Browsing by Author "Warner, Digby F"

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    Correction to: Bioaerosol sampling of patients with suspected pulmonary tuberculosis: a study protocol
    (2020-08-24) Patterson, Benjamin; Koch, Anastasia; Gessner, Sophia; Dinkele, Ryan; Gqada, Melitta; Bryden, Wayne; Cobelens, Frank; Little, Francesca; Warner, Digby F; Wood, Robin
    An amendment to this paper has been published and can be accessed via the original article
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    Developing methods to prioritize in vitro drug combinations against Mycobacterium tuberculosis: fusidic acid as potential combination partner with known antitubercular agents
    (2017) Omollo, Charles; Chibale, Kelly; Warner, Digby F
    The tuberculosis (TB) epidemic remains a major threat to public health globally, and is exacerbated by the escalating number of multi-drug resistant cases. These factors have highlighted the urgent need for new effective therapies or different approaches to augment the efficacy of current anti-TB drugs. Synergistic drug combinations present a feasible strategy towards expanding TB treatment options. Despite reported successes with combination screening, as well as the current reliance on combination therapy for TB, this approach remains largely underexplored. Evidence suggests that utilizing synergistic combinations might enable existing clinically-approved drugs to be readily re-purposed for TB treatment, including against multi-(MDR) and extensively- (XDR) drug resistant strains for current therapies are often ineffective. This thesis focused on the development and application of improved methods to identify and advance novel drug combinations for TB therapy. There were two key aspects to this work: firstly, exploring mechanisms of synergy between fusidic acid (FSA), a natural product antibiotic, and current anti-TB agents and, secondly, characterizing antibiotic action by delineating bacteriostatic and bactericidal compounds.
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    Elucidation of mechanisms of antibiotic subversion in mycobacteria
    (2015) Naran, Krupa; Warner, Digby F; Mizrahi, Valerie
    The intrinsic resistance of Mycobacterium tuberculosis ( Mtb ) to antibiotics is generally attributed to multiple factors, most significantly the low permeability of the mycobacterial cell wall, the operation of various drug inactivating systems, and the activity of efflux pumps. This study aimed to investigate the role of various components of the "intrinsic resistome" that limit the efficacy of antitubercular agents. The DNA damage response: the SOS response was hypothesized to play a role in antibiotic- mediated cellular death, and that disabling the mycobacterial SOS response, by generating non-cleavable LexA mutants (lexA Ind-), could be used as a tool to validate antibiotic-mediated cell death. To this end, the M. smegmatis (Msm) cleavable LexA was shown to be essential for induced mutagenesis and damage tolerance and that an intact DNA damage repair system is required to respond to antibiotic-mediated DNA damage. In contrast, Mtb cleavable LexA was required for induced mutagenesis but not necessarily damage sensitivity. In addition, the Mtb SOS response does not contribute significantly to remediation of antibiotic-mediated DNA damage. Collectively, these data suggest that DNA repair mechanisms differ between the mycobacterial species and despite effectively inactivating the LexA-dependent DNA repair mechanism(s) in Msm and Mtb, these organisms are able to circumvent this pathway and successfully remediate damaged DNA sustained under various conditions. Furthermore, Mtb auto-bioluminescent reporter strains were generated by introducing the lux operon downstream of the recA or radA promoters. Analysis of a panel of antimicrobials against these strains allowed for the identification of true DNA-damaging agents and the evaluation of the kinetics of the DNA-damage response, in a concentration- and time-dependent manner. Efflux-mediated drug resistance: This study aimed to evaluate the interactions between pairwise combinations of selected antimicrobials and efflux pump inhibitors (EPIs), in vitro and ex vivo, and to identify a novel verapamil (VER)-analogue with improved efficacy against Mtb. Subsequently, a candidate EPI was identified, with equivalent in vitro synergistic effects to VER when used in combination with various antibiotics but with reduced cytotoxic effects, ex vivo, when compared to VER. Mycothiol-mediated protection : It was hypothesized that undetectable levels of mycothiol ( MSH ) in Mtb would potentiate the use of current antibiotics. To investigate the contribution of the cellular antioxidant, MSH, to the mitigation of antimicrobial efficacy, this study aimed to disrupt MSH production by conditionally knocking-down expression of the essential gene, mshC. The mshC knock-down mutants (in all configurations) were not anhydrotetracycline (ATC)-regulatable in liquid or on solid medium, which was subsequently validate d with quantitative gene expression analysis. These data suggest that a tetracycline (Tet)-based conditional expression system may not be applicable to mshC. In conclusion, Mtb has a multitude of inherent mechanisms to subvert the effects of antimicrobial treatment. This study has contributed to the understanding of certain aspects of the intrinsic resistome and in doing so, established tools that can be used in future drug discovery programmes.
