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Long-chain-fatty-acid (LCFA) metabolism is a fundamental cellular process in bacteria that is involved in lipid homeostasis, energy production, and infection. However, the role of LCFA metabolism in Salmonella enterica serovar Typhimurium (S. Tm) gut infection remains unclear. Here, using a murine gastroenteritis infection model, we demonstrate involvement of LCFA metabolism in S. Tm gut colonization. The LCFA metabolism-associated transcriptional regulator FadR contributes to S. Tm gut colonization. fadR deletion alters the gene expression profile and leads to aberrant flagellar motility of S. Tm. Colonization defects in the fadR mutant are attributable to altered swimming behavior characterized by less frequently smooth swimming, resulting from reduced expression of the phase 2 flagellin FljB. Notably, changes in lipid LCFA composition by fadR deletion lead to reduced expression of fljB, which is restored by exogenous LCFA. Therefore, LCFA homeostasis may maintain proper flagellar motility by activating fljB expression, contributing to S. Tm gut colonization. Our findings improve the understanding of the effect of luminal LCFA on the virulence of enteric pathogens.

There are numerous bacteria reside within the mammalian gastrointestinal tract. Among the intestinal bacteria, Akkermansia, Bacteroides, Bifidobacterium, and Ruminococcus closely interact with the intestinal mucus layer and are, therefore, known as mucosal bacteria. Mucosal bacteria use host or dietary glycans for colonization via adhesion, allowing access to the carbon source that the host’s nutrients provide. Cell wall or membrane proteins, polysaccharides, and extracellular vesicles facilitate these mucosal bacteria-host interactions. Recent studies revealed that the physiological properties of Bacteroides and Bifidobacterium significantly change in the presence of co-existing symbiotic bacteria or markedly differ with the spatial distribution in the mucosal niche. These recently discovered strategic colonization processes are important for understanding the survival of bacteria in the gut. In this review, first, we introduce the experimental models used to study host-bacteria interactions, and then, we highlight the latest discoveries on the colonization properties of mucosal bacteria, focusing on the roles of the cell surface architecture regarding Bacteroides and Bifidobacterium.

Several bacterial moonlighting proteins act as adhesion factors, which are important for bacterial colonization of the gastrointestinal (GI) tract. However, little is known about the adherence properties of moonlighting proteins in the GI tract. Here, we describe a new approach for visualizing the localization of moonlighting protein-coated fluorescent microbeads in the whole GI tract by using a tissue optical clearing method, using elongation factor Tu (EF-Tu) as an example. As a bacterial cell surface-localized protein mimic, recombinant EF-Tu from Lactobacillus reuteri was immobilized on microbeads. EF-Tu-coating promoted the interaction of the microbeads with a Caco-2 cell monolayer. Next, the microbeads were orally administered to mice. GI whole tissues were cleared in aqueous fructose solutions of increasing concentrations. At 1 h after administration, the microbeads were diffused from the stomach up to the cecum, and after 3 h, they were diffused throughout the intestinal tract. In the lower digestive tract, EF-Tu-beads were significantly more abundant than non-coated control beads, suggesting that EF-Tu plays an important role in the persistence of the microbeads in the GI tract. The new approach will help in evaluating how moonlighting proteins mediate bacterial colonization.

Nuclear factor-kappa B (NF-κB) plays a critical role in the host defense against microbial pathogens. Many pathogens modulate NF-κB signaling to establish infection in their host. Salmonella enterica serovar Typhimurium (S. Typhimurium) possesses two type III secretion systems (T3SS-1 and T3SS-2) and directly injects many effector proteins into host cells. It has been reported that some effectors block NF-κB signaling, but the molecular mechanism of the inactivation of NF-κB signaling in S. Typhimurium is poorly understood. Here, we identified seven type III effectors—GogA, GtgA, PipA, SseK1, SseK2, SseK3, and SteE—that inhibited NF-κB activation in HeLa cells stimulated with TNF-α. We also determined that only GogA and GtgA are involved in regulation of the activation of NF-κB in HeLa cells infected with S. Typhimurium. GogA, GtgA, and PipA are highly homologous to one another and have the consensus zinc metalloprotease HEXXH motif. Our experiments demonstrated that GogA, GtgA, and PipA each directly cleaved NF-κB p65, whereas GogA and GtgA, but not PipA, inhibited the NF-κB activation in HeLa cells infected with S. Typhimurium. Further, expressions of the gogA or gtgA gene were induced under the SPI-1-and SPI-2-inducing conditions, but expression of the pipA gene was induced only under the SPI-2-inducing condition. We also showed that PipA was secreted into RAW264.7 cells through T3SS-2. Finally, we indicated that PipA elicits bacterial dissemination in the systemic stage of infection of S. Typhimurium via a T3SS-1-independent mechanism. Collectively, our results suggest that PipA, GogA and GtgA contribute to S. Typhimurium pathogenesis in different ways.

Salmonella enterica serovar Typhimurium is an important foodborne pathogen that causes diarrhea. S. Typhimurium elicits inflammatory responses and colonizes the gut lumen by outcompeting the microbiota. Although evidence is accumulating with regard to the underlying mechanism, the infectious stage has not been adequately defined. Peptidoglycan amidases are widely distributed among bacteria and play a prominent role in peptidoglycan maintenance by hydrolyzing peptidoglycans. Amidase activation is required for the regulation of at least one of two cognate activators, NlpD or EnvC (also called YibP). Recent studies established that the peptidoglycan amidase AmiC-mediated cell division specifically confers a fitness advantage on S. Typhimurium in the inflamed gut. However, it remains unknown which cognate activators are involved in the amidase activation and how the activators influence Salmonella sp. pathogenesis. Here, we characterize the role of two activators, NlpD and EnvC, in S. Typhimurium cell division and gut infection. EnvC was found to contribute to cell division of S. Typhimurium cells through the activation of AmiA and AmiC. The envC mutant exhibited impairments in gut infection, including a gut colonization defect and reduced ability to elicit inflammatory responses. Importantly, the colonization defect of the envC mutant was unrelated to the microbiota but was conferred by attenuated motility and chemotaxis of S. Typhimurium cells, which were not observed in the amiA amiC mutant. Furthermore, the envC mutant was impaired in its induction of mucosal inflammation and sustained gut colonization. Collectively, our findings provide a novel insight into the peptidoglycan amidase/cognate activator circuits and their dependent pathogenesis.

