Cell Wall Assembly by Pneumocystis carinii

Abstract

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. 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; PubMed Scopus Google Scholar). has been in from patients with P. carinii pneumonia A. K. S. S. Clin. Lab. Immunol. 1996; PubMed Google Scholar). P. also represent by (11Hoffman O.A. Standing J.E. Limper A.H. J. Immunol. 1993; 150: 3932-3940PubMed Google Scholar, J.A. J. Protozool. 1991; PubMed Scopus Google Scholar). P. carinii with of the and lung (11Hoffman O.A. Standing J.E. Limper A.H. J. Immunol. 1993; 150: 3932-3940PubMed Google Scholar). of into the lung is an to this infection A.H. Respir. Dis. 1989; PubMed Scopus Google Scholar, Respir. Dis. 1988; PubMed Scopus Google Scholar, M.L. Limper A.H. J. 1997; Google are by a the cell Gsc-1 the of uridine 5′-diphosphoglucose into the insoluble β-1,3-glucan for cell wall assembly C. J.C. R. A. J. PubMed Google Scholar). are by gsc-1 which a catalytic protein in and in other fungi F. J.A. J.A. P. M. J.A. S.A. Proc. S. A. 1994; PubMed Scopus Google Scholar, R. E. M.J. M. J. J. 1996; PubMed Scopus Google Scholar, Proc. S. A. 1994; PubMed Scopus Google Scholar, S. J. C. J. C. Microbiol. 1997; PubMed Scopus Google Scholar, T. M. Y. H. T. T. M. T. H. J. 1997; PubMed Google Scholar, G. A. K. J.A. J. K. Infect. 1996; PubMed Google Scholar). synthetase by Gsc-1-type is specifically inhibited by pneumocandin and G. A. K. J.A. J. K. Infect. 1996; PubMed Google mammalian not glucan cell wall assembly represents an attractive target for the of fungal is that pneumocandin of β-glucan synthesis been to rapidly carinii in models D.M. M.A. R.A. K.H. M.J. Proc. S. A. 1990; 87: PubMed Scopus Google Scholar, D.M. Powles M.A. D. K. P. J. Antimicrob. Agents Chemother. PubMed Scopus Google Scholar, M.A. P. J. Y. Fujioka H. Aikawa M. D. Schmatz D. Antimicrob. Agents Chemother. 1998; PubMed Google Scholar, Antimicrob. Agents Chemother. 1996; 40: PubMed Google Scholar). studies of the of β-glucan life cycle of this organism. the of β-glucan assembly in life cycle expression of this organism, in immune and as a therapeutic target for the mechanisms of β-1,3-glucan assembly by P. carinii are not was to the to P. carinii cell wall assembly of a Gsc-1 protein β-1,3-glucan to and the gsc-1 encoding this in P. and to expression of P. carinii gsc-1 over the life cycle of the carinii membrane the to incorporate into insoluble carbohydrate, which is inhibited by the pneumocandin antagonist of Gsc-1-type β-1,3-glucan synthetases. of carinii gsc-1 a protein with and unique to other fungal β-1,3-glucan synthetases. the mRNA and protein expression ofP. carinii gsc-1 are regulated over the life cycle of the by cystic forms of the organism. protein to the predicted of the cloned gsc-1 was in P. of the carinii Gsc-1 protein with a synthetic peptide yielded a capable of of into with carinii Gsc-1 glucan synthetase unique As in the domain of P. carinii Gsc-1 and C. J.C. R. A. J. PubMed Google Scholar, F. J.A. J.A. P. M. J.A. S.A. Proc. S. A. 1994; PubMed Scopus Google Scholar, R. E. M.J. M. J. J. 1996; PubMed Scopus Google Scholar). is the of to the cystic forms of the P. carinii. other fungi, ascomycetous fungi, cell wall assembly the life cycle B. R. T. E. 1997; 13: PubMed Scopus Google Scholar). and are to immune which also indicate that β-1,3-glucan is largely cysts (6Campbell W.G. Arch. Pathol. 