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Compares the Escherichia coli isolates from fecal samples of young calves in Bavaria, Germany. Identification of the resistance rate of E. coli populations; Observation of the anamnestic data of antibiotic usage; Registration of the resistance to microbials.

The diarrheal disease cholera is caused by the versatile and responsive bacterium Vibrio cholerae, which is capable of adapting to environmental changes. Among others, the alternative sigma factor RpoS activates response pathways, including regulation of motility- and chemotaxis-related genes under nutrient-poor conditions in V. cholerae. Although RpoS has been well characterised, links between RpoS and other regulatory networks remain unclear. In this study, we identified the ArcAB two-component system to control rpoS transcription and RpoS protein stability in V. cholerae. In a manner similar to that seen in Escherichia coli, the ArcB kinase not only activates the response regulator ArcA but also RssB, the anti-sigma factor of RpoS. Our results demonstrated that, in V. cholerae, RssB is phosphorylated by ArcB, which subsequently activates RpoS proteolysis. Furthermore, ArcA acts as a repressor of rpoS transcription. Additionally, we determined that the cysteine residue at position 180 of ArcB is crucial for signal recognition and activity. Thus, our findings provide evidence linking RpoS response to the anoxic redox control system ArcAB in V. cholerae., (© 2024 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

The seventh pandemic of the diarrheal cholera disease, which began in 1960, is caused by the Gram-negative bacterium Vibrio cholerae . Its environmental persistence provoking recurring sudden outbreaks is enabled by V. cholerae's rapid adaption to changing environments involving sensory proteins like ToxR and ToxS. Located at the inner membrane, ToxR and ToxS react to environmental stimuli like bile acid, thereby inducing survival strategies for example bile resistance and virulence regulation. The presented crystal structure of the sensory domains of ToxR and ToxS in combination with multiple bile acid interaction studies, reveals that a bile binding pocket of ToxS is only properly folded upon binding to ToxR. Our data proposes an interdependent functionality between ToxR transcriptional activity and ToxS sensory function. These findings support the previously suggested link between ToxRS and VtrAC-like co-component systems. Besides VtrAC, ToxRS is now the only experimentally determined structure within this recently defined superfamily, further emphasizing its significance. In-depth analysis of the ToxRS complex reveals its remarkable conservation across various Vibrio species, underlining the significance of conserved residues in the ToxS barrel and the more diverse ToxR sensory domain. Unravelling the intricate mechanisms governing ToxRS's environmental sensing capabilities, provides a promising tool for disruption of this vital interaction, ultimately inhibiting Vibrio's survival and virulence. Our findings hold far-reaching implications for all Vibrio strains that rely on the ToxRS system as a shared sensory cornerstone for adapting to their surroundings., Competing Interests: NG, TS, CB, BG, GW, LP, MG, MR, TB, IU, JR, PS, KZ, TP No competing interests declared, JS is affiliated with Redshift BioAnalytics, Inc which distributes the AQS3pro. Access to the AQS3pro instrument was provided to Nina Gubensäk as part of the RedShiftBio demo lab, (© 2023, Gubensäk et al.)

Cholera is a life-threatening diarrheal disease caused by the human pathogenic bacterium Vibrio cholerae. Regulatory elements are essential for bacterial transition between the natural aquatic environment and the human host. One of them is the alternative sigma factor RpoS and its anti-sigma factor RssB. Regulation principles seem to be conserved among RpoS/RssB interaction modes between V. cholerae and Enterobacteriaceae species, however the associated input and output pathways seem different. In Escherichia coli, RpoS/RssB is important for the activation of an emergency program to increase persistence and survival. Whereas, it activates motility and chemotaxis in V. cholerae, used strategically to escape from starvation conditions. We characterised a starvation-induced interaction model showing a negative feedback loop between RpoS and RssB expression. We showed by genotypic and phenotypic analysis that rssB influences motility, growth behaviour, colonization fitness, and post-infectious survival. Furthermore, we found that RssB itself is a substrate for proteolysis and a critical Asp mutation was identified and characterised to influence rssB phenotypes and their interaction with RpoS. In summary, we present novel information about the regulatory interaction between RpoS and RssB being active under in vivo colonization conditions and mark an extension to the feedback regulation circuit, showing that RssB is a substrate for proteolysis., (Copyright © 2022 The Authors. Published by Elsevier GmbH.. All rights reserved.)

ToxR represents an essential transcription factor of Vibrio cholerae, which is involved in the regulation of multiple, mainly virulence associated genes. Its versatile functionality as activator, repressor or coactivator suggests a complex regulatory mechanism, whose clarification is essential for a better understanding of the virulence expression system of V. cholerae. Here, we provide structural information elucidating the organization and binding behavior of the cytoplasmic DNA-binding domain of ToxR (cToxR), containing a winged helix-turn-helix (wHTH) motif. Our analysis reveals unexpected structural features of this domain expanding our knowledge of a poorly defined subfamily of wHTH proteins. cToxR forms an extraordinary long α-loop and furthermore has an additional C-terminal beta strand, contacting the N-terminus and thus leading to a compact fold. The identification of the exact interactions between ToxR and DNA contributes to a deeper understanding of this regulatory process. Our findings not only show general binding of the soluble cytoplasmic domain of ToxR to DNA, but also indicate a higher affinity for the toxT motif. These results support the current theory of ToxR being a "DNA-catcher" to enable binding of the transcription factor TcpP and thus activation of virulence-associated toxT transcription. Although, TcpP and ToxR interaction is assumed to be crucial in the activation of the toxT genes, we could not detect an interaction event of their isolated cytoplasmic domains. We therefore conclude that other factors are needed to establish this protein-protein interaction, e.g., membrane attachment, the presence of their full-length proteins and/or other intermediary proteins that may facilitate binding., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021. Published by Elsevier Inc.)

