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. 2015 Apr 10;59(5):2560–2566. doi: 10.1128/AAC.05092-14

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

A Luraschi a, O H Cissé a,b,*, M Monod c, M Pagni b, P M Hauser a,✉
PMCID: PMC4394821  PMID: 25691634

Abstract

Pneumocystis 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.

INTRODUCTION

Pneumocystis 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 >400,000 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 anti-Pneumocystis 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 prophylaxis for PCP (11–13). The impact of these mutations on PCP treatment remains controversial, but a strong effect seems unlikely because it would have been detected even in studies with small cohorts (14). However, isolates resistant to the high doses of cotrimoxazole used for treatment may emerge in the future. Cotrimoxazole presents the disadvantage that it is associated with adverse effects in patients, such as intolerance and toxicity. Because of these drawbacks of the most efficient drug available, the development of new drugs against P. jirovecii is of utmost importance.

Although P. jirovecii is an important cause of mortality in immunocompromised patients, there is still no in vitro long-term culture method available for this pathogen. A novel system of coculture on human pseudostratified airway epithelial cells was recently described (15), but it remains to be widely established. The lack of a culture method complicates the identification of new drug targets in P. jirovecii. The strategy used so far has been to identify potential drug targets in the genome of Pneumocystis carinii, the species infecting rats, which was used as model (16–20). The existing antifungal agents and their targets in P. carinii have been reviewed (21). The function of the potential targets was then characterized by complementation of the deletion mutant of the orthologous gene in the model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. This strategy proved useful but presents the drawback that P. carinii is relatively distant from P. jirovecii, with a mean divergence at the nucleotide level of ∼20% (22). Although active sites are generally more conserved than the rest of the proteins, which may ensure development of drugs across species, the drug sensitivities of the targets may vary between the two species. However, the recent release of the P. jirovecii genome sequence opens a new era in the search for new drug targets against this pathogen (23). Indeed, it offers the opportunity to identify the actual targets to inhibit within the P. jirovecii genome rather than those of the model P. carinii.

Therapeutic intervention inhibiting the biosynthesis of folic acid is used successfully against a number of human pathogens. Seven enzymes with activities involved in this pathway are ideal drug targets for antimicrobial therapy because (i) they are essential for the life of the pathogen, and (ii) they have no mammalian ortholog, which favors drug specificity and thus reduction of secondary effects in patients. These enzymes are GTP cyclohydrolase (GTP-CH), dihydroneopterin aldolase (DHNA), dihydropterin pyrophosphokinase (HPPK), DHPS, dihydrofolate synthase (DHFS), para-aminobenzoate synthase (ABZ1), and aminodeoxychorismate lyase (ABZ2) (Fig. 1). Only two enzymes have been targeted in this pathway so far: DHPS and DHFR, which is an eighth enzyme of the pathway with a human ortholog. GTP-CH may not be a good candidate because it includes a pterin binding site, which is very well conserved across all living species (24). The other five enzymes remain to be evaluated as drug targets. The DHNA, HPPK, and DHPS activities are encoded by a single trifunctional enzyme in fungi so that their study is complicated. On the other hand, DHFS, ABZ1, and ABZ2 are single enzymes.

FIG 1.

FIG 1

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Folate biosynthesis and utilization pathway (modified from reference 20).

In the present study, we reported the identification of the dhfs and abz2 genes encoding DHFS and ABZ2 within the P. jirovecii genome sequence, as well as the assessment of their function by the successful complementation of the deleted orthologous gene of S. cerevisiae.

MATERIALS AND METHODS

Strains and growth conditions.

LCY1 is an S. cerevisiae haploid strain that has a disruption of the FOL3 gene, which encodes Dhfs protein (MATa leu2-3,112 trp1 tup1 ura3-52 FOL3::URA3) (25). This strain is herein named the dhfs deletant. In the absence of folate synthesis, this strain requires methionine, adenine, histidine, and TMP. It was grown on complete yeast extract-peptone-dextrose (YEPD) medium (1% [wt/vol] Difco yeast extract, 2% Difco peptone, 2% glucose) supplemented with TMP (100 μg/ml) at 30°C.

