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.     

Unique Telomeric Expression Site of Major-Surface-Glycoprotein Genes of Pneumocystis carinii

Major cell surface glycoproteins (MSG) of Pneumocystis cariniiplay a crucial role in the host-parasite interactions involvedin P. carinii pneumonia in AIDS patients. Genes encoding MSGsare repeated, highly polymorphic, and distributed among allof the 14-15 chromosomes. Here we show, by BAL-31 exonucleasecleavage and DNA cloning experiments, that the unique expressionsite (previously termed UCS) of MSG genes located in the 500-kbchromosome is telomeric. The 11-kb genomic UCS fragment isolatedand sequenced in this study contained one MSG coding sequence(termed msg105), subtelomeric repetitive sequences and telomere-specifictandem repeats of TTAGGG oriented 5' to 3' towards the DNA end.Despite the N-terminal polymorphism, the C-terminal one-thirdsequence of MSG105 was identical to one of the known MSG-cDNAs,suggesting homologous recombination within the MSG coding sequences.These features closely resemble the Variant Surface Glycoproteinsystem of the protozoan parasite Trypanosoma brucei, suggestingthat the genetic heterogeneity of MSGs is generated by recombinationbetween the UCS expression site and multiple MSG genes by meansof reciprocal exchange or gene conversion.     

Gene arrays at Pneumocystis carinii telomeres

In the fungus Pneumocystis carinii, at least three gene families (PRT1, MSR, and MSG) have the potential to generate high-frequency antigenic variation, which is likely to be a strategy by which this parasitic fungus is able to prolong its survival in the rat lung. Members of these gene families are clustered at chromosome termini, a location that fosters recombination, which has been implicated in selective expression of MSG genes. To gain insight into the architecture, evolution, and regulation of these gene clusters, six telomeric segments of the genome were sequenced. Each of the segments began with one or more unique genes, after which were members of different gene families, arranged in a head-to-tail array. The three-gene repeat PRT1-MSR-MSG was common, suggesting that duplications of these repeats have contributed to expansion of all three families. However, members of a gene family in an array were no more similar to one another than to members in other arrays, indicating rapid divergence after duplication. The intergenic spacers were more conserved than the genes and contained sequence motifs also present in subtelomeres, which in other species have been implicated in gene expression and recombination. Long mononucleotide tracts were present in some MSR genes. These unstable sequences can be expected to suffer frequent frameshift mutations, providing P. carinii with another mechanism to generate antigen variation.     

Pneumocystis murina MSG gene family and the structure of the locus associated with its transcription

Analysis of the Pneumocystis murina MSG gene family and expression-site locus showed that, as in Pneumocystis carinii, P. murina MSG genes are arranged in head-to-tail tandem arrays located on multiple chromosomes, and that a variety of MSG genes can reside at the unique P. murina expression site. Located between the P. murina expression site and attached MSG gene is a block of 132 basepairs that is also present at the beginning of MSG genes that are not at the expression site. The center of this sequence block resembles the 28 basepair CRJE of P. carinii, but the block of conserved sequence in P. murina is nearly five times longer than in P. carinii, and much shorter than in P. wakefieldiae. These data indicate that the P. murina expression-site locus has a distinct structure.     

Characterization of Rat CD4 T Cell Clones Specific for the Major Surface Glycoprotein of Pneumocystis carinii

ABSTRACT. Pneumocystis carinii are coated by abundant heterogenous major surface glycoproteins (MSGs), which facilitate interaction with the host. We have produced MSG-specific T-cell clones from the spleens of P. carinii -exposed Lewis rats and analyzed five for antigen specificity to native MSG and a recombinant form of MSG, cell surface markers, and cytokine profiles. All five of the clones were CD4+. All of the clones proliferated specifically to both the native MSG and the recombinant MSG only in the presence of antigen presenting cells demonstrating that the response is antigen/driven rather than mitogen/driven. All five of the clones secreted IL-2 and IFN-γ, although in differing amounts, implicating a Th l response. Only one of the clones produced any detectable IL-4. This is the first report of T cell clones responsive to a specific antigen of P. carinii , MSG. We conclude that the T cell clones will be helpful in mapping protective epitopes present in MSG and in functional studies of MSG.     

A Tandem Repeat of Rat-derived Pneumocystis carinii Genes Encoding the Major Surface Glycoprotein

ABSTRACT. A fragment from the genome of rat-derived Pneumocystis carinii was found to contain two MSG genes arranged as a direct repeat. The sequences from one gene (MSG B), the region between the two genes, and part of the second gene (MSG A) were determined. The two MSG genes were not identical in sequence. The open reading frames of MSG A and MSG B encode non-identical proteins, both of which are similar to that encoded by a previously published cDNA. The MSG B gene sequence showed no evidence of introns. The 5'and 3'untranslated regions of the MSG gene pair were highly conserved, but the regions immediately upstream of the open reading frames of MSG A and B were different from the region upstream of a previously characterized MSG cDNA. Primers designed to extend upstream of the 5'end of MSG and downstream of the 3'end of MSG were used in a polymerase chain reaction with total genomic P. carinii DNA as template. Presumptive intergenic amplification products from this reaction were cloned and sequenced. The sequences of these regions were similar but distinct, indicating that tandem arrangement of MSG genes is a common organizational motif.     