The twin-arginine translocation (Tat) system is involved in not only a wide array of cellular processes but also pathogenesis in many bacterial pathogens; thus, this system is expected to become a novel therapeutic target to treat infections. To the best of our knowledge, involvement of the Tat system has not been reported in the gut infection caused by Citrobacter rodentium. Here, we studied the role of Tat in C. rodentium gut infection, which resembles human infection with enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). A C. rodentium Tat loss-of-function mutant displayed prolonged gut colonization, which was explained by reduced inflammatory responses and, particularly, neutrophil infiltration. Further, the Tat mutant had colonization defects upon coinfection with the wild-type strain of C. rodentium. The Tat mutant also became hypersensitive to bile acids, and an increase in fecal bile acids fostered C. rodentium clearance from the gut lumen. Finally, we show that the chain form of C. rodentium cells, induced by a Tat-dependent cell division defect, exhibits impaired resistance to bile acids. Our findings indicate that the Tat system is involved in gut colonization by C. rodentium, which is associated with neutrophil infiltration and resistance to bile acids. Interventions that target the Tat system, as well as luminal bile acids, might thus be promising therapeutic strategies to treat human EHEC and EPEC infections.

Summary Bifidobacterium bifidum YIT 10347 (BF-1) is adhesive in vitro. Here we studied the molecular aspects of the BF-1 adhesion process. We identified and characterized non-adhesive mutants and found that a class E housekeeping sortase was critical for the adhesion to mucin. These mutants were significantly less adhesive to GCIY cells than was the wild type (WT), which protected GCIY cells against acid treatment more than did a non-adhesive mutant. The non-adhesive mutants aberrantly accumulated precursors of putative sortase-dependent proteins (SDPs). Recombinant SDPs bound to mucin. Disruption of the housekeeping sortase influenced expression of SDPs and pilus components. Mutants defective in a pilin or in an SDP showed the same adhesion properties as WT. Therefore, multiple SDPs and pili seem to work cooperatively to achieve adhesion, and the housekeeping sortase is responsible for cell wall anchoring of its substrates to ensure their proper biological function., Graphical abstract, Highlights • Non-adhesive mutants of B. bifidum lack a functional housekeeping sortase • A non-adhesive mutant is less protective for cultured cells against acid stress • The housekeeping sortase is important for anchoring adhesion factors to cell wall • B. bifidum has species-specific sortase-dependent proteins, Microbiology; Microbiome; Evolutionary ecology

In the inflamed gut, the bactericidal lectin RegIIIβ is massively produced by intestinal mucosa. RegIIIβ binds peptidoglycan and lipid A respectively, and thus can kill certain Gram-positive and Gram-negative bacteria, including the gut commensal microbiota and enteropathogenic bacteria. Considering the expression pattern and bactericidal activity, RegIIIβ is believed to be a host defense factor for protecting against the infection with enteropathogenic bacteria. However, it was poorly understood how RegIIIβ recognizes the target bacteria and kill them, and how RegIIIβ plays role(s) in infectious diarrhea. Therefore, our recent study has focused on RegIIIβ-target recognition, killing of Gram-negative bacteria, and host protective functions of RegIIIβ for infectious diarrhea inflicted by Salmonella Typhimurium. Here, we discuss novel insights into the protective role of RegIIIβ in infectious diarrhea, and propose avenues towards novel therapeutic interventions for Salmonella diarrhea., Gut Microbes, 9 (2), ISSN:1949-0976, ISSN:1949-0984

Salmonella enterica serovar Typhimurium (S. Typhimurium) induces inflammatory changes in the ceca of streptomycin-pretreated mice. In this mouse model of colitis, the type III secretion system 1 (T3SS-1) has been shown to induce rapid inflammatory change in the cecum at early points, 10 to 24 h after infection. Five proteins, SipA, SopA, SopB, SopD, and SopE2, have been identified as effectors involved in eliciting intestinal inflammation within this time range. In contrast, a T3SS-1-deficient strain was shown to exhibit inflammatory changes in the cecum at 72 to 120 h postinfection. However, the effectors eliciting T3SS-1-independent inflammation remain to be clarified. In this study, we focused on two T3SS-2 phenotypes, macrophage proliferation and cytotoxicity, to identify the T3SS-2 effectors involved in T3SS-1-independent inflammation. We identified a mutant strain that could not induce cytotoxicity in a macrophage-like cell line and that reduced intestinal inflammation in streptomycin-pretreated mice. We also identified five T3SS-2 effectors, SifA, SpvB, SseF, SseJ, and SteA, associated with T3SS-1-independent macrophage cytotoxicity. We then constructed a strain lacking T3SS-1 and all the five T3SS-2 effectors, termed T1S5. The S. Typhimurium T1S5 strain significantly reduced cytotoxicity in macrophages in the same manner as a mutant invA spiB strain (T1T2). Finally, the T1S5 strain elicited no inflammatory changes in the ceca of streptomycin-pretreated mice. We conclude that these five T3SS-2 effectors contribute to T3SS-1-independent inflammation.

Salmonella enterica serovar Typhimurium (S. Tm) is a cause of food poisoning accompanied with gut inflammation. Although mucosal inflammation is generally thought to be protective against bacterial infection, S. Tm exploits the inflammation to compete with commensal microbiota, thereby growing up to high densities in the gut lumen and colonizing the gut continuously at high levels. However, the molecular mechanisms underlying the beneficial effect of gut inflammation on S. Tm competitive growth are poorly understood. Notably, the twin-arginine translocation (Tat) system, which enables the transport of folded proteins outside bacterial cytoplasm, is well conserved among many bacterial pathogens, with Tat substrates including virulence factors and virulence-associated proteins. Here, we show that Tat and Tat-exported peptidoglycan amidase, AmiA- and AmiC-dependent cell division contributes to S. Tm competitive fitness advantage in the inflamed gut. S. Tm tatC or amiA amiC mutants feature a gut colonization defect, wherein they display a chain form of cells. The chains are attributable to a cell division defect of these mutants and occur in inflamed but not in normal gut. We demonstrate that attenuated resistance to bile acids confers the colonization defect on the S. Tm amiA amiC mutant. In particular, S. Tm cell chains are highly sensitive to bile acids as compared to single or paired cells. Furthermore, we show that growth media containing high concentrations of NaCl and sublethal concentrations of antimicrobial peptides induce the S. Tm amiA amiC mutant chain form, suggesting that gut luminal conditions such as high osmolarity and the presence of antimicrobial peptides impose AmiA- and AmiC-dependent cell division on S. Tm. Together, our data indicate that Tat and the Tat-exported amidases, AmiA and AmiC, are required for S. Tm luminal fitness in the inflamed gut, suggesting that these proteins might comprise effective targets for novel antibacterial agents against infectious diarrhea., Author summary For proteins residing outside the bacterial cytoplasm, transport is an essential step for adequate function. The twin-arginine translocation (Tat) system enables the transport of folded proteins across the cytoplasmic membrane in prokaryotes. It has recently become clear that this system plays a pivotal role in the detrimental effects of many bacterial pathogens, suggesting Tat as a novel therapeutic target against their infection. In particular, the bacterial enteropathogen Salmonella Typhimurium causes foodborne diarrhea by colonizing the gut interior space. Here, we describe that the S. Typhimurium Tat system contributes to intestinal infection by facilitating colonization of the gut by this pathogen. We also identify that two Tat-exported enzymes, peptidoglycan amidase AmiA and AmiC, are responsible for the Tat-dependent colonization. S. Typhimurium strains having nonfunctional Tat systems or lacking these enzymes undergo filamentous growth in the gut interior owing to defective cell division. Notably, this chain form of S. Typhimurium cells is highly sensitive to bile acids, rendering it less competitive with native bacteria in the gut. The data presented here suggest that the Tat system and associated amidases may comprise promising therapeutic targets for Salmonella diarrhea, and that controlling bacterial shape might be new strategy for regulating intestinal enteropathogen infection.

