DNA gyrase plays a vital role in resolving DNA topological problems and is the target of antibiotics such as fluoroquinolones. Mycobacterium fluoroquinolone resistance protein A (MfpA) from Mycobacterium smegmatis is a newly identified DNA gyrase inhibitor that is believed to confer intrinsic resistance to fluoroquinolones. However, MfpA does not prevent drug-induced inhibition of DNA gyrase in vitro , implying the involvement of other as yet unknown factors. Here, we have identified a new factor, named Mycobacterium fluoroquinolone resistance protein B (MfpB), which is involved in the protection of DNA gyrase against drugs both in vivo and in vitro . Genetic results suggest that MfpB is necessary for MfpA protection of DNA gyrase against drugs in vivo ; an mfpB knockout mutant showed greater susceptibility to ciprofloxacin than the wild-type, whereas a strain overexpressing MfpA and MfpB showed higher loss of susceptibility. Further biochemical characterization indicated that MfpB is a small GTPase and its GTP bound form interacts directly with MfpA and influences its interaction with DNA gyrase. Mutations in MfpB that decrease its GTPase activity disrupt its protective efficacy. Our studies suggest that MfpB, a small GTPase, is required for MfpA-conferred protection of DNA gyrase.

DNA gyrase, a type II topoisomerase, introduces negative supercoils into DNA using ATP hydrolysis. The highly effective gyrase-targeted drugs, fluoroquinolones (FQs), interrupt gyrase by stabilizing a DNA-cleavage complex, a transient intermediate in the supercoiling cycle, leading to double-stranded DNA breaks. MfpA, a pentapeptide-repeat protein in mycobacteria, protects gyrase from FQs, but its molecular mechanism remains unknown. Here, we show that Mycobacterium smegmatis MfpA (MsMfpA) inhibits negative supercoiling by M. smegmatis gyrase (Msgyrase) in the absence of FQs, while in their presence, MsMfpA decreases FQ-induced DNA cleavage, protecting the enzyme from these drugs. MsMfpA stimulates the ATPase activity of Msgyrase by directly interacting with the ATPase domain (MsGyrB47), which was confirmed through X-ray crystallography of the MsMfpA–MsGyrB47 complex, and mutational analysis, demonstrating that MsMfpA mimics a T (transported) DNA segment. These data reveal the molecular mechanism whereby MfpA modulates the activity of gyrase and may provide a general molecular basis for the action of other pentapeptide-repeat proteins. Significance Pentapeptide-repeat proteins, such as MfpA and Qnr proteins, have an intriguing right-handed quadrilateral β-helical fold that gives rise to an elongated roughly cylindrical structure that resembles the shape of a double-stranded DNA helix. It has been speculated that these proteins, particularly those that interact with bacterial DNA gyrase, act as DNA mimics, competing with DNA to fulfil their functions. Until now there has been no direct evidence for this. Using enzymology and X-ray crystallography, we show the mycobacterial MfpA appears to act as a mimic of the transported (T) DNA segment during the gyrase supercoiling cycle, protecting the enzyme from fluoroquinolone antibiotics. These data suggest a mechanism to limit fluoroquinolone efficacy.

Tuberculosis (TB) is a major public health problem, with nearly 10 million new cases and millions of deaths each year. Around 10% of these cases are in children, but only a fraction receive proper diagnosis and treatment. The spread of drug-resistant (DR) strain of TB has made it difficult to control, with only 60% of patients responding to treatment. Multi-drug resistant TB (MDR-TB) is often undiagnosed in children due to lack of awareness or under-diagnosis, and the target for children’s DR-TB treatment has only been met in 15% of goals. New medications such as bedaquiline and delamanid have been approved for treating DR-TB. However, due to age and weight differences, adults and children require different dosages. The availability of child-friendly formulations is limited by a lack of clinical data in children. This paper reviews the development history of these drugs, their mechanism of action, efficacy, safety potential problems and current use in treating DR-TB in children.

