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.     

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.     

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.     

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.     

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.     

Molecular phylogeny of Pneumocystis based on 5.8S rRNA gene and the internal transcribed spacers of rRNA gene sequences

To clarify the phylogenetic relationships and species status of Pneumocystis, the 5.8S rRNA gene and the internal transcribed spacers (ITS, 1 and 2) of Pneumocystis rRNA derived from rat, gerbil and human were amplified, cloned and sequenced. The genetic distance matrix of six Pneumocystis species compared with other fungi like Taphrina and Saccharomyces indicated that the Pneumocystis genus contained multiple species including Pneumocystis from gerbil. The phylogenetic tree also showed that Pneumocystis from human and monkey formed one group and four rodent Pneumocystis formed another group. Among the four members, Pneumocystis wakefieldiae was most closely related to Pneumocystis murina and Pneumocystis carinii, and was least related to gerbil Pneumocystis.     

Sequence and variability of the 5.8S and 26S rRNA genes of Pneumocystis carinii.

The sequence of the coding region of the rRNA operon of rat-derived Pneumocystis carinii has been completed, including the genes for 5.8S and 26S rRNA. These genes show homology to the rRNA genes of yeast, and an apparent group I self-splicing intron is present in the 26S rRNA gene. Like a similar intron in the 16S rRNA gene, this intron is in a phylogenetically conserved region. Variation in the 26S rRNA sequence was noted between P. carinii organisms isolated from two different sources.     

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.     

Cloning and Identification of Arp1, an Actin-Related Protein from Pneumocystis carinii

ABSTRACT. The complete Pneumocystis carinii Arp1 gene has been sequenced from two cDNA clones. The gene encodes a protein 385 bp in length with an estimated size of 45,000 kD. The A + T% for the Arp1 gene and a 900-bp sequence upstream of the gene were 63.7% and 70.3%, respectively. These values are consistant with A + T codon preference displayed by P. carinii and are similar to values reported for other P. carinii genes. The predicted amino acid sequence of the P. carinii Arp1 protein had a similarity of 87.6% with Neurospora crassa Arp1, 82.1% similarity with vertebrate centractin, and 71.2% similarity with the Saccharomyces cerevisiae Act5p. Expression of Arp1 mRNA in P. carinii was detectable via synthesis of cDNA and subsequent PCR amplification. Affinity purified antibodies against S. cerevisiae Act5p, and canine centractin recognized both the recombinantly expressed protein and a 45,000 kD protein in P. carinii nuclear extracts. The Arp1 gene is the second member of the actin multigene family that has been identified in P. carinii .     

Pneumocystis: from a doubtful unique entity to a group of highly diversified fungal species

At the end of the 20th century the unique taxonomically enigmatic entity called Pneumocystis carinii was identified as a heterogeneous group of microscopic Fungi, constituted of multiple stenoxenic biological entities largely spread across ecosystems, closely adapted to, and coevolving in parallel with, mammal species. The discoveries and reasoning that led to the current conceptions about the taxonomy of Pneumocystis at the species level are examined here. The present review also focuses on the biological, morphological and phylogenetical features of Pneumocystis jirovecii, Pneumocystis oryctolagi, Pneumocystis murina, P. carinii and Pneumocystis wakefieldiae, the five Pneumocystis species described until now, mainly on the basis of the phylogenetic species concept. Interestingly, Pneumocystis organisms exhibit a successful adaptation enabling them to dwell and replicate in the lungs of both immunocompromised and healthy mammals, which can act as infection reservoirs. The role of healthy carriers in aerial disease transmission is nowadays recognized as a major contribution to Pneumocystis circulation, and Pneumocystis infection of nonimmunosuppressed hosts has emerged as a public health issue. More studies need to be undertaken both on the clinical consequences of the presence of Pneumocystis in healthy carriers and on the intricate Pneumocystis life cycle to better define its epidemiology, to adapt existing therapies to each clinical context and to discover new drug targets.