High-throughput functional genomics: A (myco)bacterial perspective.

Advances in sequencing technologies have enabled unprecedented insights into bacterial genome composition and dynamics. However, the disconnect between the rapid acquisition of genomic data and the (much slower) confirmation of inferred genetic function threatens to widen unless techniques for fast, high-throughput functional validation can be applied at scale. This applies equally to Mycobacterium tuberculosis, the leading infectious cause of death globally and a pathogen whose genome, despite being among the first to be sequenced two decades ago, still contains many genes of unknown function. Here, we summarize the evolution of bacterial high-throughput functional genomics, focusing primarily on transposon (Tn)-based mutagenesis and the construction of arrayed mutant libraries in diverse bacterial systems. We also consider the contributions of CRISPR interference as a transformative technique for probing bacterial gene function at scale. Throughout, we situate our analysis within the context of functional genomics of mycobacteria, focusing specifically on the potential to yield insights into M. tuberculosis pathogenicity and vulnerabilities for new drug and regimen development. Finally, we offer suggestions for future approaches that might be usefully applied in elucidating the complex cellular biology of this major human pathogen.

De Novo Cobalamin Biosynthesis, Transport, and Assimilation and Cobalamin-Mediated Regulation of Methionine Biosynthesis in Mycobacterium smegmatis.

Cobalamin is an essential cofactor in all domains of life, yet its biosynthesis is restricted to some bacteria and archaea. Mycobacterium smegmatis, an environmental saprophyte frequently used as surrogate for the obligate human pathogen M. tuberculosis, carries approximately 30 genes predicted to be involved in de novo cobalamin biosynthesis. M. smegmatis also encodes multiple cobalamin-dependent enzymes, including MetH, a methionine synthase that catalyzes the final reaction in methionine biosynthesis. In addition to metH, M. smegmatis possesses a cobalamin-independent methionine synthase, metE, suggesting that enzyme use-MetH versus MetE-is regulated by cobalamin availability. Consistent with this notion, we previously described a cobalamin-sensing riboswitch controlling metE expression in M. tuberculosis Here, we apply a targeted mass spectrometry-based approach to confirm de novo cobalamin biosynthesis in M. smegmatis during aerobic growth in vitro We also demonstrate that M. smegmatis can transport and assimilate exogenous cyanocobalamin (CNCbl; also known as vitamin B12) and its precursor, dicyanocobinamide ([CN]2Cbi). However, the uptake of CNCbl and (CN)2Cbi in this organism is restricted and seems dependent on the conditional essentiality of the cobalamin-dependent methionine synthase. Using gene and protein expression analyses combined with single-cell growth kinetics and live-cell time-lapse microscopy, we show that transcription and translation of metE are strongly attenuated by endogenous cobalamin. These results support the inference that metH essentiality in M. smegmatis results from riboswitch-mediated repression of MetE expression. Moreover, differences observed in cobalamin-dependent metabolism between M. smegmatis and M. tuberculosis provide some insight into the selective pressures which might have shaped mycobacterial metabolism for pathogenicity.IMPORTANCE Alterations in cobalamin-dependent metabolism have marked the evolution of Mycobacterium tuberculosis into a human pathogen. However, the role(s) of cobalamin in mycobacterial physiology remains poorly understood. Using the nonpathogenic saprophyte M. smegmatis, we investigated the production of cobalamin, transport and assimilation of cobalamin precursors, and the role of cobalamin in regulating methionine biosynthesis. We confirm constitutive de novo cobalamin biosynthesis in M. smegmatis, in contrast with M. tuberculosis, which appears to lack de novo cobalamin biosynthetic capacity. We also show that uptake of cyanocobalamin (vitamin B12) and its precursors is restricted in M. smegmatis, apparently depending on the cofactor requirements of the cobalamin-dependent methionine synthase. These observations establish M. smegmatis as an informative foil to elucidate key metabolic adaptations enabling mycobacterial pathogenicity.

A gender-based comparison of coracoid and glenoid anatomy: CT analysis and discussion of potential impact on the Latarjet procedure.

