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Characterization of a mitogen-activated protein kinase from Pneumocystis carinii

Charles F. Thomas Jr.1, Theodore J. Kottom1, Edward B. Leof1,2, and Andrew H. Limper1,2

1 Thoracic Diseases Research Unit, Division of Pulmonary, Critical Care and Internal Medicine and 2 Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, Minnesota 55905

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The pathogenic fungus Pneumocystis carinii causes severe pneumonia in patients with impaired immunity, particularly patients with acquired immunodeficiency syndrome. The life cycle of P. carinii is poorly understood, and the inability to continuously culture P. carinii is a major limitation in understanding its cell biology. In fungi homologous to P. carinii, pheromone mating factors signal through a mitogen-activated protein kinase (MAPK) signal transduction cascade, resulting in mitotic cell cycle arrest and entry into a pathway of conjugation, cellular differentiation, and proliferation. Using degenerate PCR and library screening, we have identified a MAPK cDNA in P. carinii that is highly homologous to fungal MAPKs involved in the pheromone mating signal transduction cascade, and we demonstrate MAPK activity in P. carinii lysates with a specific antiserum derived from the translated P. carinii MAPK cDNA sequence.

acquired immunodeficiency syndrome; serine-threonine protein kinase; signal transduction; molecular cloning

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

PNEUMOCYSTIS CARINII causes severe pneumonia in patients with an impaired immune system, particularly in patients with acquired immunodeficiency syndrome (AIDS), solid or hematological malignancies, or organ transplantation or patients requiring chronic immunosuppressive therapy for inflammatory conditions (14, 27, 31). Despite the availability of a number of effective medications for prophylaxis and treatment of P. carinii pneumonia, the morbidity and mortality remain in the range of 15-40%, being higher in patients without AIDS (14, 31). Since its discovery, investigators have been unsuccessful in attempts to continuously culture P. carinii. A variety of media, cell lines, and supplements have been tried, with limited success in sustaining growth for more than a few weeks, and none has been successful in propagating P. carinii continuously (2, 24). Currently, the immunosuppressed rat model is the most widely used source of obtaining P. carinii organisms for study; however, even this model occasionally yields isolates of P. carinii mixed with other microogranisms or host cells (2, 24). The inability to consistently grow and propagate P. carinii poses a serious hindrance to understanding the cell biology of this important fungal pathogen.

Recent investigations have classified P. carinii with the fungi (6). Molecular analysis of ribosomal RNA places P. carinii on a branch within the Ascomycetes, having homology to Saccharomyces cerevisiae and Schizosaccharomyces pombe (6). Microscopically, P. carinii has a complex life cycle composed of two distinct life forms (4, 11). Trophic forms (trophozoites) are 1-2 µm in size and are the most abundant life cycle form, outnumbering cysts 10 to 1. Trophic forms attach to the alveolar epithelium through adhesive glycoproteins (13, 15). Cysts, which are 8-10 µm in size, contain multiple intracystic bodies (up to 8 in number) that mature and subsequently leave the cyst as trophic forms (16). The mechanisms regulating growth and differentiation of the life cycle forms are currently unknown.

In fungi homologous to P. carinii, differentiation and proliferation are regulated by mitogen-activated protein kinase (MAPK) signal transduction cascades (1, 7, 19). MAPKs are serine-threonine protein kinases that phosphorylate substrates in response to specific signals. A number of mitogenic factors, such as pheromones and growth factors, and environmental factors, such as osmotic stress, have been identified as stimuli for activating MAPK signal transduction cascades (20, 28). Once a stimulus activates a particular MAPK pathway, the signal is propagated by protein kinases specific for that pathway. Perhaps the best characterized pathway is the pheromone mating response cascade in the budding yeast S. cerevisiae, where haploid cells conjugate to form diploids by the mutual secretion of peptide mating factors (or pheromones) by each of the two haploid cell types (1). The binding of a pheromone peptide to its cell surface receptor activates a heterotrimeric G protein that initiates a series of sequential protein phosphorylations that are propagated by protein kinases of a MAPK signal transduction cascade specific for the mating response (1). The events that follow binding of the pheromone peptide culminate in an activated MAPK, which is achieved by phosphorylation of two conserved threonine and tyrosine residues between kinase subdomains VII and VIII (9, 17). Once activated, MAPK controls the many cellular events the organism will undergo in response to the mating signal. One of these events is a controlled and orderly exit from the mitotic cell cycle, allowing the cells to synchronize before conjugation (18, 22, 25).