1972; 93: 312-330PubMed Google Scholar, K.H. Powles M.A. Fujioka H. Aikawa M. Schmatz D.M. Antimicrob. Agents Chemother. 1994; PubMed Scopus Google mammalian not an to inhibition of β-1,3-glucan synthesis represents an attractive target for of fungal and are that for fungal to G. A. K. J.A. J. K. Infect. 1996; PubMed Google Scholar). Schmatz and D.M. M.A. R.A. K.H. M.J. Proc. S. A. 1990; 87: PubMed Scopus Google Scholar, D.M. Powles M.A. D. K. P. J. Antimicrob. Agents Chemother. PubMed Scopus Google of in and models of P. pneumocandin in has in the P. carinii with of cysts D.M. M.A. R.A. K.H. M.J. Proc. S. A. 1990; 87: PubMed Scopus Google that cystic forms of P. carinii, of P. carinii pneumonia. The results of in animal models of P. to the D.M. Powles M.A. D. K. P. J. Antimicrob. Agents Chemother. PubMed Scopus Google Scholar). These that of trophic forms into cysts represents an of life cycle in P. carinii, an form utilized 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). also of trophic in Antimicrob. Agents Chemother. 1996; 40: PubMed Google Scholar). study of expression in trophic forms, amounts of Gsc-1 protein by to trophic and pneumocandin also other P. the of a has studies of life cycle by P. carinii E. B. B. Cushion M. A. Kovacs J.A. E. J. Eukaryot. Microbiol. 1993; 40: PubMed Scopus Google Scholar). Recent studies a cell cycle which regulated ofP. carinii trophic forms to cysts C.F. Anders R.A. Leof E.B. Limper A.H. J. Respir. Mol. Biol. 1998; PubMed Scopus (46) Google Scholar, A.H. Thomas C.F. Leof E.B. J. Eukaryot. Microbiol. 1997; PubMed Scopus Google Scholar). as to assembly of the is specifically to the cystic form of the organism. of with lung of and and the of to cyst formation. and S. H. K. R. Proc. S. A. PubMed Scopus Google of P. carinii. of this the molecular into of the P. β-1,3-glucan the of fungi also represents a target of and (11Hoffman O.A. Standing J.E. Limper A.H. J. Immunol. 1993; 150: 3932-3940PubMed Google Scholar, J.A. J. Protozool. 1991; PubMed Scopus Google Scholar). of β-glucan to in of and M.J. 1988; Google Scholar, Infect. PubMed Google Scholar). the of and from and also the of and T. 1992; PubMed Scopus Google Scholar, Proc. S. A. 82: PubMed Scopus Google Scholar, M.J. J. Immunol. Google Scholar, M. Limper A.H. Infect. 1994; PubMed Google Scholar, Standing J.E. O.A. Limper A.H. Infect. 1996; PubMed Google Scholar, R. J. Biochem. 1992; Scopus Google Scholar, O.A. Limper A.H. Immunol. 1993; 37: PubMed Scopus Google Scholar). to P. carinii, β-1,3-glucan the of P. carinii and also as a of of and (11Hoffman O.A. Standing J.E. Limper A.H. J. Immunol. 1993; 150: 3932-3940PubMed Google Scholar, R. Thomas C.F. Limper A.H. Lab. Clin. Med. Full Text Full Text PDF PubMed Scopus Google glucan synthetase cell wall membrane of P. and a β-1,3-glucan synthetase from this organism. protein of molecular was in P. carinii an to the predicted Gsc-1 The expression of P. carinii gsc-1 is regulated over the life cycle of the of its in assembly of the P. carinii cyst the Gsc-1 β-1,3-glucan synthetase is an attractive therapeutic target for the 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). 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; PubMed Scopus Google Scholar). has been in from patients with P. carinii pneumonia A. K. S. S. Clin. Lab. Immunol. 1996; PubMed Google Scholar). P. also represent by (11Hoffman O.A. Standing J.E. Limper A.H. J. Immunol. 1993; 150: 3932-3940PubMed Google Scholar, J.A. J. Protozool. 