Immunization with recombinant Pneumocystis carinii p55 antigen provides partial protection against infection: characterization of epitope recognition associated with immunization

Many therapeutic options exist for the treatment of Pneumocystis carinii pneumonia, a common fungal opportunistic pulmonary pathogen, but treatment is often complicated by side effects and toxicity and, more recently, markers of drug resistance have been described. The development of immunotherapetic modalities such as active immunization or passive immunotherapy may play an increasing important role in the prevention and treatment of infection. Passive immunotherapy with polyclonal anti-P. carinii reagents, such as serum or T cells, and monospecific reagents reactive with the major surface glycoprotein (MSG or gpA), such as monoclonal antibodies or MSG primed T cells, reduce the severity or eradicate infection. Active immunization with whole P. carinii, P. carinii extracts or MSG has afforded partial protection against the subsequent development of P. carinii pneumonia in some animal models. Identification of additional antigens with protective benefits will aid in the development of vaccines or other reagents. The p55 antigen of rat-derived P. carinii is well recognized by animals following natural exposure to the organism. This 414 amino acid residue antigen found within the cell wall of P. carinii contains 7 repeats of a glutamic acid-rich motif in the carboxyl portion of the molecule. Both humoral and cellular immune responses reactive with this repeated domain are present following natural infection while, the amino terminal portion of the molecule is immunologically silent. In this study, immunization with recombinant p55 elicited significant humoral and cellular immune responses which persisted during 10 weeks of immunosupression in corticosteroid treated rats; rp55 immunization resulted in a significant reduction in organism burden, improved histological score, lower lung weight to body weight ratio (a marker of infection or lung inflammation) and improved survival (P < 0.01). Greater protection was afforded by immunization with a peptide containing amino acid residues 1-200, than by the entire rp55 molecule. Epitope recognition by serum from animals immunized with rp55 differed from that of naturally exposed animals with oligoclonal responses to residues 22-92 and residues 196-218. This study demonstrates that protection against P. carinii can be afforded by immunization with antigen preparations other than whole extracts of P. carinii or the major surface antigen, MSG. This antigen moiety will likely be most useful as a vaccine candidate in combination with other immunogens which provide similar partial protection.     

Genetic heterogeneity of Pneumocystis carinii from rats of several regions and strains

Pneumocystis carinii is a major opportunistic pathogen which has been found in the lungs of a wide variety of mammalian host species, and the fact suggests the possibility of intraspecific variation. Until now, P. carinii from different mammalian species are differentiated as subspecies, and the rats are known to be infected by two subspecies. The present study investigated genetic heterogeneity of P. carinii isolates from two strains of rats in Korea and China by molecular karyotyping, RFLP and sequencing analysis. Karyotypes of P. carinii were grouped into three, two from two strains of rats in Korea and one from rats in China. However RFLP of PCR product of ribosomal and MSG gene of the P. carinii isolates showed same pattern. The sequence homology rates of alpha-tubulin DNA of the P. carinii isolates were 96% in Seoul Wistar rats, 93% in Seoul Sprague-Dawley rats, and 85% in Chinese Sprague-Dawley rats. The present finding confirmed that P. carinii from rats in Korea are grouped into two karyotype strains which are different from that of P. carinii from rats in China. The Chinese isolate shows a little different sequences of alpha-tubulin DNA.     

Fine analysis of the Pneumocystis carinii f. sp. carinii genome by two-dimensional pulsed-field gel electrophoresis

Pneumocystis carinii is a general designation for a group of unusual unicellular fungal parasites responsible of pneumopathy in animal hosts. Divided into several subgroups termed the 'special forms', P. carinii is prone to an extensive karyotype variation. In previous studies, the nuclear genome of these organisms has been considered to be haploid and a set of 16 chromosomes has been assigned to P. carinii f. sp. carinii, a special form known to infect rats. We report the analysis of the genome of an isolate representative of the karyotype 1 of this special form, using two-dimensional pulsed-field gel electrophoresis procedures. The 'karyotype and restriction display' (KARD) fingerprints indicated the presence of 17 different chromosomes. The haploid genome size was estimated to be 8.4 Mbp. Some homologous chromosomes were distinguished on the basis of a single restriction fragment length polymorphism, which raises the possibility of a diploid nucleus. A restriction map of the chromosome 15, characterized by two homologues with a size difference of 7 kb, was constructed. Hybridization data indicated that insertion/deletion events may have occurred within subtelomeric regions which carry genes encoding the major surface glycoprotein (MSG) of Pneumocystis.     

