Structural And Functional Studies Of MycP1from Mycobacterial ESX-1Secretion System

Tuberculosis (TB) is one of the most common causes of death globally. Mycobacterium tuberculosis, the etiological agent of TB, still remains to emerge as a major public health threat because of the high pathogenicity, a significant increase in multiple-drug-resistance and the ability of immune evasion. As a progress of studies in pathogenicity and multiple-drug-resistance of M. tuberculosis, a number of related proteins have been identified and characterized. This dissereation focuses on the regulatory protein MycPl of mycobacterial virulence factor secretion system ESX-1and the outer-membrane prion Rv1698in M. tuberculosis. The structures of these proteins are determined using X-ray diffraction method, and the releationship between structure and function is investigated. These work could be helpful to explain the molecular etiology of M. tuberculosis, which can provide the the theoretical foundation to the prevention and treatment of TB.In Chapter2, we report structural and functional studies of mycobacterial MycP1. In mycobacterium, a secretion system different from the known Type Ⅰ to Type Ⅵ secretion systems in Gram-negative bacteria was found and defined as Type Ⅶ secretion system (T7SS). The ESX-1system, a typical T7SS in M. tuberculosis, is the most extensively studied secretion system so far. M. tuberculosis use the ESX-1secretion system to deliver virulence proteins during infection. The ESX-1secretion system, therefor, was identified as a key virulence determinant in M. tuberculosis. The identity of all ESX-1substrates and the mechanism by which they affect host cells are not well understood. Various activities have been ascribed to the ESX-1substrates ESAT-6and CFP-10, including inhibition of phagosome maturation and cytokine signaling by infected macrophages, interaction with the macrophage immune receptor TLR2and inhibition of TLR signaling, and formation of pores in mycobacterial phagosomes, perhaps allowing bacterial spread. The regulation mechanism of ESX-1secretion system is an increasing concern. Ohol, Y. M. et al reported a mechanism of posttranscriptional control of ESX-1mediated by MycPl, a putative subtilisin-like serine protease with a C-terminal transmembrane helix. MycPl plays a dual role in regulating secretion activity of the ESX-1system. MycPl protein is required for the integrity and secretion activity of the ESX-1system. A mycPl deletion mutant in M. tuberculosis leads to the loss of the secretion activity for ESAT-6/CFP-10. Whereas the MycP1protein is required for secretion, abolition of MycP1protease activity by mutagenesis of the active site leads to increased secretion. Further more, EspB protein was identified as a substrate of MycPl. It was concluded that the MycP1protein is required for ESX-1secretion but that its protease activity negatively regulates secretion via EspB. As a subtilisin-like serine protease, MycP1contains a putative N-terminal inhibitory propeptide and a catalytic triad of Asp-His-Ser, classic hallmarks of a subtilase family serine protease. The MycP1propeptide was previously reported to be initially inactive and activated after prolonged incubation. In this study, we determined crystal structures of MycP1with (MycP124-422) and without (MycP163-422) the propeptide, and conducted EspB cleavage assays using the two proteins. Very high structural similarity was observed in the two crystal structures. Interestingly, protease assays demonstrated positive EspB cleavage for both proteins, indicating that the putative propeptide does not inhibit protease activity. Molecular dynamic simulations showed higher rigidity in regions guarding the entrance to the catalytic site in MycP124-422than in MycP163-422, suggesting that the putative propeptide might contribute to the conformational stability of the active site cleft and surrounding regions.Chapter3presents our work on the outer-membrane protein Rv1698from M. tuberculosis. It has been reported that mycobacteria has a complex cell wall, which forms an exceptionally strong permeability barrier because of its extremely low permeability. The protective function of cell wall is an essential virulence factor of M. tuberculosis and contributes to its intrinsic drug resistance. Cryo-electron microscopy showed that mycobacterial cell walls include an unusual outer membrane, which composes of long-chain mycolic acids and a large variety of other extractable lipids. Meanwhile, a numer of proteins were provided to locate in the outer-membrane. In Gram-negative bacteria, the outer-membrane proteins contributes to the transport processes across the outer membrane. Escherichia coli use more than60proteins to functionalize its outer membrane. While only three mycobacterial outer membrane proteins (OMPs)(OmpATb, Rv1973and Rv1698) are known in M. tuberculosis so far. Bioinformatics analysis by Song, H. et al revealed that Rv1698is an outer-membrane protein that is likely involved in transport processes across the outer membrane of M. tuberculosis. Expression of rv1698restored the sensitivity to ampicillin and chloramphenicol of a Mycobacterium smegmatis mutant lacking the main porin MspA. Uptake experiments showed that Rv1698partially complemented the permeability defect of the M. smegmatis porin mutant for glucose. Lipid bilayer experiments demonstrated that purified Rv1698is an integral membrane protein that indeed produces channels. Taken together, these experiments demonstrated that Rv1698is a channel protein that is likely involved in transport processes across the outer membrane of M. tuberculosis. In our work, crystals of Rv169827-314(with truncation of the N-terminal signal peptide) in detergent-containing buffer and aqueous solution were collected and diffracted at the resolutions of2.30and3.25A, respectively. The two crystal structures were in high similarity, indicating no pronounced conformational differences were induced by the presence of detergent. A mixed α/β-globular structure with a long helix extending away from the globular domain was observed for Rv169827-314, rather than a previously proposed integral membrane proteins, neither a beta-barrel porin like structure nor multi-span helical structure. It was also found that the predicted N-terminal signal sequence is not removed in the mature protein, and plays an improtant role in the oliogmerzation and membrane location of Rvl698. The cryo-EM showed that the purified full-length Rv1698protein forms hexamer in the presence of LDAO. An overall architecture model of Rv1698hexamer was built. The reported structures of Rv169827-314and the overall architecture model of the protein suggest alternative modes of membrane association, demanding further investigations combining structural biology and bacterial physiology.