Y00875 is an S. cerevisiae haploid strain with a deletion of the ABZ2 gene that encodes Abz2 protein (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 YMR289w::kanMX4). This strain is herein named the abz2 deletant. It was obtained from Euroscarf (European Saccharomyces cerevisiae Archive for Functional Analysis [http://web.uni-frankfurt.de/fb15/mikro/euroscarf]). The deletion of ABZ2 induces a para-aminobenzoate (PABA) auxotrophy (26). The parental strain of the abz2 deletant, strain BY4741 from Euroscarf (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0), was used as the control in the complementation tests.

Escherichia coli DH5α (Life Technologies, Basel, Switzerland) was used for gene cloning. Cells were made competent using the method of Chung and Miller (27), stored at −80°C, and transformed for resistance to 50 μg/ml ampicillin on solid LB medium (1% [wt/vol] Difco tryptone, 0.5% Difco yeast extract, 1% NaCl, 2% Gibco agar).

Source of P. jirovecii gene sequences.

The P. carinii Dhfs protein (NCBI accession number DQ128176; 20) or the S. cerevisiae Abz2 protein (NP_014016) was used as the query sequence in a BLASTp search against the P. jirovecii proteome at http://blast.ncbi.nlm.nih.gov/Blast.cgi. The P. jirovecii gene sequences encoding the proteins identified were then retrieved from the European Nucleotide Archive (http://www.ebi.ac.uk/ena) (28). The dhfs and abz2 genes correspond, respectively, to PNEJI1_000945 and PNEJI1_000496 loci in the P. jirovecii genome assembly version ASM33397v2 published previously (23). Protein multiple sequence alignments were generated using T-Coffee (29).

Cloning of P. jirovecii genes.

Since no introns are present in the P. jirovecii dhfs gene, this 1,269-bp gene was amplified by PCR directly from DNA extracted from a bronchoalveolar lavage fluid sample (BAL fluid) of a patient with PCP using the QIAamp DNA blood kit (Qiagen). PCR was carried out using the proofreading high-fidelity expand polymerase (Roche Diagnostics), a final concentration of 3 mM MgCl2, and primers 5′-GCG GGG GAT CCA TGT CGC TAA GAC TAG GTT TAT C-3′ and 5′-CCC CCC CGT CGA CTT ATA TTA TTT TTT TAT CAA AAC-3′. These primers created unique BamHI and SalI restriction sites in the PCR product (restriction sites are underlined in primers). Primers were synthesized by Microsynth (Balgach, Switzerland). The PCR program included an initial denaturation for 3 min at 94°C, followed by 35 cycles consisting of 30 s at 94°C, 30 s at 52°C, and 90 s at 72°C. The reaction ended with a 10-min extension at 72°C. The PCR product was extracted using the QIAquick gel extraction kit (Qiagen), digested with BamHI and SalI restriction enzymes, and then ligated using T4 ligase (New England BioLabs) into the p414GPD expression vector (30) previously digested with the same two restriction enzymes. After ligation, the plasmids were introduced into E. coli DH5α-competent cells. Minipreparation of plasmid DNA was carried out according to Birnboim and Doly (31).

The P. jirovecii abz2 gene without its two introns was 750 bp and was synthesized by GeneCust Europe (Dudelange, Luxembourg). It was cloned into p416GPD (27), as described above for the dhfs gene.

Transformation of S. cerevisiae deletants.

Recombinant plasmids p414GPD.Pjdhfs and p416GPD.Pjabz2 were introduced into their corresponding S. cerevisiae deletant by transformation for tryptophan or uracil prototrophy, respectively. Yeast transformations utilized the one-step method described by Chen et al. (32). Transformants were selected on solid yeast nitrogen base (YNB) medium (0.67% [wt/vol] yeast nitrogen base, 2% glucose, 2% Gibco agar) supplemented with complete supplement mixture (CSM) lacking tryptophan or uracil (MP Biomedicals). Four randomly chosen isolated colonies of transformants were purified by streaking and growth on the same selective medium.

Complementation tests.

Functional complementation of the S. cerevisiae dhfs deletant with the P. jirovecii dhfs gene was assessed by growth on YEPD medium lacking TMP. As further validation of functional complementation, the presence or absence of the P. jirovecii dhfs gene in the different strains was confirmed by PCR. The PCR conditions described above were used. S. cerevisiae genomic DNA was extracted as described previously (33).