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 σ E -pathway in V. cholerae. To understand the mechanism by which the sigma factor σ E 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 σ E 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 σ E on ompU regulation, which is critical to the pathogen's intestinal survival., (© 2021 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

The transmembrane protein ToxR plays a key role in the virulence expression system of Vibrio cholerae. The activity of ToxR is dependent on its periplasmic sensor domain (ToxRp) and on the inner membrane protein ToxS. Herein, we present the Nuclear Magnetic Resonance NMR solution structure of the sensory ToxRp containing an intramolecular disulfide bond. The presented structural and dynamic experiments with reduced and oxidized ToxRp propose an explanation for the increased proteolytic sensitivity of reduced ToxR. Additionally, for the first time, we could identify the formation of a strong heterodimer complex between the periplasmic domains of ToxR and ToxS in solution. NMR interaction studies reveal that binding of ToxS is not dependent on the redox state of ToxR cysteines, and formed complexes are structurally similar. By monitoring the proteolytic cleavage of ToxRp with NMR, we additionally provide a direct evidence of ToxS protective function. Taken together our results suggest that ToxR activity is regulated by its stability which is, on the one hand, dependent on the redox states of its cysteines, influencing the stability of its fold, and on the other hand, on its interaction with ToxS, which binds independent on the cysteines and acts as a protection against proteases., (© 2021 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Protein-protein interactions (PPIs) are key mechanisms in the maintenance of biological regulatory networks. Herein, we characterize PPIs within ToxR and its co-activator, ToxS, to understand the mechanisms of ToxR transcription factor activation. ToxR is a key transcription activator that is supported by ToxS for virulence gene regulation in Vibrio cholerae. ToxR comprises a cytoplasmic DNA-binding domain that is linked by a transmembrane domain to a periplasmic signal receiver domain containing two cysteine residues. ToxR-ToxR and ToxR-ToxS PPIs were detected using an adenylate-cyclase-based bacterial two-hybrid system approach in Escherichia coli. We found that the ToxR-ToxR PPIs are significantly increased in response to ToxR operators, the co-activator ToxS and bile salts. We suggest that ToxS and bile salts promote the interaction between ToxR molecules that ultimately results in dimerization. Upon binding of operators, ToxR-ToxR PPIs are found at the highest frequency. Moreover, disulfide-bond-dependent interaction in the periplasm results in homodimer formation that is promoted by DNA binding. The formation of these homodimers and the associated transcriptional activity of ToxR were strongly dependent on the oxidoreductases DsbA/DsbC. These findings show that protein and non-protein partners, that either transiently or stably interact with ToxR, fine-tune ToxR PPIs, and its associated transcriptional activity in changing environments., (© 2020 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Gram-negative bacteria release outer membrane vesicles into the external milieu to deliver effector molecules that alter the host and facilitate virulence. Vesicle formation is driven by phospholipid accumulation in the outer membrane and regulated by the phospholipid transporter VacJ/Yrb. We use the facultative human pathogen Vibrio cholerae to show that VacJ/Yrb is silenced early during mammalian infection, which stimulates vesiculation that expedites bacterial surface exchange and adaptation to the host environment. Hypervesiculating strains rapidly alter their bacterial membrane composition and exhibit enhanced intestinal colonization fitness. This adaptation is exemplified by faster accumulation of glycine-modified lipopolysaccharide (LPS) and depletion of outer membrane porin OmpT, which confers resistance to host-derived antimicrobial peptides and bile, respectively. The competitive advantage of hypervesiculation is lost upon pre-adaptation to bile and antimicrobial peptides, indicating the importance of these adaptive processes. Thus, bacteria use outer membrane vesiculation to exchange cell surface components, thereby increasing survival during mammalian infection., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Protein secretion plays a crucial role for bacterial pathogens, exemplified by facultative human-pathogen Vibrio cholerae , which secretes various proteinaceous effectors at different stages of its lifecycle. Accordingly, the identification of factors impacting on protein secretion is important to understand the bacterial pathophysiology. PglL Vc , a predicted oligosaccharyltransferase of V. cholerae , has been recently shown to exhibit O -glycosylation activity with relaxed glycan specificity in an engineered Escherichia coli system. By engineering V. cholerae strains to express a defined, undecaprenyl diphosphate-linked glycoform precursor, we confirmed functional O -linked protein glycosylation activity of PglL Vc in V. cholerae . We demonstrate that PglL Vc is required for the glycosylation of multiple V. cholerae proteins, including periplasmic chaperones such as DegP, that are required for efficient type II-dependent secretion. Moreover, defined deletion mutants and complementation strains provided first insights into the physiological role of O -linked protein glycosylation in V. cholerae . RbmD, a protein with structural similarities to PglL Vc and other established oligosaccharyltransferases (OTases), was also included in this phenotypical characterization. Remarkably, presence or absence of PglL Vc and RbmD impacts the secretion of proteins via the type II secretion system (T2SS). This is highlighted by altered cholera toxin (CT) secretion, chitin utilization and biofilm formation observed in Δ pglL Vc and Δ rbmD single or double mutants. This work thus establishes a unique connection between broad spectrum O -linked protein glycosylation and the efficacy of type II-dependent protein secretion critical to the pathogen's lifecycle., (Copyright © 2019 Vorkapic, Mitterer, Pressler, Leitner, Anonsen, Liesinger, Mauerhofer, Kuehnast, Toeglhofer, Schulze, Zingl, Feldman, Reidl, Birner-Gruenberger, Koomey and Schild.)

