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Abstract Vibrio cholerae, a facultative human pathogen and causative agent of the severe diarrheal disease cholera, transits between the human intestinal tract and aquatic reservoirs. Like other bacterial species, V. cholerae continuously releases bacterial extracellular vesicles (BEVs) from its surface, which have been recently characterised for their role during in vivo colonisation. However, between epidemic outbreaks, V. cholerae persists in the biofilm mode for extended periods in aquatic reservoirs, which enhances environmental fitness and host transition. In this study, we investigated the effect of V. cholerae BEVs on biofilm formation, a critical feature for ex vivo survival. In contrast to BEVs from planktonic cultures, our results show that physiological concentrations of BEVs from dynamic biofilm cultures facilitate V. cholerae biofilm formation, which could be linked to a proteinaceous factor. Comparative proteomic analyses of planktonic‐ and biofilm‐derived BEVs identified a previously uncharacterised outer membrane protein as an abundant component of dynamic biofilm‐derived BEVs, which was found to be responsible for the BEV‐dependent enhancement of biofilm production. Consequently, this protein was named outer membrane‐associated biofilm facilitating protein A (ObfA). Comprehensive molecular studies unravelled ObfA as a negative modulator of HapR activity. HapR is a key transcriptional regulator of the V. cholerae quorum sensing (QS) cascade acting as a potent repressor of biofilm formation and virulence. Consistently, obfA mutants not only exhibited reduced biofilm production but also reduced colonisation fitness. Surprisingly, our results demonstrate that ObfA does not affect HapR through the canonical QS system but via the Csr‐cascade altering the expression of the small regulatory RNAs CsrC and CsrD. In summary, this study elucidates a novel intraspecies BEV‐based communication in V. cholerae that influences biofilm formation and colonisation fitness via a new regulatory pathway involving HapR, Csr‐cascade and the BEV‐associated protein ObfA.

Abstract Background E. coli O83 (Colinfant Newborn) is a Gram-negative (G-) probiotic bacterium used in the clinic. When administered orally, it reduces allergic sensitisation but not allergic asthma. Intranasal administration offers a non-invasive and convenient delivery method. This route bypasses the gastrointestinal tract and provides direct access to the airways, which are the target of asthma prevention. G- bacteria such as E. coli O83 release outer membrane vesicles (OMVs) to communicate with the environment. Here we investigate whether intranasally administered E. coli O83 OMVs (EcO83-OMVs) can reduce allergic airway inflammation in mice. Methods EcO83-OMVs were isolated by ultracentrifugation and characterised their number, morphology (shape and size), composition (proteins and lipopolysaccharide; LPS), recognition by innate receptors (using transfected HEK293 cells) and immunomodulatory potential (in naïve splenocytes and bone marrow-derived dendritic cells; BMDCs). Their allergy-preventive effect was investigated in a mouse model of ovalbumin-induced allergic airway inflammation. Results EcO83-OMVs are spherical nanoparticles with a size of about 110 nm. They contain LPS and protein cargo. We identified a total of 1120 proteins, 136 of which were enriched in OMVs compared to parent bacteria. Proteins from the flagellum dominated. OMVs activated the pattern recognition receptors TLR2/4/5 as well as NOD1 and NOD2. EcO83-OMVs induced the production of pro- and anti-inflammatory cytokines in splenocytes and BMDCs. Intranasal administration of EcO83-OMVs inhibited airway hyperresponsiveness, and decreased airway eosinophilia, Th2 cytokine production and mucus secretion. Conclusions We demonstrate for the first time that intranasally administered OMVs from probiotic G- bacteria have an anti-allergic effect. Our study highlights the advantages of OMVs as a safe platform for the prophylactic treatment of allergy. Graphical Abstract Video Abstract