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    Identification of Antimycobacterial Natural Products from a Library of Marine Invertebrate Extracts
    (2022-01-28) Acquah, Kojo Sekyi; Beukes, Denzil R; Seldon, Ronnett; Jordaan, Audrey; Sunassee, Suthananda N; Warner, Digby F; Gammon, David W
    Tuberculosis (TB) remains a public health crisis, requiring the urgent identification of new anti-mycobacterial drugs. We screened several organic and aqueous marine invertebrate extracts for their in vitro inhibitory activity against the causative organism, Mycobacterium tuberculosis. Here, we report the results obtained for 54 marine invertebrate extracts. The chemical components of two of the extracts were dereplicated, using 1H NMR and HR-LCMS with GNPS molecular networking, and these extracts were further subjected to an activity-guided isolation process to purify the bioactive components. Hyrtios reticulatus yielded heteronemin 1 and Jaspis splendens was found to produce the bengamide class of compounds, of which bengamides P 2 and Q 3 were isolated, while a new derivative, bengamide S 5, was putatively identified and its structure predicted, based on the similarity of its MS/MS fragmentation pattern to those of other bengamides. The isolated bioactive metabolites and semi-pure fractions exhibited M. tuberculosis growth inhibitory activity, in the range
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    Investigating permeation of anti-mycobacterial agents in Mycobacterium tuberculosis and M. tuberculosis-infected macrophages in vitro as a model for early stage tuberculosis drug discovery
    (2021) Mabhula, Amanda N; Chibale, Kelly; Warner, Digby F
    Tuberculosis (TB) is the leading cause of death due to a single infectious disease and remains a major threat to global public health. The increasing emergence of multi-drug resistance to current anti-TB drugs, exacerbated by the long treatment duration, highlights the need for new effective treatments or strategies to shorten the treatment duration, improve patient adherence and curb the alarming rates of resistance. A key challenge to current strategies employed in the development of anti-TB drugs is the complexity in TB disease pathology which presents as a wide spectrum of lesions in patients presenting with the disease. These lesions occur at different anatomical loci in the same individual and at different stages as the disease progresses. In addition, the interaction between the causative agent, Mycobacterium tuberculosis, and its obligate human host induces physiologic and metabolic changes in the infecting bacillus that are specific to each lesion compartment, and dynamic. This is likely to influence M. tuberculosis susceptibility to antibiotic treatment and, consequently, affect treatment duration and possibly the development of drug resistance. A major limitation in current strategies to address this problem is translation of in vitro compound potency to in vivo efficacy. To reach the target site, a drug must first distribute and accumulate in the lesion microenvironments where bacteria reside: the macrophage host cell and the caseum. This thesis focused on the development of an in vitro infection model that could be used to predict drug penetration into M. tuberculosis-infected macrophages. Of particular interest was the extent to which host intracellular drug concentrations translate into effective antimycobacterial activity. To this end, the thesis comprised three key aspects: (i) characterization of physicochemical properties, antimycobacterial activities and M. tuberculosis-mediated metabolism of selected antiTB compounds; (ii) determination of intracellular drug permeation in resting, activated and foamy macrophages; and (iii) determination of the correlation (or not) between intracellular drug concentration and effective M. tuberculosis growth inhibition. The highly lipophilic natural product, fusidic acid (FA), its known human metabolite, 3-ketofusidic (3-ketoFA or GKFA37), and two C-3 alkyl esters (GKFA16 and GKFA17) as FA prodrugs were utilized in the study. In addition, another chemical class, the less lipophilic benzoxazole-based oxime derivatives were also investigated. Moxifloxacin (MXF), levofloxacin (LVF), bedaquiline (BDQ), rifampicin (RIF) and clofazimine (CFZ) were included for reference as known anti-TB drugs with varying lipophilicities. In chapter 2, FA and derivatives showed potent antimycobacterial