σE controlled regulation of porin OmpU in Vibrio cholerae.

Bile resistance is essential for enteric pathogens, as exemplified by Vibrio cholerae, the causative agent of cholera. The outer membrane porin OmpU confers bacterial survival and colonization advantages in the presence of host-derived antimicrobial peptides as well as bile. Expression of ompU is controlled by the virulence regulator ToxR. rpoE knockouts are accompanied by suppressor mutations causing ompU downregulation. Therefore, OmpU constitutes an intersection of the ToxR regulon and the σE -pathway in V. cholerae. To understand the mechanism by which the sigma factor σE regulates OmpU synthesis, we performed transcription studies using ompU reporter fusions and immunoblot analysis. Our data revealed an increase in ompU promoter activity in ΔrpoE strains, as well as in a ΔompU background, indicating a negative feedback regulation circuit of ompU expression. This regulation seems necessary, since elevated lethality rates of ΔrpoE strains occur upon ompU overexpression. Manipulation of OmpU's C-terminal portion revealed its relevance for protein stability and potency of σE release. Furthermore, ΔrpoE strains are still capable of elevating OmpU levels under membrane stress conditions triggered by the bile salt sodium deoxycholate. This study provides new details about the impact of σE on ompU regulation, which is critical to the pathogen's intestinal survival.

Multiple weak interactions between BvgA~P and ptx promoter DNA strongly activate transcription of pertussis toxin genes in Bordetella pertussis.

Pertussis toxin is the preeminent virulence factor and major protective antigen produced by Bordetella pertussis, the human respiratory pathogen and etiologic agent of whooping cough. Genes for its synthesis and export are encoded by the 12 kb ptx-ptl operon, which is under the control of the pertussis promoter, Pptx. Expression of this operon, like that of all other known protein virulence factors, is regulated by the BvgAS two-component global regulatory system. Although Pptx has been studied for years, characterization of its promoter architecture vis-à-vis BvgA-binding has lagged behind that of other promoters, mainly due to its lower affinity for BvgA~P. Here we take advantage of a mutant BvgA protein (Δ127-129), which enhances ptx transcription in B. pertussis and also demonstrates enhanced binding affinity to Pptx. By using this mutant protein labeled with FeBABE, binding of six head-to-head dimers of BvgA~P was observed, with a spacing of 22 bp, revealing a binding geometry similar to that of other BvgA-activated promoters carrying at least one strong binding site. All of these six BvgA-binding sites lack sequence features associated with strong binding. A genetic analysis indicated the degree to which each contributes to Pptx activity. Thus the weak/medium binding affinity of Pptx revealed in this study explains its lower responsiveness to phosphorylated BvgA, relative to other promoters containing a high affinity binding site, such as that of the fha operon.

Analyzing the adhesion mechanism of FnBPA, a surface adhesin from Staphylococcus aureus on its interaction with nanoparticle.

Staphylococcus aureus expresses many Microbial Surface Recognizing Adhesive Matrix Molecules (MSCRAMM's) to recognize host extracellular matrix (ECM) molecules to initiate colonization. The MSCRAMM, fibronectin binding protein A (FnBPA), is an important adhesin for S. aureus infection. FnBPA also binds with fibrinogen (Fg) by using a unique ligand binding mechanism called dock, lock and latch. Nanoparticles, especially nanosilver particles have been widely used in a variety of biomedical applications which includes disease diagnosis and treatment, drug delivery and implanted medical device coating. In a biological system, when protein molecules encounter nanoparticle, they can be absorbed onto its surface which results in the formation of protein corona. In the present study, we have analysed the fibrinogen binding ability of rFnBPA(189-512) in the presence of silver nanoparticles by employing techniques like gel shift assay, Western blot, size exclusion chromatography, enzyme-linked immunosorbent assay, bio-layer interferometry and circular dichroism spectroscopy. The results indicate that rFnBPA(189-512) is unable to bind to Fg in the presence of a nanoparticle. This could be due to the inaccessibility of the Fg binding site and conformational change in rFnBPA(189-512). With nanoparticles, rFnBPA(189-512) undergoes significant structural changes as the ß-sheet content has drastically reduced to 10% from the initial 60% at higher concentration of the nanoparticle. Pathogenic bacteria interact with its surrounding environment through their surface molecules which includes MSCRAMMs. Therefore MSCRAMMs play an important role when bacteria encounter nanoparticles. The results of the present study suggest that the orientation of the protein during the absorption on the surface of a nanoparticle as well as the concentration of the nanoparticle, will dictate the function of the absorbed protein and in this case the Fg binding property of rFnBPA(189-512).

