title: Glycosylation and immunoreactivity of the Histoplasma capsulatum Cfp4 yeast-phase exoantigen.
abstract: The yeast phase of Histoplasma capsulatum is the virulent form of this thermally dimorphic fungal pathogen. Among the secreted proteome of Histoplasma, culture filtrate protein 4 (Cfp4) is a heavily glycosylated factor produced abundantly and specifically by Histoplasma yeast cells, suggesting its role in pathogenesis. We have generated three monoclonal antibodies as tools for characterization and detection of Cfp4 and determined the epitope each recognizes. Through site-directed mutagenesis of Cfp4, we identified three asparagines that function as the principal sites of N-linked glycan modification. To test the function of Cfp4 in Histoplasma pathogenesis, we generated Cfp4-deficient strains by insertional mutagenesis and by RNA interference. Cfp4-deficient strains are not attenuated in virulence in human macrophages or during lung infection in a murine model of histoplasmosis. Coinfection of differentially marked Cfp4-producing and Cfp4-deficient strains demonstrates that production of Cfp4 does not confer a fitness advantage to Histoplasma yeasts during murine lung infection. Despite no apparent role in acute virulence in mice, secretion of the Cfp4 glycoprotein by yeast cells is consistent across clinical and laboratory isolates of the North American type 1 and type 2 phylogenetic groups as well as a strain from Panama. In addition, human immune sera recognize the Histoplasma Cfp4 protein, confirming Cfp4 production during infection of human hosts. These results suggest the potential utility of Cfp4 as a diagnostic exoantigen for histoplasmosis.
authors.affiliation: Department of Microbiology, Department of Microbial Infection and Immunity, Ohio State University, Columbus, Ohio, USA.
authors.forename: Eric D
authors.initials: ED
authors.lastname: Holbrook
authors.affiliation: Department of Microbiology, Department of Microbial Infection and Immunity, Ohio State University, Columbus, Ohio, USA.
authors.forename: Megan M
authors.initials: MM
authors.lastname: Kemski
authors.affiliation: MiraVista Diagnostics, Indianapolis, Indiana, USA.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Richer
authors.affiliation: MiraVista Diagnostics, Indianapolis, Indiana, USA.
authors.forename: L Joseph
authors.initials: LJ
authors.lastname: Wheat
authors.affiliation: Department of Microbiology, Department of Microbial Infection and Immunity, Ohio State University, Columbus, Ohio, USA rappleye.1@osu.edu.
authors.forename: Chad A
authors.initials: CA
authors.lastname: Rappleye
mesh_terms: D000818:Animals; D000908:Antibodies, Fungal / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D004195:Disease Models, Animal; D018604:Epitope Mapping; D005656:Fungal Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D055785:Gene Knockdown Techniques; D055786:Gene Knockout Techniques; D006031:Glycosylation; D006658:Histoplasma / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006660:Histoplasmosis / Q000382:microbiology / Q000473:pathology; D006801:Humans; D008264:Macrophages / Q000276:immunology / Q000382:microbiology; D051379:Mice; D008810:Mice, Inbred C57BL; D016254:Mutagenesis, Insertional; D011499:Protein Processing, Post-Translational; D034622:RNA Interference
chemical_list: D000908:Antibodies, Fungal; D000911:Antibodies, Monoclonal; D005656:Fungal Proteins
references.citation: Mol Microbiol. 2008 Oct;70(2):352-68
references.pmid: 18761625
