Cell Wall Assembly by Pneumocystis carinii
2000; Elsevier BV; Volume: 275; Issue: 51 Linguagem: Inglês
10.1074/jbc.m002103200
ISSN1083-351X
AutoresTheodore J. Kottom, Andrew H. Limper,
Tópico(s)Peptidase Inhibition and Analysis
ResumoPneumocystis carinii remains a persistent cause of severe pneumonia in immune compromised patients. Recent studies indicate that P. carinii is a fungal species possessing a glucan-rich cyst wall. Pneumocandin antagonists of β-1,3-glucan synthesis rapidly suppress infection in animal models ofP. carinii pneumonia. We, therefore, sought to define the molecular mechanisms of β-glucan cell wall assembly by P. carinii. Membrane extracts derived from freshly purified P. carinii incorporate uridine 5′-diphosphoglucose into insoluble carbohydrate, in a manner that was completely inhibited by the pneumocandin L733–560, an antagonist of Gsc-1-type β-glucan synthetases. Using degenerative polymerase chain reaction and library screening, the P. carinii Gsc-1 catalytic subunit of β-1,3-glucan synthetase was cloned and characterized. P. carinii gsc1 exhibited homology to phylogenetically related fungal β-1,3-glucan synthetases, encoding a predicted 214-kDa integral membrane protein with 12 transmembrane domain structure. Immunoprecipitation of P. carinii extracts, with a synthetic peptide anti-Gsc-1 antibody, specifically yielded a protein of 219.4 kDa, which was also capable of incorporating 5′-diphosphoglucose into insoluble glucan carbohydrate. As opposed to other fungi, the expression of gsc-1 mRNA is uniquely regulated over P. carinii's life cycle, having minimal expression in trophic forms, but substantial expression in the thick-walled cystic form of the organism. These results indicate thatP. carinii contains a unique catalytic subunit of β-1,3-glucan synthetase utilized in cyst wall formation. Because synthesis of β-1,3-glucan is absent in mammalian cells, inhibition of the P. carinii Gsc-1 represents an attractive molecular target for therapeutic exploitation. Pneumocystis carinii remains a persistent cause of severe pneumonia in immune compromised patients. Recent studies indicate that P. carinii is a fungal species possessing a glucan-rich cyst wall. Pneumocandin antagonists of β-1,3-glucan synthesis rapidly suppress infection in animal models ofP. carinii pneumonia. We, therefore, sought to define the molecular mechanisms of β-glucan cell wall assembly by P. carinii. Membrane extracts derived from freshly purified P. carinii incorporate uridine 5′-diphosphoglucose into insoluble carbohydrate, in a manner that was completely inhibited by the pneumocandin L733–560, an antagonist of Gsc-1-type β-glucan synthetases. Using degenerative polymerase chain reaction and library screening, the P. carinii Gsc-1 catalytic subunit of β-1,3-glucan synthetase was cloned and characterized. P. carinii gsc1 exhibited homology to phylogenetically related fungal β-1,3-glucan synthetases, encoding a predicted 214-kDa integral membrane protein with 12 transmembrane domain structure. Immunoprecipitation of P. carinii extracts, with a synthetic peptide anti-Gsc-1 antibody, specifically yielded a protein of 219.4 kDa, which was also capable of incorporating 5′-diphosphoglucose into insoluble glucan carbohydrate. As opposed to other fungi, the expression of gsc-1 mRNA is uniquely regulated over P. carinii's life cycle, having minimal expression in trophic forms, but substantial expression in the thick-walled cystic form of the organism. These results indicate thatP. carinii contains a unique catalytic subunit of β-1,3-glucan synthetase utilized in cyst wall formation. Because synthesis of β-1,3-glucan is absent in mammalian cells, inhibition of the P. carinii Gsc-1 represents an attractive molecular target for therapeutic exploitation. uridine 5′-diphosphoglucose glucan synthetase catalytic subunit tumor necrosis factor-α polymerase chain reaction guanosine 5′-O-(thiotriphosphate) base pair(s) contour-clamped homogenous field kilobase(s) Classification of Pneumocystis carinii as a fungus revolutionized study of this organism, which continues to cause life-threatening pneumonia in immune-compromised patients with AIDS, malignancy, and organ transplantation (1Edman J.C. Kovacs J.A. Masur H. Santi D.V. Elwood H.J. Sogin M.L. Nature. 1988; 334: 519-522Crossref PubMed Scopus (551) Google Scholar, 2Bruns T.D. Vilgalys R. Barns S.M. Gonzalez D. Hibbett D.S. Lane D.J. Simon L. Stickel S. Szaro T.M. Weisburg W.G. Mol. Phylogenet. Evol. 1992; 1: 231-241Crossref PubMed Scopus (234) Google Scholar, 3Van Der Peer Y. Hendriks L. Goris A. Syst. Appl. Microbiol. 1992; 15: 250-258Crossref Scopus (39) Google Scholar, 4Yale S.H. Limper A.H. Mayo Clin. Proc. 1996; 71: 5-13Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 5Thomas C.F. Limper A.H. Semin. Respir. Infect. 1998; 13: 289-295PubMed Google Scholar). Studies of P. carinii in infected lung indicate its life cycle alternates between smaller trophic forms and thick-walled cysts (6Campbell W.G. Arch. Pathol. 1972; 93: 312-330PubMed Google Scholar, 7Limper A.H. Thomas C.F. Anders R.A. Leof E.B. J. Lab. Clin. Med. 1997; 130: 132-138Abstract Full Text PDF PubMed Scopus (37) Google Scholar, 8De Stefano J.A. Cushion M.T. Sleight R.G. Walzer P.D. J. Protozool. 1990; 37: 428-435Crossref PubMed Scopus (34) Google Scholar). The origin of P. carinii cysts remains controversial, but it has been postulated they arise from sexual conjugation, analogous to ascomycetous fungi (9Itatani C.A. J. Parasitol. 1996; 82: 163-171Crossref PubMed Scopus (27) Google Scholar, 10MacNiell S.A. Fantes P.A. Hutchison C. Glover D.M. Cell Cycle Control. IRL Press at Oxford University Press, Oxford1995: 63-105Google Scholar). The mechanisms of cyst wall assembly byP. carinii are not well known, although recent studies reveal that these walls are largely composed of β-glucans, glycoprotein A, and chitins (11Hoffman O.A. Standing J.E. Limper A.H. J. Immunol. 1993; 150: 3932-3940PubMed Google Scholar, 12Lundgren B. Lipchik G.Y. Kovacs J.A. J. Clin. Invest. 1991; 87: 163-170Crossref PubMed Scopus (80) Google Scholar, 13Gigliotti F. Haidaris P.J. Haidaris C.G. Wright T.W. Van der Meid K.R. J. Infect. Dis. 1993; 168: 191-194Crossref PubMed Scopus (31) Google Scholar, 14Linke M.J. Cushion M.T. Walzer P.D. Infect. Immunol. 