Non-pathogenic Escherichia coli biofilms: effects of growth conditions and surface properties on structure and curli gene expression.

Biofilm formation is a harmful phenomenon in many areas, such as in industry and clinically, but offers advantages in the field of biocatalysis for the generation of robust biocatalytic platforms. In this work, we optimised growth conditions for the production of Escherichia coli biofilms by three strains (PHL644, a K-12 derivative with enhanced expression of the adhesin curli; the commercially-used strain BL21; and the probiotic Nissle 1917) on a variety of surfaces (plastics, stainless steel and PTFE). E. coli PHL644 and PTFE were chosen as optimal strain and substratum, respectively, and conditions (including medium, temperature, and glucose concentration) for biofilm growth were determined. Finally, the impact of these growth conditions on expression of the curli genes was determined using flow cytometry for planktonic and sedimented cells. We reveal new insights into the formation of biofilms and expression of curli in E. coli K-12 in response to environmental conditions.

Shigella flexneri Adherence Factor Expression in In Vivo-Like Conditions.

The Shigella species are Gram-negative, facultative intracellular pathogens that invade the colonic epithelium and cause significant diarrheal disease. Despite extensive research on the pathogen, a comprehensive understanding of how Shigella initiates contact with epithelial cells remains unknown. Shigella maintains many of the same Escherichia coli adherence gene operons; however, at least one critical gene component in each operon is currently annotated as a pseudogene in reference genomes. These annotations, coupled with a lack of structures upon microscopic analysis following growth in laboratory media, have led the field to hypothesize that Shigella is unable to produce fimbriae or other traditional adherence factors. Nevertheless, our previous analyses have demonstrated that a combination of bile salts and glucose induces both biofilm formation and adherence to colonic epithelial cells. The goal of this study was to perform transcriptomic and genetic analyses to demonstrate that adherence gene operons in Shigella flexneri strain 2457T are functional, despite the gene annotations. Our results demonstrate that at least three structural genes facilitate S. flexneri 2457T adherence for epithelial cell contact and biofilm formation. Furthermore, our results demonstrate that host factors, namely, glucose and bile salts at their physiological concentrations in the small intestine, offer key environmental stimuli required for adherence factor expression in S. flexneri This research may have a significant impact on Shigella vaccine development and further highlights the importance of utilizing in vivo-like conditions to study bacterial pathogenesis.IMPORTANCE Bacterial pathogens have evolved to regulate virulence gene expression at critical points in the colonization and infection processes to successfully cause disease. The Shigella species infect the epithelial cells lining the colon to result in millions of cases of diarrhea and a significant global health burden. As antibiotic resistance rates increase, understanding the mechanisms of infection is vital to ensure successful vaccine development. Despite significant gains in our understanding of Shigella infection, it remains unknown how the bacteria initiate contact with the colonic epithelium. Most pathogens harbor multiple adherence factors to facilitate this process, but Shigella was thought to have lost the ability to produce these factors. Interestingly, we have identified conditions that mimic some features of gastrointestinal transit and that enable Shigella to express adherence structural genes. This work highlights aspects of genetic regulation for Shigella adherence factors and may have a significant impact on future vaccine development.

Protocol for a controlled human infection with genetically modified Neisseria lactamica expressing the meningococcal vaccine antigen NadA: a potent new technique for experimental medicine.