1991; PubMed Scopus Google Scholar). P. carinii with of the and lung (11Hoffman O.A. Standing J.E. Limper A.H. J. Immunol. 1993; 150: 3932-3940PubMed Google Scholar). of into the lung is an to this infection A.H. Respir. Dis. 1989; PubMed Scopus Google Scholar, Respir. Dis. 1988; PubMed Scopus Google Scholar, M.L. Limper A.H. J. 1997; Google Scholar). are by a the cell Gsc-1 the of uridine 5′-diphosphoglucose into the insoluble β-1,3-glucan for cell wall assembly C. J.C. R. A. J. PubMed Google Scholar). are by gsc-1 which a catalytic protein in and in other fungi F. J.A. J.A. P. M. J.A. S.A. Proc. S. A. 1994; PubMed Scopus Google Scholar, R. E. M.J. M. J. J. 1996; PubMed Scopus Google Scholar, Proc. S. A. 1994; PubMed Scopus Google Scholar, S. J. C. J. C. Microbiol. 1997; PubMed Scopus Google Scholar, T. M. Y. H. T. T. M. T. H. J. 1997; PubMed Google Scholar, G. A. K. J.A. J. K. Infect. 1996; PubMed Google Scholar). synthetase by Gsc-1-type is specifically inhibited by pneumocandin and G. A. K. J.A. J. K. Infect. 1996; PubMed Google Scholar). Because mammalian not glucan cell wall assembly represents an attractive target for the of fungal is that pneumocandin of β-glucan synthesis been to rapidly carinii in models D.M. M.A. R.A. K.H. M.J. Proc. S. A. 1990; 87: PubMed Scopus Google Scholar, D.M. Powles M.A. D. K. P. J. Antimicrob. Agents Chemother. PubMed Scopus Google Scholar, M.A. P. J. Y. Fujioka H. Aikawa M. D. Schmatz D. Antimicrob. Agents Chemother. 1998; PubMed Google Scholar, Antimicrob. Agents Chemother. 1996; 40: PubMed Google Scholar). studies of the of β-glucan life cycle of this organism. the of β-glucan assembly in life cycle expression of this organism, in immune and as a therapeutic target for the mechanisms of β-1,3-glucan assembly by P. carinii are not The was to the to P. carinii cell wall assembly of a Gsc-1 protein β-1,3-glucan to and the gsc-1 encoding this in P. and to expression of P. carinii gsc-1 over the life cycle of the organism. carinii membrane the to incorporate into insoluble carbohydrate, which is inhibited by the pneumocandin antagonist of Gsc-1-type β-1,3-glucan synthetases. of carinii gsc-1 a protein with and unique to other fungal β-1,3-glucan synthetases. the mRNA and protein expression ofP. carinii gsc-1 are regulated over the life cycle of the by cystic forms of the organism. protein to the predicted of the cloned gsc-1 was in P. of the carinii Gsc-1 protein with a synthetic peptide yielded a capable of of into with carinii Gsc-1 glucan synthetase unique As in the domain of P. carinii Gsc-1 and C. J.C. R. A. J. PubMed Google Scholar, F. J.A. J.A. P. M. J.A. S.A. Proc. S. A. 1994; PubMed Scopus Google Scholar, R. E. M.J. M. J. J. 1996; PubMed Scopus Google Scholar). is the of to the cystic forms of the P. carinii. other fungi, ascomycetous fungi, cell wall assembly the life cycle B. R. T. E. 1997; 13: PubMed Scopus Google Scholar). and are to immune which also indicate that β-1,3-glucan is largely cysts (6Campbell W.G. Arch. Pathol. 1972; 93: 312-330PubMed Google Scholar, K.H. Powles M.A. Fujioka H. Aikawa M. Schmatz D.M. Antimicrob. Agents Chemother. 1994; PubMed Scopus Google mammalian not an to inhibition of β-1,3-glucan synthesis represents an attractive target for of fungal and are that for fungal to G. A. K. J.A. J. K. Infect. 