Spontaneous mutational patterns and novel mutations for bedaquiline and clofazimine resistance in Mycobacterium tuberculosis.

The 2022 World Health Organization guidelines recommend use of two core anti-tuberculosis (TB) drugs, bedaquiline (BDQ) and clofazimine (CFZ), for treatment of drug-resistant (DR)-TB. However, several mutated Mycobacterium tuberculosis (MTB) genes, conferring BDQ and CFZ resistance, have been reported that predominantly arose from sporadic mutations that have not been comprehensively characterized. Herein, MTB clinical isolates collected from drug-susceptible (DS)-, multidrug-resistant (MDR)-, and extensively drug-resistant (XDR)-TB patients were cultured in vitro with BDQ or CFZ to generate progeny strains with resistance to these drugs. Progeny strains exposed to CFZ exhibited increased CFZ minimum inhibitory concentrations (MICs) that exceeded MIC increases of BDQ-exposed progeny strains. Notably, mmpR and pepQ mutations accounted for 83% and 17% of BDQ-induced spontaneous gene mutations, respectively, and 86% and 14% of CFZ-induced spontaneous gene mutations, respectively. Analyses of predicted mutation-induced changes in amino acid sequences and structures of MmpR and PepQ mutants revealed several point mutations affected sequence conversation and functionality as an underlying mechanism for observed acquired BDQ/CFZ resistance. Moreover, our results revealed differences in patterns of BDQ- and CFZ-induced acquired spontaneous mutations that may enhance our understanding of MTB BDQ/CFZ-resistance mechanisms. IMPORTANCE This study of MTB drug resistance mechanisms revealed patterns of spontaneous MTB mutations associated with acquired BDQ and CFZ resistance that arose after clinical MTB isolates were cultured in vitro with BDQ or CFZ. Results of protein sequence and structural analyses provided insights into potential mechanisms underlying associations between MTB gene mutations and DR phenotypes. Taken together, these results revealed differences in acquired BDQ and CFZ resistance mechanisms as a new perspective that may enhance our understanding of BDQ/CFZ resistance mechanisms and facilitate the development of new methods for detecting MTB drug resistance genes.

Spontaneous Mutational Patterns and Novel Mutations for Delamanid Resistance in Mycobacterium tuberculosis.

Delamanid (DLM) and pretomanid (PTM) are recent additions to the anti-tuberculosis (TB) drug armamentarium, and they offer more effective options for drug-resistant TB treatment. In particular, DLM is included in Group C, which is recommended for use in longer multidrug-resistant (MDR)-TB regimens. Previous studies have shown that resistance to DLM/PTM is caused by mutations in the ddn, fgd1, fbiA, fbiB, fbiC, and fbiD genes, which are related to the F420-dependent bioactivation pathway. Herein, we conduct in vitro selection of DLM-resistant strains using clinical Mycobacterium tuberculosis (MTB) isolates with various drug resistance profiles. The spontaneous resistance frequency of drug-susceptible (DS) MTB (1.14 × 10-6 to 1.04 × 10-4) to DLM was similar to that of H37Rv (8.88 × 10-6 to 9.96 × 10-6) but higher than those of multidrug-resistant MTB (2.03 × 10-7 to 3.18 × 10-6) and extensively drug-resistant (XDR) MTB (4.67 × 10-8 to 3.60 × 10-6). Of the 100 independently selected DLM-resistant MTB mutants, 65% harbored mutations in genes associated with either DLM prodrug activation (ddn, 39.73%; fgd1, 16.44%) or the F420 biosynthetic pathway (fbiA, 16.44%; fbiB, 5.48%; fbiC, 21.92%). Of the 45 mutations we identified, 38 were not previously reported. A structure analysis revealed that several point mutations affected the ligand binding or structural stability of enzymes related to DLM resistance, which would block the enzyme activity required for prodrug activation. Our results elucidate the in vitro spontaneous DLM-resistance patterns of different clinical strains, which could improve the understanding of the causes of DLM resistance in clinical strains and of the effects on drug resistance of different mutations in genes that are related to the DLM activation pathway.

HDAC6 contributes to human resistance against Mycobacterium tuberculosis infection via mediating innate immune responses.

Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (Mtb), remains a major cause of morbidity and mortality worldwide. Increasing lines of evidence indicate that certain individuals, which are termed resisters, are naturally resistant to TB infection. The resister phenotype has been linked to host efficient innate immune responses, but the underlying mechanisms and the key immune factors remain unclear. Here, we find that upon Mtb infection, monocyte-derived macrophages (MDMs) from TB resisters exhibited distinctly higher production of TNF-α, IL-1ß and IL-6, higher ratio of bacteria in acidic vacuoles, and lower intracellular bacterial loads, as compared to that from the healthy controls, individuals with latent TB infection, and TB patients. Such enhanced anti-Mtb immune capacity of macrophages from resisters largely depends on histone deacetylase 6 (HDAC6), whose expression is specifically maintained in MDMs from TB resisters during Mtb infection. Furthermore, we demonstrate that HDAC6 is required for acidification of Mtb-containing phagosomes in macrophages, thus controlling the intracellular survival of Mtb. Taken together, these findings unravel an indispensable role of HDAC6 in human innate resistance against Mtb infection, suggesting that HDAC6 may serve as a marker for individual TB risk as well as a novel host-directed anti-TB therapeutic target.