INTRODUCTION: Neisseria lactamica is a commensal organism found in the human nasopharynx and is closely related to the pathogen N. meningitidis (meningococcus). Carriage of N. lactamica is associated with reduced meningococcal carriage and disease. We summarise an ethically approved protocol for an experimental human challenge study using a genetically modified strain of N. lactamica that expresses the meningococcal antigen NadA. We aim to develop a model to study the role of specific bacterial antigens in nasopharyngeal carriage and immunity, to evaluate vaccines for their efficacy in preventing colonisation and to provide a proof of principle for the development of bacterial medicines. METHODS AND ANALYSIS: Healthy adult volunteers aged 18-45 years will receive an intranasal inoculation of either the NadA containing strain of N. lactamica or a genetically modified, but wild-type equivalent control strain. These challenge volunteers will be admitted for 4.5 days observation following inoculation and will then be discharged with strict infection control rules. Bedroom contacts of the challenge volunteers will also be enrolled as contact volunteers. Safety, colonisation, shedding, transmission and immunogenicity will be assessed over 90 days after which carriage will be terminated with antibiotic eradication therapy. ETHICS AND DISSEMINATION: This study has been approved by the Department for Environment, Food and Rural Affairs and South Central Oxford A Research Ethics Committee (reference: 18/SC/0133). Findings will be published in peer-reviewed open-access journals as soon as possible. TRIAL REGISTRATION NUMBER: NCT03630250; Pre-results.

Production and characterization of recombinant P1 adhesin essential for adhesion, gliding, and antigenic variation in the human pathogenic bacterium, Mycoplasma pneumoniae.

Mycoplasma pneumoniae forms an attachment organelle at one cell pole, binds to the host cell surface, and glides via a unique mechanism. A 170-kDa protein, P1 adhesin, present on the organelle surface plays a critical role in the binding and gliding process. In this study, we obtained a recombinant P1 adhesin comprising 1476 amino acid residues, excluding the C-terminal domain of 109 amino acids that carried the transmembrane segment, that were fused to additional 17 amino acid residues carrying a hexa-histidine (6 × His) tag using an Escherichia coli expression system. The recombinant protein showed solubility, and chirality in circular dichroism (CD). The results of analytical gel filtration, ultracentrifugation, negative-staining electron microscopy, and small-angle X-ray scattering (SAXS) showed that the recombinant protein exists in a monomeric form with a uniformly folded structure. SAXS analysis suggested the presence of a compact and ellipsoidal structure rather than random or molten globule-like conformation. Structure model based on SAXS results fitted well with the corresponding structure obtained with cryo-electron tomography from a closely related species, M. genitalium. This recombinant protein may be useful for structural and functional studies as well as for the preparation of antibodies for medical applications.

Dual Inhibitor of Staphylococcus aureus Virulence and Biofilm Attenuates Expression of Major Toxins and Adhesins.

Staphylococcus aureus is a major bacterial pathogen that invades and damages host tissue by the expression of devastating toxins. We here performed a phenotypic screen of 35 molecules that were structurally inspired by previous hydroxyamide-based S. aureus virulence inhibitors compiled from commercial sources or designed and synthesized de novo. One of the most potent compounds, AV73, not only reduced hemolytic alpha-hemolysin production in S. aureus but also impeded in vitro biofilm formation. The effect of AV73 on bacterial proteomes and extracellular protein levels was analyzed by quantitative proteomics and revealed a significant down-regulation of major virulence and biofilm promoting proteins. To elucidate the mode of action of AV73, target identification was performed using affinity-based protein profiling (AfBPP), where among others YidC was identified as a target.

Screening and Genomic Characterization of Filamentous Hemagglutinin-Deficient Bordetella pertussis.

