Signal Transduction in Pneumocystis carinii - Infection and Immunity

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Pneumocystis carinii is a eukaryotic organism that causes pneumonia in ... signal transduction pathways in P. carinii, we cloned a G-protein alpha subunit (G- ) ...
INFECTION AND IMMUNITY, Mar. 1996, p. 691–701 0019-9567/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 64, No. 3

Signal Transduction in Pneumocystis carinii: Characterization of the Genes (pcg1) Encoding the Alpha Subunit of the G Protein (PCG1) of Pneumocystis carinii carinii and Pneumocystis carinii ratti A. GEORGE SMULIAN,1,2* MARNIE RYAN,1 CHUCK STABEN,3 1,2 AND MELANIE T. CUSHION Infectious Disease Division, University of Cincinnati College of Medicine,1 and Cincinnati VA Medical Center,2 Cincinnati, Ohio, and T. H. Morgan School of Biological Sciences, University of Kentucky, Lexington, Kentucky3 Received 5 July 1995/Returned for modification 1 September 1995/Accepted 4 December 1995

Pneumocystis carinii is a eukaryotic organism that causes pneumonia in immunocompromised hosts. The cell biology and life cycle of the organism are poorly understood primarily because of the lack of a continuous in vitro cultivation system. These limitations have prevented investigation of the organism’s infectious cycle and hindered the rational development of new antimicrobial therapies and implementation of measures to prevent exposure to the organism or transmission. The interaction of P. carinii with its host and its environment may be critical determinants of pathogenicity and life cycle. Signal transduction pathways are likely to be critical in regulating these processes. G proteins are highly conserved members of the pathways important in many cellular events, including cell proliferation and environmental sensing. To characterize signal transduction pathways in P. carinii, we cloned a G-protein alpha subunit (G-a) of P. carinii carinii and P. carinii ratti by PCR amplification and hybridization screening. The gene encoding the G-a was present in single copy on a 450-kb chromosome of P. c. carinii and on a 420-kb chromosome of P. c. ratti. The 1,062-bp G-a open reading frame is interrupted by nine introns. The predicted polypeptide showed 29 to 53% identity with known fungal G-a proteins with greatest homology to Neurospora crassa Gna-2. Northern (RNA) blot analysis and immunoprecipitation demonstrated expression of the G-a mRNA and protein in P. carinii isolated from heavily infected animals. Some alteration in the level of transcription was noted in short-term maintenance in starvation or rich medium. Characterization of signal transduction in P. carinii will permit a better understanding of the reproductive capacity and other cellular processes in this family of organisms that cannot be cultured continuously.

ter understanding of the life processes of this family of organisms. Heterotrimeric G proteins are highly conserved members of the signal transduction pathway important in many intracellular signaling pathways, including cell proliferation and sensing of environmental conditions (19, 33). The best-studied fungal G proteins are those involved in the control of mating and cell proliferation in Saccharomyces cerevisiae and Schizosaccharomyces pombe (13, 16, 17, 20, 21). These G proteins consist of three subunits (abg), with the alpha subunit interacting with membrane-bound receptors that bind mating pheromones. In the most clearly characterized systems, the subunit associations change in response to environmental stimuli. The subunits then interact with target proteins that ultimately regulate protein kinase cascades that control cell proliferation and gene regulation. In other fungal systems, the roles of heterotrimeric G proteins are less clear. Cryphonectria parasitica (a pathogen of chestnut trees) has two G-protein alpha subunit (G-a) genes (2), and down-regulation of one, cpg-1, decreases the virulence of the fungus. It is likely that similar signal transduction pathways exist in P. carinii. Such pathways may be involved in the interaction of P. carinii with its environment, with host cells, or with other members of its species. In an attempt to understand the cellular biology of P. carinii and its

Pneumocystis carinii is an opportunistic pathogen that causes pneumonia, resulting in significant morbidity and mortality among immunocompromised individuals. Very little is known about the basic biology of the organism because no isolate of the organism has been maintained continuously in vitro. Although transmitted by an airborne route, the infectious particle has not been identified (3). Similarly, whether an environmental cycle exists or whether it is necessary for infection is not known. Recently, two putative species of rat-derived P. carinii have been characterized by several molecular genetic and antigenic criteria, including electrophoretic karyotype, nuclear small-subunit RNA sequences, hybridization profiles with single-copy and multiple-copy gene probes (5), and immunoblotting techniques (30). These two candidate species have been provisionally designated P. carinii f. sp. carinii and P. carinii f. sp. ratti (22). In the present study, it was our strategy to begin characterizing genes and gene products involved in essential cellular pathways in Pneumocystis organisms to permit a bet-

* Corresponding author. Mailing address: 231 Bethesda Ave., Cincinnati, OH 45267-0560. Phone: (513) 558-4704. Fax: (513) 559-5646. Electronic mail address: [email protected]. 691

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response to the environment, we have cloned a G-a gene of P. c. carinii and P. c. ratti and analyzed its expression in vivo and in vitro.

