The small non-coding RNA B11 regulates multiple facets of Mycobacterium abscessus virulence.

Mycobacterium abscessus causes severe disease in patients with cystic fibrosis. Little is known in M. abscessus about the roles of small regulatory RNAs (sRNA) in gene regulation. 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 strain, increased pro-inflammatory signaling and virulence in multiple infection models, and increased resistance to antibiotics. Examination of clinical isolate cohorts identified 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 mutations found in clinical isolates. Over 200 genes were differentially expressed in the deletion mutant. Strains with the clinical B11 mutations showed expression trends similar to the deletion mutant, 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 B11 functioning as a negative regulator that represses translation via base-pairing to RBSs. Comparing the proteomes similarly revealed that upregulated proteins were strongly enriched for B11-complementary sequences. Intriguingly, genes upregulated in the absence of B11 included components of the ESX-4 secretion system, critical for M. abscessus virulence. Many of these genes had B11-complementary sequences at their RBSs, which we show is sufficient to mediate repression by B11 through direct binding. Altogether, our data show that B11 acts as a direct negative regulator and mediates (likely indirect) positive regulation with pleiotropic effects on gene expression and clinically important phenotypes in M. abscessus. The presence of hypomorphic B11 mutations in clinical strains is consistent with the idea that lower B11 activity may be advantageous for M. abscessus in some clinical contexts. This is the first report on an sRNA role in M. abscessus.

Virulence-Associated Secretion in Mycobacterium abscessus.

Non-tuberculous mycobacteria (NTM) are a heterogeneous group of originally environmental organi3sms, increasingly recognized as pathogens with rising prevalence worldwide. Knowledge of NTM's mechanisms of virulence is lacking, as molecular research of these bacteria is challenging, sometimes more than that of M. tuberculosis (Mtb), and far less resources are allocated to their investigation. While some of the virulence mechanisms are common to several mycobacteria including Mtb, others NTM species-specific. Among NTMs, Mycobacterium abscessus (Mabs) causes some of the most severe and difficult to treat infections, especially chronic pulmonary infections. Mabs survives and proliferates intracellularly by circumventing host defenses, using multiple mechanisms, many of which remain poorly characterized. Some of these immune-evasion mechanisms are also found in Mtb, including phagosome pore formation, inhibition of phagosome maturation, cytokine response interference and apoptosis delay. While much is known of the role of Mtb-secreted effector molecules in mediating the manipulation of the host response, far less is known of the secreted effector molecules in Mabs. In this review, we briefly summarize the knowledge of secreted effectors in Mtb (such as ESX secretion, SecA2, TAT and others), and draw the parallel pathways in Mabs. We also describe pathways that are unique to Mabs, differentiating it from Mtb. This review will assist researchers interested in virulence-associated secretion in Mabs by providing the knowledge base and framework for their studies.

Comparison of Isogenic Strains Shows No Evidence of Altered Nosocomial Transmission-Competency of Rough, GPL-Negative Mycobacterium abscessus Strains.

Mycobacterium abscessus is an emerging pathogen causing severe pulmonary infections. While environmental in origin, in the clinical setting M. abscessus often changes to a Rough phenotype associated with severe non-remitting infections. Clinical isolates baring mutations in glycopeptidolipid-synthesis genes, leading to the Rough phenotype, were suggested to have increase bacterial virulence while possibly showing reduced transmissibility on fomites. We set to determine whether an isolated glycopeptidolipid (GPL) defect affects transmissibility. We used transposon technology to create a fully isogenic Rough (GPL-defective) (Tn_4099c) and compare it to the isogenic parent strain (ATCC 19977). Survival on fomites was determined by spotting, drying, and retrieving the isolates at designated time points. This was repeated as a competition experiment using a mixture of differentially fluorescent M. abscessus 19977 (Smooth) and the Tn_4099c mutant (Rough). Survival ability in chlorhexidine solution (Septal Scrub Teva) was performed using a disinfectant killing-assay for mycobacteria. Despite significant bacterial killing in all assays, we found no survival advantage to either GPL-defected Rough or GPL-reserved Smooth morphotype-both on fomites and in chlorhexidine. Our findings suggest that while transmission fitness may be altered due to some within-host evolutionary changes, decreased transmissibility of clinical strains cannot be attributed to the GPL-synthesis defect alone. Further studies are needed to determine the effect of other mutations on the transmission potential of M. abscessus in the clinical setting. IMPORTANCE Mycobacterium abscessus is an emerging pathogen causing severe pulmonary infections. In the clinical setting, M. abscsssus undergoes molecular and genetic changes associated with increased virulence. Specifically, bacterial defects in glycopeptidolipid (GPL) synthesis, creating the "Rough" colony phenotype, have been associated with increased virulence, yet were also presumably observed to have decreased survival on fomites, leading to reduced transmissibility. We set to determine whether GPL-synthesis defects are indeed responsible for reduced transmissibility of clinical isolates. We compared fully isogenic GPL-disrupted versus GPL-preserved strains, and demonstrated no survival advantage for either strain on fomites. Additionally, neither isolate had a survival advantage in chlorhexidine, a widely used disinfectant in health care settings. Our findings suggest that reduced transmissibility of clinical isolates, should it be found, cannot be attributed to GPL-synthesis mutations. While clinical isolates may show changes in transmission potential, more studies are needed to investigate the mechanisms leading to these phenotypic changes.

