Mutually constructive roles of Ail and LPS in Yersinia pestis serum survival.

The outer membrane is a key virulence determinant of gram-negative bacteria. In Yersinia pestis, the deadly agent that causes plague, the protein Ail and lipopolysaccharide (LPS)6 enhance lethality by promoting resistance to human innate immunity and antibiotics, enabling bacteria to proliferate in the human host. Their functions are highly coordinated. Here we describe how they cooperate to promote pathogenesis. Using a multidisciplinary approach, we identify mutually constructive interactions between Ail and LPS that produce an extended conformation of Ail at the membrane surface, cause thickening and rigidification of the LPS membrane, and collectively promote Y. pestis survival in human serum, antibiotic resistance, and cell envelope integrity. The results highlight the importance of the Ail-LPS assembly as an organized whole, rather than its individual components, and provide a handle for targeting Y. pestis pathogenesis.

Complete Genome Assembly of Yersinia pestis subsp. pestis bv. Medievalis SCPM-O-B-6530, a Proline-Dependent Strain Isolated from the Central-Caucasian High-Mountain Plague Focus in Kabardino-Balkar Republic (Russia).

We report the complete genome assembly of Yersinia pestis subsp. pestis bv. Medievalis SCPM-O-B-6530, a strain belonging to the most ancient phylogenetic group (group 2.MED0) of Y. pestis subsp. pestis bv. Medievalis. This proline-dependent strain, carrying an additional plasmid (pCKF), was isolated from the Central-Caucasian high-mountain plague focus in Kabardino-Balkar Republic, Russia.

PgtE Enzyme of Salmonella enterica Shares the Similar Biological Roles to Plasminogen Activator (Pla) in Interacting With DEC-205 (CD205), and Enhancing Host Dissemination and Infectivity by Yersinia pestis.

Yersinia pestis, the cause of plague, is a newly evolved Gram-negative bacterium. Through the acquisition of the plasminogen activator (Pla), Y. pestis gained the means to rapidly disseminate throughout its mammalian hosts. It was suggested that Y. pestis utilizes Pla to interact with the DEC-205 (CD205) receptor on antigen-presenting cells (APCs) to initiate host dissemination and infection. However, the evolutionary origin of Pla has not been fully elucidated. The PgtE enzyme of Salmonella enterica, involved in host dissemination, shows sequence similarity with the Y. pestis Pla. In this study, we demonstrated that both Escherichia coli K-12 and Y. pestis bacteria expressing the PgtE-protein were able to interact with primary alveolar macrophages and DEC-205-transfected CHO cells. The interaction between PgtE-expressing bacteria and DEC-205-expressing transfectants could be inhibited by the application of an anti-DEC-205 antibody. Moreover, PgtE-expressing Y. pestis partially re-gained the ability to promote host dissemination and infection. In conclusion, the DEC-205-PgtE interaction plays a role in promoting the dissemination and infection of Y. pestis, suggesting that Pla and the PgtE of S. enterica might share a common evolutionary origin.

Identification of the lipopolysaccharide core of Yersinia pestis and Yersinia pseudotuberculosis as the receptor for bacteriophage φA1122.

φA1122 is a T7-related bacteriophage infecting most isolates of Yersinia pestis, the etiologic agent of plague, and used by the CDC in the identification of Y. pestis. φA1122 infects Y. pestis grown both at 20 °C and at 37 °C. Wild-type Yersinia pseudotuberculosis strains are also infected but only when grown at 37 °C. Since Y. pestis expresses rough lipopolysaccharide (LPS) missing the O-polysaccharide (O-PS) and expression of Y. pseudotuberculosis O-PS is largely suppressed at temperatures above 30 °C, it has been assumed that the phage receptor is rough LPS. We present here several lines of evidence to support this. First, a rough derivative of Y. pseudotuberculosis was also φA1122 sensitive when grown at 22 °C. Second, periodate treatment of bacteria, but not proteinase K treatment, inhibited the phage binding. Third, spontaneous φA1122 receptor mutants of Y. pestis and rough Y. pseudotuberculosis could not be isolated, indicating that the receptor was essential for bacterial growth under the applied experimental conditions. Fourth, heterologous expression of the Yersinia enterocolitica O:3 LPS outer core hexasaccharide in both Y. pestis and rough Y. pseudotuberculosis effectively blocked the phage adsorption. Fifth, a gradual truncation of the core oligosaccharide into the Hep/Glc (L-glycero-D-manno-heptose/D-glucopyranose)-Kdo/Ko (3-deoxy-D-manno-oct-2-ulopyranosonic acid/D-glycero-D-talo-oct-2-ulopyranosonic acid) region in a series of LPS mutants was accompanied by a decrease in phage adsorption, and finally, a waaA mutant expressing only lipid A, i.e., also missing the Kdo/Ko region, was fully φA1122 resistant. Our data thus conclusively demonstrated that the φA1122 receptor is the Hep/Glc-Kdo/Ko region of the LPS core, a common structure in Y. pestis and Y. pseudotuberculosis.

