The small non-coding RNA B11 regulates multiple facets ofMycobacterium abscessusvirulence

Abstract

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.

Conserved mycobacterial sRNA B11 regulates lipooligosaccharide synthesis at post-transcriptional level

“…Here, we explored the biological function and regulatory mechanisms of sRNA B11 in M. marinum . By using different methods to discover B11-targets, we found that B11 controls several key genes in the LOS biosynthesis locus, which is distinct from the regulatory roles reported for B11 orthologues in M. smegmatis and M. abscessus (26,27). Furthermore, we found that M. marinum strains lacking B11 display a clear colony phenotype, suggesting that B11 acts as a negative posttranscriptional regulator of LOS synthesis.…”

“…Our data showed that the seed region for regulatory base pairing by B11 is a cytosine-rich loop near its 3' end, which appears to be conserved in the B11 orthologues in M. smegmatis (26) and M. abscessus (27). Because mycobacteria have high GC-content with numerous guanine-rich motifs throughout the entire genome, computational methods using the cytosine-rich regions to predict the targets were not accurate.…”

“…Because B11 is the most widely studied mycobacterial sRNA, it is possible to compare its function across different mycobacteria species, including M. tuberculosis(19) , M. smegmatis (26), M. abscessus(27) , and M. kansasii (47). However, the phenotype of B11-deficient strains has only been described in M. abscessus , M. kansasii and here, in M. marinum .…”

“…One surprising finding is that the direct B11 targets are distinct in the three species studied with target screening: M. smegmatis (26); M. abscessus(27) ; and M. marinum . The direct B11 targets in M. smegmatis are involved with DNA replication and protein secretion, and in M. abscessus with ESX-related genes, but we found no evidence that B11 is involved with the regulation of these processes in M. marinum .…”

“…Furthermore, we identified a G-stretch close to the translation start site of the M. kansasii pks5 gene (mkan_27485, an orthologue of mmar_2340) , suggesting that B11 regulation in M. kansasii could be similar to the B11 regulation we describe in M. marinum . In M. abscessus, which does not produce LOS, disruption of B11 resulted in smooth-to-rough colony variation by reducing the biosynthesis of glycopeptidolipids (GPL) through a mechanism, perhaps indirect, that appears to be independent of base-paring (27) . In addition, overexpression of B11 in the wild-type background of M. tuberculosis and M. smegmatis resulted in elongated bacilli, a phenotype that may be associated with the composition of the cell wall (19,26).…”