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Functional and Expression Analyses of the Pneumocystis MAT Genes Suggest Obligate Sexuality Through Primary Homothallism Within Host Lungs

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Functional and Expression Analyses of the Pneumocystis MAT Genes Suggest Obligate Sexuality Through Primary Homothallism Within Host Lungs

S Richard et al. mBio.

Abstract

Fungi of the genus Pneumocystis are obligate parasites that colonize mammals' lungs and are host species specific. Pneumocystis jirovecii and Pneumocystis carinii infect, respectively, humans and rats. They can turn into opportunistic pathogens in immunosuppressed hosts, causing severe pneumonia. Their cell cycle is poorly known, mainly because of the absence of an established method of culture in vitro It is thought to include both asexual and sexual phases. Comparative genomic analysis suggested that their mode of sexual reproduction is primary homothallism involving a single mating type (MAT) locus encompassing plus and minus genes (matMc, matMi, and matPi; Almeida et al., mBio 6:e02250-14, 2015). Thus, each strain would be capable of sexual reproduction alone (self-fertility). However, this is a working hypothesis derived from computational analyses that is, in addition, based on the genome sequences of single isolates. Here, we tested this hypothesis in the wet laboratory. The function of the P. jirovecii and P. carinii matMc genes was ascertained by restoration of sporulation in the corresponding mutant of fission yeast. Using PCR, we found the same single MAT locus in all P. jirovecii isolates and showed that all three MAT genes are often concomitantly expressed during pneumonia. Extensive homology searches did not identify other types of MAT transcription factors in the genomes or cis-acting motifs flanking the MAT locus that could have been involved in MAT switching or silencing. Our observations suggest that Pneumocystis sexuality through primary homothallism is obligate within host lungs to complete the cell cycle, i.e., produce asci necessary for airborne transmission to new hosts.IMPORTANCE Fungi of the genus Pneumocystis colonize the lungs of mammals. In immunosuppressed human hosts, Pneumocystis jirovecii may cause severe pneumonia that can be fatal. This disease is one of the most frequent life-threatening invasive fungal infections in humans. The analysis of the genome sequences of these uncultivable pathogens suggested that their sexual reproduction involves a single partner (self-fertilization). Here, we report laboratory experiments that support this hypothesis. The function of the three genes responsible for sexual differentiation was ascertained by the restoration of sexual reproduction in the corresponding mutant of another fungus. As predicted by self-fertilization, all P. jirovecii isolates harbored the same three genes that were often concomitantly expressed within human lungs during infection. Our observations suggest that the sexuality of these pathogens relies on the self-fertility of each isolate and is obligate within host lungs to complete the cell cycle and allow dissemination of the fungus to new hosts.

Keywords: RT-PCR; complementation; fission yeast; gene expression; heterologous gene expression.

Figures

FIG 1
FIG 1
Complementation of the S. pombe matMc mutant by expression of the P. jirovecii (Pj), P. carinii (Pc), or control S. pombe (Sp) matMc gene on a plasmid. Tester strain P was crossed with various strains by using a plasmid expressing or not expressing a heterologous gene by mixing them in liquid, spotting an aliquot on the mating plate, and incubating it to allow sporulation. Complementation was assessed after 6 days of incubation at 30°C by iodine staining of spore wall starch, as well as by the presence of zygotes containing four spores (asci) upon microscopic observation. Triplicate recombinant strain isolates gave similar results.
FIG 2
FIG 2
Structure of the single MAT locus of P. jirovecii corresponding to a fusion of loci P and M. The approximate locations of the primers used to confirm the presence of the locus in several isolates are shown. As in S. pombe, the gene encoding the huntingtin-interacting protein (end4) located between the MAT genes might be essential under most conditions, whereas the gene encoding a heat shock protein (hsp104) is probably not. The synteny of these genes, as well those flanking the MAT locus, is fully conserved in P. carinii and P. murina (17).
FIG 3
FIG 3
Amplification of the single MAT locus from three P. jirovecii isolates by long-range PCR. PCR analysis was performed with DNA extracted from BAL fluid samples from 11 patients with PCP; 3 are shown here as examples. The identity of the relatively pure PCR product (10,157 bp) from patient 3 was confirmed by sequencing its ends.
FIG 4
FIG 4
Amplification of the MAT and β-tub mRNAs of three P. jirovecii isolates by RT-PCR. RT-PCR analysis was performed with cDNAs obtained from BAL fluid samples from 11 patients with PCP; 3 are shown here as examples. Random amplification of the cDNA proved to be necessary to obtain PCR products. The PCR products were of the expected sizes shown next to the bands. The positive control was genomic DNA extracted from another BAL fluid sample. The matMc PCR products of patients 2 and 3 were obtained during an experiment other than that during which the matMc PCR product of patient 1 was obtained. The identities of the PCR products were confirmed by sequencing.

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