Cloning of the Pneumocystis jirovecii trifunctional FAS gene and complementation of its DHPS activity in Escherichia coli

Iliades, Peter; Walker, Daniel J.; Castelli, Laura A.; Satchell, Jacqueline; Meshnick, Steven R.; Macreadie, Ian

doi:10.1016/j.fgb.2004.08.006

Cited by 12 publications

(23 citation statements)

References 43 publications

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“…In order to assess whether the mutations that conferred SMX resistance (as previously demonstrated [15]) also conferred cross-resistance to other sulfa drugs, the growth inhibition of four mutant clones (VRS, ARS, TRS, and ARP) by 15 sulfa drugs was evaluated by using an optimized agar drug diffusion assay described previously (13). The growth inhibition was analyzed as described in Materials and Methods.…”

Section: Resultsmentioning

confidence: 99%

“…However, it was not surprising that sulfa drug resistance results do not always translate from agar to liquid media. A case in point is the sulfamethoxazole resistance of the PjFAS mutants determined previously (15) by using the validated Etest methodology. The MIC of 1 to 3 g/ml determined in that study does not correlate to the MIC determined with liquid medium, which was in the order of 600 g/ml.…”

Section: Resultsmentioning

confidence: 99%

“…Sulfa drugs are inhibitors of folic acid synthesis (FAS). In fungi, including Candida albicans, Saccharomyces cerevisiae, and P. jirovecii, the FAS genes are part of a single open reading frame that encodes a trifunctional, multidomain enzyme that includes dihydroneopterin aldolase, hydroxymethyldihydropterin pyrophosphokinase, and dihydropteroate synthase (DHPS) (15,34). The FAS genes, including dihydrofolate synthase, are essential to prokaryotes and lower eukaryotes since they are dependent on de novo folate biosynthesis and do not possess the capability to actively sequester exogenous folate.…”

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confidence: 99%

“…Recently, cloning of the trifunctional PjFAS genes and their heterologous complementation in a DHPS-disrupted E. coli host strain was achieved (15). This provided an assay method that permitted a direct assessment of sulfa drug resistance conferred by mutations observed clinically (T 517 A and P 519 S) in the PjFAS genes.…”

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confidence: 99%

“…(i) pGEX.PjFAS and pET28a.PjFAS. PjFAS expression constructs in pGEX 4T-2 (glutathione S-transferase tagged) or in pET28a (six-His tagged) have been described previously (15). In this study, we employed a pET28a.PjFAS mutant construct (M 596 T) that permitted heterologous complementation in E. coli C600⌬folP::Km r and was identified following random chemical mutagenesis with ethane methyl sulfonate.…”

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Mutations in the Pneumocystis jirovecii DHPS Gene Confer Cross-Resistance to Sulfa Drugs

Iliades

Meshnick

Macreadie

2005

Antimicrob Agents Chemother

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Pneumocystis jirovecii is a major opportunistic pathogen that causes Pneumocystis pneumonia (PCP) and results in a high degree of mortality in immunocompromised individuals. The drug of choice for PCP is typically sulfamethoxazole (SMX) or dapsone in conjunction with trimethoprim. Drug treatment failure and sulfa drug resistance have been implicated epidemiologically with point mutations in dihydropteroate synthase (DHPS) of P. jirovecii. P. jirovecii cannot be cultured in vitro; however, heterologous complementation of the P. jirovecii trifunctional folic acid synthesis (PjFAS) genes with an E. coli DHPS-disrupted strain was recently achieved. This enabled the evaluation of SMX resistance conferred by DHPS mutations. In this study, we sought to determine whether DHPS mutations conferred sulfa drug cross-resistance to 15 commonly available sulfa drugs. It was established that the presence of amino acid substitutions (T 517 A or P 519 S) in the DHPS domain of PjFAS led to cross-resistance against most sulfa drugs evaluated. The presence of both mutations led to increased sulfa drug resistance, suggesting cooperativity and the incremental evolution of sulfa drug resistance. Two sulfa drugs (sulfachloropyridazine [SCP] and sulfamethoxypyridazine [SMP]) that had a higher inhibitory potential than SMX were identified. In addition, SCP, SMP, and sulfadiazine (SDZ) were found to be capable of inhibiting the clinically observed drug-resistant mutants. We propose that SCP, SMP, and SDZ should be considered for clinical evaluation against PCP or for future development of novel sulfa drug compounds.Pneumocystis jirovecii is a major opportunistic pathogen that results in Pneumocystis pneumonia (PCP) of AIDS patients and immunocompromised individuals. It accounts for 40% of all AIDS-defining conditions and is the major cause of mortality of children with AIDS in Africa (2, 16).Clinically, PCP has been treated with antifolates including combination therapy with sulfamethoxazole (SMX) and trimethoprim (TM) as the preferred first-line treatment (19). Sulfa drugs are inhibitors of folic acid synthesis (FAS). In fungi, including Candida albicans, Saccharomyces cerevisiae, and P. jirovecii, the FAS genes are part of a single open reading frame that encodes a trifunctional, multidomain enzyme that includes dihydroneopterin aldolase, hydroxymethyldihydropterin pyrophosphokinase, and dihydropteroate synthase (DHPS) (15, 34). The FAS genes, including dihydrofolate synthase, are essential to prokaryotes and lower eukaryotes since they are dependent on de novo folate biosynthesis and do not possess the capability to actively sequester exogenous folate. Sulfa drugs such as SMX are competitive inhibitors of DHPS and work synergistically with TM, which inhibits microbial dihydrofolate reductase. In P. jirovecii, however, there is some evidence to suggest that TM is ineffective and that such treatment is actually sulfamethoxazole monotherapy (26, 35). Drug treatment failure of SMX-TM has been associated with point mutations in the P. jirove...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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