Despite high vaccine coverage, pertussis cases in the United States have increased over the last decade. Growing evidence suggests that disease resurgence results, in part, from genetic divergence of circulating strain populations away from vaccine references. The United States employs acellular vaccines exclusively, and current Bordetella pertussis isolates are predominantly deficient in at least one immunogen, pertactin (Prn). First detected in the United States retrospectively in a 1994 isolate, the rapid spread of Prn deficiency is likely vaccine driven, raising concerns about whether other acellular vaccine immunogens experience similar pressures, as further antigenic changes could potentially threaten vaccine efficacy. We developed an electrochemiluminescent antibody capture assay to monitor the production of the acellular vaccine immunogen filamentous hemagglutinin (Fha). Screening 722 U.S. surveillance isolates collected from 2010 to 2016 identified two that were both Prn and Fha deficient. Three additional Fha-deficient laboratory strains were also identified from a historic collection of 65 isolates dating back to 1935. Whole-genome sequencing of deficient isolates revealed putative, underlying genetic changes. Only four isolates harbored mutations to known genes involved in Fha production, highlighting the complexity of its regulation. The chromosomes of two Fha-deficient isolates included unexpected structural variation that did not appear to influence Fha production. Furthermore, insertion sequence disruption of fhaB was also detected in a previously identified pertussis toxin-deficient isolate that still produced normal levels of Fha. These results demonstrate the genetic potential for additional vaccine immunogen deficiency and underscore the importance of continued surveillance of circulating B. pertussis evolution in response to vaccine pressure.

[Construction of recombinant strain Brevibacillus choshinensis for chimeric borrelia Dbpa antigen production.]

The aim of this work was creation of recombinant chimeric protein using TAKARA expression system Brevibacillus choshinensis with fused gene dbpAAG, which include the parts of dbpAA and dbpAG genes coding the major antigenic determinants of decorinbinding proteins А (DbpA) from two species of borreliosis agents - Borrelia afzelii and Borrelia gаrinii. Such plasmid should be able to support the synthesis of recombinant chimeric polypeptide consisting immunogenic domains of DbpA Borrelia afzelii and Borrelia gаrinii in the stable and soluble forms, that important for effective using in Lyme diseases serodiagnosis. We chose the TAKARA expression system based on the strain Brevibacillus choshinensis and plasmid pNCMO2. It give us possibilities to obtain the scale quantity of the secreted soluble target proteins with native conformation in particular with conserve antigenic determinants. As results, the plasmid pNCMO2 with a fusion gene dbpAAG was constructed. Recombinante plasmide DNA pNCMO2/dbpAAG was used for Brevibacillus choshinensis trasformation. We were able to show that during cultivation in a liquid medium recombinant cells of В. choshinensis/pNCMO2/dbpAAG produced secreted chimeric 30кD protein with high immunoreactivity to Lyme borreliosis patient's serum.

Blue Light Switchable Bacterial Adhesion as a Key Step toward the Design of Biofilms.

The control of where and when bacteria adhere to a substrate is a key step toward controlling the formation and organization in biofilms. This study shows how we engineer bacteria to adhere specifically to substrates with high spatial and temporal control under blue light, but not in the dark, by using photoswitchable interaction between nMag and pMag proteins. For this, we express pMag proteins on the surface of E. coli so that the bacteria can adhere to substrates with immobilized nMag protein under blue light. These adhesions are reversible in the dark and can be repeatedly turned on and off. Further, the number of bacteria that can adhere to the substrate as well as the attachment and detachment dynamics are adjustable by using different point mutants of pMag and altering light intensity. Overall, the blue light switchable bacteria adhesions offer reversible, tunable and bioorthogonal control with exceptional spatial and temporal resolution. This enables us to pattern bacteria on substrates with great flexibility.

Quantitative Expression Analysis of SpA, FnbA and Rsp Genes in Staphylococcus aureus: Actively Associated in the Formation of Biofilms.