activity (~1 µM) with selectivity indices (SI) >20 against the THP-1 macrophage cell line. Predicted artificial membrane permeability assay (PAMPA) results suggested that FA and derivatives would readily permeate the cell membrane. M. tuberculosis metabolized the C-3 alkyl-ester prodrug GKFA17 to form both FA and 3-ketoFA, with complete hydrolysis of the prodrug. FA was metabolized to 3-ketoFA, but the low levels of the metabolite suggested that another unidentified metabolite, presumed to be 3-epifusidic acid (3-epiFA), was formed. In vitro assays revealed that the potent benzoxazole-based oxime carbamates (PMN1-201, PMN1-136 and PMN2-09) were rapidly hydrolyzed by M. tuberculosis and were also susceptible to spontaneous degradation in media, forming the poorly active corresponding free oximes (PMN1-199, PMN1-135 and PMN1-157). In chapter 3, the in vitro macrophage drug uptake assay showed that FA C-3 alkyl prodrugs, GKFA16 and GKFA17, accumulated in significantly higher amounts in resting macrophages in comparison to FA and GKFA37. Accumulation of MXF was comparable to the least accumulated FA derivative, GKFA37, and showed steady state intracellular concentrations over a 6-day period. While GKFA16 and GKFA17 showed continued increasing accumulation, intracellular concentrations of FA and GKFA37 decreased after 48 hours, suggesting a likely susceptibility to macrophage efflux. In infected macrophages, the presence of intracellular bacteria or increasing bacterial burden did not affect the host cell ability to accumulate the drugs. FA and derivatives exhibited bacteriostatic inhibition of intracellular mycobacterial growth. MXF showed a potent bactericidal effect, reducing intracellular bacterial counts significantly at 10x MIC, with complete sterilization at 50x MIC even though MXF accumulation was significantly less than that of FA alkyl esters. These results suggested that both the inherent activity of a compound and ability to accumulate within host cells drive cellular efficacy. Given that the C-3 alkyl ester prodrugs accumulated at significantly higher concentrations than FA and GKFA37, this demonstrates the limitations of this assay in ascertaining the impact of intracellular concentration on drug efficacy for bacteriostatic drugs while highlighting its ability to correlate drug penetration and intracellular activity for cidal drugs. The prodrug GKFA17 was shown to undergo metabolism in resting host cells and during infection to form FA and then 3-ketoFA. Therefore, the prodrug strategy could be used to increase intracellular exposure of FA as GKFA17 showed superior macrophage accumulation. Benzoxazole-based oxime carbamates and their corresponding free oximes failed to accumulate in host macrophages and this was corroborated by their failure to control host cell bacterial growth despite the potent in vitro activity against M. tuberculosis of the carbamates, suggesting that they are poorly permeable. Chapter 4 investigated drug permeation in different macrophage phenotypes known to exist in the granuloma during TB disease, including foamy and activated macrophages. The activation state of the host cell did not affect the ability to accumulate anti-TB drugs such as RIF and BDQ. However, FA and its prodrug GKFA17 were significantly reduced in M1 activated macrophages. Despite the significantly reduced intracellular concentration, activated macrophages treated with FA and derivatives showed superior intracellular M. tuberculosis growth inhibition, suggesting that macrophage activation potentiates the activity of these compounds. In order to assess the effect of foamy macrophage lipid bodies (LBs) on drug uptake and intracellular localization, oleic acid-induced foamy macrophages were treated with selected antiTB drugs and experimental compounds. FA and derivatives showed early increased accumulation in foamy cells compared to resting macrophages, while MXF, BDQ and RIF levels were not significantly changed. Intracellular:extracellular (I/E) ratios increased with increase in lipophilicity, with FA C-3 alkyl prodrugs exhibiting the highest I/E ratios of >100. Despite exhibiting increased foamy macrophage concentrations, FA and derivatives exhibited a similar reduction (bacteriostatic) in bacterial counts in both resting and foamy macrophages. The intracellular activity of RIF was also not affected by presence of LBsin foamy macrophages. BDQ, LVF and MXF, however, showed reduced intracellular