Phylogenetic and Structural Significance of Dihydrofolate Synthase (folC) Mutations in Drug-Resistant Mycobacterium tuberculosis

Abstract

The aim of this study is to investigate the mutation pattern of the folC gene in drug-resistant Mycobacterium tuberculosis (MTB) clinical isolates of global and Hong Kong cohorts. The public sequence read archives of 1,124 MTB genomes from three independent studies were retrieved and folC mutations existing solely in drug-resistant MTB strains were identified. A phylogenetic tree was constructed to analyze the segregation of mutation-related amino acid residues in the FolC structure. These mutation sites were further supported by direct Sanger sequencing of the folC gene among 254 clinical MTB isolates in a Hong Kong cohort. Homology modeling of wild-type and mutated FolC was performed, and the predicted structures were docked with hydroxydihydropteroate, the metabolic derivative of para-aminosalicylic acid (PAS), to evaluate the resultant binding affinity changes. Combining the results of three previous cohorts and our cohort, E40, I43, S150, and E153 are the most frequently affected amino acid residues in resistant isolates. Based on the distribution of mutations in the genome-based phylogenetic tree, lineage-specific mutation patterns were observed. Regarding the segregation of affected amino acid residues, the four most frequently affected residues are all in close proximity of the binding pocket for the PAS derivative. Molecular modeling results showed that mutations at E40, I43, and S150 can alter the structure of FolC putative binding pocket, causing the PAS derivative to bind outside of the now deformed pocket. This might ablate the interaction between the protein and the PAS derivative. To conclude, this study is the first comprehensive mutation pattern and bioinformatics analysis of the folC gene in MTB drug-resistant isolates. The distribution of mutations in phylogenetic lineages and protein structure is reported, analyzed, and discussed.

<p>Molecular characterization of para-aminosalicylic acid resistant <em>Mycobacterium</em> <em>tuberculosis</em> clinical isolates in southwestern China</p>

“…Previously, E153A was found to be the most frequently identified mutation type in folC in PAS-resistant isolates from Hongkong, which is also common in isolates from northern China. However, in this study, we did not find any E150 mutation in PAS-resistant isolates from Chongqing 18,19…”

“…To our best knowledge, this is the first report showing that double mutations exist in folC of PAS-resistant TB. Most mutations in folC such as E40, I43, R49, S150, and E153 are located in the substrate binding pocket, which leads to reduced efficiency of H 2 PtePAS glutamination 9,19,20. Since E44 and S46 were adjacent to I43 and R49, H100 was located in the Ω-loop just adjacent to the Ser- cis -Pro motif in the active site of FolC 20.…”

Evaluation of Whole-Genome Sequence Method to Diagnose Resistance of 13 Anti-tuberculosis Drugs and Characterize Resistance Genes in Clinical Multi-Drug Resistance Mycobacterium tuberculosis Isolates From China

“…PAS resistance is mainly related to the folate metabolism pathway, and the main genes involved include thyA, folC, ribD , and dfrA (Mathys et al, 2009; Chakraborty et al, 2013; Zhao et al, 2014; Zhang et al, 2015b; Cheng et al, 2016). In this study, the seven resistant strains carried the following mutations: two strains with FolC I43T, one strain with FolC I43T+ thyA t-31c, two strains with FolC S150G, one strain with FolC S150G+ThyA H75N, and one wildtype strain.…”