Expression and Immunostaining Analyses Suggest that Pneumocystis Primary Homothallism Involves Trophic Cells Displaying Both Plus and Minus Pheromone Receptors.

The genus Pneumocystis encompasses fungal species that colonize mammals' lungs with host specificity. Should the host immune system weaken, the fungal species can cause severe pneumonia. The life cycle of these pathogens is poorly known, mainly because an in vitro culture method has not been established. Both asexual and sexual cycles would occur. Trophic cells, the predominant forms during infection, could multiply asexually but also enter into a sexual cycle. Comparative genomics revealed a single mating type locus, including plus and minus genes, suggesting that primary homothallism involving self-fertility of each strain is the mode of reproduction of Pneumocystis species. We identified and analyzed the expression of the mam2 and map3 genes encoding the receptors for plus and minus pheromones using reverse transcriptase PCR, in both infected mice and bronchoalveolar lavage fluid samples from patients with Pneumocystis pneumonia. Both receptors were most often concomitantly expressed during infection, revealing that both pheromone-receptor systems are involved in the sexual cycle. The map3 transcripts were subject to alternative splicing. Using immunostaining, we investigated the presence of the pheromone receptors at the surfaces of Pneumocystis cells from a patient. The staining tools were first assessed in Saccharomyces cerevisiae displaying the Pneumocystis receptors at their cellular surface. Both receptors were present at the surfaces of the vast majority of the cells that were likely trophic forms. The receptors might have a role in mate recognition and/or postfertilization events. Their presence at the cell surface might facilitate outbreeding versus inbreeding of self-fertile strains.IMPORTANCE The fungi belonging to the genus Pneumocystis may cause severe pneumonia in immunocompromised humans, a disease that can be fatal if not treated. This disease is nowadays one of the most frequent invasive fungal infections worldwide. Whole-genome sequencing revealed that the sexuality of these fungi involves a single partner that can self-fertilize. Here, we report that two receptors recognizing specifically excreted pheromones are involved in this self-fertility within infected human lungs. Using fluorescent antibodies binding specifically to these receptors, we observed that most often, the fungal cells display both receptors at their surface. These pheromone-receptor systems might play a role in mate recognition and/or postfertilization events. They constitute an integral part of the Pneumocystis obligate sexuality within human lungs, a cycle that is necessary for the dissemination of the fungus to new individuals.

Correction: Development of highly active anti-Pneumocystis bisbenzamidines: insight into the influence of selected substituents on the in vitro activity.

[This corrects the article DOI: 10.1039/C7MD00445A.].

Development of highly active anti-Pneumocystis bisbenzamidines: insight into the influence of selected substituents on the in vitro activity.

Here we describe the potency of 21 pentamidine analogues against the fungal pathogen, Pneumocystis carinii, in an ATP bioluminescent assay with toxicity profiles in 2 mammalian cell lines. Reduction of two 5-methyl-1,2,4-oxadiazole rings was applied to the synthesis of acid-labile bisamidines. Anti-Pneumocystis activity is discussed in the context of 3 groups of compounds depending on the main structural changes of the pentamidine lead structure. The groups include: 1) 1,4-bis(methylene)piperazine derivatives 1-5; 2) alkanediamide derivatives 6-10; 3) alkane-derived bisbenzamidines 11-21. IC50 values of 18 compounds were lower than the IC50 of pentamidine. Four bisamidines were active at nanogram concentrations. Introduction of sulfur atoms in the alkane bridge, replacement of the amidino groups with imidazoline rings, or attachment of nitro or amino groups to the benzene rings is responsible for remarkable activity of the new leading structures. The vast majority of compounds, including four highly active ones, can be classified as mild or nontoxic to host cells. These compounds show promise as candidates for new anti-Pneumocystis agents.

CAY-I, a fungicidal saponin from Capsicum sp. fruit.

Saponins are steroidal or terpenoid-based glycosides with surface active properties. A steroidal saponin, CAY-1, with a molecular weight of 1243.35 Da, was isolated and purified to homogeneity from commercially available dry, ground fruit of Capsicum frutescens. CAY-1 was shown to be a potent fungicide for the germinating conidia of Aspergillus flavus, A. fumigatus, A. parasiticus and A. niger with species-dependent LD90 values between 3 and 20 microM. Activity against some Aspergillus species was affected by the test medium used. In vitro assays, CAY-1 was effective against Pneumocystis carinii (IC50): 9.5 microM) and Candida albicans (IC90: 6.2 microM). CAY-1 had no effect on the viability of the nongerminating conidia of the two filamentous fungi, P. carinii and C. albicans, nor on the conidial type of Fusarium oxysporum. It was ineffective against the bacteria Enterobacter agglomerans, Bacillus subtilis, Escherichia coli and Staphylococcus aureus. CAY-1 was not cytotoxic to A 549 lung carcinoma cells or HeLa cells at effective fungicidal concentrations. The results indicate that CAY-1 is an effective fungicide for Aspergillus species, C. albicans and P. carinii at concentrations below the threshold for mammalian cell toxicity.