1989; 57: 1547-1555Crossref PubMed Google Scholar, 15Stringer S.L. Garbe T. Suskin S.M. Stringer J.R. J. Eukaryot. Microbiol. 1993; 40: 821-826Crossref PubMed Scopus (46) Google Scholar, 16Roth A. Wecke J. Karsten V. Janitschke K. Parasitol. Res. 1997; 83 (1997): 177-184Crossref PubMed Scopus (18) Google Scholar).β-Glucans are glucose homopolymers composed mainly of a β-1,3-linked carbohydrate core, with variable amounts of β-1,6- and β-1,4-linked glucose side chains (17Manners D.J. Masson A.J. Patterson J.C. Biochem. J. 1973; 135: 19-30Crossref PubMed Scopus (361) Google Scholar, 18Bacon J.S. Farmer V.C. Jones D. Taylor I.F. Biochem. J. 1969; 114: 557-567Crossref PubMed Scopus (118) Google Scholar, 19Kollar R. Reinhold B.B. Petrakova E. Yeh H.J. Ashwell G. Drgonova J. Kapteyn J.C. Klis F.M. Cabib E. J. Biol. Chem. 1997; 272: 17762-17775Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar). Glucans represent principal components of cell walls in fungi related to P. carinii. Ultrastructural investigations demonstrate an electron-lucent layer unique to the cystic form of P. carinii, which is specifically degraded by β-1,3-glucanases (16Roth A. Wecke J. Karsten V. Janitschke K. Parasitol. Res. 1997; 83 (1997): 177-184Crossref PubMed Scopus (18) Google Scholar). Additional studies with specific β-1,3-glucan antiserum also localize glucan to the walls of cysts (20Nollstadt K.H. Powles M.A. Fujioka H. Aikawa M. Schmatz D.M. Antimicrob. Agents Chemother. 1994; 38: 2258-2265Crossref PubMed Scopus (33) Google Scholar). β-1,3-Glucan has been detected in bronchoalveolar lavage from patients with P. carinii pneumonia (21Yasuoka A. Tachikawa N. Shimada K. Kimura S. Oka S. Clin. Diagn. Lab. Immunol. 1996; 3: 197-199Crossref PubMed Google Scholar). Furthermore, P. cariniiβ-glucans also represent important epitopes recognized by host cells (11Hoffman O.A. Standing J.E. Limper A.H. J. Immunol. 1993; 150: 3932-3940PubMed Google Scholar, 22Hidalgo H.A. Helmke R.J. German V.F. Mangos J.A. J. Protozool. 1991; 38: 30S-31SCrossref PubMed Scopus (28) Google Scholar). P. carinii β-glucans interact with alveolar macrophages mediating phagocytic uptake of the organism and lung inflammatory responses (11Hoffman O.A. Standing J.E. Limper A.H. J. Immunol. 1993; 150: 3932-3940PubMed Google Scholar). Subsequent glucan-mediated influx of neutrophils into the lung is an important contributor to respiratory impairment during this infection (23Limper A.H. Offord K.P. Smith T.F. Martin W.J. Am. Rev. Respir. Dis. 1989; 140: 1204-1209Crossref PubMed Scopus (394) Google Scholar, 24el-Sadr W. Simberkoff M.S. Am. Rev. Respir. Dis. 1988; 137: 1264-1267Crossref PubMed Scopus (76) Google Scholar, 25Yu M.L. Limper A.H. Am. J. Physiol. 1997; 273: L1103-L1111PubMed Google Scholar).Fungal β-glucans are assembled by a multisubunit enzyme complex within the organism's cell membrane. Gsc-1 proteins mediate the polymerization of uridine 5′-diphosphoglucose (UDP-Glc)1 into the insoluble β-1,3-glucan core required for cell wall assembly (26Castro C. Ribas J.C. Valdivieso M.H. Varona R. Ray F.D. Duran A. J. Bacteriol. 1995; 177: 5732-5739Crossref PubMed Google Scholar). Glucan synthetases are generally encoded by gsc-1 genes, which generate a 210-kDa catalytic protein in Saccharomyces cerevisiae and comparable proteins in other fungi (27Douglas C.M. Foor F. Marrinan J.A. Morin N. Nielsen J.B. Dahl J.A. Mazur P. Baginsky W. Li W. El-Sherbeini M. Clemas J.A. Mandela S.A. Frommer B.R. Kurtz M.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12907-12911Crossref PubMed Scopus (338) Google Scholar, 28Kelly R. Register E. Hsu M.J. Kurtz M. Nielsen J. J. Bacteriol. 1996; 178: 4381-4391Crossref PubMed Scopus (99) Google Scholar, 29Enderlin C.S. Selitrennikoff C.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9500-9504Crossref PubMed Scopus (29) Google Scholar, 30Tentler S. Palas J. Enderlin C. Campbell J. Taft C. Miller T.K. Wood R.L. Selitrennikoff C.P. Curr. Microbiol. 1997; 34: 303-330Crossref PubMed Scopus (20) Google Scholar, 31Mio T. Adachi-Shimizu M. Tachibana Y. Tabuchi H. Inoue S.B. Yabe T. Yamada-Okabe T. Arisawa M. Watanabe T. Yamada-Okabe H. J. Bacteriol. 1997; 179: 4096-4105Crossref PubMed Google Scholar, 32Kurtz M.B. Abruzzo G. Flattery A. Bartizal K. Marrinan J.A. Li W. Milligan J. Nollstadt K. Douglas C.M. Infect. Immun. 1996; 64: 3244-3251Crossref PubMed Google Scholar). Glucan synthetase activity by Gsc-1-type proteins is specifically inhibited by pneumocandin and echinocandin class compounds (32Kurtz M.B. Abruzzo G. Flattery A. Bartizal K. Marrinan J.A. Li W. Milligan J. Nollstadt K. Douglas C.M. Infect. Immun. 1996; 64: 3244-3251Crossref PubMed Google Scholar).Because mammalian cells do not possess glucan biosynthetic pathways, cell wall assembly represents an attractive target for the treatment of fungal infection. It is particularly noteworthy that pneumocandin inhibitors of β-glucan synthesis have been shown to rapidly inhibitP. carinii growth in rodent models (33Schmatz D.M. Romancheck M.A. Pittarelli L.A. Schwartz R.E. Fromtling R.A. Nollstadt K.H. Vanmiddlesworth F.L. Wilson K.E. Turner M.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5950-5954Crossref PubMed Scopus (153) Google Scholar, 34Schmatz D.M. Powles M.A. McFadden D. Nollstadt K. Bouffard F.A. Dropinski J.F. Liberator P. Andersen J. Antimicrob. Agents Chemother. 1995; 39: 1320-1323Crossref PubMed Scopus (49) Google Scholar, 35Powles M.A. Liberator P. Anderson J. Karkhanis Y. Dropinski J.F. Bouffard F.A. Balkovec J.M. Fujioka H. Aikawa M. McFadden D. Schmatz D. Antimicrob. Agents Chemother. 1998; 42: 1985-1989Crossref PubMed Google Scholar, 36Bartlett M.S. Current W.L. Goheen M.P. Boylan C.J. Lee C.H. Shaw M.M. Queener S.F. Smith J.W. Antimicrob. Agents Chemother. 