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Virulence gene expression of Yersinia pseudotuberculosis changes during the different stages of infection and this is tightly controlled by environmental cues. In this study, we show that the small protein YmoA, a member of the Hha family, is part of this process. It controls temperature- and nutrient-dependent early and later stage virulence genes in an opposing manner and co-regulates bacterial stress responses and metabolic functions. Our analysis further revealed that YmoA exerts this function by modulating the global post-transcriptional regulatory Csr system. YmoA pre-dominantly enhances the stability of the regulatory RNA CsrC. This involves a stabilizing stem-loop structure within the 5′-region of CsrC. YmoA-mediated CsrC stabilization depends on H-NS, but not on the RNA chaperone Hfq. YmoA-promoted reprogramming of the Csr system has severe consequences for the cell: we found that a mutant deficient of ymoA is strongly reduced in its ability to enter host cells and to disseminate to the Peyer’s patches, mesenteric lymph nodes, liver and spleen in mice. We propose a model in which YmoA controls transition from the initial colonization phase in the intestine toward the host defense phase important for the long-term establishment of the infection in underlying tissues.

Many bacterial pathogens use a type III secretion system (T3SS) as molecular syringe to inject effector proteins into the host cell. In the foodborne pathogen Yersinia pseudotuberculosis, delivery of the secreted effector protein cocktail through the T3SS depends on YopN, a molecular gatekeeper that controls access to the secretion channel from the bacterial cytoplasm. Here, we show that several checkpoints adjust yopN expression to virulence conditions. A dominant cue is the host body temperature. A temperature of 37 °C is known to induce the RNA thermometer (RNAT)-dependent synthesis of LcrF, a transcription factor that activates expression of the entire T3SS regulon. Here, we uncovered a second layer of temperature control. We show that another RNAT silences translation of the yopN mRNA at low environmental temperatures. The long and short 5’-untranslated region of both cellular yopN isoforms fold into a similar secondary structure that blocks ribosome binding. The hairpin structure with an internal loop melts at 37 °C and thereby permits formation of the translation initiation complex as shown by mutational analysis, in vitro structure probing and toeprinting methods. Importantly, we demonstrate the physiological relevance of the RNAT in the faithful control of type III secretion by using a point-mutated thermostable RNAT variant with a trapped SD sequence. Abrogated YopN production in this strain led to unrestricted effector protein secretion into the medium, bacterial growth arrest and delayed translocation into eukaryotic host cells. Cumulatively, our results show that substrate delivery by the Yersinia T3SS is under hierarchical surveillance of two RNATs.Author summaryTemperature serves as reliable external cue for pathogenic bacteria to recognize the entry into or exit from a warm-blooded host. At the molecular level, a temperature of 37 °C induces various virulence-related processes that manipulate host cell physiology. Here, we demonstrate the temperature-dependent synthesis of the secretion regulator YopN in the foodborne pathogen Yersinia pseudotuberculosis, a close relative of Yersinia pestis. YopN blocks secretion of effector proteins through the type III secretion system unless host cell contact is established. Temperature-specific regulation relies on an RNA structure in the 5’-untranslated region of the yopN mRNA, referred to as RNA thermometer, which allows ribosome binding and thus translation initiation only at an infection-relevant temperature of 37 °C. A mutated variant of the thermosensor resulting in a closed conformation prevented synthesis of the molecular gatekeeper YopN and led to permanent secretion and defective translocation of virulence factors into host cells. We suggest that the RNA thermometer plays a critical role in adjusting the optimal cellular concentration of a surveillance factor that maintains the controlled translocation of virulence factors.

Cytotoxic necrotizing factors (CNFs) are bacterial single‐chain exotoxins that modulate cytokinetic/oncogenic and inflammatory processes through activation of host cell Rho GTPases. To achieve this, they are secreted, bind surface receptors to induce endocytosis and translocate a catalytic unit into the cytosol to intoxicate host cells. A three‐dimensional structure that provides insight into the underlying mechanisms is still lacking. Here, we determined the crystal structure of full‐length Yersinia pseudotuberculosis CNFY. CNFY consists of five domains (D1–D5), and by integrating structural and functional data, we demonstrate that D1–3 act as export and translocation module for the catalytic unit (D4–5) and for a fused β‐lactamase reporter protein. We further found that D4, which possesses structural similarity to ADP‐ribosyl transferases, but had no equivalent catalytic activity, changed its position to interact extensively with D5 in the crystal structure of the free D4–5 fragment. This liberates D5 from a semi‐blocked conformation in full‐length CNFY, leading to higher deamidation activity. Finally, we identify CNF translocation modules in several uncharacterized fusion proteins, which suggests their usability as a broad‐specificity protein delivery tool., Structure‐function analyses of the full‐length Yersinia pseudotuberculosis toxin CNFY offer insights into individual domain contributions to stepwise receptor binding, endocytosis, and translocation into the host cell cytosol.

The dynamic conformation of RNA molecules within living cells is key to their function. Recent advances in probing the RNA structurome in vivo, including the use of SHAPE (Selective 2′-Hydroxyl Acylation analyzed by Primer Extension) or kethoxal reagents or DMS (dimethyl sulfate), provided unprecedented insights into the architecture of RNA molecules in the living cell. Here, we report the establishment of lead probing in a global RNA structuromics approach. In order to elucidate the transcriptome-wide RNA landscape in the enteric pathogen Yersinia pseudotuberculosis, we combined lead(II) acetate-mediated cleavage of single-stranded RNA regions with high-throughput sequencing. This new approach, termed ‘Lead-seq’, provides structural information independent of base identity. We show that the method recapitulates secondary structures of tRNAs, RNase P RNA, tmRNA, 16S rRNA and the rpsT 5′-untranslated region, and that it reveals global structural features of mRNAs. The application of Lead-seq to Y. pseudotuberculosis cells grown at two different temperatures unveiled the first temperature-responsive in vivo RNA structurome of a bacterial pathogen. The translation of candidate genes derived from this approach was confirmed to be temperature regulated. Overall, this study establishes Lead-seq as complementary approach to interrogate intracellular RNA structures on a global scale.

Cytotoxic necrotizing factors (CNFs) are bacterial single-chain exotoxins that modulate cytokinetic/oncogenic and inflammatory processes through activation of host cell Rho GTPases. To achieve this, they are secreted, bind surface receptors to induce endocytosis and translocate a catalytic unit into the cytosol to intoxicate host cells. A three-dimensional structure that provides insight into the underlying mechanisms is still lacking. Here, we determined the crystal structure of full-length Yersinia pseudotuberculosis CNFY. CNFY consists of five domains (D1-D5), and by integrating structural and functional data we demonstrate that D1-3 act as export and translocation module for the catalytic unit (D4-5) or fused β-lactamase reporter proteins. We further found that domain D4, which possesses structural similarity to ADP-ribosyl transferases, but had no equivalent catalytic activity, changed its position to interact extensively with D5 in the crystal structure of the free D4-5 fragment. This liberates D5 from a semi-blocked conformation in full-length CNFY, leading to higher deamidation activity. Finally, sequence comparisons identified the CNF translocation module in many uncharacterized bacterial proteins, suggesting its usability as a universal drug delivery tool.

Oral administration is a preferred model for studying infection by bacterial enteropathogens such as Yersinia spp. In the mouse model, the most frequent method for oral infection consists of oral gavage with a feeding needle directly introduced in the animal stomach via the esophagus. In this study, we compared needle gavage to bread feeding as an alternative mode of bacterial administration. Using bioluminescence-expressing strains of Yersinia pseudotuberculosis and Yersinia enterocolitica, we detected very early upon needle gavage a bioluminescent signal in the neck area together with a signal in the abdominal region, highlighting the presence of two independent sites of bacterial colonization and multiplication., Oral administration is a preferred model for studying infection by bacterial enteropathogens such as Yersinia spp. In the mouse model, the most frequent method for oral infection consists of oral gavage with a feeding needle directly introduced in the animal stomach via the esophagus. In this study, we compared needle gavage to bread feeding as an alternative mode of bacterial administration. Using bioluminescence-expressing strains of Yersinia pseudotuberculosis and Yersinia enterocolitica, we detected very early upon needle gavage a bioluminescent signal in the neck area together with a signal in the abdominal region, highlighting the presence of two independent sites of bacterial colonization and multiplication. Bacteria were often detected in the esophagus and trachea, as well as in the lymph nodes draining the salivary glands, suggesting that lesions made during needle introduction into the animal oral cavity lead to rapid bacterial draining to proximal lymph nodes. We then tested an alternative mode of bacterial administration using pieces of bread containing bacteria. Upon bread feeding infection, mice exhibited a stronger bioluminescent signal in the abdominal region than with needle gavage, and no signal was detected in the neck area. Moreover, Y. pseudotuberculosis incorporated in the bread is less susceptible to the acidic environment of the stomach and is therefore more efficient in causing intestinal infections. Based on our observations, bread feeding constitutes a natural and more efficient administration method which does not require specialized skills, is less traumatic for the animal, and results in diseases that more closely mimic foodborne intestinal infection.

