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.

Loss of RNase J leads to multi-drug tolerance and accumulation of highly structured mRNA fragments in Mycobacterium tuberculosis.

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.

An O-methylflavone from Artemisia afra kills non-replicating hypoxic Mycobacterium tuberculosis.

ETHNOPHARMACOLOGICAL RELEVANCE: African wormwood (Artemisia afra Jacq. ex Willd.) has been used traditionally in southern Africa to treat illnesses causing fever and was recently shown to possess anti-tuberculosis activity. As tuberculosis is an endemic cause of fever in southern Africa, this suggests that the anti-tubercular activity of A. afra may have contributed to its traditional medicinal use. AIM OF THE STUDY: Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is a deadly and debilitating disease globally affecting millions annually. Emerging drug-resistant Mtb strains endanger the efficacy of the current therapies employed to treat tuberculosis; therefore, there is an urgent need to develop novel drugs to combat this disease. Given the reported activity of A. afra against Mtb, we sought to determine the mechanisms by which A. afra inhibits and kills this bacterium. MATERIALS AND METHODS: We used transcriptomics to investigate the impact of Artemisia spp. extracts on Mtb physiology. We then used chromatographic fractionation and biochemometric analyses to identify a bioactive fractions of A. afra extracts and identify an active compound. RESULTS: Transcriptomic analysis revealed that A. afra exerts different effects on Mtb compared to A. annua or artemisinin, suggesting that A. afra possesses other phytochemicals with unique modes of action. A biochemometric study of A. afra resulted in the isolation of an O-methylflavone (1), 5-hydroxy-7-methoxy-2-(4-methoxyphenyl)chromen-4-one, which displayed considerable activity against Mtb strain mc26230 in both log phase growth and metabolically downshifted hypoxic cultures. CONCLUSIONS: The present study demonstrated that an O-methylflavone constituent of Artemisia afra explains part of the activity of this plant against Mtb. This result contributes to a mechanistic understanding of the reported anti-tubercular activity of A. afra and highlights the need for further study of this traditional medicinal plant and its active compounds.

Superior protection against paratuberculosis by a heterologous prime-boost immunization in a murine model.

Vaccination is the best strategy to control Paratuberculosis (PTB), which is a significant disease in cattle and sheep. Previously we showed the humoral and cellular immune response induced by a novel vaccine candidate against PTB based on the Argentinian Mycobacterium avium subspecies paratuberculosis (Map) 6611 strain. To improve 6611 immunogenicity and efficacy, we evaluated this vaccine candidate in mice with two different adjuvants and a heterologous boost with a recombinant modified vaccinia Ankara virus (MVA) expressing the antigen 85A (MVA85A). We observed that boosting with MVA85A did not improve total IgG or specific isotypes in serum induced by one or two doses of 6611 formulated with incomplete Freund's adjuvant (IFA). However, when 6611 was formulated with ISA201 adjuvant, MVA85A boost enhanced the production of IFNγ, Th1/Th17 cytokines (IL-2, TNF, IL-17A) and IL-6, IL-4 and IL-10. Also, this group showed the highest levels of IgG2b and IgG3 isotypes, both important for better protection against Map infection in the murine model. Finally, the heterologous scheme elicited the highest levels of protection after Map challenge (lowest CFU count and liver lesion score). In conclusion, our results encourage further evaluation of 6611 strain + ISA201 prime and MVA85A boost in bovines.

Artemisia afra and Artemisia annua Extracts Have Bactericidal Activity against Mycobacterium tuberculosis in Physiologically Relevant Carbon Sources and Hypoxia.

Mycobacterium tuberculosis (Mtb) is a deadly pathogen and causative agent of human tuberculosis, causing ~1.5 million deaths every year. The increasing drug resistance of this pathogen necessitates novel and improved treatment strategies. A crucial aspect of the host-pathogen interaction is bacterial nutrition. In this study, Artemisia annua and Artemisia afra dichloromethane extracts were tested for bactericidal activity against Mtb strain mc26230 under hypoxia and various infection-associated carbon sources (glycerol, glucose, and cholesterol). Both extracts showed significant bactericidal activity against Mtb, regardless of carbon source. Based on killing curves, A. afra showed the most consistent bactericidal activity against Mtb for all tested carbon sources, whereas A. annua showed the highest bactericidal activity in 7H9 minimal media with glycerol. Both extracts retained their bactericidal activity against Mtb under hypoxic conditions. Further investigations are required to determine the mechanism of action of these extracts and identify their active constituent compounds.

A methoxylated flavone from Artemisia afra kills Mycobacterium tuberculosis.

