Population structure of rat-derived Pneumocystis carinii in Danish wild rats

The rat model of Pneumocystis carinii pneumonia is frequently used to study human P. carinii infection, but there are many differences between the rat and human infections. We studied naturally acquired P. carinii in wild rats to examine the relevance of the rat model for human infection. P. carinii DNA was detected in 47 of 51 wild rats and in 10 of 12 nonimmunosuppressed laboratory rats. Evidence for three novel formae speciales of rat-derived P. carinii was found, and these were provisionally named Pneumocystis carinii f. sp. rattus-secundi, Pneumocystis carinii f. sp. rattus-tertii, and Pneumocystis carinii f. sp. rattus-quarti. Our data suggest that low-level carriage of P. carinii in wild rats and nonimmunosuppressed laboratory rats is common and that wild rats are frequently coinfected with more than one forma specialis of P. carinii. We also examined the diversity in the internally transcribed spacer (ITS) regions of the nuclear rRNA operon of Pneumocystis carinii f. sp. carinii by using samples from wild rats and laboratory rats and spore trap samples. We report a lack of variation in the ITS1 and ITS2 regions that is consistent with an evolutionary bottleneck in the P. carinii f. sp. carinii population. This study shows that human- and rat-derived P. carinii organisms are very different, not only in genetic composition but also in population structure and natural history.     

Pneumocystis carinii and Pneumocystis wakefieldiae in Wild Rattus norvegicus Trapped in Thailand

ABSTRACT. This work reports for the first time the presence of two Pneumocystis species in wild Rattus norvegicus specimens from Thailand. Pneumocystis DNA was detected in 57.7% (15/26) wild rats without apparent association with typical pneumocystosis. Pneumocystis carinii was found alone in five rats (19.2%), Pneumocystis wakefieldiae was detected alone in six rats (23.1%), and two rats were infected by both species (7.7%). In addition, a new P. wakefieldiae variant sequence has been identified in three wild R. norvegicus specimens caught in the same geographical area. The high frequency of Pneumocystis in wild rats documented in this study and the apparent scarcity of severe pneumocystosis were consistent with an efficient circulation of rat Pneumocystis species in ecosystems.     

Molecular Genetic Distinction of Pneumocystis carinii from Rats and Humans

Pneumocystis carinii from rats and from humans were compared with respect to electrophoretic karyotype, presence of DNA sequences known to be repeated in rat-derived P. carinii, overall DNA sequence homology, and the sequences at two genetic loci. The organisms from each host species were different in each respect. Neither of two repeated DNAs from rat-derived P. carinii was found in the genome of human-derived organisms, and total DNA from rat-derived P. carinii failed to hybridize to human-derived P. carinii DNA. The sequences of the α-tubulin genes from the two P. carinii were strikingly different and the base composition of the α-tubulin gene from rat-derived P. carinii was rich in adenine and thymine, while the base composition of this gene from human-derived P. carinii was rich in guanine and cytosine. The sequence from the 18S rRNA gene of human-derived P. carinii was twice as divergent from that of rat-derived P. carinii as the sequence from the corresponding region of Candida albicans was from that of Candida tropicalis. These data show that rats and humans can harbor distinct types of P. carinii that are sufficiently different to suggest that P. carinii from the two hosts could be different species.     