1996; 40: 1811-1816Crossref PubMed Google Scholar). Such studies provide evidence of the importance of β-glucan generation during life cycle progression of this organism. Despite the considerable importance of β-glucan assembly in life cycle expression of this organism, in immune recognition during infection, and as a potential therapeutic target for pneumonia, the mechanisms of β-1,3-glucan assembly by P. carinii are not yet fully understood.The current investigation was undertaken to accomplish the following: 1) to establish whether P. carinii cell wall assembly occurs through action of a Gsc-1 protein mediating β-1,3-glucan synthesis; 2) to clone and characterize the respective gsc-1 encoding this activity in P. carinii; and finally 3) to evaluate expression of P. carinii gsc-1 over the life cycle of the organism.DISCUSSIONP. carinii membrane isolates possess the ability to incorporate UDP-[14C]glucose into insoluble carbohydrate, which is inhibited by the pneumocandin L-733,560 antagonist of Gsc-1-type β-1,3-glucan synthetases. Molecular cloning of theP. carinii gsc-1 gene predicts a mature protein with both similarities and unique differences to other fungal β-1,3-glucan synthetases. Specifically, the mRNA and protein expression ofP. carinii gsc-1 are highly regulated over the life cycle of the organism being predominantly expressed by cystic forms of the organism. A protein corresponding to the predicted sequence of the cloned gsc-1 gene was present in P. cariniimembrane isolates. Furthermore, immunoprecipitation of the putativeP. carinii Gsc-1 protein with a synthetic peptide antibody yielded a product capable of mediating incorporation of UDP-Glc into trichloroacetic acid-insoluble material, consistent with glucan.P. carinii Gsc-1 glucan synthetase exhibits several unique features. As discussed, structural differences were detected in the domain configuration of P. carinii Gsc-1 compared withS. cerevisiae and Aspergillus (26Castro C. Ribas J.C. Valdivieso M.H. Varona R. Ray F.D. Duran A. J. Bacteriol. 1995; 177: 5732-5739Crossref PubMed Google Scholar, 27Douglas C.M. Foor F. Marrinan J.A. Morin N. Nielsen J.B. Dahl J.A. Mazur P. Baginsky W. Li W. El-Sherbeini M. Clemas J.A. Mandela S.A. Frommer B.R. Kurtz M.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12907-12911Crossref PubMed Scopus (338) Google Scholar, 28Kelly R. Register E. Hsu M.J. Kurtz M. Nielsen J. J. Bacteriol. 1996; 178: 4381-4391Crossref PubMed Scopus (99) Google Scholar). Of further contrast, is the significant restriction of gsc-1expression predominantly to the cystic forms of the P. carinii. Most other fungi, including ascomycetous fungi, exhibit cell wall assembly constitutively throughout the life cycle (55Carlson C.R. Grallert B. Bernander R. Stokke T. Boye E. Yeast. 1997; 13: 1329-1335Crossref PubMed Scopus (30) Google Scholar). Our immunoblot and Northern analyses are complementary to previous immune localization studies, which also indicate that β-1,3-glucan is largely found within cysts (6Campbell W.G. Arch. Pathol. 1972; 93: 312-330PubMed Google Scholar, 20Nollstadt K.H. Powles M.A. Fujioka H. Aikawa M. Schmatz D.M. Antimicrob. Agents Chemother. 1994; 38: 2258-2265Crossref PubMed Scopus (33) Google Scholar).Because mammalian hosts do not possess an equivalent to Gsc-1, inhibition of β-1,3-glucan synthesis represents an attractive target for treatment of fungal infections. Echinocandins and pneumocandins are selective lipopeptide inhibitors that may expand our armamentaria for fungal infections, including those organisms resistant to standard agents (32Kurtz M.B. Abruzzo G. Flattery A. Bartizal K. Marrinan J.A. Li W. Milligan J. Nollstadt K. Douglas C.M. Infect. Immun. 1996; 64: 3244-3251Crossref PubMed Google Scholar). Schmatz and colleagues (33Schmatz D.M. Romancheck M.A. Pittarelli L.A. Schwartz R.E. Fromtling R.A. Nollstadt K.H. Vanmiddlesworth F.L. Wilson K.E. Turner M.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5950-5954Crossref PubMed Scopus (153) Google Scholar, 34Schmatz D.M. Powles M.A. McFadden D. Nollstadt K. Bouffard F.A. Dropinski J.F. Liberator P. Andersen J. Antimicrob. Agents Chemother. 1995; 39: 1320-1323Crossref PubMed Scopus (49) Google Scholar) have shown rapid reduction of organisms in rat and mouse models of P. cariniipneumonia. One pneumocandin in particular, L-671,329 has shown remarkable activity in the P. carinii rat model with >98% of cysts being eliminated (33Schmatz D.M. Romancheck M.A. Pittarelli L.A. Schwartz R.E. Fromtling R.A. Nollstadt K.H. Vanmiddlesworth F.L. Wilson K.E. Turner M.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5950-5954Crossref PubMed Scopus (153) Google Scholar).Concern had arisen that pneumocandins might only be effective against cystic forms of P. carinii, thereby limiting efficacy of such compounds during P. carinii pneumonia. The striking results of pneumocandins in animal models of P. cariniipneumonia strongly argue to the contrary (34Schmatz D.M. Powles M.A. McFadden D. Nollstadt K. Bouffard F.A. Dropinski J.F. Liberator P. Andersen J. Antimicrob. Agents Chemother. 1995; 39: 1320-1323Crossref PubMed Scopus (49) Google Scholar). These findings do suggest, however, that progression of trophic forms into cysts represents an essential component of life cycle progression in P. carinii, rather than an elective form utilized only under hostile conditions (7Limper A.H. Thomas C.F. Anders R.A. Leof E.B. J. Lab. Clin. Med. 1997; 130: 132-138Abstract Full Text PDF PubMed Scopus (37) Google Scholar). Other investigators have also found some effect of echinocandins on trophic structure after in vitro exposure (36Bartlett M.S. Current W.L. Goheen M.P. Boylan C.J. Lee C.H. Shaw M.M. Queener S.F. Smith J.W. Antimicrob. Agents Chemother. 1996; 40: 1811-1816Crossref PubMed Google Scholar). Although our study demonstrates low levels of gsc-1mRNA expression in trophic forms, small residual amounts of Gsc-1 protein were detected by Western analysis to remain within trophic forms. In addition, echinocandin and pneumocandin compounds may also effect other targets within P. carinii.Until recently, the lack of a reliable culture system has hindered studies of life cycle regulation by P. carinii (56Sloand E. Laughon B. Armstrong B. Bartlett M.S. Blumenfeld W. Cushion M. Kalica A. Kovacs J.A. Martin W.J. Pitt E. J. Eukaryot. Microbiol. 1993; 40: 188-195Crossref PubMed Scopus (86) Google Scholar). Recent studies implicate a cyclin-dependent kinase cell cycle control system, which exhibits regulated activity during progression ofP. carinii trophic forms to cysts (44Thomas Jr., C.F. Anders R.A. Gustafson M.P. Leof E.B. Limper A.H. Am. J. Respir. Cell. Mol. Biol. 1998; 18: 297-306Crossref PubMed Scopus (46) Google Scholar, 57Limper A.H. Thomas C.F. Mubarak K.K. Gustafson M.P. Kottom T.J. Leof E.B. J. Eukaryot. Microbiol. 