The Effect Of MIP_XCC Protein On The Extracellular Proteases Activity In Xanthomonas Campestris PV. Campestris

Mip （macrophage infectivity potentiator） proteins are a family of FKBP domain-containing proteins exhibiting peptidyl-prolyl cis/trans isomerase activity. Mip proteins have been identified as important virulence factor in several animal pathogens such as Legionella pneumophila, Chlamydia trachomatis, Neisseria gonorrhoeae. Our previous work has shown that inactivation of the mip-like gene （thereinafter mipXcc） of Xanthomonas campestris pathovar campestris（Xcc） led to almost loss of extracellular protease activity, a significant reduction in exopolysaccharide production and virulence, indicating that the Mip-like protein of Xcc （thereinafter MipXcc） plays an important role in extracellular proteases activity, exopolysaccharide production and pathogenesis. But the the mechanisms by which MipXcc involved in these cellular processes is still unknown. In this study, we investigated the exact role of the MipXcc in the full extracellular protease activity of Xcc.The genome of Xcc strain8004（Xcc8004） has six ORFs encoding extracellular proteases （XC₁514, XC₁515, XC₃376, XC₃377, XC₃378, and XC₃379）. One of them, XC₃379（also known as prtA）, has already been characterized as the major extracellular protease gene. Inactivation of prtA lost95%extracellular protease activities of Xcc8004. To study the role of MipXcc in the extracellular protease production, we detected the mRNA level of prtA in mipxcc mutant NK2699and wild-type strain8004by using semi-quantitative RT-PCR, and the result showed that the transcription level of prtA in mipXcc mutant is identical to that in the wild type strain. Consistent with this, introducing of a plasmid constitutively expressing prtA into the mipXcc mutant NK2699could not restore its extracellular protease activity. These results demonstrated that MipXcc did not regulate the expressing of prtA at the transcriptional level. It has been demonstrated that the extracellular hydrolytic enzymes including extracellular proteases of Xcc are secreted outside of cells through the type Ⅱ secretion system （T2SS）. To determine whether the fuction of T2SS was affected by the mipXcc mutation, we employed chloroform vapor treatment to release the perilasmic proteins in situ, and the result reveals that no mature protease was accumulated in the periplasm of the mipXcc mutant. This combined with our previous observations that the extracellular cellulose and amylase activity did not affected by mipXcc mutation, we conclude that MipXcc is not required for the normal T2SS function of Xcc.It is well known that PPIase can act as a foldase, which accelerate the correct folding of polypeptides, and that the extracellular protease folded in the periplasm. These information promotes us to speculate that MipXcc may function as a folding factor for the extracellular proteases. If this is true, the MipXcc protein should located in the periplasm of the Xcc cells. To confirm this, we used Western Blotting to determine the subcellular location of MipXcc. The result shows that MipXcc is indeed located in the periplasmic space.If Mipxcc functions as a folding factor for Xcc extracellular proteases, there should exist physical interaction between MipXcc and extracellular proteases. To confirm this, bacterial two-hybrid analysis were carried out to test the interactions between MipXcc and each of the six extracellular proteases, as well as a extracellular cellulose （XC₀639） and a extracellular amylase （XC₀141）, respectively. The bacterial two-hybrid analysis results show that MipXcc interacts with four extracellular serine proteases including PrtA. Far-Western blotting analysis and Co-Immunoprecipitation further confirmed that MipXcc protein specifically bound to PrtA in vitro as well as in Xcc cell. Further more, we employed’protease activity rescue experiment’to further confirm that MipXcc is able to assist PrtA precursor to fold into its active mature protease. These datas demonstrated that the correct folding and maturation of extracellular protease PrtA depend on the MipXcc protein in the periplasm of Xcc. To the best of our knowledge, the PrtA is the first identified native substrate （target） for a Mip protein. A typical Mip protein consists of two parts:an N-terminal dimerization region and a C-terminal PPIase region. We further determined, by using bacterial two-hybrid analysis, that the C-terminus but not the N-terminus is essential for the interaction of MipXcc with PrtA. Furthermore, we found that mutation in each of the five highly conserved amino-acid residues （Met192, Ala207, Tyr208, Gly209, Pro218） within the PPIase region of MipXcc lost the ability of MipXcc interaction with PrtA. This indicates that the five conserved amino-acid residues are required for MipXcc binding to PrtA.We speculated that in addition to the four extracellular serine proteases, there may exist other targets for MipXcc. To identified other binding targets of MipXcc, we constructed a genomic DNA expressing library of Xcc8004, and carried out two-hybrid screening the expressing library. As a result, we obtained16positive clones, and two of which, conserved hypothetical protein （XC₂962） and alkaline phosphatase （XC₂462）, may be the binding target of MipXcc.In summary, our results clarified the reason why mutation in mipXcc resulted in the near lost the extracellular protease activity of Xcc, that is, the correct folding of the major extracellular protease PrtA depends on the MipXcc protein, and in the mipXcc mutant, PrtA precursor can not fold into active mature protease due to lacking MipXcc. In addition, our works also provide new clue and idea towards the understanding the role of MipXcc in Xcc pathogenesis.