Intervening along the spectrum of tuberculosis: meeting report from the World TB Day nanosymposium in the Institute of Infectious Disease and Molecular Medicine at the University of Cape Town [version 4; peer review: 2 approved]

Tuberculosis (TB), caused by the highly infectious Mycobacterium tuberculosis, remains a leading cause of death worldwide, with an estimated 1.6 million associated deaths reported in 2017. In South Africa, an estimated 322,000 (range 230,000-428,000) people were infected with TB in 2017, and a quarter of them lost their lives due to the disease. Bacille Calmette-Guérin (BCG) remains the only effective vaccine against disseminated TB, but its inability to confer complete protection against pulmonary TB in adolescents and adults calls for an urgent need to develop new and better vaccines. There is also a need to identify markers of disease protection and develop novel drugs. It is within this backdrop that we convened a nanosymposium at the Institute of Infectious Disease and Molecular Medicine at the University of Cape Town to commemorate World TB Day and showcase recent findings generated by early career scientists in the institute. The speakers spoke on four broad topics: identification of novel drug targets, development of host-directed drug therapies, transmission of TB and immunology of TB/HIV co-infections

Intervening along the spectrum of tuberculosis: meeting report from the World TB Day nanosymposium in the Institute of Infectious Disease and Molecular Medicine at the University of Cape Town [version 1; peer review: 1 approved]

Tuberculosis, caused by the highly infectious Mycobacterium tuberculosis, remains a leading cause of death worldwide, with an estimated 1.6 million associated deaths reported in 2017. In South Africa, an estimated 322,000 people were infected with TB in 2017, and a quarter of them lost their lives due to the disease. Bacille Calmette-Guérin remains the only effective vaccine against disseminated TB, but its inability to confer complete protection against pulmonary TB in adolescents and adults calls for an urgent need to develop new and better vaccines. There is also a need to identify markers of disease protection and develop novel drugs. On March 25th 2019, the Institute of Infectious Disease and Molecular Medicine at the University of Cape Town hosted the second annual World TB Day nanosymposium. The theme of the nanosymposium was “Intervening across the spectrum of TB II” and the goal was to commemorate World TB Day by showcasing research insights shared by early-career scientists and researchers in the field. The speakers spoke on four broad topics: identification of novel drug targets, development of host-directed drug therapies, transmission of tuberculosis and immunology of TB/HIV co-infections. Assistant Professor Bryan Bryson gave a highly interesting keynote address that showcased the application of engineering tools to answer fundamental biological questions, particularly in the context of tuberculosis

Modulation of riboflavin biosynthesis and utilization in mycobacteria

ABSTRACT Riboflavin (vitamin B2) is the precursor of the flavin coenzymes, FAD and FMN, which play a central role in cellular redox metabolism. While humans must obtain riboflavin from dietary sources, certain microbes, including Mycobacterium tuberculosis (Mtb), can biosynthesize riboflavin de novo. Riboflavin precursors have also been implicated in the activation of mucosal-associated invariant T (MAIT) cells which recognize metabolites derived from the riboflavin biosynthesis pathway complexed to the MHC-I-like molecule, MR1. To investigate the biosynthesis and function of riboflavin and its pathway intermediates in mycobacterial metabolism and physiology, we constructed conditional knockdowns (hypomorphs) in riboflavin biosynthesis and utilization genes in Mycobacterium smegmatis (Msm) and Mtb by inducible CRISPR interference. Using this comprehensive panel of hypomorphs, we analyzed the impact of gene silencing on viability, on the transcription of (other) riboflavin pathway genes, on the levels of the pathway proteins, and on riboflavin itself. Our results revealed that (i) despite lacking a canonical transporter, both Msm and Mtb assimilate exogenous riboflavin when supplied at high concentration; (ii) there is functional redundancy in lumazine synthase activity in Msm; (iii) silencing of ribA2 or ribF is profoundly bactericidal in Mtb; and (iv) in Msm, ribA2 silencing results in concomitant knockdown of other pathway genes coupled with RibA2 and riboflavin depletion and is also bactericidal. In addition to their use in genetic validation of potential drug targets for tuberculosis, this collection of hypomorphs provides a useful resource for future studies investigating the role of pathway intermediates in MAIT cell recognition of mycobacteria.IMPORTANCEThe pathway for biosynthesis and utilization of riboflavin, precursor of the essential coenzymes, FMN and FAD, is of particular interest in the flavin-rich pathogen, Mycobacterium tuberculosis (Mtb), for two important reasons: (i) the pathway includes potential tuberculosis (TB) drug targets and (ii) intermediates from the riboflavin biosynthesis pathway provide ligands for mucosal-associated invariant T (MAIT) cells, which have been implicated in TB pathogenesis. However, the riboflavin pathway is poorly understood in mycobacteria, which lack canonical mechanisms to transport this vitamin and to regulate flavin coenzyme homeostasis. By conditionally disrupting each step of the pathway and assessing the impact on mycobacterial viability and on the levels of the pathway proteins as well as riboflavin, our work provides genetic validation of the riboflavin pathway as a target for TB drug discovery and offers a resource for further exploring the association between riboflavin biosynthesis, MAIT cell activation, and TB infection and disease

Bacterial and host determinants of cough aerosol culture positivity in patients with drug-resistant versus drug-susceptible tuberculosis

A burgeoning epidemic of drug-resistant tuberculosis threatens to derail global control efforts. Although the mechanisms remain poorly clarified, drug-resistant strains are widely believed to be less infectious than drug-susceptible strains. Consequently, we hypothesised that lower proportions of drug-resistant TB patients would have culturable Mycobacterium tuberculosis from respirable cough-generated aerosols compared to drug-susceptible TB patients, and that multiple factors, including mycobacterial genomic variation, would predict culturable cough aerosol production. We enumerated colony forming units (CFU) in aerosols (≤10μm) from 500 tuberculosis patients (227 with drug-resistance), compared clinical characteristics, and performed mycobacterial whole genome sequencing, dormancy phenotyping, and drug susceptibility analyses on M. tuberculosis from sputum. After considering treatment duration, we found that almost half of drug-resistant tuberculosis patients were cough aerosol culture-positive. Surprisingly, neither mycobacterial genomic variants, lineage, nor dormancy status predicted cough aerosol culture-positivity. However, mycobacterial sputum bacillary load and clinical characteristics, including a lower symptom score and stronger cough, were strongly predictive; thereby supporting targeted transmission-limiting interventions. Effective treatment largely abrogated cough aerosol culture-positivity, however, this was not always rapid. These data question current paradigms, inform public health strategies, and suggest the need to redirect tuberculosis transmission-associated research efforts towards host-pathogen interactions