Interspecies Interaction of Bacteria Listeria monocytogenes, Yersinia pseudotuberculosis, and Marine Saprotrophs during Long-Term Culturing in Biofilms.

All bacterial strains studied retained the viability and ability to form both mono- and polycultural biofilms under conditions of long-term culturing in artificial seawater at 6°C and without addition of nutrients. Bacillus sp. and Pseudomonas japonica presumably stimulated the growth and reproduction of the pathogenic bacteria Listeria monocytogenes and Yersinia pseudotuberculosis. Preserved cell viability in a monoculture biofilm for a long period without adding a food source can indicate allolysis. At the same time, in a polycultural biofilm, the metabolites secreted by saprotrophic strains can stimulate the growth of L. monocytogenes and Y. pseudotuberculosis.

Modifying TIMER to generate a slow-folding DsRed derivative for optimal use in quickly-dividing bacteria.

It is now well appreciated that members of pathogenic bacterial populations exhibit heterogeneity in growth rates and metabolic activity, and it is known this can impact the ability to eliminate all members of the bacterial population during antibiotic treatment. It remains unclear which pathways promote slowed bacterial growth within host tissues, primarily because it has been difficult to identify and isolate slow growing bacteria from host tissues for downstream analyses. To overcome this limitation, we have developed a novel variant of TIMER, a slow-folding fluorescent protein, named DsRed42, to identify subsets of slowly dividing bacteria within host tissues. The original TIMER folds too slowly for fluorescence accumulation in quickly replicating bacterial species (Escherichia coli, Yersinia pseudotuberculosis), however DsRed42 accumulates red fluorescence in late stationary phase cultures of E. coli and Y. pseudotuberculosis. We show DsRed42 signal also accumulates during exposure to sources of nitric oxide (NO), suggesting DsRed42 signal detects growth-arrested bacterial cells. In a mouse model of Y. pseudotuberculosis deep tissue infection, DsRed42 signal was detected, and primarily accumulates in bacteria expressing markers of stationary phase growth. There was no significant overlap between DsRed42 signal and NO-exposed subpopulations of bacteria within host tissues, suggesting NO stress was transient, allowing bacteria to recover from this stress and resume replication. This novel DsRed42 variant represents a tool that will enable additional studies of slow-growing subpopulations of bacteria, specifically within bacterial species that quickly divide.

Modeling of Interspecific Interaction of Yersinia pseudotuberculosis and Listeria monocytogenes in a Mixed-Species Biofilms with Microorganisms Isolated from Coastal Waters of the Sea of Japan.

We studied the possibility of formation of mixed-species biofilms by Yersinia pseudotuberculosis and Listeria monocytogenes with marine saprotrophic bacteria Flavobacterium sp. and Micrococcus luteus isolated from the coastal waters of the Sea of Japan in summer. Model experiments showed that Flavobacterium sp. and Micrococcus luteus can form both single- and mixed-species biofilms with the specified pathogenic bacteria thus stimulating their growth. This can contribute to the preservation of the pathogens in the marine environment.

A Trimeric Autotransporter Enhances Biofilm Cohesiveness in Yersinia pseudotuberculosis but Not in Yersinia pestis.