efficacy against M. tuberculosis in foamy macrophages compared to resting macrophages, suggesting a role for LBs to impact intracellular drug distribution. In conclusion, this thesis demonstrates the potential utility in combining advanced analytical methods and an in vitro infection model to determine cellular drug permeation profiles that might be applied to prioritize compounds and combinations optimized for distribution to target bacterial populations. This will facilitate well-informed decision-making processes in progression of lead compounds in pre-clinical development and, therefore, may offer the potential to reduce high rates of attrition of compounds which enter clinical phase of development.
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    Molecular mechanisms of DNA repair in Mycobacterium tuberculosis
    (2017) Gessner, Sophia Johanna; Warner, Digby F; Mizrahi, Valerie
    The mycobacterial DNA damage and repair pathways involved in the emergence of drug-resistance during host infection remain poorly understood, yet are critical to any efforts to develop novel "anti-evolution" drugs aimed at reducing the capacity of Mycobacterium tuberculosis to adapt genetically during tuberculosis (TB) treatment. The thesis presented here aimed to investigate the contribution of the DNA damage (SOS) response in adaptive mutagenesis, and focused on two specific components: the role of the specialist translesion synthesis DNA polymerase, DnaE2, in mutagenesis under stress and, secondly, the function of the mycobacterial homologue of a putative SOS response associated peptidase (SRAP) protein which has been identified in comparative genomics analyses of organisms possessing a DnaE2-type C family DNA polymerase. This work focused on the putative SRAP protein which was predicted to form part of the mycobacterial DNA damage response as a functional switch by binding DNA in an autoproteolytic dependent manner. To this end, SRAP deletion mutants were generated for both M. smegmatis (MSMEG_1891) and M. tuberculosis (Rv3226c). Despite the fact that SRAP was upregulated in both M. smegmatis and M. tuberculosis following genotoxic stress, no DNA damage phenotype was detected in any SRAP deletion mutant using a variety of DNA damaging agents. In parallel, an eGFP-tagged M. smegmatis SRAP allele was constructed to enable visualisation of SRAP upregulation and sub-cellular recruitment using fluorescent microscopy; however no eGFP expression could be visualised after MMC treatment. It was not clear whether this was due to faulty eGFP expression in the fusion protein, or to low-level induction of SRAP. In a biochemical approach to elucidate SRAP function, soluble M. smegmatis SRAP protein was expressed and purified using a N-terminal hexa-histidine tag. No proteolytic activity was detected in gelatine or casein zymography, perhaps indicating that SRAP has a very specific substrate. Moreover, while it was predicted that autocatalytic cleavage of the C-terminus was required for activation of SRAP, no such cleavage was detected using hexa-histidine tag staining, possibly pointing to a set of very specific conditions for activation. In combination, therefore, neither microbiological nor biochemical assays could elucidate a definitive role for SRAP in the mycobacterial DNA damage response. DnaE2 has been directly implicated in induced mutagenesis to rifampicin (Rif) resistance in Mycobacterium tuberculosis following exposure of bacilli to genotoxic stress. In previous work in our group, a vitamin B₁₂-sensitive ΔmetH strain was found to form "B₁₂-resistant" suppressor mutants at a frequency higher than could be explained by spontaneous mutagenesis alone. The first part of this thesis investigated the potential role of DnaE2 in the high-frequency emergence of B₁₂-resistance by mutating DnaE2 in the ΔmetH background. Whereas elimination of polymerase function in a DnaE2ᴬᴵᴬ mutant abrogated DNA damage-induced mutagenesis to Rif resistance, no change in B₁₂ sensitivity was detected in a ΔmetH dnaE2ᴬᴵᴬ double mutant. PCR sequencing of spontaneous B₁₂-resistant mutants revealed mutations in genes previously associated with the suppressor phenotype; moreover, there was no apparent difference in the nature of mutations observed in both parental and dnaE2ᴬᴵᴬ mutant strains. Instead, these results suggest that an alternative mechanism must exist to enable adaptive mutagenesis in methionine-starved mycobacteria.
