Growth of calcium-blind mutants of Yersinia pestis at 37 degrees C in permissive Ca2+-deficient environments.

Cells of wild-type Yersinia pestis exhibit a low-calcium response (LCR) defined as bacteriostasis with expression of a pCD-encoded type III secretion system (T3SS) during cultivation at 37 degrees C without added Ca(2+) versus vegetative growth with downregulation of the T3SS with Ca(2+) (>or=2.5 mM). Bacteriostasis is known to reflect cumulative toxicity of Na(+), l-glutamic acid and culture pH; control of these variables enables full-scale growth ('rescue') in the absence of Ca(2+). Several T3SS regulatory proteins modulate the LCR, because their absence promotes a Ca(2+)-blind phenotype in which growth at 37 degrees C ceases and the T3SS is constitutive even with added Ca(2+). This study analysed the connection between the LCR and Ca(2+) by determining the response of selected Ca(2+)-blind mutants grown in Ca(2+)-deficient rescue media containing Na(+) plus l-glutamate (pH 5.5), where the T3SS is not expressed, l-glutamate alone (pH 6.5), where l-aspartate is fully catabolized, and Na(+) alone (pH 9.0), where the electrogenic sodium pump NADH : ubiquinone oxidoreductase becomes activated. All three conditions supported essentially full-scale Ca(2+)-independent growth at 37 degrees C of wild-type Y. pestis as well as lcrG and yopN mutants (possessing a complete but dysregulated T3SS), indicating that bacteriostasis reflects a Na(+)-dependent lesion in bioenergetics. In contrast, mutants lacking the negative regulator YopD or the YopD chaperone (LcrH) failed to grow in any rescue medium and are therefore truly temperature-sensitive. The Ca(2+)-blind yopD phenotype was fully suppressed in a Ca(2+)-independent background lacking the injectisome-associated inner-membrane component YscV but not peripheral YscK, suggesting that the core translocon energizes YopD.

yadBC of Yersinia pestis, a new virulence determinant for bubonic plague.

In all Yersinia pestis strains examined, the adhesin/invasin yadA gene is a pseudogene, yet Y. pestis is invasive for epithelial cells. To identify potential surface proteins that are structurally and functionally similar to YadA, we searched the Y. pestis genome for open reading frames with homology to yadA and found three: the bicistronic operon yadBC (YPO1387 and YPO1388 of Y. pestis CO92; y2786 and y2785 of Y. pestis KIM5), which encodes two putative surface proteins, and YPO0902, which lacks a signal sequence and likely is nonfunctional. In this study we characterized yadBC regulation and tested the importance of this operon for Y. pestis adherence, invasion, and virulence. We found that loss of yadBC caused a modest loss of invasiveness for epithelioid cells and a large decrease in virulence for bubonic plague but not for pneumonic plague in mice.

Yersinia pestis YadC: a novel vaccine candidate against plague.

Current subunit vaccines provide partial protection against pneumonic plague if the infecting Y. pestis strain is encapsulated (F1+). Here we describe YadC, a novel Y. pestis outer membrane protein that provides partial protection against a F1(-) Y. pestis strain. Swiss-Webster mice were immunized subcutaneously with glutathione S-transferase (GST) or His6-tagged (HT) purified fusion proteins (GST-YadC137-409 or HT-LcrV) or buffer emulsified with Alhydrogel. Intravenous challenge with 1 x 10(4) F1(-) Deltapgm Y. pestis CO99-3015 revealed no protection for those mice immunized with GST-Alhydrogel alone, full protection for HT-LcrV-immunized mice, and partial protection for GST-YadC137-409-immunized mice. Similarly, C57BL/6 mice were immunized with GST-YadC137-409, HT-LcrV, or GST all with Alhydrogel adjuvant. After intranasal challenge with 3 x 10(3) F1(-) Y. pestis CO99-3015, 87% of GST-YadC137-409-immunized mice survived pneumonic plague. This is compared to the GST control group (0 surviving mice) and the LcrV-immunized group where 50% survived the challenge. This protection was correlated with a predominantly IgG1 response in LcrV-immunized mice and an IgG1/IgG3 antibody response in YadC-immunized mice. Additionally, we report the cytokine response from HT-LcrV- and GST-YadC137-409-stimulated peripherally derived macrophages. YadC-stimulated cells demonstrated a predominant pro-inflammatory cytokine production. This mixed Thl/Th2 response suggests that YadC's protection may involve a different adaptive immune response than the LcrV protein that currently is part of plague vaccines.

A surface-focused biotinylation procedure identifies the Yersinia pestis catalase KatY as a membrane-associated but non-surface-located protein.

This study identified major surface proteins of the plague bacterium Yersinia pestis. We applied a novel surface biotinylation method, followed by NeutrAvidin (NA) bead capture, on-bead digestion, and identification by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The use of stachyose during biotinylation focused the reaction to the surface. Coupled with NA pulldown and immunoblot analysis, this method determined whether a protein was accessible to the surface. We applied the method to test the hypothesis that the catalase KatY is a surface protein of the plague bacterium Y. pestis. A rabbit serum recognized the catalase KatY as a major putative outer membrane-associated antigen expressed by Y. pestis cells grown at 37 degrees C. Similar findings by other groups had led to speculations that this protein might be exposed to the surface and might be a candidate for evaluation as a protective antigen for an improved plague vaccine. KatY was obtained only in the total membrane fraction, and stachyose greatly reduced its biotinylation as well as that of the periplasmic maltose binding protein, indicating that KatY is not on the bacterial surface. LC-MS-MS analysis of on-bead digests representing ca. 10(9) cells identified highly abundant species, including KatY, Pal, and OmpA, as well as the lipoprotein Pcp, all of which bound in a biotin-specific manner. Pla, Lpp, and OmpX (Ail) bound to the NA beads in a non-biotin-specific manner. There was no contamination from abundant cytoplasmic proteins. We hypothesize that OmpX and Pcp are highly abundant and likely to be important for the Y. pestis pathogenic process. We speculate that a portion of KatY associates with the outer membrane in intact cells but that it is located on the periplasmic side. Consistent with this idea, it did not protect C57BL/6 mice against bubonic plague.

Mice naturally resistant to Yersinia pestis Delta pgm strains commonly used in pathogenicity studies.

We report that females of some substrains of inbred mouse strain 129 are resistant to systemic plague due to conditionally virulent Deltapgm strains of Yersinia pestis; however, fully virulent Y. pestis is not attenuated in these mice. Therefore, these mice offer a powerful system in which to map in parallel host resistance traits and opposing bacterial virulence properties for plague.