Resistance of Wolbachia to Trimethoprim: Insights into Genes Encoding Dihydrofolate Reductase, Thymidylate Synthase and Serine Hydroxymethyltransferase in the Rickettsiales

Bacterial and eukaryotic dihydrofolate reductase (DHFR) enzymes are essential for DNA synthesis and are differentially sensitive to the competitive inhibitors trimethoprim and methotrexate. Unexpectedly, trimethoprim did not reduce <i>Wolbachia</i> abundance, and the <i>w</i>Stri DHFR homolog contained amino acid substitutions associated with trimethoprim resistance in <i>E. coli</i>. A phylogenetic tree showed good association of DHFR protein sequences with supergroup A and B assignments. In contrast, DHFR is not encoded by <i>w</i>Fol (supergroup E) and <i>w</i>Bm (supergroup D) or by genomes of the closely related genera <i>Anaplasma</i>, <i>Ehrlichia</i>, <i>Neorickettsia</i>, and possibly <i>Orientia</i>. In <i>E. coli</i> and humans, DHFR participates in a coupled reactions with the conventional thymidylate synthase (TS) encoded by <i>thyA</i> to produce the dTMP required for DNA synthesis. In contrast, <i>Wolbachia</i> and other Rickettsiales express the unconventional FAD-TS enzyme encoded by <i>thyX</i>, even when <i>folA</i> is present. The exclusive use of FAD-TS suggests that <i>Wolbachia</i> DHFR provides a supplementary rather than an essential function for de novo synthesis of dTMP, possibly reflecting the relative availability of, and competing demands for, FAD and NAD coenzymes in the diverse intracellular environments of its hosts. Whether encoded by <i>thyA</i> or <i>thyX</i>, TS produces dTMP by transferring a methyl group from methylene tetrahydrofolate to dUMP. In the Rickettsiales, serine hydroxymethyltransferase (SMHT), encoded by a conserved <i>glyA</i> gene, regenerates methylene tetrahydrofolate. Unlike <i>thyA</i>, <i>thyX</i> lacks a human counterpart and thus provides a potential target for the treatment of infections caused by pathogenic members of the Rickettsiales.