We are investigating the role of MAPK in the regulation of the P. carinii life cycle. As a first step in our studies, we have isolated a full-length cDNA from P. carinii that is highly homologous to fungal MAPKs involved in the pheromone mating signal transduction cascade. Additionally, we have identified its chromosomal location within the P. carinii genome and demonstrated that this cDNA is expressed as a functional MAPK in P. carinii organisms.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Materials. All reagents were from Sigma Chemical (St. Louis, MO) unless otherwise specified. Restriction endonucleases, Taq polymerase, myelin basic protein, and H1 histone were from GIBCO-BRL (Life Technologies). [gamma -32P]ATP and [alpha -32P]dATP were obtained from ICN Pharmaceuticals.

Preparation of P. carinii organisms. All studies described in this report were approved by the institutional animal care facility. P. carinii pneumonia was induced in Harlan Sprague-Dawley rats by immunosuppression with dexamethasone, as reported previously (2, 13, 21). Rats received drinking water containing dexamethasone (2 mg/l) and were fed a diet containing 8% protein to intensify the infection. After 1 wk, rats were anesthetized with ether and inoculated with 1 × 106 P. carinii by intratracheal injection. Six weeks later, rats were killed by injection with 26% pentobarbital sodium. Lungs from moribund rats were minced in HBSS and homogenized in a Stomacher Tissue Blender (Tekmar) for 10 min. P. carinii were purified from this homogenate by filtration through a 10-µm filter (Millipore). These preparations contained ~98% P. carinii organisms, with the remainder being mostly nonviable host cells. P. carinii organisms were confirmed by staining smears of the fluid with Wright-Giemsa, and samples containing contaminating bacterial or fungal organisms were discarded. P. carinii enumeration was performed by counting air-dried samples stained with Diff-Quik, as described (13, 21).

Molecular cloning and chromosomal location of P. carinii MAPK. Oligonucleotides were made at the Mayo Clinic Molecular Biology Core Facility. Genomic DNA was obtained from purified P. carinii organisms as previously described (26). We designed degenerate PCR primers based on the conserved amino acid sequences of two fungal MAPKs involved in the pheromone mating cascade, S. cerevisiae FUS3 and S. pombe spk1. The codon bias for P. carinii was used to limit the amount of degeneracy in the third position of the codon (8). PCR amplification was performed with 1 µM of each degenerate primer GAYGVVC, 5'-GG(A/T)GC(A/T)TATGG(A/T)GT(A/T)GT(A/T)TG-3', and TAVYETM, 5'-G(A/T)GT(A/T)GC(A/T)ACATATTC(A/T)GTCAT-3' using P. carinii genomic DNA as the template. An initial 4-min hot start at 94°C was followed by 30 cycles of 94°C for 60 s, 56°C for 60 s, 72°C for 60 s, and a final 72°C 15-min extension. A single 617-bp amplification product was identified by electrophoresis on 2% agarose after ethidium bromide staining and was subcloned into the pCR2.1 vector (Invitrogen). The amplified product was sequenced by cycle sequencing from both directions with SP6 and T7 sequencing primers from pCR2.1 and internal primers from the gene. Nucleotide sequence comparisons to the GenBank database were done with Basic Local Alignment Search Tool (BLAST) [National Center for Biotechnology Information (NCBI)].

Separation of P. carinii chromosomes by contour-clamped homogeneous electrical field electrophoresis (CHEF) was performed as reported previously (5). The 617-bp PCR amplification product was labeled with [alpha -32P]dATP using the random-primer method (Rediprime System; Amersham). After prehybridization for 30 min (ExpressHyb solution; Clontech), the CHEF nitrocellulose membrane was incubated with the probe (1.5 × 106 counts · min-1 · ml-1) at 60°C for 1 h, washed one time at 37°C for 40 min in 2× saline-sodium citrate (SSC) buffer containing 0.05% SDS, washed one time at 50°C for 40 min in 2× SSC buffer containing 0.1% SDS, and examined by autoradiography.

P. carinii MAPK cDNA was cloned from a rat-derived P. carinii cDNA library in the bacteriophage Uni-Zap XR. This library was screened using the 617-bp PCR amplicon as a probe using standard methods. Excision of the phagemid was performed with M13 helper phage following the manufacturer's instructions. A 1,341-bp insert was identified by restriction endonuclease digestion with EcoR I and Xho I and was completely sequenced on both strands using cycle sequencing with primers derived from the plasmid and the gene. Nucleotide sequence comparisons to the GenBank database were done with BLAST (NCBI).

