Despite the existence of well-characterized, canonical mutations that confer high-level drug resistance to Mycobacterium tuberculosis (Mtb), there is evidence that drug resistance mechanisms are more complex than simple acquisition of such mutations. Recent studies have shown that Mtb can acquire non-canonical resistance-associated mutations that confer survival advantages in the presence of certain drugs, likely acting as stepping-stones for acquisition of high-level resistance. Rv2752c/rnj, encoding RNase J, is disproportionately mutated in drug-resistant clinical Mtb isolates. Here we show that deletion of rnj confers increased tolerance to lethal concentrations of several drugs. RNAseq revealed that RNase J affects expression of a subset of genes enriched for PE/PPE genes and stable RNAs and is key for proper 23S rRNA maturation. Gene expression differences implicated two sRNAs and ppe50-ppe51 as important contributors to the drug tolerance phenotype. In addition, we found that in the absence of RNase J, many short RNA fragments accumulate because they are degraded at slower rates. We show that the accumulated transcript fragments are targets of RNase J and are characterized by strong secondary structure and high G+C content, indicating that RNase J has a rate-limiting role in degradation of highly structured RNAs. Taken together, our results demonstrate that RNase J indirectly affects drug tolerance, as well as reveal the endogenous roles of RNase J in mycobacterial RNA metabolism.

Mycobacterium abscessuscauses severe, virtually incurable disease in young patients with cystic fibrosis. Little is known inM. abscessusabout the roles of small regulatory RNAs (sRNA) in gene expression regulation. Here, we show that the sRNA B11 controls gene expression and virulence-associated phenotypes in this pathogen. B11 deletion from the smooth strain ATCC_19977 produced a rough colony morphology, increased pro-inflammatory signaling and virulence in in-vivo infection models, and increased resistance to clinically relevant antibiotics. Examination of clinical isolate cohorts revealed some isolates with B11 mutations or reduced expression. We used RNAseq and proteomics to investigate the effects of B11 on gene expression and test the impact of two mutations found in clinical isolates. Approximate 230 genes were differentially expressed in the B11 deletion mutant. Strains with the clinical B11 mutations showed similar expression trends to the deletion mutant but of a lesser magnitude, suggesting partial loss of function. Among genes upregulated in the B11 mutant, there was a strong enrichment for genes with B11-complementary sequences in their predicted ribosome binding sites (RBS), consistent with a model of translational repression via base-pairing of B11 to RBSs. Comparing the proteomes similarly revealed that upregulated proteins were strongly enriched for B11-complementary sequences in their RBS, consistent with B11 functioning as a negative regulator through direct binding of target mRNAs. Intriguingly, the genes upregulated in the absence of B11 included components of the ESX-4 secretion system, known to be critical forM. abscessusvirulence. One of these genes had a B11-complementary sequence at its RBS, and fusing the UTR of this gene to a reporter was sufficient to make the reporter suppressible by B11. Taken together, our data show that B11 may act as either a negative or positive regulator with pleiotropic effects on gene expression and clinically important phenotypes inM. abscessus. The presence of hypomorphic B11 mutations in clinical strains supports the idea that lower B11 activity may be advantageous forM. abscessusin some clinical contexts. To our knowledge, this is the first report of the role of an sRNA inM. abscessus.

ABSTRACT. We constructed a prokaryotic expression vector expressing the Mycobacterium tuberculosis protein TB16.3, as well as 3 other proteins, including TB15.3, CFP-10, and Rv2626C, which were purified and analyzed for their effectiveness as detection antibodies. The TB16.3 genes of M. tuberculosis H37Rv genomic DNA were amplified by polymerase chain reaction, inserted into the expression vector pET-30a, and expressed in Escherichia coli. An enzyme-linked immunosorbent assay was used to detect the 4 M. tuberculosis antibodies. Engineered E. coli bacteria expressing TB16.3 and the 3 other proteins were constructed and found mainly to be soluble. For recombinant TB16.3 proteins, serum samples of 118 tuberculosis (TB) patients and 96 healthy controls were analyzed. Sensitivity, specificity, and adjusted concordance rate for the TB16.3 antibody were 72.9, 86.5, and 79.6%, respectively. The positive rate of Rv2626C antibody in TB patients (44.1%) was significantly lower than that in normal controls (75.0%, χ 2 = 20.8, P < 0.01). TB15.3 and TB16.3 were used for simultaneous detection and showed sensitivity, specificity, and repeatability rates of 69.4, 96.9, and 83.7%. The antibody positive rate and specificity for patients with lung disease was 9.6 and 90.4%, respectively. TB15.3 and TB16.3 were mixed and detected simultaneously. Combined with the results for TB15.3, the sensitivity, specificity, and concordance rates were 82.2, 95.9, and 88.9%, respectively. The concordance rate was the highest value observed. Target genes were cloned into a host strain and expressed successfully. The TB16.3 recombinant protein may be used as a new serological antigen for tuberculosis diagnosis.

Despite the existence of well-characterized, canonical mutations that confer high-level drug resistance to Mycobacterium tuberculosis (Mtb), there is evidence that drug resistance mechanisms are more complex than simple acquisition of such mutations. Recent studies have shown that Mtb can acquire non-canonical resistance-associated mutations that confer survival advantages in the presence of certain drugs, likely acting as stepping-stones for acquisition of high-level resistance. Rv2752c/rnj, encoding RNase J, is disproportionately mutated in drug-resistant clinical Mtb isolates. Here we show that deletion of rnj confers increased tolerance to lethal concentrations of several drugs. RNAseq revealed that RNase J affects expression of a subset of genes enriched for PE/PPE genes and stable RNAs and is key for proper 23S rRNA maturation. Gene expression differences implicated two sRNAs and ppe50-ppe51 as important contributors to the drug tolerance phenotype. In addition, we found that in the absence of RNase J, many short RNA fragments accumulate because they are degraded at slower rates. We show that the accumulated transcript fragments are targets of RNase J and are characterized by strong secondary structure and high G+C content, indicating that RNase J has a rate-limiting role in degradation of highly structured RNAs. Taken together, our results demonstrate that RNase J indirectly affects drug tolerance, as well as reveal the endogenous roles of RNase J in mycobacterial RNA metabolism.

No abstract