Rapid PCR-single-strand conformation polymorphism method to differentiate and estimate relative abundance of Pneumocystis carinii special forms infecting rats.

A rapid method that uses PCR-single-strand conformation polymorphism analysis of the intron of the nuclear 26S rRNA gene was shown to differentiate the two Pneumocystis carinii special forms that infect rats, P. carinii f. sp. carinii and P. carinii f. sp. ratti. The method also provides a means for estimation of the relative abundance of the two special forms in the case of a coinfected rat. The results suggest that the method described will help to further standardize the immunosuppressed rat model of P. carinii infection and, thus, contribute to a better understanding of P. carinii infection in humans.

In vitro and in vivo effects of quinupristin-dalfopristin against Pneumocystis carinii.

Quinupristin-dalfopristin (Q-D), which is active against bacteria and Toxoplasma gondii, was examined for its activity against Pneumocystis carinii. After 72 h of incubation with rat P. carinii in an ATP cytotoxicity assay, the 50% inhibitory concentration of Q-D was 10.6 microg/ml, a level that can be achieved in serum with high-dose administration. Q-D administered intraperitoneally at doses of 50 to 200 mg per kg of body weight per day in the treatment and 100 mg/kg/day three times per week in the prophylaxis of pneumocystosis in immunosuppressed mice reduced the organism burden up to 15- and 302-fold, respectively. We conclude that Q-D has activity against P. carinii in vitro and in vivo.

Anti-human immunodeficiency virus drugs are ineffective against Pneumocystis carinii in vitro and in vivo.

Human immunodeficiency virus (HIV) protease inhibitors (PIs) recently have been reported to be active against Pneumocystis carinii in cell culture. Twelve anti-HIV drugs were analyzed for their effects against rat P. carinii by an ATP cytotoxicity assay. Indinavir and saquinavir exhibited slight anti-P. carinii activity at concentrations above those that can be clinically achieved in serum; other PIs and nucleoside and nonnucleoside reverse-transcriptase inhibitors were inactive against the organism. Anti-HIV drugs, alone or in combination, did not materially reduce the organism count in the treatment of P. carinii pneumonia in immunosuppressed mice. Thus, anti-HIV drugs have little or no activity against P. carinii in these in vitro and in vivo systems. Caution should be used when interpreting reports of the susceptibility of P. carinii to anti-HIV drugs on the basis of in vitro testing only.

Widespread occurrence of Pneumocystis carinii in commercial rat colonies detected using targeted PCR and oral swabs.

The genus Pneumocystis contains a family of fungal organisms that infect a wide variety of mammalian species. Although it is a cause of pneumonia in immunocompromised hosts, recent evidence suggests that these organisms colonize nonimmunosuppressed hosts. Detection of cryptic colonization with Pneumocystis becomes important in animal studies when infection-free animals are necessary. Provocation by chronic immunosuppression, histology, and serology has been widely used to detect the presence of Pneumocystis in rat colonies, requiring lengthy time periods and/or postmortem tissue. We conducted a study to evaluate the use of PCR amplification of oral swabs for the antemortem detection of Pneumocystis in 12 rat groups from three commercial vendors. Sera were collected upon arrival, and the oral cavity was swabbed for PCR analysis. Ten of these groups of rats were then housed in pairs under barrier and immunosuppressed to provoke Pneumocystis growth. Once moribund, the rats were sacrificed, and the lungs were collected to evaluate the presence of Pneumocystis by PCR and microscopic enumeration. DNA was extracted from oral swabs and lung homogenates, and PCR was performed using primers targeting a region within the mitochondrial large-subunit rRNA of Pneumocystis carinii f. sp. carinii. Upon receipt, 64% of rats were positive for P. carinii f. sp. carinii-specific antibodies, while P. carinii f. sp. carinii DNA was amplified from 98% of oral swabs. Postmortem PCR analysis of individual lungs revealed P. carinii f. sp. carinii DNA in all rat lungs, illustrating widespread occurrence of Pneumocystis in commercial rat colonies. Thus, oral swab/PCR is a rapid, nonlethal, and sensitive method for the assessment of Pneumocystis exposure.

The ste3 pheromone receptor gene of Pneumocystis carinii is surrounded by a cluster of signal transduction genes.