In Staphylococcus aureus, adherence and secretory proteins play chief role in the formation of biofilms. This mode of growth exhibits resistance to a variety of antibiotics and spreads its infections. In the present study, secretary and adherence proteins, Protein-A, Fibronectin-binding protein-A (FnbA) and Rsp (a transcription regulator encoding proteolytic property) expression levels were evaluated at different stages of growth in S. aureus ATCC12600 a drug-sensitive strain and multidrug-resistant strains of S. aureus. Initially, the SpA, FnbA and Rsp genes of S. aureus ATCC12600 were cloned, sequenced, expressed and characterized. The proteolytic property of recombinant Rsp was conspicuously shown when this pathogen was grown in aerobic conditions correlating with reduced biofilm units. In anaerobic mode of growth, S. aureus exhibited a higher expression of SpA and FnbA in early and mid adherence phases and finally stabilized at 48 h of incubation. This expression was more pronounced in methicillin-resistant strains (LMV1-8 and D1-4) of S. aureus. In all these stages, Rsp gene expression was at the lowest level and these results concur with the increased biofilm units. The results of the present study explain proteins chiefly contribute in the formation of biofilms.

Host Determinants of Expression of the Helicobacter pylori BabA Adhesin.

Expression of the Helicobacter pylori blood group antigen binding adhesin A (BabA) is more common in strains isolated from patients with peptic ulcer disease or gastric cancer, rather than asymptomatic colonization. Here we used mouse models to examine host determinants that affect H. pylori BabA expression. BabA expression was lost by phase variation as frequently in WT mice as in RAG2-/- mice that do not have functional B or T cells, and in MyD88-/-, TLR2-/- and TLR4-/- mice that are defective in toll like receptor signaling. The presence of other bacteria had no effect on BabA expression as shown by infection of germ free mice. Moreover, loss of BabA expression was not dependent on Leb expression or the capacity of BabA to bind Leb. Surprisingly, gender was the host determinant most associated with loss of BabA expression, which was maintained to a greater extent in male mice and was associated with greater bacterial load. These results suggest the possibility that loss of BabA expression is not driven by adaptive immunity or toll-like receptor signaling, and that BabA may have other, unrecognized functions in addition to serving as an adhesin that binds Leb.

In Vitro and In Vivo Antibacterial Activities of Patchouli Alcohol, a Naturally Occurring Tricyclic Sesquiterpene, against Helicobacter pylori Infection.

This study further evaluated the in vitro and in vivo anti-Helicobacter pylori activities and potential underlying mechanism of patchouli alcohol (PA), a tricyclic sesquiterpene. In the in vitro assay, the capacities of PA to inhibit and kill H. pylori were tested on three standard strains at different pH values and on 12 clinical isolates. The effects of PA on H. pylori adhesion (and its alpA, alpB, and babA genes), motility (and its flaA and flaB genes), ultrastructure, and flagellation were investigated. Moreover, the H. pylori resistance to and postantibiotic effect (PAE) of PA were determined. Furthermore, the in vivo effects of PA on H. pylori eradication and gastritis were examined. Results showed that MICs of PA against three standard strains (pH 5.3 to 9) and 12 clinical isolates were 25 to 75 and 12.5 to 50 µg/ml, respectively. The killing kinetics of PA were time and concentration dependent, and its minimal bactericidal concentrations (MBCs) were 25 to 75 µg/ml. In addition, H. pylori adhesion, motility, ultrastructure, and flagellation were significantly suppressed. PA also remarkably inhibited the expression of adhesion genes (alpA and alpB) and motility genes (flaA and flaB). Furthermore, PA treatment caused a longer PAE and less bacterial resistance than clarithromycin and metronidazole. The in vivo study showed that PA can effectively eradicate H. pylori, inhibit gastritis, and suppress the expression of inflammatory mediators (COX-2, interleukin 1ß, tumor necrosis factor alpha, and inducible nitric oxide synthase [iNOS]). In conclusion, PA can efficiently kill H. pylori, interfere with its infection process, and attenuate gastritis with less bacterial resistance, making it a potential candidate for new drug development.

Surface expression of Helicobacter pylori HpaA adhesion antigen on Vibrio cholerae, enhanced by co-expressed enterotoxigenic Escherichia coli fimbrial antigens.