Whole-Genome Assembly of Yersinia pestis 231, the Russian Reference Strain for Testing Plague Vaccine Protection.

We report the whole-genome sequence of Yersinia pestis subsp. pestis bv. Antiqua strain 231 belonging to the 0.ANT3 phylogroup, the reference strain for testing plague vaccine protection in Russia. Genome sequencing was completed using the Oxford Nanopore MinION and Illumina platforms.

Lipopolysaccharide of the Yersinia pseudotuberculosis Complex.

Lipopolysaccharide (LPS), localized in the outer leaflet of the outer membrane, serves as the major surface component of the Gram-negative bacterial cell envelope responsible for the activation of the host's innate immune system. Variations of the LPS structure utilized by Gram-negative bacteria promote survival by providing resistance to components of the innate immune system and preventing recognition by TLR4. This review summarizes studies of the biosynthesis of Yersinia pseudotuberculosis complex LPSs, and the roles of their structural components in molecular mechanisms of yersiniae pathogenesis and immunogenesis.

Peptidoglycan-Free Bacterial Ghosts Confer Enhanced Protection against Yersinia pestis Infection.

To develop a modern plague vaccine, we used hypo-endotoxic Yersinia pestis bacterial ghosts (BGs) with combinations of genes encoding the bacteriophage ɸX174 lysis-mediating protein E and/or holin-endolysin systems from λ or L-413C phages. Expression of the protein E gene resulted in the BGs that retained the shape of the original bacterium. Co-expression of this gene with genes coding for holin-endolysin system of the phage L-413C caused formation of structures resembling collapsed sacs. Such structures, which have lost their rigidity, were also formed as a result of the expression of only the L-413C holin-endolysin genes. A similar holin-endolysin system from phage λ containing mutated holin gene S and intact genes R-Rz coding for the endolysins caused generation of mixtures of BGs that had (i) practically preserved and (ii) completely lost their original rigidity. The addition of protein E to the work of this system shifted the equilibrium in the mixture towards the collapsed sacs. The collapse of the structure of BGs can be explained by endolysis of peptidoglycan sacculi. Immunizations of laboratory animals with the variants of BGs followed by infection with a wild-type Y. pestis strain showed that bacterial envelopes protected only cavies. BGs with maximally hydrolyzed peptidoglycan had a greater protectivity compared to BGs with a preserved peptidoglycan skeleton.

Temperature-induced changes in the lipopolysaccharide of Yersinia pestis affect plasminogen activation by the pla surface protease.

The Pla surface protease of Yersinia pestis activates human plasminogen and is a central virulence factor in bubonic and pneumonic plague. Pla is a transmembrane beta-barrel protein and member of the omptin family of outer membrane proteases which require bound lipopolysaccharide (LPS) to be proteolytically active. Plasminogen activation and autoprocessing of Pla were dramatically higher in Y. pestis cells grown at 37 degrees C than in cells grown at 20 degrees C; the difference in enzymatic activity by far exceeded the increase in the cellular content of the Pla protein. Y. pestis modifies its LPS structure in response to growth temperature. We purified His(6)-Pla under denaturing conditions and compared various LPS types for their capacity to enhance plasmin formation by His(6)-Pla solubilized in detergent. Reactivation of His(6)-Pla was higher with Y. pestis LPSs isolated from bacteria grown at 37 degrees C than with LPSs from cells grown at 25 degrees C. Lack of O antigens and the presence of the outer core region as well as a lowered level of acylation in LPS were found to enhance the Pla-LPS interaction. Genetic substitution of arginine 138, which is part of a three-dimensional protein motif for binding to lipid A phosphates, decreased both the enzymatic activity of His(6)-Pla and the amount of Pla in Y. pestis cells, suggesting the importance of the Pla-lipid A phosphate interaction. The temperature-induced changes in LPS are known to help Y. pestis to avoid innate immune responses, and our results strongly suggest that they also potentiate Pla-mediated proteolysis.