Salmonella enterica , a common cause of diarrhea, has to colonize the gut lumen to elicit disease. In the gut, the pathogen encounters a vast array of environmental stresses that cause perturbations in the bacterial envelope. The CpxRA two-component system monitors envelope perturbations and responds by altering the bacterial gene expression profile. This allows Salmonella to survive under such harmful conditions. Therefore, CpxRA activation is likely to contribute to Salmonella gut infection. However, the role of the CpxRA-mediated envelope stress response in Salmonella -induced diarrhea is unclear. Here, we show that CpxRA is dispensable for the induction of colitis by S. enterica serovar Typhimurium, whereas it is required for gut colonization. We prove that CpxRA is expressed during gut infection and that the presence of antimicrobial peptides in growth media activates the expression of CpxRA-regulated genes. In addition, we demonstrate that a S . Typhimurium strain lacking the cpxRA gene is able to cause colitis but is unable to continuously colonize the gut. Finally, we show that CpxRA-dependent gut colonization requires the host gut inflammatory response, while DegP, a CpxRA-regulated protease, is dispensable. Our findings reveal that the CpxRA-mediated envelope stress response plays a crucial role in Salmonella gut infection, suggesting that CpxRA might be a promising therapeutic target for infectious diarrhea.

We investigated the roles of extracellular sialidases (SiaBb1 and SiaBb2) in cross-feeding between sialidase-carrying Bifidobacterium bifidum and sialic acid-utilizing Bifidobacterium breve. Using 6ʹ sialyllactose (6ʹSL) as a carbon source, the number of wild-type B. bifidum cells increased while that of a siabb2-inactivated strain (Δsiabb2) did not. Coculture of these two strains in the presence of 6ʹSL resulted in similar increase in cell numbers. Coculture of wild-type B. bifidum, but not the Δsiabb2 strain, with sialic acid-utilizing Bifidobacterium breve, which cannot release sialic acids from carbohydrates, in the presence of 6ʹSL increased the number of B. breve cells. Moreover, when mucin was used as a carbon source, B. breve growth was increased in cocultures with B. bifidum wild-type and Δsiabb2 strains, suggesting that SiaBb1 may be involved. Additionally, B. breve cell numbers increased during cultivation with recombinant SiaBb1-and SiaBb2-treated mucin as the sole carbon source. These results indicated that B. bifidum SiaBb2 liberated sialic acid from sialyl-human milk oligosaccharides and -mucin glycans, supporting the growth of B. breve through sialic acid cross-feeding. SiaBb1 may assist in the degradation of mucin glycan. Collectively, our results revealed that both the B. bifidum extracellular sialidases promote the utilization of sialylated carbohydrates and supply free sialic acid to other Bifidobacterium strains.

Ten specific primer sets, for Lactobacillus gasseri, Lactobacillus crispatus, Atopobium vaginae, Gardnerella vaginalis, Mobiluncus curtisii, Chlamydia trachomatis/muridarum, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium adolescentis, and Bifidobacterium angulatum, were developed for quantitative analysis of vaginal microbiota. rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) analysis of the vaginal samples from 12 healthy Japanese volunteers using the new primer sets together with 25 existing primer sets revealed the diversity of their vaginal microbiota: Lactobacilli such as L. crispatus, L. gasseri, Lactobacillus jensenii, Lactobacillus iners, and Lactobacillus vaginalis, as the major populations at 10(7) cells/ml vaginal fluid, were followed by facultative anaerobes such as Streptococcus and strict anaerobes at lower population levels of 10(4) cells/ml or less. Certain bacterial vaginosis (BV)-related bacteria, such as G. vaginalis, A. vaginae, M. curtisii, and Prevotella, were also detected in some subjects. Especially in one subject, both G. vaginalis and A. vaginae were detected at high population levels of 10(8.8) and 10(8.9) cells/ml vaginal fluid, suggesting that she is an asymptomatic BV patient. These results suggest that the RT-qPCR system is effective for accurate analysis of major vaginal commensals and diagnosis of several vaginal infections.

Here, we report a case of pneumonia possibly caused by the aspiration of runoff water containing Chromobacterium haemolyticum. A 69-year-old man became intoxicated with alcohol, fell into a ditch, and lost consciousness after suffering a blow to the head. The lower half of his body was completely paralyzed because of damage to his spinal cord, and he aspirated runoff water from the ditch. Chest computed tomography scans revealed consolidation in the right upper lobe and bilateral lower lobes. A sputum culture detected gram-negative bacteria that was identified as C. haemolyticum. Antibacterial chemotherapy was initiated, and the clinical course was favorable. To the best of our knowledge, this is the first report of C. haemolyticum pneumonia in the literature.

Mucosal barrier formed by cationic antimicrobial peptides (CAMPs) is believed to be crucial for host protection from pathogenic gut infection. However, some pathogens can develop resistance to the CAMPs to survive in hosts. Salmonella enterica is a common cause of acute diarrhea. During the course of this disease, the pathogen must continuously colonize the gut lumen, which contains CAMPs. However, it is incompletely understood whether the resistance of Salmonella strains to CAMPs contributes to the development of gut infections. PhoPQ two-component system-dependent lipid A modifications confer resistance to CAMPs in S. enterica serovar Typhimurium. Therefore, we introduced mutations into the PhoPQ-regulated genes in an S. Typhimurium strain, obtaining pagP ugtL and pmrA mutant strains. Each mutant strain demonstrated a distinct spectrum of the resistance to CAMPs. Using streptomycin mouse model for Salmonella diarrhea, we show that the pagP ugtL, but not pmrA, mutant strain had a gut colonization defect. Furthermore, the pagP ugtL, but not pmrA, mutant strain had decreased outer membrane integrity and susceptibility to magainin 2, an alpha-helical CAMP. Taken together, the PagP- and UgtL-dependent resistance to CAMPs was demonstrated to contribute to sustained colonization in the gut. This may be due to the robust outer membrane of S. Typhimurium, inducing the resistance to alpha-helical CAMPs such as α-defensins. Our findings indicate that the development of resistance to CAMPs is required for the S. Typhimurium gut infection.