Yersinia pseudotuberculosis is a Gram-negative bacterium and zoonotic pathogen responsible for a wide range of diseases, ranging from mild diarrhea, enterocolitis, lymphatic adenitis to persistent local inflammation. The Y. pseudotuberculosis invasin D (InvD) molecule belongs to the invasin (InvA)-type autotransporter proteins, but its structure and function remain unknown. In this study, we present the first crystal structure of InvD, analyzed its expression and function in a murine infection model, and identified its target molecule in the host. We found that InvD is induced at 37 °C and expressed in vivo 2–4 days after infection, indicating that InvD is a virulence factor. During infection, InvD was expressed in all parts of the intestinal tract, but not in deeper lymphoid tissues. The crystal structure of the C-terminal adhesion domain of InvD revealed a distinct Ig-related fold that, apart from the canonical β-sheets, comprises various modifications of and insertions into the Ig-core structure. We identified the Fab fragment of host-derived IgG/IgA antibodies as the target of the adhesion domain. Phage display panning and flow cytometry data further revealed that InvD exhibits a preferential binding specificity toward antibodies with VH3/VK1 variable domains and that it is specifically recruited to a subset of B cells. This finding suggests that InvD modulates Ig functions in the intestine and affects direct interactions with a subset of cell surface-exposed B-cell receptors. In summary, our results provide extensive insights into the structure of InvD and its specific interaction with the target molecule in the host.

Many bacterial pathogens use a type III secretion system (T3SS) as molecular syringe to inject effector proteins into the host cell. In the foodborne pathogen Yersinia pseudotuberculosis, delivery of the secreted effector protein cocktail through the T3SS depends on YopN, a molecular gatekeeper that controls access to the secretion channel from the bacterial cytoplasm. Here, we show that several checkpoints adjust yopN expression to virulence conditions. A dominant cue is the host body temperature. A temperature of 37°C is known to induce the RNA thermometer (RNAT)-dependent synthesis of LcrF, a transcription factor that activates expression of the entire T3SS regulon. Here, we uncovered a second layer of temperature control. We show that another RNAT silences translation of the yopN mRNA at low environmental temperatures. The long and short 5’-untranslated region of both cellular yopN isoforms fold into a similar secondary structure that blocks ribosome binding. The hairpin structure with an internal loop melts at 37°C and thereby permits formation of the translation initiation complex as shown by mutational analysis, in vitro structure probing and toeprinting methods. Importantly, we demonstrate the physiological relevance of the RNAT in the faithful control of type III secretion by using a point-mutated thermostable RNAT variant with a trapped SD sequence. Abrogated YopN production in this strain led to unrestricted effector protein secretion into the medium, bacterial growth arrest and delayed translocation into eukaryotic host cells. Cumulatively, our results show that substrate delivery by the Yersinia T3SS is under hierarchical surveillance of two RNATs., Author summary Temperature serves as reliable external cue for pathogenic bacteria to recognize the entry into or exit from a warm-blooded host. At the molecular level, a temperature of 37°C induces various virulence-related processes that manipulate host cell physiology. Here, we demonstrate the temperature-dependent synthesis of the secretion regulator YopN in the foodborne pathogen Yersinia pseudotuberculosis, a close relative of Yersinia pestis. YopN blocks secretion of effector proteins through the type III secretion system unless host cell contact is established. Temperature-specific regulation relies on an RNA structure in the 5’-untranslated region of the yopN mRNA, referred to as RNA thermometer, which allows ribosome binding and thus translation initiation only at an infection-relevant temperature of 37°C. A mutated variant of the thermosensor resulting in a closed conformation prevented synthesis of the molecular gatekeeper YopN and led to permanent secretion and defective translocation of virulence factors into host cells. We suggest that the RNA thermometer plays a critical role in adjusting the optimal cellular concentration of a surveillance factor that maintains the controlled translocation of virulence factors.

Frequent transitions of bacterial pathogens between their warm-blooded host and external reservoirs are accompanied by abrupt temperature shifts. A temperature of 37°C serves as reliable signal for ingestion by a mammalian host, which induces a major reprogramming of bacterial gene expression and metabolism. Enteric Yersiniae are Gram-negative pathogens accountable for self-limiting gastrointestinal infections. Among the temperature-regulated virulence genes of Yersinia pseudotuberculosis is cnfY coding for the cytotoxic necrotizing factor (CNFY), a multifunctional secreted toxin that modulates the host’s innate immune system and contributes to the decision between acute infection and persistence. We report that the major determinant of temperature-regulated cnfY expression is a thermo-labile RNA structure in the 5’-untranslated region (5’-UTR). Various translational gene fusions demonstrated that this region faithfully regulates translation initiation regardless of the transcription start site, promoter or reporter strain. RNA structure probing revealed a labile stem-loop structure, in which the ribosome binding site is partially occluded at 25°C but liberated at 37°C. Consistent with translational control in bacteria, toeprinting (primer extension inhibition) experiments in vitro showed increased ribosome binding at elevated temperature. Point mutations locking the 5’-UTR in its 25°C structure impaired opening of the stem loop, ribosome access and translation initiation at 37°C. To assess the in vivo relevance of temperature control, we used a mouse infection model. Y. pseudotuberculosis strains carrying stabilized RNA thermometer variants upstream of cnfY were avirulent and attenuated in their ability to disseminate into mesenteric lymph nodes and spleen. We conclude with a model, in which the RNA thermometer acts as translational roadblock in a two-layered regulatory cascade that tightly controls provision of the CNFY toxin during acute infection. Similar RNA structures upstream of various cnfY homologs suggest that RNA thermosensors dictate the production of secreted toxins in a wide range of pathogens., Author summary Bacterial pathogens closely survey the ambient conditions and induce virulence genes only at appropriate conditions. Upon host contact, many pathogens secrete toxins in order to subvert host defense systems. We find that such a secreted toxin in enteropathogenic Yersinia pseudotuberculosis is produced only at host body temperature. This regulation depends on a temperature-responsive RNA structure, an RNA thermometer, in the 5’-untranslated region of the toxin mRNA, which prevents translation at low temperatures when the bacterium is outside the host. Preventing melting of the RNA structure at 37°C by nucleotide substitutions that stabilize base pairing resulted in avirulent Yersinia strains unable to infect mice. Given that similar RNA thermometer-like structures exist upstream of related toxin genes in various bacterial pathogens, we propose that RNA thermometer-mediated toxin production is an evolutionary conserved mechanism. Interfering with opening of such regulatory structures might thus be a promising strategy targeting a broad spectrum of bacterial pathogens.

Oral administration is a preferred model for studying infection by bacterial enteropathogens such asYersinia. In the mouse model, the most frequent method for oral infection consists of oral gavage with a feeding needle directly introduced in the animal stomach via the esophagus. In this study, we compared needle gavage to bread feeding as an alternative mode of bacterial administration. Using a bioluminescence-expressing strain ofYersinia pseudotuberculosis, we detected very early upon needle gavage a bioluminescent signal in the neck area together with a signal in the abdominal region, highlighting the presence of two independent sites of bacterial colonization and multiplication. Bacteria were often detected in the esophagus and trachea, as well as in the lymph nodes draining the salivary glands, suggesting that lesions made during needle introduction into the animal oral cavity lead to rapid bacterial draining to proximal lymph nodes. We then tested an alternative mode of bacterial administration using small pieces of white bread containing bacteria. Upon bread feeding infection, mice exhibited a stronger bioluminescent signal in the abdominal region as compared to needle gavage, and no signal was detected in the neck area. Moreover,Y. pseudotuberculosisincorporated in the bread is less susceptible to the acidic environment of the stomach and is therefore more efficient in causing intestinal infections. Based on our observations, bread feeding constitutes a natural and more efficient administration method which does not require specialized skills, is less traumatic for the animal, and results in diseases that more closely mimic food-borne intestinal infection.