Although the clinical aspects of Pneumocystis carinii pneumonia are well characterized, the basic biology of the causative organism is poorly understood. Most proposed life cycles of P. carinii include both asexual and sexual replicative cycles. The two most prominent morphological forms are a trophic form, thought to undergo asexual replication by binary fission, and a cystic form or ascus containing intracystic bodies or ascospores, the products of sexual replication. To facilitate the Pneumocystis genome project, a P. carinii f. sp. carinii genomic cosmid library and an additional lambda cDNA library were generated. A partial expressed sequence tag database, created as part of the genome project, revealed the transcription of meiosis-specific genes and other genes related to sexual reproduction. The ortholog of Ste3, an a-factor pheromone receptor, was cloned and genes surrounding the ste3 locus were examined. Clustered around the ste3 gene are genes encoding elements functional in the pheromone response signal transduction cascade of model fungal organisms. These include the Ste20 protein kinase, the Ste12 homoeodomain transcriptional regulator, a potential pheromone mating factor, and other DNA-binding proteins. The genomic organization of the ste3 locus bears significant similarity to that of the mating locus recently described in Cryptococcus neoformans. The P. carinii genome contains much of the genetic machinery necessary for pheromone responsiveness, and these data support the existence of a sexual replication cycle.

Time between inoculations and karyotype forms of Pneumocystis carinii f. sp. Carinii influence outcome of experimental coinfections in rats.

The prevalence of Pneumocystis carinii pneumonia (PCP) in humans caused by more than a single genotype has been reported to range from 10 to 67%, depending on the method used for detection (3, 19). Most coinfections were associated with primary rather than recurrent disease. To better understand the factors influencing the development of coinfections, the time periods between inoculations and the genotype of the infecting organisms were evaluated in the chronically immunosuppressed-inoculated rat model of PCP. P. carinii f. sp. carinii infecting rats differentiated by karyotypic profiles exhibit the same low level of genetic divergence manifested by organisms infecting humans. P. carinii f. sp. carinii karyotype forms 1, 2, and 6 were inoculated into immunosuppressed rats, individually and in dual combinations, spaced 0, 10, and 20 days apart. Infections comprised of both organism forms resulted from admixtures inoculated at the same time. In contrast, coinfections did not develop in most rats, where a 10- or 20-day gap was inserted between inoculations; only the first organism form inoculated was detected by pulsed-field gel electrophoresis in the resultant infection. Organism burdens were reduced with combinations of forms 1 and 2 spaced 20 days apart but not in rats inoculated with forms 1 and 6. A role for the host response in the elimination of the second population and in reduction of the organism burden was suggested by the lack of direct killing of forms 1 and 2 in an in vitro ATP assay, by reduction of the burden by autoclaved organisms, and by the specific reactions of forms 1 and 2 but not forms 1 and 6. These studies showed that the time between inoculations was critical in establishing coinfections and P. carinii f. sp. carinii karyotype profiles were associated with differences in biological responses. This model provides a useful method for the study of P. carinii coinfections and their transmission in humans.

Inhibitors of sterol biosynthesis and amphotericin B reduce the viability of pneumocystis carinii f. sp. carinii.

Pneumocystis carinii synthesizes sterols with a double bond at C-7 of the sterol nucleus and an alkyl group with one or two carbons at C-24 of the side chain. Also, some human-derived Pneumocystis carinii f. sp. hominis strains contain lanosterol derivatives with an alkyl group at C-24. These unique sterols have not been found in other pathogens of mammalian lungs. Thus, P. carinii may have important differences in its susceptibility to drugs known to block reactions in ergosterol biosynthesis in other fungi. In the present study, inhibitors of 3-hydroxy-3-methyglutaryl coenzyme A reductase, squalene synthase, squalene epoxidase, squalene epoxide-lanosterol cyclase, lanosterol demethylase, Delta(8) to Delta(7) isomerase, and S-adenosylmethionine:sterol methyltransferase were tested for their effects on P. carinii viability as determined by quantitation of cellular ATP levels in a population of organisms. Compounds within each category varied in inhibitory effect; the most effective included drugs targeted at squalene synthase, squalene epoxide-lanosterol cyclase, and Delta(8) to Delta(7) isomerase. Some drugs that are potent against ergosterol-synthesizing fungi had little effect against P. carinii, suggesting that substrates and/or enzymes in P. carinii sterol biosynthetic reactions are distinct. Amphotericin B is ineffective in clearing P. carinii infections at clinical doses; however, this drug apparently binds to sterols and causes permeability changes in P. carinii membranes, since it reduced cellular ATP levels in a dose-dependent fashion.