Pesticinogenic and Ca2+-dependent strains of Yersiniapestis harbored plasmids of about 6 and 45 megadaltons, respectively. In addition, most isolates examined possessed a cryptic 65-megadalton plasmid.
LcrV (V antigen), a known unstable 37.3-kDa monomeric peptide encoded on the ca. 70-kb Lcr plasmid of Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica, has been implicated as a regulator of the low-calcium response, virulence factor, and protective antigen. In this study, IcrV of Y. pestis was cloned into protease-deficient Escherichia coli BL21. The resulting recombinant V antigen underwent marked degradation from the C-terminal end during purification, yielding major peptides of 36, 35, 34, and 32 to 29 kDa. Rabbit gamma globulin raised against this mixture of cleavage products provided significant protection against 10 minimum lethal doses of Y. pestis (P < 0.01) and Y. pseudotuberculosis (P < 0.02). To both stabilize V antigen and facilitate its purification, plasmid pPAV13 was constructed so as to encode a fusion of krV and the structural gene for protein A (i.e., all but the first 67 N-terminal amino acids of V antigen plus the signal sequence and immunoglobulin G-binding domains but not the cell wall-associated region of protein A). The resulting fusion peptide, termed PAV, could be purified to homogeneity in one step by immunoglobulin G affinity chromatography and was stable thereafter. Rabbit polyclonal gamma globulin directed against PAV provided excellent passive immunity against 10 minimum lethal doses of Y. pestis (P < 0.005) and Y. pseudotuberculosis (P < 0.005) but was ineffective against Y. enterocolitica. Protection failed after absorption with excess PAV, cloned whole V antigen, or a large (31.5-kDa) truncated derivative of the latter but was retained (P < 0.005) upon similar absorption with a smaller (19.3-kDa) truncated variant, indicating that at least one protective epitope resides internally between amino acids 168 and 275.
A 37 but not 26 degrees C virulent Yersinia pestis is known to require at least 2.5 mM Ca2+ for growth; this requirement is potentiated by Mg2+. After shift of log-phase cells (doubling time of 2 h) from 26 to 37 degrees C in Ca2+-deficient medium, shutoff of net ribonucleic acid synthesis preceded that of protein and cell mass. With 2.5 mM Mg2+, about two doublings in cell mass and number occurred before restriction with synthesis of sufficient deoxyribonucleic acid to account for initiation and termination of two postshift rounds of chromosome replication. Temperature shift with 20 mMMg2+ resulted in a single doubling of cell mass and number with one round of chromosome replication. Subsequent to shutoff of ribonucleic acid accumulation, ribonucleoside but not deoxyribonucleoside triphosphate pools became reduced to about 50% of normal values and the adenylate energy change fell from about 0.8, typical of growing cells, to about 0.6. Excretion of significant concentrations of adenine nucleotides under both permissive and restrictive conditions was observed. Only trace levels (less than 0.01 microM ol/g [dry weight]) of guaninosine 5'-diphosphate 3'-diphosphate accumulated under restrictive or permissive conditions; guanosine 5'-triphosphate 3'-diphosphate was not detected. Return of fully restricted cells from 37 to 26 degrees C with Ca2+ resulted in prompt growth, whereas addition of Ca2+ at 37 degrees C was ineffective. This finding indicates that the observed temperature-sensitive lesion in ribonucleic acid synthesis that results in restriction can be prevented but not reversed by cultivation with Ca2+.
An avirulent guanine auxotroph of wild-type Yersinia pestis was used to select isogenic mutants lacking invasive determinants of virulence including V and W antigens (Vwa-), genetically linked fibrinolysin, coagulase, and pesticin activities (Pst-), and the capacity to absorb exogenous pesticin and pigments including hemin (Pgm-). After growth in environments known to favor expression of these factors by the parent, cells were converted to spheroplasts and disrupted to obtain preparations of cytoplasm; particulate matter was separated into inner and outer membranes by sucrose gradient centrifugation. Peptides present in these fractions were then solubilized and compared by two-dimensional polyacrylamide gel electrophoresis. Components unique to Vwa+ cells, including V antigen, were restricted to the cytoplasmic fraction. In contrast, peptides possibly corresponding to fibrinolysin and coagulase were located primarily within the outer membrane of the Pst+ parent; pesticin was not identified. Similarly, a major outer membrane peptide, possibly representing the pesticin and pigment receptor, was peculiar to the Pgm+ parent. Accordingly, two of the virulence factors examined (Pst+ and Pgm+) can interact directly with host cells or fluids by virtue of their location on the bacterial surface. The remaining cytoplasmic Vwa+ determinant remains a candidate for a regulatory system whose role in pathogenicity is expression of functions required for intracellular survival.
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