The Characters And Functions Of Mycobacterium Tuberculosis PE/PPE Family Proteins In Pathogen-Host Interaction

Tuberculosis(TB), caused by Mycobacterium tuberculosis infection, remains a formidable threat to global public health. M. tuberculosis can modulate and elude host immune responses and persist for prolonged periods. One hallmark of the M. tuberculosis genome is the presence of the multi-genic PE/PPE family proteins, which consist of PPE, PE and PE_PGRS subfamilies and account for about 10% of the coding capacity of the M. tuberculosis genome. There are about 100 genes encoding PE subfamily, which harbor a conserved N-terminal domain with 110 amino acid residues with the Pro-Glu(PE)motif, while the C-termini vary significantly in size and protein-specific polymorphic GC-rich repeats PGRS. The PE subfamily contains 37 PE genes with a conserved N terminal and 61 PE_PGRS genes with G-G-A and G-G-N tandem repeats in their C terminal. The 69 genes encoding PPE subfamily which are named after its N terminal Pro(P)-Pro(P)-Glu(E) motif. PPE subfamily proteins are mainly classified into PPE_SVP, PPE_PPW and PPE_MPTR subfamily. The unique sequences of these proteins might underlie the specific physiological role of this family during M. tuberculosis infection.The exclusive presence of PE/PPE family among pathogenic mycobacteria has attracted many researchers. The primary origin, regulation and physiological role of some PE/PPE family proteins have been well characterized and reviewed. The origin of the PE/PPE genes is associated with the Type VII secretion system(T7S). Several transcriptional regulators involved in the regulation of PE/PPE family proteins have been characterized, including the stringent response mediator Rel A, ESX-1 secreted protein regulator Esp R and global nucleoid-associated transcriptional inhibitor Lsr2 and sigma factors, such as Sig F, Sig B and Sig D. PE/PPE family proteins are cell wall associated, suggesting that a role in directly interaction with host targets such as the cell surface receptor TLR2, or even interfering the host immunity. Macrophages are the first line of defense against bacteria infection, which can secrete various cytokines to mediate the inflammatory response. The varied transcription level of several PE/PPE genes within macrophages or mouse during M. tuberculosis infection suggested a role in manipulation the host macrophage activity. It has been shown that several members of PE/PPE proteins are immunogenic and might contribute to the antigenic diversity. PE/PPE proteins might be linked to virulence and responsible for its ability to grow in a macrophage, resulting in immune evasion of mycobacteria.M. tuberculosis rv1169 c encodes for PE11, a prototypical member of PE/PPE family protein, was named after Lip X as the potential lipase. The up-regulation of rv1169 c in starvation or transient acid exposure, suggested a role in pathogen persistence or dormancy. To study the function of Rv1169 c protein and underlying mechanism, M. smegmatis expressing Rv1169c(Ms_Rv1169c) and M. smegmatis harboring the vector only(Ms_Vec) were constructed and confirmed rv1169 c gene encoded protein is cell wall associated. GC-MS has used for identification distinct cell wall fatty acid component in recombinant strains, we found the cell wall fatty acid in Ms_Rv1169c is significantly different from Ms_Vec and wild type M. smegmatis strain. We identified recombinant Ms_Rv1169c is more susceptible to surface stress SDS and acid stress compared to Ms_Vec, implicating a role of Rv1169 c in stress response. Further evidence proved that Ms_Rv1169c is more sensitive to antibiotics including rifampicin andnorfloxacin. These results indicate that Rv1169 c protein may increase the cell wall permeability of mycobacteria by affecting their stability. Moreover, this phenotype is not correlated with increased survival within macrophage, as Rv1169 c protein enhance s the intracellular survival of M. smegmatis. We demonstrated that Rv1169 c protein helps M. smegmatis enhance cell death with unknown mechanism. We demonstrated that Rv1169 c affects the expression of IL-6. However, how Rv1169 c protein selectively regulates host pleiotropic pro-inflammatory cytokine IL-6 remains to be determined, as Rv1169 c promotes the relative expression of IL6 m RNA while decreases the IL-6 secretion. Moreover, we found Rv1169 c protein may helps M. smegmatis to induce the cell death of macrophage. In summary, we provide here evidence that M. tuberculosis Rv1169 c protein regulates macrophage IL-6 secretion and plays an important role in cell death of macrophage. It’s first reported that Rv1169 c protein may directly or indirectly involves in fatty acid metabolism, resulting in the remarkable changes in fatty acid component of mycobacterial cell wall. Moreover, a role of Rv1169 c protein in stability of mycobacterial cell wall integrity was confirmed as evidenced by the declined resistance of recombinant M. smegmatis to anti-mycobacterial factors including SDS, acid stress and antibiotics.We also focus on another M. tuberculosis PPE subfamily protein PPE32, which encoded by rv1808 gene. The recombinant M. smegmatis(Ms_Rv1808 and Ms_Vec) were constructed, the recombinant Rv1808 protein(r Rv1808) was expressed and purified from recombinant E.coli. We found r Rv1808 protein localized to cell wall of M. smegmatis.Pull-down assay identified r Rv1808 protein interacts with TLR-2 of macrophage. The secretion of TNF-α,IL-6 and IL-10 of macrophages was increased after incubation with Rv1808 protein. We indentified r Rv1808 protein specifically induce these cytokines as protease treated r Rv1808 protein can’t induce the production of TNF-α, IL-6 and IL-10. On the other hand, the production of TNF-α, IL-6 and IL-10 was dependent on the concentration of r Rv1808 protein. In order to study which signaling pathway involved in the secretion of TNF-α, IL-6 and IL-10, a series of specific inhibitors were used. We found the signaling pathways such as NF-κB, ERK1/2, p38 and JNK were necessary to the production of TNF-α, IL-6 and IL-10. In addition, r Rv1808 protein can enhance the survival of M. smegmatis in macrophage with unknown mechanism. LDH assay has identified that there is no difference LDH release between Ms_Rv1808 and Ms_Vec infected macrophage, suggesting that Rv1808 has no effect oncell lysis, as the same result was get when we use the r Rv1808 protein incubate with macrophage. Interesting, we noticed that MTT assay showed that r Rv1808 effect the cell viability of macrophage in dose-dependent response. Therefore, we suspect that r Rv1808 may play an important role in apoptotic cell death. In summary, we found that the cell wall associated Rv1808 protein interacts with TLR-2 and induces TNF-α, IL-6 and IL-10 production from macrophage through NF-κB, ERK1/2, p38 and JNK signaling pathway, resulting in the enhanced intracellular survival.