1997; 44: 32SCrossref PubMed Scopus (3) Google Scholar). Considerable questions remain as to how assembly of the thickened β-glucan-rich pellicle is specifically limited to the cystic form of the organism. A variety of potential environmental signals, including interaction with lung epithelium, availability of nitrogen and lipid substrates, and the presence of differential mating types might initiate progression to cyst formation. Recently, Merali and Clarkson (58Merali S. Frevert U. Williams H. Chin K. Bryan R. Clarkson Jr., A.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2402-2407Crossref PubMed Scopus (87) Google Scholar) have reported continuous axenic culture of P. carinii. Further exploitation of this system using the molecular tools we report should provide essential insights into regulation of the P. cariniilife cycle.Exposed β-1,3-glucan on the surface of fungi also represents a major target of host recognition and inflammatory response (11Hoffman O.A. Standing J.E. Limper A.H. J. Immunol. 1993; 150: 3932-3940PubMed Google Scholar, 22Hidalgo H.A. Helmke R.J. German V.F. Mangos J.A. J. Protozool. 1991; 38: 30S-31SCrossref PubMed Scopus (28) Google Scholar). Binding of β-glucan to receptors on macrophages participates in phagocytic uptake of Candida albicans and Cryptococcus neoformans (59Janusz M.J. Austen K.F. Czop J.K. Immunology. 1988; 65: 181-185PubMed Google Scholar, 60Cross C.E. Bancroft G.J. Infect. Immun. 1995; 63: 2604-2611Crossref PubMed Google Scholar). Fungal β-glucans further stimulate the release of TNFα and IL-1β from monocytes, and also promotes the liberation of eicosanoids and lysozymal enzymes (61Daum T. Rohrbach M.S. FEBS Lett. 1992; 309: 119-122Crossref PubMed Scopus (33) Google Scholar, 62Czop J.K. Austen K.F. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 2751-2755Crossref PubMed Scopus (91) Google Scholar, 63Janusz M.J. Austen K.F. Czop J.K. J. Immunol. 1987; 138: 3897-3901PubMed Google Scholar, 64Castro M. Ralston N.V. Morgenthaler T.I. Rohrbach M.S. Limper A.H. Infect. Immun. 1994; 62: 3138-3145Crossref PubMed Google Scholar, 65Olson E.J. Standing J.E. Griego-Harper N. Hoffman O.A. Limper A.H. Infect. Immun. 1996; 64: 3548-3554Crossref PubMed Google Scholar, 66Rassmussen L.T. Seljelid R. J. Cell. Biochem. 1992; 46: 60-68Crossref Scopus (25) Google Scholar, 67Hoffman O.A. Olson E.J. Limper A.H. Immunol. Lett. 1993; 37: 19-25Crossref PubMed Scopus (57) Google Scholar). With respect to P. carinii, surface β-1,3-glucan on the organism can mediate alveolar macrophage uptake of P. carinii and also serve as a potent stimulant of release of TNFα and reactive oxidants (11Hoffman O.A. Standing J.E. Limper A.H. J. Immunol. 1993; 150: 3932-3940PubMed Google Scholar, 68Vassallo R. Thomas C.F. Vuk-Pavlovic Z. Limper A.H. J Lab. Clin. Med. 1999; 133: 535-540Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar).In summary, we have observed pneumocandin-inhibitable glucan synthetase activity within cell wall membrane isolates of P. cariniiand have identified and characterized a gsc-1-type β-1,3-glucan synthetase gene from this organism. A corresponding protein of appropriate 219.5-kDa molecular mass was present in P. carinii using an antibody generated to the predicted Gsc-1 protein. The expression of P. carinii gsc-1 is regulated over the life cycle of the organisms. In view of its central role in assembly of the P. carinii cyst wall, the Gsc-1 β-1,3-glucan synthetase is an attractive therapeutic target for the treatment of P. carinii pneumonia. Classification of Pneumocystis carinii as a fungus revolutionized study of this organism, which continues to cause life-threatening pneumonia in immune-compromised patients with AIDS, malignancy, and organ transplantation (1Edman J.C. Kovacs J.A. Masur H. Santi D.V. Elwood H.J. Sogin M.L. Nature. 1988; 334: 519-522Crossref PubMed Scopus (551) Google Scholar, 2Bruns T.D. Vilgalys R. Barns S.M. Gonzalez D. Hibbett D.S. Lane D.J. Simon L. Stickel S. Szaro T.M. Weisburg W.G. Mol. Phylogenet. Evol. 1992; 1: 231-241Crossref PubMed Scopus (234) Google Scholar, 3Van Der Peer Y. Hendriks L. Goris A. Syst. Appl. Microbiol. 1992; 15: 250-258Crossref Scopus (39) Google Scholar, 4Yale S.H. Limper A.H. Mayo Clin. Proc. 1996; 71: 5-13Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 5Thomas C.F. Limper A.H. Semin. Respir. Infect. 1998; 13: 289-295PubMed Google Scholar). Studies of P. carinii in infected lung indicate its life cycle alternates between smaller trophic forms and thick-walled cysts (6Campbell W.G. Arch. Pathol. 1972; 93: 312-330PubMed Google Scholar, 7Limper A.H. Thomas C.F. Anders R.A. Leof E.B. J. Lab. Clin. Med. 1997; 130: 132-138Abstract Full Text PDF PubMed Scopus (37) Google Scholar, 8De Stefano J.A. Cushion M.T. Sleight R.G. Walzer P.D. J. Protozool. 1990; 37: 428-435Crossref PubMed Scopus (34) Google Scholar). The origin of P. carinii cysts remains controversial, but it has been postulated they arise from sexual conjugation, analogous to ascomycetous fungi (9Itatani C.A. J. Parasitol. 1996; 82: 163-171Crossref PubMed Scopus (27) Google Scholar, 10MacNiell S.A. Fantes P.A. Hutchison C. Glover D.M. Cell Cycle Control. IRL Press at Oxford University Press, Oxford1995: 63-105Google Scholar). The mechanisms of cyst wall assembly byP. carinii are not well known, although recent studies reveal that these walls are largely composed of β-glucans, glycoprotein A, and chitins (11Hoffman O.A. Standing J.E. Limper A.H. J. Immunol. 1993; 150: 3932-3940PubMed Google Scholar, 12Lundgren B. Lipchik G.Y. Kovacs J.A. J. Clin. Invest. 1991; 87: 163-170Crossref PubMed Scopus (80) Google Scholar, 13Gigliotti F. Haidaris P.J. Haidaris C.G. Wright T.W. Van der Meid K.R. J. Infect. Dis. 1993; 168: 191-194Crossref PubMed Scopus (31) Google Scholar, 14Linke M.J. Cushion M.T. Walzer P.D. Infect. Immunol. 1989; 57: 1547-1555Crossref PubMed Google Scholar, 15Stringer S.L. Garbe T. Suskin S.M. Stringer J.R. J. Eukaryot. Microbiol. 