The Gram-negative bacterium Vibrio cholerae encodes two nucleases, Dns and Xds, which play a major role during the human pathogen's lifecycle. Dns and Xds control three-dimensional biofilm formation and bacterial detachment from biofilms via degradation of extracellular DNA and thus contribute to the environmental, inter-epidemic persistence of the pathogen. During intestinal colonization the enzymes help evade the innate immune response, and therefore promote survival by mediating escape from neutrophil extracellular traps. Xds has the additional function of degrading extracellular DNA down to nucleotides, which are an important nutrient source for V. cholerae . Thus, Xds is a key enzyme for survival fitness during distinct stages of the V. cholerae lifecycle and could be a potential therapeutic target. This study provides detailed information about the enzymatic properties of Xds using purified protein in combination with a real time nuclease activity assay. The data define an optimal buffer composition for Xds activity as 50 mM Tris/HCl pH 7, 100 mM NaCl, 10 mM MgCl 2 , and 20 mM CaCl 2 . Moreover, maximal activity was observed using substrate DNA with low GC content and ambient temperatures of 20-25°C. In silico analysis and homology modeling predicted an exonuclease domain in the C-terminal part of the protein. Biochemical analyses with truncated variants and point mutants of Xds confirm that the C-terminal region is sufficient for nuclease activity. We also find that residues D787 and H837 within the predicted exonuclease domain are key to formation of the catalytic center.

The lifecycle of the causative agent of the severe secretory diarrheal disease cholera, Vibrio cholerae , is characterized by the transition between two dissimilar habitats, i.e., as a natural inhabitant of aquatic ecosystems and as a pathogen in the human gastrointestinal tract. Vibrio cholerae faces diverse stressors along its lifecycle, which require effective adaptation mechanisms to facilitate the survival fitness. Not surprisingly, the pathogen's transcriptome undergoes global changes during the different stages of the lifecycle. Moreover, recent evidence indicates that several of the transcription factors (i.e., ToxR, TcpP, and ToxT) and alternative sigma factors (i.e., FliA, RpoS, and RpoE) involved in transcriptional regulations along the lifecycle are controlled by regulated proteolysis. This post-translational control ensures a fast strategy by the pathogen to control cellular checkpoints and thereby rapidly respond to changing conditions. In this review, we discuss selected targets for regulated proteolysis activated by various stressors, which represent a key feature for fast adaptation of V. cholerae .

In Vibrio cholerae, virulence gene expression is regulated by a transmembrane-localized transcription factor complex designated as ToxRS. ToxR harbours two cysteines in the periplasmic domain that can form inter- and intramolecular disulfide bonds. In this study, we investigated the σ E -dependent inner membrane proteolysis of ToxR, which occurs via the periplasmic-localized proteases DegS and DegP. Both proteases respond to the redox state of the two cysteine thiol groups of ToxR. Interestingly, in the presence of sodium deoxycholate, ToxR proteolysis is blocked independently of ToxS, whereas ToxR activation by bile salts requires ToxS function. From these data, we identified at least two levels of control for ToxR activation by sodiumdeoxycholate. First, bile inhibits ToxR degradation under starvation and alkaline pH or under conditions in which DegPS responds to the reduced disulfide bonds of ToxR. The second level links bile to ToxRS complex formation and further activation of its transcription factor activity. Overall, our data suggest a comprehensive bile sensory function for the ToxRS complex during host colonization., (© 2018 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.)

