1887

Abstract

is an emerging pathogen that mostly affects immunocompromised patients, causing infections that are often difficult to manage therapeutically. Current diagnostic strategies are limited to the detection of fungal growth using radiological methods or biopsy, which often does not enable species-specific identification. There is thus a critical need for diagnostic techniques to enable early and specific identification of the causative agent. In this study, we describe monoclonal antibodies (mAbs) developed to a previously described recombinant form of the haemolysin terrelysin. Sixteen hybridomas of various IgG isotypes were generated to the recombinant protein, of which seven demonstrated reactivity to the native protein in hyphal extracts. Cross-reactivity analysis using hyphal extracts from 29 fungal species, including 12 species and five strains of , showed that three mAbs (13G10, 15B5 and 10G4) were -specific. Epitope analysis demonstrated mAbs 13G10 and 10G4 recognize the same epitope, PSNEFE, while mAb 15B5 recognizes the epitope LYEGQFHS. Time-course studies showed that terrelysin expression was highest during early hyphal growth and dramatically decreased after mycelial expansion. Immunolocalization studies demonstrated that terrelysin was not only localized within the cytoplasm of hyphae but appeared to be more abundant at the hyphal tip. These findings were confirmed in cultures grown at room temperature as well as at 37 °C. Additionally, terrelysin was detected in the supernatant of cultures. These observations suggest that terrelysin may be a candidate biomarker for infection.

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2012-04-01
2019-12-08
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References

  1. Andrew S. M. , Titus J. A. . ( 1997; ). Purification and fragmentation of antibodies. . In Current Protocols in Immunology, pp. 2.7.1–2.7.12. Somerset, NJ:: Wiley;.
    [Google Scholar]
  2. Baddley J. W. , Pappas P. G. , Smith A. C. , Moser S. A. . ( 2003; ). Epidemiology of Aspergillus terreus at a university hospital. . J Clin Microbiol 41:, 5525–5529. [CrossRef] [PubMed]
    [Google Scholar]
  3. Balajee S. A. . ( 2009a; ). Aspergillus terreus complex. . Med Mycol 47: (Suppl. 1), S42–S46. [CrossRef] [PubMed]
    [Google Scholar]
  4. Balajee S. A. , Baddley J. W. , Peterson S. W. , Nickle D. , Varga J. , Boey A. , Lass-Flörl C. , Frisvad J. C. , Samson R. A. . ISHAM Working Group on A. terreus ( 2009; b). Aspergillus alabamensis, a new clinically relevant species in the section Terrei . . Eukaryot Cell 8:, 713–722. [CrossRef] [PubMed]
    [Google Scholar]
  5. Berne S. , Krizaj I. , Pohleven F. , Turk T. , Macek P. , Sepcić K. . ( 2002; ). Pleurotus and Agrocybe hemolysins, new proteins hypothetically involved in fungal fruiting. . Biochim Biophys Acta 1570:, 153–159. [CrossRef] [PubMed]
    [Google Scholar]
  6. Berne S. , Pohleven J. , Vidic I. , Rebolj K. , Pohleven F. , Turk T. , Macek P. , Sonnenberg A. , Sepcić K. . ( 2007; ). Ostreolysin enhances fruiting initiation in the oyster mushroom (Pleurotus ostreatus). . Mycol Res 111:, 1431–1436. [CrossRef] [PubMed]
    [Google Scholar]
  7. Berne S. , Lah L. , Sepcić K. . ( 2009; ). Aegerolysins: structure, function, and putative biological role. . Protein Sci 18:, 694–706.[PubMed]
    [Google Scholar]
  8. Bhakdi S. , Bayley H. , Valeva A. , Walev I. , Walker B. , Kehoe M. , Palmer M. . ( 1996; ). Staphylococcal alpha-toxin, streptolysin-O, and Escherichia coli hemolysin: prototypes of pore-forming bacterial cytolysins. . Arch Microbiol 165:, 73–79. [CrossRef] [PubMed]
    [Google Scholar]
  9. Blum G. , Perkhofer S. , Haas H. , Schrettl M. , Würzner R. , Dierich M. P. , Lass-Flörl C. . ( 2008; ). Potential basis for amphotericin B resistance in Aspergillus terreus . . Antimicrob Agents Chemother 52:, 1553–1555. [CrossRef] [PubMed]
    [Google Scholar]
  10. Chen C. Y. , Séguin-Swartz G. . ( 2002; ). A rapid method for assessing the viability of fungal spores. . Can J Plant Pathol 24:, 230–232. [CrossRef]
    [Google Scholar]
  11. Chowdhury H. H. , Rebolj K. , Kreft M. , Zorec R. , Macek P. , Sepcić K. . ( 2008; ). Lysophospholipids prevent binding of a cytolytic protein ostreolysin to cholesterol-enriched membrane domains. . Toxicon 51:, 1345–1356. [CrossRef] [PubMed]
    [Google Scholar]
  12. Chu L. , Bramanti T. E. , Ebersole J. L. , Holt S. C. . ( 1991; ). Hemolytic activity in the periodontopathogen Porphyromonas gingivalis: kinetics of enzyme release and localization. . Infect Immun 59:, 1932–1940.[PubMed]
    [Google Scholar]