references.citation: Mol Ecol. 2003 Dec;12(12):3383-401
references.pmid: 14629354
references.citation: Infect Immun. 1998 Apr;66(4):1697-707
references.pmid: 9529100
references.citation: FEMS Microbiol Lett. 2011 Nov;324(1):1-9
references.pmid: 22092757
references.citation: PLoS Pathog. 2012;8(5):e1002713
references.pmid: 22615571
references.citation: Mol Microbiol. 2006 Nov;62(4):970-83
references.pmid: 17038119
references.citation: Nat Methods. 2011 Sep 29;8(10):785-6
references.pmid: 21959131
references.citation: Infect Immun. 2013 Jul;81(7):2334-46
references.pmid: 23589579
references.citation: Eukaryot Cell. 2007 Apr;6(4):609-15
references.pmid: 17337639
references.citation: J Clin Lab Anal. 1991;5(2):121-6
references.pmid: 2023057
references.citation: J Med Vet Mycol. 1990;28(6):479-85
references.pmid: 2093121
references.citation: Clin Chest Med. 2009 Jun;30(2):217-25, v
references.pmid: 19375629
references.citation: Braz J Microbiol. 2006 Jan;37(1):1-13
references.pmid: 20445761
references.citation: Biotechniques. 1990 May;8(5):528-35
references.pmid: 2357375
references.citation: BMC Microbiol. 2009 Nov 17;9:236
references.pmid: 19919692
references.citation: J Immunol. 1993 Apr 1;150(7):2920-30
references.pmid: 8454864
references.citation: Proc Natl Acad Sci U S A. 2007 Jan 23;104(4):1366-70
references.pmid: 17227865
references.citation: Mol Microbiol. 2004 Jul;53(1):153-65
references.pmid: 15225311
references.citation: Clin Infect Dis. 2011 Sep;53(5):448-54
references.pmid: 21810734
references.citation: Gene. 1989 Apr 15;77(1):51-9
references.pmid: 2744487
references.citation: Proc Natl Acad Sci U S A. 2008 Mar 25;105(12):4880-5
references.pmid: 18339808
references.citation: Science. 2000 Nov 17;290(5495):1368-72
references.pmid: 11082066
references.citation: Expert Opin Biol Ther. 2006 Nov;6(11):1207-21
references.pmid: 17049017
references.citation: Am Rev Respir Dis. 1969 Apr;99(4):Suppl:1-132
references.pmid: 5767603
references.citation: J Bacteriol. 1998 Apr;180(7):1786-92
references.pmid: 9537376
references.citation: Methods Mol Biol. 2012;845:51-66
references.pmid: 22328367
references.citation: PLoS Pathog. 2008 Apr 11;4(4):e1000044
references.pmid: 18404210
references.citation: Antimicrob Agents Chemother. 2001 Aug;45(8):2354-7
references.pmid: 11451696
references.citation: Clin Vaccine Immunol. 2014 Feb;21(2):143-6
references.pmid: 24285817
references.citation: MMWR Morb Mortal Wkly Rep. 2012 Sep 21;61:747-8
references.pmid: 22992573
references.citation: Diagn Microbiol Infect Dis. 2002 May;43(1):29-37
references.pmid: 12052626
references.citation: Med Mycol. 2011 Aug;49(6):633-42
references.pmid: 21341981
references.citation: PLoS One. 2013 Jun 28;8(6):e67008
references.pmid: 23840574
references.citation: Infect Immun. 2011 Aug;79(8):3348-57
references.pmid: 21606189
references.citation: Eukaryot Cell. 2011 Jan;10(1):87-97
references.pmid: 21037179
references.citation: Science. 2006 Apr 28;312(5773):583-8
references.pmid: 16645097
references.citation: J Clin Microbiol. 1999 Mar;37(3):675-80
references.pmid: 9986830
references.citation: Curr Opin Microbiol. 2008 Aug;11(4):318-24
references.pmid: 18573684
references.citation: J Med Vet Mycol. 1988 Jun;26(3):137-43
references.pmid: 3171821
references.citation: Infect Immun. 2014 Jan;82(1):393-404
references.pmid: 24191299
references.citation: Science. 1986 Jan 31;231(4737):476-9
references.pmid: 3001938
references.citation: J Proteome Res. 2011 Apr 1;10(4):1929-43
references.pmid: 21291285