BACKGROUND: The success of the modified Latarjet procedure depends on proper sizing of the coracoid graft. There is no information available regarding the morphometric relationship between the glenoid cavity and the coracoid process for the South African population. This study aims at measuring the relationship between the glenoid and coracoid morphometries and investigates their gender-related differences. METHODS: Glenohumeral computerized tomography scans of 100 consecutive patients were considered for this study. Morphometric measurements were performed after aligning the coracoid and glenoid in their optimum orientation. These measurements were performed by 2 independent observers. The ratio between glenoid and coracoid measurements was calculated and statistically compared using the Mann-Whitney U test. Intraclass correlation coefficients were calculated to analyze interobserver reliability. All the statistical tests were performed in SPSS v.26, and power calculations in G∗Power v.3.1. RESULTS: An average intraclass correlation coefficient value of 0.79 suggested that the interobserver reliability was good. Except for coracoid length, statistically significant (P < .05) gender differences were observed for all the other morphometries. The coracoid width (16.5 ± 1.4 mm vs. 14.7 ± 1.4 mm) and height (13.6 ± 1.6 mm vs. 10.5 ± 1.5 mm) differed between genders by 1.8 and 3.1 mm, respectively. The glenoid anteroposterior (AP) (25.3 ± 2.9 mm vs. 23.2 ± 2.4 mm) and superioinferior (36.9 ± 1.9 mm vs. 33.7 ± 2.6 mm) measurements differed by 2.1 and 3.3 mm, respectively, between the males and the females. The ratio between the AP width of the glenoid and the coracoid height was also found to be significantly different (P < .05) between the gender groups. These morphometric ratios for the coracoid width (0.66 ± 0.09 mm vs. 0.64 ± 0.08 mm) and the coracoid height (0.55 ± 0.09 mm vs. 0.46 ± 0.07 mm) differed between genders by 0.02 and 0.09, respectively. CONCLUSION: Measurements taken from computerized tomography scans showed significant differences between genders in absolute measurements and in the ratio of the coracoid height to the glenoid AP distance. This could have implications on the ability of the Latarjet procedure to compensate for bone loss in female patients.

Harnessing Biological Insight to Accelerate Tuberculosis Drug Discovery.

Tuberculosis (TB) is the leading cause of mortality globally resulting from an infectious disease, killing almost 1.6 million people annually and accounting for approximately 30% of deaths attributed to antimicrobial resistance (AMR). This despite the widespread administration of a neonatal vaccine, and the availability of an effective combination drug therapy against the causative agent, Mycobacterium tuberculosis (Mtb). Instead, TB prevalence worldwide is characterized by high-burden regions in which co-epidemics, such as HIV, and social and economic factors, undermine efforts to control TB. These elements additionally ensure conditions that favor the emergence of drug-resistant Mtb strains, which further threaten prospects for future TB control. To address this challenge, significant resources have been invested in developing a TB drug pipeline, an initiative given impetus by the recent regulatory approval of two new anti-TB drugs. However, both drugs have been reserved for drug-resistant disease, and the seeming inevitability of new resistance plus the recognized need to shorten the duration of chemotherapy demands continual replenishment of the pipeline with high-quality "hits" with novel mechanisms of action. This represents a massive challenge, which has been undermined by key gaps in our understanding of Mtb physiology and metabolism, especially during host infection. Whereas drug discovery for other bacterial infections can rely on predictive in vitro assays and animal models, for Mtb, inherent metabolic flexibility and uncertainties about the nutrients available to infecting bacilli in different host (micro)environments instead requires educated predictions or demonstrations of efficacy in animal models of arguable relevance to human disease. Even microbiological methods for enumeration of viable mycobacterial cells are fraught with complication. Our research has focused on elucidating those aspects of mycobacterial metabolism that contribute to the robustness of the bacillus to host immunological defenses and applied antibiotics and that, possibly, drive the emergence of drug resistance. This work has identified a handful of metabolic pathways that appear vulnerable to antibiotic targeting. Those highlighted, here, include the inter-related functions of pantothenate and coenzyme A biosynthesis and recycling and nucleotide metabolism-the last of which reinforces our view that DNA metabolism constitutes an under-explored area for new TB drug development. Although nonessential functions have traditionally been deprioritized for antibiotic development, a common theme emerging from this work is that these very functions might represent attractive targets because of the potential to cripple mechanisms critical to bacillary survival under stress (for example, the RelMtb-dependent stringent response) or to adaptability under unfavorable, potentially lethal, conditions including antibiotic therapy (for example, DnaE2-dependent SOS mutagenesis). The bar, however, is high: demonstrating convincingly the likely efficacy of this strategy will require innovative models of human TB disease. In the concluding section, we focus on the need for improved techniques to elucidate mycobacterial metabolism during infection and its impact on disease outcomes. Here, we argue that developments in other fields suggest the potential to break through this barrier by harnessing chemical-biology approaches in tandem with the most advanced technologies. As researchers based in a high-burden country, we are impelled to continue participating in this important endeavor.

