Sara Schesser Bartra

Resistance of Yersinia pestis to Complement-Dependent Killing Is Mediated by the Ail Outer Membrane Protein

ABSTRACT Yersinia pestis, the causative agent of plague, must survive in blood in order to cause disease and to be transmitted from host to host by fleas. Members of the Ail/Lom family of outer membrane proteins provide protection from complement-dependent killing for a number of pathogenic bacteria. The Y. pestis KIM genome is predicted to encode four Ail/Lom family proteins. Y. pestis mutants specifically deficient in expression of each of these proteins were constructed using lambda Red-mediated recombination. The Ail outer membrane protein was essential for Y. pestis to resist complement-mediated killing at 26 and 37°C. Ail was expressed at high levels at both 26 and 37°C, but not at 6°C. Expression of Ail in Escherichia coli provided protection from the bactericidal activity of complement. High-level expression of the three other Y. pestis Ail/Lom family proteins (the y1682, y2034, and y2446 proteins) provided no protection against complement-mediated bacterial killing. A Y. pestis ail deletion mutant was rapidly killed by sera obtained from all mammals tested except mouse serum. The role of Ail in infection of mice, Caenorhabditis elegans, and fleas was investigated.

Plasminogen Activator Pla of Yersinia pestis Utilizes Murine DEC-205 (CD205) as a Receptor to Promote Dissemination

Yersinia pestis, a Gram-negative bacterium that causes bubonic and pneumonic plague, is able to rapidly disseminate to other parts of its mammalian hosts. Y. pestis expresses plasminogen activator (PLA) on its surface, which has been suggested to play a role in bacterial dissemination. It has been speculated that Y. pestis hijacks antigen-presenting cells, such as macrophages (MΦs) and dendritic cells, to be delivered to lymph nodes to initiate dissemination and infection. Both alveolar MΦs and pulmonary dendritic cells express a C-type lectin receptor, DEC-205 (CD205), which mediates antigen uptake and presentation. However, no ligand has been identified for DEC-205. In this study, we show that the invasion of alveolar MΦsby Y. pestis depends both in vitro and in vivo on the expression of PLA. DEC-205-expressing MΦs and transfectants, but not their negative counterparts, phagocytosed PLA-expressing Y. pestis and Escherichia coli K12 more efficiently than PLA-negative controls. The interactions between PLA-expressing bacteria and DEC-205-expressing transfectants or alveolar MΦs could be inhibited by an anti-DEC-205 antibody. Importantly, the blockage of the PLA-DEC-205 interaction reduced the dissemination of Y. pestis in mice. In conclusion, murine DEC-205 is a receptor for PLA of Y. pestis, and this host-pathogen interaction appears to play a key role in promoting bacterial dissemination.

Yersinia pestis kills Caenorhabditis elegans by a biofilm-independent process that involves novel virulence factors

It is known that Yersinia pestis kills Caenorhabditis elegans by a biofilm‐dependent mechanism that is similar to the mechanism used by the pathogen to block food intake in the flea vector. Using Y. pestis KIM5, which lacks the genes that are required for biofilm formation, we show that Y. pestis can kill C. elegans by a biofilm‐independent mechanism that correlates with the accumulation of the pathogen in the intestine. We used this novel Y. pestis–C. elegans pathogenesis system to show that previously known and unknown virulence‐related genes are required for full virulence in C. elegans. Six Y. pestis mutants with insertions in genes that are not related to virulence before were isolated using C. elegans. One of the six mutants carried an insertion in a novel virulence gene and showed significantly reduced virulence in a mouse model of Y. pestis pathogenesis. Our results indicate that the Y. pestis–C. elegans pathogenesis system that is described here can be used to ide.jpgy and study previously uncharacterized Y. pestis gene products required for virulence in mammalian systems.