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    Real-time investigation of tuberculosis transmission: developing the Respiratory Aerosol Sampling Chamber (RASC)
    (Public Library of Science, 2016) Wood, Robin; Morrow, Carl; III, Clifton E Barry; Bryden, Wayne A; Call, Charles J; Hickey, Anthony J; Rodes, Charles E; Scriba, Thomas J; Blackburn, Jonathan; Issarow, Chacha; Mulder, Nicola; Woodward, Jeremy; Moosa, Atica; Singh, Vinayak; Mizrahi, Valerie; Warner, Digby F
    Knowledge of the airborne nature of respiratory disease transmission owes much to the pioneering experiments of Wells and Riley over half a century ago. However, the mechanical, physiological, and immunopathological processes which drive the production of infectious aerosols by a diseased host remain poorly understood. Similarly, very little is known about the specific physiological, metabolic and morphological adaptations which enable pathogens such as Mycobacterium tuberculosis ( Mtb ) to exit the infected host, survive exposure to the external environment during airborne carriage, and adopt a form that is able to enter the respiratory tract of a new host, avoiding innate immune and physical defenses to establish a nascent infection. As a first step towards addressing these fundamental knowledge gaps which are central to any efforts to interrupt disease transmission, we developed and characterized a small personal clean room comprising an array of sampling devices which enable isolation and representative sampling of airborne particles and organic matter from tuberculosis (TB) patients. The complete unit, termed the Respiratory Aerosol Sampling Chamber (RASC), is instrumented to provide real-time information about the particulate output of a single patient, and to capture samples via a suite of particulate impingers, impactors and filters. Applying the RASC in a clinical setting, we demonstrate that a combination of molecular and microbiological assays, as well as imaging by fluorescence and scanning electron microscopy, can be applied to investigate the identity, viability, and morphology of isolated aerosolized particles. Importantly, from a preliminary panel of active TB patients, we observed the real-time production of large numbers of airborne particles including Mtb , as confirmed by microbiological culture and polymerase chain reaction (PCR) genotyping. Moreover, direct imaging of captured samples revealed the presence of multiple rod-like Mtb organisms whose physical dimensions suggested the capacity for travel deep into the alveolar spaces of the human lung.
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    Replication fidelity in the mircroevolution of mycobacteria
    (2015) Ditse, Zanele; Warner, Digby F; Mizrahi, Valerie
    This thesis aimed to elucidate the structure-function relationships determining the differential fidelities of the dnaE1- and dnaE2-encoded mycobacterial PolIIIα subunits under conditions of genotoxic stress. To this end, the role in DnaE1 intrinsic fidelity of highly conserved PHP domain residues was explored by site-directed replacement of targeted amino acids, resulting in a panel of Mycobacterium smegmatis mutants carrying selected dnaE1 alleles. A complementary approach investigated the contribution of the mycobacterial proofreading DnaQ subunit homolog to the maintenance of DnaE1-dependent replicative fidelity by generating a targeted dnaQ knockout mutant. The third component of this study focused on the inferred role of a highly-conserved N-terminal extension and C-terminal pentapeptide motif in the function of the alternative, error-prone DNA PolIIIα subunit, DnaE2.
