Structure of major surface determinants and DNA diagnosis of Pneumocystis carinii

Pneumocystis carinii is a pathogen which causes fatal pneumonia in patients with the acquired immune deficiency syndrome (AIDS). To facilitate the basic study of P. carinii, we have analyzed its major surface proteins by both immunochemical and biochemical methods. The major protein components of both cysts and trophozoites are a group of proteins called "P115" with apparent masses of 105-120 kd. It includes 6 isoelectric variants. A monoclonal antibody raised against cysts recognizes all 6 variants and reacts with epitopes located in the cell wall indicating that P115 is an immunoreactive surface component. The isoelectric variants contain identical or closely related protein components and they are mannose-rich glycoproteins. The isoelectric variation may be due primarily to differences in glycosylation. The majority of sera from humans with diagnosed pneumocystosis that were tested reacted strongly with the P115 proteins. To develop probes for DNA diagnosis and to facilitate molecular studies, a genomic DNA library of P. carinii has been constructed. Some of these clones were used for DNA hybridization analysis of rat and human lungs.     

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 enyzmes, 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.     

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.     

Characterisation of human and murine snRNP proteins by two-dimensional gel electrophoresis and phosphopeptide analysis of U1-specific 70K protein variants.

The proteins of the major human snRNPs U1, U2, U4/U6 and U5 were characterised by two-dimensional electrophoresis, with isoelectric focussing in the first dimension and SDS-polyacrylamide gel electrophoresis in the second. With the exception of protein F, which exhibits an acidic pl value (pl = 3.3), the snRNP proteins are basic. Post-translational modification was found among the proteins associated specifically with the U1 and U2 particles. The most complex modification pattern was observed for the U1-specific 70K protein. This was found in at least 13 isoelectric variants, with pl values ranging from 6.7 to 8.7; these variants differed also in molecular weight. All of the 70K variants are phosphorylated in the cell. Thin-layer analysis of their tryptic phosphopeptides revealed that the 70K variants have four major phosphopeptides in common, in addition to which at least four additional serine residues are phosphorylated to different extents. The comparative phosphopeptide analysis shows that differential phosphorylation alone is not sufficient to explain the occurrence of the many isoelectric variants of 70K, so that the final charge of the 70K variants is determined both by phosphorylation and by other, as yet unidentified posttranslational modifications. By two-dimensional separation of snRNP proteins obtained from mouse Ehrlich ascites tumour cells, it was shown that the pattern of pl values of the mouse proteins was almost identical with the corresponding pattern for human proteins. Even the complex modification patterns of the 70K protein are identical in mouse and man, indicating that the presence in the cell of so many variants of this protein may have functional importance. The major difference between murine and human snRNP proteins is the absence of protein B' from mouse snRNPs. This suggests that the homologous protein B may be able to carry out the task of protein B'.     

Structure, Expression and Phylogenetic Analysis of the Gene Encoding Actin I in Pneumocystis Carinii

Actin is a major component of the cytoskeleton and one of the most abundant proteins found in eukaryotic cells. Comparative sequence analysis shows that this essential gene has been highly conserved throughout eukaryotic evolution making it useful for phylogenetic analysis. Complete cDNA clones for the actin-encoding gene were isolated and characterized from Pneumocystis carinii purified from immunosuppressed rat lungs. The nucleotide sequence encodes a protein of 376 amino acids. The predicted actin protein of P. carinii shares a high degree of conservation to other known actins. Only one major actin gene was found in P. carinii. The P. carinii actin sequence was compared with 30 other actin sequences. Gene phylogenies constructed using both neighbor-joining and protein parsimony methods places the P. carinii actin sequence closest to the majority of the fungi. Since the phylogenetic relationship of P. carinii to fungi and protists has been questioned, these data on the actin gene phylogeny support the grouping of P. carinii with the fungi.     

Characterization of the expression site of the major surface glycoprotein of human-derived Pneumocystis carinii

The major surface glycoprotein (MSG) of Pneumocystis carinii, a pathogen responsible for pulmonary infection in AIDS and other immunocompromised patients, is an abundant surface protein that potentially allows the organism to evade host defences by antigenic variation. MSG is encoded by a multicopy gene family; in two specific forms of rat-derived P. carinii, regulation of MSG expression uses a single expression site, termed the upstream conserved sequence (UCS), through two related but distinct mechanisms. In the current study, the UCS of the MSG from human-derived P. carinii was obtained using an RNA ligase-mediated rapid amplification of cDNA ends technique. Southern blot analysis demonstrated that the UCS was present in a single copy per genome, whereas multiple copies of the downstream MSG gene were present. Sequencing and restriction fragment length polymorphism analysis of polymerase chain reaction products amplified from pulmonary samples of patients with P. carinii pneumonia demonstrated that multiple MSG genes were expressed in a given host, and that different patterns of MSG expression were seen among different patients. Tandem repeats present in the single intron occurred with varying frequency in different patient isolates, potentially providing a new method for typing human isolates. Thus, human-derived P. carinii regulates MSG expression in a manner similar to P. carinii f. sp. carinii and, in immunosuppressed patients, in whom immune pressures that probably drive antigenic variation are functioning inadequately, P. carinii can express a broad repertoire of MSG variants.     