ABSTRACT Although the immunomodulatory potency of bacterial membrane vesicles (MVs) is widely acknowledged, their interactions with host cells and the underlying signaling pathways have not been well studied. Herein, we provide a comparative analysis of the proinflammatory cytokine profile secreted by human intestinal epithelial cells exposed to MVs derived from 32 gut bacteria. In general, outer membrane vesicles (OMVs) from Gram-negative bacteria induced a stronger proinflammatory response than MVs from Gram-positive bacteria. However, the quality and quantity of cytokine induction varied between MVs from different species, highlighting their unique immunomodulatory properties. OMVs from enterotoxigenic Escherichia coli (ETEC) were among those showing the strongest proinflammatory potency. In depth analyses revealed that the immunomodulatory activity of ETEC OMVs relies on a so far unprecedented two-step mechanism, including their internalization into host cells followed by intracellular recognition. First, OMVs are efficiently taken up by intestinal epithelial cells, which mainly depends on caveolin-mediated endocytosis as well as the presence of the outer membrane porins OmpA and OmpF on the MVs. Second, lipopolysaccharide (LPS) delivered by OMVs is intracellularly recognized by novel caspase- and RIPK2-dependent pathways. This recognition likely occurs via detection of the lipid A moiety as ETEC OMVs with underacylated LPS exhibited reduced proinflammatory potency but similar uptake dynamics compared to OMVs derived from wild-type (WT) ETEC. Intracellular recognition of ETEC OMVs in intestinal epithelial cells is pivotal for the proinflammatory response as inhibition of OMV uptake also abolished cytokine induction. The study signifies the importance of OMV internalization by host cells to exercise their immunomodulatory activities. IMPORTANCE The release of membrane vesicles from the bacterial cell surface is highly conserved among most bacterial species, including outer membrane vesicles (OMVs) from Gram-negative bacteria as well as vesicles liberated from the cytoplasmic membrane of Gram-positive bacteria. It is becoming increasingly evident that these multifactorial spheres, carrying membranous, periplasmic, and even cytosolic content, contribute to intra- and interspecies communication. In particular, gut microbiota and the host engage in a myriad of immunogenic and metabolic interactions. This study highlights the individual immunomodulatory activities of bacterial membrane vesicles from different enteric species and provides new mechanistic insights into the recognition of ETEC OMVs by human intestinal epithelial cells.

Summary: The DNA-alkylating metabolite tilimycin is a microbial genotoxin. Intestinal accumulation of tilimycin in individuals carrying til+ Klebsiella spp. causes apoptotic erosion of the epithelium and colitis. Renewal of the intestinal lining and response to injury requires the activities of stem cells located at the base of intestinal crypts. This study interrogates the consequences of tilimycin-induced DNA damage to cycling stem cells. We charted the spatial distribution and luminal quantities of til metabolites in Klebsiella-colonized mice in the context of a complex microbial community. Loss of marker gene G6pd function indicates genetic aberrations in colorectal stem cells that became stabilized in monoclonal mutant crypts. Mice colonized with tilimycin-producing Klebsiella displayed both higher frequencies of somatic mutation and more mutations per affected individual than animals carrying a non-producing mutant. Our findings imply that genotoxic til+ Klebsiella may drive somatic genetic change in the colon and increase disease susceptibility in human hosts.

Bacterial membrane vesicles (MVs) are nanosized lipid particles secreted by lysis or blebbing mechanisms from Gram-negative and -positive bacteria. It is becoming increasingly evident that MVs can promote antimicrobial resistance but also provide versatile opportunities for therapeutic exploitation. As non-living facsimiles of parent bacteria, MVs can carry multiple bioactive molecules such as proteins, lipids, nucleic acids, and metabolites, which enable them to participate in intra- and interspecific communication. Although energetically costly, the release of MVs seems beneficial for bacterial fitness, especially for pathogens. In this review, we briefly discuss the current understanding of diverse MV biogenesis routes affecting MV cargo. We comprehensively highlight the physiological functions of MVs derived from human pathogens covering in vivo adaptation, colonization fitness, and effector delivery. Emphasis is given to recent findings suggesting a vicious cycle of MV biogenesis, pathophysiological function, and antibiotic therapy. We also summarize potential therapeutical applications, such as immunotherapy, vaccination, targeted delivery, and antimicrobial potency, including their experimental validation. This comparative overview identifies common and unique strategies for MV modification used along diverse applications. Thus, the review summarizes timely aspects of MV biology in a so far unprecedented combination ranging from beneficial function for bacterial pathogen survival to future medical applications.