MATERIALS AND METHODS Isolation of P. carinii organisms and nucleic acids. P. carinii organisms were isolated from infected lung homogenates prepared from corticosteroid-treated Lewis and Sprague-Dawley rats (1, 31). Briefly, lungs were homogenized, host cells were lysed with NH4Cl (0.85% aqueous solution), and organisms were purified by repeated centrifugation. The preparations were then visualized microscopically to determine the extent of contamination by host cells or other microbes and to enumerate the organisms. To obtain P. carinii genomic DNA, organisms were embedded in low-melting-point agarose and treated with Sarkosyl and proteinase K (4, 5). Plugged DNA was utilized for pulse-field gel electrophoresis, restriction digestion, and Southern blotting under standard conditions (24). Template DNA for PCR was obtained by b-agarase treatment of plugged DNA followed by ethanol precipitation. RNA was isolated from P. carinii organisms by extraction in guanidine isothiocyanate-acid phenol-chloroform (Trizol; Bethesda Research Laboratories, Gaithersburg, Md.). Oligonucleotide primers. Oligonucleotide primers for cDNA synthesis, rapid amplification of cDNA ends (RACE), and PCR were supplied as part of the Marathon cDNA amplification kit (Promega, Madison, Wis.; cDNA synthesis primer and AP1 primer) or synthesized by the DNA core facility of the Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine. Primers synthesized by the DNA core facility were as follows: primer RRKWIH, AA(A/G)AA(A/G)TGGAT(T/A/C)CA(T/C) TG(T/C)TT(T/C)G; LFLNKI, TC(A/G/T)AT(C/T)TT(A/G)TTIA(G/A)(A/G) AAIA(G/A)(A/G/T)AT; LGAGE, T(T/G/C/A)(C/T)T(T/C/G/A)GG(A/G/T)G CIGG(A/G/T)GAI(A/T)(G/C)IGG; GS 0, TTTTAAGAATTCGGATGTTGTT TTTCTGC; GS 1, AGGCGCTGGAGAAAGTGG; GS 2, CGACGAGAAAA CGAAGTAATTTG; GS 3, AATATCCATAGACTTTTTGAAAGG; GS 4, CG AATGCAAGAGGCTCTTG; GAS 1, CACGTTCTTTCAATGTTAAGCC; GAS 2, CAATTATCATGCAAAGCA; GAS 3, CATACAGCGCCAATATCTTCTG; GAS 4, AGCGTTGTATCAGTCGC. PCR amplification of P. c. carinii G-a. Amplification was performed under nonstringent conditions with P. c. carinii (form 6) genomic DNA as the template with degenerate primers to the conserved amino acid sequences RRKWIH and LFLNKI (see Fig. 1). The amplification conditions used consisted of a 5-min hot start at 948C followed by 30 cycles of 948C for 60 s, 378C for 60 s, and 728C for 60 s. A single dominant 280-bp product identified on agarose gel electrophoresis was purified and cloned into the vector pGEM-T (Promega). The amplified product was sequenced by a modified dideoxy nucleotide chain termination sequencing technique (Sequenase v2.0; Amersham Life Science, Arlington Heights, Ill.). Additional primers were designed on the basis of the highly conserved amino acid sequence LGAGE and P. carinii-based sequence (GAS 3) within the initial 280-bp amplified product. These were used to amplify the major portion of the gene (30 cycles of 948C for 60 s, 458C for 60 s, and 728C for 120 s). Subsequently, the 59 and 39 portions of the gene were amplified from P. c. carinii genomic template DNA with primers targeted to sequences within the 59 and 39 untranslated portion of the cDNA. Cloning of P. c. carinii G-a cDNA. First-strand cDNA was reverse transcribed from 1 mg of total RNA from freshly isolated P. c. carinii (form 1) organisms by use of an oligo(dT) primer and avian myeloblastosis virus reverse transcriptase (Marathon cDNA amplification kit; Promega). Following second-strand synthesis, the cDNA was blunt-ended and ligated to oligonucleotide adapters. The 59 portion of the G-a cDNA was amplified (by a hot start followed by 30 cycles of 948C for 30 s, 508C for 30 s, and 728C for 120 s) with a gene-specific primer (GAS 3) located within the initial amplicon and a primer (AP1) complementary to the adapter ligated during cDNA synthesis. The resulting amplification product was gel purified and digested with BclI within the known gene sequence and NotI within the adapter primer. The digested product was cloned into pSKII1 digested with NotI and BamHI. The cDNA was sequenced by dideoxy chain termination with vector primers T3 and T7 and internal gene-specific primers. Similarly, the 39 portion of the cDNA was amplified by the RACE technique with the cDNA synthesis primer (cDNAp) and a gene-specific primer (GS 3). Successful amplification was achieved by initial PCR amplification with primers AP1 and GS 0 under the following conditions: 30 cycles of 948C for 60 s, 508C for 60 s, and 728C for 120 s, followed by nested PCR with a 1:100 dilution of the initial amplification reaction. Amplification with internal primers GS 3 and AP2 for 30 cycles of 948C for 60 s, 508C for 60 s, and 728C for 90 s followed. The single

