| Shigella flexneri, the highly infectious and severely harmful gram-netative bacteria, is responsible for bacillary dysentery in humans. Comparative genomics clearly indicates that Shigella evolved from multiple E. coli strains by convergent evolution and acquired a large virulence plasmid by horizontal gene transfer. Most proteins needed for invasion and intracellular survival are encoded on this large virulence plasmid, including membrane protein complex Type III Secretion System(T3SS) which plays a key role in the pathogenic process. Our question is that are there more membrane protein complexes in the membrane of S. flexneri.In order to answer this question, the membrane protein complexes from S. flexneri and ApWR100were extracted. Finally, we identified a unique membrane protein complex in M90T by blue native electrophoresis, which was not present in ApWR100, indicating that it might be related to the virulence of wild-type strain. According to the results of TOF/TOF identification of the protein bands separated by SDS-PAGE, M90T-290might be a290kDa protein complex composed of the DnaK, Apyrase, ClpB, YdgA, EF-Tu, and GapA. Since two of the components of M90T-290is a known virulence-related protein Apyrase and dnaK, which is essential for proper unipolar VirG localization, we speculate that this complex might be a virulence-related complex.To test whether different potential components of the M90T-290complex is the true subunits of this complex, a serial of gene deletion mutants (△ydgA, AphoN2, AdnaK and△clpB) was constructed. Then, the natural membrane protein complexes from△ydgA,△phoN2,△dnaK and△clpB were extracted and separated by BN PAGE. M90T-290was disappeared in AydgA,△phoN2, AdnaK mutants, while it remained in△clpB mutant, just like in the wild-type strain. That is, protein ClpB might be not one part of this complex. These data suggested that DnaK, YdgA and Apyrase are the components of the290kDa complex that existed in M90T. To verify interaction between different components of M90T-290complex, we performed a serial of pull-down experiments. Based on the results of pull-down assays, components of M90T-290had wide interaction.To find which subunits of this complex is indispensable part for its fully function, a serial of Sereny tests and HeLa cell invasion assays using above different mutants were carried out. According to the results of Sereny tests, for guinea pigs infected by△ydgA,△phoN2, and AdnaK mutants, keratoconjunctivitis developed significantly later than that infected by wild-type strain. When complemented with corresponding genes in these deletion mutants, the bacteria could regain the ability to evoke a strong inflammatory reaction of host. HeLa cell invasion assays showed similar results. It has been reported that two subunits of complex M90T-290, Apyrase and DnaK, might facilitate the function of known virulence factor VirG(IcsA). Firstly, to check whether the loss of complex M90T-290affects the asymmetric distribution of VirG, immunofluorescence assays were carried out using wild-type strain andM90T-290deletion mutant strain△ydgA,△phoN2,△dnaK. Asymmetric distribution of VirG was not changed in△ydgA, which means that M90T-290does not affect asymmetric distribution of VirG.Secondly, to check whether M90T-290have effect on the actin accumulation, immunofluorescence assays were carried out to observe the actin form in HeLa cells infected with wild-type,, mutant and their complemented strain. Actin accumulation was observed in the HeLa cells infected with wild-typeand complemented strain, but not in the cells infected with mutant strain. The reasons of Apyrase, DnaK and YdgA impacted actin accumulation have two:1the complex including Apyrase, DnaK and YdgA influenced actin accumulation.2there was only one protein impacted actin accumulation, while other protein regulated the expression of the former. We investigated the regulating relation among the three protein by western blotting experiment. There are not regulating relation among the three protein. These results showed that the integrity of complex M90T-290is essential for the actin-based intracellular mobility of S. flexneri.After further investigation of composition of M90T-290and BN-PAGE profiles, we identified the complex M90T-260, which composed of the DnaK, YdgA, EF-Tu, and GapA. In vivo assay and in vitro assay, M90T-290might be formed by virulence protein Apyrase recruiting chromosome M90T-260. Taken together, here we showed a novel virulence related complex with a MW about290kDa was found to be composed of virulence protein Apyrase and holo-complex M90T-260(DnaK, GapA, EF-TU and YdgA). Mutation experiments of these proteins showed that loss of this complex would lead to inability of S. flexneri to stimulate actin accumulation of host cell after invasion, which is essential for its intracellular motility and cell-to-cell spread. These findings provide new evidence for the co-evolution between bacterial chromosome and exogenous virulence plasmid. |
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