Molecular characterization of KEX1, a kexin-like protease in mouse Pneumocystis carinii

Expression screening of a Pneumocystis carinii-infected mouse lung cDNA library with specific monoclonal antibodies (mAbs) led to the identification of a P. carinii cDNA with extensive homology to subtilisin-like proteases, particularly fungal kexins and mammalian prohormone convertases. The 3.1 kb cDNA contains a single open reading frame encoding 1011 amino acids. Structural similarities to fungal kexins in the deduced primary amino acid sequence include a putative proenzyme domain delineated by a consensus autocatalytic cleavage site (Arg-Glu-Lys-Arg), conserved Asp, His, Asn and Ser residues in the putative catalytic domain, a hydrophobic transmembrane spanning domain, and a carboxy-terminal cytoplasmic domain with a conserved tyrosine motif thought to be important for localization of the protease in the endoplasmic reticulum and/or Golgi apparatus. Based on these structural similarities and the classification of P. carinii as a fungus, the protease was named KEX1. Southern blotting of mouse P. carinii chromosomes localized kex1 to a single chromosome of approximately 610 kb. Southern blotting of restriction enzyme digests of genomic DNA from P. carinii-infected mouse lung demonstrated that kex1 is a single copy gene. The function of kexins in other fungi suggests that KEX1 may be involved in the post-translational processing and maturation of other P. carinii proteins.     

Mitochondrial gene sequences show fungal homology for Pneumocystis carinii

A 6.8 kilobase fragment of mitochondrial DNA from Pneumocystis carinii encodes for apocytochrome b, NADH dehydrogenase subunits 1, 2, 3, and 6, cytochrome oxidase subunit II, and the small subunit of ribosomal RNA. Comparative sequence analysis with a series of organisms representative of the fungal and protozoan groups shows that P. carinii has, consistently, an average similarity of 60% with the fungi but only 20% with the protozoa. The data indicate homology with the fungi for this opportunistic pathogen.     

Pneumocystis carinii BCK1 functions in a mitogen-activated protein kinase cascade regulating fungal cell-wall assembly

Pneumocystis pneumonia remains the most common AIDS-defining opportunistic infection in people with HIV. The process by which Pneumocystis carinii constructs its cell wall is not well known, although recent studies reveal that molecules such as beta-1-3-glucan synthetase (GSC1) and environmental pH-responsive genes such as PHR1 are important for cell-wall integrity. In closely related fungi, a specific mitogen-activated protein kinase (MAPK) cascade regulates cell-wall assembly in response to elevated temperature. The upstream mitogen-activated protein kinase kinase kinase (MAPKKK, or MEKK), BCK1, is an essential component in this pathway for maintaining cell-wall integrity and preventing fungal cell lysis. We have identified a P. carinii MEKK gene and have expressed it in Saccharomyces cerevisiae to gain insights into its function. The P. carinii MEKK, PCBCK1, corrects the temperature-sensitive cell lysis defect of bck1Delta yeast. Further, at elevated temperature PCBCK1 restored the signaling defect in bck1Delta yeast to maintain expression of the temperature-inducible beta-1-3-glucan synthetase gene, FKS2. PCBCK1, as a functional kinase, is capable of autophosphorylation and substrate phosphorylation. Since glucan machinery is not present in mammals, a better understanding of this pathway in P. carinii might aid in the development of novel medications which interfere with the integrity of the Pneumocystis cell wall.     