INFECT. IMMUN. dominant 700-bp band was gel purified and cloned in the vector pGEM-T. The sequence of the amplification product was obtained by the dideoxy chain termination method. To obtain a full-length cDNA, the 59 RACE and 39 RACE products were fused by annealing the overlapping 200-bp portion common to both products. The 59 RACE PCR product was digested with BclI to remove the primer sequences which included three bases extending into intron 5 at the 59 end of the primer. Equimolar quantities of the 59 RACE product and 39 RACE were combined, denatured at 948C for 2 min, allowed to anneal at 658C for 2 min, and filled in by a mixture of Taq and Pwo DNA polymerase (Expand Long Template PCR System; Boehringer Mannheim, Indianapolis, Ind.) in the presence of deoxynucleoside triphosphates at 728C for 5 min. Five cycles of annealing and extension were performed, followed by the addition of primers at the 59 end of the coding sequence and within the 39 cDNA synthesis primer (GS 0 and AP1, respectively). Full-length cDNA was amplified for 35 cycles of 948C for 60 s, 558C for 60 s, and 728C for 300 s. The amplification product was digested with EcoRI and NotI, which cleave at sites within the amplification primers, and cloned into EcoRI-NotI-digested pSKII1. Isolation of the G-a gene of P. c. ratti. A rat-derived P. carinii genomic library constructed in lgt11 was obtained as a generous gift from J. R. Stringer, University of Cincinnati College of Medicine, and screened by hybridization with the initial 280-bp amplification product as a probe. Multiple clones were isolated and plaque purified, and a single clone was characterized. A 3.1-kb EcoRI fragment was excised from the lgt11 clone and subcloned into pSKII1. Single-strand phagemid template DNA was generated with helper phage VCSM13 and sequenced by the dideoxy chain termination method. Southern and Northern (RNA) blot analysis. For pulsed-field gel electrophoresis and restriction digestion, P. carinii organisms were embedded in 1.2% low-gelling agarose as described above. Chromosomal-size DNA bands were separated by contour-clamped homogeneous electrical field (CHEF) electrophoresis as described previously (4). Restriction digestion was performed on DNA embedded in agarose for conventional agarose electrophoresis and Southern blotting. Hybridization was performed under standard conditions (24). Total RNA was isolated from freshly isolated P. c. carinii organisms with guanidine isothiocyanate-acid phenol-chloroform. Five micrograms of total RNA was separated by formaldehyde agarose gel electrophoresis and transferred under neutral conditions to a nylon membrane. The blot was prehybridized and hybridized in the presence of 10% sodium dodecyl sulfate (SDS)–50 mM Na phosphate (pH 7.0)–50 mM PIPES [piperazine-N,N9-bis(2-ethanesulfonic acid)]–100 mM NaCl–1 mM EDTA (pH 8.0) at 658C. Antiserum production. A 14-amino-acid peptide, DICFERRENEQYLD, corresponding to residues 92 to 105 of the predicted P. carinii G-a protein sequence was synthesized (Genemed Biotechnologies, San Francisco, Calif.). The peptide was conjugated to maleimide-activated keyhole limpet hemocyanin as described in the manufacturer’s instructions (Pierce, Rockford, Ill.). A female New Zealand White rabbit was immunized with 100 mg of antigen in complete Freund’s adjuvant. The animal’s immunity was boosted with 50 mg of antigen in incomplete Freund’s adjuvant 14, 21, and 49 days following the initial immunization. In addition, an antipeptide antiserum raised against a carboxy-terminal peptide of Cryphonectria parasitica CPG-1 was obtained as a generous gift from Donald Nuss (2). Immunoprecipitation. P. carinii organisms isolated from heavily infected immunosuppressed animals were isolated as described previously (31). Approximately 108 organisms suspended in 100 ml of solubilization buffer (190 mM NaCl, 6 mM EDTA, 60 mM Tris [pH 7.4], 4% SDS) were sonicated for 30 s. The suspension was immediately heated at 1008C for 4 min, and 1 volume of distilled water and 4 volumes of dilution buffer (190 mM NaCl, 6 mM EDTA, 50 mM Tris [pH 7.4], 2.5% Triton X-100) were added. After a brief centrifugation, the supernatant was removed and incubated with 10 ml of P. c. carinii G-protein antiserum or prebleed serum from the immunized rabbit overnight at 48C. Thirty microliters of 1:1 protein G-agarose (Sigma Chemical Co., St. Louis, Mo.) in water was added, and the mixture was incubated at room temperature for 2 h with rotation. After centrifugation, the pellet was washed four times in 150 mM NaCl–50 mM Tris (pH 7.4)–5 mM EDTA–0.1% Triton X-100–0.02% SDS and then twice in 150 mM NaCl–50 mM Tris (pH 7.4)–50 mM EDTA, resuspended in 40 ml of SDS-lysis buffer of 2% SDS–0.06 M Tris (pH 6.8)–1% glycerol–5% 2-mercaptoethanol, boiled for 3 min, cooled, and briefly centrifuged at 15,000 3 g. The supernatants were electrophoresed in 0.1% SDS–discontinuous 12.5% polyacrylamide gels (14). Separated proteins were transferred to nitrocellulose (28). Duplicate samples (proteins immunoprecipitated by prebleed serum or P. c. carinii G-protein antiserum) were incubated with P. c. carinii G-protein antiserum (diluted 1:500) or Cryphonectria parasitica G-protein antiserum (diluted 1:500) overnight at 48C. Strips were washed, incubated for 2 h at room temper-

