Hierarchy and interconnected networks in the WhiB7 mediated transcriptional response to antibiotic stress in Mycobacterium abscessus.

Mycobacterium abscessus is intrinsically resistant to antibiotics effective against other pathogenic mycobacteria largely due to the drug-induced expression of genes that confer resistance. WhiB7 is a major hub controlling the induction of resistance to ribosome-targeting antibiotics. It activates the expression of >100 genes, 7 of which are known determinants of drug resistance; the function of most genes within the regulon is however unknown, but some conceivably encode additional mechanisms of resistance. Furthermore, the hierarchy of gene expression within the regulon, if any, is poorly understood. In the present work we have identified 56 WhiB7 binding sites using chromatin immunoprecipitation sequencing (CHIP-Seq) which accounts for the WhiB7-dependent upregulation of 72 genes, and find that M. abscessus WhiB7 functions exclusively as a transcriptional activator at promoters recognized by σA/σB. We have investigated the role of 18 WhiB7 regulated genes in drug resistance. Our results suggest that while some genes within the regulon (eg. erm41, hflX, eis2 and the ABCFs) play a major role in resistance, others make smaller contributions (eg. MAB_4324c and MAB_1409c) and the observed hypersensitivity ΔMabwhiB7 is a cumulative effect of these individual contributions. Moreover, our CHIP-Seq data implicate additional roles of WhiB7 induced genes beyond antibiotic resistance. Finally, we identify a σH-dependent network in aminoglycoside and tigecycline resistance which is induced upon drug exposure and is further activated by WhiB7 demonstrating the existence of a crosstalk between components of the WhiB7-dependent and -independent circuits.

Dynamin-like proteins mediate extracellular vesicle secretion in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) secretes extracellular vesicles (EVs) containing a variety of proteins, lipoproteins, and lipoglycans. While emerging evidence suggests that EVs contribute to tuberculosis pathogenesis, the factors and molecular mechanisms involved in mycobacterial EV production have not been identified. In this study, we use a genetic approach to identify Mtb proteins that mediate vesicle release in response to iron limitation and antibiotic exposure. We uncover a critical role for the isoniazid-induced, dynamin-like proteins, IniA and IniC, in mycobacterial EV biogenesis. Further characterization of a Mtb iniA mutant shows that the production of EVs enables intracellular Mtb to export bacterial components into the extracellular environment to communicate with host cells and potentially modulate the immune response. The findings advance our understanding of the biogenesis and functions of mycobacterial EVs and provide an avenue for targeting vesicle production in vivo.

Hierarchy and networks in the transcriptional response of Mycobacterium abscessus to antibiotics.

Mycobacterium abscessus causes acute and chronic pulmonary infection in patients with chronic lung damage. It is intrinsically resistance to antibiotics effective against other pathogenic mycobacteria largely due to the drug-induced expression of genes that confer resistance. Induction of genes upon exposure to ribosome targeting antibiotics proceeds via WhiB7-dependent and -independent pathways. WhiB7 controls the expression of >100 genes, a few of which are known determinants of drug resistance. The function of the vast majority of genes within the regulon is unknown, but some conceivably encode additional mechanisms of resistance. Furthermore, the hierarchy of gene expression within the regulon, if any, is poorly understood. In the present work we have identified 56 WhiB7 binding sites using chromatin immunoprecipitation sequencing (CHIP-Seq) which accounts for the WhiB7-dependent upregulation of 70 genes, and find that M. abscessus WhiB7 functions exclusively as a transcriptional activator at promoters recognized by σ A /σ B We have investigated the role of 18 WhiB7 regulated genes in drug resistance and demonstrated the role of MAB_1409c and MAB_4324c in aminoglycoside resistance. Further, we identify a σ H -dependent pathway in aminoglycoside and tigecycline resistance which is induced upon drug exposure and is further activated by WhiB7 demonstrating the existence of a crosstalk between components of the WhiB7-dependent and -independent circuits. Abstract Importance: The induction of multiple genes that confer resistance to structurally diverse ribosome-targeting antibiotics is funneled through the induction of a single transcriptional activator, WhiB7, by antibiotic-stalled ribosomes. This poses a severe restriction in M. abscessus therapy as treatment with one ribosome-targeting antibiotic confers resistance to all other ribosome-targeting antibiotics. Here we uncover the intricacies of the WhiB7 regulatory circuit, identify three previously unknown determinants of aminoglycoside resistance and unveil a communication between WhiB7 dependent and independent components. This not only expands our understanding of the antibiotic resistance potential of M. abscessus but can also inform the development of much needed therapeutic options.