Treatment and prevention of Pneumocystis carinii pneumonia and further elucidation of the P. carinii life cycle with 1,3-beta-glucan synthesis inhibitor L-671,329.

Two different classes of 1,3-beta-glucan synthesis inhibitors, the echinocandins and papulacandins, have anti-Pneumocystis activity in an immunosuppressed rat model for acute P. carinii pneumonia (PCP). This activity combined with potent anti-Candida activity makes the echinocandins attractive agents for treating both Pneumocystis and candidiasis in the immunocompromised patient. Natural product echinocandin L-671,329 rapidly eliminates greater than 99% of the P. carinii cysts after 4 days of treatment at a dose of 1 mg/kg twice daily while 2-3 weeks of therapy with trimethoprimsulfamethoxazole (TMP-SMZ) or pentamidine was required to achieve the same degree of cyst clearance. Effects of L-671,329, TMP-SMZ and pentamidine on the trophozoite stage of P. carinii were also explored using a P. carinii-specific DNA probe to quantitate organism load. Although L-671,329 was not as effective as the known agents against the trophozoite stage, prophylactic use of L-671,329 at a daily dose of 1 mg/kg prevented the development of cysts and trophozoites in the rat model. The foamy exudate commonly seen in lungs of animals with PCP is also absent in rats receiving L-671,329 prophylaxis. In addition to demonstrating the potential of L-671,329 as a prophylactic agent these studies also help in elucidating the life cycle of P. carinii. The observation that L-671,329 prophylaxis prevents the appearance of trophozoites, while acute therapy does not directly affect trophozoites, provides the first evidence that the cyst stage is required for trophozoite proliferation. The rapid elimination of cysts by L-671,329 in animals with acute PCP also indicates that all cysts are turning over within 4 days since it is the development of new cysts which is prevented with this compound.     

High copy display of large proteins on phage for functional selections

We have isolated mutations in the major coat protein P8 of M13 phage that greatly increase the surface display of monomeric or oligomeric proteins. The monomeric protein, human growth hormone (hGH), was fused to the N terminus of P8; libraries of P8 variants were constructed and variants that increased hGH display were selected by binding to the extracellular domain of the hGH receptor. The hGH-P8 fusion protein was found to be extremely tolerant of mutations, and a number of P8 variants were found that increased display to levels that improved detection of the hGH-P8 fusion by almost 100-fold. The increased display likely results from better accommodation of the hGH-P8 fusion protein in the phage coat. Using this high copy display format, it was possible for the first time to detect variants of hGH with very weak affinities for the hGHbp (K(d)>1 microM). The display of a tetrameric protein, streptavidin (approximately 50 kDa), was also increased, suggesting the approach may be general to many proteins. The initial product of a natural or invented selection from a naive library is often a weakly functioning protein. These improvements in high copy display should facilitate the broader goal for selection of proteins with novel functions.     

Cell surface protease PRT1 identified in the fungal pathogen Pneumocystis carinii

The subtelomeric regions of the chromosomes of many organisms contain gene families that allow adaptation to a changing environment. In a number of parasites, these subtelomeric gene families encode cell surface proteins that undergo antigenic variation. Proteases are another important virulence determinant in pathogenic microorganisms. We report the localization of the PRT1 protease of the pathogenic fungus Pneumocystis carinii sp. f. carinii, encoded by a subtelomeric gene family, to the cell surface of both the trophozoite and the cyst phase of the organism. Using anti-PRT1 antiserum, we demonstrated specificity to P. carinii sp. f. carinii in sections of infected rat lungs and, using immunofluorescence, we showed that the PRT1 protease has the characteristic distribution of a surface protein. The anti-PRT1 antiserum showed cross-reactivity with a number of P. carinii sp. f. carinii proteins migrating between 185 kDa and 28 kDa, the majority migrating between 42 kDa and 52 kDa, a region that has been shown by serological studies to contain important immunodominant P. carinii proteins. Cross-reactivity was also observed with P. carinii sp. f. hominis proteins. We have also cloned a portion of the catalytic domain of PRT1 from P. carinii sp. f. hominis, P. carinii sp. f. muris and P. carinii sp. f. rattus. Our data suggest that the PRT1 protease plays an important role in the pathogenicity of P. carinii.     

