Biological Effects Of Yersinia Pestis YscW On Host Murine Macrophages

Plague is an ancient deadly disease caused by Yersinia pestis, which is one of the most devastating diseases in human history. In the early stage of plague development, intracellular survival is an important mechanism for the bacterial proliferation and virulence. The virulence of the pathogenic Yersinia species depends on a plasmid- encoded type III secretion system (T3SS) that transfers six Yop effector proteins into the cytosol of macrophages, leading to disruption of host defense mechanisms.It is shown in this report that Yersinia pestis YscW contribute to the induction of apoptosis in murine macrophages and significantly related to caspase-3 signal pathways. The yscW mutant strain decreased more significantly the apoptotic percentages of macrophages than that of wild type Yersinia pestis, and the yscW complement could significantly recover the apoptotic changes induced by yscW mutation both in vivo and in vitro. Meanwhile, expression of caspase-3, the key apoptosis inducing protein, showed coincident results with the macrophages apoptosis changes separated infection with infections by different strains. However, YscW over-expression in RAW264.7 can not increase the macrophages apoptosis and death. These results indicated Yersinia pestis YscW only could indirectly induce macrophages apoptosis, and not the direct macrophage toxicity effects. To study the mechanism of this phenomenon further, we observe the effect of wild, mutant and complement strains respectively on the secretion of YopJ, which was thought the only effector related to apoptosis. Results showed that in yscW mutant strain, secretion of YopJ was decreased significantly in the supernatant. This means although YscW does not induce apoptosis directly, it can indirectly affect apoptosis through reducing the secretion of YopJ.Further, the functional study of infected macrophage showed that Yersinia pestis YscW contributes to the induction of deficiency in phagocytosis and reduction of antigen presenting capacity of host macrophages. Yersinia pestis strain lacking yscW had no effect on uptake of host macrophages. When infected with wild type, yscW mutant or complement strain, immunodeficiency was observed in the host macrophages compared to those from uninfected mice. However, the phagocytosis and antigen presenting capacity to OVA (ovalbumin) of macrophages infected by yscW mutant strain both in vivo and in vitro were significantly higher than those by wild type strain. In consistent, when the yscW gene was over-expressed in the RAW264.7 cells, the phagocytosis capacity and antigen presenting capacity were also significantly lower than the control groups. These results indicated that Y. pestis YscW could directly induce immunodeficiency in murine macrophages by crippling its phagocytosis and antigen presenting capacity. It provides further evidences to Y. pestis pathogenesis that some proteins in T3SS injectisome, such as YscW protein, might play independent roles in disrupting the host defense apart from their known functions.In addition, two proteins of YscW and Rnf149 with GST tag or His tag respectively, were expressed and purified. The assay of GST-Pull Down clear showed the interaction between these two proteins in vitro, which proved the evidence for the further assays.This work studied the biological effects of Yersinia pestis YscW on host murine macrophages and RAW264.7 cells, and evaluated the direct role of YscW during the infection, especially on the phagocytosis and antigen presenting capacity of macrphages. GST-Pull down indentified the interaction between YscW and macrophage Rnf149 in vitro. This study showed new roles of institute protein of Yersina pestis T3SS, which was important for another relative researches.