The facultative human pathogen Vibrio cholerae changes its transcriptional profile upon oral ingestion by the host to facilitate survival and colonization fitness. Here, we used a modified version of recombination-based in vivo expression technology to investigate gene silencing during the in vivo passage, which has been understudied. Using a murine model of cholera, we screened a V. cholerae transposon library composed of 10,000 randomly generated reporter fusions and identified 101 in vivo repressed ( ivr ) genes. Our data indicate that constitutive expression of ivr genes reduces colonization fitness, highlighting the necessity to down-regulate these genes in vivo. For example, the ivr gene clcA , encoding an H + /Cl - transporter, could be linked to the acid tolerance response against hydrochloric acid. In a chloride-dependent manner, ClcA facilitates survival under low pH (e.g., the stomach), but its presence becomes detrimental under alkaline conditions (e.g., lower gastrointestinal tract). This pH-dependent clcA expression is controlled by the LysR-type activator AphB, which acts in concert with AphA to initiate the virulence cascade in V. cholerae after oral ingestion. Thus, transcriptional networks dictating induction of virulence factors and the repression of ivr genes overlap to regulate in vivo colonization dynamics. Overall, the results presented herein highlight the impact of spatiotemporal gene silencing in vivo. The molecular characterization of the underlying mechanisms can provide important insights into in vivo physiology and virulence network regulation., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Vibrio cholerae can colonize the gastrointestinal track of humans and cause the disease cholera. During colonization, the alternative sigma factor, RpoS, controls a process known as "mucosal escape response," defining a specific spatial and temporal response and effecting chemotaxis and motility. In this report, the expression and proteolytic control of RpoS in V. cholerae was characterized. To date, aspects of proteolysis control, the involved components, and proteolysis regulation have not been addressed for RpoS in V. cholerae. Similar to Escherichia coli, we find that the RpoS protein is subjected to regulated intracellular proteolysis, which is mediated by homologues of the proteolysis-targeting factor RssB and the protease complex ClpXP. As demonstrated, RpoS expression transiently peaks after cells are shifted from rich to minimal growth medium. This peak level is dependent on (p)ppGpp-activated rpoS transcription and controlled RpoS proteolysis. The RpoS peak level also correlates with induction of a chemotaxis gene, encoding a methyl-accepting chemotaxis protein, earlier identified to belong to the mucosal escape response pathway. These results suggest that the RpoS expression peak is linked to (p)ppGpp alarmone increase, leading to enhanced motility and chemotaxis, and possibly contributing to the mucosal escape response., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Haemophilus influenzae harbours a complex array of factors to resist human complement attack. As non-typeable H. influenzae (NTHi) strains do not possess a capsule, their serum resistance mainly depends on other mechanisms including LOS decoration. In this report, we describe the identification of a highly serum resistant, nasopharyngeal isolate (NTHi23) by screening a collection of 77 clinical isolates. For NTHi23, we defined the MLST sequence type 1133, which matches the profile of a previously published invasive NTHi isolate. A detailed genetic analysis revealed that NTHi23 shares several complement evading mechanisms with invasive disease isolates. These mechanisms include the functional expression of a retrograde phospholipid trafficking system and the presumable decoration of the LOS structure with sialic acid. By screening the NTHi23 population for spontaneous decreased serum resistance, we identified a clone, which was about 10 3 -fold more sensitive to complement-mediated killing. Genome-wide analysis of this isolate revealed a phase variation in the N'-terminal region of lpsA, leading to a truncated version of the glycosyltransferase (LpsA). We further showed that a NTHi23 lpsA mutant exhibits a decreased invasion rate into human alveolar basal epithelial cells. Since only a small proportion of the NTHi23 population expressed the serum sensitive phenotype, resulting from lpsA phase-off, we conclude that the nasopharyngeal environment selected for a population expressing the intact and functional glycosyltransferase., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

The facultative human pathogen Vibrio cholerae has to adapt to different environmental conditions along its lifecycle by means of transcriptional, translational and post-translational regulation. This study provides a first comprehensive analysis regarding the contribution of the cytoplasmic AAA+ proteases Lon, ClpP and HslV to distinct features of V. cholerae behaviour, including biofilm formation, motility, cholera toxin expression and colonization fitness in the mouse model. While absence of HslV did not yield to any altered phenotype compared to wildtype, absence of Lon or ClpP resulted in significantly reduced colonization in vivo. In addition, a Δlon deletion mutant showed altered biofilm formation and increased motility, which could be correlated with higher expression of V. cholerae flagella gene class IV. Concordantly, we could show by immunoblot analysis, that Lon is the main protease responsible for proteolytic control of FliA, which is required for class IV flagella gene transcription, but also downregulates virulence gene expression. FliA becomes highly sensitive to proteolytic degradation in absence of its anti-sigma factor FlgM, a scenario reported to occur during mucosal penetration due to FlgM secretion through the broken flagellum. Our results confirm that the high stability of FliA in the absence of Lon results in less cholera toxin and toxin corgulated pilus production under virulence gene inducing conditions and in the presence of a damaged flagellum. Thus, the data presented herein provide a molecular explanation on how V. cholerae can achieve full expression of virulence genes during early stages of colonization, despite FliA getting liberated from the anti-sigma factor FlgM., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Recent research has focused on the biological role of outer membrane vesicles (OMVs), which are derived from the outer membranes (OMs) of Gram-negative bacteria, and their potential exploitation as therapeutics. OMVs have been characterized in many ways and functions. Until recently, research focused on hypothetical and empirical models that addressed the molecular mechanisms of OMV biogenesis, such as vesicles bulging from the OM in various ways. The recently reported study by Elhenawy et al. (mBio 7:e00940-16, 2016, http://dx.doi.org/10.1128/mBio.00940-16) provided further insights into OMV biogenesis of Salmonella enterica serovar Typhimurium. That study showed that deacylation of lipopolysaccharides (LPS) influences the level of OMV production and, furthermore, determines a sorting of high versus low acylated LPS in OMs and OMVs, respectively. Interestingly, deacylation may inversely correlate with other LPS modifications, suggesting some synergy toward optimized host resistance via best OM compositions for S Typhimurium., (Copyright © 2016 Reidl.)

As it became evident recently, extracellular DNA could be a versatile nutrient source of the facultative pathogen Vibrio cholerae along the different stages of its life cycle. By the use of two extracellular nucleases and periplasmic phosphatases, V. cholerae degrades extracellular DNA to nucleosides. In this study, we investigated the nucleoside uptake via identification and characterization of VCA0179, VC1953 and VC2352 representing the three nucleoside transport systems in V. cholerae. Based on our results VC2352 seems to be the dominant nucleoside transporter. Nevertheless, all three transporters are functional and can contribute to the utilization of nucleosides as a sole source of carbon or nitrogen. We found that the transcriptional activity of these three distal genes is equally promoted or antagonized by CRP or CytR respectively. Finally, mutants impaired for nucleoside uptake exhibit decreased transition fitness from the host into low carbon environments along the life cycle of V. cholerae., (© 2015 John Wiley & Sons Ltd.)

