Identification and Characterization of the Virulence Determinant of the 9.5 Kilobase Plasmid of Yersinia Pestis: a Thesis

Abstract

The pathogenicity of Yersinia pestis, the causative agent of plague, is specified by chromosomal and plasmid encoded genes. At least two plasmids, with sizes of 9.5 and 75 kilobases, are indispensable to the full expression of virulence. Loss of the 75 kb plasmid results in outright avirulence. Strains lacking the 9.5 kb plasmid exhibit LD50s at least six orders of magnitude greater than wild-type following subcutaneous or intraperitoneal infection of mice or guinea-pigs but have LD50s as low as wild-type when injected intravenously. Four biochemical properties are associated with the 9.5 kb plasmid. These include plasminogen activator and coagulase activities in addition to the bacteriocin pesticin, and its immunity determinant. A genetic analysis of this plasmid was undertaken as a first step towards the identification and characterization of its virulence determinant(s). This led to the construction of a physical and genetic map of the plasmid. Four loci were mapped to the plasmid: pst and pim, which encode pesticin and its immunity determinant respectively; pla, which encodes both plasminogen activator and coagulase activities; and ori/inc, the locus containing both the origin of replication and the region responsible for the control of plasmid incompatibility. pst was shown to encode a 45 kD protein but the pim gene product was not identified. pla encodes two outer membrane proteins, α- and β-Pla of 37 and 35 kD, respectively, the latter being derived from the former most probably by a proteolytic processing event. At least one of these proteins is responsible for the highly specific degradation of the YOPs, a set of virulence-associated outer membrane proteins encoded by the 75 kb plasmid. The nucleotide sequence of pla revealed that it possessed significant homology to both prtA (geneE) of Salmonella typhimurium and ompT of Escherichia coli. Subcutaneous infection of mice with isogenic strains of Y. pestis harboring well defined mutations in the genes that reside on the 9.5 kb plasmid revealed that pla is a virulence determinant of Y. pestis, and also that of the genes harbored by the plasmid, pla is both necessary and sufficient to account for the high degree of virulence of Y. pestis for mice from subcutaneous sites of infection. pla encodes an activator of human, rat, and mouse plasminogen but does not induce coagulation of plasma obtained from these species. Treatment of mice with the antifibrinolytic agent, trans-4(aminomethyl)-cyclohexanecarboxylic acid did not affect the outcome of plague infection, indicating that fibrinolysis per se does not play a role in plague pathogenesis.