  13. Crissy J. T. , Lang H. , Parish L. C. . ( 1995; ). Manual of Medical Mycology. Cambridge, MA:: Blackwell Sciences;.
    [Google Scholar]
  14. Ebina K. , Yokota K. , Sakaguchi O. . ( 1982; ). Studies on toxin of Aspergillus fumigatus XIV: Relationship between Asp-hemolysin and experimental infection in mice. . Jpn J Med Mycol 23:, 246–252. [CrossRef]
    [Google Scholar]
  15. Garg P. , Mahesh S. , Bansal A. K. , Gopinathan U. , Rao G. N. . ( 2003; ). Fungal infection of sutureless self-sealing incision for cataract surgery. . Ophthalmology 110:, 2173–2177. [CrossRef] [PubMed]
    [Google Scholar]
  16. Gravelat F. N. , Doedt T. , Chiang L. Y. , Liu H. , Filler S. G. , Patterson T. F. , Sheppard D. C. . ( 2008; ). In vivo analysis of Aspergillus fumigatus developmental gene expression determined by real-time reverse transcription-PCR. . Infect Immun 76:, 3632–3639. [CrossRef] [PubMed]
    [Google Scholar]
  17. Hachem R. Y. , Kontoyiannis D. P. , Boktour M. R. , Afif C. , Cooksley C. , Bodey G. P. , Chatzinikolaou I. , Perego C. , Kantarjian H. M. , Raad I. I. . ( 2004; ). Aspergillus terreus: an emerging amphotericin B-resistant opportunistic mold in patients with hematologic malignancies. . Cancer 101:, 1594–1600. [CrossRef] [PubMed]
    [Google Scholar]
  18. Han M. J. , Kim N. J. , Lee S. Y. , Chang H. N. . ( 2010; ). Extracellular proteome of Aspergillus terreus grown on different carbon sources. . Curr Genet 56:, 369–382. [CrossRef] [PubMed]
    [Google Scholar]
  19. Hill T. W. , Kafer E. . ( 2001; ). Improved protocols for Aspergillus minimal medium: trace element and minimal medium stock solutions. . Fungal Genet Newsl 48:, 20–21.
    [Google Scholar]
  20. Hilmioğlu-Polat S. , Metin D. Y. , Inci R. , Dereli T. , Kilinç I. , Tümbay E. . ( 2005; ). Non-dermatophytic molds as agents of onychomycosis in Izmir, Turkey – a prospective study. . Mycopathologia 160:, 125–128. [CrossRef] [PubMed]
    [Google Scholar]
  21. Iwen P. C. , Rupp M. E. , Langnas A. N. , Reed E. C. , Hinrichs S. H. . ( 1998; ). Invasive pulmonary aspergillosis due to Aspergillus terreus: 12-year experience and review of the literature. . Clin Infect Dis 26:, 1092–1097. [CrossRef] [PubMed]
    [Google Scholar]
  22. Köhler G. , Milstein C. . ( 1975; ). Continuous cultures of fused cells secreting antibody of predefined specificity. . Nature 256:, 495–497. [CrossRef] [PubMed]
    [Google Scholar]
  23. Kudo Y. , Fukuchi Y. , Kumagai T. , Ebina K. , Yokota K. . ( 2001; ). Oxidized low-density lipoprotein-binding specificity of Asp-hemolysin from Aspergillus fumigatus . . Biochim Biophys Acta 1568:, 183–188. [CrossRef] [PubMed]
    [Google Scholar]
  24. Kudo Y. , Ootani T. , Kumagai T. , Fukuchi Y. , Ebina K. , Yokota K. . ( 2002; ). A novel oxidized low-density lipoprotein-binding protein, Asp-hemolysin, recognizes lysophosphatidylcholine. . Biol Pharm Bull 25:, 787–790. [CrossRef] [PubMed]
    [Google Scholar]
