Novel Virulence Factors in the Pathogenesis of Yersinia pestis Infection, the Causative Agent of Plague
Fitts, Eric Charles
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Identification of new virulence factors in Yersinia pestis, the causative agent of plague, and understanding their molecular mechanisms during an infection process are necessary in designing a better vaccine or to formulate an appropriate therapeutic intervention. By using a high-throughput, signature-tagged mutagenic approach, we screened 5,088 mutants of Y. pestis CO92. From this screen, 118 clones showing impairment in disseminating to spleen were obtained. In a subsequent screen, 20/118 mutants exhibited attenuation when tested individually in a mouse model of bubonic plague, with 10/20 aforementioned mutants providing 40% or higher survival rates at an infectious dose of 40 LD50. Upon sequencing, six of the attenuated mutants carried interruptions in genes encoding hypothetical proteins or proteins with putative functions. In-frame deletion mutation of two of the genes identified from the screen were also found to exhibit some attenuation at 11-12 LD50 in a mouse model of pneumonic plague. Likewise, among the remaining 18 signature-tagged mutants, 9 were also attenuated (40-100%) at 12 LD50 in a pneumonic plague mouse model. Combinatorial deletions including the newly identified genes, rbsA and vasK, were significantly attenuated in pneumonic plague models. Interestingly, rbsA gene products have been associated with a highly conserved inter-bacterial signaling system mediated by autoinducer-2 (AI-2) quorum-sensing molecule. Deletion of the gene encoding the synthetic enzyme for AI-2 substrate, luxS, leads to either no change or, paradoxically, an increase in in vivo bacterial virulence. Deletion of rbsA and lsrA genes, ABC transport components interacting with AI-2, synergistically disrupted AI-2 signaling patterns and resulted in an over 50-fold decrease in Y. pestis CO92 virulence in a mouse model. Deletion of luxS from the ΔrbsAΔlsrA strain reverted the virulence phenotype similar to wild-type CO92. Administration of AI-2 in mice infected with the ΔrbsAΔlsrAΔluxS mutant strain attenuated this triple mutant. Role of AI-2 signaling genes that modulated bacterial virulence was determined by RNAseq. Characterization of AI-2 signaling in Y. pestis should lead to re-examination of AI-2 systems in other pathogens and may represent a broad-spectrum therapeutic target to combat antibiotic-resistant bacteria.