Cohesion of biofilms made by Yersinia pestis and Yersinia pseudotuberculosis has been attributed solely to an extracellular polysaccharide matrix encoded by the hms genes (Hms-dependent extracellular matrix [Hms-ECM]). However, mutations in the Y. pseudotuberculosis BarA/UvrY/CsrB regulatory cascade enhance biofilm stability without dramatically increasing Hms-ECM production. We found that treatment with proteinase K enzyme effectively destabilized Y. pseudotuberculosiscsrB mutant biofilms, suggesting that cell-cell interactions might be mediated by protein adhesins or extracellular matrix proteins. We identified an uncharacterized trimeric autotransporter lipoprotein (YPTB2394), repressed by csrB, which has been referred to as YadE. Biofilms made by a ΔyadE mutant strain were extremely sensitive to mechanical disruption. Overexpression of yadE in wild-type Y. pseudotuberculosis increased biofilm cohesion, similar to biofilms made by csrB or uvrY mutants. We found that the Rcs signaling cascade, which represses Hms-ECM production, activated expression of yadE The yadE gene appears to be functional in Y. pseudotuberculosis but is a pseudogene in modern Y. pestis strains. Expression of functional yadE in Y. pestis KIM6+ weakened biofilms made by these bacteria. This suggests that although the YadE autotransporter protein increases Y. pseudotuberculosis biofilm stability, it may be incompatible with the Hms-ECM production that is essential for Y. pestis biofilm production in fleas. Inactivation of yadE in Y. pestis may be another instance of selective gene loss in the evolution of flea-borne transmission by this species.IMPORTANCE The evolution of Yersinia pestis from its Y. pseudotuberculosis ancestor involved gene acquisition and gene losses, leading to differences in biofilm production. Characterizing the unique biofilm features of both species may provide better understanding of how each adapts to its specific niches. This study identifies a trimeric autotransporter, YadE, that promotes biofilm stability of Y. pseudotuberculosis but which has been inactivated in Y. pestis, perhaps because it is not compatible with the Hms polysaccharide that is crucial for biofilms inside fleas. We also reveal that the Rcs signaling cascade, which represses Hms expression, activates YadE in Y. pseudotuberculosis The ability of Y. pseudotuberculosis to use polysaccharide or YadE protein for cell-cell adhesion may help it produce biofilms in different environments.

Bread Feeding Is a Robust and More Physiological Enteropathogen Administration Method Compared to Oral Gavage.

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.

Temperature-responsive in vitro RNA structurome of Yersinia pseudotuberculosis.

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 mRNAs.

YopN Is Required for Efficient Effector Translocation and Virulence in Yersinia pseudotuberculosis.

Type III secretion systems (T3SSs) are used by various Gram-negative pathogens to subvert the host defense by a host cell contact-dependent mechanism to secrete and translocate virulence effectors. While the effectors differ between pathogens and determine the pathogenic life style, the overall mechanism of secretion and translocation is conserved. T3SSs are regulated at multiple levels, and some secreted substrates have also been shown to function in regulation. In Yersinia, one of the substrates, YopN, has long been known to function in the host cell contact-dependent regulation of the T3SS. Prior to contact, through its interaction with TyeA, YopN blocks secretion. Upon cell contact, TyeA dissociates from YopN, which is secreted by the T3SS, resulting in the induction of the system. YopN has also been shown to be translocated into target cells by a T3SS-dependent mechanism. However, no intracellular function has yet been assigned to YopN. The regulatory role of YopN involves the N-terminal and C-terminal parts, while less is known about the role of the central region of YopN. Here, we constructed different in-frame deletion mutants within the central region. The deletion of amino acids 76 to 181 resulted in an unaltered regulation of Yop expression and secretion but triggered reduced YopE and YopH translocation within the first 30 min after infection. As a consequence, this deletion mutant lost its ability to block phagocytosis by macrophages. In conclusion, we were able to differentiate the function of YopN in translocation and virulence from its function in regulation.

High-throughput analysis of Yersinia pseudotuberculosis gene essentiality in optimised in vitro conditions, and implications for the speciation of Yersinia pestis.

BACKGROUND: Yersinia pseudotuberculosis is a zoonotic pathogen, causing mild gastrointestinal infection in humans. From this comparatively benign pathogenic species emerged the highly virulent plague bacillus, Yersinia pestis, which has experienced significant genetic divergence in a relatively short time span. Much of our knowledge of Yersinia spp. evolution stems from genomic comparison and gene expression studies. Here we apply transposon-directed insertion site sequencing (TraDIS) to describe the essential gene set of Y. pseudotuberculosis IP32953 in optimised in vitro growth conditions, and contrast these with the published essential genes of Y. pestis. RESULTS: The essential genes of an organism are the core genetic elements required for basic survival processes in a given growth condition, and are therefore attractive targets for antimicrobials. One such gene we identified is yptb3665, which encodes a peptide deformylase, and here we report for the first time, the sensitivity of Y. pseudotuberculosis to actinonin, a deformylase inhibitor. Comparison of the essential genes of Y. pseudotuberculosis with those of Y. pestis revealed the genes whose importance are shared by both species, as well as genes that were differentially required for growth. In particular, we find that the two species uniquely rely upon different iron acquisition and respiratory metabolic pathways under similar in vitro conditions. CONCLUSIONS: The discovery of uniquely essential genes between the closely related Yersinia spp. represent some of the fundamental, species-defining points of divergence that arose during the evolution of Y. pestis from its ancestor. Furthermore, the shared essential genes represent ideal candidates for the development of novel antimicrobials against both species.