FIG. 1. (A) Schematic representation of the amplification and cloning strategies for the P. c. carinii (Pcc) G-a gene pcg1. (B) Diagrammatic representation of 59 and 39 RACE to generate the pcg1 cDNA. DNA sequencing of both strands of the cDNA was performed with the identified oligonucleotide primers. (C) Genetic organization of the G-as of P. c. carinii and P. c. ratti (Pcr) pcg1 genes, mRNA, and ORF. Recognition sequences for the restriction enzymes BclI, ClaI, and HindIII are identified as B, C, and H, respectively.

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FIG. 2. Comparison of P. c. carinii and P. c. ratti G-a amino acid sequences with known fungal G-a proteins. Sequences compared are as follows: PccG, P. c. carinii PCG1; PcrG, P. c. ratti PCG1; NcG-2, N. crassa Gna-2; CpG-1, Cryphonectria parasitica CPG-1; NcG-1, N. crassa Gna-1; SpG-1, Schizosaccharomyces pombe Gpa1; CaG-1, Candida albicans CAG1 (23); ScG-1, Saccharomyces cerevisiae GPA1 (6); CpG-2, Cryphonectria parasitica CPG-2; CnG-1, Cryptococcus neoformans GPA1; ScG-2, Saccharomyces cerevisiae GPA2 (18); SpG-2, Schizosaccharomyces pombe Gpa2; RGa; Rat Gai. Nonconserved sequence inserts at positions 1 to 5 were excluded for purposes of comparison. Amino acid identity is depicted by an asterisk and similarity is depicted by a period below the sequence.

ature with affinity-purified peroxidase-conjugated goat antibody to rabbit immunoglobulin G (Kirkegaard & Perry, Gaithersburg, Md.), washed, and developed with 4-chloro-naphthol hydrogen peroxide substrate. In vitro modulation of G-a mRNA. Freshly isolated P. c. carinii organisms from a single heavily infected animal were enumerated (1). One aliquot containing 2 3 109 P. c. carinii organisms were harvested immediately, and total RNA was isolated, while the remaining organisms were placed in Pneumocystis maintenance media consisting of RPMI 1640, 20% fetal bovine serum, and vitamin, mineral, and amino acid supplements (1) or in low-nitrogen medium based on Cryptococcus neoformans mating agar (American Type Culture Collection) containing (per liter) 2.0 g of sucrose, 100 g of KH2PO4, 0.5 g of MgSO4, 0.1 g of CaCl2, 0.1 g of NaCl, 0.03 g of leucine, and 5.0 mg of biotin (pH 6.2). Aliquots containing 2 3 109 organisms were harvested from each medium after 1, 2, 4, 24, and 48 h, and total RNA was isolated. At each time point, organisms were examined microscopically, and ATP content was determined by bioluminescence to assess viability (1). Total RNA was quantitated, normalized, and analyzed by Northern blot assay. Sample load was verified by hybridization with a constitutive housekeeping gene probe, thymidylate synthase (9). Autoradiographic signal intensity was measured by densitometric scanning on a FOTOanalyst II documentation system with Collage 4.0 software (Fotodyne, Hartland, Wis.). G-a mRNA levels were normalized for sample load differences by expressing the G-a mRNA signal intensity relative to the thymidylate synthase signal intensity. Sequence analysis and nucleotide sequence accession numbers. Nucleotide

and amino acid sequences were analyzed and compared with the Nalign, Palign, and Clustal programs of PCGENE software (IntelliGenetics, Inc., Mountain View, Calif.). These sequences have been deposited in GenBank under the accession numbers U30790, U30791, and U30792.