The Mycobacterium smegmatis HesB Protein, MSMEG_4272, Is Required for In Vitro Growth and Iron Homeostasis.

A-type carrier (ATC) proteins are proposed to function in the biogenesis of Fe-S clusters, although their exact role remains controversial. The genome of Mycobacterium smegmatis encodes a single ATC protein, MSMEG_4272, which belongs to the HesB/YadR/YfhF family of proteins. Attempts to generate an MSMEG_4272 deletion mutant by two-step allelic exchange were unsuccessful, suggesting that the gene is essential for in vitro growth. CRISPRi-mediated transcriptional knock-down of MSMEG_4272 resulted in a growth defect under standard culture conditions, which was exacerbated in mineral-defined media. The knockdown strain displayed reduced intracellular iron levels under iron-replete conditions and increased susceptibility to clofazimine, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), and isoniazid, while the activity of the Fe-S containing enzymes, succinate dehydrogenase, and aconitase were not affected. This study suggests that MSMEG_4272 plays a role in the regulation of intracellular iron levels and is required for in vitro growth of M. smegmatis, particularly during exponential growth.

Investigating the composition and recruitment of the mycobacterial ImuA'-ImuB-DnaE2 mutasome.

A DNA damage-inducible mutagenic gene cassette has been implicated in the emergence of drug resistance in Mycobacterium tuberculosis during anti-tuberculosis (TB) chemotherapy. However, the molecular composition and operation of the encoded 'mycobacterial mutasome' - minimally comprising DnaE2 polymerase and ImuA' and ImuB accessory proteins - remain elusive. Following exposure of mycobacteria to DNA damaging agents, we observe that DnaE2 and ImuB co-localize with the DNA polymerase III ß subunit (ß clamp) in distinct intracellular foci. Notably, genetic inactivation of the mutasome in an imuBAAAAGG mutant containing a disrupted ß clamp-binding motif abolishes ImuB-ß clamp focus formation, a phenotype recapitulated pharmacologically by treating bacilli with griselimycin and in biochemical assays in which this ß clamp-binding antibiotic collapses pre-formed ImuB-ß clamp complexes. These observations establish the essentiality of the ImuB-ß clamp interaction for mutagenic DNA repair in mycobacteria, identifying the mutasome as target for adjunctive therapeutics designed to protect anti-TB drugs against emerging resistance.

Changing character and waning impact of COVID-19 at a tertiary centre in Cape Town, South Africa.

Background: The emergence of genetic variants of SARS-CoV-2 was associated with changing epidemiological characteristics throughout coronavirus disease 2019 (COVID-19) pandemic in population-based studies. Individual-level data on the clinical characteristics of infection with different SARS-CoV-2 variants in African countries is less well documented. Objectives: To describe the evolving clinical differences observed with the various SARS-CoV-2 variants of concern and compare the Omicron-driven wave in infections to the previous Delta-driven wave. Method: We performed a retrospective observational cohort study among patients admitted to a South African referral hospital with COVID-19 pneumonia. Patients were stratified by epidemiological wave period, and in a subset, the variants associated with each wave were confirmed by genomic sequencing. Outcomes were analysed by Cox proportional hazard models. Results: We included 1689 patients were included, representing infection waves driven predominantly by ancestral, Beta, Delta and Omicron BA1/BA2 & BA4/BA5 variants. Crude 28-day mortality was 25.8% (34/133) in the Omicron wave period versus 37.1% (138/374) in the Delta wave period (hazard ratio [HR] 0.68 [95% CI 0.47-1.00] p = 0.049); this effect persisted after adjustment for age, gender, HIV status and presence of cardiovascular disease (adjusted HR [aHR] 0.43 [95% CI 0.28-0.67] p < 0.001). Hospital-wide SARS-CoV-2 admissions and deaths were highest during the Delta wave period, with a decoupling of SARS-CoV-2 deaths and overall deaths thereafter. Conclusion: There was lower in-hospital mortality during Omicron-driven waves compared with the prior Delta wave, despite patients admitted during the Omicron wave being at higher risk. Contribution: This study summarises clinical characteristics associated with SARS-CoV-2 variants during the COVID-19 pandemic at a South African tertiary hospital, demonstrating a waning impact of COVID-19 on healthcare services over time despite epidemic waves driven by new variants. Findings suggest the absence of increasing virulence from later variants and protection from population and individual-level immunity.

Serial measurement of M. tuberculosis in blood from critically-ill patients with HIV-associated tuberculosis.