The relationship between polyribosomal and latent membrane-bound messenger ribonucleoprotein particles in Artemia embryos

Polysomal messenger ribonucleoprotein (mRNP) particles from developing Artemia cysts were isolated, characterized and compared with latent membrane-bound mRNP particles isolated from dormant cysts. The polyribosomal mRNP particles sedimented between 25-35 S in a sucrose gradient and had a buoyant density of 1.33 g/cm3 in Cs2So4. Latent particles had a higher sedimentation coefficient and lower buoyant density. The poly(A) + RNA in the two kinds of particles was comparable in size, 10-20 S. The protein composition of the particles, as determined by electrophoresis, was different. Polyribosomal particles contained 9 major and 6 minor proteins; a 72 k poly(A)-associated protein was present. Latent particles were characterized by a complex protein pattern ranging in apparent mol. wt between 14,000-140,000. Some proteins with similar molecular weight and isoelectric point were probably common to both kinds of particles.     

Analysis of Pneumocystis carinii Cyst Wall. II. Sugar Composition

ABSTRACT. Pneumocystis carinii cysts are capable of resisting host defenses and antimicrobial drugs and are therefore thought to be responsible for relapses of P. carinii pneumonia in AIDS and other immunocompromised patients. The interaction of P. carinii with its host, and other P. carinii , might be mediated by molecules which form the outer surfaces of this organism. Carbohydrates are known to play many roles in cell-cell adhesion, and have been detected on the surface of P. carinii by lectin labeling experiments. In this study P. carinii cyst wall material was obtained from Zymolyase treatment. Alditol acetate derivatives of neutral and amino sugars or trimethylsilyl derivatives of methyl glycosides were prepared from the monosaccharides released from the sample by acid hydrolysis. Analyses were done by a combination of gas chromatography and mass spectrometry. Glucose was found to be the major sugar constituent. Mannose and galactose were present in equal ratios. A lesser amount of N-acetyl-D-glucosamine, and trace amounts of ribose and sialic acid were present in the cyst wall samples analyzed. These sugars may mediate P. carinii -host interaction and play an important protective role by creating a permeability barrier around the cyst.     

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.     

Cytoplasmic microtubule-associated proteins: Phosphorylation at novel sites is correlated with their incorporation into assembled microtubules

We have analyzed the detailed structure and cytoplasmic distribution of cytoplasmic microtubule-associated proteins. The procedure used to identify these proteins, based on preparation of detergent-extracted cytoskeletons, permits separation of fractions containing assembled and unassembled microtubule proteins. We show that two of these proteins, 69 and 80 kd, are closely related to one another and that each protein is present as a set of structurally related polypeptides with differing isoelectric points. In both neuroblastoma and pheochromocytoma cells, several of the isoelectric variants are greatly enriched in the fraction containing assembled microtubule components. Their differential distribution is correlated with phosphorylation at novel sites on the protein. These results support the possibility that covalent modification of a cytoskeletal component may specify its functional state.     

Cloning and Characterization of an ATPase Gene from Pneumocystis carinii which Closely Resembles Fungal H+ ATPases

ABSTRACT. A gene encoding a P-type cation translocating ATPase was cloned from a genomic library of rat-derived Pneumocystis carinii. The nucleotide sequence of the gene contains a 2781 base-pair open reading frame that is predicted to encode a 101, 401 dalton protein composed of 927 amino acids. The P. carinii ATPase protein (pcal) is 69–75% identical when compared with eight proton pumps from six fungal species. The Pneumocystis ATPase is less than 34% identical to ATPase proteins from protozoans, vertebrates or the Ca⁺⁺ ATPases of yeast. The P. carinii ATPase contains 115 of 121 residues previously identified as characteristic of H⁺ ATPases. Alignment of the Pneumocystis and fungal proton pumps reveals five homologous domains specific for fungal H⁺ ATPases.     

