Construction Of β-lactamases Reporter System For Type Ⅲ Secretion System Associated Proteins Of Yersinia Pestis

Plague is a deadly natural focus-based disease. There were three human plaguepandemics in history. The causative agent of plague is Yersinia pestis, and the mainhost is rodent. Plague is transmited among different hosts and ultimately transmited tohuman by flea biting or other routes, leading bubonic plague, pneumonic plague andsepticaemia plague.Y. pestis, which can translocate effector proteins into host cells through type IIIsecretion system during infection. Yops can disrupt the host defense mechanisms andenable bacteria to resist phagocytosis, to inhibit innate immune response to benefit thebacterial survival and proliferation, and finally to establish persistent infection.Therefore, it is crucial for can be used to detect whether type III secretion systemassociated proteins of Y. pestis can be translocated in host cell during infection andcan be helpful for the research pathogenesis mechanism of Y. pestis.To establish a TEM-1β-lactamases based reporter system that can be used todetermine whether an interested Y. pestis protein can be translocated into the hostcells during infection. TEM gene was cloned into pACYC184vector, while targetgenes and their promoter sequences were inserted upstream of TEM gene toconstruct the vectors that can express the fusion proteins of target protein andβ-lactamases. The constructed vectors were introduced into Y. pestis via electrotransformation. HeLa cells were then infected with individual Y. pestis strainsexpressing different T3SS genes fused with β-lactamases (Bla). After2-hour’sinfection CCF2-AM substrate was added into the cell culture, and was incubated forone more hour. The results were analyzed by laser scanning confocal microscope. Inthis study, YopE was used as a positive control because it is a well-known effectorprotein of Yersinae T3SS that can be translocated into host cells. Our resultsconfirmed that HeLa cells infected by strain expressing YopE-Bla show blue color,indicating that we successfully established the TEM reporter system that can be usedto detect the translocation of a tested Y. pestis protein into host cells during infection.We further detected whether YopK-Bla, YscK-Bla, YscL-Bla and YscX-Bla could be translocated into the HeLa cell during infection. Our results indicated that HeLa cellsinfected with the strain of YopK-Bla or YscX-Bla showed the blue color, while thoseinfected with the strain expressing YscL-Bla or YscK-Bla showed the green color,implying that YopK and YscX can be injected into the host cell, whereas YscK andYscL cannot. TEM reporter system constructed in this study was successfully appliedto detect the translocation of the target proteins of Y. pe stis into HeLa cell. Thisreporter system can be used as a tool to determine whether other proteins of Y. pestiscan be translocated into the host cell during infection and can be helpful for theresearch about T3SS-associated protein pathogenic mechanism.