1996; PubMed Google Scholar). Schmatz and D.M. M.A. R.A. K.H. M.J. Proc. S. A. 1990; 87: PubMed Scopus Google Scholar, D.M. Powles M.A. D. K. P. J. Antimicrob. Agents Chemother. PubMed Scopus Google of in and models of P. pneumocandin in has in the P. carinii with of cysts D.M. M.A. R.A. K.H. M.J. Proc. S. A. 1990; 87: PubMed Scopus Google that cystic forms of P. carinii, of P. carinii pneumonia. The results of in animal models of P. to the D.M. Powles M.A. D. K. P. J. Antimicrob. Agents Chemother. PubMed Scopus Google Scholar). These that of trophic forms into cysts represents an of life cycle in P. carinii, an form utilized 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). also of trophic in Antimicrob. Agents Chemother. 1996; 40: PubMed Google Scholar). study of expression in trophic forms, amounts of Gsc-1 protein by to trophic and pneumocandin also other P. the of a has studies of life cycle by P. carinii E. B. B. Cushion M. A. Kovacs J.A. E. J. Eukaryot. Microbiol. 1993; 40: PubMed Scopus Google Scholar). Recent studies a cell cycle which regulated ofP. carinii trophic forms to cysts C.F. Anders R.A. Leof E.B. Limper A.H. J. Respir. Mol. Biol. 1998; PubMed Scopus (46) Google Scholar, A.H. Thomas C.F. Leof E.B. J. Eukaryot. Microbiol. 1997; PubMed Scopus Google Scholar). as to assembly of the is specifically to the cystic form of the organism. of with lung of and and the of to cyst formation. and S. H. K. R. Proc. S. A. PubMed Scopus Google of P. carinii. of this the molecular into of the P. β-1,3-glucan the of fungi also represents a target of and (11Hoffman O.A. Standing J.E. Limper A.H. J. Immunol. 1993; 150: 3932-3940PubMed Google Scholar, J.A. J. Protozool. 1991; PubMed Scopus Google Scholar). of β-glucan to in of and M.J. 1988; Google Scholar, Infect. PubMed Google Scholar). the of and from and also the of and T. 1992; PubMed Scopus Google Scholar, Proc. S. A. 82: PubMed Scopus Google Scholar, M.J. J. Immunol. Google Scholar, M. Limper A.H. Infect. 1994; PubMed Google Scholar, Standing J.E. O.A. Limper A.H. Infect. 1996; PubMed Google Scholar, R. J. Biochem. 1992; Scopus Google Scholar, O.A. Limper A.H. Immunol. 1993; 37: PubMed Scopus Google Scholar). to P. carinii, β-1,3-glucan the of P. carinii and also as a of of and (11Hoffman O.A. Standing J.E. Limper A.H. J. Immunol. 1993; 150: 3932-3940PubMed Google Scholar, R. Thomas C.F. Limper A.H. Lab. Clin. Med. Full Text Full Text PDF PubMed Scopus Google glucan synthetase cell wall membrane of P. and a β-1,3-glucan synthetase from this organism. protein of molecular was in P. carinii an to the predicted Gsc-1 The expression of P. carinii gsc-1 is regulated over the life cycle of the of its in assembly of the P. carinii cyst the Gsc-1 β-1,3-glucan synthetase is an attractive therapeutic target for the of P. carinii pneumonia. P. carinii membrane the to incorporate into insoluble carbohydrate, which is inhibited by the pneumocandin antagonist of Gsc-1-type β-1,3-glucan synthetases. of carinii gsc-1 a protein with and unique to other fungal β-1,3-glucan synthetases. the mRNA and protein expression ofP. carinii gsc-1 are regulated over the life cycle of the by cystic forms of the organism. protein to the predicted of the cloned gsc-1 was in P. of the carinii Gsc-1 protein with a synthetic peptide yielded a capable of of into with P. carinii Gsc-1 glucan synthetase unique As in the domain of P. carinii Gsc-1 and C. J.C. R. A. J. PubMed Google Scholar, F. J.A. J.A. P. M. J.A. S.A. Proc. S. A. 1994; PubMed Scopus Google Scholar, R. E. M.J. M. J. J. 1996; PubMed Scopus Google Scholar). is the of to the cystic forms of the P. carinii. other fungi, ascomycetous fungi, cell wall assembly the life cycle B. R. T. E. 1997; 13: PubMed Scopus Google Scholar). and are to immune which also indicate that β-1,3-glucan is largely cysts (6Campbell W.G. Arch. Pathol. 