Transcriptomic profiling of Yersinia pseudotuberculosis reveals reprogramming of the Crp regulon by temperature and uncovers Crp as a master regulator of small RNAs.

One hallmark of pathogenic yersiniae is their ability to rapidly adjust their life-style and pathogenesis upon host entry. In order to capture the range, magnitude and complexity of the underlying gene control mechanisms we used comparative RNA-seq-based transcriptomic profiling of the enteric pathogen Y. pseudotuberculosis under environmental and infection-relevant conditions. We identified 1151 individual transcription start sites, multiple riboswitch-like RNA elements, and a global set of antisense RNAs and previously unrecognized trans-acting RNAs. Taking advantage of these data, we revealed a temperature-induced and growth phase-dependent reprogramming of a large set of catabolic/energy production genes and uncovered the existence of a thermo-regulated 'acetate switch', which appear to prime the bacteria for growth in the digestive tract. To elucidate the regulatory architecture linking nutritional status to virulence we also refined the CRP regulon. We identified a massive remodelling of the CRP-controlled network in response to temperature and discovered CRP as a transcriptional master regulator of numerous conserved and newly identified non-coding RNAs which participate in this process. This finding highlights a novel level of complexity of the regulatory network in which the concerted action of transcriptional regulators and multiple non-coding RNAs under control of CRP adjusts the control of Yersinia fitness and virulence to the requirements of their environmental and virulent life-styles.

Yersinia pseudotuberculosis†YopD mutants that genetically separate effector protein translocation from host membrane disruption.

The Yersinia type III secretion system (T3SS) translocates Yop effector proteins into host cells to manipulate immune defenses such as phagocytosis and reactive oxygen species (ROS) production. The T3SS translocator proteins YopB and YopD form pores in host membranes, facilitating Yop translocation. While the YopD amino and carboxy termini participate in pore formation, the role of the YopD central region between amino acids 150-227 remains unknown. We assessed the contribution of this region by generating Y. pseudotuberculosis yopD(Δ150-170) and yopD(Δ207-227) mutants and analyzing their T3SS functions. These strains exhibited wild-type levels of Yop secretion in vitro and enabled robust pore formation in macrophages. However, the yopDΔ150-170 and yopD(Δ207-227) mutants were defective in Yop translocation into CHO cells and splenocyte-derived neutrophils and macrophages. These data suggest that YopD-mediated host membrane disruption and effector Yop translocation are genetically separable activities requiring distinct protein domains. Importantly, the yopD(Δ150-170) and yopD(Δ207-227) mutants were defective in Yop-mediated inhibition of macrophage cell death and ROS production in neutrophil-like cells, and were attenuated in disseminated Yersinia infection. Therefore, the ability of the YopD central region to facilitate optimal effector protein delivery into phagocytes, and therefore robust effector Yop function, is important for Yersinia virulence.

YPTB3816 of Yersinia pseudotuberculosis strain IP32953 is a virulence-related metallo-oligopeptidase.

BACKGROUND: Although bacterial peptidases are known to be produced by various microorganisms, including pathogenic bacteria, their role in bacterial physiology is not fully understood. In particular, oligopeptidases are thought to be mainly involved in degradation of short peptides e.g. leader peptides released during classical protein secretion pathways. The aim of this study was to investigate effects of inactivation of an oligopeptidase encoding gene opdA gene of Yersinia pseudotuberculosis on bacterial properties in vivo and in vitro, and to test dependence of the enzymatic activity of the respective purified enzyme on the presence of different divalent cations. RESULTS: In this study we found that oligopeptidase OpdA of Yersinia pseudotuberculosis is required for bacterial virulence, whilst knocking out the respective gene did not have any effect on bacterial viability or growth rate in vitro. In addition, we studied enzymatic properties of this enzyme after expression and purification from E. coli. Using an enzyme depleted of contaminant divalent cations and different types of fluorescently labelled substrates, we found strong dependence of its activity on the presence of particular cations. Unexpectedly, Zn2+ showed stimulatory activity only at low concentrations, but inhibited the enzyme at higher concentrations. In contrast, Co2+, Ca2+ and Mn2+ stimulated activity at all concentrations tested, whilst Mg2+ revealed no effect on the enzyme activity at all concentrations used. CONCLUSIONS: The results of this study provide valuable contribution to the investigation of bacterial peptidases in general, and that of metallo-oligopeptidases in particular. This is the first study demonstrating that opdA in Yersinia pseudotuberculsosis is required for pathogenicity. The data reported are important for better understanding of the role of OpdA-like enzymes in pathogenesis in bacterial infections. Characterisation of this protein may serve as a basis for the development of novel antibacterials based on specific inhibition of this peptidase activity.