Global warming is increasing human exposure to pathogens and has already had an impact on human health. Vibrio parahaemolyticus , a major pathogen causing foodborne illness, accumulates in numerous aquatic organisms and can be affected by environmental stressors such as increased replication errors or DNA damage resulting in point mutations. This could lead to an elevation in the mutation rate, which influences the expansion of Vibrio spp. habitats and the spread of associated diseases. In this study, a total of 241 strains were isolated from aquatic products imported or exported through China Customs between 2005 and 2010. The whole genomes of those strains were sequenced, revealing a highly significant level of genetic diversity. Our analysis identified 27 new sequence types (STs) ranging from ST2950 to ST2976. The global temperature trend since 1950 affects the thermolabile hemolysin gene ( tlh ) found in all Vibrio parahaemolyticus leading to mutant sites exhibiting similar trends as temperatures rise; seven high-frequency mutation hotspots (A180G, T552G, G657T, T858C, C1062T, A1137G, and T1179C) were identified along with two clinically specific sites (T259C and A951T) that may indicate adaptation over time due to climate change, leading to increased virulence potential for this bacterial species. These results provide insight into the genetics of Vibrio parahaemolyticus and provide a reference for subsequent research, identification, and monitoring efforts related to its spread. IMPORTANCE: In this study, Vibrio parahaemolyticus strains were collected from a large number of aquatic products globally and found that temperature has an impact on the virulence of these bacteria. As global temperatures rise, mutations in a gene marker called thermolabile hemolysin ( tlh ) also increase. This suggests that environmental isolates adapt to the warming environment and become more pathogenic. The findings can help in developing tools to analyze and monitor these bacteria as well as assess any link between climate change and vibrio-associated diseases, which could be used for forecasting outbreaks associated with them.

Hemerythrin-like proteins are oxygen-carrying non-heme di-iron binding proteins and their functions have effect on oxidation-reduction regulation and antibiotic resistance. Recent studies using bioinformatic analyses suggest that multiple hemerythrin-like protein coding sequences might have been acquired by lateral gene transfer and the number of hemerythrin-like proteins varies amongst different species. Mycobacterium smegmatis contains three hemerythrin-like proteins, MSMEG_3312, MSMEG_2415 and MSMEG_6212. In this study, we have systematically analyzed all three hemerythrin-like proteins in M. smegmatis and our results identified and characterized two functional classes: MSMEG_2415 plays an important role in H₂O₂ susceptibility and MSMEG_3312 and MSMEG_6212 are associated with erythromycin susceptibility. Phylogenetic analysis indicated that these three proteins have different evolutionary origins, possibly explaining their different physiological functions. Here, combined with biological and phylogenetic analyses, our results provide new insights into the evolutionary divergence of the hemerythrin-like proteins in M. smegmatis.

Hydrogen peroxide (H₂O₂) is a natural oxidant produced by aerobic organisms and gives rise to oxidative damage, including DNA mutations, protein inactivation and lipid damage. The genus Mycobacterium utilizes redox sensors and H₂O₂ scavenging enzymes for the detoxification of H₂O₂. To date, the precise response to oxidative stress has not been fully elucidated. Here, we compared the effects of different levels of H₂O₂ on transcription in M. smegmatis using RNA-sequencing. A 0.2 mM H₂O₂ treatment had little effect on the growth and viability of M. smegmatis whereas 7 mM H₂O₂ was lethal. Analysis of global transcription showed that 0.2 mM H₂O₂ induced relatively few changes in gene expression, whereas a large proportion of the mycobacterial genome was found to be differentially expressed after treatment with 7 mM H₂O₂. Genes differentially expressed following treatment with 0.2 mM H₂O₂ included those coding for proteins involved in glycolysis-gluconeogenesis and fatty acid metabolism pathways, and expression of most genes encoding ribosomal proteins was lower following treatment with 7 mM H₂O₂. Our analysis shows that M. smegmatis utilizes the sigma factor MSMEG_5214 in response to 0.2 mM H₂O₂, and the RpoE1 sigma factors MSMEG_0573 and MSMEG_0574 in response to 7 mM H₂O₂. In addition, different transcriptional regulators responded to different levels of H₂O₂: MSMEG_1919 was induced by 0.2 mM H₂O₂, while high-level induction of DevR occurred in response to 7 mM H₂O₂. We detected the induction of different detoxifying enzymes, including genes encoding KatG, AhpD, TrxB and Trx, at different levels of H₂O₂ and the detoxifying enzymes were expressed at different levels of H₂O₂. In conclusion, our study reveals the changes in transcription that are induced in response to different levels of H₂O₂ in M. smegmatis.