RESULTS PCR amplification of a heterotrimeric G-a gene of P. c. carinii. The G-a contains sequences involved in guanine nucleotide binding that are particularly well conserved among eukaryotes and have been used successfully to design degenerate amplification primers for cross-species amplification (27). These conserved sequences and P. carinii codon bias were used to design degenerate oligonucleotides to target the conserved GTP binding sequences RRKWIH and LFLNKI (Fig. 1A). A 280-bp DNA segment was amplified from purified P. c. carinii DNA with these primers under nonstringent conditions. This 280-bp fragment contained a potential open reading frame (ORF) interrupted by a presumed 46-bp intron. The

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FIG. 2—Continued.

predicted peptide showed 47 to 80% similarity to known fungal G-a proteins (Fig. 2). The amplified sequence hybridized at high stringency to a 450-kb P. c. carinii chromosome Southern blot of P. carinii chromosomal bands separated by CHEF electrophoresis (Fig. 3). The amplified sequence did not hybridize to rat host DNA (data not shown). These observations indicated that the amplified sequence was a portion of a G-a gene, pcg1, of P. c. carinii origin. Isolation of the G-a cDNA by 5* and 3* RACE. A complete cDNA encoding the G-a protein, PCG1, was produced by RACE (Fig. 1B). This cDNA included gene expression signals typical of P. carinii genes and encoded a G-a protein homolog most closely related to Neurospora crassa Gna-2 (29). The cDNA contained a 1,062-bp ORF predicted to encode a 354amino-acid protein homologous to the G-a family (Fig. 2). The sequence included a predicted translational start site and 90 bases of 59 untranslated sequence. The predicted translational start was the first AUG within the mRNA. This AUG initiated a long continuous ORF, and the sequence surrounding the AUG strongly resembled the eukaryotic consensus sequence for efficient translational initiation (8, 12). The codon usage

within the ORF was typical of that seen in other P. c. carinii genes, with 79% A or T usage in the third position within codons (25). The polypeptide encoded by the amplified cDNA is clearly a member of the G-a family. The deduced amino acid sequence showed 29 to 53% identity with known fungal G-as. The regions implicated in guanine nucleotide binding showed much higher identity (Fig. 2). The sequence contained the arginine residue sensitive to ADP-ribosylation by cholera toxin but lacked the cysteine residue sensitive to pertussis toxin-mediated ADP-ribosylation. The NH-terminal glycine important in myristylation and membrane association was conserved as in other G-a proteins. Characterization of the genes encoding G-as of P. c. carinii and P. c. ratti. The entire region encoding the G-a was amplified from genomic P. c. carinii template DNA with primers located at the translational start site and near the stop codon (Fig. 1A). The template DNA used for amplification was isolated from organisms with a karyotypic pattern different from those used to isolate the RNA for cDNA synthesis (form 1 versus form 6) (4). The amplified DNA sequences

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were sequenced directly or subcloned and sequenced. Comparison of the cDNA and genomic sequences revealed nine introns ranging in size from 42 to 48 bases flanked by eukaryotic consensus donor and acceptor sequences, GT and AG, respectively (Fig. 1C). The amplified genomic sequence differed from the cDNA sequence at 8 of 956 (0.8%) nucleotides within the coding region. This minor difference was thought to represent differences between form 1 and form 6 organisms. This hypothesis was verified by amplifying and directly sequencing a variable region from form 1 and form 6 organisms. The genomic sequence from form 1 organisms was identical to that of the cDNA, while that from form 6 organisms matched the previously sequenced genomic DNA (data not shown). To obtain a full genomic clone, the G-protein-encoding sequence was labeled and used to screen a lgt11 P. carinii genomic library. This library was constructed from DNA isolated from a rat with a mixed infection of P. c. carinii (form 1) and P. c. ratti. The number of plaques identified was appropriate for a low-copy-number gene. The single plaque analyzed, l10-1-1, contained a 3.1-kb insert which was subcloned into pSKII1. The restriction map of this DNA differed from that predicted from the amplified genomic sequence. The sequence of this cloned genomic DNA indicated that this DNA was slightly different from the initial P. c. carinii G-a gene. The gene differed from the initial P. c. carinii G-a gene by 17.5% at a nucleotide level and 7.9% at a predicted amino acid sequence level (Fig. 4). The ORF was interrupted by nine introns, which ranged in size between 41 and 50 bp. The introns were in positions identical to those within the initial P. c. carinii G-a gene. We hypothesized that this gene encoded the G-a of P. c. ratti rather than P. c. carinii. This hypothesis was verified in two ways. First, the putative P. c. ratti G-a probe hybridized more strongly to P. c. ratti DNA than to P. c. carinii DNA under stringent conditions. In mixed P. c. carinii-P. c. ratti samples, the putative P. c. ratti pcg1 probe demonstrated stronger hybridization to a P. c. ratti chromosomal band of 420 kb than to the 450-kb P. c. carinii chromosomal band. The converse was true when a putative P. c. carinii probe was used to probe the same blot (Fig. 3). Second, a portion of the G-a amplified from DNA isolated from a rat known to harbor only P. c. ratti organisms was identical to the sequence obtained from the lambda genomic clone l10-1-1 (data not shown). Copy number of G-a genes. Hybridization of G-a probes from either P. c. carinii or P. c. ratti to Southern blots of CHEF gels of P. carinii DNA demonstrated hybridization with only a single chromosomal size band, except in mixed P. c. cariniiP. c. ratti infections, where there were two (Fig. 3). To exclude the presence of two linked genes within this band or genes on two comigrating chromosomal size bands, Southern hybridization of restriction enzyme-digested P. c. carinii DNA was performed. The presence of a single band suggests that the signal detected on the CHEF Southern blot is produced by hybridization to the cloned G-a gene but does not

