Mycobacterium tuberculosis Rv0431 expressed in Mycobacterium smegmatis, a potentially mannosylated protein, mediated the immune evasion of RAW 264.7 macrophages. (November 2016)

• Record Type: Journal Article
• Authors: Deng, Guoying; Zhang, Fei; Yang, Shufeng; Kang, Jian; Sha, Shanshan; Ma, Yufang
• Is Part Of: Microbial pathogenesis. Volume 100(2016)
• Page Start: 285
• Page End: 292
• Publication Date: 2016-11
• Subjects: Mycobacterium tuberculosis -- Rv0431 -- Mannosylated protein -- Macrophage -- Cytokine; Pathogenic microorganisms -- Periodicals; Pathology, Molecular -- Periodicals; Communicable Diseases -- microbiology -- Periodicals; Communicable Diseases -- parasitology -- Periodicals; Micro-organismes pathogènes -- Périodiques; Pathologie moléculaire -- Périodiques; Electronic journals; 616.9041
• DOI: 10.1016/j.micpath.2016.10.013
• Languages: English
• ISSNs: 0882-4010

Abstract

Abstract: Tuberculosis remains a global major problem. The immune responses of host against Mycobacterium tuberculosis ( M . tuberculosis ) are complicated. M . tuberculosis lives mainly within host cells, usually macrophages which constitute the first line of host defense. Mycobacterial proteins, especially cell wall-associated proteins, interact with macrophages of host to regulate the functions and cytokine production. Recent studies indicate that glycoproteins are involved in this process. Here, we investigated the function of Rv0431, a cell wall-associated protein in the M . tuberculosis H37Rv strain. Rv0431 protein was heterologously overexpressed in the fast-growing and nonpathogenic Mycobacterium smegmatis ( M . smegmatis ). Binding assay to concanavalin A (ConA) lectin was performed and the result indicated that Rv0431 protein was a potentially mannosylated protein. M . smegmatis MSMEG_5447 gene encoding a polyprenol-phosphate-mannose-protein mannosyl-transferase (PMT) which catalyzes the O-mannosylation of protein was knocked out. The Rv0431 protein overexpressed in MSMEG_5447 gene knockout stain, ΔM5447, lost its reactivity to ConA, providing evidence that Rv0431 was likely O-mannosylated. M . smegmatis overexpressed Rv0431 evaded the killing of RAW264.7 macrophages and altered the cytokine production of macrophages compared to M . smegmatis carrying empty vector. These results suggested that Rv0431, a probably mannosylated protein might promote the evasion of immune responses during mycobacterial infection. Graphical abstract: Prediction of glycosylation sites and analysis of mutant Rv0431 proteins and Rv0431 protein. A. Prediction of glycosylation sites. 59 Thr, 60 Thr, 61 Thr, 62 Thr were predicted to be glycosylated with possibility. B. SDS-PAGE of purified mutant Rv0431 proteins and purified Rv0431 protein in ΔM5447. M. PageRuler Prestained Protein Ladder (Fermentas); Lane 1. Rv0431m3 /M; lane 2. Rv0431 expressed in ΔM5447; lane 3. Rv0431m1 /M; lane 4. Rv0431m2 /M; lane 5. Rv0431m4 /M; lane 6. Rv0431/M. C. Western blot of purified mutant Rv0431 protein and Rv0431 protein in ΔM5447 using anti-His tag antibody. M. PageRuler Prestained Protein Ladder (Fermentas); Lane 1. Rv0431m3 /M; lane 2. Rv0431 expressed in ΔM5447; lane 3. Rv0431m1 /M; lane 4. Rv0431m2 /M; lane 5. Rv0431m4 /M; lane 6. Rv0431/M. D. Western blot of purified Rv0431 protein and Rv0431 protein in ΔM5447 using ConA. M. PageRuler Prestained Protein Ladder (Fermentas); Lane 1. Rv0431m3 /M; lane 2. Rv0431 expressed in ΔM5447; lane 3. Rv0431m1 /M; lane 4. Rv0431m2 /M; lane 5. Rv0431m4 /M; lane 6. Rv0431/M. Highlights: Rv0431 was a potentially O-mannosylated protein. The protein mediated the immune evasion of macrophages. Mannosyl modification of protein contributed to the immune evasion.