Dihydropteroate synthase mutations in Pneumocystis jiroveci can affect sulfamethoxazole resistance in a Saccharomyces cerevisiae model

Abstract

Dihydropteroate synthase (DHPS) mutations in Pneumocystis jiroveci have been associated epidemiologically with resistance to sulfamethoxazole (SMX). Since P. jiroveci cannot be cultured, inherent drug resistance cannot be measured. This study explores the effects of these mutations in a tractable model organism, Saccharomyces cerevisiae. Based on the sequence conservation between the DHPS enzymes of P. jiroveci and S. cerevisiae, together with the structural conservation of the three known DHPS structures, DHPS substitutions commonly observed in P. jiroveci were reverse engineered into the S. cerevisiae DHPS. Those mutations, T(597)A and P(599)S, can occur singly but are most commonly found together and are associated with SMX treatment failure. Mutations encoding the corresponding changes in the S. cerevisiae dhps were made in a yeast centromere vector, p414FYC, which encodes the native yeast DHPS as part of a trifunctional protein that also includes the two enzymes upstream of DHPS in the folic acid synthesis pathway, dihydroneopterin aldolase and 2-amino-4-hydroxymethyl dihydropteridine pyrophosphokinase. A yeast strain with dhps deleted was employed as the host strain, and transformants having DHPS activity were recovered. Mutants having both T(597) and P(599) substitutions had a requirement for p-aminobenzoic acid (PABA), consistent with resistance being associated with altered substrate binding. These mutants could be adapted for growth in the absence of PABA, which coincided with increased sulfa drug resistance. Upregulated PABA synthesis was thus implicated as a mechanism for sulfa drug resistance for mutants having two DHPS substitutions.

FIG. 1.

Alignment of DHPS sequences by using ClustalW. Displayed are sequences from E. coli (Ec), Mycobacterium tuberculosis (Mt), and Staphylococcus aureus (Sa), whose structures have been solved, aligned to those of S. cerevisiae (Sc) and P. jiroveci (Pj). The asterisks indicate totally conserved amino acids, the colons indicate very high conservation, and the dots indicate weak conservation. Amino acids associated with SMX resistance in P. jiroveci DHPS (T→A and P→S) are highlighted and in boldface. Dashes represent gaps introduced by the alignment.

FIG. 2.

Growth of transformants on minimal medium supplemented with various levels of PABA.

FIG. 3.

Determination of SMX resistance by agar drug diffusion assay. SMX was solubilized in DMSO (100 mg/ml), and 25 μl was applied to the centers of agar plates. Additional plates were supplemented with increasing concentrations of PABA. Transformants were harvested in log-phase growth, washed in PBS, and normalized to 6 × 10⁶ CFU/ml. Each strain was then streaked radially from the center of the plate. The plates were grown for 3 to 5 days, and the zones of inhibition for each isolate, TRP, VRS, ARS, TRS, and ARP, were then scored. The results are averages of triplicate experiments; error bars represent the standard deviations of the means.

FIG. 4.

Generation time of adapted transformants supplemented with increasing concentrations of SMX in MM₂₀₀. The results are averages of four growth experiments.

FIG. 5.

MICs for naïve transformants growing in MM₀ supplemented with increasing concentrations of SMX and PABA. The results are averages of three experiments. Note that the higher seeding density for this experiment permitted the growth of all naïve transformants.