Human antibodies targeting a Mycobacterium transporter protein mediate protection against tuberculosis.

Mycobacterium tuberculosis (Mtb) exposure drives antibody responses, but whether patients with active tuberculosis elicit protective antibodies, and against which antigens, is still unclear. Here we generate monoclonal antibodies from memory B cells of one patient to investigate the B cell responses during active infection. The antibodies, members of four distinct B cell clones, are directed against the Mtb phosphate transporter subunit PstS1. Antibodies p4-36 and p4-163 reduce Mycobacterium bovis-BCG and Mtb levels in an ex vivo human whole blood growth inhibition assay in an FcR-dependent manner; meanwhile, germline versions of p4-36 and p4-163 do not bind Mtb. Crystal structures of p4-36 and p4-170, complexed to PstS1, are determined at 2.1 Å and 2.4 Å resolution, respectively, to reveal two distinctive PstS1 epitopes. Lastly, a prophylactic p4-36 and p4-163 treatment in Mtb-infected Balb/c mice reduces bacterial lung burden by 50%. Our study shows that inhibitory anti-PstS1 B cell responses arise during active tuberculosis.

The Unexpected Essentiality of glnA2 in Mycobacterium smegmatis Is Salvaged by Overexpression of the Global Nitrogen Regulator glnR, but Not by L-, D- or Iso-Glutamine.

Nitrogen metabolism plays a central role in the physiology of microorganisms, and Glutamine Synthetase (GS) genes are present in virtually all bacteria. In M. tuberculosis, four GS genes are present, but only glnA1 is essential, whereas glnA2 was shown to be non-essential for in-vitro as well as in-vivo growth and pathogenesis, and is postulated to be involved in D-glutamine and iso-glutamine synthesis. Whilst investigating the activity of an antimicrobial compound in M. smegmatis, we found a spontaneous temperature-sensitive mutant in glnA2 (I133F), and used it to investigate the role of glnA2 in M. smegmatis. We deleted the native glnA2 and replaced it with a mutated allele. This re-created the temperature sensitivity-as after 3-4 seemingly normal division cycles, glnA2 became essential for growth. This essentiality could not be salvaged by neither L, D- nor iso-glutamine, suggesting an additional role of glnA2 in M. smegmatis over its role in M. tuberculosis. We also found that overexpression of the global nitrogen regulator glnR enabled bypassing the essentiality of glnA2, allowing the creation of a complete deletion mutant. The discrepancy between the importance of glnA2 in Mtb and M. smegmatis stresses the caution in which results in one are extrapolated to the other.

A signal amplification probe enhances sensitivity of antibodies and aptamers based Immuno-diagnostic assays.

One major unmet need is improving the sensitivity of immune-diagnostic assays. This is particularly important in the field of biomarker discoveries and monitoring. We have established a novel signal amplification probe system enabling a highly sensitive target detection platform to be used in immuno-assays. The probe consists of a double stranded DNA that can carry a large number of signaling elements such as biotin or fluorescent molecules. The DNA probe anchors to the recognition unit, whether an antibody or an aptamer, by covalent conjugation or by a simple and rapid molecular association process. Binding curves obtained by using the DNA amplification probe are dose dependent and linear over a wide range of antigen concentration. The optimal slopes are characterized by high signals and low background increasing the assay sensitivity and reducing the limit of detection by up to 10-fold compared to biotinylated antibodies commonly used in ELISA systems. When using aptamers in combination with the amplification probe for antigen recognition, the limit of detection is comparable to that obtained by biotinylated antibodies. Biotin labeled aptamers practically cannot be used for detection of low target levels. The DNA amplification probe system enables to expand the range of diagnostic assays including clinical samples and meet research needs.

Acetylation reduces SOX9 nuclear entry and ACAN gene transactivation in human chondrocytes.

Changes in the content of aggrecan, an essential proteoglycan of articular cartilage, have been implicated in the pathophysiology of osteoarthritis (OA), a prevalent age-related, degenerative joint disease. Here, we examined the effect of SOX9 acetylation on ACAN transactivation in the context of osteoarthritis. Primary chondrocytes freshly isolated from degenerated OA cartilage displayed lower levels of ACAN mRNA and higher levels of acetylated SOX9 compared with cells from intact regions of OA cartilage. Degenerated OA cartilage presented chondrocyte clusters bearing diffused immunostaining for SOX9 compared with intact cartilage regions. Primary human chondrocytes freshly isolated from OA knee joints were cultured in monolayer or in three-dimensional alginate microbeads (3D). SOX9 was hypo-acetylated in 3D cultures and displayed enhanced binding to a -10 kb ACAN enhancer, a result consistent with higher ACAN mRNA levels than in monolayer cultures. It also co-immunoprecipitated with SIRT1, a major deacetylase responsible for SOX9 deacetylation. Finally, immunofluorescence assays revealed increased nuclear localization of SOX9 in primary chondrocytes treated with the NAD SIRT1 cofactor, than in cells treated with a SIRT1 inhibitor. Inhibition of importin ß by importazole maintained SOX9 in the cytoplasm, even in the presence of NAD. Based on these data, we conclude that deacetylation promotes SOX9 nuclear translocation and hence its ability to activate ACAN.