Application of whole genome sequence analysis to the study of <i>Mycobacterium tuberculosis</i> in Nunavut, Canada

<div><p>Canada has one of the lowest rates of tuberculosis (TB) in the world, however, among certain sub-populations, disease incidence rates approach those observed in sub-Saharan Africa, and other high incidence regions. In this study, we applied mycobacterial interspersed repetitive unit (MIRU) variable number of tandem repeat (VNTR) and whole genome sequencing (WGS) to the analysis of <i>Mycobacterium tuberculosis</i> isolates obtained from Northern communities in the territory of Nunavut. WGS was carried out using the Illumina MiSeq, with identified variants used to infer phylogenetic relationships and annotated to infer functional implications. Additionally, the sequencing data from these isolates were augmented with publically available WGS to evaluate data from the Nunavut outbreak in the broader Canadian context. In this study, isolates could be classified into four major clusters by MIRU-VNTR analysis. These could be further resolved into sub-clusters using WGS. No evidence for antimicrobial resistance, either genetic or phenotypic, was observed in this cohort. Among most subjects with multiple samples, reactivation/incomplete treatment likely contributed to recurrence. However, isolates from two subjects appeared more likely to have occurred via reinfection, based on the large number of genomic single nucleotide variants detected. Finally, although quite distinct from previously reported Canadian MTB strains, isolates obtained from Nunavut clustered most closely with a cohort of samples originating in the Nunavik region of Northern Quebec. This study demonstrates the benefit of using WGS for discriminatory analysis of MTB in Canada, especially in high incidence regions. It further emphasizes the importance of focusing epidemiological intervention efforts on interrupting transmission chains of endemic TB throughout Northern communities, rather than relying on strategies applied in regions where the majority of TB cases result from importation of foreign strains.</p></div

Repeat sampling of individuals from whom multiple isolates were obtained.

<p>Repeat sampling of individuals from whom multiple isolates were obtained.</p

Pairwise intra- and inter-cluster SNV variability in four MIRU groups.

<p>Pairwise intra- and inter-cluster SNV variability in four MIRU groups.</p

Nonsense single nucleotide variants (resulting in premature stop, or abrogation of start).

<p>Loci based on genomic position and numbering in the H37Rv reference genome (NC_00962.3). All described alternate alleles at specified loci are in relation to the reference sequence at that position. Included loci are those with at least 5 isolates possessing the variant genotype. All were significantly associated with a MIRU cluster (p_FDR <0.05).</p

Broken-Symmetry DFT Computations for the Reaction Pathway of IspH, an Iron–Sulfur Enzyme in Pathogenic Bacteria

The recently discovered methylerythritol phosphate (MEP) pathway provides new targets for the development of antibacterial and antimalarial drugs. In the final step of the MEP pathway, the [4Fe–4S] IspH protein catalyzes the 2<i>e</i>^–/2H⁺ reductive dehydroxylation of (<i>E</i>)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) to afford the isoprenoid precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). Recent experiments have attempted to elucidate the IspH catalytic mechanism to drive inhibitor development. Two competing mechanisms have recently emerged, differentiated by their proposed HMBPP binding modes upon 1<i>e</i>^– reduction of the [4Fe–4S] cluster: (1) a Birch reduction mechanism, in which HMBPP remains bound to the [4Fe–4S] cluster through its terminal C₄–OH group (ROH-bound) until the −OH is cleaved as water; and (2) an organometallic mechanism, in which the C₄–OH group rotates away from the [4Fe–4S] cluster, allowing the HMBPP olefin group to form a metallacycle complex with the apical iron (η²-bound). We perform broken-symmetry density functional theory computations to assess the energies and reduction potentials associated with the ROH- and η²-bound states implicated by these competing mechanisms. Reduction potentials obtained for ROH-bound states are more negative (−1.4 to −1.0 V) than what is typically expected of [4Fe–4S] ferredoxin proteins. Instead, we find that η²-bound states are lower in energy than ROH-bound states when the [4Fe–4S] cluster is 1<i>e</i>^– reduced. Furthermore, η²-bound states can already be generated in the oxidized state, yielding reduction potentials of ca. −700 mV when electron addition occurs after rotation of the HMBPP C₄–OH group. We demonstrate that such η²-bound states are kinetically accessible both when the IspH [4Fe–4S] cluster is oxidized and 1<i>e</i>^– reduced. The energetically preferred pathway gives 1<i>e</i>^– reduction of the cluster after substrate conformational change, generating the 1<i>e</i>^– reduced intermediate proposed in the organometallic mechanism

Use of Broken-Symmetry Density Functional Theory To Characterize the IspH Oxidized State: Implications for IspH Mechanism and Inhibition

With current therapies becoming less efficacious due to increased drug resistance, new inhibitors of both bacterial and malarial targets are desperately needed. The recently discovered methylerythritol phosphate (MEP) pathway for isoprenoid synthesis provides novel targets for the development of such drugs. Particular attention has focused on the IspH protein, the final enzyme in the MEP pathway, which uses its [4Fe–4S] cluster to catalyze the formation of the isoprenoid precursors IPP and DMAPP from HMBPP. IspH catalysis is achieved via a 2<i>e</i>^–/2H⁺ reductive dehydroxylation of HMBPP; the mechanism by which catalysis is achieved, however, is highly controversial. The work presented herein provides the first step in assessing different routes to catalysis by using computational methods. By performing broken-symmetry density functional theory (BS–DFT) calculations that employ both the conductor-like screening solvation model (DFT/COSMO) and a finite-difference Poisson–Boltzmann self-consistent reaction field methodology (DFT/SCRF), we evaluate geometries, energies, and Mössbauer signatures of the different protonation states that may exist in the oxidized state of the IspH catalytic cycle. From DFT/SCRF computations performed on the oxidized state, we find a state where the substrate, HMBPP, coordinates the apical iron in the [4Fe–4S] cluster as an alcohol group (ROH) to be one of two, isoenergetic, lowest-energy states. In this state, the HMBPP pyrophosphate moiety and an adjacent glutamate residue (E126) are both fully deprotonated, making the active site highly anionic. Our findings that this low-energy state also matches the experimental geometry of the active site and that its computed isomer shifts agree with experiment validate the use of the DFT/SCRF method to assess relative energies along the IspH reaction pathway. Additional studies of IspH catalytic intermediates are currently being pursued