FIG. 3. Localization of the G-a gene to chromosomal size bands of P. c. carinii and P. c. ratti. (A) P. carinii chromosomal size DNA from P. c. carinii forms 1 to 4 and 6 to 8 (lanes 1 to 7, respectively), P. c. ratti DNA (lane 9), and a mixed infection containing P. c. carinii form 1 and P. c. ratti (lane 8) were separated by CHEF electrophoresis and stained with ethidium bromide. (B and C) A Southern blot of the CHEF gel was probed with a 32P-labeled P. c. carinii G-a DNA probe (bases 120 to 990) (B) or with a P. c. ratti G-a probe (bases 300 to 970) (C). The specificities of the two probes are most clearly visible in the mixed infection in lane 8. The probe used in panel C had a higher specific activity than that used in panel B. Sizes in kilobases are indicated on the left.

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exclude the possibility of other distantly related G-a genes (Fig. 5). Expression of the G-a gene in P. c. carinii. The G-a gene was transcribed and translated in P. carinii. Northern blot analysis showed that pcg1 is transcribed in P. c. carinii (Fig. 6). A single 1.4-kb message was detected. To determine whether the message was translated, P. c. carinii G-protein antiserum was used to immunoprecipitate proteins from P. c. carinii organisms. Immunoprecipitated proteins were subjected to Western blot (immunoblot) analysis and reacted with a monospecific polyclonal antiserum raised against a P. c. carinii G-a peptide and a monospecific polyclonal antiserum raised against a C-terminal peptide from the G-a of Cryphonectria parasitica CPG-1 (2). P. c. carinii anti-G-protein antiserum precipitated a 40kDa protein that reacted with both the antiserum used for precipitation and the Cryphonectria parasitica G-protein antiserum (Fig. 7). Preimmune serum failed to precipitate immunoreactive proteins. A second smaller protein of unknown significance was also precipitated by the P. c. carinii G-protein antiserum. In vitro modulation of G-a mRNA. Both freshly isolated and cultured P. c. carinii expressed pcg1 mRNA. RNA from P. c. carinii freshly isolated from rat lung contained a 1.4-kb mRNA. G-a mRNA increased twofold over 4 h in organisms maintained in standard medium compared with the baseline and with organisms maintained in starvation medium used to induce fungal mating (Fig. 6). Organisms maintained .90% viability in both rich and starvation media during this time period. After 24 h in starvation medium, organism viability dropped significantly, and no intact RNA could be isolated from these organisms. DISCUSSION Heterotrimeric G proteins are highly conserved elements of the signal transduction pathway of fungi and higher eukaryotes. In higher eukaryotes, specificity of response to external stimuli is maintained by the presence of many (.20) G-as that interact with specific G-protein-coupled transmembrane receptors (19). Among the fungi studied, the G-a diversity is more limited, with only one or two G-a genes having been identified in a given organism. Some heterotrimeric G proteins of fungi have clear roles in the pheromone response and mating cascade and in response to nitrogen starvation (nutritional response). We have taken advantage of the high level of conservation among G proteins to clone and characterize P. carinii genes encoding the G-a. The deduced G-a polypeptide sequence was homologous to the sequence of other fungal and mammalian G-a subunit proteins. This similarity was greatest in regions functionally important in GTP binding but ranged from 29 to 53% identity among fungal G-as. Like all G-a polypeptides, it contains a consensus myristylation site near the NH terminus. The P. carinii G-a is not a member of the G-ai subclass because it lacks the carboxyl-terminal cysteine residue sensitive to ADPribosylation by pertussis toxin. P. carinii exposed to pertussis toxin in short-term maintenance culture did not decrease in viability (data not shown). The P. carinii G-a does, however, contain the arginine residue required for ADP-ribosylation by cholera toxin. Although not a G-ai subclass member, the P. c. carinii G-a protein bears close resemblance to the two G-ais N. crassa Gna-1 (52.4% identity) and Cryphonectria parasitica CPG-1 (52.7% identity). The role of these G-a proteins is not known (2, 11). The structure of the pcg1 gene resembles that of other

