Study Of Yersinia Pestis Degrading Its Secreted Murine Toxin

Plague is an awful and natural infectious focus disease caused by Yersinia pestis. In human history, three major plague pandemics have ever occurred, which once brought hideous disaster to humans. In China, plague is defined as Class A infectious diseases, plague natural infectious foci range widely, accounting for about 15% of our country’s territory area, and animal plague epidemic regions continue to grow, and human plague cases increased steadily in our country. In addition, Y. pestis is a typical biological warfare and bioterrorism agent. Therefore, study of Y. pestis pathogenicity is very important in maintaining the health of the people, economic construction and national security.A full virulence of Y.pestis contains three virulence plasmids(p CD1, p MT1, p PCP1), of which p CD1 plasmid is considered to play the most important role in virulence, proliferation, and diffusion of Y. pestis. p CD1 encodes Type Ⅲ secretion system(T3SS) that injects Yops protein into the host cell, resulting in death of the host by interfering with the normal function and immuno-suppression of the host cells. p MT1 plasmid encodes the murine toxin, which is an important factor in the survival of Y. pestis in flea guts and the formation of the bacterial embolus, play an important role in the spread of Y. pestis. p PCP1 plasmid encodes the pla gene that has proteolytic activity and can degrade Yops protein,and is an important factor leading to high invasiveness of Y. pestis. Therefore, p MT1, p PCP1 plasmid played a role in the transmission and spread of Y. pestis. We conserved a wild-strain of Y.pestis 201 separated from Inner Mongolia Brandt vole(Microtus brandii) in our Laboratory, which contains p PCP1, p CD1, p MT1 and p CRY plasmids. Then Dr. Ni Bin used Y.pestis 201 to construct plasmid-deficientmutants(201-p PCP1 + p CD1 + p MT1 +, 201-p PCP1 + p MT1 +, 201-p PCP1 + p CD1 +, 201-p CD1 + p MT1 +, 201-p CD1 +, 201-p PCP1 +, 201-p MT1 +, 201-p).After mice were challenged with each plasmid-deficient mutant, only p MT1 strain(201-p MT1+) showed unusual virulence to mice, which do not coincide with the fact that p MT1 plasmid is not a virulence plasmid in Y.pestis. SDS-PAGE electrophoresis analysis of each plasmid-mutant strain showed that a Ymt band was only observed in the bacterial pellet of the strains containing p MT1 plasmid(201-p PCP1 + p CD1 + p MT1 +, 201-p PCP1 + p MT1 +, 201-p CD1 + p MT1 +, 201-p MT1 +,). By contrast, Ymt strips could not be detected in the culture supernatant of the strains contains the p MT1 and p PCP1 plasmid(201-p PCP1 + p CD1 + p MT1 +, 201-p PCP1 + p MT1+), but its degradation fragments can be detected. Ymt strips could be detected in the culture supernatant of the strains lacking p PCP1 plasmid(201-p CD1 + p MT1 +, 201-p MT1 +). Ymt has long been recognized as an intracellular protein. However, in this study, a secreted Ymt was observed in the culture supernatant of the strains lacking p PCP1 plasmid. This implies that when mice was infected with Y. pestis, Ymt did not show high toxicity to mice, which is likely because Ymt protein was degraded in the process of secretion by certain genes encoded on p PCP1.To further explore the target gene degrading Ymt protein during the secretory process, and the degradation mode, we built pla knockout strains(201-p CD1+-p MT1+-p PCP1+-Δpla、201-p MT1+-p PCP1+-Δpla) using the Red recombinant system; we built comparatory strains(201-p CD1+-p MT1+-p PCP1+-Δpla-p ACYC184-pla、201-p MT1+-p PCP1+-Δpla-p ACYC184-pla) using p ACYC184 plasmid. The culture supernatant of both pla knockout strains and its comparatory strains were identified by SDS-PAGE and WB. Results showed that Ymt protein was detected in the culture supernatant of pla knockout strains(201- p CD1 +-p MT1 +-p PCP1 +-Δpla, 201-p MT1 +-p PCP1 +-Δpla), but not detected in the culture supernatant of the comparatory strains, but Ymt protein degradation fragments can be detected, indicating that pla encoded by p PCP1 plasmid degraded Ymt protein. In addition, we constructed E. coli BL21-p ET28a-pla and BL21-p ET28a-ymt and obtained recombinant Pla protein and Ymt protein that have activities. We detected the toxicity of Ymt protein in mice, observed the pathological changes caused by the Ymt protein in mice organs, which lay a foundation for later study of pathogenic mechanism of Ymt protein. Then we incubated the protein Pla and Ymt protein under 37 ℃ in vitro, no degradation of Ymt by Pla was observed. We transform recombinant plasmid p ACYC184-pla gene into plasmid-free Y.pestis to construct P?-p ACYC184-pla, and then incubated recombinant strains P?-p ACYC184-pla with purified protein Ymt under 37 ℃,and identified by SDS-PAGE electrophoresis. Then we further transform p ACYC184-pla or p ET28a-pla into E.coli K12 to construct E.coli K12-p ACYC184-pla、E.coli K12-p ET28a-pla. SDS-PAGE results showed that Ymt degradation fragments were detected in supernatant of the two strains, indicating that Pla protein can degrade Ymt protein.Our study found that the Ymt protein is a secreted protein, and can be degraded by the pla encoded on the plasmid p PCP1 during the secretory process. However, here, we did not observe the recombinant Pla protein can degrade Ymt protein in vitro. This result indicated that degradation of Ymt may need the help of other factors, or Pla protein having a special spatial structure, or a special mechanism. Our studies lay a foundation for further studing the pathogenesis of Y.pestis, and provide robust evidence for the research of the evolution of Y.pestis.