Lectins as probes to Pneumocystis carinii surface glycocomplexes

The binding characteristics of a panel of commercially available FITC-conjugated lectins to Pneumocystis carinii (Pc) were assessed by fluorescence microscopy and flow cytometry. Rat Pc obtained from infected lung homogenates were incubated with FITC-conjugated lectins in a series of concentrations, counterstained with propidium iodide, and analyzed for percent fluorescence and fluorescence intensity. All organisms bound concanavalin A and Wisteria floribunda agglutinin, 2 representatives of the glucose/mannose-binding group. From the lectin group specific for N-acetylglucosamine, Pc reacted more strongly with wheat germ agglutinin than with Solanum tuberosum agglutinin or Griffonia simplicifolia II lectin. Pneumocystis treated with lectins specific for N-acetyl-D-galactosamine and galactose exhibited much variation; the cells reacted moderately well to soybean agglutinin and less to Bauhinia purpurea, Maclura pomifera and Dolichos biflorus agglutinins and Griffonia simplicifolia I lectin. Arachis hypogaea agglutinin, Viscum album agglutinin and Griffonia simplicifolia I-beta 4 lectin had not effect. The organisms reacted weakly with Ulex europeus I agglutinin which is specific for fucose and did not react with Limax flavus lectin, which is specific for sialic acid. Competitive inhibition studies using relevant carbohydrates were performed to indicate that the positive reactions were specific. These studies should help to elucidate the mechanisms of attachment and pathogenesis of this organism.     

Analysis of a surface antigen of Pneumocystis carinii

The 115 kd band in polyacrylamide gels is a major antigen of Pneumocystis carinii. Data obtained from treatment with enzymes, binding to lectins, and labelling the surface with biotin suggest that this moiety is a glycoprotein containing mannosyl/glucosyl and N-acetylglucosamine residues, and that it is located on the cell wall of the organism. Other rat and human P. carinii antigens also are glycoproteins but differ in specific protein or carbohydrate residues or location on the organism.     

Cell population obtained by bronchoalveolar lavage in Pneumocystis carinii pneumonitis

In the course of bronchoalveolar lavages performed in 115 immunocompromised patients in order to investigate the occurrences of pneumonitis, Pneumocystis carinii pneumonia was diagnosed by demonstration of cysts in bronchoalveolar lavage specimens from 11 patients. The cellular phenomena associated with P. carinii infection at the level of the alveolar space were evaluated. Differential cell counts on bronchoalveolar lavage preparations stained by the May-Grünwald-Giemsa method were performed in immunocompromised patients and in ten nonimmunocompromised patients without any respiratory disease. A decrease in the alveolar macrophage count associated with an increase in the polymorphonuclear neutrophil count and the presence of plasma cells and/or immunoblasts was highly suggestive of P. carinii pneumonia. These cellular changes in bronchoalveolar lavage specimens are discussed in relation to the pathologic features usually described in P. carinii pneumonia.     

Propagation and Purification of Rat Pneumocystis carinii in Short-Term Cell

A short-term cell culture is used to propagate and purify rat-derived Pneumocystis carinii (Pc). An aliquot of pelleted material washed out of the lungs of rats with moderate to severe Pc pneumonia is cultured for 7 to 10 days on the adherent mink lung cell line Mv 1 Lu, and the rest of the material is frozen down in medium with 10% glycerol. Although it has not been established that substantial multiplication of Pc occurs in culture, the Pc organisms harvested from the supernatant at the end of the culture period arc relatively free of both host and feeder cells. This is in marked contrast with the lung wash inoculum in which the Pc organisms arc heavily contaminated with rat cells and enmeshed in a highly sticky material. Lung wash preparations frozen down in glycerol and stored at −70.°C for as long as 6 months or more can be successfully cultured upon thawing with no apparent loss of viability of the Pc organisms.     

The Challenge of Pneumocystis carinii Culture1

ABSTRACT. Published and unpublished data on the cultivation of P. carinii were reviewed by a panel of investigators convened by the National Institutes of Health. Although several cell culture systems allow propagation of P. carinii for a limited time with modest rates of replication, these have not proved adequate for isolation of P. carinii in sufficient quantity to explore important basic biological investigation. Attempts at cell-free culture have yielded only transient proliferation. Because much of the unsuccessful work on cultivation of the organism has been unpublished, the panel agreed that these data may be useful to other investigators in designing experimental strategies for cultivation. Therefore, the purpose of this report is to make available this information to researchers, lest others unknowingly repeat unsuccessful methods. It is hoped that by documenting the history and the complexities of Pneumocystis culture, renewed interest and efforts will be directed toward this fundamental scientific challenge.     