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cloned P. carinii genes. The ORF has a marked AT bias in the third position of codons. The translational start site is also consistent with consensus sequences. The ORF is interrupted by nine short introns flanked by consensus eukaryotic donor and acceptor site sequences. Introns within the G-a genes are uncommon in ascomycetes. The gpa2 gene of Schizosaccharomyces pombe has one intron; the published report of N. crassa gna-2 contains one intron in the portion examined (10, 29). In contrast, GPA1 of Cryptococcus neoformans contains seven introns (27). Multiple introns have been found in several P. carinii genes such as the TATA binding protein and alphatubulin genes (26, 34). The untranslated regions, while rich in adenine and thymidine residues, are less than 100 bases in length. Our finding of variant G-a DNA sequences suggests important experimental and biological considerations. Recently, two genetic variants of P. carinii residing within a single mammalian host, the rat, were identified by several genetic and antigenic criteria (5, 30). Genetic differences were apparent in the sequence of a portion of the nuclear small rRNA gene (i.e., ;7%), the presence or absence of a type I self-splicing intron in the small rRNA gene, electrophoretic karyotype, and homology with a repetitive probe containing genes for the major surface glycoproteins of P. carinii (5). Antigenic differences between the two putative species were detected by probing the separated proteins of the organism with a panel of monoclonal antibodies and polyclonal antisera (30). The differences between the two types of organisms were compatible with those observed between other species of yeast (e.g., Candida tropicalis and Candida albicans), and it was recommended at a recent workshop on P. carinii that the nomenclature be changed to reflect these differences (22). The organism most frequently found in the lungs of rats was termed P. carinii f. sp. carinii (formerly, prototype), while the other type was named P. carinii f. sp. ratti (formerly, variant). Of the P. c. carinii organisms, eight individual forms have been identified by the technique of electrophoretic karyotyping. Five have been identified previously (4, 32), while the other three have been characterized during a recent survey of rat colonies in the United States (3a). Genetic differences among the karyotypic forms were minimal (,0.5%) when portions of the small rRNA and mitochondrial largesubunit RNA genes were sequenced and compared (3b), suggesting that these karyotypic forms did not differ at a species level but were representative of strain differences or genetic drift. The G-a gene sequences differed by 17.5%, while the predicted polypeptides differed by 7.9%. The magnitude of these differences is similar to the species-level sequence differences and support the designation of P. c. carinii and P. c. ratti as separate Pneumocystis species (5). The differences may predict functionally important domains since variant regions are unlikely to be critical domains. The hybridization and amplification data strongly suggest that the two cloned genes are homologs of the same G-a gene from P. c. carinii and P. c. ratti rather than two genes from the same species. A G-a probe predicted to be of P. c. carinii origin hybridized more strongly to a P. c. carinii chromosome than to a P. c. ratti chromosome, and the converse was true with a P. c. ratti G-a probe. G-a segments amplified from a genomic DNA template from known P. c. carinii- or P. c. ratti-infected animals contained only a single sequence corresponding to that predicted by the karyotype of the infecting organisms. The similarity in the polypeptides encoded by P. c. carinii and P. c. ratti pcg1 genes is much higher (92%) than the similarity between two G-a’s encoded in other fungal genomes (30 to 46%). Rat colonies are

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FIG. 4. Comparison of the nucleotide sequences of the G-a genes of P. c. carinii and P. c. ratti. The regions encoding pcg1 of P. c. ratti (Pcr) and P. c. carinii (Pcc) are shown. Intron sequences are depicted in lowercase letters. Identity between the nucleotide sequences is indicated by a vertical line.

frequently infected with different strains of P. carinii defined by electrophoretic karyotype as well as with the two species (5). Small differences observed between the G-a sequences from form 1 and form 6 P. c. carinii organisms are in keeping with strain-level differences. The isolation of two G-a genes emphasizes the necessity of careful documentation of the source of organisms and DNA especially when amplifying gene sequences. This report is the first characterization of a gene involved in the intracellular signaling systems of P. carinii. The detection of both G-a mRNA and protein within P. carinii provide evidence that serpentine receptor–G-protein signal transduction pathways in Pneumocystis organisms are functional. Immunoelectron microscopy demonstrated a pleomorphic distribution

of the G-a within the organisms (data not shown). The ease with which mRNA and protein were detected and the level of expression in freshly isolated organisms could be explained by a high level of constitutive expression, by up-regulation due to the alveolar environment in terminally ill animals, or by upregulation of expression by factors within the animal model such as corticosteroids. Expression of mammalian G-a0 proteins can increase in the presence of corticosteroids (15). Although the stimulus for transcription in P. carinii is not known, the repertoire of stimuli to which G-protein-coupled receptors (and G proteins) respond in fungi is limited to pheromones, nitrogen starvation, nutritional stimuli, and mating-type regulatory genes (10, 13). Within the environment of the mammalian alveoli, it is conceivable that any one or more of these