Cell Host & Microbe, 21 (2), ISSN:1931-3128, ISSN:1934-6069

Salmonella phosphothreonine lyase SpvC inactivates the dual-phosphorylated host mitogen-activated protein kinases (MAPK) through β-elimination. While SpvC can be secreted in vitro by both Salmonella pathogenicity island (SPI)-1 and SPI-2 type III secretion systems (T3SSs), translocation of this protein into the host cell cytosol has only been demonstrated by SPI-2 T3SS. In this study, we show that SpvC can be delivered into the host cell cytoplasm by both SPI-1 and SPI-2 T3SSs. Dephosphorylation of the extracellular signal-regulated protein kinases (ERK) was detected in an SPI-1 T3SS-dependent manner 2 h post infection. Using a mouse model for Salmonella enterocolitis, which was treated with streptomycin prior to infection, we observed that mice infected with Salmonella enterica serovar Typhimurium strains lacking the spvC gene showed pronounced colitis when compared with mice infected with the wild-type strain 1 day after infection. The effect of SpvC on the development of colitis was characterized by reduced mRNA levels of the pro-inflammatory cytokines and chemokines, and reduced inflammation with less infiltration of neutrophils. Furthermore, the reduction in inflammation by SpvC resulted in increased bacterial dissemination in spleen of mice infected with Salmonella. Collectively, our findings suggest that SpvC exerts as an anti-inflammatory effector and the attenuation of intestinal inflammatory response by SpvC is involved in systemic infection of Salmonella.

SlyA protein plays a key role in virulence in Salmonella enterica. In this study, we evaluated the ability of the slyA mutant strain of S. enterica serovar Choleraesuis (S. choleraesuis) to protect against swine salmonellosis. Using a murine model infected with S. enterica serovar Typhimurium (S. typhimurium), we showed that the Salmonella strain with a deletion of slyA could be used as a highly immunogenic, effective and safe vaccine in mice. Based on these data, a slyA mutant of S. enterica serovar Choleraesuis strain RF-1 was constructed, and the ability of this mutant to protect immunized pigs from S. choleraesuis infection was examined. As with the S. typhimurium slyA mutant, immunization of pigs with the S. choleraesuis slyA mutant strain provided significant protection against subsequent challenge by the wild-type RF-1. These results demonstrate that SlyA is a potential target in the development of a novel live attenuated vaccine against S. enterica.

The type III secretion system (T3SS) encoded by Chromobacterium pathogenicity islands 1 and 1a (Cpi-1/-1a) is critical for Chromobacterium violaceum pathogenesis. T3SS-dependent virulence is commonly characterized by type III effector virulence function, but the full repertoire of the effector proteins of Cpi-1/-1a T3SS is unknown. In this study, we showed that expression of Cpi-1/-1a T3SS is controlled by the master regulator CilA. We used transcriptional profiling with DNA microarrays to define CilA regulon and identified genes encoding T3SS effectors whose translocation into host cells was dependent on Cpi-1/-1a T3SS. From these effectors, we found that CopE (CV0296) has similarities to a guanine nucleotide exchange factor (GEF) for Rho GTPases in its C-terminal portion. The N-terminal portions (1-81 amino acids) of CopE and a CivB as a putative chaperone were required for its translocation. CopE specifically activates Rac1 and Cdc42 followed by the induction of actin cytoskeletal rearrangement. Interestingly, C. violaceum invades human epithelial HeLa cells in a Cpi-1/-1a-encoded T3SS- and CopE-dependent manner. Finally, C. violaceum strains lacking copE and expressing a CopE-G168V deficient in GEF activity were attenuated for virulence in mice, suggesting that CopE contributes to the virulence of this pathogen.

Listeria monocytogenes, the causative agent of listeriosis, has strong osmotolerance and is able to grow in severe circumstances. Many studies of the mechanisms of listerial osmotolerance have been performed. However, there is much which remains unknown. In previous studies we constructed two kinds of mutant in L. monocytogenes EGD strain to analyse the mechanisms of osmotolerance in L. monocytogenes by molecular genetic methods. In this paper, we summarized the genetical studies of osmotolerance in this bacterium by many researchers and ourselves. First, a transposon-insertional mutant strain was constructed that showed reduced growth in high osmotic agar compared with the parental strain. The results of cloning and sequencing analysis showed that the rel gene, which encodes guanosine tetra- and pentaphosphate synthesis and hydrolysis protein, is involved in osmotolerance in L. monocytogenes. Next, the expression levels of five sigma factor coding genes in L. monocytogenes were examined using real-time polymerase chain reaction (PCR) and it was found that the rpoN gene (the alternative sigma factor RpoN (sigma54)-encoding gene) was activated under high osmotic conditions. A deletion mutant of rpoN was constructed and its response to osmotic stress was analysed. In minimal medium with NaCl and carnitine, an osmoprotectant, the mutant showed deficient growth to that of the parental strain when the starting optical density was high, though the expression level of carnitine transporter operon, opuC, and the rate of carnitine uptake in the mutant was similar to that of EGD. These results suggest that the rpoN mutant may need larger amounts of carnitine which might be needed for its growth under high osmolarity. Through the analysis of these mutants, new insights have been obtained into osmotolerance in L. monocytogenes.

