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Antimicrobial Agents and Chemotherapy, July 2004, p. 2617-2623, Vol. 48, No. 7
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.7.2617-2623.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Dihydropteroate Synthase Mutations in Pneumocystis jiroveci Can Affect Sulfamethoxazole Resistance in a Saccharomyces cerevisiae Model

Peter Iliades,^1* Steven R. Meshnick,² and Ian G. Macreadie¹

CSIRO Health Sciences and Nutrition, Parkville, Victoria, Australia,¹ Department of Epidemiology, University of North Carolina, Chapel Hill, North Carolina²

Received 1 September 2003/ Returned for modification 6 October 2003/ Accepted 31 March 2004

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, T₅₉₇A and P₅₉₉S, 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 T₅₉₇ and P₅₉₉ 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.

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* Corresponding author. Mailing address: CSIRO Health Sciences and Nutrition, 343 Royal Parade, Parkville, Victoria 3052, Australia. Phone: (613) 9662 7259. Fax: (613) 9662 7266. E-mail: peter.iliades{at}csiro.au.

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Antimicrobial Agents and Chemotherapy, July 2004, p. 2617-2623, Vol. 48, No. 7
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.7.2617-2623.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Iliades, P., Meshnick, S. R., Macreadie, I. G. (2005). Mutations in the Pneumocystis jirovecii DHPS Gene Confer Cross-Resistance to Sulfa Drugs. Antimicrob. Agents Chemother. 49: 741-748 [Abstract] [Full Text]