Tetraacylated lipopolysaccharide of Yersinia pestis can inhibit multiple Toll-like receptor-mediated signaling pathways in human dendritic cells.

BACKGROUND: Yersinia pestis, the causative agent of plague, showed a temperature-dependent change in lipid A composition, with a reduced degree of acylation when bacteria were grown at 37 degrees C (tetraacylated) versus ambient temperature (hexaacylated). METHODS: Human monocytes and monocyte-derived dendritic cells (DCs) were exposed to Y. pestis grown at 26 degrees C or 37 degrees C, to their corresponding lipopolysaccharides (LPS-26 degrees C or LPS-37 degrees C), and to ligands of different Toll-like receptors (TLRs), such as LPS from Escherichia coli (TLR4), lipoprotein (TLR2), polyinosinic-polycytidylic acid (poly-IC) (TLR9), and their combinations. Production of cytokines was measured, along with expression of surface markers of DC maturation. RESULTS: Y. pestis grown at 37 degrees C or LPS-37 degrees C induced much lower production of cytokines (such as tumor necrosis factor alpha and interleukins 1beta, 10, and 12) by DCs than did Y. pestis grown at 26 degrees C or LPS-26 degrees C. Expression of the surface markers HLA-DR, CD86, and CD40 by DCs was also reduced in response to treatment with LPS-37 degrees C compared with LPS-26 degrees C. Pretreatment of DCs with LPS-37 degrees C inhibited subsequent stimulation with LPS-26 degrees C, control LPS from E. coli, lipoprotein, or poly-IC. CONCLUSIONS: LPS-37 degrees C can inhibit stimulation of DCs not only via TLR4 signaling but also via TLR2 and TLR3. [corrected]

Yersinia pestis Plasminogen Activator.

The Gram-negative bacterium Yersinia pestis causes plague, a fatal flea-borne anthropozoonosis, which can progress to aerosol-transmitted pneumonia. Y. pestis overcomes the innate immunity of its host thanks to many pathogenicity factors, including plasminogen activator, Pla. This factor is a broad-spectrum outer membrane protease also acting as adhesin and invasin. Y. pestis uses Pla adhesion and proteolytic capacity to manipulate the fibrinolytic cascade and immune system to produce bacteremia necessary for pathogen transmission via fleabite or aerosols. Because of microevolution, Y. pestis invasiveness has increased significantly after a single amino-acid substitution (I259T) in Pla of one of the oldest Y. pestis phylogenetic groups. This mutation caused a better ability to activate plasminogen. In paradox with its fibrinolytic activity, Pla cleaves and inactivates the tissue factor pathway inhibitor (TFPI), a key inhibitor of the coagulation cascade. This function in the plague remains enigmatic. Pla (or pla) had been used as a specific marker of Y. pestis, but its solitary detection is no longer valid as this gene is present in other species of Enterobacteriaceae. Though recovering hosts generate anti-Pla antibodies, Pla is not a good subunit vaccine. However, its deletion increases the safety of attenuated Y. pestis strains, providing a means to generate a safe live plague vaccine.

Proteus mirabilis Targets Atherosclerosis Plaques in Human Coronary Arteries via DC-SIGN (CD209).