Bacterial outer membrane vesicles (OMVs) have important biological roles in pathogenesis and intercellular interactions, but a general mechanism of OMV formation is lacking. Here we show that the VacJ/Yrb ABC (ATP-binding cassette) transport system, a proposed phospholipid transporter, is involved in OMV formation. Deletion or repression of VacJ/Yrb increases OMV production in two distantly related Gram-negative bacteria, Haemophilus influenzae and Vibrio cholerae. Lipidome analyses demonstrate that OMVs from VacJ/Yrb-defective mutants in H. influenzae are enriched in phospholipids and certain fatty acids. Furthermore, we demonstrate that OMV production and regulation of the VacJ/Yrb ABC transport system respond to iron starvation. Our results suggest a new general mechanism of OMV biogenesis based on phospholipid accumulation in the outer leaflet of the outer membrane. This mechanism is highly conserved among Gram-negative bacteria, provides a means for regulation, can account for OMV formation under all growth conditions, and might have important pathophysiological roles in vivo.

Haemophilus influenzae is a Gram-negative pathogen colonizing the upper respiratory tract mucosa. H. influenzae is one of several human-restricted bacteria, which bind to carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) on the epithelium leading to bacterial uptake by the eukaryotic cells. Adhesion to CEACAMs is thought to be mediated by the H. influenzae outer membrane protein (OMP) P5. However, CEACAMs still bound to H. influenzae lacking OMP P5 expression, and soluble CEACAM receptor ectodomains failed to bind to OMP P5, when heterologously expressed in Escherichia coli. Screening of a panel of H. influenzae OMP mutants revealed that lack of OMP P1 completely abrogated CEACAM binding and supressed CEACAM-mediated engulfment of H. influenzae by epithelial cells. Moreover, ectopic expression of OMP P1 in E. coli was sufficient to induce CEACAM binding and to promote attachment to and internalization into CEACAM-expressing cells. Interestingly, OMP P1 selectively recognizes human CEACAMs, but not homologs from other mammals and this binding preference is preserved upon expression in E. coli. Together, our data identify OMP P1 as the bona fide CEACAM-binding invasin of H. influenzae. This is the first report providing evidence for an involvement of the major OMP P1 of H. influenzae in pathogenesis., (© 2015 John Wiley & Sons Ltd.)

Enteric infections induced by pathogens like Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) remain a massive burden in developing countries with increasing morbidity and mortality rates. Previously, we showed that the immunization with genetically detoxified outer membrane vesicles (OMVs) derived from V. cholerae elicits a protective immune response based on the generation of O antigen antibodies, which effectively block the motility by binding to the sheathed flagellum. In this study, we investigated the potential of lipopolysaccharide (LPS)-modified and toxin negative OMVs isolated from V. cholerae and ETEC as a combined OMV vaccine candidate. Our results indicate that the immunization with V. cholerae or ETEC OMVs induced a species-specific immune response, whereas the combination of both OMV species resulted in a high-titer, protective immune response against both pathogens. Interestingly, the immunization with V. cholerae OMVs alone resulted in a so far uncharacterized and cholera toxin B-subunit (CTB) independent protection mechanism against an ETEC colonization. Furthermore, we investigated the potential use of V. cholerae OMVs as delivery vehicles for the heterologously expression of the ETEC surface antigens, CFA/I, and FliC. Although we induced a detectable immune response against both heterologously expressed antigens, none of these approaches resulted in an improved protection compared to a simple combination of V. cholerae and ETEC OMVs. Finally, we expanded the current protection model from V. cholerae to ETEC by demonstrating that the inhibition of motility via anti-FliC antibodies represents a relevant protection mechanism of an OMV-based ETEC vaccine candidate in vivo.

Antibiotic therapy in patients currently treated with corticosteroids is common in chronic respiratory diseases when exacerbation symptoms attributable to infection appear. Among them, obstructive diseases such as asthma and chronic obstructive pulmonary disease (COPD) are major health issues affecting hundreds of million people worldwide that are frequently treated with inhaled corticosteroids. Systemic corticosteroids are also used for idiopathic pulmonary fibrosis, a less prevalent chronic respiratory disease. In this issue of EMBO Molecular Medicine, Earl et al (2015) report a potentially baleful relationship between steroid and antibiotic treatment in chronic respiratory diseases, affecting colonization persistence and antibiotic tolerance for Haemophilus influenzae, one of the leading potentially pathogenic microorganisms (PPMs) of the respiratory system.

Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released from the outer membrane (OM) of Gram-negative bacteria. They have been proposed to possess several biological roles in pathogenesis and interbacterial interactions. Additionally, OMVs have been suggested as potential vaccine candidates against infections caused by pathogenic bacteria like Haemophilus influenzae, a human pathogen of the respiratory tract. Unfortunately, there is still a lack of fundamental knowledge regarding OMV biogenesis, protein sorting into OMVs, OMV size and quantity, as well as OMV composition in H. influenzae. Thus, this study comprehensively characterized and compared OMVs and OMs derived from heterologous encapsulated as well as nonencapsulated H. influenzae strains. Semiquantitative immunoblot analysis revealed that certain OM proteins are enriched or excluded in OMVs suggesting the presence of regulated protein sorting mechanisms into OMVs as well as interconnected OMV biogenesis mechanisms in H. influenzae. Nanoparticle tracking analysis, transmission electron microscopy, as well as protein and lipooligosaccharide quantifications demonstrated that heterologous H. influenzae strains differ in their OMV size and quantity. Lipidomic analyses identified palmitic acid as the most abundant fatty acid, while phosphatidylethanolamine was found to be the most dominant phospholipid present in OMVs and the OM of all strains tested. Proteomic analysis confirmed that H. influenzae OMVs contain vaccine candidate proteins as well as important virulence factors. These findings contribute to the understanding of OMV biogenesis as well as biological roles of OMVs and, in addition, may be important for the future development of OMV based vaccines against H. influenzae infections., (Copyright © 2015. Published by Elsevier GmbH.)