BACKGROUND: Despite being highly prevalent in hospitalised patients with severe HIV-associated tuberculosis (TB) and sepsis, little is known about the mycobacteriology of Mycobacterium tuberculosis bloodstream infection (MTBBSI). We developed methods to serially measure bacillary load in blood and used these to characterise MTBBSI response to anti-TB therapy (ATT) and relationship with mortality. METHODS: We established a microscopy method for direct visualisation of M. tuberculosis bacilli in blood using a novel lysis-concentration protocol and the fluorescent probe, 4-N,N-dimethylaminonaphthalimide-trehalose (DMN-Tre). We tested blood using GeneXpert® MTB/RIF-Ultra (Xpert-ultra) and Myco/F lytic culture after processing blood through lysis-wash steps to remove PCR inhibitors and anti-microbial drug carry-over. HIV-positive patients predicted to have MTBBSI gave blood samples 0, 4, 24, 48 and 72 h after ATT initiation. Bacillary loads were quantified using microscopy, Xpert-ultra cycle threshold, and culture time-to-positivity. Pharmacodynamics were modelled using these measures combined on an ordinal scale, including association with 12-week mortality. FINDINGS: M. tuberculosis was detected in 27 of 28 recruited participants; 25 (89%) by blood Xpert-ultra, 22 (79%) by DMN-Tre microscopy, and 21 (75%) by Myco/F lytic blood culture. Eight (29%) participants died by 12-week follow-up. In a combined pharmacodynamic model, predicted probabilities of negative DMN-Tre microscopy, blood Xpert-ultra, or blood culture after 72 h treatment were 0·64, 0·27, and 0·94, respectively, in those who survived, compared with 0·23, 0·06, and 0·71 in those who died (posterior probability of slower clearance of MTBBSI in those that died >0·99). DMN-Tre microscopy of blood demonstrated heterogenous bacillary morphologies, including microcolonies and clumps. Bacillary cell-length varied significantly with ATT exposure (mean cell-length increase 0·13 log-µm/day; 95%CrI 0·10-0·16). INTERPRETATION: Pharmacodynamics of MTBBSI treatment can be captured using DMN-Tre microscopy, blood Xpert-ultra and culture. This could facilitate interventional trials in severe HIV-associated TB. FUNDING: Wellcome Trust, NIH Fogarty International Center, South African MRC, NIHR(UK), National Research Foundation of South Africa.

Intracellular Accumulation of Novel and Clinically Used TB Drugs Potentiates Intracellular Synergy.

The therapeutic repertoire for tuberculosis (TB) remains limited despite the existence of many TB drugs that are highly active in in vitro models and possess clinical utility. Underlying the lack of efficacy in vivo is the inability of TB drugs to penetrate microenvironments inhabited by the causative agent, Mycobacterium tuberculosis, including host alveolar macrophages. Here, we determined the ability of the phenoxazine PhX1 previously shown to be active against M. tuberculosis in vitro to differentially penetrate murine compartments, including plasma, epithelial lining fluid, and isolated epithelial lining fluid cells. We also investigated the extent of permeation into uninfected and M. tuberculosis-infected human macrophage-like Tamm-Horsfall protein 1 (THP-1) cells directly and by comparing to results obtained in vitro in synergy assays. Our data indicate that PhX1 (4,750 ± 127.2 ng/ml) penetrates more effectively into THP-1 cells than do the clinically used anti-TB agents, rifampin (3,050 ± 62.9 ng/ml), moxifloxacin (3,374 ± 48.7 ng/ml), bedaquiline (4,410 ± 190.9 ng/ml), and linezolid (770 ± 14.1 ng/ml). Compound efficacy in infected cells correlated with intracellular accumulation, reinforcing the perceived importance of intracellular penetration as a key drug property. Moreover, we detected synergies deriving from redox-stimulatory combinations of PhX1 or clofazimine with the novel prenylated amino-artemisinin WHN296. Finally, we used compound synergies to elucidate the relationship between compound intracellular accumulation and efficacy, with PhX1/WHN296 synergy levels shown to predict drug efficacy. Collectively, our data support the utility of the applied assays in identifying in vitro active compounds with the potential for clinical development. IMPORTANCE This study addresses the development of novel therapeutic compounds for the eventual treatment of drug-resistant tuberculosis. Tuberculosis continues to progress, with cases of Mycobacterium tuberculosis (M. tuberculosis) resistance to first-line medications increasing. We assess new combinations of drugs with both oxidant and redox properties coupled with a third partner drug, with the focus here being on the potentiation of M. tuberculosis-active combinations of compounds in the intracellular macrophage environment. Thus, we determined the ability of the phenoxazine PhX1, previously shown to be active against M. tuberculosis in vitro, to differentially penetrate murine compartments, including plasma, epithelial lining fluid, and isolated epithelial lining fluid cells. In addition, the extent of permeation into human macrophage-like THP-1 cells and H37Rv-infected THP-1 cells was measured via mass spectrometry and compared to in vitro two-dimensional synergy and subsequent intracellular efficacy. Collectively, our data indicate that development of new drugs will be facilitated using the methods described herein.