Bacterial DNAs are constantly detected in atherosclerotic plaques (APs), suggesting that a combination of chronic infection and inflammation may have roles in AP formation. A series of studies suggested that certain Gram-negative bacteria were able to interact with dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin [DC-SIGN; cluster of differentiation (CD) 209] or langerin (CD207), thereby resulting in deposition of CD209s at infection sites. We wondered if Proteus mirabilis (a member of Proteobacteria family) could interact with APs through CD209/CD207. In this study, we first demonstrated that CD209/CD207 were also receptors for P. mirabilis that mediated adherence and phagocytosis by macrophages. P. mirabilis interacted with fresh and CD209s/CD207-expressing APs cut from human coronary arteries, rather than in healthy and smooth arteries. These interactions were inhibited by addition of a ligand-mimic oligosaccharide and the coverage of the ligand, as well as by anti-CD209 antibody. Finally, the hearts from an atherosclerotic mouse model contained higher numbers of P. mirabilis than that of control mice during infection-challenging. We therefore concluded that the P. mirabilis interacts with APs in human coronary arteries via CD209s/CD207. It may be possible to slow down the progress of atherosclerosis by blocking the interactions between CD209s/CD207 and certain atherosclerosis-involved bacteria with ligand-mimic oligosaccharides.

Yersinia Outer Membrane Vesicles as Potential Vaccine Candidates in Protecting against Plague.

Despite the relatively low incidence of plague, its etiological agent, Yersinia pestis, is an exceptional epidemic danger due to the high infectivity and mortality of this infectious disease. Reports on the isolation of drug-resistant Y. pestis strains indicate the advisability of using asymmetric responses, such as phage therapy and vaccine prophylaxis in the fight against this problem. The current relatively effective live plague vaccine is not approved for use in most countries because of its ability to cause heavy local and system reactions and even a generalized infectious process in people with a repressed immune status or metabolic disorders, as well as lethal infection in some species of nonhuman primates. Therefore, developing alternative vaccines is of high priority and importance. However, until now, work on the development of plague vaccines has mainly focused on screening for the potential immunogens. Several investigators have identified the protective potency of bacterial outer membrane vesicles (OMVs) as a promising basis for bacterial vaccine candidates. This review is aimed at presenting these candidates of plague vaccine and the results of their analysis in animal models.

The subcutaneous inoculation of pH 6 antigen mutants of Yersinia pestis does not affect virulence and immune response in mice.

Two isogenic sets of Yersinia pestis strains were generated, composed of wild-type strains 231 and I-1996, their non-polar pH 6(-) mutants with deletions in the psaA gene that codes for its structural subunit or the whole operon, as well as strains with restored ability for temperature- and pH-dependent synthesis of adhesion pili or constitutive production of pH 6 antigen. The mutants were generated by site-directed mutagenesis of the psa operon and subsequent complementation in trans. It was shown that the loss of synthesis or constitutive production of pH 6 antigen did not influence Y. pestis virulence or the average survival time of subcutaneously inoculated BALB/c naïve mice or animals immunized with this antigen.

Yersinia pestis Interacts With SIGNR1 (CD209b) for Promoting Host Dissemination and Infection.

Yersinia pestis, a Gram-negative bacterium and the etiologic agent of plague, has evolved from Yersinia pseudotuberculosis, a cause of a mild enteric disease. However, the molecular and biological mechanisms of how Y. pseudotuberculosis evolved to such a remarkably virulent pathogen, Y. pestis, are not clear. The ability to initiate a rapid bacterial dissemination is a characteristic hallmark of Y. pestis infection. A distinguishing characteristic between the two Yersinia species is that Y. pseudotuberculosis strains possess an O-antigen of lipopolysaccharide (LPS) while Y. pestis has lost the O-antigen during evolution and therefore exposes its core LPS. In this study, we showed that Y. pestis utilizes its core LPS to interact with SIGNR1 (CD209b), a C-type lectin receptor on antigen presenting cells (APCs), leading to bacterial dissemination to lymph nodes, spleen and liver, and the initiation of a systemic infection. We therefore propose that the loss of O-antigen represents a critical step in the evolution of Y. pseudotuberculosis into Y. pestis in terms of hijacking APCs, promoting bacterial dissemination and causing the plague.

Reinvestigation of the O-antigens of Yersinia pseudotuberculosis: revision of the O2c and confirmation of the O3 antigen structures.