Functional complementation of the S. cerevisiae abz2 deletant with the P. jirovecii abz2 gene was assessed by the growth rate at 30°C in YNB medium lacking PABA and folic acid that was supplemented with CSM. Overnight cultures were diluted at an absorbance at 540 nm of 0.1 (∼1.5 × 106 cells/ml), and growth was determined by optical density at 540 nm. In order to express its auxotrophy phenotype, the deletant was subcultured twice overnight in the medium lacking PABA and folic acid before the experiment. To confirm the presence or absence of the P. jirovecii abz2 gene, primers 5′-GCG ATG AAA AAA ACA GAA AAG C-3′ and 5′-CCC CTA TTC GAA GAA TGC CTG-3′ were used to amplify the complete gene (GCG or CCC were added at the 5′ end of the primers before the start and stop codons of the open reading frame [ORF] in order to obtain similar melting temperatures). The PCR conditions were as described above for the dhfs gene, except that the final concentration of MgCl2 was 4.5 mM, the temperature of hybridization was 58°C, and the elongation time was 1 min at 72°C.

Assessment of the extracted DNAs was done by amplification of the unrelated S. cerevisiae BRL1 gene encoding an essential nuclear membrane protein (18). The primers used were 5′-GAA ACT CTT GGT ACA GAG G-3′ and 5′-TGA TCT GTC CCA GTT GTG-3′. The PCR conditions were as described above for the P. jirovecii dhfs gene, except that the temperature of hybridization was 52°C and the elongation time was 2 min at 72°C. The PCR product was 2,008 bp.

RESULTS

Identification and cloning of the P. jirovecii dhfs gene.

The Dhfs protein was identified within the P. jirovecii proteome by a homology search using the Dhfs protein of P. carinii as the query sequence. The gene encompasses no introns. The translation product of the ORF bears the highest degree of amino acid similarity with the Dhfs protein of P. carinii (72%) and a lower degree of amino acid similarity with those of S. cerevisiae (36%) and S. pombe (40%) (Fig. 2A). Because of the absence of introns, the P. jirovecii dhfs gene was directly amplified by PCR from the genomic DNA extracted from a BAL fluid sample from a patient with PCP and cloned into the expression vector p414GPD.

FIG 2.

FIG 2

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Multiple-sequence alignment of Dhfs (A) and Abz2 (B) proteins. T-Coffee was used (29). The identical and strongly and weakly conserved residues are indicated by asterisks, double points, and single points, respectively. Dashes indicate gaps. (A) Alignment of Dhfs proteins of P. jirovecii (locus tag PNEJI1_000945), P. carinii (GenBank accession number DQ128176), S. cerevisiae (NP_013831), and S. pombe (NM_001018363.2). Also shown is the P loop (phosphate binding), the Ω loop (involved in the folate binding site), and the linker that connects the N- and C-terminal domains. (B) Alignment of Abz2 proteins of P. jirovecii (locus tag PNEJI1_000496), S. cerevisiae (NP_014016.1), and S. pombe (NM_001021876.2). Also shown is the pyridoxal (Py) binding site located at the interface of the N-terminal and C-terminal domains of the enzyme, which is a hallmark of an aminotransferase-like enzyme. Conserved residues of both domains that form the active site are underlined.

Functional complementation of the S. cerevisiae dhfs deletant with the P. jirovecii dhfs gene.

The recombinant plasmid p414GPD.Pjdhfs and the empty p414GPD vector were introduced into the S. cerevisiae dhfs deletant. Transformant isolates were then grown on rich medium supplemented with or without TMP. Growth occurred on the medium lacking TMP only in the presence of p414GPD.Pjdhfs but not in the presence of p414GPD (Fig. 3). This proved that expression of the P. jirovecii gene rescued the function of the deleted FOL3 gene encoding the Dhfs protein. However, the growth rate of the rescued deletant proved to be lower than that of the wild-type strain (results not shown; notably, the deletant rescued with the P. carinii Dhfs protein constructed in reference 20 also showed a similar reduced growth rate). The presence or absence of the P. jirovecii dhfs gene in the different strains was assessed by PCR analysis. As expected, P. jirovecii dhfs was present in the functionally complemented strains but not in the deletant (Fig. 4A). To confirm that the DNA from which the P. jirovecii dhfs gene could not be amplified was valid, the unrelated S. cerevisiae gene BRL1 was amplified (Fig. 4A).