Helicobacter pylori infection can cause peptic ulceration and is associated with gastric adenocarcinoma. This study aimed to construct and characterize a non-virulent Vibrio cholerae O1 strain, which grows more rapidly than H. pylori, as vector for H. pylori antigens for possible use as a vaccine strain against H. pylori. This was done by recombinant expression of the H. pylori adhesion antigen HpaA alone or, as a proof of principle, together with different colonization factor (CF) antigens of enterotoxigenic Escherichia coli (ETEC) which may enhance immune responses against HpaA. A recombinant V. cholerae strain co-expressing HpaA and a fimbrial CF antigens CFA/I or CS5, but not the non-fimbrial CF protein CS6, was shown to express larger amounts of HpaA on the surface when compared with the same V. cholerae strain expressing HpaA alone. Mutations in the CFA/I operon showed that the chaperon, possibly together with the usher, was involved in enhancing the surface expression of HpaA. Oral immunization of mice with formaldehyde-inactivated recombinant V. cholerae expressing HpaA alone or together with CFA/I induced significantly higher serum antibody responses against HpaA than mice similarly immunized with inactivated HpaA-expressing H. pylori bacteria. Our results demonstrate that a non-virulent V. cholerae strain can be engineered to allow strong surface expression of HpaA, and that the expression can be further increased by co-expressing it with ETEC fimbrial antigens. Such recombinant V. cholerae strains expressing HpaA, and possibly also other H. pylori antigens, may have the potential as oral inactivated vaccine candidates against H. pylori.

Discovery of a novel periplasmic protein that forms a complex with a trimeric autotransporter adhesin and peptidoglycan.

Trimeric autotransporter adhesins (TAAs), fibrous proteins on the cell surface of Gram-negative bacteria, have attracted attention as virulence factors. However, little is known about the mechanism of their biogenesis. AtaA, a TAA of Acinetobacter sp. Tol 5, confers nonspecific, high adhesiveness to bacterial cells. We identified a new gene, tpgA, which forms a single operon with ataA and encodes a protein comprising two conserved protein domains identified by Pfam: an N-terminal SmpA/OmlA domain and a C-terminal OmpA_C-like domain with a peptidoglycan (PGN)-binding motif. Cell fractionation and a pull-down assay showed that TpgA forms a complex with AtaA, anchoring it to the outer membrane (OM). Isolation of total PGN-associated proteins showed TpgA binding to PGN. Disruption of tpgA significantly decreased the adhesiveness of Tol 5 because of a decrease in surface-displayed AtaA, suggesting TpgA involvement in AtaA secretion. This is reminiscent of SadB, which functions as a specific chaperone for SadA, a TAA in Salmonella species; however, SadB anchors to the inner membrane, whereas TpgA anchors to the OM through AtaA. The genetic organization encoding the TAA-TpgA-like protein cassette can be found in diverse Gram-negative bacteria, suggesting a common contribution of TpgA homologues to TAA biogenesis.

Molecular Basis of Ligand-Dependent Regulation of NadR, the Transcriptional Repressor of Meningococcal Virulence Factor NadA.

Neisseria adhesin A (NadA) is present on the meningococcal surface and contributes to adhesion to and invasion of human cells. NadA is also one of three recombinant antigens in the recently-approved Bexsero vaccine, which protects against serogroup B meningococcus. The amount of NadA on the bacterial surface is of direct relevance in the constant battle of host-pathogen interactions: it influences the ability of the pathogen to engage human cell surface-exposed receptors and, conversely, the bacterial susceptibility to the antibody-mediated immune response. It is therefore important to understand the mechanisms which regulate nadA expression levels, which are predominantly controlled by the transcriptional regulator NadR (Neisseria adhesin A Regulator) both in vitro and in vivo. NadR binds the nadA promoter and represses gene transcription. In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. NadR also mediates ligand-dependent regulation of many other meningococcal genes, for example the highly-conserved multiple adhesin family (maf) genes, which encode proteins emerging with important roles in host-pathogen interactions, immune evasion and niche adaptation. To gain insights into the regulation of NadR mediated by 4-HPA, we combined structural, biochemical, and mutagenesis studies. In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of 'conformational selection' by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. Overall, this study deepens our molecular understanding of the sophisticated regulatory mechanisms of the expression of nadA and other genes governed by NadR, dependent on interactions with niche-specific signal molecules that may play important roles during meningococcal pathogenesis.

