Double mutation in DNA gyrase confers moxifloxacin resistance and decreased fitness of Mycobacterium smegmatis.

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.

PPE27 in Mycobacterium smegmatis Enhances Mycobacterial Survival and Manipulates Cytokine Secretion in Mouse Macrophages.

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)-1ß, 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.

Mycobacterium tuberculosis hypoxic response protein 1 (Hrp1) augments the pro-inflammatory response and enhances the survival of Mycobacterium smegmatis in murine macrophages.

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-1ß (IL-1ß), 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.