Numerous Gram-negative pathogens use a Type III Secretion System (T3SS) to promote virulence by injecting effector proteins into targeted host cells, which subvert host cell processes. Expression of T3SS and the effectors is triggered upon host cell contact, but the underlying mechanism is poorly understood. Here, we report a novel strategy of Yersinia pseudotuberculosis in which this pathogen uses a secreted T3SS translocator protein (YopD) to control global RNA regulators. Secretion of the YopD translocator upon host cell contact increases the ratio of post-transcriptional regulator CsrA to its antagonistic small RNAs CsrB and CsrC and reduces the degradosome components PNPase and RNase E levels. This substantially elevates the amount of the common transcriptional activator (LcrF) of T3SS/Yop effector genes and triggers the synthesis of associated virulence-relevant traits. The observed hijacking of global riboregulators allows the pathogen to coordinate virulence factor expression and also readjusts its physiological response upon host cell contact., Author summary Many bacterial pathogens sense contact to host cells and respond by inducing expression of crucial virulence factors. This includes the type III secretion systems (T3SSs) and their substrates, which manipulate different host cell functions to promote colonization and survival of the pathogen within its host. In this study, we used enteropathogenic Yersinia pseudotuberculosis to elucidate the molecular mechanism of how cell contact is transmitted and translated to trigger this process. We found that multiple global riboregulators control the decay and/or translation of the major transcriptional activator of the T3SS. In the absence of cell contact, these important RNA regulators are coopted by one of the substrate proteins of the T3SS to repress expression of the secretion machinery. Translocation of the substrate protein upon cell contact relieves riboregulator-mediated repression. This leads to a strong induction of the master regulator of T3SS/effector gene expression promoting an increase of the virulence potential and provokes a fast adaptation of the pathogen's fitness, e.g. to compensate for the imposed energetic burden.

The twin-arginine translocation (Tat) system mediates the secretion of folded proteins that are identified via an N-terminal signal peptide in bacteria, plants, and archaea. Tat systems are associated with virulence in many bacterial pathogens, and our previous studies revealed that Tat-deficient Yersinia pseudotuberculosis was severely attenuated for virulence. Aiming to identify Tat-dependent pathways and phenotypes of relevance for in vivo infection, we analyzed the global transcriptome of parental and Δ tatC mutant strains of Y. pseudotuberculosis during exponential and stationary growth at 26°C and 37°C. The most significant changes in the transcriptome of the Δ tatC mutant were seen at 26°C during stationary-phase growth, and these included the altered expression of genes related to virulence, stress responses, and metabolism. Subsequent phenotypic analysis based on these transcriptome changes revealed several novel Tat-dependent phenotypes, including decreased YadA expression, impaired growth under iron-limited and high-copper conditions, as well as acidic pH and SDS. Several functionally related Tat substrates were also verified to contribute to these phenotypes. Interestingly, the phenotypic defects observed in the Tat-deficient strain were generally more pronounced than those in mutants lacking the Tat substrate predicted to contribute to that specific function. Altogether, this provides new insight into the impact of Tat deficiency on in vivo fitness and survival/replication of Y. pseudotuberculosis during infection. IMPORTANCE In addition to its established role in mediating the secretion of housekeeping enzymes, the Tat system has been recognized as being involved in infection. In some clinically relevant bacteria, such as Pseudomonas spp., several key virulence determinants can readily be identified among the Tat substrates. In enteropathogens, such as Yersinia spp., there are no obvious virulence determinants among the Tat substrates. Tat mutants show no growth defect in vitro but are highly attenuated in in vivo . This makes Tat an attractive target for the development of novel antimicrobials. Therefore, it is important to establish the causes of the attenuation. Here, we show that the attenuation is likely due to synergistic effects of different Tat-dependent phenotypes that each contributes to lowered in vivo fitness.

PLoS pathogens 16(9), e1008552 (1-37) - (2020). doi:10.1371/journal.ppat.1008552, Type VI secretion systems (T6SSs) are complex macromolecular injection machines which are widespread in Gram-negative bacteria. They are involved in host-cell interactions and pathogenesis, required to eliminate competing bacteria, or are important for the adaptation to environmental stress conditions. Here we identified regulatory elements controlling the T6SS4 of Yersinia pseudotuberculosis and found a novel type of hexameric transcription factor, RovC. RovC directly interacts with the T6SS4 promoter region and activates T6SS4 transcription alone or in cooperation with the LysR-type regulator RovM. A higher complexity of regulation was achieved by the nutrient-responsive global regulator CsrA, which controls rovC expression on the transcriptional and post-transcriptional level. In summary, our work unveils a central mechanism in which RovC, a novel key activator, orchestrates the expression of the T6SS weapons together with a global regulator to deploy the system in response to the availability of nutrients in the species' native environment., Published by PLoS, Lawrence, Kan.

The Gram-negative enteropathogen Yersinia pseudotuberculosis possesses a number of regulatory systems that detect cell envelope damage caused by noxious extracytoplasmic stresses. The CpxA sensor kinase and CpxR response regulator two-component regulatory system is one such pathway. Active Cpx signalling upregulates various factors designed to repair and restore cell envelope integrity. Concomitantly, this pathway also down-regulates key determinants of virulence. In Yersinia, cpxA deletion accumulates high levels of phosphorylated CpxR (CpxR~P). Accumulated CpxR~P directly repressed rovA expression and this limited expression of virulence-associated processes. A second transcriptional regulator, RovM, also negatively regulates rovA expression in response to nutrient stress. Hence, this study aimed to determine if CpxR~P can influence rovA expression through control of RovM levels. We determined that the active CpxR~P isoform bound to the promoter of rovM and directly induced its expression, which naturally associated with a concurrent reduction in rovA expression. Site-directed mutagenesis of the CpxR~P binding sequence in the rovM promoter region desensitised rovM expression to CpxR~P. These data suggest that accumulated CpxR~P inversely manipulates the levels of two global transcriptional regulators, RovA and RovM, and this would be expected to have considerable influence on Yersinia pathophysiology and metabolism.

The genus Yersinia includes three human pathogenic species, Yersinia pestis, the causative agent of the bubonic and pneumonic plague, and enteric pathogens Y. enterocolitica and Y. pseudotuberculosis that cause a number of gut-associated diseases. Over the past years a large repertoire of RNA-based regulatory systems has been discovered in these pathogens using different RNA-seq based approaches. Among them are several conserved or species-specific RNA-binding proteins, regulatory and sensory RNAs as well as various RNA-degrading enzymes. Many of them were shown to control the expression of important virulence-relevant factors and have a very strong impact on Yersinia virulence. The precise targets, the molecular mechanism and their role for Yersinia pathogenicity is only known for a small subset of identified genus- or species-specific RNA-based control elements. However, the ongoing development of new RNA-seq based methods and data analysis methods to investigate the synthesis, composition, translation, decay, and modification of RNAs in the bacterial cell will help us to generate a more comprehensive view of Yersinia RNA biology in the near future.

Gastrointestinal infections caused by enteric yersiniae can become persistent and complicated by relapsing enteritis and severe autoimmune disorders. To establish a persistent infection, the bacteria have to cope with hostile surroundings when they transmigrate through the intestinal epithelium and colonize underlying gut-associated lymphatic tissues. How the bacteria gain a foothold in the face of host immune responses is poorly understood. Here, we show that the CNFY toxin, which enhances translocation of the antiphagocytic Yop effectors, induces inflammatory responses. This results in extensive tissue destruction, alteration of the intestinal microbiota and bacterial clearance. Suppression of CNFY function, however, increases interferon-γ-mediated responses, comprising non-inflammatory antimicrobial activities and tolerogenesis. This process is accompanied by a preterm reprogramming of the pathogen's transcriptional response towards persistence, which gives the bacteria a fitness edge against host responses and facilitates establishment of a commensal-type life style., Author summary A complex cascade of events is triggered by the mammalian host upon a bacterial infection to prevent pathogen-induced damage. Many pathogens are successfully eliminated by the host immune system. However, some bacterial pathogens, including enteric yersiniae, evolved strategies to efficiently evade immune surveillance. This enables the pathogen to colonize specific sites in the host in which they can persist for years. These persistent infections are often asymptomatic for long time periods, but can undergo reactivation and cause clinical symptoms. In order to understand how enteric bacteria can persist in gut-associated host niches, we investigated changes of bacterial and host responses from an acute to a persistent infection. We found that persistency of Y. pseudotuberculosis in the cecum is characterized by suppression of pathogen-induced inflammation and tissue destruction, and an induction of interferon-γ promoted tolerogenic responses. We further show that the removal of a single bacterial virulence factor, the CNFY toxin, can trigger this process and drives the bacteria into persistency.

