Lang Bao

探究结核分枝杆菌（Mycobacterium tuberculosis, Mtb）higBA基因对细菌应激反应及胞内感染免疫的作用。 从Mtb H37Rv基因组上扩增获得目的基因，与载体连接后电转化入耻垢分枝杆菌（Mycobacterium smegmatis, Ms）构建重组菌，对空载菌 Ms_vec和重组菌 Ms_ higBA进行应激实验和Raw264.7小鼠巨噬细胞感染实验，检测细菌菌落形成单位（CFU）和细胞因子白介素（interleukin, IL）-1β、IL-6、IL-10、IL-12p40，干扰素（interferon, IFN）-γ，肿瘤坏死因子（tumor necrosis factor, TNF）-α和诱导型一氧化氮合酶（inductible nitric oxide synthase, iNOS ）的转录水平变化。 成功构建Ms_higBA重组菌。应激实验结果表明higBA确能提高细菌在体外培养特定条件下的生存能力。胞内感染实验证明higBA能提高细菌在巨噬细胞内存活能力，影响细胞因子的转录水平。 Mtb的higBA基因在细菌应激反应和胞内感染免疫中发挥了作用。

Mycobacterium tuberculosis has been observed to develop resistance to the frontline anti-tuberculosis drug rifampicin, primarily through mutations in the rifampicin resistance-determining region (RRDR) of rpoB. While these mutations have been determined to confer a fitness cost, compensatory mutations in rpoA and rpoC that may enhance the fitness of resistant strains have been demonstrated. Recent genomic studies identified several rpoB non-RRDR mutations that co-occurred with RRDR mutations in clinical isolates without rpoA/rpoC mutations and may confer fitness compensation. In this study, we identified 33 evolutionarily convergent rpoB non-RRDR mutations through phylogenomic analysis of public genomic data for clinical M. tuberculosis isolates. We found that none of these mutations, except V170F and I491F, can cause rifampin resistance in Mycolicibacterium smegmatis. The compensatory effects of five representative mutations across rpoB were evaluated by an in vitro competition assay, through which we observed that each of these mutations can significantly improve the relative fitness of the initial S450L mutant (0.97–1.08 vs 0.87). Furthermore, we observed that the decreased RNAP transcription efficiency introduced by S450L was significantly alleviated by each of the five mutations. Structural analysis indicated that the fitness compensation observed for the non-RRDR mutations might be achieved by modification of the RpoB active centre or by changes in interactions between RNAP subunits. Our results provide experimental evidence supporting that compensatory effects are exerted by several rpoB non-RRDR mutations, which could be utilized as additional molecular markers for predicting the fitness of clinical rifampin-resistant M. tuberculosis strains.

Mycobacterium tuberculosis PE/PPE family proteins play a vital role in antigenic diversity, host–pathogen interactions, and immune evasion. As secreted by ESX-5 system, M. tuberculosis PPE27 is related to the growth and virulence of the bacilli. In this study, we expressed PPE27 in the nonpathogenic fast growing Mycobacterium smegmatis. We found that the recombinant strain exhibits higher survival rate under several hostile conditions in vitro and longer persistence in mouse tissues. The survival of the recombinant strains was also enhanced in the mouse macrophage ANA-1, accompanied by higher level of host cell death, higher secretion of tumor necrosis factor-alpha, and a slightly higher amount of interleukin (IL)-1β, which could be abolished by the nuclear factor-κB, p38, and ERK inhibitors. In addition, the recombinant strain robustly induced larger amount of nitric oxide (NO) and lower amount of IL-6 after infection of mouse macrophages. In brief, our data suggest that PPE27 promotes the survival of nonpathogenic M. smegmatis in vitro by manipulating the expression of multiple cytokines, NO, and affecting host cell necrosis, which provide a new insight to understand the functions of this gene.

Purpose. The DosR/DosS two-component regulatory system of Mycobacterium tuberculosis regulates the expression of numerous genes under stress conditions and is important for the long-term survival of M. tuberculosis in the host. The rv2626c gene of M. tuberculosis is one of the most strongly induced transcripts of the dormancy regulon. This study focused on the immunological effects and possible function of Rv2626c in maintaining mycobacterial survival under various stress conditions. Methodology. We heterologously expressed the Rv2626c protein in Mycobacterium smegmatis by constructing a recombinant strain Ms_rv2626c. The viability of Ms_rv2626c was evaluated both in vivo and ex vivo. Different stress conditions, including acidified sodium nitrite, malachite green, low pH, SDS and lysozyme, were used to evaluate the effect of Rv2626c on bacterial resistance. An in vitro assay using a macrophage infection model was utilized to investigate the potential effect of Rv2626c to alter the immune response of host cell and its associated pathways. The effect of Rv2626c on cell necrosis was also explored. Results. The expression of Rv2626c-enhanced M. smegmatis survival under hypoxia and nitric oxide stress in vitro, and this enhancement was maintained within macrophages and in mouse tissues. In addition, macrophages infected with M. smegmatis expressing Rv2626c showed significantly higher interleukin-1β (IL-1β), IL-6, tumour necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) expression, as well as a higher level of cell necrosis, compared with the control. Conclusion. M. tuberculosis protein Rv2626c plays a significant role in stimulating macrophages to provoke a pro-inflammatory response and in mycobacterial survival during infection.

Objectives: Ofloxacin and moxifloxacin are the most commonly used fluoroquinolones (FQs) for the treatment of tuberculosis. As a new generation FQ, moxifloxacin has been recommended for the treatment of ofloxacin-resistant TB. However, the mechanism by which ofloxacin-resistant Mycobacterium tuberculosis further gains resistance to moxifloxacin remains unclear. Methods: We used Mycobacterium smegmatis as a model for studying FQ resistance in M. tuberculosis. Moxifloxacin-resistant M. smegmatis was selected in vitro based on strains with primary ofloxacin resistance. The gyrA and gyrB genes of the resistant strains were sequenced to identify resistance-associated mutations. An in vitro competition assay was applied to explore the influence of gyrA/gyrB mutations on bacterial fitness. Finally, we evaluated the clinical relevance of our findings by analysing the WGS data of 1984 globally collected M. tuberculosis strains. Results: A total of 57 moxifloxacin-resistant M. smegmatis strains based on five ofloxacin-resistant strains were obtained. Sequencing results revealed that all moxifloxacin-resistant strains harboured second-step mutations in gyrA or gyrB. The relative fitnesses of the double-mutation strains varied from 0.65 to 0.93 and were mostly lower than those of their mono-mutation parents. From the genomic data, we identified 37 clinical M. tuberculosis strains harbouring double mutations in gyrA and/or gyrB and 36 of them carried at least one low-level FQ-resistance mutation. Conclusions: Double mutation in DNA gyrase leads to moxifloxacin resistance and decreased fitness in M. smegmatis. Under current dosing of moxifloxacin, double mutations mainly happened in M. tuberculosis strains with primary low-level resistance mutations.