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is a deadly and debilitating disease globally affecting millions annually. Emerging drug-resistant Mtb strains endanger the efficacy of the current combination therapies employed to treat tuberculosis; therefore, there is an urgent need to develop novel drugs to combat this disease. Artemisia afra is used traditionally in southern Africa to treat malaria and recently has shown anti tuberculosis activity. This genus synthesizes a prodigious number of phytochemicals, many of which have demonstrated human health effects. Transcriptomic analysis revealed that A. afra exerts different effects on Mtb compared to A. annua or the well-known antimalarial artemisinin, suggesting other phytochemicals present in A. afra with unique modes of action. A biochemometric study of A. afra resulted in the isolation of a methoxylated flavone (1), which displayed considerable activity against Mtb strain mc26230. Compound 1 had an MIC of 312.5 µg/mL and yielded no viable colonies after 6 days of treatment. In addition, 1 was effective in killing hypoxic Mtb cultures, with no viable cultures after 2 days of treatment. This suggested that A. afra is a source of potentially powerful anti-Mtb phytochemicals with novel mechanisms of action.

Development and Validation of a Novel ELISA for the Specific Detection of Antibodies against Mycobacterium avium Subspecies paratuberculosis Based on a Chimeric Polyprotein.

Bovine paratuberculosis (PTB) is caused by Mycobacterium avium subsp. paratuberculosis (MAP). The optimization of detection tests specific for MAP is crucial to improve PTB control. In this work, we aimed to develop and validate a diagnostic tool based on an ELISA to specifically detect anti-MAP antibodies from bovine serum samples. For that purpose, we designed a recombinant polyprotein containing four specific antigens from MAP and optimized the ELISA. The validation consisted of the assessment of 10 sera from PTB-infected and healthy bovines with different OD values. The diagnostic performance of the polyprotein-ELISA was evaluated by testing 130 bovine serum samples (47 healthy, 48 MAP-infected, and 35 M. bovis-infected bovines). The ELISA using the polyprotein yielded an area under the ROC curve (AUC) of 0.9912 (95% CI, 0.9758-1.007; P < 0.0001). Moreover, for this ELISA, the cut-off selected from the ROC curve based on the point with a sensitivity of 95.56% (95% CI, 0.8485-0.9946) and specificity of 97.92 (95% CI, 0.8893-0.9995) was 0.3328. Similar results were obtained with an ELISA using the commercial Paratuberculosis Protoplasmatic Antigen (PPA). However, the ELISA with the polyprotein antigen showed a better performance against sera from animals infected with Mycobacterium bovis compared to the ELISA with PPA: lower cross-reactivity (2.85% versus 25.71%). These results demonstrate a very low cross-reactivity of the polyprotein with antibodies present in serum samples from animals infected with M. bovis. The designed polyprotein and the validated ELISA could be very useful for the specific identification of MAP-infected animals in herds.

Evaluation of a virulent strain of Mycobacterium avium subsp. Paratuberculosis used as a heat-killed vaccine.

Bovine paratuberculosis is one of the most important chronic infectious diseases in livestock. This disease is difficult to control because of its inefficient management (test and cull strategy and inadequate biosecurity). Thus, the development of an effective vaccine is essential. In this study, we evaluated a local virulent strain (6611) of Mycobacterium avium subsp. paratuberculosis as an inactivated vaccine in comparison with the Silirum vaccine in mouse model and cattle. Regarding the mice model, only the groups vaccinated with 6611 showed lower colony forming unit (CFU) counts with a lower lesion score in the liver in comparison to the control group at 6 and 12 weeks post-challenge (wpc). The immune response was predominantly humoral (IgG1), although both vaccinated groups presented a cellular response with IFNγ production as well, but the 6611 group had also significant production of IL-2, IL-6, IL-17a, TNF, and IL-10. In cattle, the 6611 vaccinated group was the only one that maintained significant antibody values at the end of the trial, with significant production of IgG2 and IFNγ. No PPDb reactor was detected in the vaccinated animals, according to the intradermal caudal fold tuberculin test. Our results indicate that the 6611 local strain protected mice from challenge with a virulent strain, by inducing a humoral and cellular immune response. In the bovine, the natural host, the evaluated vaccine also induced humoral and cellular immune responses, with higher levels of CD4 + CD25+ and CD8 + CD25+ T cells populations than the commercial vaccine. Despite the encouraging results obtained in this study, an experimental challenge trial in cattle is mandatory to evaluate the efficacy of our candidate vaccine in the main host.

Efficient method for targeted gene disruption by homologous recombination in Mycobacterium avium subspecie paratuberculosis.

Targeted gene disruption by homologous recombination, has been widely used in mycobacterium species to understand the genetic basis of virulence and persistence in the host and to develop efficacious potential live vaccines. However, in slow growing pathogenic mycobacteria as Mycobacterium avium subsp paratuberculosis (MAP), these methods have been inefficient, in part due to the low frequency of legitimate homologous recombination. Another feature of mycobacteria is the low efficiency of transformation; therefore, some years ago, a phage-mediated transduction process was developed to introduce DNA into mycobacteria. This strategy is very efficient, due to the high rate of infection of the phage. This report describes a genetic method for the generation of targeted deletion mutations in MAP by allelic exchange using in vitro-generated specialized transducing mycobacteriophages, which does not require the critical packaging step and that could also be applied to other mycobacteria. We provide a detailed gene deletion methodology and demonstrate the use of this genetic system by deleting the mce4 operon of MAP. Finally, our results showed that the deletion of mce4 in MAP induces triacylglycerol accumulation; alter morphology and aggregation in liquid culture.