Morphogenesis of Experimental Infection Caused by Plasmid Variants of Yersinia pseudotuberculosis.

The dynamics of pathomorphological changes in response to infection with plasmid variants of Yersinia pseudotuberculosis was studied in experimental animals. Variability of cell injuries in pseudotuberculosis histopathology depended on the plasmid-associated virulence of the infection agent. Infection with highly virulent two-plasmid strain pYV48:pVM82 MDa and Y. pseudotuberculosis strain with low virulence with the only plasmid pVM82 MDa led to the development of cell destruction (necrosis and apoptosis) in the target organs. Apoptosis predominated in response to infection by plasmid variant pVM82 MDa with low virulence.

The pyruvate-tricarboxylic acid cycle node: a focal point of virulence control in the enteric pathogen Yersinia pseudotuberculosis.

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 [(13)C]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.

[IMMUNOCHEMICAL ACTIVITY OF YERSINIA PSEUDOTUBERCULOSIS B-ANTIGEN].

A hybridoma panel producing monoclonal antibodies to immunochemically non-identical antigenic epitopes of the protein nature located in outer membrane of Yersinia pseudotuberculosis was obtained. It was revealed that the previously identified B-antigen protecting laboratory animals from experimental plague was detected using both monoclonal antibodies against mentioned protein determinants and the determinants of lipopolysaccharide O-side chains. The B-antigen is a component of the Y. pseudotuberculosis outer membrane, egested in the form of the vesicles (OMVs) by bacteria cultivated in fluid nutrient medium.

[CHARACTERISTICS OF FORMATION, INHIBITION AND DESTRUCTION OF YERSINIA PSEUDOTUBERCULOSIS BIOFILMS FORMING ON ABIOTIC SURFACES].

AIM: Detection of conditions of Yersinia pseudotuberculosis biofilm formation, their quantitative testing. MATERIALS AND METHODS: Y. pseudotuberculosis strains, nutrient media, standard 96-well polystyrene plates, crystal violet dye as well as bacteriologic, spectrophotometric, statistical methods were used. RESULTS: All the studied Y pseudotuberculosis strains formed a well expressed biofilm on abiotic surface during cultivation of bacteria in 200 µl of a plate well at a temperature of 20-22°C for 4-7 days. Bacteria CFU number in biofilm reduced by day 10 of incubation. DNAse I was found to inhibit biofilm formation, and also partially destroyed mature Y. pseudotuberculosis biofilm. The presence of DNA in extra-cellular matrix of biofilm was shown. CONCLUSION: An ability of Y. pseudotuberculosis to form biofilm on abiotic surface was established. The conditions of biofilm formation were determined. Inhibiting effect of DNAse I on Y. pseudotuberculosis was shown.

The importance of the magnesium transporter MgtB for virulence of Yersinia pseudotuberculosis and Yersinia pestis.

Mg(2+) has been shown to be an important signal controlling gene regulation via the PhoPQ two-component regulatory system for a range of Gram-negative bacteria, including Yersinia pestis and Yersinia pseudotuberculosis. The magnesium ion transporter MgtB is part of the complex PhoPQ regulon, being upregulated in response to low Mg(2+). Despite the presence of other Mg(2+) transport systems in Yersinia, inactivation of mgtB had a significant effect on the ability of the bacteria to scavenge this crucial ion. Whereas inactivation of PhoPQ is reported to adversely affect intracellular survival, we show that Y. pestis and Y. pseudotuberculosis ΔmgtB mutants survived equally as well as the respective parent strain within macrophages, although they were more sensitive to killing in the Galleria model of infection. Surprisingly, despite MgtB being only one member of the Mg(2+) stimulon and PhoPQ controlling the expression levels of a range of genes including mgtB, the Yersinia ΔmgtB mutants were more highly attenuated than the equivalent Yersinia ΔphoP mutants in mouse models of infection. MgtB may be a suitable target for development of novel antimicrobials, and investigation of its role may help elucidate the contribution of this component of the PhoPQ regulon to pathogenesis.

