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.     

Azospirillum DNA shows homology with Agrobacterium chromosomal virulence genes

DNA from Azospirillum brasilense Sp7 (ATCC 29145) was hybridized with probes containing the T-region and the vir-region of a nopaline Ti-plasmid, and the chromosomal virulence region (chv) of Agrobacterium tumefaciens. Homology to chv was found. Hybridization signals with the chv probe were detected in 7 A. brasilense strains and 1 A. lipoferum strain tested.     

Complementation of a manganese-dependent superoxide dismutase-deficient yeast strain with Pneumocystis carinii sod2 gene

Manganese-dependent superoxide dismutase (MnSOD) is one of the key enzymes involved in the cellular defense against oxidative stress. Previously, the Pneumocystis carinii sod2 gene (Pcsod2) was isolated and characterized. Based on protein sequence comparison, Pcsod2 was suggested to encode a putative MnSOD protein likely to be targeted into the mitochondrion. In this work, the Pcsod2 was cloned and expressed as a recombinant protein in EG110 Saccharomyces cerevisiae strain lacking the MnSOD-coding gene (Scsod2) in order to investigate the function and subcellular localization of P. carinii MnSOD (PcMnSOD). The Pcsod2 gene was amplified by PCR and cloned into the pYES2.1/V5-His-TOPO^® expression vector. The recombinant construct was then transformed into EG110 strain. Once its expression had been induced, PcMnSOD was able to complement the growth defect of EG110 yeast cells that had been exposed to the redox-cycling compound menadione. N-term sequencing of the PcMnSOD protein allowed identifying the cleavage site of a mitochondrial targeting peptide. Immune-colocalization of PcMnSOD and yeast CoxIV further confirmed the mitochondrial localization of the PcMnSOD.Heterologous expression of PcMnSOD in yeast indicates that Pcsod2 encodes an active MnSOD, targeted to the yeast mitochondrion that allows the yeast cells to grow in the presence of reactive oxygen species (ROS).     

Proteinase yscE of yeast shows homology with the 20 S cylinder particles of Xenopus laevis

Proteinase yscE of the yeast Saccharomyces cerevisiae has been compared with the 20 S cylinder particles of Xenopus laevis. Both proteins are characterized by a similar group of 10-12 polypeptides with molecular masses ranging between 21 and 38 kDa. Antibodies generated against the 20 S Xenopus cylinder particles show cross-reactivity with yeast proteinase yscE subunits. The Xenopus particles and yeast proteinase yscE exhibit an identical image in electron microscopy. Both proteins appear as hollow cylinders mostly composed of four stacked annuli. The Xenopus 20 S particles exhibit proteolytic activity against the three peptide derivatives known to be substrates of proteinase yscE. The pH optimum for activity and the inhibition spectrum of the proteolytic activities of Xenopus 20 S particles and of yeast proteinase yscE are identical. The RNA content of the cylinder particles and of proteinase yscE is below 0.1 RNA chain per molecule. Our data suggest that proteinase yscE from yeast and the 20 S cylinder particles of X. laevis are homologous, highly conserved proteins carrying the catalytic character of a peptidase.     

Detection of Pneumocystis carinii DNA in clinical samples from children with respiratory diseases

Method of Pneumocystis carinii DNA detection in clinical samples (sputum) is presented. Primers to one fragment of 16S rRNA gene were used for detection of DNA. Isolation and amplification of DNA were performed in presence of internal DNA control. Analytical sensitivity of method was 200 copies of DNA per 1 ml. Analytical and diagnostic specificities were 100% and 95% accordingly. Sputum from 176 children with frequent respiratory infections were sampled before start of antibacterial therapy and studied simultaneously by the polymerase-chain reaction (PCR) and immunofluorescent assay (IFA). Results of PCR and IFA coincided in 167 (94.89%) children. From them, P.carinii was detected in sputum in 5 (2.85%) children. All children with positive results were treated with antibiotics. Repeated tests of sputum 8 days after start of treatment were all negative. PCR could be recommended as part of complex of clinical diagnostics and control of treatment.     

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 isoelcclric 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 immunorcactive 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.     

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.     

Synthetic p55 tandem DNA vaccine against Pneumocystis carinii in rats

Pneumocystis spp. are opportunistic fungal pathogens that are closely associated with severe pneumonia and pulmonary complications in patients with impaired immunity. In this study, the antigenic epitopes of the gene encoding the 55 kDa antigen fragment of Pneumocystis (p55), which may play an important role in Pneumocystis pneumonia, were analyzed. A gene containing tandem variants of the p55 antigen was synthesized and named the tandem antigen gene (TAG). TAG's potential as a DNA vaccine was assessed in immunosuppressed rats. Immunization with p55‐TAG DNA vaccine significantly reduced both the pathogen burden and lung–weight to body–weight ratios. Additionally, p55‐TAG vaccination in immunosuppressed rats elicited both cell‐mediated and humoral immunity.     

Active immunization against Pneumocystis carinii with p55-v3 DNA vaccine in rats

Pneumocystis pneumonia (PCP) occurs predominately in patients with impaired immunity. Because standard PCP chemoprophylaxis and chemotherapies have limitations, immunotherapy, particularly vaccination, offers an attractive alternative approach for PCP prevention and treatment. The goal of this study was to evaluate the potential of DNA vaccines targeting two closely related antigens, p55-v0 and p55-v3, in an immunosuppressed rat PCP model. We found that immunization with p55-v0 and p55-v3 DNA vaccines afforded a similar level of protection to rats against PCP, as evidenced by significant reductions in organism burdens, improved histological scores, and lower lung weight to body weight ratios. Additionally, vaccination elicited both cellular and humoral immunity in immunosuppressed rats. Our data suggest the potential of p55 DNA vaccines to protect against PCP in rats. Future work should focus on epitope mapping and identifying protective moieties in each gene.