S-adenosylmethionine and Pneumocystis carinii

We previously reported that S-adenosylmethionine (AdoMet), a key molecule in methylation reactions and polyamine biosynthesis, enhances axenic culture of the AIDS-associated opportunistic fungal pathogen Pneumocystis carinii. Here we report that AdoMet is absolutely required for continuous growth. Two transporters are present, one high affinity, K(m) = 4.5 microm, and one low affinity, K(m) = 333 microm. The physiologically relevant high affinity transporter has a pH optimum of 7.5 and no related natural compounds compete for uptake. Transport is 98% inhibited at 4 degrees C, 24% inhibited by 20 mm sodium azide, and 95% inhibited by the combination of 20 mm sodium azide and 1 mm salicylhydroxamic acid; thus transport is active and dependent on both a cytochrome chain and an alternative oxidase. In vitro, AdoMet is used at a rate of 1. 40 x 10(7) molecules cell(-1) min(-1). AdoMet synthetase activity was not detected by a sensitive radiolabel incorporation assay capable of detecting 0.1% of the activity in rat liver. In addition, the AdoMet plasma concentration of rats is inversely correlated with the number of P. carinii in the lungs. These findings demonstrate that P. carinii is an AdoMet auxotroph. The uptake and metabolism of this compound are rational chemotherapeutic targets.     

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.     

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.     

Diagnosis of Pneumocystis carinii pneumonia

Pneumocystis carinii is the prime opportunistic pathogen of our time, the leading cause of fatal pneumonia in the increasing number of immunosuppressed subjects encountered on oncology and transplant programmes' and in subjects with the acquired immuno-deficiency syndrome (AIDS).     

Immunopathogenesis of Pneumocystis carinii pneumonia

Pneumocystis carinii pneumonia (PCP) is a life-threatening infection that occurs in immunocompromised individuals, particularly those with advanced human immunodeficiency virus (HIV) infection. Interestingly, morbidity and mortality is related to the underlying cause of immunosuppression, with AIDS patients faring better than oncology patients for example. In addition, the prognosis of PCP has been correlated with markers of inflammation rather than with organism numbers. There is now increasing evidence that lung damage occurring during PCP is a result of the type and extent of the host inflammatory response to P. carinii rather than a result of direct damage by the organism. This review will discuss the experimental and clinical data demonstrating how the host-mediated inflammatory response to infection with P. carinii determines the ultimate outcome of PCP. A better understanding of the pathophysiology of PCP should lead to the development of improved therapies for the treatment of PCP.     

Uptake and Metabolism of L-Serine by Pneumocystis carinii carinii

ABSTRACT. Serine is an important amino acid that is utilized in the biosyntheses of proteins and lipids. It is directly incorporated into the head group of phosphatidylserine, which in turn can be converted to other phospholipids. Also, it is required for the formation of long chain bases, precursors of sphingolipids. Uptake and incorporation of radiolabeled serine into both lipids and acid-precipitable material were demonstrated in Pneumocystis carinii carinii organism preparations freshly isolated from infected rat lungs. Radioactivity in proteins was about double that observed in lipids. Liquid scintillation spectrometry of metabolically radiolabeled lipids separated by thin-layer chromatography showed 53% of the total radioactivity were in phosphatidylserine, 12% in phosphatidylethanolamine, 24% in ceramides, and 11% in long chain bases and other compounds. Four long chain bases were detected by thin-layer chromatography in hydrolyzed P. carinii ceramides metabolically labeled with radioactive serine. Phytosphingosine and dihydrosphingosine were tentatively identified by their migrations on thin-layer plates. Radiolabeled ethanolamine was incorporated into P. carinii phosphatidylethanolamine, but relatively low incorporation of radiolabeled choline into phosphatidylcholine occurred. The observations made in this study indicated that P. carinii has the biosynthetic capacity to metabolize phospholipid head groups and to de novo synthesize sphingolipids. L-Cycloserine and β-CI-D-alanine, inhibitors of long chain base synthesis, reduced the incorporation of serine into P. carinii long chain bases and ceramides, which supported the conclusion that the pathogen synthesizes sphingolipids.     

Glucan synthesis in Pneumocystis carinii.

Rat-derived Pneumocystis carinii lysed with sodium deoxycholate catalysed the incorporation of uridine diphosphoglucose 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 alpha 1----4 glucan, which is either unbranched or has relatively few branches. Polymer formation was completely inhibited by the addition of alpha amyloglucohydrolase to the supernatant. Polymer formation in the pellet of deoxycholate P. carinii preparations, unlike that in the supernatant, was partially resistant to alpha 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.