Determination of the copy number of the nuclear rDNA and beta-tubulin genes of Pneumocystis carinii f. sp. hominis using PCR multicompetitors

Multiple copies of a gene may lead to difficulty in the interpretation of typing results because polymorphism of the copies may wrongly lead to the conclusion that different types are present in a specimen. To determine the copy number per genome of the nuclear rDNA and beta-tubulin genes analyzed for the typing of Pneumocystis carinii f. sp. hominis, we developed a strategy based on the use of the same multicompetitor molecule in two different quantitative-competitive PCRs, one for the gene under study and the other for a reference single copy gene, allowing direct comparison of the results of both PCRs. Control experiments showed that the strategy was sensitive enough to detect duplication of a gene. The copy number of the nuclear rDNA operon was determined by amplification of the intron of the 26S rDNA gene and that of the beta-tubulin by amplification of the region surrounding the intron no. 6. The method was first tested on P. c. carinii, the special form commonly infecting rats. Pneumocystis c. carinii was found to contain a single copy of the rDNA operon. The method was then applied to P. c. hominis. The results confirmed that P. c. hominis genome contains a single copy of the nuclear rDNA and beta-tubulin genes.     

Inversion and crossover recombination contributions to the spacing between two functionally linked genes

The roles of inversion and crossover recombination in determining the spacing between two functionally linked genes on an individual strand of DNA and the resulting genetic organization throughout the population is not well understood. We employ a computer simulation to look at the spacing between functionally linked genes after many generations of a population of haploid individuals, each with a single chromosome. Simulations show that inversion and crossover recombination combine to create four attractors in gene spacing. The two major attractors include one in which the linked genes are forced to be near each other and one in which the linked genes are forced to be separated by one third of the chromosome length. Multiplicative functional linkage between two linked genes also causes a decreased average spacing compared to additive and random functional linkage.     

Clonality Despite Sex: The Evolution of Host-Associated Sexual Neighborhoods in the Pathogenic Fungus Penicillium marneffei

Molecular genetic approaches typically detect recombination in microbes regardless of assumed asexuality. However, genetic data have shown the AIDS-associated pathogen Penicillium marneffei to have extensive spatial genetic structure at local and regional scales, and although there has been some genetic evidence that a sexual cycle is possible, this haploid fungus is thought to be genetically, as well as morphologically, asexual in nature because of its highly clonal population structure. Here we use comparative genomics, experimental mixed-genotype infections, and population genetic data to elucidate the role of recombination in natural populations of P. marneffei. Genome wide comparisons reveal that all the genes required for meiosis are present in P. marneffei, mating type genes are arranged in a similar manner to that found in other heterothallic fungi, and there is evidence of a putatively meiosis-specific mutational process. Experiments suggest that recombination between isolates of compatible mating types may occur during mammal infection. Population genetic data from 34 isolates from bamboo rats in India, Thailand and Vietnam, and 273 isolates from humans in China, India, Thailand, and Vietnam show that recombination is most likely to occur across spatially and genetically limited distances in natural populations resulting in highly clonal population structure yet sexually reproducing populations. Predicted distributions of three different spatial genetic clusters within P. marneffei overlap with three different bamboo rat host distributions suggesting that recombination within hosts may act to maintain population barriers within P. marneffei.     

Purification of a 68-kDa cysteine proteinase from crude extract of Pneumocystis carinii

The present study intended to verify activities of cysteine proteinase of Pneumocystis carinii from rats and to purify the enzyme. In order to exclude the contamination of host-derived enzymes, concentrates of P. carinii was primarily treated with a mixture of proteinase inhibitors before lysis of P. carinii. A 68-kDa cysteine proteinase was finally purified from the crude extract of P. carinii by 4 sequential chromatographic methods. The enzyme showed an optimal activity at pH 5.5 in 0.1 M sodium acetate, and its activity was specifically inhibited by L-trans-epoxy-succinylleucylamido (4-guanidino) butane (E-64) and iodoacetic acid, suggesting that the enzyme is a cysteine proteinase. The 68-kDa proteinase weakly digested macromolecules such as collagen, hemoglobin and fibronectin. The present study demonstrated the activity of cysteine proteinase at the 68-kDa band of P. carinii, and purified and characterized the molecule.     

Karyotypes of Pneumocystis carinii derived from several mammals

Pneumocystis carinii is the most important opportunistic pathogen of humans in the world. Pneumocystis carinii is experimentally detected in the lungs of rats, mice, rabbits, and monkeys, however, the organisms from different mammals are identical in microscopic morphology. The present study tried to find out more mammalian hosts of P. carinii and also to differentiate the organisms from different mammals by karyotyping. Rats, mice, hamsters, rabbits, cats, and dogs were successfully infected by P. carinii, but guinea pigs and pigs were not. Karyotype of P. carinii from rabbits showed similar size range of chromosomes with that of the prototype, but in different pattern. The patterns from cats and dogs were also different from that of rats. The present study confirms that cats and dogs are infected by P. carinii and at least total three karyotype strains of P. carinii are proven in Korea.