Arrayed CRISPRi and quantitative imaging describe the morphotypic landscape of essential mycobacterial genes.

Mycobacterium tuberculosis possesses a large number of genes of unknown or predicted function, undermining fundamental understanding of pathogenicity and drug susceptibility. To address this challenge, we developed a high-throughput functional genomics approach combining inducible CRISPR-interference and image-based analyses of morphological features and sub-cellular chromosomal localizations in the related non-pathogen, M. smegmatis. Applying automated imaging and analysis to 263 essential gene knockdown mutants in an arrayed library, we derive robust, quantitative descriptions of bacillary morphologies consequent on gene silencing. Leveraging statistical-learning, we demonstrate that functionally related genes cluster by morphotypic similarity and that this information can be used to inform investigations of gene function. Exploiting this observation, we infer the existence of a mycobacterial restriction-modification system, and identify filamentation as a defining mycobacterial response to histidine starvation. Our results support the application of large-scale image-based analyses for mycobacterial functional genomics, simultaneously establishing the utility of this approach for drug mechanism-of-action studies.

Caused by the microorganism Mycobacterium tuberculosis, tuberculosis kills more people around the world than any other infectious disease. M. tuberculosis is also becoming increasingly resistant to treatments, which are particularly difficult for patients to complete. The M. tuberculosis genome carries about four thousand genes, with several hundred being vital for survival. Finding new ways to fight tuberculosis relies on understanding the exact role of these essential genes, but they are difficult to study in living bacteria. To investigate this question, de Wet et al. used the related, fast-dividing bacterial species called M. smegmatis as a model. Microscopic imaging was combined with CRISPR-interference ­ a method that temporarily disrupts expression of a specific gene ­ to examine how blocking an essential gene would affect the shape of the living microorganism. Experiments were conducted on a collection of 270 mutants, capturing single-cell data for hundreds of thousands of live bacteria. To analyze the data, a computational pipeline was built, which automatically clustered similar-shaped bacteria. These groups, or 'phenoprints', brought together genes of known and unknown roles; this indicated that these genes participate in similar biological networks ­ and, if unknown, hinted at their function. Finally, targeting essential genes with CRISPR-interference often yielded the same shape changes as blocking their encoded proteins with antibiotics. This suggests that phenoprints could be useful to understand the mode of action of potential new tuberculosis treatments. When applied to M. tuberculosis and other deadly bacteria, the approach developed by de Wet et al. might speed up drug development.

Mycobacterium tuberculosis Metabolism.

Mycobacterium tuberculosis is the cause of tuberculosis (TB), a disease which continues to overwhelm health systems in endemic regions despite the existence of effective combination chemotherapy and the widespread use of a neonatal anti-TB vaccine. For a professional pathogen, M. tuberculosis retains a surprisingly large proportion of the metabolic repertoire found in nonpathogenic mycobacteria with very different lifestyles. Moreover, evidence that additional functions were acquired during the early evolution of the M. tuberculosis complex suggests the organism has adapted (and augmented) the metabolic pathways of its environmental ancestor to persistence and propagation within its obligate human host. A better understanding of M. tuberculosis pathogenicity, however, requires the elucidation of metabolic functions under disease-relevant conditions, a challenge complicated by limited knowledge of the microenvironments occupied and nutrients accessed by bacilli during host infection, as well as the reliance in experimental mycobacteriology on a restricted number of experimental models with variable relevance to clinical disease. Here, we consider M. tuberculosis metabolism within the framework of an intimate host-pathogen coevolution. Focusing on recent advances in our understanding of mycobacterial metabolic function, we highlight unusual adaptations or departures from the better-characterized model intracellular pathogens. We also discuss the impact of these mycobacterial "innovations" on the susceptibility of M. tuberculosis to existing and experimental anti-TB drugs, as well as strategies for targeting metabolic pathways. Finally, we offer some perspectives on the key gaps in the current knowledge of fundamental mycobacterial metabolism and the lessons which might be learned from other systems.