1972; 93: 312-330PubMed Google Scholar, K.H. Powles M.A. Fujioka H. Aikawa M. Schmatz D.M. Antimicrob. Agents Chemother. 1994; PubMed Scopus Google Scholar). Because mammalian not an to inhibition of β-1,3-glucan synthesis represents an attractive target for of fungal and are that for fungal to G. A. K. J.A. J. K. Infect. 1996; PubMed Google Scholar). Schmatz and D.M. M.A. R.A. K.H. M.J. Proc. S. A. 1990; 87: PubMed Scopus Google Scholar, D.M. Powles M.A. D. K. P. J. Antimicrob. Agents Chemother. PubMed Scopus Google of in and models of P. pneumocandin in has in the P. carinii with of cysts D.M. M.A. R.A. K.H. M.J. Proc. S. A. 1990; 87: PubMed Scopus Google Scholar). that cystic forms of P. carinii, of P. carinii pneumonia. The results of in animal models of P. to the D.M. Powles M.A. D. K. P. J. Antimicrob. Agents Chemother. PubMed Scopus Google Scholar). These that of trophic forms into cysts represents an of life cycle in P. carinii, an form utilized 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). also of trophic in Antimicrob. Agents Chemother. 1996; 40: PubMed Google Scholar). study of expression in trophic forms, amounts of Gsc-1 protein by to trophic and pneumocandin also other P. carinii. the of a has studies of life cycle by P. carinii E. B. B. Cushion M. A. Kovacs J.A. E. J. Eukaryot. Microbiol. 1993; 40: PubMed Scopus Google Scholar). Recent studies a cell cycle which regulated ofP. carinii trophic forms to cysts C.F. Anders R.A. Leof E.B. Limper A.H. J. Respir. Mol. Biol. 1998; PubMed Scopus (46) Google Scholar, A.H. Thomas C.F. Leof E.B. J. Eukaryot. Microbiol. 1997; PubMed Scopus Google Scholar). as to assembly of the is specifically to the cystic form of the organism. of with lung of and and the of to cyst formation. and S. H. K. R. Proc. S. A. PubMed Scopus Google of P. carinii. of this the molecular into of the P. β-1,3-glucan the of fungi also represents a target of and (11Hoffman O.A. Standing J.E. Limper A.H. J. Immunol. 1993; 150: 3932-3940PubMed Google Scholar, J.A. J. Protozool. 1991; PubMed Scopus Google Scholar). of β-glucan to in of and M.J. 1988; Google Scholar, Infect. PubMed Google Scholar). the of and from and also the of and T. 1992; PubMed Scopus Google Scholar, Proc. S. A. 82: PubMed Scopus Google Scholar, M.J. J. Immunol. Google Scholar, M. Limper A.H. Infect. 1994; PubMed Google Scholar, Standing J.E. O.A. Limper A.H. Infect. 1996; PubMed Google Scholar, R. J. Biochem. 1992; Scopus Google Scholar, O.A. Limper A.H. Immunol. 1993; 37: PubMed Scopus Google Scholar). to P. carinii, β-1,3-glucan the of P. carinii and also as a of of and (11Hoffman O.A. Standing J.E. Limper A.H. J. Immunol. 1993; 150: 3932-3940PubMed Google Scholar, R. Thomas C.F. Limper A.H. Lab. Clin. Med. Full Text Full Text PDF PubMed Scopus Google Scholar). glucan synthetase cell wall membrane of P. and a β-1,3-glucan synthetase from this organism. protein of molecular was in P. carinii an to the predicted Gsc-1 The expression of P. carinii gsc-1 is regulated over the life cycle of the of its in assembly of the P. carinii cyst the Gsc-1 β-1,3-glucan synthetase is an attractive therapeutic target for the of P. carinii pneumonia.