Modulation of riboflavin biosynthesis and utilization in mycobacteria

Author:

Chengalroyen Melissa D.1ORCID,Mehaffy Carolina2,Lucas Megan2,Bauer Niel2,Raphela Mabule L.1,Oketade Nurudeen2,Warner Digby F.13ORCID,Lewinsohn Deborah A.4,Lewinsohn David M.45,Dobos Karen M.2ORCID,Mizrahi Valerie13ORCID

Affiliation:

1. Molecular Mycobacteriology Research Unit, Institute of Infectious Disease and Molecular Medicine & Department of Pathology, University of Cape Town, Cape Town, South Africa

2. Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA

3. Wellcome Centre for Infectious Disease Research in Africa, University of Cape Town, Cape Town, South Africa

4. Oregon Health and Science University, Portland, Oregon, USA

5. Portland VA Medical Center, Portland, Oregon, USA

Abstract

ABSTRACT Riboflavin (vitamin B 2 ) 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. IMPORTANCE The 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.

Funder

Ernest Oppenheimer Memorial Trust

Bill and Melinda Gates Foundation

South African Medical Research Council

National Research Foundation

Department of Science and Innovation, South Africa

HHS | National Institutes of Health

University of Cape Town

Colorado State University

HHS | NIH | National Institute of Allergy and Infectious Diseases

Publisher

American Society for Microbiology

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