Generation of P. carinii MAPK antisera. Analysis of the translated P. carinii MAPK cDNA revealed a unique amino acid sequence, SAVSTEDSSS, between kinase subdomains VII and VIII. The peptide CSAVSTEDSSS was synthesized at the Mayo Protein Core Facility and coupled to keyhole limpet hemocyanin (KLH) at the NH2-terminal cysteine. Rabbits received a primary subcutaneous injection of the antigen mixed with complete Freunds adjuvant, followed by intramuscular boosts of the antigen mixed with incomplete Freunds adjuvant using standard antibody protocols. Reactivity of the immune sera compared with the preimmune sera was verified by ELISA using the cognate peptide as a target antigen. ELISA plates were coated with the target antigen in coupling buffer (0.1 M NaHCO3, pH 8.3) and blocked with 1% BSA, and serial dilutions of preimmune or immune serum were added. Specific binding was detected with a goat anti-rabbit secondary antibody coupled to horseradish peroxidase using o-phenylenediamine dihydrochloride as a substrate, with measurement at 450 nm.

MAPK assays. Immunoprecipitation kinase assays were performed using the specific P. carinii MAPK antiserum (23). P. carinii was purified from immunosuppressed rats by lung homogenization and differential filtration as described above. P. carinii were disrupted in ice-cold kinase lysis buffer [50 mM Tris · HCl, pH 7.5, 100 mM NaCl, 50 mM NaF, 1% (vol/vol) Triton X-100, 5 mM EDTA, 1 mM EGTA, 200 µM sodium orthovanadate, 100 µM phenylmethylsulfonyl fluoride, 1 g/ml leupeptin, and 0.1 tissue inhibitor units aprotinin] and vortexed repeatedly over 5 min. The sample was sonicated for 30 s, and the debris was pelleted by centrifugation at 12,000 g at 4°C for 10 min. The protein concentration of the lysate was determined spectrophotometrically using the bicinchoninic acid method (Pierce). Protein lysate (500 µg) was precleared with a 50% slurry of protein A-Sepharose at 4°C for 30 min, centrifuged at 12,000 g for 5 min, and then transferred to clean tubes; immune or nonimmune serum (1:250 dilution) was added; and the mixture was rocked at 4°C for 4 h. Immune complexes were precipitated with a 50% slurry of protein A-Sepharose at 4°C for 60 min and collected by centrifugation at 12,000 g for 5 min. The pellet was washed four times with 750 µl of kinase lysis buffer and two times with kinase buffer (50 mM HEPES, pH 7.5, 10 mM magnesium acetate, and 1 mM dithiothreitol). After the final wash, the pellet was resuspended in 50 µl of kinase reaction buffer (50 mM HEPES, pH 7.5, 10 mM magnesium acetate, 1 mM dithiothreitol, 20 µM cold ATP, 0.3 mg/ml of myelin basic protein or H1 histone, and 10 µCi [gamma -32P]ATP) and incubated at 30°C for 30 min. The reaction was stopped with the addition of Laemmli buffer, boiled for 5 min, and centrifuged at 12,000 g for 5 min, and the supernatant was resolved by 12% SDS-PAGE. The gel was fixed in 40% methanol-10% acetic acid for 30 min, washed in distilled water for 15 min, and exposed to autoradiography film at -70°C. To control for host cells that may pass through the filtration process, protein lysates from healthy rat lungs were prepared and filtered in an identical manner as described above and were immunoprecipitated with the immune or nonimmune serum (1:250 dilution) using the identical methods described.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

P. carinii MAPK gene is homologous to fungal MAPKs involved in differentiation and proliferation. Cloning of the P. carinii MAPK was performed using a degenerate PCR cloning strategy and traditional library screening (Fig. 1). The 617-bp PCR amplicon was found to be unique on GenBank analysis but homologous to fungal MAPKs involved in mating. To verify that the 617-bp PCR amplicon was part of the P. carinii genome, it was hybridized to P. carinii chromosomes separated by CHEF. The probe hybridized to a single chromosomal band (Fig. 2). With the use of the 617-bp PCR amplicon as a probe, a 1,341-bp full-length cDNA was obtained by screening a P. carinii cDNA library in the bacteriophage Uni-Zap XR (Fig. 1).


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Fig. 1.   Nucleotide and predicted amino acid sequence of Pneumocystis carinii mitogen-activated protein kinase (MAPK). The 5'- and 3'-untranslated regions are in lowercase letters. The single open reading frame encodes a 351-amino acid protein with predicted molecular mass of 40,571. The T and Y amino acids are the conserved putative phosphorylation sites for MAPK activation. (The sequence data are available from GenBank accession number AF043941.)