Notably, bacterial biofilm formation is increasingly recog-nized as a passive virulence factor facilitating many infec-tious disease processes. In this review we will focus on bacterial biofilms formed by human pathogens and high-light their relevance for diverse diseases. Along biofilm composition and regulation emphasis is laid on the inten-sively studied biofilms of Vibrio cholerae, Pseudomonas aeruginosa and Staphylococcus spp., which are commonly used as biofilm model organisms and therefore contribute to our general understanding of bacterial biofilm (patho-)physiology. Finally, therapeutical intervention strategies targeting biofilms will be discussed.

Vibrio cholerae, the etiological agent of cholera, is a facultative intestinal pathogen which can also survive in aquatic ecosystems in the form of biofilms, surface-associated microbial aggregates embedded in an extracellular matrix, which protects them from predators and hostile environmental factors. Biofilm-derived bacteria and biofilm aggregates are considered a likely source for cholera infections, underscoring the importance of V. cholerae biofilm research not just to better understand bacterial ecology, but also cholera pathogenesis in the human host. While several studies focused on factors induced during biofilm formation, genes repressed during this persistence stage have been fairly neglected. In order to complement these previous studies, we used a single cell-based transcriptional reporter system named TetR-controlled recombination-based in-biofilm expression technology (TRIBET) and identified 192 genes to be specifically repressed by V. cholerae during biofilm formation. Predicted functions of in-biofilm repressed (ibr) genes range from metabolism, regulation, surface association, transmembrane transport as well as motility and chemotaxis. Constitutive (over)-expression of these genes affected static and dynamic biofilm formation of V. cholerae at different stages. Notably, timed expression of one candidate in mature biofilms induced their rapid dispersal. Thus, genes repressed during biofilm formation are not only dispensable for this persistence stage, but their presence can interfere with ordered biofilm development. This work thus contributes new insights into gene silencing during biofilm formation of V. cholerae.

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.

The prevailing pandemic of SARS-CoV-2 highlights the desperate need of alternative vaccine-platforms, which are safe, effective, and can be modified to carry antigens of emerging pathogens. The current SARS-CoV-2 vaccines based on mRNA and adenoviral vector technology meet some of these criteria but still face limitations regarding administration route, mass production, stability, and storage. Herein, we introduce a novel SARS-CoV-2 vaccine candidate based on bacterial outer membrane vesicles (OMVs). Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) have been genetically modified to produce increased amounts of detoxified OMVs decorated with the receptor binding domain (RBD) of the SARS-CoV-2 Spike protein. Intranasal immunization with RBD-decorated OMVs induced not only a robust immune response against the bacterial outer membrane components but also detectable antibody titers against the Spike protein. Cell culture infection assays using a Spike-pseudotyped lentivirus confirmed the presence of SARS-CoV-2 neutralizing antibodies. Highest titers against the SARS-CoV-2 Spike protein and most potent neutralization activity were observed for an alternating immunization regimen using RBD-decorated OMVs from ETEC and V. cholerae in turn. These results highlight the versatile vaccine applications offered by OMVs via expression of heterologous antigens in the donor bacterium.

Cholera is still a massive global health burden because it causes large outbreaks with millions of infections and thousands of deaths every year. Several studies have contributed to the knowledge of this pathogen, although key parts are still missing.