Probiotic bacteria are microorganisms that benefit the host through improvement of the balance of intestinal microflora and possibly by augmentation of host defense systems. We examined the mechanisms for the up-regulation of innate immune responses by a probiotic Lactobacillus casei ATCC27139, in vivo. Using mouse models of systemic Listeria monocytogenes infection and MethA fibrosarcoma tumorigenesis in combination with BALB/c and SCID mice, we found that parenteral administration of L. casei ATCC27139 confers a protective effect against L. monocytogenes infection and anti-tumor activity against MethA fibrosarcoma by activation of innate immunity, while L. casei ATCC27139-J1R strains, which are J1 phage-resistant strains that have been selected from MNNG-treated clones, lacked these activities. Substantial differences between ATCC27139 and ATCC27139-J1R strains were observed in the capacity to induce innate cytokines such as TNF-alpha, IL-12, IL-18, and IFN-gamma, and pathogen-associated molecular pattern receptors, TLR2 and Nod2, by spleen cells. In addition, although phosphorylation of NF-kappaB p65 in spleen was equally enhanced in the ATCC27139- and the ATCC27139-J1R-treated groups, phosphorylation of both p38 MAPK and MAPKAPK-2 was significantly induced only by ATCC27139. Furthermore, inhibitors of NF-kappaB (sulfasalazine) and p38 MAPK (SB203580) significantly reduced cytokine production by the spleen cells of the mice treated with L. casei ATCC27139, suggesting that both NF-kappaB and p38 MAPK signaling pathways play important roles in the augmentation of innate immunity by the probiotic L. casei.

The nucleotide sequence of a small plasmid, designated pRF-1, isolated from Salmonella enterica serovar Choleraesuis, was determined. We identified seven open reading frames (ORFs) encoded by 6066 nucleotides with a total G + C content of 53.6%. Analysis of the complete nucleotide sequence revealed a replicon of pRF-1 to have high similarity to the p15A origin of replication, with a possible cer-like region. ORF1, which is composed of 816 nucleotides, shows a high degree of similarity to dihydropteroate synthetase encoded by the sulII gene from plasmids in several enteropathogenic bacteria, which functions as the sulfonamide resistance determinant. In fact, Salmonella and Escherichia coli strains carrying pRF-1 were found to show strong resistance to sulfathiazole, suggesting that orf1 is a functional gene. Four of seven ORFs were found to encode putative proteins of unknown function.

The spv genes carried on the Salmonella virulence plasmid are commonly associated with severe systemic infection in experimental animals. The SpvB virulence-associated protein has been shown to ADP-ribosylate actin, and this enzymatic activity is essential for virulence in mice. Here, we present evidence that intracellular expression of SpvB protein induces not only disruption of actin filaments but also apoptotic cell death in eukaryotic cells.

Shiga toxin 1 (Stx1) of enterohemorrhagic Escherichia coli O157:H7 was cloned, and four mutant Stx1s were constructed by site-directed mutagenesis with PCR. The wild-type and mutant Stx1s with amino acid replacements at positions 167 and 170 of the A subunit were purified by one-step affinity chromatography with commercially available Globotriose Fractogel, and the mutant Stxs were used for the immunization of mice. The mutant toxins were nontoxic to Vero cells in vitro and to mice in vivo and induced the immunoglobulin G antibody against the wild-type Stx1, which neutralized the cytotoxicity of Stx1. The induced antibody titers depended on the mutation at position 170 of the A subunit. The mice immunized with the mutant Stx1s were protected against a challenge of approximately 100 times the 50% lethal dose of the wild-type Stx1, suggesting that the mutant toxins are good candidates for toxoid vaccines for infection by Stx1-producing E. coli.

Nontyphoid Salmonella enterica requires the plasmid-encoded spv genes to establish successful systemic infection in experimental animals. The SpvB virulence-associated protein has recently been shown to contain the ADP-ribosyltransferase domain. SpvB ADP-ribosilates actin and depolymerizes actin filaments when expressed in cultured epithelial cells. However, spontaneous secretion or release of SpvB has not been observed under in vitro growth conditions. In the present study we investigated the secretion of SpvB from Salmonella using in vitro and in vivo assay systems. We showed that SpvB is secreted into supernatant from Salmonella strains that contain the cloned spvB gene on a plasmid when they grew in intracellular salts medium (ISM), a minimal medium mimicing the intracellular iron concentrations of eukaryotic cells. A series of mutant SpvB proteins revealed that an N-terminal region of SpvB located at amino acids 1-229 was sufficient to promote secretion into extracellular milieu. Confocal immunofluorescence microscopy also demonstrated efficient localization of the N-terminal domain of SpvB(1-360) tagged with biotinylated peptide within infected host cell cytosol but not truncated SpvB(1-179) fusion protein. In addition, mutations that inactivate genes within Salmonella pathogenicity island 1 or Salmonella pathogenicity island 2 that encode type III secretion systems (TTSS) could secrete the SpvB protein into the culture medium. These results indicate that SpvB protein is transported from the bacteria and into the host cytoplasm independent of TTSS.

We studied the effect of fosfomycin (FOM) on the intracellular growth of two different facultative intracellular bacteria, Salmonella enterica serovar Typhimurium and Listeria monocytogenes, in an enterocyte-like cell line, Caco-2. These bacteria replicate in different compartments within the host cells; Salmonella serovar Typhimurium grow inside phagosomes, whereas L. monocytogenes escape the phagosomal environment and multiply in the cytosol of the host cells. At concentrations equal to 0.25 of the MIC and 4 times the MIC, respectively, FOM effectively decreased the number of intracellular Salmonella serovar Typhimurium. Although FOM was ineffective at inhibiting the extracellular growth of L. monocytogenes in vitro, this antibiotic induced a marked reduction in intracellular L. monocytogenes, indicating that, comparatively, FOM may be more effectively taken up by L. monocytogenes in the intracellular environment.

Chromobacterium haemolyticum is a Gram-negative bacterium displaying remarkable hemolysis against human and sheep erythrocytes. In addition, C. haemolyticum infects humans, in which the infection mechanism remains unknown. We report here the draft genome sequence of C. haemolyticum strain T124, isolated from a young patient with sepsis in Japan.

The slyA gene, which has been implicated in the virulence of Salmonella serovar Typhimurium and its survival in macrophages, is widely distributed among different Salmonella serovars. In this study, we cloned and sequenced the translational initiation region of the slyA gene from nine different serovars and found sequence differences in the previously proposed ATG initiation codon but not in a TTG triplet, another putative initiation codon in the slyA gene. Therefore, we determined the actual translational initiation site of the slyA gene by analyzing slyA genes with defined mutation in either the ATG or TTG sequences in an in vitro translation assay and a quantitative hemolytic assay in Escherichia coli. The replacement of TTG by TTC in the slyA gene significantly reduced both the amount of protein synthesized and the hemolytic activity of a transformed strain of E. coli, while replacement of ATG by ATC had no effect in these assays. In addition, the amino acid sequence analysis of the His-tagged SlyA protein showed that it was identical with the amino acid sequence deduced from the 5' end of the slyA gene with a TTG initiation codon. Our results suggest that TTG serves as the translational initiation codon for the slyA gene of Salmonella.