Identification and type III‐dependent secretion of the Yersinia pestis insecticidal‐like proteins

Plague, or the Black Death, is a zoonotic disease that is spread from mammal to mammal by fleas. This mode of transmission demands that the causative agent of this disease, Yersinia pestis, is able to survive and multiply in both mammals and insects. In recent years the complete genome sequence of a number of Y. pestis strains have been determined. This sequence information indicates that Y. pestis contains a cluster of genes with homology to insecticidal toxin encoding genes of the insect pathogen Photorhabdus luminescens. Here we demonstrate that Y. pestis KIM strains produced the encoded proteins. Production of the locus‐encoded proteins was dependent on a gene (yitR) encoding a member of the LysR family of transcriptional activators. Evidence suggests the proteins are type III secretion substrates. N terminal amino acids (100 to 367) of each protein fused to an epitope tag were secreted by the virulence plasmid type III secretion type. A fusion protein comprised of the N‐terminus of YipB and the enzymatic active component of Bordetella pertussis adenylate cyclase (Cya) was translocated into both mammalian and insect cells. In conclusion, a new class of Y. pestis type III secreted and translocated proteins has been identified. We hypothesize that these proteins function to promote transmission of and infection by Y. pestis.

Na +/H + Antiport Is Essential for Yersinia pestis Virulence

ABSTRACT Na+/H+ antiporters are ubiquitous membrane proteins that play a central role in the ion homeostasis of cells. In this study, we examined the possible role of Na+/H+ antiport in Yersinia pestis virulence and found that Y. pestis strains lacking the major Na+/H+ antiporters, NhaA and NhaB, are completely attenuated in an in vivo model of plague. The Y. pestis derivative strain lacking the nhaA and nhaB genes showed markedly decreased survival in blood and blood serum ex vivo. Complementation of either nhaA or nhaB in trans restored the survival of the Y. pestis nhaA nhaB double deletion mutant in blood. The nhaA nhaB double deletion mutant also showed inhibited growth in an artificial serum medium, Opti-MEM, and a rich LB-based medium with Na+ levels and pH values similar to those for blood. Taken together, these data strongly suggest that intact Na+/H+ antiport is indispensable for the survival of Y. pestis in the bloodstreams of infected animals and thus might be regarded as a promising noncanonical drug target for infections caused by Y. pestis and possibly for those caused by other blood-borne bacterial pathogens.

Calcium-Regulated Type III Secretion of Yop Proteins by an Escherichia coli hha Mutant Carrying a Yersinia pestis pCD1 Virulence Plasmid

ABSTRACT A series of four large deletions that removed a total of ca. 36 kb of DNA from the ca. 70-kb Yersinia pestis pCD1 virulence plasmid were constructed using lambda Red-mediated recombination. Escherichia coli hha deletion mutants carrying the virulence plasmid with the deletions expressed a functional calcium-regulated type III secretion system. The E. coli hha/pCD1 system should facilitate molecular studies of the type III secretion process.

The outer membrane protein A (OmpA) of Yersinia pestis promotes intracellular survival and virulence in mice.

The plague bacterium Yersinia pestis has a number of well-described strategies to protect itself from both host cells and soluble factors. In an effort to identify additional anti-host factors, we employed a transposon site hybridization (TraSH)-based approach to screen 10(5)Y. pestis mutants in an in vitro infection system. In addition to loci encoding various components of the well-characterized type III secretion system (T3SS), our screen unambiguously identified ompA as a pro-survival gene. We go on to show that an engineered Y. pestis ΔompA strain, as well as a ΔompA strain of the closely related pathogen Yersinia pseudotuberculosis, have fully functioning T3SSs but are specifically defective in surviving within macrophages. Additionally, the Y. pestis ΔompA strain was out competed by the wild-type strain in a mouse co-infection assay. Unlike in other bacterial pathogens in which OmpA can promote adherence, invasion, or serum resistance, the OmpA of Y. pestis is restricted to enhancing intracellular survival. Our data show that OmpA of the pathogenic Yersinia is a virulence factor on par with the T3SS.