Lactobacilli Reduce Helicobacter pylori Attachment to Host Gastric Epithelial Cells by Inhibiting Adhesion Gene Expression.

The human gastrointestinal tract, including the harsh environment of the stomach, harbors a large variety of bacteria, of which Lactobacillus species are prominent members. The molecular mechanisms by which species of lactobacilli interfere with pathogen colonization are not fully characterized. In this study, we aimed to study the effect of lactobacillus strains upon the initial attachment of Helicobacter pylori to host cells. Here we report a novel mechanism by which lactobacilli inhibit adherence of the gastric pathogen H. pylori In a screen with Lactobacillus isolates, we found that only a few could reduce adherence of H. pylori to gastric epithelial cells. Decreased attachment was not due to competition for space or to lactobacillus-mediated killing of the pathogen. Instead, we show that lactobacilli act on H. pylori directly by an effector molecule that is released into the medium. This effector molecule acts on H. pylori by inhibiting expression of the adhesin-encoding gene sabA Finally, we verified that inhibitory lactobacilli reduced H. pylori colonization in an in vivo model. In conclusion, certain Lactobacillus strains affect pathogen adherence by inhibiting sabA expression and thereby reducing H. pylori binding capacity.

The bvg-repressed gene brtA, encoding biofilm-associated surface adhesin, is expressed during host infection by Bordetella bronchiseptica.

Bordetella species display phase modulation between Bvg(+) and Bvg(-) phases. Because expression of known virulence factors is up-regulated in the Bvg(+) phase, bacteria in this phase are considered competent for infection. However, the Bvg(-) phase is of negligible importance for infection. No studies have shown that bacterial factors specific to the Bvg(-) phase (bvg-repressed factors) are expressed in the course of Bordetella infection. In the present study, the gene brtA (Bordetella RTX-family Adhesin), which is a typical bvg-repressed gene but is expressed in B. bronchiseptica infecting hosts, was characterized. BrtA is composed of repeated pairs of the VCBS unit and dystroglycan-type cadherin-like unit, the von Willebrand Factor A domain, RTX motif and type I secretion target signal. It is herein demonstrated that BrtA is secreted by the type I secretion system and is essential for Ca(2+) -dependent bacteria-to-substrate adherence, followed by biofilm formation. Although the contribution of BrtA to bacterial colonization of the rat trachea currently remains unclear, this is the first study to present concrete evidence for the expression of a bvg-repressed gene during infection, which may provide a novel aspect for analyses of Bordetella pathogenesis.

Impact of OmpR on the membrane proteome of Yersinia enterocolitica in different environments: repression of major adhesin YadA and heme receptor HemR.

Enteropathogenic Yersinia enterocolitica is able to grow within or outside the mammalian host. Previous transcriptomic studies have indicated that the regulator OmpR plays a role in the expression of hundreds of genes in enterobacteria. Here, we have examined the impact of OmpR on the production of Y. enterocolitica membrane proteins upon changes in temperature, osmolarity and pH. Proteomic analysis indicated that the loss of OmpR affects the production of 120 proteins, a third of which are involved in uptake/transport, including several that participate in iron or heme acquisition. A set of proteins associated with virulence was also affected. The influence of OmpR on the abundance of adhesin YadA and heme receptor HemR was examined in more detail. OmpR was found to repress YadA production and bind to the yadA promoter, suggesting a direct regulatory effect. In contrast, the repression of hemR expression by OmpR appears to be indirect. These findings provide new insights into the role of OmpR in remodelling the cell surface and the adaptation of Y. enterocolitica to different environmental niches, including the host.