A type III secretion system (T3SS) is utilized by a large number of gram-negative bacteria to deliver effectors directly into the cytosol of eukaryotic host cells. One essential component of a T3SS is an ATPase that catalyzes the unfolding of proteins, which is followed by the translocation of effectors through an injectisome. Here we demonstrate a functional role of the ATPase SsaN, a component of Salmonella pathogenicity island 2 T3SS (T3SS-2) in Salmonella enterica serovar Typhimurium. SsaN hydrolyzed ATP in vitro and was essential for T3SS function and Salmonella virulence in vivo. Protein-protein interaction analyses revealed that SsaN interacted with SsaK and SsaQ to form the C ring complex. SsaN and its complex co-localized to the membrane fraction under T3SS-2 inducing conditions. In addition, SsaN bound to Salmonella pathogenicity island 2 (SPI-2) specific chaperones, including SsaE, SseA, SscA, and SscB that facilitated translocator/effector secretion. Using an in vitro chaperone release assay, we demonstrated that SsaN dissociated a chaperone-effector complex, SsaE and SseB, in an ATP-dependent manner. Effector release was dependent on a conserved arginine residue at position 192 of SsaN, and this was essential for its enzymatic activity. These results strongly suggest that the T3SS-2-associated ATPase SsaN contributes to T3SS-2 effector translocation efficiency.

We have developed a series of shuttle vectors based on the conjugative transposon Tn916 that have been designed for the analysis of transcriptional regulation in Streptococcus pyogenes and other gram-positive bacteria. Designated the pVIT vectors (vectors for integration into Tn916), the vectors are small, stable plasmids in Escherichia coli to facilitate the fusion of promoters from cloned S. pyogenes genes to a promoterless gene which encodes chloramphenicol acetyltransferase. The vectors each contain one or more small regions of Tn916 to direct the integration of the transcriptional fusion into the transposon via homologous recombination following transformation of S. pyogenes or other suitable gram-positive hosts. Integration can be monitored by the inactivation or replacement of an antibiotic resistance determinant in modified derivatives of Tn916. Promoter activity can then be quantitated by the determination of chloramphenicol acetyltransferase-specific activity. In addition, since integration is into loci that do not disrupt the conjugative transpositional functions of Tn916, the vectors are useful for analysis of regulation in strains that are difficult or impossible to transform and can be introduced into these strains by conjugation following transformation of an intermediate host. The promoters for the genes which encode both the M protein and protein F of S. pyogenes were active in pVIT vectors, as was the region which controls transcription of mry, a trans-acting positive regulator of M protein expression. However, neither of the two characterized promoters for mry demonstrated activity when independently analyzed in pVIT-generated partial diploid strains, suggesting that regulation of mry is more complex than predicted by current models. The broad host range of Tn916 should make the pVIT vectors useful for analysis of regulation in numerous other bacterial species.

Background The global ppGpp-mediated stringent response in pathogenic bacteria plays an important role in the pathogenesis of bacterial infections. In Salmonella enterica serovar Typhimurium (S. Typhimurium), several genes, including virulence genes, are regulated by ppGpp when bacteria are under the stringent response. To understand the control of virulence genes by ppGpp in S. Typhimurium, agarose 2-dimensional electrophoresis (2-DE) combined with mass spectrometry was used and a comprehensive 2-DE reference map of amino acid-starved S. Typhimurium strain SH100, a derivative of ATCC 14028, was established. Results Of the 366 examined spots, 269 proteins were successfully identified. The comparative analysis of the wild-type and ppGpp0 mutant strains revealed 55 proteins, the expression patterns of which were affected by ppGpp. Using a mouse infection model, we further identified a novel virulence-associated factor, STM3169, from the ppGpp-regulated and Salmonella-specific proteins. In addition, Salmonella strains carrying mutations in the gene encoding STM3169 showed growth defects and impaired growth within macrophage-like RAW264.7 cells. Furthermore, we found that expression of stm3169 was controlled by ppGpp and SsrB, a response regulator of the two-component system located on Salmonella pathogenicity island 2. Conclusions A proteomic approach using a 2-DE reference map can prove a powerful tool for analyzing virulence factors and the regulatory network involved in Salmonella pathogenesis. Our results also provide evidence of a global response mediated by ppGpp in S. enterica.

The purpose of this study was to investigate the effects of heat-killed Lactobacillus plantarum strain b240 (b240) on systemic infection by Salmonella enterica serovar Typhimurium (S. Typhimurium) and to determine the mechanism by which b240 protects against infection. Mice were administered either b240 or saline orally for 3 weeks, and then inoculated with S. Typhimurium. The mice treated with b240 were significantly protected against S. Typhimurium as compared to those fed saline. Moreover, translocation of S. Typhimurium into each organ tested in the mice that received b240 tended to be less than in the control mice. An important mechanism of protection against infection was demonstrated by the ability of b240 to inhibit both binding by and invasion of S. Typhimurium into cells. These results indicate that nonviable lactic acid bacteria also play important roles in preventing infection by enteric pathogens.

Chromobacterium violaceum is a Gram-negative bacterium that causes fatal septicaemia in humans and animals. C. violaceum ATCC 12472 possesses genes associated with two distinct type III secretion systems (T3SSs). One of these systems is encoded by Chromobacterium pathogenicity islands 1 and 1a (Cpi-1/-1a), another is encoded by Chromobacterium pathogenicity island 2 (Cpi-2). Here we show that C. violaceum causes fulminant hepatitis in a mouse infection model, and Cpi-1/-1a-encoded T3SS is required for its virulence. In addition, using C. violaceum strains with defined mutations in the genes that encode the Cpi-1/-1a or Cpi-2 locus in combination with cultured mammalian cell lines, we found that C. violaceum is able to induce cytotoxicity in a Cpi-1/-1a-dependent manner. Characterization of Chromobacterium-induced cytotoxicity revealed that cell lysis by C. violaceum infection involves the formation of pore structures on the host cell membrane, as demonstrated by protection by cytotoxicity in the presence of osmoprotectants. Finally, we demonstrated that CipB, a Cpi-1/-1a effector, is implicated in translocator-mediated pore formation and the ability of CipB to form a pore is essential for Chromobacterium-induced cytotoxicity. These results strongly suggest that Cpi-1/-1a-encoded T3SS is a virulence determinant that causes fatal infection by the induction of cell death in hepatocytes.