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FIG. 4—Continued.

factors were present and provoked the mRNA levels that were detected. One stimulus known to initiate G-protein-coupled responses in fungi, i.e., nitrogen starvation, had no apparent effect on pcg1 mRNA levels in vitro. The starvation medium used to deprive the organisms of nitrogen and other nutrients was based on culture media known to induce mating in fungi such as Cryptococcus neoformans, but such conditions were not appropriate for P. carinii and caused a dramatic decline in viability. Varied responses of G-a transcription from different fungi have also been noted. G-a transcription increases under conditions supportive of mating in Cryptococcus neoformans (27), while in Saccharomyces cerevisiae, little change in the mRNA level is detected, but increased activity of the Saccharomyces cerevisiae G-a is associated with an alteration in the myristylation state of the G-a protein (7). Production of a different-size transcript is seen in response to stimulation of the pathway in Schizosaccharomyces pombe (21). The failure to detect changes in mRNA levels under the starvation conditions studied will be interpretable only in conjunction with additional data on changes in the functional activity of the G proteins under these and other conditions. Despite the inability to sustain long-term culture of P. carinii, the short-term maintenance culture does allow evaluation

of the organism’s response to mating or sporulation formulations. Characterization of the signal transduction pathway in P. carinii will be valuable in determining the response of the organism to its microenvironment within the lung. This is especially relevant given the inability to sustain long-term in vitro culture of the organism. Response to the microenvironment may identify factors that lead to successful cultivation of the organism and may provide a tool with which to rapidly assess experimental conditions. Full characterization of the pathway and its interaction with specific receptors-ligands may further delineate the role of sexual reproduction in this class of organisms. ACKNOWLEDGMENTS This work was supported by a Research Challenge grant from the University of Cincinnati, by the Medical Research Service, Department of Veterans Affairs, and by grants AI-29839 and AI-25139 from the National Institutes of Health. We thank Mike Linke for assistance and advice, Natalie Kloepfer for excellent technical assistance, Jim Stringer for the lgt11 P. carinii genomic library and the cloned P. carinii thymidylate synthase gene, and Donald Nuss for antisera.

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FIG. 7. Detection of immunoprecipitated P. c. carinii G-a protein by immunoblotting. Proteins immunoprecipitated by preimmune serum (lane 1) or P. c. carinii G-protein antiserum (lanes 2 and 3) were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose. Strips were reacted with P. c. carinii G-protein antiserum (lanes 1 and 2) or Cryphonectria parasitica G-protein antiserum. A protein of approximately 40 kDa precipitated by the P. c. carinii G-protein antiserum reacted with both the P. c. carinii G-protein antiserum and the Cryphonectria parasitica G-protein antiserum. The heavy immunoreactive bands of 52 kDa and larger represent rabbit immunoglobulin in the antiserum used for immunoprecipitation that was precipitated by the protein G-agarose. Sizes in kilodaltons are indicated on the left.

FIG. 5. Southern blot analysis of P. c. carinii DNA. P. c. carinii DNA digested with the restriction enzymes listed below was separated by agarose gel electrophoresis and transferred to a nylon membrane. Lanes: 1, SalI; 2, PstI; 3, EcoRI; 4, EagI. The Southern blot probed with a 32P-labeled G-a cDNA probe revealed single bands in all lanes with the exception of the lane containing EcoRI-digested DNA. The gene sequence contains an EcoRI site at position 636. Sizes in kilobase pairs are indicated on the left.

FIG. 6. Northern blot analysis of P. c. carinii RNA. Hybridization of a 32Plabeled G-a probe (bases 120 to 990) to a Northern blot of P. c. carinii total RNA revealed a single mRNA transcript of approximately 1.4 kb (upper panel). Total RNA isolated from freshly isolated P. c. carinii organisms (lane 1) or nitrogendeficient starvation medium for 1, 2, or 4 h (lanes 2, 4, and 6) showed no apparent change in G-a mRNA level, while organisms maintained in short-term maintenance culture in rich medium for 1, 2, and 4 h (lanes 3, 5, and 7) increased the level of G-a mRNA transcription. The Northern blot was probed with thymidylate synthase, a constitutive housekeeping gene (lower panel), to normalize for differences in mRNA loads. Sizes in kilobases are indicated on the left.

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