Analysis of Pneumocystis carinii cyst wall. II. Sugar composition

Pneumocystis carinii cysts are capable of resisting host defenses and antimicrobial drugs and are therefore thought to be responsible for relapses of P. carinii pneumonia in AIDS and other immunocompromised patients. The interaction of P. carinii with its host, and other P. carinii, might be mediated by molecules which form the outer surfaces of this organism. Carbohydrates are known to play many roles in cell-cell adhesion, and have been detected on the surface of P. carinii by lectin labeling experiments. In this study P. carinii cyst wall material was obtained from Zymolyase treatment. Alditol acetate derivatives of neutral and amino sugars or trimethylsilyl derivatives of methyl glycosides were prepared from the monosaccharides released from the sample by acid hydrolysis. Analyses were done by a combination of gas chromatography and mass spectrometry. Glucose was found to be the major sugar constituent. Mannose and galactose were present in equal ratios. A lesser amount of N-acetyl-D-glucosamine, and trace amounts of ribose and sialic acid were present in the cyst wall samples analyzed. These sugars may mediate P. carinii-host interaction and play an important protective role by creating a permeability barrier around the cyst.     

DNA-binding proteins from Novikoff hepatoma cells.

In 0.05 M NaCl, 6-8% of the total soluble proteins from Novikoff hepatoma cells bind rapidly and reversibly to columns containing either heterologous or homologous DNA adsorbed to cellulose. These proteins can be eluted by buffer containing 2.0 M NaCl. 0.5-1% of the total protein exhibits a 7-17-fold preference for rat DNA over Escherichia coli DNA. 1-1.5% of the proteins bind DNA so strongly that elution cannot be effected by 4.0 M NaCl but can be accomplished by deoxyribonuclease I treatment of the columns. DNA-binding proteins eluted by 2.0 M NaCl were labeled with 125I or 131I and characterized by sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing. These experiments indicate that DNA-binding proteins represent a discrete subset of the total soluble protein. Many similarities were noted between the major components of the homologous and heterologous DNA-binding fractions.     

Cell Wall Antigens of Pneumocystis carinii Trophozoites and Cysts: Purification and Carbohydrate Analysis of these Glycoproteins

We have purified and biochemically analyzed individual cell wall glycoproteins of Pneumocystis carinii. Our results show that corresponding core glycoproteins constitute the cell wall antigens in both trophozoites and cysts, and glycosylation of these glycoproteins does not appear to be significantly altered during development. Cysts and trophozoites in rat-derived organism preparations were separated from each other by counterflow centrifugal elutriation, then treated with Zymolyase to obtain the cell wall fractions. Gel electrophoresis patterns of these fractions from both life-cycle stages were qualitatively similar. Ten major antigenic glycoproteins in these fractions were purified by preparative continuous elution gel electrophoresis. All ten glycoproteins from cysts and trophozoites contained mannose, glucose, galactose. and N-acetylglucosamine, and some contained traces of fucose. The glycoproteins of cysts had more mannose than their trophozoite counterparts. The trophozoite glycoproteins differed from those of the cyst by the presence of xylose. To examine the species-specificity of glycoprotein glycosylation, preparations of human-derived P. carinii (comprised of mixed life-cycle stages) were also examined and found to contain the same sugars as those found in rat-derived organisms. Most of the purified rat-derived glycoproteins bound Concanavalin A, which was abolished by treatment with N-glycanase. This suggested that the majority of the oligosaccharides were N-linked to the proteins, but attempts to identify carbohydrate linkage sites by amino acid sequencing were hampered by apparent modifications of residues. The peptides derived by cyanogen bromide cleavage revealed distinct size patterns for each glycoprotein, suggesting that they were distinct proteins. Most of the glycoproteins reacted with monoclonal antibodies which recognize a highly conserved epitope on rat P. carinii. Four of the individually purified glycoprotein preparations elicited in vitro cellular immune responses, implicating their involvement in the recognition of P. carinii by host T cells. The identification and characterization of P. carinii cell wall proteins will be helpful in analyzing the relationship of the organism to its mammalian host. Supplementary key words. Biochemical analysis, developmental stages, opportunistic pathogen, structure.     

Pneumocystis carinii telomere repeats are composed of TTAGGG and the subtelomeric sequence contains a gene encoding the major surface glycoprotein

We have cloned a telomere and adjacent sequences from rat-derived Pneumocystis carinii using the ability of foreign telomeres to complement a yeast artificial chromosome (YAC) deficient by one telomere in Saccharomyces cerevisiae . Characterization of the cloned DNA in the recombinant YAC demonstrated that it was a chimera of two P. carinii sequences, namely a 13.5 kb fragment of mitochondrial DNA and an 8.3 kb distal portion consisting of subtelomeric DNA. The P. carinii telomere repeat was demonstrated to be TTAGGG, the most common telomere repeat found in organisms from the animal and fungal kingdoms. Karyotype analysis confirmed that this sequence was present on all the P. carinii chromosomes. Sequence adjacent to the telomere repeats was shown by Bal 31 exonuclease digestion to be located at the chromosome ends. Analysis of the subtelomeric fragment revealed homology to the gene encoding the major surface glycoprotein of P. carinii