Structures of the O-antigens of Yersinia pseudotuberculosis O2c and O3 were reinvestigated by NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY, (1)H,(13)C HSQC, and HMBC experiments. The following revised structure of the O2c tetrasaccharide repeating unit was established, which differs from the structure proposed earlier in the glycosylation pattern of the mannose residue at the branching point: [carbohydrate structure: see text] where Abe stands for 3,6-dideoxy-d-xylo-hexose. The structure of the Y. pseudotuberculosis O3 antigen reported earlier was confirmed.

Nine Whole-Genome Assemblies of Yersinia pestis subsp. microtus bv. Altaica Strains Isolated from the Altai Mountain Natural Plague Focus (No. 36) in Russia.

We report here the draft genome sequences of nine Yersinia pestis subsp. microtus bv. Altaica strains isolated from the Altai Mountain plague focus (no. 36), which represent the 0.PE4 phylogroup circulating in populations of Mongolian pika (Ochotona pallasi).

Six Whole-Genome Assemblies of Yersinia pestis subsp. microtus bv. ulegeica (Phylogroup 0.PE5) Strains Isolated from Mongolian Natural Plague Foci.

Here, we report the draft genome sequences of six Yersinia pestis subsp. microtus bv. ulegeica strains isolated from the territory of Mongolia and representing the 0.PE5 phylogroup circulating in populations of voles and picas.

Effect of deletion of the lpxM gene on virulence and vaccine potential of Yersinia pestis in mice.

Yersinia pestis undergoes an obligate flea-rodent-flea enzootic life cycle. The rapidly fatal properties of Y. pestis are responsible for the organism's sustained survival in natural plague foci. Lipopolysaccharide (LPS) plays several roles in Y. pestis pathogenesis, prominent among them being resistance to host immune effectors and induction of a septic-shock state during the terminal phases of infection. LPS is acylated with 4-6 fatty acids, the number varying with growth temperature and affecting the molecule's toxic properties. Y. pestis mutants were constructed with a deletion insertion in the lpxM gene in both virulent and attenuated strains, preventing the organisms from synthesizing the most toxic hexa-acylated lipid A molecule when grown at 25 degrees C. The virulence and/or protective potency of pathogenic and attenuated Y. pestis DeltalpxM mutants were then examined in a mouse model. The DeltalpxM mutation in a virulent strain led to no change in the LD(50) value compared to that of the parental strain, while the DeltalpxM mutation in attenuated strains led to a modest 2.5-16-fold reduction in virulence. LPS preparations containing fully hexa-acylated lipid A were ten times more toxic in actinomycin D-treated mice then preparations lacking this lipid A isoform, although this was not significant (P>0.05). The DeltalpxM mutation in vaccine strain EV caused a significant increase in its protective potency. These studies suggest there is little impact from lipid A modifications on the virulence of Y. pestis strains but there are potential improvements in the protective properties in attenuated vaccine strains.

Variability of the protein sequences of lcrV between epidemic and atypical rhamnose-positive strains of Yersinia pestis.

Sequencing of lcrV genes and comparison of the deduced amino acid sequences from ten Y. pestis strains belonging mostly to the group of atypical rhamnose-positive isolates (non-pestis subspecies or pestoides group) showed that the LcrV proteins analyzed could be classified into five sequence types. This classification was based on major amino acid polymorphisms among LcrV proteins in the four "hot points" of the protein sequences. Some additional minor polymorphisms were found throughout these sequence types. The "hot points" corresponded to amino acids 18 (Lys --> Asn), 72 (Lys --> Arg), 273 (Cys --> Ser), and 324-326 (Ser-Gly-Lys --> Arg) in the LcrV sequence of the reference Y. pestis strain CO92. One possible explanation for polymorphism in amino acid sequences of LcrV among different strains is that strain-specific variation resulted from adaptation of the plague pathogen to different rodent and lagomorph hosts.

Relationship of the lipopolysaccharide structure of Yersinia pestis to resistance to antimicrobial factors.

Disruption of lipopolysaccharide (LPS) biosynthesis genes in an epidemiologically significant Yersinia pestis strain showed that the ability to synthesize the full inner core of the LPS is crucial for resistances to the bactericidal action of antimicrobial peptides and to complement-mediated serum killing. Resistance to polymyxin B also requires a high content of the cationic sugar, 4-amino-4-deoxy-L-arabinose, in lipid A.