Activation and Regulation of NLRP3 by Sterile and Infectious Insults.

Nod-Like Receptor (NLR) is the largest family of Pathogen Recognition Receptors (PRRs) that patrols the cytosolic environment. NLR engagement drives caspase-1 activation that cleaves pro-IL-1B which then gets secreted. Released IL-1B recruits immune cells to the site of infection/injury. Caspase-1 also cleaves Gasdermin-D (GSDM-D) that forms pores within the plasma membrane driving inflammatory cell death called pyroptosis. NLRP3 is the most extensively studied NLR. The NLRP3 gene is encoded by 9 exons, where exon 1 codes for pyrin domain, exon 3 codes for NACHT domain, and Leucine Rich Repeat (LRR) domain is coded by exon 4-9. Exon 2 codes for a highly disorganized loop that connects the rest of the protein to the pyrin domain and may be involved in NLRP3 regulation. The NLRP3 inflammasome is activated by many structurally divergent agonists of microbial, environmental, and host origin. Activated NLRP3 interacts with an adaptor protein, ASC, that bridges it to pro-Caspase-1 forming a multi-protein complex called inflammasome. Dysregulation of NLRP3 inflammasome activity is a hallmark of pathogenesis in several human diseases, indicating its highly significant clinical relevance. In this review, we summarize the existing knowledge about the mechanism of activation of NLRP3 and its regulation during activation by infectious and sterile triggers.

Extracellular vesicles in the context of Mycobacterium tuberculosis infection.

The production of extracellular vesicles (EVs) has emerged as an important process in bacterial biology and host-pathogen interactions. Like many other bacteria, mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis (TB), produces EVs in vitro and in vivo. These membrane-enclosed nanoparticles enable Mtb to secrete hydrophobic molecules, proteins, lipids and glycolipids in a concentrated and protected manner and engage in remote interactions with the host. The nature of the material secreted in mycobacterial EVs, the functional attributes of these vesicles and their potential as protective antigens have stimulated great interest in the mycobacterial field. Although the field of EVs in mycobacterial infections is developing, it has already uncovered a whole new dimension for Mtb-host interactions potentially relevant to TB pathogenesis. In this mini-review, we discuss the current evidence supporting an important role of mycobacterial EVs in modulating cellular immune response, the challenges and recent advances in understanding the mechanisms of vesicle biogenesis and the implications for development of new preventive and therapeutic tools against TB.

Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria.

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) containing immunologically active molecules. Knowledge regarding the molecular mechanisms of vesicle biogenesis, the content of the vesicles, and their functions at the pathogen-host interface is very limited. Addressing these questions requires rigorous procedures for isolation, purification, and validation of EVs. Previously, vesicle production was found to be enhanced when M. tuberculosis was exposed to iron restriction, a condition encountered by Mtb in the host environment. Presented here is a complete and detailed protocol to isolate and purify EVs from iron-deficient mycobacteria. Quantitative and qualitative methods are applied to validate purified EVs.

Mycobacterial extracellular vesicles and host pathogen interactions.

Mycobacteria, like other bacteria, archaea and eukaryotic cells, naturally release extracellular vesicles (EVs) to interact with their environment. EVs produced by pathogenic bacteria are involved in many activities including cell-cell communication, immunomodulation, virulence and cell survival. Although EVs released by thick cell wall microorganisms like mycobacteria were recognized only recently, studies of Mycobacterium tuberculosis EVs already point to their important roles in host pathogen interactions, opening exciting new areas of investigation. This minireview will summarize the current understanding of mycobacterial EV biology and roles in pathogenesis and will discuss their potential therapeutic applications.

