Mycobacterium tuberculosis thymidylate synthase gene thyX is essential and potentially bifunctional, while thyA deletion confers resistance to p-aminosalicylic acid

Abstract

Thymidylate synthase (TS) enzymes catalyse the biosynthesis of deoxythymidine monophosphate (dTMP or thymidylate), and so are important for DNA replication and repair. Two different types of TS proteins have been described (ThyA and ThyX), which have different enzymic mechanisms and unrelated structures. Mycobacteria are unusual as they encode both thyA and thyX, and the biological significance of this is not yet understood. Mycobacterium tuberculosis ThyX is thought to be essential and a potential drug target. We therefore analysed M. tuberculosis thyA and thyX expression levels, their essentiality and roles in pathogenesis. We show that both thyA and thyX are expressed in vitro, and that this expression significantly increased within murine macrophages. Under all conditions tested, thyA expression exceeded that of thyX. Mutational studies show that M. tuberculosis thyX is essential, confirming that the enzyme is a plausible drug target. The requirement for M. tuberculosis thyX in the presence of thyA implies that the essential function of ThyX is something other than dTMP synthase. We successfully deleted thyA from the M. tuberculosis genome, and this deletion conferred an in vitro growth defect that was not observed in vivo. Presumably ThyX performs TS activity within M. tuberculosis ΔthyA at a sufficient rate in vivo for normal growth, but the rate in vitro is less than optimal. We also demonstrate that thyA deletion confers M. tuberculosis p-aminosalicylic acid resistance, and show by complementation studies that ThyA T202A and V261G appear to be functional and non-functional, respectively.

Binding Pocket Alterations in Dihydrofolate Synthase Confer Resistance to para -Aminosalicylic Acid in Clinical Isolates of Mycobacterium tuberculosis

“…Consistent with this model for the PAS mode of action, overexpression of an alternate enzyme with dihydrofolate reductase activity, RibD, confers resistance to this drug (8). Interestingly, loss-of-function mutations in thyA, which encodes a folate-dependent thymidylate synthase, have been associated with PAS resistance by an undefined mechanism in both laboratory and clinical isolates of M. tuberculosis (9,10). As only one-third of PAS-resistant clinical isolates have been found to harbor thyA mutations, and not all thyA mutations are associated with PAS resistance (10)(11)(12), additional PAS resistance alleles have yet to be identified.…”

“…Interestingly, loss-of-function mutations in thyA, which encodes a folate-dependent thymidylate synthase, have been associated with PAS resistance by an undefined mechanism in both laboratory and clinical isolates of M. tuberculosis (9,10). As only one-third of PAS-resistant clinical isolates have been found to harbor thyA mutations, and not all thyA mutations are associated with PAS resistance (10)(11)(12), additional PAS resistance alleles have yet to be identified. Very recently, Zheng et al (8) described a spontaneous PAS-resistant isolate of M. tuberculosis H37Rv harboring a substitution mutation in folC that was responsible for PAS resistance, suggesting that PAS resistance can also be mediated by alterations in dihydrofolate synthase.…”

Genetic Determinants Involved in p -Aminosalicylic Acid Resistance in Clinical Isolates from Tuberculosis Patients in Northern China from 2006 to 2012

“…In addition, mutations causing an overexpression of RibD, which can act as an alternative dihydrofolate reductase (DHFR/DfrA), confer resistance (5, 6). Finally, loss-of-function mutations in thymidylate synthase ThyA, which is a major consumer of tetrahydrofolate, result in PAS resistance (7,8).Other studies reported that one-third of PAS-resistant isolates had thyA mutations; however, little is known about the antibiotic resistance mechanism(s) in the remaining samples (9, 10). Here, we first randomly selected PAS-resistant isolates to determine their genotypes for folC, thyA, and ribD.…”

“…Phylogenetic polymorphisms exist within antibiotic resistance genes, which might lead to false associations made between certain polymorphisms and phenotypic resistance (23,24). For example, in our study, isolates carried the mutation T202A in thyA, a marker for the LAM lineage of M. tuberculosis that is not responsible for PAS resistance (8,25). Theoretically, these false associations would be identified by detecting the occurrence of mutations in both susceptible and resistant strains from within the same genotype (26).…”

“…In addition, mutations causing an overexpression of RibD, which can act as an alternative dihydrofolate reductase (DHFR/DfrA), confer resistance (5, 6). Finally, loss-of-function mutations in thymidylate synthase ThyA, which is a major consumer of tetrahydrofolate, result in PAS resistance (7,8).…”