Investigation of bacterial gene regulation upon environmental changes is still a challenging task. For example, Vibrio cholerae, a pathogen of the human gastrointestinal tract, faces diverse transient conditions in different compartments upon oral ingestion. Genetic reporter systems have been demonstrated to be extremely powerful tools to unravel gene regulation events in complex conditions, but so far focused mainly on gene induction. Herein, we describe the TetR-controlled recombination-based in vivo expression technology TRIVET, which allows detection of gene silencing events. TRIVET resembles a modified variant of the in vivo expression technology (IVET) as well as recombination-based in vivo expression technology (RIVET), which were used to identify conditional gene induction in several bacteria during host colonization. Like its predecessors, TRIVET is a single cell based reporter system, which allows the analysis of bacterial gene repression in a spatiotemporal manner via phenotypical changes in the resistance profile. Briefly, a promoterless tetR (encoding the transcriptional repressor TetR) can be integrated randomly into the bacterial genome via transposon mutagenesis or site-specific downstream of a promoter of interest via homologous recombination. Reduction of transcriptional expression of TetR results in a de-repression of the TetR-controlled resolvase TnpR, which in turn leads to excision of an antibiotic resistance cassette (also known as res-cassette) and altered resistance profile observable via streaking on ampicillin and kanamycin plates. This alteration can then be quantified as the ratio between resistant and non-resistant isolates. Furthermore, the newly introduced second reporter gene, a promoterless phoA (encoding the alkaline phosphatase PhoA) offers an additional validation step of the results via an independent colorimetric assay to measure enzyme activity. The protocol presented herein also offers an approach to identify the gene locus in case of the random screen for gene repression as well as a quantification of the conditional repression of a gene of interest. Although the current protocol is established for gene repression during host colonization, it can likely be adapted to study gene silencing under various conditions faced by a bacterium.

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. PglLVc, 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 PglLVc in V. cholerae. We demonstrate that PglLVc 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 PglLVc and other established oligosaccharyltransferases (OTases), was also included in this phenotypical characterization. Remarkably, presence or absence of PglLVc 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 ΔpglLVc and ΔrbmD 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.

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 MgCl2, and 20 mM CaCl2. 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.

ABSTRACT During its life cycle, the facultative human pathogen Vibrio cholerae, which is the causative agent of the diarrheal disease cholera, needs to adapt to a variety of different conditions, such as the human host or the aquatic environment. Importantly, cholera infections originate from the aquatic reservoir where V. cholerae persists between the outbreaks. In the aquatic environment, bacteria are constantly threatened by predatory protozoa and nematodes, but our knowledge of the response pathways and adaptation strategies of V. cholerae to such stressors is limited. Using a temporally controlled reporter system of transcription, we identified more than 100 genes of V. cholerae induced upon exposure to the nematode Caenorhabditis elegans, which emerged recently as a valuable model for environmental predation during the aquatic lifestyle of V. cholerae. Besides others, we identified and validated the genes encoding the mannose-sensitive hemagglutinin (MSHA) type IV pilus to be significantly induced upon exposure to the nematode. Subsequent analyses demonstrated that the mannose-sensitive hemagglutinin is crucial for attachment of V. cholerae in the pharynx of the worm and initiation of colonization, which results in growth retardation and developmental delay of C. elegans. Thus, the surface adhesion factor MSHA could be linked to a fitness advantage of V. cholerae upon contact with bacterium-grazing nematodes. IMPORTANCE The waterborne diarrheal disease cholera is caused by the bacterium Vibrio cholerae. The facultative human pathogen persists as a natural inhabitant in the aquatic ecosystem between outbreaks. In contrast to the human host, V. cholerae requires a different set of genes to survive in this hostile environment. For example, predatory micrograzers are commonly found in the aquatic environment and use bacteria as a nutrient source, but knowledge of the interaction between bacterivorous grazers and V. cholerae is limited. In this study, we successfully adapted a genetic reporter technology and identified more than 100 genes activated by V. cholerae upon exposure to the bacterium-grazing nematode Caenorhabditis elegans. This screen provides a first glimpse into responses and adaptational strategies of the bacterial pathogen against such natural predators. Subsequent phenotypic characterization revealed the mannose-sensitive hemagglutinin to be crucial for colonization of the worm, which causes developmental delay and growth retardation.