Background To successfully limit pathogen dissemination, an immunological link between the entry tissue of the pathogen and the underlying secondary lymphoid organs (SLOs) needs to be established to prime adaptive immune responses. Here, the prerequisite of CCR7 to mount host immune responses within SLOs during gastrointestinal Yersinia pseudotuberculosis infection to limit pathogen spread was investigated. Methods Survival, bacterial dissemination, and intestinal and systemic pathology of wild-type and CCR7-/- mice were assessed and correlated to the presence of immune cell subsets and cytokine responses throughout the course of infection. Results The CCR7-/- mice show a significantly higher morbidity and are more prone to pathogen dissemination and intestinal and systemic inflammation during the oral route of infection. Significant impact of CCR7 deficiency over the course of infection on several immunological parameters were observed (ie, elevated neutrophil-dominated innate immune response in Peyer's patches, limited dendritic cell migration to mesenteric lymph nodes [mLNs] causing reduced T cell-mediated adaptive immune responses (in particular Th17-like responses) in mLNs). Conclusions Our work indicates that CCR7 is required to mount a robust immune response against enteropathogenic Y. pseudotuberculosis by promoting Th17-like responses in mLNs.

Adaptive immunity critically contributes to control acute infection with enteropathogenic Yersinia pseudotuberculosis; however, the role of CD4+ T cell subsets in establishing infection and allowing pathogen persistence remains elusive. Here, we assessed the modulatory capacity of Y. pseudotuberculosis on CD4+ T cell differentiation. Using in vivo assays, we report that infection with Y. pseudotuberculosis resulted in enhanced priming of IL-17-producing T cells (Th17 cells), whereas induction of Foxp3+ regulatory T cells (Tregs) was severely disrupted in gut-draining mesenteric lymph nodes (mLNs), in line with altered frequencies of tolerogenic and proinflammatory dendritic cell (DC) subsets within mLNs. Additionally, by using a DC-free in vitro system, we could demonstrate that Y. pseudotuberculosis can directly modulate T cell receptor (TCR) downstream signaling within naïve CD4+ T cells and Tregs via injection of effector molecules through the type III secretion system, thereby affecting their functional properties. Importantly, modulation of naïve CD4+ T cells by Y. pseudotuberculosis resulted in an enhanced Th17 differentiation and decreased induction of Foxp3+ Tregs in vitro. These findings shed light to the adjustment of the Th17-Treg axis in response to acute Y. pseudotuberculosis infection and highlight the direct modulation of CD4+ T cell subsets by altering their TCR downstream signaling. Electronic supplementary material The online version of this article (doi:10.1007/s00018-017-2516-y) contains supplementary material, which is available to authorized users.

Despite our increasing knowledge of the specific pathogenicity factors in bacteria, the contribution of metabolic processes to virulence is largely unknown. Here, we elucidate a tight connection between pathogenicity and core metabolism in the enteric pathogen Yersinia pseudotuberculosis by integrated transcriptome and [13C]fluxome analysis of the wild type and virulence-regulator mutants. During aerobic growth on glucose, Y. pseudotuberculosis reveals an unusual flux distribution with a high level of secreted pyruvate. The absence of the transcriptional and post-transcriptional regulators RovA, CsrA, and Crp strongly perturbs the fluxes of carbon core metabolism at the level of pyruvate metabolism and the tricarboxylic acid (TCA) cycle, and these perturbations are accompanied by transcriptional changes in the corresponding enzymes. Knock-outs of regulators of this metabolic branch point and of its central enzyme, pyruvate kinase (ΔpykF), result in mutants with significantly reduced virulence in an oral mouse infection model. In summary, our work identifies the pyruvate-TCA cycle node as a focal point for controlling the host colonization and virulence of Yersinia.

RNA structures are fundamentally important for RNA function. Dynamic, condition-dependent structural changes are able to modulate gene expression as shown for riboswitches and RNA thermometers. By parallel analysis of RNA structures, we mapped the RNA structurome of Yersinia pseudotuberculosis at three different temperatures. This human pathogen is exquisitely responsive to host body temperature (37 ° C), which induces a major metabolic transition. Our analysis profiles the structure of more than 1,750 RNAs at 25 ° C, 37 ° C, and 42 ° C. Average mRNAs tend to be unstructured around the ribosome binding site. We searched for 5?-UTRs that are folded at low temperature and identified novel thermoresponsive RNA structures from diverse gene categories. The regulatory potential of 16 candidates was validated. In summary, we present a dynamic bacterial RNA structurome and find that the expression of virulence-relevant functions in Y. pseudotuberculosis and reprogramming of its metabolism in response to temperature is associated with a restructuring of numerous mRNAswindow into the immense complexity of human brain function.

Plasmid copy number promotes plague The virulence of the plague bacterium, Yersinia pestis , is encoded on a plasmid. Normally, the bacterium only tolerates one copy of the plasmid, which provides the molecular machinery for injecting toxins into host cells. Wang et al. have found that Y. pestis can only be pathogenic if plasmid numbers are boosted and express enough protein components to assemble a functional virulence apparatus. Problematically, the synthetic activity of the plasmid has such a high metabolic cost that it can impede growth. Thus, the bacterium only allows the plasmid to replicate when it senses a potential host cell. Science , this issue p. 492

The Yersinia pseudotuberculosis Ifp and InvC molecules are putative autotransporter proteins with a high homology to the invasin (InvA) protein. To characterize the function of these surface proteins, we expressed both factors in Escherichia coli K-12 and demonstrated the attachment of Ifp- and InvC-expressing bacteria to human-, mouse-, and pig-derived intestinal epithelial cells. Ifp also was found to mediate microcolony formation and internalization into polarized human enterocytes. The ifp and invC genes were not expressed under in vitro conditions but were found to be induced in the Peyer's patches of the mouse intestinal tract. In a murine coinfection model, the colonization of the Peyer's patches and the mesenteric lymph nodes of mice by the ifp -deficient strain was significantly reduced, and considerably fewer bacteria reached liver and spleen. The absence of InvC did not have a severe influence on bacterial colonization in the murine infection model, and it resulted in only a slightly reduced number of invC mutants in the Peyer's patches. The analysis of the host immune response demonstrated that the presence of Ifp and InvC reduced the recruitment of professional phagocytes, especially neutrophils, in the Peyer's patches. These findings support a role for the adhesins in modulating host-pathogen interactions that are important for immune defense.

The outer membrane proteins YadA and invasin of Yersinia pseudotuberculosis promote invasion into mammalian cells through beta(1)-integrins and trigger the production of interleukin (IL)-8. FAK, c-Src and the PI3 kinase were previously found to be important for both YadA- and invasin-promoted uptake. Here, we demonstrate that two different downstream effectors of PI3 kinase, Akt and phospholipase Cgamma1 are required for efficient cell invasion. Inhibition of Akt or phospholipase C-gamma (PLC-gamma)1 by pharmaceutical agents as well as reduced expression of the isoforms Akt1 and Akt2, and of PLC-gamma1 by RNA interference decreased entry of YadA- and Inv-expressing bacteria significantly. In addition, we report that the conventional protein kinases C (PKC)alpha and -beta, positioned downstream of PLC-gamma1, are activated upon Inv- or YadA-promoted cell entry. They colocalize with intracellular bacteria and their depletion by siRNA treatment also resulted in a strong reduction of cell entry. In contrast, neither Akt nor PLC-gamma1, and the PKCs are essential for YadA- and Inv-mediated IL-8 synthesis and release. We conclude that YadA and invasin of Y. pseudotuberculosis both trigger similar signal transduction pathways during integrin-mediated phagocytosis into epithelial cells, which lead to the activation of Akt, PLC-gamma1, PKCalpha and -beta downstream of PI3 kinase, separate from the MAPK-dependent pathway that triggers IL-8 production.

Different biomolecules have been identified in bacterial pathogens that sense changes in temperature and trigger expression of virulence programs upon host entry. However, the dynamics and quantitative outcome of this response in individual cells of a population, and how this influences pathogenicity are unknown. Here, we address these questions using a thermosensing virulence regulator of an intestinal pathogen (RovA of Yersinia pseudotuberculosis) as a model. We reveal that this regulator is part of a novel thermoresponsive bistable switch, which leads to high- and low-invasive subpopulations within a narrow temperature range. The temperature range in which bistability is observed is defined by the degradation and synthesis rate of the regulator, and is further adjustable via a nutrient-responsive regulator. The thermoresponsive switch is also characterized by a hysteretic behavior in which activation and deactivation occurred on vastly different time scales. Mathematical modeling accurately mirrored the experimental behavior and predicted that the thermoresponsiveness of this sophisticated bistable switch is mainly determined by the thermo-triggered increase of RovA proteolysis. We further observed RovA ON and OFF subpopulations of Y. pseudotuberculosis in the Peyer’s patches and caecum of infected mice, and that changes in the RovA ON/OFF cell ratio reduce tissue colonization and overall virulence. This points to a bet-hedging strategy in which the thermoresponsive bistable switch plays a key role in adapting the bacteria to the fluctuating conditions encountered as they pass through the host’s intestinal epithelium and suggests novel strategies for the development of antimicrobial therapies., Author Summary The ability of pathogens to sense temperature changes when they enter their mammalian hosts from the environment is crucial to optimize their fitness and adjust expression of their virulence programs. Until now it has been assumed that all cells within a population participate in the thermo-triggered adaptive response. Here, we show that a small subpopulation of an enteric pathogen does not follow thermo-induced reprogramming when the bacteria pass the intestinal epithelial layer. Observed heterogeneity is promoted by a new type of bistable switch, implicating a highly precise, thermoresponsive control element. Moreover, we demonstrate that this regulatory implement is important for virulence as it prepares the pathogen for sudden, unpredictable fluctuations encountered during host entry and exit.

