| Shigella spp. is a gram-negative enteric pathogen,which children and old people are susceptible to. To prevent and cure dysentery is a great challenge due to the extremely low doses of infection and cross-resistance. Thus, it seems that to develop a proper vaccine is urgent.Type Three Secretion System of Shigella spp. is a powerful weapon against the host immune system. Virulence factor Ics A(Vir G) is required for Shigella spp. to spread between the host cells. Studies have shown that Htr A facilitates Vir G surface expression and acts as a virulence factor. In addition, the transmission mode of Shigella spp. is fecal-oral route. Shigella spp. needs to be able to withstand the acid stomach environment and other extreme environments before its final infection in the large intestine. It has been reported in a variety of pathogens that Htr A plays an important role in the growth in high temperature, acidic or alkaline and other extreme environments. Therefore, the study of the role of protease of Htr A protein will help us find some novel virulence-associated proteins, understand the specific pathogenesis of Shigella spp., and provide us a theoretical basis for the development of a vaccine basis.The sequence of Htr A protein keeps conserved in most pathogens. It owns both chaperone and protease activities and exists in the periplasm. Htr A is a significant regulator of protein quality. Current reports of Htr A protein mostly focused on its chaperone or protease activity.However, during the research of the function of Arg T protein in our lab, we have found that Htr A protein will form two spots on the two-dimensional electrophoresis gel map, and there will be an abundance "shift" phenomenon between the two spots in accordance with the changes of its substrate. So we speculate that Htr A may be undergone some kind of post-translational modifications. Currently, there is no related research about this phenomenon.In this paper, we will study on two aspects of Htr A protein, protease activity and post-translational modification, and hope to identify potential substrates of proteinHtr A to get a better understanding of the pathogenesis of Shigella app. At the same time, based on the resolved crystal structure and combined with experimental analysis,we hope to identify potential modification sites and determine the modified form to open a new field of Htr A protein.In the study the protease activity of Htr A, we first constructed up-regulated and down-regulated strains of htr A. Then use the comparative proteomic method to compare protein expression profiles in different strains to find out the potential substrates.We imported an arabinose operon to control the expression of Htr A in up-regulated strain; and chose the popular CRISPRi techniques to silence the gene htr A. For the existence of many mobile elements in the genome of Shigella spp.,especially the high rate of spontaneous mutation of the virulence region, we need to validate the virulence gene by PCR after successful construction of the up-regulated and down-regulated strains to make sure the further research is scientific and accurate.After sequencing and PCR, we detected the abundance changes of Htr A in different stains by Western Blot with self-made Htr A antibody. Western Blot results confirm that the strains were successfully constructed.After successfully constructing the strains, we chose two-dimensional electrophoresis to separate the proteins and used the comparative proteomic method coupled with MALDI-TOF/TOF to identify the potential substrate proteins. Because Htr A is a periplasmic protein, the most direct way to look for its natural substrate proteins is extracting the periplasmic proteins. On the other side, Htr A protein is also involved in the synthesis of outer membrane proteins, so the outer membrane protein extraction is also needed. Finally, comparing the differential expression of whole-cell proteins between different strains will help to confirm above results.Firstly, we compared the periplasmic proteins between up-regulated and down-regulated strain of htr A under the condition of 30 o Cand 37 o C. The results showed that protein Pho N1, Pho N2 and Gln H might be the potential natural substrate proteins. Pho N1 and Pho N2,periplasmic non-specific acid phosphatase, share about50% sequence homology. Both of them are down-regulated at 37 o C when the expression of Htr A is uplifted. While at 30 o C, the abundance of Pho N1 and Pho N2 have no significant difference between different strains, suggesting that Pho N2 and Pho N1 were both substrate proteins of Htr A. In addition, we also found that under the condition of 37 o C, the abundance of periplasmic glutamine binding protein Gln Hwere changed in opposite directions against Htr A, but with no obvious difference in30 o C, indicating that Gln H protein was also a substrate of Htr A.Comparison of the protein expression profiles of outer membrane proteins extraction in 37 o C showed that the expression of outer membrane protein Omp A was also decreased when the amount of Htr A protein decreased. This phenomenon verified the previous reported that Htr A proteins was involved in the synthesis of Omp A. In addition, comparison of the protein expression profiles of the whole cell protein in37 o C, we found Pho N2 changed in the same trend as it did in periplasmic protein extraction. So we further confirmed Pho N2 was one of substrate proteins of Htr A.Since the PI of Pho N1 is 6.9, or the like, while the whole cell protein using p H 4-7and p H 6-11 IPG strips. Therefore in the final gel maps, we could not identify Pho N1 protein.As far as the post translational modification of protein Htr A is concerned, S210 was first excluded to be the modification site because there were still two spots on the two-dimensional electrophoresis gel map when this amino acid were mutated to alanine. Then we digested the two separated spots on the 2DE gel of wild-type strain with Typsin, Glu-C and Chymotrypsin. LC-MS/MS results showed that the putative modified segments might be the amino acids between sites 57-63. This section is located in the Q-linker according to the crystal structure of Htr A. Because of its flexibility, it could not be predicted in the 3-dimensional structure. Therefore,we performed alanine scanning analysis. The 301/C57 A, 301/Q58 A, 301/E59 A,301/S61 A site-mutants were constructed. Two-dimensional electrophoresis results showed that there were still two spots when the protein profiles of these mutants were analyzed. Therefore, we can exclude the possibility of modifications on C57 Q58,E59 and S61 amino acids.In summary, we constructed up-regulated htr A strains by imported arabinose operon and down-regulated htr A strains by CRISPRi technique, and analyzed the differences of protein expression profiles between different strains under 37 o C and 30 o C conditions with comparative proteomics method. Finally we concluded that periplasmic protein Pho N1, Pho N2 and Gln H were the possible substrate proteins.Furthermore, we also verified that Htr A involved in the synthesis of Omp A.For the possible post-translational modifications, we mainly ruled out the possibility of S210, C57, Q58, E59 and S61 through this study. But the specific modification sites and form are still not clear. |
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