The impact of genotype on the phenotype of Mycobacterium tuberculosis ΔsufR mutants.

Iron-sulphur (FeS) cluster biogenesis is a tightly regulated process in vivo. In Mycobacterium tuberculosis (Mtb), SufR functions as a transcriptional repressor of the operon encoding the primary FeS cluster biogenesis system. Previously, three independently isolated mutants (ΔRv1460stop_1.19, ΔRv1460stop _5.19 and ΔRv1460stop _5.20) harbouring the same deletion in sufR, displayed different growth kinetics in OADC supplemented 7H9 media. To investigate this discrepancy, we performed whole genome sequencing of the 3 mutants and the wild-type progenitor. Single nucleotide polymorphisms (SNPs) were identified in 3 genes in the ΔRv1460stop_1.19 mutant and one gene in the ΔRv1460stop_5.20 mutant. Phenotyping of the ΔRv1460stop_5.19 mutant, which had no additional SNPs, revealed increased susceptibility to clofazimine, DMNQ and menadione, while uptake and survival in THP-1 cells were not significantly different from the wild-type strain. Given that these results differ from those reported for other sufR deletion mutants (ΔSufRMTB and MtbΔSufR), they suggest that the position of the sufR deletion and the genotype of the progenitor strain impact the resulting phenotype.

Transcriptional Response of Mycobacterium tuberculosis to Cigarette Smoke Condensate.

Smoking is known to be an added risk factor for tuberculosis (TB), with nearly a quarter of the TB cases attributed to cigarette smokers in the 22 countries with the highest TB burden. Many studies have indicated a link between risk of active TB and cigarette smoke. Smoking is also known to significantly decrease TB cure and treatment completion rate and increase mortality rates. Cigarette smoke contains thousands of volatile compounds including carcinogens, toxins, reactive solids, and oxidants in both particulate and gaseous phase. Yet, to date, limited studies have analyzed the impact of cigarette smoke components on Mycobacterium tuberculosis (Mtb), the causative agent of TB. Here we report the impact of cigarette smoke condensate (CSC) on survival, mutation frequency, and gene expression of Mtb in vitro. We show that exposure of virulent Mtb to cigarette smoke increases the mutation frequency of the pathogen and strongly induces the expression of the regulon controlled by SigH-a global transcriptional regulator of oxidative stress. SigH has previously been shown to be required for Mtb to respond to oxidative stress, survival, and granuloma formation in vivo. A high-SigH expression phenotype is known to be associated with greater virulence of Mtb. In patients with pulmonary TB who smoke, these changes may therefore play an important, yet unexplored, role in the treatment efficacy by potentially enhancing the virulence of tubercle bacilli.

Using genomic DNA copies to enumerate Mycobacterium tuberculosis load in macaque tissue samples.

It is important to accurately quantify Mycobacterium tuberculosis (Mtb) load in laboratory-based tuberculosis (TB) research. This study aims to determine if real-time quantitative PCR (qPCR) and digital PCR (dPCR) can be used instead of colony forming unit (CFU) enumeration, to quantify Mtb load in rhesus and cynomolgus macaque tissue samples. Tissue samples of actively infected high Mtb-burden rhesus and cynomolgus macaques were selected from historic sample collections. CFUs were enumerated by plating, and Chelex-extracted genomic DNA used to quantify bacterial load by qPCR and dPCR. Three genes, sigA, 16S and CFP10, were assessed for their ability to quantify Mtb. All genes showed comparable quantification of Mtb between 2 and 20 000 copies/µl in the qPCR and 5-4000 copies/µl in the dPCR assay. The highest bacterial load was observed with dPCR, followed by qPCR, and CFU enumeration. Although the CFU count was consistently lower than the genomic copy numbers predicted by qPCR and dPCR, a significant correlation was observed. Quantification of Mtb by PCR was, however, only possible in higher-Mtb-load samples, suggesting that qPCR and dPCR quantification assays can predict bacterial load in actively infected and higher-Mtb-burden macaque tissue samples.

SufT is required for growth of Mycobacterium smegmatis under iron limiting conditions.

Iron-sulphur (FeS) clusters are versatile cofactors required for a range of biological processes within cells. Due to the reactive nature of the constituent molecules, assembly and delivery of these cofactors requires a multi-protein machinery in vivo. In prokaryotes, SufT homologues are proposed to function in the maturation and transfer of FeS clusters to apo-proteins. This study used targeted gene deletion to investigate the role of SufT in the physiology of mycobacteria, using Mycobacterium smegmatis as a model organism. Deletion of the sufT gene in M. smegmatis had no impact on growth under standard culture conditions and did not significantly alter activity of the FeS cluster dependent enzymes succinate dehydrogenase (SDH) and aconitase (ACN). Furthermore, the ΔsufT mutant was no more sensitive than the wild-type strain to the redox cycler 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), or the anti-tuberculosis drugs isoniazid, clofazimine or rifampicin. In contrast, the ΔsufT mutant displayed a growth defect under iron limiting conditions, and an increased requirement for iron during biofilm formation. This data suggests that SufT is an accessory factor in FeS cluster biogenesis in mycobacteria which is required under conditions of iron limitation.

Rv1460, a SufR homologue, is a repressor of the suf operon in Mycobacterium tuberculosis.

Iron-sulphur (Fe-S) clusters are ubiquitous co-factors which require multi-protein systems for their synthesis. In Mycobacterium tuberculosis, the Rv1460-Rv1461-Rv1462-Rv1463-csd-Rv1465-Rv1466 operon (suf operon) encodes the primary Fe-S cluster biogenesis system. The first gene in this operon, Rv1460, shares homology with the cyanobacterial SufR, which functions as a transcriptional repressor of the sufBCDS operon. Rv1460's function in M. tuberculosis has however not been determined. In this study, we demonstrate that M. tuberculosis mutants lacking a functional Rv1460 protein are impaired for growth under standard culture conditions. Elevated expression of Rv1460 and Rv1461 was observed in the mutant, implicating Rv1460 in the regulation of the suf operon. Binding of an Fe-S cluster to purified recombinant Rv1460 was confirmed by UV-visible spectroscopy and circular dichroism. Furthermore, three conserved cysteine residues, C203, C216 and C244, proposed to provide ligands for the coordination of an Fe-S cluster, were shown to be required for the function of Rv1460 in M. tuberculosis. Rv1460 therefore seems to be functionally analogous to cyanobacterial SufR.