Bacteria release outer membrane vesicles (OMVs) that play important roles in pathogenesis and intercellular interactions. Here, Roier et al. provide evidence supporting that phospholipid accumulation in the outer leaflet of the outer membrane participates in OMV formation in Gram-negative bacteria.

Outer membrane vesicle (OMV) release by Gram-negative bacteria has been observed and studied for decades. First considered as a by-product of cell lysis, it soon became evident that OMVs are actively secreted from the outer membrane (OM) of Gram-negative bacteria. Accordingly, these small particles (~ 10-300 nm in diameter) consist mainly of OM components like phospholipids (PLs), OM proteins, and lipopolysaccharides or lipooligosaccharides. However, OMVs may also comprise periplasmic, inner membrane, or cytoplasmic components. Since the shedding of substantial amounts of OM material represents a significant energy cost to the bacterial cell, OMV production must have some vital biological functions for Gram-negative bacteria. Indeed, intense research on that topic revealed that OMVs play important roles in bacterial physiology and pathogenesis, ranging from secretion and delivery of biomolecules (for example, toxins, DNA, or quorum sensing molecules) over stress response and biofilm formation to immunomodulation and adherence to host cells. Only recently researchers have begun to elucidate the mechanistic aspects of OMV formation, but a general mechanism for the biogenesis of these vesicles is still lacking. Here we review the findings and implications of our recent study published in Nature Communications (Roier S, et al. (2016) Nat. Commun. 7:10515), where we propose a novel and highly conserved bacterial OMV biogenesis mechanism based on PL accumulation in the outer leaflet of the OM. This mechanism might not only have important pathophysiological roles in vivo, but also represents the first general mechanism of OMV formation applicable to all Gram-negative bacteria.

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.

Acinetobacter baumannii is an emerging cause of nosocomial infections. The isolation of strains resistant to multiple antibiotics is increasing at alarming rates. Although A. baumannii is considered as one of the more threatening "superbugs" for our healthcare system, little is known about the factors contributing to its pathogenesis. In this work we show that A. baumannii ATCC 17978 possesses an O-glycosylation system responsible for the glycosylation of multiple proteins. 2D-DIGE and mass spectrometry methods identified seven A. baumannii glycoproteins, of yet unknown function. The glycan structure was determined using a combination of MS and NMR techniques and consists of a branched pentasaccharide containing N-acetylgalactosamine, glucose, galactose, N-acetylglucosamine, and a derivative of glucuronic acid. A glycosylation deficient strain was generated by homologous recombination. This strain did not show any growth defects, but exhibited a severely diminished capacity to generate biofilms. Disruption of the glycosylation machinery also resulted in reduced virulence in two infection models, the amoebae Dictyostelium discoideum and the larvae of the insect Galleria mellonella, and reduced in vivo fitness in a mouse model of peritoneal sepsis. Despite A. baumannii genome plasticity, the O-glycosylation machinery appears to be present in all clinical isolates tested as well as in all of the genomes sequenced. This suggests the existence of a strong evolutionary pressure to retain this system. These results together indicate that O-glycosylation in A. baumannii is required for full virulence and therefore represents a novel target for the development of new antibiotics.

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.

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.