1993; 40: 821-826Crossref PubMed Scopus (46) Google Scholar, 16Roth A. Wecke J. Karsten V. Janitschke K. Parasitol. Res. 1997; 83 (1997): 177-184Crossref PubMed Scopus (18) Google Scholar). β-Glucans are glucose homopolymers composed mainly of a β-1,3-linked carbohydrate core, with variable amounts of β-1,6- and β-1,4-linked glucose side chains (17Manners D.J. Masson A.J. Patterson J.C. Biochem. J. 1973; 135: 19-30Crossref PubMed Scopus (361) Google Scholar, 18Bacon J.S. Farmer V.C. Jones D. Taylor I.F. Biochem. J. 1969; 114: 557-567Crossref PubMed Scopus (118) Google Scholar, 19Kollar R. Reinhold B.B. Petrakova E. Yeh H.J. Ashwell G. Drgonova J. Kapteyn J.C. Klis F.M. Cabib E. J. Biol. Chem. 1997; 272: 17762-17775Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar). Glucans represent principal components of cell walls in fungi related to P. carinii. Ultrastructural investigations demonstrate an electron-lucent layer unique to the cystic form of P. carinii, which is specifically degraded by β-1,3-glucanases (16Roth A. Wecke J. Karsten V. Janitschke K. Parasitol. Res. 1997; 83 (1997): 177-184Crossref PubMed Scopus (18) Google Scholar). Additional studies with specific β-1,3-glucan antiserum also localize glucan to the walls of cysts (20Nollstadt K.H. Powles M.A. Fujioka H. Aikawa M. Schmatz D.M. Antimicrob. Agents Chemother. 1994; 38: 2258-2265Crossref PubMed Scopus (33) Google Scholar). β-1,3-Glucan has been detected in bronchoalveolar lavage from patients with P. carinii pneumonia (21Yasuoka A. Tachikawa N. Shimada K. Kimura S. Oka S. Clin. Diagn. Lab. Immunol. 1996; 3: 197-199Crossref PubMed Google Scholar). Furthermore, P. cariniiβ-glucans also represent important epitopes recognized by host cells (11Hoffman O.A. Standing J.E. Limper A.H. J. Immunol. 1993; 150: 3932-3940PubMed Google Scholar, 22Hidalgo H.A. Helmke R.J. German V.F. Mangos J.A. J. Protozool. 1991; 38: 30S-31SCrossref PubMed Scopus (28) Google Scholar). P. carinii β-glucans interact with alveolar macrophages mediating phagocytic uptake of the organism and lung inflammatory responses (11Hoffman O.A. Standing J.E. Limper A.H. J. Immunol. 1993; 150: 3932-3940PubMed Google Scholar). Subsequent glucan-mediated influx of neutrophils into the lung is an important contributor to respiratory impairment during this infection (23Limper A.H. Offord K.P. Smith T.F. Martin W.J. Am. Rev. Respir. Dis. 1989; 140: 1204-1209Crossref PubMed Scopus (394) Google Scholar, 24el-Sadr W. Simberkoff M.S. Am. Rev. Respir. Dis. 1988; 137: 1264-1267Crossref PubMed Scopus (76) Google Scholar, 25Yu M.L. Limper A.H. Am. J. Physiol. 1997; 273: L1103-L1111PubMed Google Scholar). Fungal β-glucans are assembled by a multisubunit enzyme complex within the organism's cell membrane. Gsc-1 proteins mediate the polymerization of uridine 5′-diphosphoglucose (UDP-Glc)1 into the insoluble β-1,3-glucan core required for cell wall assembly (26Castro C. Ribas J.C. Valdivieso M.H. Varona R. Ray F.D. Duran A. J. Bacteriol. 1995; 177: 5732-5739Crossref PubMed Google Scholar). Glucan synthetases are generally encoded by gsc-1 genes, which generate a 210-kDa catalytic protein in Saccharomyces cerevisiae and comparable proteins in other fungi (27Douglas C.M. Foor F. Marrinan J.A. Morin N. Nielsen J.B. Dahl J.A. Mazur P. Baginsky W. Li W. El-Sherbeini M. Clemas J.A. Mandela S.A. Frommer B.R. Kurtz M.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12907-12911Crossref PubMed Scopus (338) Google Scholar, 28Kelly R. Register E. Hsu M.J. Kurtz M. Nielsen J. J. Bacteriol. 1996; 178: 4381-4391Crossref PubMed Scopus (99) Google Scholar, 29Enderlin C.S. Selitrennikoff C.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9500-9504Crossref PubMed Scopus (29) Google Scholar, 30Tentler S. Palas J. Enderlin C. Campbell J. Taft C. Miller T.K. Wood R.L. Selitrennikoff C.P. Curr. Microbiol. 1997; 34: 303-330Crossref PubMed Scopus (20) Google Scholar, 31Mio T. Adachi-Shimizu M. Tachibana Y. Tabuchi H. Inoue S.B. Yabe T. Yamada-Okabe T. Arisawa M. Watanabe T. Yamada-Okabe H. J. Bacteriol. 1997; 179: 4096-4105Crossref PubMed Google Scholar, 32Kurtz M.B. Abruzzo G. Flattery A. Bartizal K. Marrinan J.A. Li W. Milligan J. Nollstadt K. Douglas C.M. Infect. Immun. 1996; 64: 3244-3251Crossref PubMed Google Scholar). Glucan synthetase activity by Gsc-1-type proteins is specifically inhibited by pneumocandin and echinocandin class compounds (32Kurtz M.B. Abruzzo G. Flattery A. Bartizal K. Marrinan J.A. Li W. Milligan J. Nollstadt K. Douglas C.M. Infect. Immun. 1996; 64: 3244-3251Crossref PubMed Google Scholar). Because mammalian cells do not possess glucan biosynthetic pathways, cell wall assembly represents an attractive target for the treatment of fungal infection. It is particularly noteworthy that pneumocandin inhibitors of β-glucan synthesis have been shown to rapidly inhibitP. carinii growth in rodent models (33Schmatz D.M. Romancheck M.A. Pittarelli L.A. Schwartz R.E. Fromtling R.A. Nollstadt K.H. Vanmiddlesworth F.L. Wilson K.E. Turner M.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5950-5954Crossref PubMed Scopus (153) Google Scholar, 34Schmatz D.M. Powles M.A. McFadden D. Nollstadt K. Bouffard F.A. Dropinski J.F. Liberator P. Andersen J. Antimicrob. Agents Chemother. 1995; 39: 1320-1323Crossref PubMed Scopus (49) Google Scholar, 35Powles M.A. Liberator P. Anderson J. Karkhanis Y. Dropinski J.F. Bouffard F.A. Balkovec J.M. Fujioka H. Aikawa M. McFadden D. Schmatz D. Antimicrob. Agents Chemother. 1998; 42: 1985-1989Crossref PubMed Google Scholar, 36Bartlett M.S. Current W.L. Goheen M.P. Boylan C.J. Lee C.H. Shaw M.M. Queener S.F. Smith J.W. Antimicrob. Agents Chemother. 1996; 40: 1811-1816Crossref PubMed Google Scholar). Such studies provide evidence of the importance of β-glucan generation during life cycle progression of this organism. Despite the considerable importance of β-glucan assembly in life cycle expression of this organism, in immune recognition during infection, and as a potential therapeutic target for pneumonia, the mechanisms of β-1,3-glucan assembly by P. carinii are not yet fully understood. The current investigation was undertaken to accomplish the following: 1) to establish whether P. carinii cell wall assembly occurs through action of a Gsc-1 protein mediating β-1,3-glucan synthesis; 2) to clone and characterize the respective gsc-1 encoding this activity in P. carinii; and finally 3) to evaluate expression of P. carinii gsc-1 over the life cycle of the organism. DISCUSSIONP. carinii membrane isolates possess the ability to incorporate UDP-[14C]glucose into insoluble carbohydrate, which is inhibited by the pneumocandin L-733,560 antagonist of Gsc-1-type