FIG 3.

FIG 3

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Complementation of the S. cerevisiae dhfs deletant by expression of P. jirovecii dhfs gene on plasmid. Four single colonies were isolated from the original transformation petri dish, purified by streaking on the same selection medium, and grown on rich YEPD medium with TMP (A) or without TMP (B) for 3 days at 30°C. Number 1 corresponds to the control strain bearing the empty p414GPD vector. Numbers 2 to 5 correspond to the four isolates bearing p414GPD.Pjdhfs.

FIG 4.

FIG 4

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PCR assessment of the presence or absence of the P. jirovecii dhfs and abz2 genes. (A) The presence of the P. jirovecii dhfs gene (PCR product of 1,293 bp) was confirmed in the DNA from the BAL fluid of a patient with PCP (lane 1) and in one isolate of S. cerevisiae dhfs deletant bearing p414GPD.Pjdhfs (lane 3), whereas the gene was absent in the dhfs deletant without plasmid (lane 2). As a control, the unrelated S. cerevisiae BRL1 gene was amplified (PCR product of 2,008 bp) from the dhfs deletant bearing p414GPD.Pjdhfs (lane 5) or without plasmid (lane 4). (B) The presence of the P. jirovecii abz2 gene was confirmed in the DNA from the BAL fluid of a patient with PCP (lane 1; PCR product with introns of 829 bp) and in one isolate of S. cerevisiae abz2 deletant bearing p416GPD.Pjabz2 (lane 3; PCR product without introns of 756 bp), whereas the gene was absent in the DNA of the abz2 deletant (lane 2). The unrelated S. cerevisiae BRL1 gene was amplified from the abz2 deletant bearing p416GPD.Pjabz2 (lane 5) or without plasmid (lane 4).

Identification and cloning of the P. jirovecii abz2 gene.

The P. jirovecii abz2 gene was retrieved as described above for the dhfs gene, except that the S. cerevisiae Abz2 protein was used as the initial query sequence. The gene encompasses two introns. The translation product of the ORF bears the highest degree of similarity with the Abz2 protein of S. pombe (33%) and a lower degree of similarity with that of S. cerevisiae (20%) (Fig. 2A). We identified only a truncated P. carinii abz2 gene (locus PNECA1_004600), possibly because of the known incompleteness of the genome sequence; this truncated gene was 240 bp long, and its translation product consistently shared 59% identity with the corresponding region of the P. jirovecii abz2 gene. Because S. cerevisiae does not process Pneumocystis introns, a synthetic P. jirovecii abz2 gene without introns was cloned into p416GPD.

Functional complementation of the S. cerevisiae abz2 deletant with the P. jirovecii abz2 gene.

The recombinant plasmid p416GPD.Pjabz2 and the empty p416GPD vector were introduced into the S. cerevisiae abz2 deletant. Transformant isolates, the parental wild-type strain of the abz2 deletant, and the abz2 deletant were grown in minimal medium lacking PABA and folic acid. A growth rate similar to that of the parental wild-type strain was observed in the presence of p416GPD.Pjabz2 but not in the presence of p416GPD (Fig. 5). This proved that the P. jirovecii gene rescued the function of the deleted ABZ2 gene. The presence or absence of the P. jirovecii abz2 gene in the different strains was assessed by PCR analysis. As expected, the P. jirovecii abz2 was present in the functionally complemented strains but not in the deletant (Fig. 4B). To confirm that the DNA from which the P. jirovecii abz2 could not be amplified was valid, the unrelated S. cerevisiae gene BRL1 was amplified (Fig. 4B).

FIG 5.

FIG 5

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Complementation of the S. cerevisiae abz2 deletant by expression of P. jirovecii abz2 gene on plasmid. Strains were grown overnight in YNB medium lacking PABA and folic acid that was supplemented with CSM. The cultures were diluted in the same medium at an optical density of 0.1 (time zero) and incubated at 30°C. The optical density at time zero was normalized at 0.1 for each strain. Standard deviations of triplicate optical density measurements were small (<0.005). The four complemented isolates were analyzed and had similar results; one representative experiment of one complemented isolate is shown. The labels of the curves are explicated on the top left side of the figure.

