| Tuberculosis(TB) caused by Mycobacterium tuberculosis infection is one of the major causes of morbidity and mortality worldwide, which remains a serious threat to global public health, with approximately one third of the world’s population infected and nearly nine million new cases and 1.5 million deaths annually. M. tuberculosis is considered to be one of the most successful and widespread intracellular pathogens, is transmitted through inhalation of aerosolized droplets that effectively establish pulmonary infection. Although vaccine and anti-Tuberculosis drugs appeared constantly, as the appearance of multi-drug-resistant strains and the co-infection with human immunodeficiency virus(HIV), depth exploration of M. tuberculosis pathogenicity and development of new vaccines or new Therapeutic drugs are urgently needed.M. tuberculosis enters the lung alveoli will face a variety of hostile environment stresses from the host. Macrophages in the alveoli are thought to serve as the main effector cells during the early stages of infection with M. tuberculosis, which constitutes the primary line of immune defense. Once the M. tuberculosis enters the lung via small aerosolized droplets, which will be internalized by host macrophages into a phagosome. Within the phagosome, the invading microbe is exposed to elevated levels of reactive oxygen intermediates(ROI), reactive nitrogen intermediates(RNI) and nutrient deficiencies in an increasingly hostile environment. The phagosome matures and fuses with organelles of the endocytic pathway acquiring molecular markers that act to acidify the phagocytic compartment to pH 5, and hydrolytic enzymes that digest its contents. During transmission from infected patient to a new host,M. tuberculosis will face other environmental stresses such as low temperature, UV exposure, and dehydration. M. tuberculosis have developed numerous strategies to evade the antimicrobial actions of the macrophage and to survive within this normally inhospitable cell eventually resulting in disease, this also depends on the complex expression regulation networks of multiple genes.PPE family genes were attended more and more based on the known function ofthem in the interaction of host and M. tuberculosis. The genes in this family were reported to involve in the virulence, as a source of antigenic variation and a mechanism for immune evasion. We found many genes of PPE family are regulated by Sigma factors, which plays an important role in the stress response in bacteria. Sigma Deficient strains are more sensitive to SDS-induced surface stress, heat shock and oxidative stress. Hence we speculate that Rv2770 c may also be regulated by Sigma factors and have a function response to those environmental stresses.M. tuberculosis can secrete virulence factors, which can fight for them a more favorable survive environment. mRNA Differential Display(DD) assay show that Rv2770c(PPE44) is likely to be a virulence factor, its toxicity has been verified from different angles:(1)Rv2770c, now termed ppe44, is downregulated in attenuated H37Ra;(2)Variation of the expression of ppe44 gene among clinical isolates, ppe44 expression of the Beijing strains was significantly higher than the non-Beijing strains, nucleotide sequences of ppe44 were found only in species belonging to the M. tuberculosis complex;(3)90 days after infection in guinea pigs lung whose expression was significantly upregulated. Infection of mice with BCG or with M. tuberculosis induced PPE44-specific humoral and cellular immune responses, and most importantly, vaccination of mice with PPE44-based subunit vaccines followed by an intratracheal challenge with virulent M. tuberculosis resulted in protective efficacy comparable to that afforded by BCG. Therefore, we speculate ppe44 may play a crucial role in the manipulation of the host immune response. Also, the expression of the ppe protein Rv2770 c is found to be increased under stress condition. While some members of this family have been studied, the function of most PPE proteins remains unknown.To investigate whether Rv2770 c has a role in host-pathogen interaction and response to environmental stresses, we firstly constructed a recombinant M. smegmatis mc2155 strain overexpressing Rv2770 c protein with shuttle plasmid pNIT(Myc), and verified the expression of Rv2770 c in recombinant strains by Western-blotting. We detected the localization of Rv2770 c in M. smegmatis with subcellular fractionation and detected the effect of Rv2770 c protein on growth, colony morphology, biofilm formation and sliding motility. We also investigated the resistance of the recombinant strains to acid stress, surface stress,oxidative stress and acidic NaNO2. Murine macrophage cell line RAW264.7 and human acute monocyte leukemia cell line THP-1were challenged by the resultant strains, the survival of recombinant strains in macrophages and the cytokines secretion on transcription by qRT-PCR were detected.In this study, we found that Rv2770 c protein mainly located on the surface of M. smegmatis is associated with the cell wall and is exposed at the cell surface, which alters the colony morphology, but has no effect on biofilm formation, sliding motility and fatty acids component. Overexpression of Rv2770 c greatly increased the tolerance of M. smegmatis to acid stress, SDS,diamide,H2O2 and acid NO. We also found that Rv2770 c was able to enhance the intracellular survival of recombinant M. smegmatis and promote the death of host cell. The mRNA level of IL-6 and IL-12p40 in THP-1 cells were upregulated by stimulation with M. smegmatis expressing Rv2770 c compared with controls. M. smegmatis expressing Rv2770c-induced IL-6 and IL-12p40 secretion was found to be mediated by NF-κB, ERK1/2 and p38 pathway in macrophages by pharmacological inhibitors of signaling pathways assay. Taken together, our findings demonstrate that Rv2770 c delivered in a live M. smegmatis vehicle can modify the cytokines profile of macrophages, promote host cell death and enhance the intracellular survival of bacteria within host cells. We hope that the research may provide new insights for revealing the mechanism how PPE family protein interact with the host cells and new clues into the mycobacteria pathogenesis. |
PDF Full Text Request