β-1,3-glucan synthetases. Molecular cloning of theP. carinii gsc-1 gene predicts a mature protein with both similarities and unique differences to other fungal β-1,3-glucan synthetases. Specifically, the mRNA and protein expression ofP. carinii gsc-1 are highly regulated over the life cycle of the organism being predominantly expressed by cystic forms of the organism. A protein corresponding to the predicted sequence of the cloned gsc-1 gene was present in P. cariniimembrane isolates. Furthermore, immunoprecipitation of the putativeP. carinii Gsc-1 protein with a synthetic peptide antibody yielded a product capable of mediating incorporation of UDP-Glc into trichloroacetic acid-insoluble material, consistent with glucan.P. carinii Gsc-1 glucan synthetase exhibits several unique features. As discussed, structural differences were detected in the domain configuration of P. carinii Gsc-1 compared withS. cerevisiae and Aspergillus (26Castro C. Ribas J.C. Valdivieso M.H. Varona R. Ray F.D. Duran A. J. Bacteriol. 1995; 177: 5732-5739Crossref PubMed Google Scholar, 27Douglas C.M. Foor F. Marrinan J.A. Morin N. Nielsen J.B. Dahl J.A. Mazur P. Baginsky W. Li W. El-Sherbeini M. Clemas J.A. Mandela S.A. Frommer B.R. Kurtz M.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12907-12911Crossref PubMed Scopus (338) Google Scholar, 28Kelly R. Register E. Hsu M.J. Kurtz M. Nielsen J. J. Bacteriol. 1996; 178: 4381-4391Crossref PubMed Scopus (99) Google Scholar). Of further contrast, is the significant restriction of gsc-1expression predominantly to the cystic forms of the P. carinii. Most other fungi, including ascomycetous fungi, exhibit cell wall assembly constitutively throughout the life cycle (55Carlson C.R. Grallert B. Bernander R. Stokke T. Boye E. Yeast. 1997; 13: 1329-1335Crossref PubMed Scopus (30) Google Scholar). Our immunoblot and Northern analyses are complementary to previous immune localization studies, which also indicate that β-1,3-glucan is largely found within cysts (6Campbell W.G. Arch. Pathol. 1972; 93: 312-330PubMed Google Scholar, 20Nollstadt K.H. Powles M.A. Fujioka H. Aikawa M. Schmatz D.M. Antimicrob. Agents Chemother. 1994; 38: 2258-2265Crossref PubMed Scopus (33) Google Scholar).Because mammalian hosts do not possess an equivalent to Gsc-1, inhibition of β-1,3-glucan synthesis represents an attractive target for treatment of fungal infections. Echinocandins and pneumocandins are selective lipopeptide inhibitors that may expand our armamentaria for fungal infections, including those organisms resistant to standard agents (32Kurtz M.B. Abruzzo G. Flattery A. Bartizal K. Marrinan J.A. Li W. Milligan J. Nollstadt K. Douglas C.M. Infect. Immun. 1996; 64: 3244-3251Crossref PubMed Google Scholar). Schmatz and colleagues (33Schmatz D.M. Romancheck M.A. Pittarelli L.A. Schwartz R.E. Fromtling R.A. Nollstadt K.H. Vanmiddlesworth F.L. Wilson K.E. Turner M.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5950-5954Crossref PubMed Scopus (153) Google Scholar, 34Schmatz D.M. Powles M.A. McFadden D. Nollstadt K. Bouffard F.A. Dropinski J.F. Liberator P. Andersen J. Antimicrob. Agents Chemother. 1995; 39: 1320-1323Crossref PubMed Scopus (49) Google Scholar) have shown rapid reduction of organisms in rat and mouse models of P. cariniipneumonia. One pneumocandin in particular, L-671,329 has shown remarkable activity in the P. carinii rat model with >98% of cysts being eliminated (33Schmatz D.M. Romancheck M.A. Pittarelli L.A. Schwartz R.E. Fromtling R.A. Nollstadt K.H. Vanmiddlesworth F.L. Wilson K.E. Turner M.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5950-5954Crossref PubMed Scopus (153) Google Scholar).Concern had arisen that pneumocandins might only be effective against cystic forms of P. carinii, thereby limiting efficacy of such compounds during P. carinii pneumonia. The striking results of pneumocandins in animal models of P. cariniipneumonia strongly argue to the contrary (34Schmatz D.M. Powles M.A. McFadden D. Nollstadt K. Bouffard F.A. Dropinski J.F. Liberator P. Andersen J. Antimicrob. Agents Chemother. 1995; 39: 1320-1323Crossref PubMed Scopus (49) Google Scholar). These findings do suggest, however, that progression of trophic forms into cysts represents an essential component of life cycle progression in P. carinii, rather than an elective form utilized only under hostile conditions (7Limper A.H. Thomas C.F. Anders R.A. Leof E.B. J. Lab. Clin. Med. 1997; 130: 132-138Abstract Full Text PDF PubMed Scopus (37) Google Scholar). Other investigators have also found some effect of echinocandins on trophic structure after in vitro exposure (36Bartlett M.S. Current W.L. Goheen M.P. Boylan C.J. Lee C.H. Shaw M.M. Queener S.F. Smith J.W. Antimicrob. Agents Chemother. 1996; 40: 1811-1816Crossref PubMed Google Scholar). Although our study demonstrates low levels of gsc-1mRNA expression in trophic forms, small residual amounts of Gsc-1 protein were detected by Western analysis to remain within trophic forms. In addition, echinocandin and pneumocandin compounds may also effect other targets within P. carinii.Until recently, the lack of a reliable culture system has hindered studies of life cycle regulation by P. carinii (56Sloand E. Laughon B. Armstrong B. Bartlett M.S. Blumenfeld W. Cushion M. Kalica A. Kovacs J.A. Martin W.J. Pitt E. J. Eukaryot. Microbiol. 1993; 40: 188-195Crossref PubMed Scopus (86) Google Scholar). Recent studies implicate a cyclin-dependent kinase cell cycle control system, which exhibits regulated activity during progression ofP. carinii trophic forms to cysts (44Thomas Jr., C.F. Anders R.A. Gustafson M.P. Leof E.B. Limper A.H. Am. J. Respir. Cell. Mol. Biol. 1998; 18: 297-306Crossref PubMed Scopus (46) Google Scholar, 57Limper A.H. Thomas C.F. Mubarak K.K. Gustafson M.P. Kottom T.J. Leof E.B. J. Eukaryot. Microbiol. 1997; 44: 32SCrossref PubMed Scopus (3) Google Scholar). Considerable questions remain as to how assembly of the thickened β-glucan-rich pellicle is specifically limited to the cystic form of the organism. A variety of potential environmental signals, including interaction with lung epithelium, availability of nitrogen and lipid substrates, and the presence of differential mating types might initiate progression to cyst formation. Recently, Merali and Clarkson (58Merali S. Frevert U. Williams H. Chin K. Bryan R. Clarkson Jr., A.