Intracellular bacteria invade mammalian cells to establish an infectious niche. The current work models adhesion and subsequent internalization strategy of pathogenic bacteria into mammalian cells to design a bacteriomimetic bioinvasive delivery system. We report on the surface functionalization of liposomes with a C-terminal fragment of invasin (InvA497), an invasion factor in the outer membrane of Yersinia pseudotuberculosis. InvA497-functionalized liposomes adhere to mammalian epithelial HEp-2 cell line at different infection stages with a significantly higher efficiency than liposomes functionalized with bovine serum albumin. Covalent attachment of InvA497 results in higher cellular adhesion than liposomes with physically adsorbed InvA497 with non-specific surface protein alignment. Uptake studies in HEp-2 cells indicate active internalization of InvA497-functionalized liposomes via β1-integrin receptor-mediated uptake mechanism mimicking the natural invasion strategy of Y. pseudotuberculosis. Uptake studies in Caco-2 cells at different polarization states demonstrate specific targeting of the InvA497-functionalized liposomes to less polarized cells reflecting the status of inflamed cells. Moreover, when loaded with the anti-infective agent gentamicin and applied to HEp-2 cells infected with Y. pseudotuberculosis, InvA497-functionalized liposomes are able to significantly reduce the infection load relative to non-functionalized drug-loaded liposomes. This indicates a promising application of such a bacteriomimetic system for drug delivery to intracellular compartments.

RovA is a MarR-type transcriptional regulator that controls transcription of rovA, the expression of the primary invasive factor invasin and other virulence genes of Yersinia pseudotuberculosis in response to environmental signals. Using a genetic approach to identify regulatory components that negatively influence rovA expression, we identified a new LysR-type regulatory protein, designated RovM, which exhibits homology to the virulence regulator PecT/HexA of plant pathogenic Erwinia species. DNA-binding studies revealed that RovM interacts specifically with a short binding site between promoters P1 and P2 within the rovA regulatory region and negatively modulates rovA transcription in cooperation with the histone-like protein H-NS. The rovM gene itself is under positive autoregulatory control and is significantly induced during growth in minimal media as shown in regulation studies. Disruption of the rovM gene leads to a significant increase of RovA and invasin synthesis and enhances internalization of Y. pseudotuberculosis into host cells. Finally, we show that a Y. pseudotuberculosis rovM mutant is more virulent than wild type and higher numbers of the bacteria are detectable in gut-associated lymphatic tissues and organs in the mouse infection model system. In contrast, elevated levels of the RovM protein, which exert a positive effect on flagellar motility, severely attenuate the ability of Y. pseudotuberculosis to disseminate to deeper tissues. Together, our data show, that RovM is a key regulator implicated in the environmental control of virulence factors, which are crucial for the initiation of a Yersinia infection.

The transcription factor RovA of Yersinia pseudotuberculosis and analogous proteins in other Enterobacteriaceae activate the expression of virulence genes that play a crucial role in stress adaptation and pathogenesis. In this study, we demonstrate that the RovA protein forms dimers independent of DNA binding, stimulates RNA polymerase, most likely via its C-terminal domain, and counteracts transcriptional repression by the histone-like protein H-NS. As the molecular function of the RovA family is largely uncharacterized, random mutagenesis and terminal deletions were used to identify functionally important domains. Our analysis showed that a winged-helix motif in the center of the molecule is essential and directly involved in DNA binding. Terminal deletions and amino acid changes within both termini also abrogate RovA activation and DNA-binding functions, most likely due to their implication in dimer formation. Finally, we show that the last four amino acids of RovA are crucial for activation of gene transcription. Successive deletions of these residues result in a continuous loss of RovA activity. Their removal reduced the capacity of RovA to activate RNA polymerase and abolished transcription of RovA-activated promoters in the presence of H-NS, although dimerization and DNA binding functions were retained. Our structural model implies that the final amino acids of RovA play a role in protein-protein interactions, adjusting RovA activity.

Summary The transcriptional activator RovA of Yersinia pseudotuberculosis, a member of the SlyA/Hor family, activates its own expression and that of the virulence factor invasin in response to moderate growth temperature, but not at 37°C. In this work, we analysed the mechanism of RovA-dependent transcription of the rovA and inv genes. We found that rovA is transcribed by two different promoters. Sequences located upstream and downstream of the promoters were involved in rovA autoregulation and interacted specifically with the RovA protein. To define the nucleotides recognized by the RovA protein, we determined the RovA binding sites in the rovA and the inv regulatory region and revealed related AT-rich sequence motifs at diverse positions relative to the transcriptional start sites. We also showed that rovA and the RovA-dependent inv gene were both subject to silencing by the nucleoid-associated H-NS protein of Y. pseudotuberculosis. The binding sites of the H-NS and RovA proteins in the rovA and inv regulatory sequences were superimposed, and the presence of the RovA protein alleviated H-NS-mediated repression of the rovA and inv promoter. Moreover, loss of H-NS function led to a significant increase in rovA and inv transcription nearly independently of RovA, indicating that RovA acts mainly as an antirepressor. We therefore hypothesize that the transcription level of RovA-dependent genes reflects the outcome of the RovA/H-NS competition and the rovA autoregulatory mechanism.

We recently found that Yersinia pseudotuberculosis can be used as a model of persistent bacterial infections. We performed in vivo RNA-seq of bacteria in small cecal tissue biopsies at early and persistent stages of infection to determine strategies associated with persistence. Comprehensive analysis of mixed RNA populations from infected tissues revealed that Y. pseudotuberculosis undergoes transcriptional reprogramming with drastic down-regulation of T3SS virulence genes during persistence when the pathogen resides within the cecum. At the persistent stage, the expression pattern in many respects resembles the pattern seen in vitro at 26oC, with for example, up-regulation of flagellar genes and invA. These findings are expected to have impact on future rationales to identify suitable bacterial targets for new antibiotics. Other genes that are up-regulated during persistence are genes involved in anaerobiosis, chemotaxis, and protection against oxidative and acidic stress, which indicates the influence of different environmental cues. We found that the Crp/CsrA/RovA regulatory cascades influence the pattern of bacterial gene expression during persistence. Furthermore, arcA, fnr, frdA, and wrbA play critical roles in persistence. Our findings suggest a model for the life cycle of this enteropathogen with reprogramming from a virulent to an adapted phenotype capable of persisting and spreading by fecal shedding., Author Summary To establish infection and colonize within a host, infecting pathogens have to cope with a variety of destructive surroundings. The food-borne pathogen Y. pseudotuberculosis can cause persistent infection in mice. Upon infection, Y. pseudotuberculosis passes the anti-microbial gastrointestinal milieu and finally remains associated with lymphoid follicles in cecal tissue surrounded by polymorphonuclear leukocytes, indicating that the bacteria are exposed to multiple environmental cues. We performed complex RNA-seq of small cecal biopsies of infected mice to reveal Y. pseudotuberculosis gene expression in vivo. We found that Y. pseudotuberculosis underwent reprogramming from a virulent phenotype, expressing virulence genes during early infection, to an adapted phenotype capable of persisting in the harsh cecal environment. Persistence was characterized by a novel expression pattern with down-regulation of virulence genes and up-regulation of genes involved in anaerobiosis, chemotaxis, and protection against oxidative and acidic stress. Mutagenesis of selected genes revealed that the regulator rovA was critical for the establishment of infection, and that arcA, fnr, frdA, and wrbA play critical roles in maintaining infection for long periods of time. Our study shows the power of RNA deep sequencing, which can be used to reveal the in vivo expression patterns of small amounts of bacteria in complex intestinal environments.