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Fig. 2.   Chromosomal location of the P. carinii MAPK gene. The P. carinii MAPK gene is present on a single chromosome within the P. carinii genome. The P. carinii MAPK probe hybridized to a single P. carinii chromosome. M, DNA ladder. Lanes 1-10: samples of P. carinii DNA resolved by contour-clamped homogeneous electrical field electrophoresis (CHEF). A: ethidium bromide-stained CHEF gel shows separated samples of P. carinii chromosomes. B: hybridization blot of 32P-labeled MAPK probe to separated samples of P. carinii chromosomes transferred to nitrocellulose.

Analysis of the P. carinii MAPK cDNA against the GenBank database revealed this gene to be unique. Homology on the amino acid level (BLASTX) revealed 80% identity to Fusarium solani and Magnaporthe grisea MAPKs, 78% identity to Candida albicans Cek1, 63% to S. pombe Spk1, 64% to S. cerevisiae FUS3 and KSS1, 58% to Homo sapiens ERK1 and ERK2, and 57% to Rattus norvegicus ERK1 (Fig. 3). The single open reading frame encodes a 351-amino acid protein with predicted molecular weight of 40,571. The putative regulatory phosphorylation sites necessary for P. carinii MAPK activity are present in amino acids Thr(T)-176 and Tyr(Y)-178, and the putative active site is present in amino acid Asp(D)-140 (Fig. 1). The kinase domains HRDLKPSN [containing the active site Asp(D)-140] and TEYVATRWRAPE [containing the phosphorylation sites Thr(T)-176 and Tyr(Y)-178] are 100% conserved between P. carinii and the other MAPKs listed in Fig. 3 with the exception of S. cerevisiae Kss1 (Fig. 3).


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Fig. 3.   P. carinii MAPK is homologous to other eukaryotic MAPK proteins involved in growth regulation. Amino acid alignments are as follows: Pc, P. carinii; Fs, Fusarium solani; Mg, Magnaporthe grisea; Ca, Candida albicans; Sp, Schizosaccharomyces pombe; Sc, Saccharomyces cerevisiae; Hs, Homo sapiens; Rn, Rattus norvegicus. Periods are used to maximize alignment.

Demonstration of characteristic MAPK activity from P. carinii. To phosphorylate substrates, MAPK needs to be activated by phosphorylation on two conserved threonine and tyrosine amino acids; this occurs in response to a specific signal propagated by the protein kinase cascade. To determine whether P. carinii organisms possess an activated MAPK capable of kinase activity, P. carinii protein extracts were immunoprecipitated with the specific P. carinii MAPK antiserum and reacted with [gamma -32P]ATP and myelin basic protein or H1 histone as substrates. An immune serum was generated in rabbits against a unique amino acid region in the P. carinii MAPK between kinase domains VII and VIII. The specificity of this immune serum was verified by ELISA against the target antigen (Fig. 4A). Next, kinase activity was detected in P. carinii lysates immunoprecipitated with this specific P. carinii MAPK antiserum but not with the nonimmune serum (Fig. 4B). Because we used the immunosuppressed rat model to obtain P. carinii organisms, control experiments were performed to account for the possibility of host cells that might come through the purification process of separating P. carinii from host lung. These failed to demonstrate significant protein kinase activity, thus confirming that the observed kinase activity was specifically derived from P. carinii.


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Fig. 4.   P. carinii MAPK exhibits functional in vitro kinase activity. A: ELISA comparing preimmune serum with immune serum using the peptide antigen as the target. A dilution of 1:250 was used in the immunoprecipitation kinase assays. OD 450, optical density at 450 nm. B: immunoprecipitation kinase assays. Shown is myelin basic protein (MBP) and H1 histone phosphorylated with [gamma -32P]ATP. Lanes 1 and 2: MBP as a phosphorylation substrate; P. carinii lysate immunoprecipitated with P. carinii MAPK antiserum (lane 1) reveals phosphorylation of the MBP, whereas nonimmune serum (lane 2) does not yield substantial phosphorylation. Lanes 3 and 4: MBP as a phosphorylation substrate; control experiments with uninfected rat lung lysate immunoprecipitated with P. carinii MAPK antiserum (lane 3) or nonimmune serum (lane 4) do not demonstrate substantial phosphorylation of MBP, thereby confirming that the observed kinase activity is from P. carinii and not as a result of host cell contamination. Lanes 5 and 6: H1 histone as a phosphorylation substrate; P. carinii lysate immunoprecipitated with P. carinii MAPK antiserum (lane 5) is able to phosphorylate H1 histone, whereas nonimmune serum (lane 6) is not.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