Macrolide antibiotics, such as azithromycin and erythromycin, are in widespread use for the treatment of bacterial infections. Macrolides are taken up and excreted mainly by bile. Additionally, they have been implicated in biliary system diseases and to modify the excretion of other drugs through bile. Despite mounting evidence for the interplay between macrolide antibiotics and bile acids, the molecular details of this interaction remain unknown. Herein, we show by NMR measurements that macrolides directly bind to bile acid micelles. The topology of this interaction has been determined by solvent paramagnetic relaxation enhancements (solvent PREs). The macrolides were found to be bound close to the surface of the micelle. Increasing hydrophobicity of both the macrolide and the bile acid strengthen this interaction. Both bile acid and macrolide molecules show similar solvent PREs across their whole structures, indicating that there are no preferred orientations of them in the bile micelle aggregates. The binding to bile aggregates does not impede macrolide antibiotics from targeting bacteria. In fact, the toxicity of azithromycin towards enterotoxic E. coli (ETEC) is even slightly increased in the presence of bile, as was shown by effective concentration (EC50 ) values., (© 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.)

Vibrio cholerae and enterotoxic Escherichia coli (ETEC) remain two dominant bacterial causes of severe secretory diarrhea and still a significant cause of death, especially in developing countries. In order to investigate new effective and inexpensive therapeutic approaches, we analyzed nanoparticles synthesized by a green approach using corresponding salt (silver or zinc nitrate) with aqueous extract of Caltropis procera fruit or leaves. We characterized the quantity and quality of nanoparticles by UV-visible wavelength scans and nanoparticle tracking analysis. Nanoparticles could be synthesized in reproducible yields of approximately 10(8) particles/ml with mode particles sizes of approx. 90-100 nm. Antibacterial activity against two pathogens was assessed by minimal inhibitory concentration assays and survival curves. Both pathogens exhibited similar resistance profiles with minimal inhibitory concentrations ranging between 5×10(5) and 10(7) particles/ml. Interestingly, zinc nanoparticles showed a slightly higher efficacy, but sublethal concentrations caused adverse effects and resulted in increased biofilm formation of V. cholerae. Using the expression levels of the outer membrane porin OmpT as an indicator for cAMP levels, our results suggest that zinc nanoparticles inhibit adenylyl cyclase activity. This consequently deceases the levels of this second messenger, which is a known inhibitor of biofilm formation. Finally, we demonstrated that a single oral administration of silver nanoparticles to infant mice colonized with V. cholerae or ETEC significantly reduces the colonization rates of the pathogens by 75- or 100-fold, respectively., (Copyright © 2014 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The facultative human pathogen Vibrio cholerae, the causative agent of the severe secretory diarrheal disease cholera, persists in its aquatic reservoirs in biofilms during interepidemic periods. Biofilm is a likely form in which clinically relevant V. cholerae is taken up by humans, providing an infective dose. Thus, a better understanding of biofilm formation of V. cholerae is relevant for the ecology and epidemiology of cholera as well as a target to control the disease. Most previous studies have investigated static biofilms of V. cholerae and elucidated structural prerequisites like flagella, pili and a biofilm matrix including extracellular DNA, numerous matrix proteins and exopolysaccharide, as well as the involvement of regulatory pathways like two-component systems, quorum sensing and c-di-GMP signaling. However, aquatic environments are more likely to reflect an open, dynamic system. Hence, we used a biofilm system with constant medium flow and a temporal controlled reporter-system of transcription to identify genes induced during dynamic biofilm formation. We identified genes known or predicted to be involved in c-di-GMP signaling, motility and chemotaxis, metabolism, and transport. Subsequent phenotypic characterization of mutants with independent mutations in candidate dynamic biofilm-induced genes revealed novel insights into the physiology of static and dynamic biofilm conditions. The results of this study also reinforce the hypotheses that distinct differences in regulatory mechanisms governing biofilm development are present under dynamic conditions compared to static conditions., (Copyright © 2014 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Haemophilus influenzae is a Gram-negative bacillus and a frequent commensal of the human nasopharynx. Earlier work demonstrated that in H. influenzae type b, l-lactate metabolism is associated with serum resistance and in vivo survival of the organism. To further gain insight into lactate utilization of the non-typeable (NTHi) isolate 2019 and laboratory prototype strain Rd KW20, deletion mutants of the l-lactate dehydrogenase (lctD) and permease (lctP) were generated and characterized. It is shown, that the apparent KM of l-lactate uptake is 20.1μM as determined for strain Rd KW20. Comparison of the COPD isolate NTHi 2019-R with the corresponding lctP knockout strain for survival in human serum revealed no lactate dependent serum resistance. In contrast, we observed a 4-fold attenuation of the mutant strain in a murine model of nasopharyngeal colonization. Characterization of lctP transcriptional control shows that the lactate utilization system in H. influenzae is not an inductor inducible system. Rather negative feedback regulation was observed in the presence of l-lactate and this is dependent on the ArcAB regulatory system. Additionally, for 2019 it was found that lactate may have signaling function leading to increased cell growth in late log phase under conditions where no l-lactate is metabolized. This effect seems to be ArcA independent and was not observed in strain Rd KW20. We conclude that l-lactate is an important carbon-source and may act as host specific signal substrate which fine tunes the globally acting ArcAB regulon and may additionally affect a yet unknown signaling system and thus may contribute to enhanced in vivo survival., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Pasteurella multocida is able to cause disease in humans and in a wide range of animal hosts, including fowl cholera in birds, atrophic rhinitis in pigs, and snuffles in rabbits. Together with Mannheimia haemolytica, P. multocida also represents a major bacterial causative agent of bovine respiratory disease (BRD), which is one of the most important causes for economic losses for the cattle backgrounding and feedlot industry. Commercially available vaccines only partially prevent infections caused by P. multocida and M. haemolytica. Thus, this study characterized the immunogenicity of P. multocida and M. haemolytica outer membrane vesicles (OMVs) upon intranasal immunization of BALB/c mice. Enzyme-linked immunosorbent assays (ELISA) revealed that OMVs derived from P. multocida or M. haemolytica are able to induce robust humoral and mucosal immune responses against the respective donor strain. In addition, also significant cross-immunogenic potential was observed for both OMV types. Colonization studies showed that a potential protective immune response against P. multocida is not only achieved by immunization with P. multocida OMVs, but also by immunization with OMVs derived from M. haemolytica. Immunoblot and immunoprecipitation analyses demonstrated that M. haemolytica OMVs induce a more complex immune response compared to P. multocida OMVs. The outer membrane proteins OmpA, OmpH, and P6 were identified as the three major immunogenic proteins of P. multocida OMVs. Amongst others, the serotype 1-specific antigen, an uncharacterized outer membrane protein, as well as the outer membrane proteins P2 and OmpA were found to be the most important antigens of M. haemolytica OMVs. These findings are useful for the future development of broad-spectrum OMV based vaccines against BRD and other infections caused by P. multocida or M. haemolytica., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