Propagation and Purification of Rat Pneumocystis carinii in Short-Term Cell Culture

ABSTRACT. A short-term cell culture is used to propagate and purify rat-derived Pneumocystis carinii (Pc). An aliquot of pelleted material washed out of the lungs of rats with moderate to severe Pc pneumonia is cultured for 7 to 10 days on the adherent mink lung cell line Mv I Lu, and the rest of the material is frozen down in medium with 10% glycerol. Although it has not been established that substantial multiplication of Pc occurs in culture, the Pc organisms harvested from the supernatant at the end of the culture period are relatively free of both host and feeder cells. This is in marked contrast with the lung wash inoculum in which the Pc organisms are heavily contaminated with rat cells and enmeshed in a highly sticky material. Lung wash preparations frozen down in glycerol and stored at −70° C for as long as 6 months or more can be successfully cultured upon thawing with no apparent loss of viability of the Pc organisms.     

Pneumocystis carinii: surface reactive carbohydrates detected by lectin probes

Pneumocystis carinii obtained from rat lungs (RLH) and in vitro culture (RTC) were reacted with a panel of 14 fluorescein isothiocyanate conjugated lectins. Percentage fluorescence and fluorescent intensity were determined for both trophic and cyst forms. All RLH and RTC derived organisms bound strongly concanavalin A (Con A), and wheat germ agglutinin (WGA). However, differences in soybean agglutinin (SBA) binding between RLH and RTC organisms was observed. Different subsets of the organism bound lectins from Griffonia simplicifolia I, Maclura pomifera, and Bauhinia purpurea, indicating heterogeneity in the surface carbohydrates within each of the RLH and RTC populations. Eight lectins reacted very weakly or not at all: Dolichos biflorus, Arachis hypogaea, Griffonia simplicifolia I-beta 4, Solanum tuberosum, Ulex europeus, Griffonia simplicifolia II, Viscum album, and Limax flavus. The results indicate that P. carinii trophic and cyst forms have surface constituents containing mannose, N-acetylglucosamine and N-acetylgalactosamine as the predominant carbohydrates. Molecules resembling sialic acid and beta-galactose are absent or inaccessible. The surface glycoconjugates identified in these studies may play a role in the adherent properties of P. carinii.     

Antigenic variation of a major surface glycoprotein of Pneumocystis carinii

The mannosylated surface glycoprotein (gp) of Pneumocystis carinii has one known conserved epitope that is recognized by the monoclonal antibody 85-1-5E12. The gp exhibits host species-specific antigenic variation, exhibits host species-specific collagenase sensitivity, and varies in size depending on the host of origin and the method of preparation. These data support the existence of host species-specific serotypes of P. carinii.     

Glucan Synthesis in Pneumocystis carinii

Rat-derived Pneumocystis carinii lysed with sodium deoxycholate catalysed the incorporation of uridine diphospho-glucose into an insoluble polymer. This enzyme activity was present in both the pellet and the supernatant when the P. carinii preparations were centrifuged. The polymer whose production was catalysed by the supernatant was examined by mass spectrometry and found to be an α 1→4 glucan, which is either unbranched or has relatively few branches. Polymer formation was completely inhibited by the addition of α amyloglucohydrolase to the supernatant. Polymer formation in the pellet of deoxycholate P. carinii preparations, unlike that in the supernatant, was partially resistant to α amyloglucohydrolase. The soluble glucan synthase activity in the supernatant was stable for more than 30 h at room temperature and was approximately 50 times more active on a cell-to-cell basis than the supernatant from deoxycholate preparations of the yeast Saccharomyces cerevisae.