Intracellular Yersinia pestis expresses general stress response and tellurite resistance proteins in mouse macrophages

Yersinia pestis inoculated subcutaneously via fleabite or experimental injection in natural rodent hosts multiply initially in macrophage phagolysosomes. Survival and multiplication of Y. pestis in this acidic low [Ca2+] and [Mg2+] environment likely necessitates compensatory mechanisms involving expression of specific proteins compared to those expressed during extracellular growth. A proteomics approach was used to identify these proteins using mouse macrophage RAW264.7 cells infected with Y. pestis strain KIM6-2053.1+ for 8 h. Intracellular Y. pestis protein samples were prepared by detergent lysis of infected RAW264.7 cells, isolation of intracellular Y. pestis by differential centrifugation, and sonication of isolated Y. pestis. Protein samples were similarly prepared from Y. pestis grown extracellularly in tissue culture media. Two intracellular and extracellular Y. pestis protein samples were analyzed by two-dimensional polyacrylamide gel electrophoresis and compared in silico identifying 12 protein spots present in both intracellular samples but absent in extracellularly grown Y. pestis. Mass spectrometry analysis of these identified nine proteins at a high level of confidence in the Y. pestis genome: superoxide dismutase-A (sodA), inorganic pyrophosphatase, autonomous glycyl radical cofactor GrcA, molecular chaperone DnaK, serine endoprotease GsrA, global DNA-binding transcriptional dual regulator H-NS, urease subunit gamma UreA, and tellurite resistance proteins TerD and TerE. These results support the involvement of various general stress response regulators of Y. pestis during the intracellular parasitism of host macrophages as well as identification of UreA, TerD and TerE with as yet unknown roles in the process of intracellular survival of Y. pestis.