The type III secretion system (T3SS) encoded by Salmonella pathogenicity island 2 (SPI-2) is involved in systemic infection and intracellular replication of Salmonella enterica serovar Typhimurium. In this study, we investigated the function of SsaE, a small cytoplasmic protein encoded within the SPI-2 locus, which shows structural similarity to the T3SS class V chaperones. An S. enterica serovar Typhimurium ssaE mutant failed to secrete SPI-2 translocator SseB and SPI-2-dependent effector PipB proteins. Coimmunoprecipitation and mass spectrometry analyses using an SsaE-FLAG fusion protein indicated that SsaE interacts with SseB and a putative T3SS-associated ATPase, SsaN. A series of deleted and point-mutated SsaE-FLAG fusion proteins revealed that the C-terminal coiled-coil domain of SsaE is critical for protein-protein interactions. Although SseA was reported to be a chaperone for SseB and to be required for its secretion and stability in the bacterial cytoplasm, an sseA deletion mutant was able to secrete the SseB in vitro when plasmid-derived SseB was overexpressed. In contrast, ssaE mutant strains could not transport SseB extracellularly under the same assay conditions. In addition, an ssaE ( I55G ) point-mutated strain that expresses the SsaE derivative lacking the ability to form a C-terminal coiled-coil structure showed attenuated virulence comparable to that of an SPI-2 T3SS null mutant, suggesting that the coiled-coil interaction of SsaE is absolutely essential for the functional SPI-2 T3SS and for Salmonella virulence. Based on these findings, we propose that SsaE recognizes translocator SseB and controls its secretion via SPI-2 type III secretion machinery.

Salmonella pathogenicity islands are inserted into the genome by horizontal gene transfer and are required for expression of full virulence. Here, we performed tRNA scanning of the genome of Salmonella enterica serovar Typhimurium and compared it with that of nonpathogenic Escherichia coli in order to identify genomic islands that contribute to Salmonella virulence. Using deletion analysis, we identified four genomic islands that are required for virulence in the mouse infection model. One of the newly identified pathogenicity islands was the pheV-tRNA-located genomic island, which is comprised of 26 126 bp, and encodes 22 putative genes, including STM3117-STM3138. We also showed that the pheV tRNA-located genomic island is widely distributed among different nontyphoid Salmonella serovars. Furthermore, genes including STM3118-STM3121 were identified as novel virulence-associated genes within the pheV-tRNA-located genomic island. These results indicate that a Salmonella-specific pheV-tRNA genomic island is involved in Salmonella pathogenesis among the nontyphoid Salmonella serovars.

The formation of disulfide bond is essential for the folding, activity, and stability of many secreted proteins of Gram-negative bacteria. The disulfide oxidoreductase, DsbA, introduces disulfide bonds into exported proteins from the cytoplasm. In pathogenic bacteria, DsbA is required to process virulence determinants for their folding and assembly. In this study, we investigated the role of DsbA in enteropathogenic Escherichia coli. Here, we show that the DsbA is required for stable expression of outer membrane secretin EscC. DsbA has no effect on LEE transcription as measured with LEE-lacZ fusions. Replacement of either cysteine residue 136 or 155 of EscC with a serine resulted in reduced level of EscC, similar to the effect of the dsbA mutation. These results demonstrate the role of DsbA in assembly of the type III secretion apparatus.

The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI-2) is essential for the intracellular survival and replication of Salmonella enterica. The expression of SPI-2 genes is dependent on a two-component regulatory system, SsrA (SpiR)/SsrB, encoded in the SPI-2 region. This paper shows that SlyA regulates transcription of the sensor kinase SsrA by binding to the ssrA promoter, indicating that SlyA is directly involved in the regulation of SPI-2 gene expression. A structure model of the SlyA dimer in complex with DNA was constructed. The model of SlyA indicated that its structure is very similar to that of other MarR family proteins. Based on this model, site-directed mutagenesis of residues located in the winged-helix region required for DNA binding and in the alpha-helices of the N-terminal and C-terminal regions required for dimerization of the SlyA protein was performed to identify the residues that are critical for SlyA function. Nine mutants of SlyA with single substitutions were unable to activate ssrA transcription in vivo. These mutant SlyA proteins revealed that the residues Leu-63, Val-64, Arg-65, Leu-67, Leu-70, Arg-86 and Lys-88 within the winged-helix region are required for DNA binding, and residues Leu-12 and Leu-126 within the alpha-helices of the N-terminal and C-terminal regions are required for efficient dimer formation. A Salmonella slyA mutant strain carrying a plasmid expressing SlyA derivatives containing mutations at these amino acid positions did not exhibit restored SlyA function in infected HeLa cells, thereby confirming the structural and functional relationships of the SlyA protein.

Listeria monocytogenes is able to grow under conditions of high osmolarity. We constructed a deletion mutant of rpoN , encoding the alternative sigma factor RpoN, and analyzed its response to osmotic stress. In a minimal medium with 4% NaCl and 1 mM betaine, the mutant showed a similar growth to that of the parental strain, EGD. In the same medium with 4% NaCl and 1 M carnitine, the growth rate of the mutant was greatly reduced, when the optical density at 600 nm (OD600) at the starting point of growth, was 0.15. However, when growth of the culture was started at an OD600 of 0.025, the growth of the mutant was similar to that of EGD. The mutant's expression of two betaine transporter genes, betL and gbuB , and the carnitine transporter gene opuCA , was osmotically induced at a level similar to EGD, and its rate of carnitine uptake was similar to that of EGD. These results suggest that the growth defect from the rpoN mutant is caused not by the transcriptional regulation of opuCA or by a decrease in carnitine uptake, but possibly by larger amounts of carnitine being needed for growth of the mutant in minimal medium when NaCl is present.

Serum resistance is a crucial virulence factor for the development of systemic infections, including bacteraemia, by many pathogenic bacteria. Salmonella enterica serovar Choleraesuis is an important enteric pathogen that causes serious systemic infections in swine and humans. Here, it was found that, when introduced into Escherichia coli, a recombinant plasmid carrying the pagC gene from a plasmid-based genomic library of S. enterica serovar Choleraesuis conferred a high-level resistance to the bactericidal activity of pooled normal swine serum. The resistance was equal to the level conferred by rck, a gene encoding a 17 kDa outer-membrane protein which promotes the serum resistance phenotype in S. enterica serovar Typhimurium. Insertional mutagenesis of the cloned pagC gene generated a mutation that resulted in the loss of the serum resistance phenotype in E. coli. When this mutation was introduced into the chromosome of S. enterica serovar Choleraesuis by homology recombination with the wild-type allele, the resulting strain could not produce PagC, and it showed a decreased level of resistance to complement-mediated killing. The mutation could be restored by introduction of the intact pagC gene on a plasmid, but not by introduction of the point-mutated pagC gene. In addition, PagC was able to promote serum resistance in the S. enterica serovar Choleraesuis LPS mutant strain, which is highly sensitive to serum killing. Although PagC is not thought to confer serum resistance directly, these results strongly suggest that PagC is an important outer-membrane protein that plays an important role in the serum resistance of S. enterica serovar Choleraesuis.

