Functional Characterization of the Pneumocystis jirovecii Potential Drug Targets dhfs and abz2 Involved in Folate Biosynthesis

Abstract

cPneumocystis species are fungal parasites colonizing mammal lungs with strict host specificity. Pneumocystis jirovecii is the human-specific species and can turn into an opportunistic pathogen causing severe pneumonia in immunocompromised individuals. This disease is currently the second most frequent life-threatening invasive fungal infection worldwide. The most efficient drug, cotrimoxazole, presents serious side effects, and resistance to this drug is emerging. The search for new targets for the development of new drugs is thus of utmost importance. The recent release of the P. jirovecii genome sequence opens a new era for this task. It can now be carried out on the actual targets to be inhibited instead of on those of the relatively distant model Pneumocystis carinii, the species infecting rats. We focused on the folic acid biosynthesis pathway because (i) it is widely used for efficient therapeutic intervention, and (ii) it involves several enzymes that are essential for the pathogen and have no human counterparts. In this study, we report the identification of two such potential targets within the genome of P. jirovecii, the dihydrofolate synthase (dhfs) and the aminodeoxychorismate lyase (abz2). The function of these enzymes was demonstrated by the rescue of the null allele of the orthologous gene of Saccharomyces cerevisiae. P neumocystis organisms are extracellular fungi that colonize the lungs of mammals (1, 2). Each species displays strict host specificity for a given mammalian species. These fungi are thought to be obligate biotrophic parasites whose evolution has been marked by gene losses (3-7). Pneumocystis jirovecii is the human-specific species whose reservoir would be only humans (8). P. jirovecii can turn into an opportunistic pathogen that causes severe pneumonia in immunocompromised individuals (Pneumocystis jirovecii pneumonia [PCP]). This disease is currently the second most frequent life-threatening invasive fungal infection worldwide, with Ͼ400,000 cases annually (9).The current drug of choice for prophylaxis and treatment of PCP is cotrimoxazole, a combination of sulfamethoxazole and trimethoprim. The two latter drugs are inhibitors of dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR), respectively. These two enzymes are involved in the biosynthesis of folic acid, a metabolite that is required for the biosynthesis of crucial cellular components. Organisms such as Pneumocystis and other lower eukaryotes can synthesize their own folic acid, whereas this compound is a vitamin obtained from food for mammals. Experiments in the rat animal model suggested that the antiPneumocystis activity of cotrimoxazole might be mainly due to the sulfamethoxazole (10). The widespread use of cotrimoxazole for the prevention of PCP since 1989 has been correlated with an increase in the prevalence of specific mutations within the putative active site of DHPS, similar to those observed in other pathogens resistant to cotrimoxazole. These mutations were associated with a breakthrough of prophy...

Identification and Functional Ascertainment of the Pneumocystis jirovecii Potential Drug Targets Gsc1 and Kre6 Involved in Glucan Synthesis

“…; Luraschi et al. ). Partial complementation may result from a poor fitness of the enzyme in the heterologous cellular environment.…”

Lausannevirus Encodes a Functional Dihydrofolate Reductase Susceptible to Proguanil

“…This procedure could improve the understanding of the functions of specific genes belonging to the poorly characterized family Marseilleviridae. Yeast complementation assays have been widely used to characterize fungal and protozoal enzymes involved in the folate pathway (12, 16, 17). Hume et al (18) demonstrated by yeast complementation that the UL97 protein of human cytomegalovirus (HCMV) is a functional ortholog of cellular cyclin–cyclin-dependent kinase (CDK) complexes.…”

Site-Directed Mutagenesis of the 1,3-β-Glucan Synthase Catalytic Subunit of Pneumocystis jirovecii and Susceptibility Assays Suggest Its Sensitivity to Caspofungin

“…To perform a control of sensitivity of our heterologous expression model, the GSC1 genes of C. albicans (GenBank accession number D88815) and C. parapsilosis (European Nucleotide Archive accession number EU221325) were amplified by PCR from yeast genomic DNA extracted as described previously (46). The detailed procedures for PCR amplification using the proofreading high-fidelity Expand polymerase (Roche Diagnostics) and cloning were described previously (47). Their sizes are, respectively, 5,694 and 5,730 bp.…”