| Yersinia pestis is the etiological agent of plague, a deadly infectious disease that has lead to millions of deaths in history. In recent years, small outbreaks of plague in different places, including Africa, Asia, Eurasia and America, have been reported almost every year. The World Health Organization (WHO) has reclassified plague as a re-emerging disease because the number of annually reported cases has risen throughout the world since the mid-1980s. Due to the high contagiousness, high mobility and mortality of plague, Y. pestis has gained much attention as a possible biological warfare agent that could be misused by bioterrorists.Typeâ…¢secretion system (T3SS) is a virulence mechanism widely distributed in Gram-negative bacterium, which can translocate effector proteins into host cells during infection. Three pathogenic Yersinia species share a common T3SS encoded by a 70kb-plasmid called pCD1 in Y. pestis. Yesinia T3SS is composed of more than 20 proteins, and this sophisticated secretion machine can deliver the effector proteins, known as Yops (Yersinia outer proteins), into the eukaryocyte cells. It has been shown that once inside the host cell cytosol, Yops can disrupt the host defense mechanisms and enable bacteria to resist phagocytosis, to inhibit innate immune response to benefit the bacterial survival and proliferation, and finally to establish persistent infection. YopK is one of the effectors and had been shown to be related with the virulence of Y. pestis, although the pathogenic mechanism is still unclear. Based on our previous studies, YopK was identified to interact with a human protein MATN2 (Matrilin-2) using the yeast two-hybrid screening against human cDNA library. MATN2 has been implicated in the interaction of extracellular matrix components and cells by binding to collagens, proteoglycans, laminins, fibronectins and other members of extracellular matrix (ECM) molecules and it also plays prominent role in the extracellular assemblies of ECM components as well as in the formation of collagen network. There is no evidence supporting that MATN2 has any relation with infectious disease. The aim of this project is to clarify the function ofYopK in pathogenesis of Y. pestis, to verify the interaction between YopK and MATN2, and to reveal the contribution of YopK and YopK-MATN2 interaction to the pathogenesis of Y. pestis.The yopK mutant strain ((?)yopK) was constructed by (?)-Red recombination system and the yopK complementary strain ((?)yopK- pACYC184yopK) was constructed by introducing pACYC184-yopK into the (?)yopK. The mutant strain and the complementary strains were successfully constructed as shown by specific PCR amplification and immunoblotting analysis. BALB/c mice were intravenously and intraperitoneally challenged with the wide type strain 201, (?)yopK and (?)yopK- pACYC184yopK, and the animals were observed for 14 days. LD50 of different strains were calculated, and the results showed that yopK mutant is severely attenuated when the mice were intravenously infected and the (?)yopK-pACYC184yopK regained the virulence. The bacterial load in the spleens, livers and lungs of the BALB/c mice were calculated after 24, 48 and 72 hours of infection by 201 or (?)yopK via the route of intravenous injection. The results showed that mice infected with YopK-deficient bacteria had reduced bacterial loads in all the three tissues compared to mice infected with the wide type strains. After infection for 72 hours, the mice infected by (?)yopK began to recover from infection because that the bacterial load dropped greatly.Both wide type and yopK-deficient bacteria showed the highest bacterial loads in livers, and then in spleens, the lowest in lungs.We purified recombinant YopK protein expressed in E. coil and produced the specific antibody against YopK in rabbit. The secretion and the translocation of YopK into HeLa cells were analyzed by detecting YopK in different cellular fractions of HeLa cells after they were infected by Y. pestis. The results revealed that YopK were secreted into the supernatant of culture medium and the precipitation of HeLa cells lysate, however, no bands of YopK appeared in the supernatant of cells lysate that represents the cytosol of HeLa cells. This indicated that YopK could be secreted out of the bacteria by T3SS but could not be translocated into the host cells, implying that YopK might function at the extracellular environment and thus could be related with the adhesion and invasion procedure of Y. pestis infection. Therefore, we continued to investigate whether the mutation of yopK influence the adhesion of Y. pestis to HeLa cell and the anti-phagocytic ability of Y. pestis to RAW264.7. Cytochalasin-D was added into the culture medium to inhibit the engulfment of cells, then HeLa cells were infected with 201 or (?)yopK followed by the labeling with fluorescent antibody. Finally, the flow cytometry technology was used to evaluate the adhesion ability of Y. pestis to the host cells. The results showed that the adhesion ability of (?)yopK decreased strikingly compared to the wild type strain 201. The results in RAW264.7 cells showed that the anti-phagocytic ability of (?)yopK was similar to that of 201, indicating that YopK deficient did not affect the phagocytosis of Y. pestis by RAW264.7. In the early stage of infection, Y. pestis bacillus have to adhere to the host cells before they were engulfed by phagocytes, where they can escape form the host immune surveillance and clearance by the host innate immunity. After proliferation in the host cells, virulence factors including F1 and pH6 antigen began to be expressed in large amount to benefit the bacterial extracellularly survival. We conjectured that YopK might play an important role in the early stage of infection.We further confirmed the YopK-MATN2 interaction identified in our previous yeast two hybrid screenings and investigated how this interaction contributes to the pathogenic mechanism of Y. pestis. MATN2 is the largest protein in the matrilins family with a molecular weight about 100,000 Da (956 amino acids) and is composed of two vWA (von Willebrand factor A) like domains followed by 10 EGF (epidermal growth factor) like modules and one unique sequence. We used human cDNA as template to construct the expressing plasmids of MATN2 and its various truncates using pCMV-Myc. These proteins were successfully expressed in HEK293T cells. Firstly, GST pull-down was used to verify the interaction between YopK and MATN2, and it was found that the interaction domain essential for YopK-MATN2 interaction located in 574 to 903 residues of MATN2, corresponding to the last 2 EGF domains and the second vWA domain. There is a MIDAS (metal ion dependent adhesion site) motif in the vWA domain which contains a consensus sequence of DXSXSXnTXnD. To investigate whether YopK-MATN2 interaction is mediated by MIDAS, we construct 3 mutants of MATN2 by replacing 5 critical amino acids in MIDAS to A and then test the binding activities between YopK and the various MATN2 mutants. The results showed that all the 3 mutants could bind to MATN2 with similar affinity. Further works have to be done to elucidate the mechanism and the physiological functions of the interaction between YopK and MATN2.In summary, our results revealed that YopK was intimately related with the adhesion ability to the host cells and the deletion of yopK severely impaired the adherence of Y. pestis to HeLa cells. YopK can interact with MATN2 and this interaction depended on the vWA2 domain of MATN2. We will continue this work to clarify weather and how the interaction between YopK and MATN2 interaction contribute to the pathogenesis mechanism of Y. pestis.
|
PDF Full Text Request