Natural killer cells play a crucial role in the initial defense against bacterial pathogens. The crosstalk between host cells infected with intracellular pathogens and NK cells has been studied intensively, but not much attention has been given to characterize the role of NK cells in the response to extracellular bacterial pathogens such as yersiniae. In this study we used antibody-mediated NK cell depletion to address the importance of this immune cell type in controlling a Y. pseudotuberculosis infection. Analysis of the bacterial counts was used to follow the infection and flow cytometry was performed to characterize the composition and dynamic of immune cells. Depletion of NK cells led to higher bacterial loads within the mesenteric lymph nodes. We further show that in particular CD11b+ CD27+ NK cells which express higher levels of the activation marker CD69 increase within the mesenteric lymph nodes during a Y. pseudotuberculosis infection. Moreover, in response to the activation NK cells secrete higher levels of IFNy, which in turn triggers the production of the proinflammatory cytokine TNFα. These results suggest, that NK cells aid in the clearance of Y. pseudotuberculosis infections mainly by triggering the expression of proinflammatory cytokines manipulating the host immune response.

Deciphering the principles how pathogenic bacteria adapt their metabolism to a specific host microenvironment is critical for understanding bacterial pathogenesis. The enteric pathogenic Yersinia species Yersinia pseudotuberculosis and Yersinia enterocolitica and the causative agent of plague, Yersinia pestis, are able to survive in a large variety of environmental reservoirs (e.g., soil, plants, insects) as well as warm-blooded animals (e.g., rodents, pigs, humans) with a particular preference for lymphatic tissues. In order to manage rapidly changing environmental conditions and interbacterial competition, Yersinia senses the nutritional composition during the course of an infection by special molecular devices, integrates this information and adapts its metabolism accordingly. In addition, nutrient availability has an impact on expression of virulence genes in response to C-sources, demonstrating a tight link between the pathogenicity of yersiniae and utilization of nutrients. Recent studies revealed that global regulatory factors such as the cAMP receptor protein (Crp) and the carbon storage regulator (Csr) system are part of a large network of transcriptional and posttranscriptional control strategies adjusting metabolic changes and virulence in response to temperature, ion and nutrient availability. Gained knowledge about the specific metabolic requirements and the correlation between metabolic and virulence gene expression that enable efficient host colonization led to the identification of new potential antimicrobial targets.

In this study we investigated the influence of the global response regulator PhoP on the complex regulatory cascade controlling expression of early stage virulence genes of Yersinia pseudotuberculosis via the virulence regulator RovA. Our analysis revealed the following novel features: (1) PhoP activates expression of the CsrC RNA in Y. pseudotuberculosis, leading to activation of RovA synthesis through the CsrABC-RovM cascade, (2) activation of csrC transcription is direct and PhoP is shown to bind to two separate PhoP box-like sites, (3) PhoP-mediated activation results in transcription from two different promoters closely downstream of the PhoP binding sites, leading to two distinct CsrC RNAs, and (4) the stability of the CsrC RNAs differs significantly between the Y. pseudotuberculosis strains YPIII and IP32953 due to a 20 nucleotides insertion in CsrC(IP32953), which renders the transcript more susceptible to degradation. In summary, our study showed that PhoP-mediated influence on the regulatory cascade controlling the Csr system and RovA in Y. pseudotuberculosis varies within the species, suggesting that the Csr system is a focal point to readjust and adapt the genus to different hosts and reservoirs.

The binding of the Yersinia pseudotuberculosis and Yersinia enterocolitica invasin proteins to β 1 integrin receptors allows internalization of these organisms by cultured cells. The C-terminal 192-residue superdomain of the Y. pseudotuberculosis invasin is necessary and sufficient for integrin recognition, while a region located outside, and N-terminal to, this superdomain strongly enhances the efficiency of bacterial uptake. Within the enhancer region is a domain called D2 that allows invasin-invasin interaction. To investigate the role of the enhancer region, bacterial cell binding and entry mediated by the Y. pseudotuberculosis invasin protein (invasin pstb ) was compared to that of Y. enterocolitica invasin (invasin ent ), which lacks the D2 self-association domain. Invasin ent was shown to be unable to promote self-interaction, using the DNA binding domain of λ repressor as a reporter. Furthermore, two genetically engineered in-frame deletion mutations that removed D2 from invasin pstb were significantly less proficient than wild-type invasin pstb at promoting uptake, although the amount of surface-exposed invasin as well as the cell binding capacity of the recombinant Escherichia coli strains remained similar. Competitive uptake assays showed that E. coli cells expressing invasin pstb had a significant advantage in the internalization process versus either E. coli cells expressing invasin ent or the invasin pstb derivatives deleted for D2, further demonstrating the importance of invasin self-interaction for the efficiency of invasin-mediated uptake.

To determine if recognition of the Yersinia pseudotuberculosis invasin protein and natural substrates requires identical integrin residues, a region of the human alpha3 integrin chain predicted to be involved in substrate adhesion was targeted for mutation. One point mutation located in a region of the third N-terminal repeat of the alpha3 chain, alpha3-W220A, failed to promote adhesion to the natural alpha3 beta1 substrate epiligrin but maintained near wild type levels of adhesion to invasin. A second nearby mutation, alpha3-Y218A, which showed no detectable adhesion to epiligrin, was only partially attenuated for invasin binding as well as invasin-mediated bacterial uptake. A third substitution, alpha3-D154A, predicted to be in the second N-terminal repeat not known to be implicated in cell adhesion, was competent for invasin-promoted adhesion events and appeared to encode a receptor of increased activity, as it had a higher efficiency than wild type receptor for adhesion to epiligrin. Cell lines expressing this derivative were not recognized by a function blocking anti-alpha3 antibody, indicating that the second and third repeats of the alpha3 chain are either closely linked in space or the second repeat can modulate activity of the third. Differential effects on substrate adhesion do not appear to be associated with all integrin alpha chain mutations, as alpha4 chain mutations affecting the divalent cation binding domains depressed adhesion to invasin to a significant extent.

The two-component regulatory system PhoP/PhoQ has been shown to (i) control expression of virulence-associated traits, (ii) confer survival and growth within macrophages and (iii) play a role in Yersinia infections. However, the influence of PhoP on virulence varied greatly between different murine models of infection and its role in natural oral infections with frequently used representative isolates of Y. pseudotuberculosis was unknown. To address this issue, we constructed an isogenic set of phoP + and phoP − variants of strain IP32953 and YPIII and analyzed the impact of PhoP using in vitro functionality experiments and a murine oral infection model, whereby we tested for bacterial dissemination and influence on the host immune response. Our results revealed that PhoP has a low impact on virulence, lymphatic and systemic organ colonization, and on immune response modulation by IP32953 and YPIII, indicating that PhoP is not absolutely essential for oral infections but may be involved in fine-tuning the outcome. Our work further revealed certain strain-specific differences in virulence properties, which do not strongly rely on the function of PhoP, but affect tissue colonization, dissemination and/or persistence of the bacteria. Highlighted intra-species variations may provide a potential means to rapidly adjust to environmental changes inside and outside of the host.

Des bacteries pathogenes ont adopte des strategies de survie chez leur hote vivant, en produisant des facteurs d'adhesion qui medient une association etroite avec les cellules hotes et leur internalisation. L'invasine, proteine multimerique de Y pseudotuberculosis , permet l'invasion de ce microorganisme, par l'intermediaire de sa liaison avec un sous-ensemble de recepteurs de l'integrine β1 de la cellule eucaryote. Nous presentons ici des modeles complementaires illustrant le mecanisme moleculaire et cellulaire de la penetration de Yersinia , mediee par l'invasine, dans les cellules de mammiferes.

Some isolates of Yersinia pseudotuberculosis produce the cytotoxic necrotizing factor (CNFY), but the functional consequences of this toxin for host-pathogen interactions during the infection are unknown. In the present study we show that CNFY has a strong influence on virulence. We demonstrate that the CNFY toxin is thermo-regulated and highly expressed in all colonized lymphatic tissues and organs of orally infected mice. Most strikingly, we found that a cnfY knock-out variant of a naturally toxin-expressing Y. pseudotuberculosis isolate is strongly impaired in its ability to disseminate into the mesenteric lymph nodes, liver and spleen, and has fully lost its lethality. The CNFY toxin contributes significantly to the induction of acute inflammatory responses and to the formation of necrotic areas in infected tissues. The analysis of the host immune response demonstrated that presence of CNFY leads to a strong reduction of professional phagocytes and natural killer cells in particular in the spleen, whereas loss of the toxin allows efficient tissue infiltration of these immune cells and rapid killing of the pathogen. Addition of purified CNFY triggers formation of actin-rich membrane ruffles and filopodia, which correlates with the activation of the Rho GTPases, RhoA, Rac1 and Cdc42. The analysis of type III effector delivery into epithelial and immune cells in vitro and during the course of the infection further demonstrated that CNFY enhances the Yop translocation process and supports a role for the toxin in the suppression of the antibacterial host response. In summary, we highlight the importance of CNFY for pathogenicity by showing that this toxin modulates inflammatory responses, protects the bacteria from attacks of innate immune effectors and enhances the severity of a Yersinia infection., Author Summary Various toxins and effector proteins of bacterial pathogens have been found to manipulate eukaryotic cell machineries to promote persistence and proliferation within their hosts. Many of these virulence factors target small Rho GTPases, but their role in pathogenesis is often unknown. Here, we addressed the expression and functional consequences of the CNFY toxin found in some isolates of Y. pseudotuberculosis. We found that CNFY besides modulating the cell cytoskeleton by activation of the GTPases RhoA, Rac1 and Cdc42, contributes to increased inflammation and tissue damage. Moreover, CNFY increases the ability of Yersinia to prevent the attack of the immune system, by enhancing the delivery of antiphagocytic and cytotoxic effectors into professional phagocytes. Our findings provide the first insights into the multi-functional action and severe consequences of the CNFY toxin on the inflammatory response and disease-associated tissue damage during the natural course of the infection.

