Modulation of riboflavin biosynthesis and utilization in mycobacteria

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, physiology and MAIT cell recognition, 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 investigating the role of pathway intermediates in MAIT cell recognition of mycobacteria.


FIG. S2.
Approximate positions of sgRNAs used to create hypomorphs in RF pathway genes.sgRNAs next to one another represent those sharing overlapping sequences.The sgRNA sequences are provided in Tables S1 and S2.Asterisks denote the sgRNA selected for detailed characterization.Not drawn to scale.
3 FIG.S3.Impact of RF pathway gene silencing on growth and viability of Msm in liquid culture.Cultures were monitored every 3 h by measuring OD 600 (solid lines) and enumerating CFUs by plating serial dilutions on 7H10 agar (columns).Cultures were grown without ATc (black solid line and black column) or in the presence of 100 ng/ml ATc (red solid line and red column).Error bars represent the SD derived from two biological replicates.Statistical comparisons were performed using a two-way ANOVA and Sidak's multiple comparison test whereby statistical significance is represented by p < 0.0001, denoted by an asterisk and no statistical difference represented as ns.

4
FIG. S4.Impact of RF pathway gene silencing on growth and viability of Mtb in liquid culture.Cultures were monitored every 24 h by measuring OD 600 (solid lines) and enumerating CFUs by plating serial dilutions on 7H10 agar (columns).Cultures were grown without ATc (black solid line and black column) or in the presence of 100 ng/ml ATc (red solid line and red column).Error bars represent the SD derived from two biological replicates.Statistical comparisons were performed using a two-way ANOVA and Sidak's multiple comparison test whereby statistical significance is represented by p < 0.0001, denoted by an asterisk and no statistical difference represented as ns.

FIG. S7.
Impact of exogenous RF on growth of Msm RF pathway hypomorphs.The RF dose-responsiveness of growth was determined using a microbroth dilution alamar blue (MABA) colorimetric assay at a fixed concentration of ATc (50 ng/ml) and a RF gradient (1.3 µM -2.7 mM).The minimum concentration of RF required to restore 80% of wildtype growth is shown in red.Error bars represent the SD derived from three technical replicates.

FIG. S8.
Impact of exogenous RF on growth of Mtb RF pathway hypomorphs.The RF dose-responsiveness of growth was determined using a microbroth dilution alamar blue (MABA) colorimetric assay at a fixed concentration of ATc (50 ng/ml) and a RF gradient (1. FIG. S5.Effective knockdown of RF pathway genes in Mtb by induced CRISPRi.The fold change in sigA-normalized expression of the targeted gene transcripts was determined by qRT-PCR analysis from cultures of Mtb hypomorphs grown in the absence (black) or presence of ATc (red).Error bars represent the SD derived from three biological replicates.Statistical comparisons were performed using a two-way ANOVA and Sidak's multiple comparison test whereby statistical significance is represented by p < 0.0001, denoted by an asterisk.

FIG. S6 .
FIG. S6.Efficacy of CRISPRi-dependent growth repression of Msm hypomorphs as a function of inoculum size.Kinetics of growth of Msm hypomorph cultures inoculated with cells at an OD 600 value of 0.004 (red), 0.016 (green), 0.06 (blue) or 0.25 (purple).The solid line represents growth in the absence of ATc and the dashed line in the presence of 100 ng/ml ATc.Error bars represent the SD derived from three biological replicates.
FIG. S8.Impact of exogenous RF on growth of Mtb RF pathway hypomorphs.The RF dose-responsiveness of growth was determined using a microbroth dilution alamar blue (MABA) colorimetric assay at a fixed concentration of ATc (50 ng/ml) and a RF gradient (1.3 µM -2.7 mM).The minimum concentration of RF required to restore growth to 80% of the wildtype is shown in red.Error bars represent the SD derived from three technical replicates.Values from the first four wells have been excluded owing to the dense RF pellet obscuring the spectrophotometric reading.

TABLE S1 .
Genes involved in RF biosynthesis and utilization in Mtb and Msm

TABLE S4 .
ORBIT oligo to create targeted knockout in Msm ribH.

TABLE S5 .
Plasmids used in this study.

TABLE S6 .
Strains used in this study.

TABLE S9 .
Peptides used for targeted proteomics by MRM-MS protein names used.RibF peptide 'YIH' was not able to be validated with a labeled synthetic peptide, so it was not included for sample testing. ¥Mtb