In the course of evolution, Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, has developed sophisticated strategies to evade host immune response, including the synthesis of small non-coding RNAs (sRNAs), which regulate post-transcriptional pathways involved in the stress adaptation of mycobacteria. sRNA MTS1338 is upregulated in Mtb during its infection of cultured macrophages and in the model of chronic tuberculosis, suggesting involvement in host–pathogen interactions. Here, we analyzed the role of MTS1338 in the Mtb response to macrophage-like stresses in vitro. The Mtb strain overexpressing MTS1338 demonstrated enhanced survival ability under low pH, nitrosative, and oxidative stress conditions simulating the antimicrobial environment inside macrophages. Transcriptomic analysis revealed that in MTS1338-overexpressing Mtb, the stress factors led to the activation of a number of transcriptional regulators, toxin–antitoxin modules, and stress chaperones, about half of which coincided with the genes induced in Mtb phagocytosed by macrophages. We determined the MTS1338 “core regulon”, consisting of 11 genes that were activated in all conditions under MTS1338 overexpression. Our findings indicate that MTS1338 is a stress-induced sRNA that promotes Mtb survival in macrophages by triggering adaptive transcriptional mechanisms in response to host antimicrobial defense reactions.

Small non-coding RNAs play a key role in bacterial adaptation to various stresses. Mycobacterium tuberculosis small RNA MTS1338 is upregulated during mycobacteria infection of macrophages, suggesting its involvement in the interaction of the pathogen with the host. In this study, we explored the functional effects of MTS1338 by expressing it in non-pathogenic Mycobacterium smegmatis that lacks the MTS1338 gene. The results indicated that MTS1338 slowed the growth of the recombinant mycobacteria in culture and increased their survival in RAW 264.7 macrophages, where the MTS1338-expressing strain significantly (p < 0.05) reduced the number of mature phagolysosomes and changed the production of cytokines IL-1β, IL-6, IL-10, IL-12, TGF-β, and TNF-α compared to those of the control strain. Proteomic and secretomic profiling of recombinant and control strains revealed differential expression of proteins involved in the synthesis of main cell wall components and in the regulation of iron metabolism (ESX-3 secretion system) and response to hypoxia (furA, whiB4, phoP). These effects of MTS1338 expression are characteristic for M. tuberculosis during infection, suggesting that in pathogenic mycobacteria MTS1338 plays the role of a virulence factor supporting the residence of M. tuberculosis in the host.

Mycobacterium abscessus has recently emerged as the cause of an increasing number of human infections worldwide. Unfortunately, it is highly resistant to existing drugs, and new specific agents to combat M. abscessus have not yet been found. The discovery of antibiotics that are effective not only against replicating but also against dormant and often recalcitrant cells is a daunting challenge. In this study, we developed a model of non-replicating M. abscessus, which represents a valuable screening tool for antibacterial agents. Thus, we demonstrated that, under a deficiency of potassium ions in the growth media and prolonged incubation, M. abscessus entered a ‘non-culturable’ state with a significant loss of colony-forming ability, but it retained viability, as confirmed using the most-probable-number (MPN) assay. The ‘non-culturable’ mycobacteria possessed decelerated cellular metabolism and noticeable differences in cell morphology from actively growing mycobacteria. ‘Non-culturable’ cells were used in a comprehensive screening of the efficacy of antibiotics, along with actively growing cells. Both CFU and MPN tests confirmed the prominent bactericidal effect of moxifloxacin on actively growing and ‘non-culturable’ M. abscessus, as proven by less than 0.01% of cells surviving after antibiotic treatment and prolonged storage. Bedaquiline exhibited a comparable bactericidal effect only on metabolically inactive non-culturable cells aged for 44 days. There were reductions ranging from 1000 to 10,000-fold in CFU and MPN, but it was not so efficient with respect to active cells, resulting in a bacteriostatic effect. The demonstrated specificity of bedaquiline in relation to inert non-replicating M. abscessus offers a new and unexpected result. Based on the findings of this research, moxifloxacin and bedaquiline can be regarded as potential treatments for infections caused by M. abscessus. In addition, a key outcome is the proposal to include the combination of viability assays for comprehensive testing of drug candidates. Relying on CFU-based assays alone resulted in overestimates of antibacterial efficacy, as demonstrated in our experiments.

The regulatory mechanisms of pathogenic bacteria contribute to their survival under stress conditions in the host environment, which allows them to avoid the immune system of the macroorganism. Small, noncoding RNAs were previously found to regulate some metabolic processes of Mycobacterium tuberculosis. We have revealed that the viability of M. tuberculosis in host-like conditions depends on the expression level of small RNAs. Strains overexpressing small RNA MTS1338 and MTS0997 in M. tuberculosis were produced, and their survival under stressful conditions in vitro and in infected human macrophages were studied. We found that overexpression of the small, noncoding RNA MTS1338 increased bacterial resistance to the stressful effects of hydrogen peroxide, nitric oxide, an acidic environment, and a long-term lack of nutrients at different growth phases and contributed to the viability of bacteria in infected macrophages. An overexpression of the small, noncoding RNA MTS0997 did not significantly affect cell viability under stress conditions. Thus, the two studied small RNAs play different roles in the mycobacterial adaptation to intracellular stresses during infection.

The role of small non-coding RNAs Mcr11 and DrrS with its possible synergy in the metabolism of M. tuberculosis was studied. There were no noticeable differences in the growth dynamics of both strains with a single deletion of small RNAs ΔMcr11 and ΔDrrS and a strain with a double deletion ΔΔMcr11_DrrS compared to the wild-type M. tuberculosis strain during growth on standard media in vitro. However, it was found that after virulence restoration of these strains by in vivo passage through B6 mice, they showed a growth defect on Sauton’s medium with a high concentration of glycerol (6 vol %) in vitro, more pronounced in the strain with a double deletion. As it is known, growth inhibition in the presence of high concentrations of glycerol in M. tuberculosis cells was caused by the deletion of the Rv3679-3680 genes, which was due to the accumulation of the toxic metabolite methylglyoxal. According to bioinformatic predictions, the mRNA of the Rv3679 gene in the 5'-untranslated region contains two potential binding sites for the DrrS, but not for the Mcr11, suggesting that Rv3679 may be a DrrS target. The result may indicate a common regulatory activity for small RNAs DrrS and Mcr11 of M. tuberculosis with the occurrence of a synergistic effect in the development of “glycerol toxicity” in the ΔΔMcr11_DrrS strain. Thus, small RNAs Mcr11 and DrrS are involved in the regulation of M. tuberculosis glycerol metabolism pathways.