Pathogens often rely on thermosensing to adjust virulence gene expression. In yersiniae, important virulence-associated traits are under the control of the master regulator RovA, which uses a built-in thermosensor to control its activity. Thermal upshifts encountered upon host entry induce conformational changes in the RovA dimer that attenuate DNA binding and render the protein more susceptible to proteolysis. Here, we report the crystal structure of RovA in the free and DNA-bound forms and provide evidence that thermo-induced loss of RovA activity is promoted mainly by a thermosensing loop in the dimerization domain and residues in the adjacent C-terminal helix. These determinants allow partial unfolding of the regulator upon an upshift to 37 °C. This structural distortion is transmitted to the flexible DNA-binding domain of RovA. RovA contacts mainly the DNA backbone in a low-affinity binding mode, which allows the immediate release of RovA from its operator sites. We also show that SlyA, a close homolog of RovA from Salmonella with a very similar structure, is not a thermosensor and remains active and stable at 37 °C. Strikingly, changes in only three amino acids, reflecting evolutionary replacements in SlyA, result in a complete loss of the thermosensing properties of RovA and prevent degradation. In conclusion, only minor alterations can transform a thermotolerant regulator into a thermosensor that allows adjustment of virulence and fitness determinants to their thermal environment.

This review emphasizes the function and regulation of the Csr regulatory system in the human enteropathogen Yersinia pseudotuberculosis and compares its features with the homologous Csr/Rsm systems of related pathogens. The Csr/Rsm systems of eubacteria form a complex regulatory network in which redundant non-translated Csr/Rsm-RNAs bind the RNA-binding protein CsrA/RsmA, thereby preventing its interaction with mRNA targets. The Csr system is controlled by the BarA/UvrY-type of two-component sensor-regulator systems. Apart from that, common or pathogen-specific regulators control the abundance of the Csr components. The coordinate control of virulence factors and infection-linked physiological traits by the Csr/Rsm systems helps the pathogens to adapt individually to rapidly changing conditions to which they are exposed during the different stages of an infection. As Csr/Rsm function is relevant for full virulence, it represents a target suitable for antimicrobial drug development.

The enteropathogenic bacterium Yersinia pseudotuberculosis produces different sets of virulence factors that guarantee efficient colonization during the early stages, and persistence and dissemination during the late stages of the infection. Alterations of the temperature and the nutrient availability encountered upon host entry and during the ongoing infection lead to global changes of the virulence gene expression profile. Expression of virulence factors (e.g., invasin) that promote colonization and penetration of the intestinal epithelium during the very early phase of the infection is activated by the MarR-type regulator RovA. RovA is a protein thermometer harboring an intrinsic thermosensor. Thermal upshifts to 37°C lead to a reversible conformational change which reduces the DNA binding capacity of the autoactivator and increased susceptibility of RovA for proteolysis by the Lon protease. Furthermore, regulation of rovA expression depends on the post-transcriptional carbon storage regulator system (Csr) implicating the differentially regulated small RNAs CsrB and CsrC and the RNA-binding protein CsrA. Influence of the Csr system occurs through the LysR-type regulator RovM and depends on the nutrient content of the growth medium. After Y. pseudotuberculosis has crossed the intestinal epithelial layer, synthesis of the early virulence genes is repressed and later stage virulence genes, including the type III secretion system and yop genes crucial for immune defense are induced. Activation of these genes is only promoted at 37°C and requires the AraC-like regulator LcrF. Translation of the lcrF mRNA is controlled by a thermoswitch located in the 5′-UTR of the lcrF gene. At moderate temperatures, a stable hairpin structure prevents access of the ribosome and translation of the lcrF mRNA, whereas partial melting of the structure allows ribosome binding at 37°C.

Colonization of the intestinal tract and dissemination into deeper tissues by the enteric pathogen Yersinia pseudotuberculosis demands expression of a special set of virulence factors important for the initiation and the persistence of the infection. In this study we demonstrate that many virulence-associated functions are coregulated with the carbohydrate metabolism. This link is mediated by the carbon storage regulator (Csr) system, including the regulatory RNAs CsrB and CsrC, and the cAMP receptor protein (Crp), which both control virulence gene expression in response to the nutrient composition of the medium. Here, we show that Crp regulates the synthesis of both Csr RNAs in an opposite manner. A loss of the crp gene resulted in a strong upregulation of CsrB synthesis, whereas CsrC levels were strongly reduced leading to downregulation of the virulence regulator RovA. Switching of the Csr RNA involves Crp-mediated repression of the response regulator UvrY which activates csrB transcription. To elucidate the regulatory links between virulence and carbon metabolism, we performed comparative metabolome, transcriptome, and phenotypic microarray analyses and found that Crp promotes oxidative catabolism of many different carbon sources, whereas fermentative patterns of metabolism are favored when crp is deleted. Mouse infection experiments further demonstrated that Crp is pivotal for a successful Y. pseudotuberculosis infection. In summary, placement of the Csr system and important virulence factors under control of Crp enables this pathogen to link its nutritional status to virulence in order to optimize biological fitness and infection efficiency through the infectious life cycle.

Many enteric pathogens are equipped with multiple cell adhesion factors which are important for host tissue colonization and virulence. Y. enterocolitica, a common food-borne pathogen with invasive properties, uses the surface proteins invasin and YadA for host cell binding and entry. In this study, we demonstrate unique cell adhesion and invasion properties of Y. enterocolitica serotype O:3 strains, the most frequent cause of human yersiniosis, and show that these differences are mainly attributable to variations affecting the function and expression of invasin in response to temperature. In contrast to other enteric Yersinia strains, invasin production in O:3 strains is constitutive and largely enhanced compared to other Y. enterocolitica serotypes, in which invA expression is temperature-regulated and significantly reduced at 37°C. Increase of invasin levels is caused by (i) an IS1667 insertion into the invA promoter region, which includes an additional promoter and RovA and H-NS binding sites, and (ii) a P98S substitution in the invA activator protein RovA rendering the regulator less susceptible to proteolysis. Both variations were shown to influence bacterial colonization in a murine infection model. Furthermore, we found that co-expression of YadA and down-regulation of the O-antigen at 37°C is required to allow efficient internalization by the InvA protein. We conclude that even small variations in the expression of virulence factors can provoke a major difference in the virulence properties of closely related pathogens which may confer better survival or a higher pathogenic potential in a certain host or host environment., Author Summary Bacterial infections are generally initiated by molecular interactions that occur between the pathogen and its host cell. These interactions are usually mediated by adhesion and invasion factors exposed from the surface of the bacteria which are necessary for the colonization of host tissues and fundamental to pathogenesis. It is well known that many bacterial species contain several different adhesin determinants, which often vary between bacteria of the same species, reflecting the fact that each microbe has adapted to a distinct ecological niche. Here, we show that also small alterations changing the expression pattern of adhesins and virulence gene regulators in response to environmental factors (e.g. temperature) lead to fundamental differences in pathogen-host cell interactions and pathogenesis. Modulation of virulence gene expression constitutes an ideal mechanism to adjust virulence-associated processes of pathogens to different hosts (e.g. with varying body temperature) as it allows the bacteria to readjust expression of certain gene subsets of regulatory networks controlling virulence, stress and metabolic adaptation to their demands in individual hosts.