Yersinia pseudotuberculosis disseminates directly from a replicating bacterial pool in the intestine.

Dissemination of Yersinia pseudotuberculosis within mice after oral inoculation was analyzed. Y. pseudotuberculosis translocated to organs such as the liver and spleen shortly after oral inoculation, but was quickly cleared. In contrast, a second temporally distinct bacterial translocation event resulted in successful hepatosplenic replication of the bacteria. Replicating pools of bacteria could be established in these organs in mouse mutants that lacked Peyer's patches. These animals frequently had sterile mesenteric lymph nodes, a finding consistent with translocation taking place independently of regional lymph node colonization. In further contradiction to accepted models for dissemination of enteropathogens, clonal analysis revealed that bacteria causing disease in the spleen and liver of C57BL/6J mice were derived from populations located outside the intestinal lymph nodes. Replication of bacteria in the intestine before translocation appeared critical for dissemination, as transient selective suppression by streptomycin of bacterial growth in the intestine delayed dissemination of Y. pseudotuberculosis. These results collectively indicate that hepatosplenic colonization appears intimately connected with the ability of Y. pseudotuberculosis to successfully establish replication in the intestinal lumen and does not result from ordered spread leading from the intestine to regional lymph nodes before dissemination.

Requirement for RNA helicase CsdA for growth of Yersinia pseudotuberculosis IP32953 at low temperatures.

The expression of csdA, encoding an RNA helicase, was induced at 3°C in Yersinia pseudotuberculosis. The role of CsdA in Y. pseudotuberculosis under cold conditions was confirmed by impaired growth of insertional csdA mutants at 3°C. The results suggest that CsdA is crucial for Y. pseudotuberculosis survival in the chilled food chain.

[Effect of volatile metabolites from germinating seeds on the reproduction of the bacteria Listeria monocytogenes and Yersinia pseudotuberculosis].

The biological activity of volatile metabolites of germinating seeds of cabbage (Brassica oleacia), carrot (Daukus carota), salad (Lactuca sativa), and corn (Zea mays L.) against Listeria monocytogenes and Yersinia pseudotuberculosis was studied. It was shown that volatile metabolites are transfer factors and can be the sole carbon and energy source for these bacteria. Methanol is the main substance affecting their growth and reproduction.

YopD self-assembly and binding to LcrV facilitate type III secretion activity by Yersinia pseudotuberculosis.

YopD-like translocator proteins encoded by several Gram-negative bacteria are important for type III secretion-dependent delivery of anti-host effectors into eukaryotic cells. This probably depends on their ability to form pores in the infected cell plasma membrane, through which effectors may gain access to the cell interior. In addition, Yersinia YopD is a negative regulator essential for the control of effector synthesis and secretion. As a prerequisite for this functional duality, YopD may need to establish molecular interactions with other key T3S components. A putative coiled-coil domain and an alpha-helical amphipathic domain, both situated in the YopD C terminus, may represent key protein-protein interaction domains. Therefore, residues within the YopD C terminus were systematically mutagenized. All 68 mutant bacteria were first screened in a variety of assays designed to identify individual residues essential for YopD function, possibly by providing the interaction interface for the docking of other T3S proteins. Mirroring the effect of a full-length yopD gene deletion, five mutant bacteria were defective for both yop regulatory control and effector delivery. Interestingly, all mutations clustered to hydrophobic amino acids of the amphipathic domain. Also situated within this domain, two additional mutants rendered YopD primarily defective in the control of Yop synthesis and secretion. Significantly, protein-protein interaction studies revealed that functionally compromised YopD variants were also defective in self-oligomerization and in the ability to engage another translocator protein, LcrV. Thus, the YopD amphipathic domain facilitates the formation of YopD/YopD and YopD/LcrV interactions, two critical events in the type III secretion process.