage arising from NO·–exposure. This provides genomic stability to a pathogenic microorganism that has evolved to survive within the genotoxic intracellular environment of host phagocytes.

Many of the most virulent strains of Salmonella enterica produce two distinct Cu,Zn-superoxide dismutases (SodCI and SodCII). The bacteriophage-encoded SodCI enzyme makes the greater contribution to Salmonella virulence. We have performed a detailed comparison of the functional, structural, and regulatory properties of the Salmonella SodC enzymes. Here we demonstrate that SodCI and SodCII differ with regard to specific activity, protease resistance, metal affinity, and peroxidative activity, with dimeric SodCI exhibiting superior stability and activity. In particular, monomeric SodCII is unable to retain its catalytic copper ion in the absence of zinc. We have also found that SodCI and SodCII are differentially affected by oxygen, zinc availability, and the transcriptional regulator FNR. SodCII is strongly down-regulated under anaerobic conditions and dependent on the high affinity ZnuABC zinc transport system, whereas SodCI accumulation in vitro and within macrophages is FNR-dependent. We have confirmed earlier findings that SodCII accumulation in intracellular Salmonella is negligible, whereas SodCI is strongly up-regulated in macrophages. Our observations demonstrate that differences in expression, activity, and stability help to account for the unique contribution of the bacteriophage-encoded SodCI enzyme to Salmonella virulence.

Staphylococcus aureus is one of the most successful human pathogens, colonizing 2 billion individuals worldwide and causing invasive infections even in immunocompetent hosts. S. aureus can evade multiple components of host innate immunity, including the antimicrobial radical nitric oxide (NO ⚫ ) produced by activated phagocytes. We show that S. aureus is capable of metabolically adapting to nitrosative stress by expressing an NO ⚫ -inducible l -lactate dehydrogenase ( ldh 1, SACOL0222) divergently transcribed from the NO ⚫ -detoxifying flavohemoglobin ( hmp ). l -Lactate production allows S. aureus to maintain redox homeostasis during nitrosative stress and is essential for virulence. NO ⚫ -inducible lactate dehydrogenase activity and NO ⚫ resistance distinguish S. aureus from the closely related commensal species S. epidermidis and S. saprophyticus .