ExsA and LcrF Recognize Similar Consensus Binding Sites, but Differences in Their Oligomeric State Influence Interactions with Promoter DNA

ExsA activates type III secretion system (T3SS) gene expression in Pseudomonas aeruginosa and is a member of the AraC family of transcriptional regulators. AraC proteins contain two helix-turn-helix (HTH) DNA binding motifs. One helix from each HTH motif inserts into the major groove of the DNA to make base-specific contacts with the promoter region. The amino acids that comprise the HTH motifs of ExsA are nearly identical to those in LcrF/VirF, the activators of T3SS gene expression in the pathogenic yersiniae. In this study, we tested the hypothesis that ExsA/LcrF/VirF recognize a common nucleotide sequence. We report that Yersinia pestis LcrF binds to and activates transcription of ExsA-dependent promoters in P. aeruginosa and that plasmid-expressed ExsA complements a Y. pestis lcrF mutant for T3SS gene expression. Mutations that disrupt the ExsA consensus binding sites in both P. aeruginosa and Y. pestis T3SS promoters prevent activation by ExsA and LcrF. Our combined data demonstrate that ExsA and LcrF recognize a common nucleotide sequence. Nevertheless, the DNA binding properties of ExsA and LcrF are distinct. Whereas two ExsA monomers are sequentially recruited to the promoter region, LcrF binds to promoter DNA as a preformed dimer and has a higher capacity to bend DNA. An LcrF mutant defective for dimerization bound promoter DNA with properties similar to ExsA. Finally, we demonstrate that the activators of T3SS gene expression from Photorhabdus luminescens, Aeromonas hydrophila, and Vibrio parahaemolyticus are also sensitive to mutations that disrupt the ExsA consensus binding site.

Yersinia pestis uses the Ail outer membrane protein to recruit vitronectin.

Yersinia pestis, the agent of plague, requires the Ail (attachment invasion locus) outer membrane protein to survive in the blood and tissues of its mammalian hosts. Ail is important for both attachment to host cells and for resistance to complement-dependent bacteriolysis. Previous studies have shown that Ail interacts with components of the extracellular matrix, including fibronectin, laminin and heparan sulfate proteoglycans, and with the complement inhibitor C4b-binding protein. Here, we demonstrate that Ail-expressing Y. pestis strains bind vitronectin - a host protein with functions in cell attachment, fibrinolysis and inhibition of the complement system. The Ail-dependent recruitment of vitronectin resulted in efficient cleavage of vitronectin by the outer membrane Pla (plasminogen activator protease). Escherichia coli DH5α expressing Y. pestis Ail bound vitronectin, but not heat-treated vitronectin. The ability of Ail to directly bind vitronectin was demonstrated by ELISA using purified refolded Ail in nanodiscs.

Host Langerin (CD207) is a receptor for Yersinia pestis phagocytosis and promotes dissemination

Yersinia pestis is a Gram‐negative bacterium that causes plague. After Y. pestis overcomes the skin barrier, it encounters antigen‐presenting cells (APCs), such as Langerhans and dendritic cells. They transport the bacteria from the skin to the lymph nodes. However, the molecular mechanisms involved in bacterial transmission are unclear. Langerhans cells (LCs) express Langerin (CD207), a calcium‐dependent (C‐type) lectin. Furthermore, Y. pestis possesses exposed core oligosaccharides. In this study, we show that Y. pestis invades LCs and Langerin‐expressing transfectants. However, when the bacterial core oligosaccharides are shielded or truncated, Y. pestis propensity to invade Langerhans and Langerin‐expressing cells decreases. Moreover, the interaction of Y. pestis with Langerin‐expressing transfectants is inhibited by purified Langerin, a DC‐SIGN (DC‐specific intercellular adhesion molecule 3 grabbing nonintegrin)‐like molecule, an anti‐CD207 antibody, purified core oligosaccharides and several oligosaccharides. Furthermore, covering core oligosaccharides reduces the mortality associated with murine infection by adversely affecting the transmission of Y. pestis to lymph nodes. These results demonstrate that direct interaction of core oligosaccharides with Langerin facilitates the invasion of LCs by Y. pestis. Therefore, Langerin‐mediated binding of Y. pestis to APCs may promote its dissemination and infection.