DISCUSSION

Because of the emergence of drug resistance in P. jirovecii toward the most efficient drug available and because of the side effects of this drug, the development of new drugs against this fungal pathogen is crucial. Publication of the P. jirovecii genome sequence opens a new era in the search for potential new drug targets because the actual genes to inhibit, rather than those of models, can be studied. Most enzymes involved in the biosynthesis of folic acid are ideal drug targets because of their essentiality and absence in humans. Accordingly, there are many drugs inhibiting this pathway that are currently used against many human pathogens. We focused on two enzymes involved in this pathway that were poorly investigated so far, i.e., DHFS (dihydrofolate synthase) and ABZ2 (deoxychorismate lyase). In this study, we identified the two P. jirovecii genes encoding these enzymes and demonstrated their function by their ability to rescue the null allele of their respective S. cerevisiae orthologous gene. These are steps required in the search of new targets. The P. jirovecii enzymes identified bear a higher homology with the S. pombe orthologs than with those of S. cerevisiae. This is consistent with the fact that P. jirovecii and S. pombe, but not S. cerevisiae, are members of the Taphrinomycotina subphylum.

The DHFS enzyme carries out the final step of folic acid biosynthesis, namely, the addition of a glutamate to dihydropteroate to make folic acid (i.e., dihydrofolate; Fig. 1). The DHFS enzyme shares a high degree of similarity with the enzyme folypolyglutamate synthase (FPGS), which stabilizes folic acid by the addition of several glutamates (Fig. 1). The specificity of these two enzymes for the addition of single or multiple glutamates is noteworthy. Humans have only FPGS that has no DHFS activity (34), while S. cerevisiae and other fungi have both FPGS and DHFS activities encoded by two different genes (35). Other organisms, such as E. coli and Plasmodium falciparum, have only one gene that encodes a single bifunctional polypeptide enzyme (36). The molecular basis for the mono- versus bifunctional activity remains to be elucidated. DHFS enzymes act in the cytoplasm of eukaryotes and do not include an N-terminal targeting signal sequence in order to be transferred into other cell compartments. On the other hand, eukaryotic FPGS enzymes are working in the cytoplasm and in the mitochondrion and possess a mitochondrial targeting signal sequence. Comparison of P. carinii DHFS to S. cerevisiae DHFS and FPGS showed that P. carinii DHFS is devoid of a mitochondrial targeting signal sequence and thus probably has no FPGS activity (20). The P. jirovecii DHFS isolated in the present study is close to that of P. carinii without a supplementary N-terminal sequence (72% identity; Fig. 2A), strongly suggesting that it also has no FPGS activity. Consistently, the P. carinii and P. jirovecii DHFSs share more identity with the S. cerevisiae DHFS (35% and 36%, respectively) than with the S. cerevisiae FPGS (20% and 19%, respectively). The essentiality of its activity in many organisms together with its absence in humans suggests that DHFS is a good candidate drug target against P. jirovecii.

The ABZ2 encoded by the abz2 gene is required for the biosynthesis of PABA, which in turn is necessary to produce folic acid (Fig. 1). The S. cerevisiae abz2 deletant has a reduced growth rate in minimal medium lacking PABA and folate (Fig. 5) (26). This is probably due to a cellular pool of PABA sufficient to allow survival for several generations. Subculturing the abz2 deletant in the absence of PABA leads to exhaustion of this pool of PABA, allowing the expression of PABA auxotrophy (26). Although an external source of PABA is possible by scavenging from the host, the product of the P. jirovecii abz2 gene might be required to allow survival of the pathogen during infection, rendering this gene a potential new drug target. This is plausible because antifolate drugs are effective against P. falciparum despite this pathogen's ability to scavenge folic acid from its human host (37, 38). This is also supported by the fact that the PABA synthase of Aspergillus fumigatus is essential for pathogenicity (39).

In conclusion, we characterized two new potential drug targets in P. jirovecii that deserve future investigation. They can be involved in a strategy taking advantage of the synergism provided by combination therapy, a strategy that is widely and successfully used against important human pathogens.

ACKNOWLEDGMENTS

This work was supported by Swiss National Science Foundation grant 310030-146135 to P.M.H. and M.P. O.H.C. is supported by Swiss National Science Foundation fellowship grant 151780.

We thank Sophie Chevalley for excellent technical assistance.

The present work was submitted by A. Luraschi as fulfilment for a master's degree at the University of Lausanne.

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