Most studies of antigens of Pneumocystis carinii have focused on an abundant, immunogenic 95- to 140-kDa surface glycoprotein referred to as gpA. Expression cloning of gpA from P. carinii obtained from ferrets resulted in isolation of colinear fragments of gpA cDNA encoding approximately 87 kDa of the core protein. Northern hybridization detected an abundant, single species of gpA-specific mRNA of 3600 nucleotides. Southern hybridization revealed gpA-specific bands only in P. carinii-infected lung genomic DNA, suggesting that gpA cDNA did not result from induction of a host lung gene. Antiserum raised against a fragment of recombinant gpA detected P. carinii cysts and isolated native P. carinii gpA, indicating retention of epitopes between the nonglycosylated recombinant gpA and glycosylated native gpA. The deduced amino acid sequence is hydrophilic and contains 12 potential N-linked glycosylation sites and 47 cysteine residues, consistent with the surface orientation of gpA on the organism and other known characteristics of the native molecule.

In a series of five experiments, we attempted to transmit Pneumocystis carinii from ferrets to SCID mice by intratracheal inoculation. Using highly specific and sensitive assay techniques, we could not document infection of SCID mice by P. carinii isolated from ferrets. In contrast, under identical inoculation conditions, P. carinii was easily transmissible from one SCID mouse to another. These results indicate that P. carinii organisms, at least those isolated from ferrets, have a restricted host range. The finding of restricted transmission ofP. carinii is consistent with the increasing evidence for host species-specific antigenic variation among isolates of P. carinii. If restricted host range is a consistent biological feature of animal-derived P. carinii, it would suggest that P. carinii pneumonitis in humans may not be a zoonosis as previously speculated.

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Although the liver is the primary site of fibrinogen (FBG) synthesis, epithelial cells from diverse tissues have been shown to express one or more of the FBG Aα, Bβ, and γ chain genes. In contrast, marrow megakaryocytes, which store FBG in the α-granules, are thought not to express the FBG genes. Our earlier studies have shown that epithelial cells in a variety of extrahepatic tissues express the γ chain gene ubiquitously, while the mRNAs for the Aα and Bβ chain genes are essentially undetectable. During systemic inflammation, the liver secretes increased levels of FBG into the circulation, and lung epithelium responds to local inflammation during pulmonary infection by increased transcription of the γ-FBG gene. Therefore, to determine whether extrahepatic epithelial cells express the Aα, Bβ, and γ chain genes in response to proinflammatory mediators, cultured lung epithelial cells were treated with interleukin-6 (IL-6) and dexamethasone (DEX). Northern blot analysis demonstrated that the levels of γ-FBG mRNA in cultured lung (A549) and liver (HepG2) epithelial cells increased 2- to 10-fold in response to treatment. Reverse-transcriptase-polymerase chain reaction amplification demonstrated increased accumulation of steady state levels of FBG Aα, Bβ, and γ chain mRNAs in lung epithelial cells after treatment. The basal level of lung cell γ-FBG gene transcription was not accompanied by appreciable levels of Aα and Bβ chain gene transcription; however, nuclear run-on analysis suggested that the increase in lung cell FBG mRNAs in response to DEX ± IL-6 was due to new transcription. Furthermore, stimulation of lung epithelial cells with IL-6 + DEX resulted in maximal secretion of intact FBG (340 kD) composed of the characteristic Aα, Bβ, and γ chain polypeptides. The data suggest that basal expression of the γ-FBG gene in extrahepatic tissue occurs ubiquitously in the absence of detectable levels of Aα- and Bβ-FBG gene expression, which are then upregulated on induction of an inflammatory response. This would result in local synthesis and secretion of FBG in the injured tissue, supporting the hypothesis that production of FBG by extrahepatic epithelial cells in response to inflammation plays a role in wound repair.

Two ferret P. carinii gpA cDNA clones were identified that reacted identically with a panel of anti-gpA monoclonal antibodies, although their nucleotide sequences were 22% divergent. Each clone hybridized to a single mRNA species of 3,600 nucleotides only in P. carinii-infected lung mRNA, but RT-PCR analysis demonstrated that these cDNA clones were derived from two distinct gpA mRNA transcripts. Further PCR analysis demonstrated that the ferret P. carinii genome contains at least two gpA genes lying in tandem on a single chromosome separated by a 329-bp intergenic region. Based on the terminal gene sequences of this tandem repeat and the cDNA clones, a composite full-length ferret P. carinii gpA coding sequence was constructed. The intergenic region immediately downstream of the stop codon of the first gpA gene contains three putative polyadenylation signals, and constitutes the 3' untranslated region (UTR) of the gpA mRNA. Primer extension of the gpA mRNA resulted in products extending 74 and 244 nucleotides into the 5' UTR. However, the intergenic region lying greater than 25 nucleotides upstream of the first methionine of the second gpA gene was found to be absent from the 5' UTR.