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2402-2407Crossref PubMed Scopus (87) Google Scholar) have reported continuous axenic culture of P. carinii. Further exploitation of this system using the molecular tools we report should provide essential insights into regulation of the P. cariniilife cycle.Exposed β-1,3-glucan on the surface of fungi also represents a major target of host recognition and inflammatory response (11Hoffman O.A. Standing J.E. Limper A.H. J. Immunol. 1993; 150: 3932-3940PubMed Google Scholar, 22Hidalgo H.A. Helmke R.J. German V.F. Mangos J.A. J. Protozool. 1991; 38: 30S-31SCrossref PubMed Scopus (28) Google Scholar). Binding of β-glucan to receptors on macrophages participates in phagocytic uptake of Candida albicans and Cryptococcus neoformans (59Janusz M.J. Austen K.F. Czop J.K. Immunology. 1988; 65: 181-185PubMed Google Scholar, 60Cross C.E. Bancroft G.J. Infect. Immun. 1995; 63: 2604-2611Crossref PubMed Google Scholar). Fungal β-glucans further stimulate the release of TNFα and IL-1β from monocytes, and also promotes the liberation of eicosanoids and lysozymal enzymes (61Daum T. Rohrbach M.S. FEBS Lett. 1992; 309: 119-122Crossref PubMed Scopus (33) Google Scholar, 62Czop J.K. Austen K.F. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 2751-2755Crossref PubMed Scopus (91) Google Scholar, 63Janusz M.J. Austen K.F. Czop J.K. J. Immunol. 1987; 138: 3897-3901PubMed Google Scholar, 64Castro M. Ralston N.V. Morgenthaler T.I. Rohrbach M.S. Limper A.H. Infect. Immun. 1994; 62: 3138-3145Crossref PubMed Google Scholar, 65Olson E.J. Standing J.E. Griego-Harper N. Hoffman O.A. Limper A.H. Infect. Immun. 1996; 64: 3548-3554Crossref PubMed Google Scholar, 66Rassmussen L.T. Seljelid R. J. Cell. Biochem. 1992; 46: 60-68Crossref Scopus (25) Google Scholar, 67Hoffman O.A. Olson E.J. Limper A.H. Immunol. Lett. 1993; 37: 19-25Crossref PubMed Scopus (57) Google Scholar). With respect to P. carinii, surface β-1,3-glucan on the organism can mediate alveolar macrophage uptake of P. carinii and also serve as a potent stimulant of release of TNFα and reactive oxidants (11Hoffman O.A. Standing J.E. Limper A.H. J. Immunol. 1993; 150: 3932-3940PubMed Google Scholar, 68Vassallo R. Thomas C.F. Vuk-Pavlovic Z. Limper A.H. J Lab. Clin. Med. 1999; 133: 535-540Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar).In summary, we have observed pneumocandin-inhibitable glucan synthetase activity within cell wall membrane isolates of P. cariniiand have identified and characterized a gsc-1-type β-1,3-glucan synthetase gene from this organism. A corresponding protein of appropriate 219.5-kDa molecular mass was present in P. carinii using an antibody generated to the predicted Gsc-1 protein. The expression of P. carinii gsc-1 is regulated over the life cycle of the organisms. In view of its central role in assembly of the P. carinii cyst wall, the Gsc-1 β-1,3-glucan synthetase is an attractive therapeutic target for the treatment of P. carinii pneumonia. P. carinii membrane isolates possess the ability to incorporate UDP-[14C]glucose into insoluble carbohydrate, which is inhibited by the pneumocandin L-733,560 antagonist of Gsc-1-type β-1,3-glucan synthetases. Molecular cloning of theP. carinii gsc-1 gene predicts a mature protein with both similarities and unique differences to other fungal β-1,3-glucan synthetases. Specifically, the mRNA and protein expression ofP. carinii gsc-1 are highly regulated over the life cycle of the organism being predominantly expressed by cystic forms of the organism. A protein corresponding to the predicted sequence of the cloned gsc-1 gene was present in P. cariniimembrane isolates. Furthermore, immunoprecipitation of the putativeP. carinii Gsc-1 protein with a synthetic peptide antibody yielded a product capable of mediating incorporation of UDP-Glc into trichloroacetic acid-insoluble material, consistent with glucan. P. carinii Gsc-1 glucan synthetase exhibits several unique features. As discussed, structural differences were detected in the domain configuration of P. carinii Gsc-1 compared withS. cerevisiae and Aspergillus (26Castro C. Ribas J.C. Valdivieso M.H. Varona R. Ray F.D. Duran A. J. Bacteriol. 1995; 177: 5732-5739Crossref PubMed Google Scholar, 27Douglas C.M. Foor F. Marrinan J.A. Morin N. Nielsen J.B. Dahl J.A. Mazur P. Baginsky W. Li W. El-Sherbeini M. Clemas J.A. Mandela S.A. Frommer B.R. Kurtz M.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12907-12911Crossref PubMed Scopus (338) Google Scholar, 28Kelly R. Register E. Hsu M.J. Kurtz M. Nielsen J. J. Bacteriol. 1996; 178: 4381-4391Crossref PubMed Scopus (99) Google Scholar). Of further contrast, is the significant restriction of gsc-1expression predominantly to the cystic forms of the P. carinii. Most other fungi, including ascomycetous fungi, exhibit cell wall assembly constitutively throughout the life cycle (55Carlson C.R. Grallert B. Bernander R. Stokke T. Boye E. Yeast. 1997; 13: 1329-1335Crossref PubMed Scopus (30) Google Scholar). Our immunoblot and Northern analyses are complementary to previous immune localization studies, which also indicate that β-1,3-glucan is largely found within cysts (6Campbell W.G. Arch. Pathol. 1972; 93: 312-330PubMed Google Scholar, 20Nollstadt K.H. Powles M.A. Fujioka H. Aikawa M. Schmatz D.M. Antimicrob. Agents Chemother. 1994; 38: 2258-2265Crossref PubMed Scopus (33) Google Scholar). Because mammalian hosts do not possess an equivalent to Gsc-1, inhibition of β-1,3-glucan synthesis represents an attractive target for treatment of fungal infections. Echinocandins and pneumocandins are selective lipopeptide inhibitors that may expand our armamentaria for fungal infections, including those organisms resistant to standard agents (32Kurtz M.B. Abruzzo G. Flattery A. Bartizal K. Marrinan J.A. Li W. Milligan J. Nollstadt K. Douglas C.M. Infect. Immun. 1996; 64: 3244-3251Crossref PubMed Google Scholar). Schmatz and colleagues (33Schmatz D.M. Romancheck M.A. Pittarelli L.A. Schwartz R.E. Fromtling R.A. Nollstadt K.H. Vanmiddlesworth F.L. Wilson K.E. Turner M.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5950-5954Crossref PubMed Scopus (153) Google Scholar, 34Schmatz D.M. Powles M.A. McFadden D. Nollstadt K. Bouffard F.A. Dropinski J.F. Liberator P. Andersen J. Antimicrob. Agents Chemother. 