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    Repositioning fusidic acid for tuberculosis: semi-synthesis of analogues and impact of mycobacterial biotransformation on antibiotic activity
    (2018) Wasuna, Antonina; Chibale, Kelly; Warner, Digby F
    Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is one of the leading causes of death globally, especially in low and middle-income countries. TB is primarily a curable disease, with chemotherapy predicated on a combination of four drugs. The increase in multiple forms of drug-resistant TB is a major cause for concern, underpinning the importance of a continuous pipeline of new anti-TB agents. Drug repositioning - that is, the optimization of existing drugs for new therapeutic indications - has shown promise in expanding the therapeutic options for TB chemotherapy. Fusidic acid (FA), a natural product-derived antibiotic, has modest in vitro antimycobacterial activity. Through a multi-disciplinary approach combining aspects of chemistry and biology, this study investigated the pharmacological and physicochemical properties of FA that might be exploited for optimization of FA as a lead compound for TB drug discovery. FA is a weak carboxylic acid, and it was hypothesised that the carboxylic acid moiety limits its permeation of the complex mycobacterial cell wall. Therefore, this study aimed to identify novel FA analogues with improved permeation properties and designed to act as potential prodrugs. By modifying the C-3 hydroxyl and the carboxylic acid moiety, alkyl and aminoquinoline derivatives were covalently fused to FA through ester and amide coupling reactions to generate hybrids and/or potential prodrugs.
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    Repurposing chlorpromazine and its metabolites for antituberculosis drug discovery
    (2015) Kigondu, Elizabeth Victoria Mumbi; Chibale, Kelly; Warner, Digby F
    New chemotherapeutics are urgently needed to combat Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). The development of compounds that could potentiate the activity of known antimycobacterial drugs is a relatively unexplored approach to new TB drug discovery. This study aimed to generate metabolites of chlorpromazine (CPZ), a phenothiazine with demonstrated in vitro activity against Mtb, and to investigate their potential utility in combination with anti-TB drugs. 7-HydroxyCPZ (M2), CPZ-N-oxide (M3), CPZ sulfoxide (M1), nor-CPZ (M5), nor-CPZ sulfoxide (M6b) and CPZ-N-S-dioxide (M4b) were generated from CPZ using various biotransformation systems and identified by Liquid Chromatography - Mass Spectrometry (LC/MS). The identity of M2 was confirmed with reference to a 7-hydroxyCPZ standard. M3, M1, M5, M6b and M4b were synthesized de novo and used to identify the metabolites generated in the biotransformation samples. Individually, CPZ and its metabolites (M2, M3, M5) were weakly active (MIC99 >50μM) against M. smegmatis (Msm) and Mtb while M1, M6b & M4b did not exhibit a MIC99 even at very high concentrations. Generally, an improvement in activity was observed where CPZ or its metabolites were used in combination with known anti-TB drugs. The combinations that exhibited a fractional inhibition concentration index (FICI) of < 0.5 were defined as synergistic. A combination of M2 and spectinomycin (SPEC) exhibited the highest synergism against Msm (FICI 0.19) and Mtb (FICI 0.13). In vitro assays established that CPZ and M2 are bactericidal against Mtb whereas M3 and M5 are bacteriostatic on their own. In combination assays, the use of RIF with M3 and M5, bedaquiline (BDQ) with M2, and SPEC with M3 were bactericidal. At 140μM, CPZ and M1, M2, M3 treated samples exhibited a 2-fold up-regulation of the cydA (Rv1623c) gene which encodes an essential subunit of the cytochrome bd-type menaquinol oxidase in Mtb. The same observation was made for RIF/M2 and RIF/M5 treated samples. These results suggest that the metabolites retain the mechanism of action (MoA) as the parental CPZ. The Mtb 16S rRNA gene, rrs (MTB000019) was identified as the biological target for SPEC. This brought into perspective the underlying mechanisms at play when SPEC is used in combination with CPZ, its metabolites or other drugs, against mycobacteria. This study establishes the utility of combination assays in confirming the active metabolite(s) of known drugs and provides proof of concept data to support follow-up investigations of CPZ and its metabolites as potential compounds for novel combination therapies for anti-TB drug development.