MAPKs are highly conserved serine-threonine protein kinases involved in regulating cellular differentiation and proliferation. The fungal MAPK active in the pheromone mating cascade is essential for the transition from mitotic growth into a pathway of conjugation and cellular differentiation (1, 7, 19). MAPK phosphorylates and thereby activates proteins required for conjugation, cell fusion, and for the direct inhibition of Cdc2 kinase activity, resulting in mitotic cell cycle arrest (18, 22, 25). Loss of fungal MAPK activity results in sterility, with the inability to enter meiosis and proliferate (7). Given its essential role in an organism's life cycle, MAPK is regulated at many levels. Dual-activating phosphorylations by the upstream kinase MEK occur on conserved threonine and tyrosine amino acid residues in the TEY sequence between kinase subdomains VII and VIII, and both phosphorylations are required for enzymatic function (9, 17).

The P. carinii MAPK is most closely homologous to fungal MAPKs involved in differentiation and proliferation, which suggests that P. carinii may use similar pathways. Amino acid sequence comparisons revealed homology to two phytopathogenic filamentous fungi of the ascomycetes family, M. grisea and F. solani (80% identity on the amino acid level), to the opportunistic yeast C. albicans (78% identity), and to S. pombe spk1 and S. cerevisiae FUS3 and KSS1.

M. grisea is a phytopathogenic fungus that causes rice blast disease. The M. grisea MAPK Pmk1 controls differentiation of a specialized invasion structure, the appressorium, which is required for the fungus to infect the host rice plant. M. grisea strains containing mutations in Pmk1 fail to form appressoria and thus cannot grow invasively in rice plants. Pmk1 has also been found to complement the mating defect in S. cerevisiae Fus3/Kss1 double-mutant yeast (30). F. solani, which has historically been recognized as a phytopathogen, has recently been identified as a cause of invasive human infections in immunocompromised patients with hematological malignancies (3, 10). An F. solani MAPK has recently been identified that is homologous to MAPKs involved in growth regulation, but further investigation is needed to determine its function within this fungus (12). The P. carinii MAPK shares homology to the C. albicans MAPK Cek1, which was cloned as a gene that interfered with the pheromone-mediated cell cycle arrest of S. cerevisiae and conferred a phenotype similar to the S. cerevisiae MAPK Kss1 (29) and to S. cerevisiae Fus3/Kss1 and S. pombe Spk1 MAPKs, which are involved in regulating the pheromone mating signal transduction cascade in these yeasts.

In summary, we have identified an MAPK from P. carinii that is homologous to fungal MAPKs that have important roles in regulating growth, differentiation, and proliferation pathways. Chromosomal analysis reveals that the P. carinii MAPK is present on a single chromosome within the P. carinii genome. In addition, we are able to demonstrate MAPK activity from P. carinii lysates using a specific antiserum derived from the translated P. carinii MAPK cDNA sequence, suggesting that this gene is expressed as a functional protein kinase in P. carinii organisms. We believe that these observations represent an essential first step in identifying the regulatory pathways of growth and proliferation of P. carinii. Further investigations of the regulation and activity of the P. carinii MAPK should lead to an enhanced understanding of the complex life cycle of this pulmonary pathogen.

    ACKNOWLEDGEMENTS

We thank Dr. M. Cushion, University of Cincinnati College of Medicine, for providing the contour-clamped homogeneous electrical field electrophoresis blot of Pneumocystis carinii chromosomes and photograph of the ethidium bromide-stained gel. The following reagent was obtained through the acquired immunodeficiency syndrome (AIDS) Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH): P. carinii cDNA library from Biotechnology General.

    FOOTNOTES

This work was supported by National Institutes of Health Grants HL-55934-03, AI-34336-05, and HL-57125-01 to A. H. Limper. C. F. Thomas, Jr., is a 1996 Glaxo Wellcome Pulmonary Fellowship Award Recipient.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: C. F. Thomas, Thoracic Diseases Research Unit, E18B Mayo Bldg., 200 First St. S.W., Mayo Clinic and Foundation, Rochester, MN 55905.

Received 4 February 1998; accepted in final form 1 April 1998.

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Abstract
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Results
Discussion
References

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Am J Physiol Lung Cell Mol Physiol 275(1):L193-L199
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society