April 21, 2020  |  

Mce3R Stress-Resistance Pathway Is Vulnerable to Small-Molecule Targeting That Improves Tuberculosis Drug Activities.

One-third of the world’s population carries Mycobacterium tuberculosis ( Mtb), the infectious agent that causes tuberculosis (TB), and every 17 s someone dies of TB. After infection, Mtb can live dormant for decades in a granuloma structure arising from the host immune response, and cholesterol is important for this persistence of Mtb. Current treatments require long-duration drug regimens with many associated toxicities, which are compounded by the high doses required. We phenotypically screened 35 6-azasteroid analogues against Mtb and found that, at low micromolar concentrations, a subset of the analogues sensitized Mtb to multiple TB drugs. Two analogues were selected for further study to characterize the bactericidal activity of bedaquiline and isoniazid under normoxic and low-oxygen conditions. These two 6-azasteroids showed strong synergy with bedaquiline (fractional inhibitory concentration index = 0.21, bedaquiline minimal inhibitory concentration = 16 nM at 1 µM 6-azasteroid). The rate at which spontaneous resistance to one of the 6-azasteroids arose in the presence of bedaquiline was approximately 10-9, and the 6-azasteroid-resistant mutants retained their isoniazid and bedaquiline sensitivity. Genes in the cholesterol-regulated Mce3R regulon were required for 6-azasteroid activity, whereas genes in the cholesterol catabolism pathway were not. Expression of a subset of Mce3R genes was down-regulated upon 6-azasteroid treatment. The Mce3R regulon is implicated in stress resistance and is absent in saprophytic mycobacteria. This regulon encodes a cholesterol-regulated stress-resistance pathway that we conclude is important for pathogenesis and contributes to drug tolerance, and this pathway is vulnerable to small-molecule targeting in live mycobacteria.


April 21, 2020  |  

Mitochondrial DNA Variants in Patients with Liver Injury Due to Anti-Tuberculosis Drugs.

Hepatotoxicity is the most severe adverse effect of anti-tuberculosis therapy. Isoniazid’s metabolite hydrazine is a mitochondrial complex II inhibitor. We hypothesized that mitochondrial DNA variants are risk factors for drug-induced liver injury (DILI) due to isoniazid, rifampicin or pyrazinamide.We obtained peripheral blood from tuberculosis (TB) patients before anti-TB therapy. A total of 38 patients developed DILI due to anti-TB drugs. We selected 38 patients with TB but without DILI as controls. Next-generation sequencing detected point mutations in the mitochondrial DNA genome. DILI was defined as ALT =5 times the upper limit of normal (ULN), or ALT =3 times the ULN with total bilirubin =2 times the ULN.In 38 patients with DILI, the causative drug was isoniazid in eight, rifampicin in 14 and pyrazinamide in 16. Patients with isoniazid-induced liver injury had more variants in complex I’s NADH subunit 5 and 1 genes, more nonsynonymous mutations in NADH subunit 5, and a higher ratio of nonsynonymous to total substitutions. Patients with rifampicin- or pyrazinamide-induced liver injury had no association with mitochondrial DNA variants.Variants in complex I’s subunit 1 and 5 genes might affect respiratory chain function and predispose isoniazid-induced liver injury when exposed to hydrazine, a metabolite of isoniazid and a complex II inhibitor.


April 21, 2020  |  

Whole genome sequencing identifies bacterial factors affecting transmission of multidrug-resistant tuberculosis in a high-prevalence setting.

Whole genome sequencing (WGS) can elucidate Mycobacterium tuberculosis (Mtb) transmission patterns but more data is needed to guide its use in high-burden settings. In a household-based TB transmissibility study in Peru, we identified a large MIRU-VNTR Mtb cluster (148 isolates) with a range of resistance phenotypes, and studied host and bacterial factors contributing to its spread. WGS was performed on 61 of the 148 isolates. We compared transmission link inference using epidemiological or genomic data and estimated the dates of emergence of the cluster and antimicrobial drug resistance (DR) acquisition events by generating a time-calibrated phylogeny. Using a set of 12,032 public Mtb genomes, we determined bacterial factors characterizing this cluster and under positive selection in other Mtb lineages. Four of the 61 isolates were distantly related and the remaining 57 isolates diverged ca. 1968 (95%HPD: 1945-1985). Isoniazid resistance arose once and rifampin resistance emerged subsequently at least three times. Emergence of other DR types occurred as recently as within the last year of sampling. We identified five cluster-defining SNPs potentially contributing to transmissibility. In conclusion, clusters (as defined by MIRU-VNTR typing) may be circulating for decades in a high-burden setting. WGS allows for an enhanced understanding of transmission, drug resistance, and bacterial fitness factors.


April 21, 2020  |  

Extended insight into the Mycobacterium chelonae-abscessus complex through whole genome sequencing of Mycobacterium salmoniphilum outbreak and Mycobacterium salmoniphilum-like strains.

Members of the Mycobacterium chelonae-abscessus complex (MCAC) are close to the mycobacterial ancestor and includes both human, animal and fish pathogens. We present the genomes of 14 members of this complex: the complete genomes of Mycobacterium salmoniphilum and Mycobacterium chelonae type strains, seven M. salmoniphilum isolates, and five M. salmoniphilum-like strains including strains isolated during an outbreak in an animal facility at Uppsala University. Average nucleotide identity (ANI) analysis and core gene phylogeny revealed that the M. salmoniphilum-like strains are variants of the human pathogen Mycobacterium franklinii and phylogenetically close to Mycobacterium abscessus. Our data further suggested that M. salmoniphilum separates into three branches named group I, II and III with the M. salmoniphilum type strain belonging to group II. Among predicted virulence factors, the presence of phospholipase C (plcC), which is a major virulence factor that makes M. abscessus highly cytotoxic to mouse macrophages, and that M. franklinii originally was isolated from infected humans make it plausible that the outbreak in the animal facility was caused by a M. salmoniphilum-like strain. Interestingly, M. salmoniphilum-like was isolated from tap water suggesting that it can be present in the environment. Moreover, we predicted the presence of mutational hotspots in the M. salmoniphilum isolates and 26% of these hotspots overlap with genes categorized as having roles in virulence, disease and defense. We also provide data about key genes involved in transcription and translation such as sigma factor, ribosomal protein and tRNA genes.


April 21, 2020  |  

Genome-wide mutational biases fuel transcriptional diversity in the Mycobacterium tuberculosis complex.

The Mycobacterium tuberculosis complex (MTBC) members display different host-specificities and virulence phenotypes. Here, we have performed a comprehensive RNAseq and methylome analysis of the main clades of the MTBC and discovered unique transcriptional profiles. The majority of genes differentially expressed between the clades encode proteins involved in host interaction and metabolic functions. A significant fraction of changes in gene expression can be explained by positive selection on single mutations that either create or disrupt transcriptional start sites (TSS). Furthermore, we show that clinical strains have different methyltransferases inactivated and thus different methylation patterns. Under the tested conditions, differential methylation has a minor direct role on transcriptomic differences between strains. However, disruption of a methyltransferase in one clinical strain revealed important expression differences suggesting indirect mechanisms of expression regulation. Our study demonstrates that variation in transcriptional profiles are mainly due to TSS mutations and have likely evolved due to differences in host characteristics.


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