The causative agent of the life-threatening gastrointestinal infectious disease cholera is the Gram-negative, facultative human pathogen Vibrio cholerae. We recently started to investigate the potential of outer membrane vesicles (OMVs) derived from V. cholerae as an alternative approach for a vaccine candidate against cholera and successfully demonstrated the induction of a long-lasting, high-titer, protective immune response upon immunization with OMVs using the mouse model. In this study, we present immunization data using lipopolysaccharide (LPS)-modified OMVs derived from V. cholerae, which allowed us to improve and identify the major protective antigen of the vaccine candidate. Our results indicate that reduction of endotoxicity can be achieved without diminishing the immunogenic potential of the vaccine candidate by genetic modification of lipid A. Although the protective potential of anti-LPS antibodies has been suggested many times, this is the first comprehensive study that uses defined LPS mutants to characterize the LPS-directed immune response of a cholera vaccine candidate in more detail. Our results pinpoint the O antigen to be the essential immunogenic structure and provide a protective mechanism based on inhibition of motility, which prevents a successful colonization. In a detailed analysis using defined antisera, we can demonstrate that only anti-O antigen antibodies, but not antibodies directed against the major flagellar subunit FlaA or the most abundant outer membrane protein, OmpU, are capable of effectively blocking the motility by binding to the sheathed flagellum and provide protection in a passive immunization assay.

The facultative human pathogen Vibrio cholerae transits between the gastrointestinal tract of its host and aquatic reservoirs. V. cholerae adapts to different situations by the timely coordinated expression of genes during its life cycle. We recently identified a subclass of genes that are induced at late stages of infection. Initial characterization demonstrated that some of these genes facilitate the transition of V. cholerae from host to environmental conditions. Among these genes are uptake systems lacking detailed characterization or correct annotation. In this study, we comprehensively investigated the function of the VCA0682-to-VCA0687 gene cluster, which was previously identified as in vivo induced. The results presented here demonstrate that the operon encompassing open reading frames VCA0685 to VCA0687 encodes an ABC transport system for hexose-6-phosphates with Km values ranging from 0.275 to 1.273 μM for glucose-6P and fructose-6P, respectively. Expression of the operon is induced by the presence of hexose-6P controlled by the transcriptional activator VCA0682, representing a UhpA homolog. Finally, we provide evidence that the operon is essential for the utilization of hexose-6P as a C and P source. Thereby, a physiological role can be assigned to hexose-6P uptake, which correlates with increased fitness of V. cholerae after a transition from the host into phosphate-limiting environments.

The Gram negative bacterium Vibrio cholerae is the causative agent of the secretory diarrheal disease cholera, which has traditionally been classified as a noninflammatory disease. However, several recent reports suggest that a V. cholerae infection induces an inflammatory response in the gastrointestinal tract indicated by recruitment of innate immune cells and increase of inflammatory cytokines. In this study, we describe a colonization defect of a double extracellular nuclease V. cholerae mutant in immunocompetent mice, which is not evident in neutropenic mice. Intrigued by this observation, we investigated the impact of neutrophils, as a central part of the innate immune system, on the pathogen V. cholerae in more detail. Our results demonstrate that V. cholerae induces formation of neutrophil extracellular traps (NETs) upon contact with neutrophils, while V. cholerae in return induces the two extracellular nucleases upon presence of NETs. We show that the V. cholerae wild type rapidly degrades the DNA component of the NETs by the combined activity of the two extracellular nucleases Dns and Xds. In contrast, NETs exhibit prolonged stability in presence of the double nuclease mutant. Finally, we demonstrate that Dns and Xds mediate evasion of V. cholerae from NETs and lower the susceptibility for extracellular killing in the presence of NETs. This report provides a first comprehensive characterization of the interplay between neutrophils and V. cholerae along with new evidence that the innate immune response impacts the colonization of V. cholerae in vivo. A limitation of this study is an inability for technical and physiological reasons to visualize intact NETs in the intestinal lumen of infected mice, but we can hypothesize that extracellular nuclease production by V. cholerae may enhance survival fitness of the pathogen through NET degradation.