A number of bacteria that are pathogenic for animals and plants possess a type III secretion system (TTSS) to translocate virulence-associated proteins into host cells. In several bacteria, it has been reported that the TTSS is correlated with an ability to cause contact-dependent hemolysis in vitro. Here, we showed that the Salmonella enterica serovar Typhimurium strain SL1344 exhibited Salmonella pathogenicity island 1 type III secretion-dependent, contact-mediated, hemolytic activity. Mutations with a single deletion in genes encoding putative pore-forming proteins, SipB and SipC, secreted by the TTSS abolished hemolytic activity. In addition, the osmoprotection studies revealed that molecules larger than PEG2000 conferred significant protection against lysis induced by Salmonella. These results indicate that the hemolysis generated by Salmonella is due to the formation of pores within the erythrocyte membrane.

The complete nucleotide sequence of pKDSC50, a large virulence plasmid from Salmonella enterica serovar Choleraesuis strain RF-1, has been determined. We identified 48 of the open reading frames (ORFs) encoded by the 49,503-bp molecule. pKDSC50 encodes a known virulence-associated operon, the spv operon, which is composed of genes essential for systemic infection by nontyphoidal Salmonella . Analysis of the genetic organization of pKDSC50 suggests that the plasmid is composed of several virulence-associated genes, which include the spvRABCD genes, plasmid replication and maintenance genes, and one insertion sequence element. A second virulence-associated region including the pef (plasmid-encoded fimbria) operon and rck (resistance to complement killing) gene, which has been identified on the virulence plasmid of S. enterica serovar Typhimurium, was absent. Two different replicon regions, similar to the RepFIIA and RepFIB replicons, were found. Both showed high similarity to those of the pO157 plasmid of enterohemorrhagic Escherichia coli O157:H7 and the enteropathogenic E. coli (EPEC) adherence factor plasmid harbored by EPEC strain B171 (O111:NM), as well as the virulence plasmids of Salmonella serovars Typhimurium and Enteritidis. Comparative analysis of the nucleotide sequences of the 50-kb virulence plasmid of serovar Choleraesuis and the 94-kb virulence plasmid of serovar Typhimurium revealed that 47 out of 48 ORFs of the virulence plasmid of serovar Choleraesuis are highly homologous to the corresponding ORFs of the virulence plasmid of serovar Typhimurium, suggesting a common ancestry.

Lactobacillus casei ATCC 27139 enhances host innate immunity, and the J1 phage-resistant mutants of this strain lose the activity. A transposon insertion mutant library of L. casei ATCC 27139 was constructed, and nine J1 phage-resistant mutants out of them were obtained. Cloning and sequencing analyses identified three independent genes that were disrupted by insertion of the transposon element: asnH, encoding asparagine synthetase, and dnaJ and dnaK, encoding the molecular chaperones DnaJ and DnaK, respectively. Using an in vivo mouse model of Listeria infection, only asnH mutant showed deficiency in their ability to enhance host innate immunity, and complementation of the mutation by introduction of the wild-type asnH in the mutant strain recovered the immuno-augmenting activity. AsnH protein exhibited asparagine synthetase activity when the lysozyme-treated cell wall extracts of L. casei ATCC 27139 was added as substrate. The asnH mutants lost the thick and rigid peptidoglycan features that are characteristic to the wild-type cells, indicating that AsnH of L. casei is involved in peptidoglycan biosynthesis. These results indicate that asnH is required for the construction of the peptidoglycan composition involved in the immune-activating capacity of L. casei ATCC 27139.

化膿連鎖球菌は上気道および皮膚において急性化膿性炎を起こすほか, 近年においては毒素ショック様症候群の起因菌として注目されている。本菌の病原性を規定する重要なビルレンス因子の1つとしてM蛋白が知られている。M蛋白はトロポミオシン様構造を呈する菌体表層蛋白で, その役割として多形核白血球による食菌作用から菌を防御することが古くから知られているほか, 菌の皮膚・表皮細胞への付着に直接関与し, このとき表皮細胞上の membrance cofactor protein (MCPあるいはCD46) を宿主レセプターとして認識すること, さらにヒト血奬蛋白であるH因子, キニノーゲン, アルブミンおよび免疫グロブリンとの結合能を持つこと, などがわかってきている。また, その発現は外界の二酸化炭素濃度に依存することが明らかにされている。

Summary The surface-located M protein functions to protect Streptococcus pyogenes (the group A streptococcus) from phagocytosis by polymorphonuclear leukocytes. It has been suggested that this protection results from the ability of M protein to bind factor H, a serum protein that can inhibit the activation of complement. Among different serological variants of M protein, the C-repeat domain is highly conserved and is exposed on the bacterial surface. This domain has been implicated in binding to complement factor H and in M-protein-mediated adherence of streptococci to human keratinocytes in the cutaneous epithelium. In this study, we constructed an S. pyogenes mutant strain which expresses an M6 protein from which the entire C-repeat domain was deleted. As predicted, this mutant did not adhere well to human keratinocytes and was unable to bind to factor H. Unexpectedly, the mutant was able to survive and multiply in human blood. Therefore, while the binding of factor H and the facilitation of adherence to keratinocytes appear to involve recognition of the C-repeat domain, a region of the M-protein molecule distinct from the C-repeat domain confers upon S. pyogenes its ability to resist phagocytosis.

Summary Transcription of the antiphagocytic M protein in the group A streptococcus (Streptococcus pyogenes) is environmentally regulated in response to CO2 and requires Mry, a trans-acting positive regulatory protein. We have examined the role of Mry in environmental regulation by analysing the factors that regulate expression of the gene that encodes Mry (mry). By employing a strategy that utilizes integrational plasmids, it was found that expression of mry requires the participation of DNA sequences that extend 473 base pairs upstream of the Mry coding region. Transcription of mry, as analysed in S1 nuclease protection assays, is initiated from two separate promoters located within this extended regulatory region. Construction and analysis of transcriptional fusions between the mry promoters and a promoterless chloramphenicol acetyltransferase gene demonstrated that mry is autoregulated and environmentally regulated in response to the level of CO2. These data suggest a model for the regulation of virulence in S. pyogenes where positive transcriptional control of mry in response to environmental stimuli regulates the expression of the M protein.