1995; 39: 1320-1323Crossref PubMed Scopus (49) Google Scholar) have shown rapid reduction of organisms in rat and mouse models of P. cariniipneumonia. One pneumocandin in particular, L-671,329 has shown remarkable activity in the P. carinii rat model with >98% of cysts being eliminated (33Schmatz D.M. Romancheck M.A. Pittarelli L.A. Schwartz R.E. Fromtling R.A. Nollstadt K.H. Vanmiddlesworth F.L. Wilson K.E. Turner M.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5950-5954Crossref PubMed Scopus (153) Google Scholar). Concern had arisen that pneumocandins might only be effective against cystic forms of P. carinii, thereby limiting efficacy of such compounds during P. carinii pneumonia. The striking results of pneumocandins in animal models of P. cariniipneumonia strongly argue to the contrary (34Schmatz D.M. Powles M.A. McFadden D. Nollstadt K. Bouffard F.A. Dropinski J.F. Liberator P. Andersen J. Antimicrob. Agents Chemother. 1995; 39: 1320-1323Crossref PubMed Scopus (49) Google Scholar). These findings do suggest, however, that progression of trophic forms into cysts represents an essential component of life cycle progression in P. carinii, rather than an elective form utilized only under hostile conditions (7Limper A.H. Thomas C.F. Anders R.A. Leof E.B. J. Lab. Clin. Med. 1997; 130: 132-138Abstract Full Text PDF PubMed Scopus (37) Google Scholar). Other investigators have also found some effect of echinocandins on trophic structure after in vitro exposure (36Bartlett M.S. Current W.L. Goheen M.P. Boylan C.J. Lee C.H. Shaw M.M. Queener S.F. Smith J.W. Antimicrob. Agents Chemother. 1996; 40: 1811-1816Crossref PubMed Google Scholar). Although our study demonstrates low levels of gsc-1mRNA expression in trophic forms, small residual amounts of Gsc-1 protein were detected by Western analysis to remain within trophic forms. In addition, echinocandin and pneumocandin compounds may also effect other targets within P. carinii. Until recently, the lack of a reliable culture system has hindered studies of life cycle regulation by P. carinii (56Sloand E. Laughon B. Armstrong B. Bartlett M.S. Blumenfeld W. Cushion M. Kalica A. Kovacs J.A. Martin W.J. Pitt E. J. Eukaryot. Microbiol. 1993; 40: 188-195Crossref PubMed Scopus (86) Google Scholar). Recent studies implicate a cyclin-dependent kinase cell cycle control system, which exhibits regulated activity during progression ofP. carinii trophic forms to cysts (44Thomas Jr., C.F. Anders R.A. Gustafson M.P. Leof E.B. Limper A.H. Am. J. Respir. Cell. Mol. Biol. 1998; 18: 297-306Crossref PubMed Scopus (46) Google Scholar, 57Limper A.H. Thomas C.F. Mubarak K.K. Gustafson M.P. Kottom T.J. Leof E.B. J. Eukaryot. Microbiol. 1997; 44: 32SCrossref PubMed Scopus (3) Google Scholar). Considerable questions remain as to how assembly of the thickened β-glucan-rich pellicle is specifically limited to the cystic form of the organism. A variety of potential environmental signals, including interaction with lung epithelium, availability of nitrogen and lipid substrates, and the presence of differential mating types might initiate progression to cyst formation. Recently, Merali and Clarkson (58Merali S. Frevert U. Williams H. Chin K. Bryan R. Clarkson Jr., A.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2402-2407Crossref PubMed Scopus (87) Google Scholar) have reported continuous axenic culture of P. carinii. Further exploitation of this system using the molecular tools we report should provide essential insights into regulation of the P. cariniilife cycle. Exposed β-1,3-glucan on the surface of fungi also represents a major target of host recognition and inflammatory response (11Hoffman O.A. Standing J.E. Limper A.H. J. Immunol. 1993; 150: 3932-3940PubMed Google Scholar, 22Hidalgo H.A. Helmke R.J. German V.F. Mangos J.A. J. Protozool. 1991; 38: 30S-31SCrossref PubMed Scopus (28) Google Scholar). Binding of β-glucan to receptors on macrophages participates in phagocytic uptake of Candida albicans and Cryptococcus neoformans (59Janusz M.J. Austen K.F. Czop J.K. Immunology. 1988; 65: 181-185PubMed Google Scholar, 60Cross C.E. Bancroft G.J. Infect. Immun. 1995; 63: 2604-2611Crossref PubMed Google Scholar). Fungal β-glucans further stimulate the release of TNFα and IL-1β from monocytes, and also promotes the liberation of eicosanoids and lysozymal enzymes (61Daum T. Rohrbach M.S. FEBS Lett. 1992; 309: 119-122Crossref PubMed Scopus (33) Google Scholar, 62Czop J.K. Austen K.F. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 2751-2755Crossref PubMed Scopus (91) Google Scholar, 63Janusz M.J. Austen K.F. Czop J.K. J. Immunol. 1987; 138: 3897-3901PubMed Google Scholar, 64Castro M. Ralston N.V. Morgenthaler T.I. Rohrbach M.S. Limper A.H. Infect. Immun. 1994; 62: 3138-3145Crossref PubMed Google Scholar, 65Olson E.J. Standing J.E. Griego-Harper N. Hoffman O.A. Limper A.H. Infect. Immun. 1996; 64: 3548-3554Crossref PubMed Google Scholar, 66Rassmussen L.T. Seljelid R. J. Cell. Biochem. 1992; 46: 60-68Crossref Scopus (25) Google Scholar, 67Hoffman O.A. Olson E.J. Limper A.H. Immunol. Lett. 1993; 37: 19-25Crossref PubMed Scopus (57) Google Scholar). With respect to P. carinii, surface β-1,3-glucan on the organism can mediate alveolar macrophage uptake of P. carinii and also serve as a potent stimulant of release of TNFα and reactive oxidants (11Hoffman O.A. Standing J.E. Limper A.H. J. Immunol. 1993; 150: 3932-3940PubMed Google Scholar, 68Vassallo R. Thomas C.F. Vuk-Pavlovic Z. Limper A.H. J Lab. Clin. Med. 1999; 133: 535-540Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). In summary, we have observed pneumocandin-inhibitable glucan synthetase activity within cell wall membrane isolates of P. cariniiand have identified and characterized a gsc-1-type β-1,3-glucan synthetase gene from this organism. A corresponding protein of appropriate 219.5-kDa molecular mass was present in P. carinii using an antibody generated to the predicted Gsc-1 protein. The expression of P. carinii gsc-1 is regulated over the life cycle of the organisms. In view of its central role in assembly of the P. carinii cyst wall, the Gsc-1 β-1,3-glucan synthetase is an attractive therapeutic target for the treatment of P. carinii pneumonia.
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