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    Targeted depletion of RibF, a putative bifunctional FAD synthetase/ flavokinase in Mycobacterium smegmatis using CRISPR interference
    (2020) Raphela, Mabule Lucas; Warner, Digby F; Chengalroyen, Melissa D
    Tuberculosis (TB) is the leading killer globally owing to an infectious disease. There is consequently an urgent need to develop novel TB drugs and shorter regimens to treat the causative agent, Mycobacterium tuberculosis, an imperative which demands the identification of new drug targets in essential mycobacterial pathways. To that end, the work presented in this dissertation aimed to functionally characterize ribF, an essential gene in the mycobacterial riboflavin (RF; vitamin B2) biosynthetic pathway. Given the role of RF as a core component of the essential flavin cofactors, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), it was hypothesized that silencing ribF would disrupt the biosynthesis of all flavoproteins, crippling numerous (essential) processes within the organism. Moreover, based on previous observations in Bacillus subtilis, it was predicted that the mycobacterial ribF homolog might play a role in regulating the rib operon (comprising a cluster of RF pathway genes) – either directly by binding to the FMN riboswitch, or indirectly through the production of FMN from RF, in turn enabling riboswitch-mediated repression of downstream genes. CRISPR interference (CRISPRi) technology was used to generate an anhydrotetracycline (ATc)-inducible ribF hypomorph of M. smegmatis, a widely exploited mycobacterial model. Consistent with other organisms, ribF was shown to be essential for in vitro growth of M. smegmatis: CRISPRi-mediated depletion of ribF was bacteriostatic, resulting in a 10-fold growth inhibition in liquid media and corresponding to no reduction (0 log-fold change) in colony forming units (CFU). Moreover, targeted metabolomic analyses revealed that ribF depletion was associated with accumulation of 6,7-Dimethylribityllumazine (DMRL), suggesting that the disruption of RibF function blocked conversion of RF to the essential cofactors, FMN and FAD, in turn inhibiting cell growth. Notably, the lethality of ribF depletion could not be complemented chemically by exogenous supplementation of growth media with RF, FMN or FAD. Downregulation of ribF also caused enhanced susceptibility to the known cell wall-targeting agent, vancomycin, but not to the putative RibF domain inhibitor, thonzonium bromide, suggesting an alternative mechanism of action or impaired bacillary permeation. In summary, these data support the inferred essentiality of ribF in mycobacteria, in turn supporting future work which aims to target this enzyme for new TB drug discovery.
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    Translating genomics research into control of tuberculosis: lessons learned and future prospects
    (BioMed Central, 2014-11-07) Warner, Digby F; Mizrahi, Valerie
    Genomics research has enabled crucial insights into the adaptive evolution of Mycobacterium tuberculosis as an obligate human pathogen. Here, we highlight major recent advances and evaluate the potential for genomics approaches to inform tuberculosis control efforts in high-burden settings.
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    Translating genomics research into control of tuberculosis: lessons learned and future prospects
    (2014-11-07) Warner, Digby F; Mizrahi, Valerie
    Abstract Genomics research has enabled crucial insights into the adaptive evolution of Mycobacterium tuberculosis as an obligate human pathogen. Here, we highlight major recent advances and evaluate the potential for genomics approaches to inform tuberculosis control efforts in high-burden settings.