Cholera outbreaks are proposed to propagate in explosive cycles powered by hyperinfectious Vibrio cholerae and quenched by lytic vibriophage. However, studies to elucidate how these factors affect transmission are lacking because the field experiments are almost intractable. One reason for this is that V. cholerae loses the ability to culture upon transfer to pond water. This phenotype is called the active but non-culturable state (ABNC; an alternative term is viable but non-culturable) because these cells maintain the capacity for metabolic activity. ABNC bacteria may serve as the environmental reservoir for outbreaks but rigorous animal studies to test this hypothesis have not been conducted. In this project, we wanted to determine the relevance of ABNC cells to transmission as well as the impact lytic phage have on V. cholerae as the bacteria enter the ABNC state. Rice-water stool that naturally harbored lytic phage or in vitro derived V. cholerae were incubated in a pond microcosm, and the culturability, infectious dose, and transcriptome were assayed over 24 h. The data show that the major contributors to infection are culturable V. cholerae and not ABNC cells. Phage did not affect colonization immediately after shedding from the patients because the phage titer was too low. However, V. cholerae failed to colonize the small intestine after 24 h of incubation in pond water-the point when the phage and ABNC cell titers were highest. The transcriptional analysis traced the transformation into the non-infectious ABNC state and supports models for the adaptation to nutrient poor aquatic environments. Phage had an undetectable impact on this adaptation. Taken together, the rise of ABNC cells and lytic phage blocked transmission. Thus, there is a fitness advantage if V. cholerae can make a rapid transfer to the next host before these negative selective pressures compound in the aquatic environment.

Vitamin B12 is a critical nutrient in vegan and vegetarian lifestyles as plant-based vitamin sources are rare. Traditional fermented foods could be enriched by adding vitamin B12-producing bacteria to offer non-animal vitamin sources. The aim was to isolate a vitamin B12 producer that is capable of producing the human-active vitamin even at low pH values so that it can be used in fruit juice fortification. Therefore, fermented foods (homemade and industrial) and probiotics were screened for vitamin B12 production strains. A modified microbiological vitamin B12 assay based on Lactobacillus delbrueckii subsp. lactis DSM 20355 was used to identify vitamin B12-containing samples and the presence of vitamin B12-producing strains. The screening resulted in isolating several positive strains for vitamin B12 formation derived from sourdough. Mass spectrometry confirmed the biosynthesis of solely the human physiologically active form. Species identification carried out by the German Strain Collection of Microorganisms and Cell Cultures resulted in two species: Acetobacter orientalis and Acetobacter malorum, of which two isolates were further characterised. The potential for cobalamin biosynthesises in food matrixes was demonstrated for A. malorum HFD 3141 and A. orientalis HFD 3031 in apple juice at different pH values (2.85–3.80). The isolates synthesised up to 18.89 µg/L and 7.97 µg/L vitamin B12 at pH 3.80. The results of this study suggest that acetic acid bacteria (AAB) and fermented acetic acid foods are promising resources for vitamin B12 and its producers, which might have been overlooked in the past.

The activation of ferroptosis is being pursued in cancer research as a strategy to target apoptosis-resistant cells. By contrast, in various diseases that affect the cardiovascular system, kidneys, liver, and central and peripheral nervous systems, attention is directed toward interventions that prevent ferroptotic cell death. Mechanistic insights into both research areas stem largely from studies using cellular in vitro models. However, intervention strategies that show promise in cellular test systems often fail in clinical trials, which raises concerns regarding the predictive validity of the utilized in vitro models.In this study, the human LUHMES cell line, which serves as a model for human dopaminergic neurons, was used to characterize factors influencing the activation of ferroptosis. Erastin and RSL-3 induced cell death that was distinct from apoptosis. Parameters such as the differentiation state of LUHMES cells, cell density, and the number and timing of medium changes were identified as determinants of sensitivity to ferroptosis activation. In differentiated LUHMES cells, interventions at mechanistically divergent sites (iron chelation, coenzyme Q10, peroxidase mimics, or inhibition of 12/15-lipoxygenase) provide almost complete protection from ferroptosis. LUHMES cells allowed the experimental modulation of intracellular iron concentrations and demonstrated a correlation between intracellular iron levels, the rate of lipid peroxidation, as well as the sensitivity of the cells to ferroptotic cell death.These findings underscore the importance of understanding the various factors that influence ferroptosis activation and highlight the need for well-characterized in vitro models to enhance the reliability and predictive value of observations in ferroptosis research, particularly when translating findings into in vivo contexts.