System-Wide Analysis Unravels the Differential Regulation and In Vivo Essentiality of Virulence-Associated Proteins B and C Toxin-Antitoxin Systems of Mycobacterium tuberculosis.

Toxin-antitoxin (TA) systems are bicistronic genetic modules that are ubiquitously present in bacterial genomes. The Mycobacterium tuberculosis genome encodes 90 putative TA systems, and these are considered to be associated with maintenance of bacterial genomic stability or bacterial survival under unfavorable environmental conditions. The majority of these in M. tuberculosis have been annotated as belonging to the virulence-associated protein B and C (VapBC) family. However, their precise role in bacterial physiology has not been elucidated. Here, we functionally characterized VapC toxins from M. tuberculosis and show that overexpression of some homologs inhibits growth of Mycobacterium bovis bacillus Calmette-Guérin in a bacteriostatic manner. Expression profiling of messenger RNA revealed that these VapC toxins were differentially induced upon exposure of M. tuberculosis to stress conditions. We also unraveled that transcriptional cross-activation exists between TA systems in M. tuberculosis. This study provides the first evidence for the essentiality of VapBC3 and VapBC4 systems in M. tuberculosis virulence.

Essential protein SepF of mycobacteria interacts with FtsZ and MurG to regulate cell growth and division.

Coordinated bacterial cell septation and cell wall biosynthesis require formation of protein complexes at the sites of division and elongation, in a temporally controlled manner. The protein players in these complexes remain incompletely understood in mycobacteria. Using in vitro and in vivo assays, we showed that Rv2147c (or SepF) of Mycobacterium tuberculosis interacts with the principal driver of cytokinesis, FtsZ. SepF also interacts with itself both in vitro and in vivo. Amino acid residues 189A, 190K and 215F are required for FtsZ-SepF interaction, and are conserved across Gram-positive bacteria. Using Mycobacterium smegmatis as a surrogate system, we confirmed that sepFMSMEG is essential. Knockdown of SepF led to cell elongation, defective growth and failure of FtsZ to localize to the site of division, suggesting that SepF assists FtsZ localization at the site of division. Furthermore, SepF interacted with MurG, a peptidoglycan-synthesizing enzyme, both in vitro and in vivo, suggesting that SepF could serve as a link between cell division and peptidoglycan synthesis. SepF emerges as a newly identified essential component of the cell division complex in mycobacteria.

MtrA, an essential response regulator of the MtrAB two-component system, regulates the transcription of resuscitation-promoting factor B of Mycobacterium tuberculosis.

The resuscitation-promoting factors of Mycobacterium tuberculosis are hydrolytic enzymes, which are required for resuscitation of dormant cells. RpfB, a peptidoglycan remodelling enzyme similar to the lytic transglycosylase of Escherichia coli, is required for reactivation of M. tuberculosis from chronic infection in vivo, underscoring the need to understand its transcriptional regulation. Here, we identified the transcriptional and translational start points of rpfB, and suggested from rpf promoter-driven GFP expression and in vitro transcription assays that its transcription possibly occurs in a SigB-dependent manner. We further demonstrated that rpfB transcription is regulated by MtrA - the response regulator of the essential two-component system MtrAB. Association of MtrA with the rpfB promoter region in vivo was confirmed by chromatin immunoprecipitation analysis. Electrophoretic mobility shift assays (EMSAs) revealed a loose direct repeat sequence associated with MtrA binding. Binding of MtrA was enhanced upon phosphorylation. MtrA could be pulled down from lysates of M. tuberculosis using a biotinylated DNA fragment encompassing the MtrA-binding site on the rpfB promoter, confirming that MtrA binds to the rpfB promoter. Enhanced GFP fluorescence driven by the rpfB promoter, upon deletion of the MtrA-binding site, and repression of rpfB expression, upon overexpression of MtrA, suggested that MtrA functions as a repressor of rpfB transcription. This was corroborated by EMSAs showing diminished association of RNA polymerase (RNAP) with the rpfB promoter in the presence of MtrA. In vitro transcription assays confirmed that MtrA inhibits RNAP-driven rpfB transcription.