The Role Of Mycobacterium Tuberculosis PPE Family Gene Rv3136 Effecting On Cell Wall Remodeling And Underlying Molecular Mechanisms

Mycobacterium tuberculosis is considered as one of the most successful and widespread intracellular pathogens, with approximately one-third of the world’s population infected and 1.5 million deaths annually. According to Global Tuberculosis Report 2015（World Health Organization Tuberculosis Data and Statistics, 2015）, there are 1.5 million people died from TB in 2014, which contains 40,000 HIV-positive patients and 1.1million HIV-negative patients. In addtition, 9.6 million people are estimated to have fallen ill with TB in 2014 and the toll comprised 5.4 million men, 3.2 million women and 1 million children. Terribly, 12% of the 9.6 million new TB cases in 2014 were HIV-positive. WHO’s global estimates that India, Indonesia and China had the largest number of cases: 23%, 10% and 10% of the global total, respectively. Of the 480 000 cases of multidrug-resistant TB（MDR-TB） estimated to have occurred in 2014, only about a quarter of these were detected and reported. Despite of the vaccines and anti-tuberculosis drugs, the emergence of multi-drug-resistant（MDR） strains and co-infection with HIV has prompted the need for new drugs and drug targets.M. tuberculosis possesses an unusual cell wall dominated by lipids and carbohydrates that provides a permeability barrier against hydrophilic drugs and is crucial for its survival and virulence. The large macromolecular structure, termed the mycolylarabinogalactan-peptidoglycan complex, and the phosphatidyl-myo-inositolbased lipoglycans are key features of the mycobacterial cell wall. Assembly of these cell wall components are an attractive target for the development of chemotherapeutics against tuberculosis. Furthermore, regulation pathway of the cell wall components is also a potential drug targetPPE represent a peculiar family of mycobacterial proteins characterized by a 180 amino acids conserved N-terminal domain, and contains about 69 members. Most of PPE proteins characterized to date were shown to be surface exposed because of its specific PPE domain. Although some PPE proteins have been shown to be involved in the modulation of the immune response and/or to be essential for virulence, their precise function has been elucidated or proposed only for a few of them. Anyway, one member of PPE family, rv3136（PPE51） gene encodes a hypothetical protein. There is no any description about the function of this gene in M. tuberculosis. To explore its function, we heterologously expressed Rv3136 gene in the non-pathogenic fast-growing M. smegmatis strain（ MS_Rv3136）.In this study, we firstly described the role of PPE51（Rv3136） involved in remodeling of the mycobacterial cell wall. We showed that PPE51（Rv3136） is not secreted but surface exposed. Rv3136 gene altered the phenotypic of M. smegmatis including morphology, sliding motility and stability of biofilm formation, which might be due to changes in the cell wall components. MS_Rv3136 showed growth advantage over the vector control under different stress conditions such as SDS, acidic stress, H2O2 and diamide. In addition, when exposed to the antibiotic associated with the cell wall, the recombinant MS_Rv3136 showed a tolerance phenotype. To gain further insight into whether Rv3136 will enhance intracellular survival of M. smegmatis, we infected macrophages by the MS_Rv3136 as well as the vector control strains. The data showed that Rv3136 gene can enhance the viability of M. smegmatis in vivo. Moreover, We examined the effect of Rv3136 in expression of several genes involved in cell wall biosynthesis, our results showed that Rv3136 significantly up-regulated glfT2（MSMEG₆403）, glfT1（MSMEG₆367）, mptA（MSMEG₄241）, mgtA（MSMEG₁113）, ubiA（MSMEG₆401）, pimB(MSMEG₄253 and sigF（MSMEG₁804）, which are involved in the cell wall formation.Taken together, our data suggested that M. tuberculosis Rv3136（PPE51） might be a novel factor involved in the remodeling of mycobacterial cell wall, and this added an new function to the PPE family. This research laid a foundation for the further study of Rv3136（PPE51） in the pathogenesis of M. tuberculosis.