Haemophilus influenzae is a Gram-negative human-restricted bacterium that can act as a commensal and a pathogen of the respiratory tract. Especially nontypeable H. influenzae (NTHi) is a major threat to public health and is responsible for several infectious diseases in humans, such as pneumonia, sinusitis, and otitis media. Additionally, NTHi strains are highly associated with exacerbations in patients suffering from chronic obstructive pulmonary disease. Currently, there is no licensed vaccine against NTHi commercially available. Thus, this study investigated the utilization of outer membrane vesicles (OMVs) as a potential vaccine candidate against NTHi infections. We analyzed the immunogenic and protective properties of OMVs derived from various NTHi strains by means of nasopharyngeal immunization and colonization studies with BALB/c mice. The results presented herein demonstrate that an intranasal immunization with NTHi OMVs results in a robust and complex humoral and mucosal immune response. Immunoprecipitation revealed the most important immunogenic proteins, such as the heme utilization protein, protective surface antigen D15, heme binding protein A, and the outer membrane proteins P1, P2, P5 and P6. The induced immune response conferred not only protection against colonization with a homologous NTHi strain, which served as an OMV donor for the immunization mixtures, but also against a heterologous NTHi strain, whose OMVs were not part of the immunization mixtures. These findings indicate that OMVs derived from NTHi strains have a high potential to act as a vaccine against NTHi infections.

Virulence factor production in Vibrio cholerae is complex, with ToxRS being an important part of the regulatory cascade. Additionally, ToxR is the transcriptional regulator for the genes encoding the major outer membrane porins OmpU and OmpT. ToxR is a transmembrane protein and contains two cysteine residues in the periplasmic domain. This study addresses the influence of the thiol-disulfide oxidoreductase system DsbAB, ToxR cysteine residues and ToxR/ToxS interaction on ToxR activity. The results show that porin production correlates with ToxR intrachain disulfide bond formation, which depends on DsbAB. In contrast, formation of ToxR intrachain or interchain disulfide bonds is dispensable for virulence factor production and in vivo colonization. This study further reveals that in the absence of ToxS, ToxR interchain disulfide bond formation is facilitated, whereat cysteinyl dependent homo- and oligomerization of ToxR is suppressed if ToxS is coexpressed. In summary, new insights into gene regulation by ToxR are presented, demonstrating a mechanism by which ToxR activity is linked to a DsbAB dependent intrachain disulfide bond formation.

Biofilms are a preferred mode of survival for many microorganisms including Vibrio cholerae, the causative agent of the severe secretory diarrhoeal disease cholera. The ability of the facultative human pathogen V. cholerae to form biofilms is a key factor for persistence in aquatic ecosystems and biofilms act as a source for new outbreaks. Thus, a better understanding of biofilm formation and transmission of V. cholerae is an important target to control the disease. So far the Vibrio exopolysaccharide was the only known constituent of the biofilm matrix. In this study we identify and characterize extracellular DNA as a component of the Vibrio biofilm matrix. Furthermore, we show that extracellular DNA is modulated and controlled by the two extracellular nucleases Dns and Xds. Our results indicate that extracellular DNA and the extracellular nucleases are involved in diverse processes including the development of a typical biofilm architecture, nutrient acquisition, detachment from biofilms and the colonization fitness of biofilm clumps after ingestion by the host. This study provides new insights into biofilm development and transmission of biofilm-derived V. cholerae., (© 2011 Blackwell Publishing Ltd.)

Due to its lifestyle as a commensal and occasional pathogen in the upper and lower respiratory tracts of humans, Haemophilus influenzae needs to protect itself from endogenously and exogenously generated reactive oxygen species. To better understand the oxygen radical resistance and to investigate a correlation with virulence, randomly generated paraquat-sensitive H. influenzae transposon mutants were analyzed in an infant rat model of infection. Among 25 different paraquat-sensitive mutants only one mutant harbouring a Tn-insertion within the tRNA-Ser1 gene specific for the rare serine codon UCC, was highly attenuated for intraperitoneal infectivity. Compared to the wild-type strain, the tRNA-Ser1 mutant was also more sensitive to neutrophil-mediated killing, deficient for DNA transformation but showed similar growth rates under laboratory conditions. However, by comparative analysis using an oxyR mutant strain, we could show that neutrophil-mediated killing might not be relevant for intraperitoneal infectivity. Therefore, the increased ROS sensitivity observed for tRNA-Ser1 mutant may not be directly responsible for the observed virulence deficiency in the intraperitoneal infection. We speculate that a reduced translation efficiency of several UCC containing mRNAs results in a delay of protein synthesis and consequently in the loss of cellular mechanisms which are necessary for ROS resistance and virulence., ((c) 2009 Elsevier GmbH. All rights reserved.)