Small RNAs GlmY and GlmZ compose a cascade that feedback-regulates synthesis of enzyme GlmS in Enterobacteriaceae. Here, we analyzed the transcriptional regulation of glmY/glmZ from Yersinia pseudotuberculosis, Salmonella typhimurium and Escherichia coli, as representatives for other enterobacterial species, which exhibit similar promoter architectures. The GlmY and GlmZ sRNAs of Y. pseudotuberculosis are transcribed from p54-promoters that require activation by the response regulator GlrR through binding to three conserved sites located upstream of the promoters. This also applies to glmY/glmZ of S. typhimurium and glmY of E. coli, but as a difference additional p70-promoters overlap the p54-promoters and initiate transcription at the same site. In contrast, E. coli glmZ is transcribed from a single p70- promoter. Thus, transcription of glmY and glmZ is controlled by p54 and the two-component system GlrR/GlrK (QseF/QseE) in Y. pseudotuberculosis and presumably in many other Enterobacteria. However, in a subset of species such as E. coli this relationship is partially lost in favor of p70- dependent transcription. In addition, we show that activity of the p54-promoter of E. coli glmY requires binding of the integration host factor to sites upstream of the promoter. Finally, evidence is provided that phosphorylation of GlrR increases its activity and thereby sRNA expression. peerReviewed

A family of versatile promoter-probe plasmids for gene expression analysis was developed based on a modular expression plasmid system (pZ). The vectors contain different replicons with exchangeable antibiotic cassettes to allow compatibility and expression analysis on a low-, midi- and high-copy number basis. Suicide vector variants also permit chromosomal integration of the reporter fusion and stable vector derivatives can be used for in vivo or in situ expression studies under non-selective conditions. Transcriptional and translational fusions to the reporter genes gfp(mut3.1), amCyan, dsRed2, luxCDABE, phoA or lacZ can be constructed, and presence of identical multiple cloning sites in the vector system facilitates the interchange of promoters or reporter genes between the plasmids of the series. The promoter of the constitutively expressed gapA gene of Escherichia coli was included to obtain fluorescent and bioluminescent expression constructs. A combination of the plasmids allows simultaneous detection and gene expression analysis in individual bacteria, e.g. in bacterial communities or during mouse infections. To test our vector system, we analyzed and quantified expression of Yersinia pseudotuberculosis virulence genes under laboratory conditions, in association with cells and during the infection process.

In enteropathogenic Yersinia, the expression of several early-phase virulence factors such as invasin is tightly regulated in response to environmental cues. The responsible regulatory network is complex, involving several regulatory RNAs and proteins such as the LysR-type transcription regulator (LTTR) RovM. In this study, the crystal structure of the effector-binding domain (EBD) of RovM, the first LTTR protein described as being involved in virulence regulation, was determined at a resolution of 2.4 A. Size-exclusion chromatography and comparison with structures of full-length LTTRs show that RovM is most likely to adopt a tetrameric arrangement with two distant DNA-binding domains (DBDs), causing the DNA to bend around it. Additionally, a cavity was detected in RovM which could bind small inducer molecules.

The outer membrane proteins YadA and invasin of Yersinia pseudotuberculosis promote invasion into mammalian cells through beta(1)-integrins and trigger the production of interleukin (IL)-8. FAK, c-Src and the PI3 kinase were previously found to be important for both YadA- and invasin-promoted uptake. Here, we demonstrate that two different downstream effectors of PI3 kinase, Akt and phospholipase Cgamma1 are required for efficient cell invasion. Inhibition of Akt or phospholipase C-gamma (PLC-gamma)1 by pharmaceutical agents as well as reduced expression of the isoforms Akt1 and Akt2, and of PLC-gamma1 by RNA interference decreased entry of YadA- and Inv-expressing bacteria significantly. In addition, we report that the conventional protein kinases C (PKC)alpha and -beta, positioned downstream of PLC-gamma1, are activated upon Inv- or YadA-promoted cell entry. They colocalize with intracellular bacteria and their depletion by siRNA treatment also resulted in a strong reduction of cell entry. In contrast, neither Akt nor PLC-gamma1, and the PKCs are essential for YadA- and Inv-mediated IL-8 synthesis and release. We conclude that YadA and invasin of Y. pseudotuberculosis both trigger similar signal transduction pathways during integrin-mediated phagocytosis into epithelial cells, which lead to the activation of Akt, PLC-gamma1, PKCalpha and -beta downstream of PI3 kinase, separate from the MAPK-dependent pathway that triggers IL-8 production.

Background All human pathogenic Yersinia species share a virulence-associated type III secretion system that translocates Yersinia effector proteins into host cells to counteract infection-induced signaling responses and prevent phagocytosis. Dictyostelium discoideum has been recently used to study the effects of bacterial virulence factors produced by internalized pathogens. In this study we explored the potential of Dictyostelium as model organism for analyzing the effects of ectopically expressed Yersinia outer proteins (Yops). Results The Yersinia pseudotuberculosis virulence factors YopE, YopH, YopM and YopJ were expressed de novo within Dictyostelium and their effects on growth in axenic medium and on bacterial lawns were analyzed. No severe effect was observed for YopH, YopJ and YopM, but expression of YopE, which is a GTPase activating protein for Rho GTPases, was found to be highly detrimental. GFP-tagged YopE expressing cells had less conspicuous cortical actin accumulation and decreased amounts of F-actin. The actin polymerization response upon cAMP stimulation was impaired, although chemotaxis was unaffected. YopE also caused reduced uptake of yeast particles. These alterations are probably due to impaired Rac1 activation. We also found that YopE predominantly associates with intracellular membranes including the Golgi apparatus and inhibits the function of moderately overexpressed RacH. Conclusion The phenotype elicited by YopE in Dictyostelium can be explained, at least in part, by inactivation of one or more Rho family GTPases. It further demonstrates that the social amoeba Dictyostelium discoideum can be used as an efficient and easy-to-handle model organism in order to analyze the function of a translocated GAP protein of a human pathogen.

Pathogens, which alternate between environmental reservoirs and a mammalian host, frequently use thermal sensing devices to adjust virulence gene expression. Here, we identify the Yersinia virulence regulator RovA as a protein thermometer. Thermal shifts encountered upon host entry lead to a reversible conformational change of the autoactivator, which reduces its DNA-binding functions and renders it more susceptible for proteolysis. Cooperative binding of RovA to its target promoters is significantly reduced at 37°C, indicating that temperature control of rovA transcription is primarily based on the autoregulatory loop. Thermally induced reduction of DNA-binding is accompanied by an enhanced degradation of RovA, primarily by the Lon protease. This process is also subject to growth phase control. Studies with modified/chimeric RovA proteins indicate that amino acid residues in the vicinity of the central DNA-binding domain are important for proteolytic susceptibility. Our results establish RovA as an intrinsic temperature-sensing protein in which thermally induced conformational changes interfere with DNA-binding capacity, and secondarily render RovA susceptible to proteolytic degradation., Author Summary Temperature is one of the most crucial environmental signals sensed by pathogens to adjust expression of their virulence factors and host survival programs after entry from a cold external environment into a warm-blooded host. Thermo-induced structural changes in bent or supercoiled DNA or mRNA secondary structures are frequently used to modulate virulence gene transcription or translation. Here we introduce a unique alternative mechanism, in which a central regulator of Yersinia virulence (RovA) uses an in-built thermosensor to control its activity in order to modulate virulence gene expression. According to our results, small thermo-induced structural alterations reduce the DNA-binding capacity of the virulence regulator and render the protein more susceptible to proteolytic degradation by ATP-dependent proteases. Amino acids in the vicinity of the DNA-binding region appear to comprise the information required for proteolysis. We therefore postulate a model in which proteolytic degradation is in direct competition with the thermo-sensitive DNA-binding function of the regulator. This regulatory concept constitutes a new example of how microbial pathogens are able to rapidly adjust virulence-associated processes in the course of an infection.

The MarR-type regulator RovA controls expression of virulence genes of Yersinia pseudotuberculosis in response to environmental signals. Using a genetic strategy to discover components that influence rovA expression, we identified new regulatory factors with homology to components of the carbon storage regulator system (Csr). We showed that overexpression of a CsrB- or a CsrC-type RNA activates rovA, whereas a CsrA-like protein represses RovA synthesis. We further demonstrate that influence of the Csr system on rovA is indirect and occurs through control of the LysR regulator RovM, which inhibits rovA transcription. The CsrA protein had also a major influence on the motility of Yersinia, which was independent of RovM. The CsrB and CsrC RNAs are differentially expressed in Yersinia. CsrC is highly induced in complex but not in minimal media, indicating that medium-dependent rovM expression is mediated through CsrC. CsrB synthesis is generally very low. However, overexpression of the response regulator UvrY was found to activate CsrB production, which in turn represses CsrC synthesis independent of the growth medium. In summary, the post-transcriptional Csr-type components were shown to be key regulators in the co-ordinated environmental control of physiological processes and virulence factors, which are crucial for the initiation of Yersinia infections.