  25. Kumagai T. , Nagata T. , Kudo Y. , Fukuchi Y. , Ebina K. , Yokota K. . ( 1999; ). Cytotoxic activity and cytokine gene induction of Asp-hemolysin to murine macrophages. . Nippon Ishinkin Gakkai Zasshi 40:, 217–222. [CrossRef] [PubMed]
    [Google Scholar]
  26. Maličev E. , Chowdhury H. H. , Macek P. , Sepcic K. . ( 2007; ). Effect of ostreolysin, an Asp-hemolysin isoform, on human chondrocytes and osteoblasts, and possible role of Asp-hemolysin in pathogenesis. . Med Mycol 45:, 123–130. [CrossRef] [PubMed]
    [Google Scholar]
  27. Medina M. L. , Haynes P. A. , Breci L. , Francisco W. A. . ( 2005; ). Analysis of secreted proteins from Aspergillus flavus . . Proteomics 5:, 3153–3161. [CrossRef] [PubMed]
    [Google Scholar]
  28. Natesan S. , Abraham G. , Mathew M. , Lalitha M. K. , Srinivasan C. N. . ( 2007; ). Secondary sternal Aspergillus osteomyelitis in a diabetic hemodialysis patient with previous allograft rejection. . Hemodial Int 11:, 403–405. [CrossRef] [PubMed]
    [Google Scholar]
  29. Nayak A. P. , Blachere F. M. , Hettick J. M. , Lukomski S. , Schmechel D. , Beezhold D. H. . ( 2011a; ). Characterization of recombinant terrelysin, a hemolysin of Aspergillus terreus . . Mycopathologia 171:, 23–34. [CrossRef] [PubMed]
    [Google Scholar]
  30. Nayak A. P. , Green B. J. , Janotka E. , Hettick J. M. , Friend S. , Vesper S. J. , Schmechel D. , Beezhold D. H. . ( 2011; b). Monoclonal antibodies to hyphal exoantigens derived from the opportunistic pathogen Aspergillus terreus . . Clin Vaccine Immunol 18:, 1568–1576. [CrossRef] [PubMed]
    [Google Scholar]
  31. Nickel W. . ( 2010; ). Pathways of unconventional protein secretion. . Curr Opin Biotechnol 21:, 621–626. [CrossRef] [PubMed]
    [Google Scholar]
  32. Osmani S. A. , Engle D. B. , Doonan J. H. , Morris N. R. . ( 1988; ). Spindle formation and chromatin condensation in cells blocked at interphase by mutation of a negative cell cycle control gene. . Cell 52:, 241–251. [CrossRef] [PubMed]
    [Google Scholar]
  33. Perfect J. R. , Cox G. M. , Lee J. Y. , Kauffman C. A. , de Repentigny L. , Chapman S. W. , Morrison V. A. , Pappas P. , Hiemenz J. W. . & other authors ( 2001; ). The impact of culture isolation of Aspergillus species: a hospital-based survey of aspergillosis. . Clin Infect Dis 33:, 1824–1833. [CrossRef] [PubMed]
    [Google Scholar]
  34. Pore R. S. , Larsh H. W. . ( 1967; ). Aleuriospore formation in four related Aspergillus species. . Mycologia 59:, 318–325. [CrossRef]
    [Google Scholar]
  35. Rao J. , DiGiandomenico A. , Unger J. , Bao Y. , Polanowska-Grabowska R. K. , Goldberg J. B. . ( 2008; ). A novel oxidized low-density lipoprotein-binding protein from Pseudomonas aeruginosa . . Microbiology 154:, 654–665. [CrossRef] [PubMed]
    [Google Scholar]
  36. Rebolj K. , Ulrih N. P. , Macek P. , Sepcić K. . ( 2006; ). Steroid structural requirements for interaction of ostreolysin, a lipid-raft binding cytolysin, with lipid monolayers and bilayers. . Biochim Biophys Acta 1758:, 1662–1670. [CrossRef] [PubMed]
    [Google Scholar]
  37. Rebolj K. , Batista U. , Sepcić K. , Cestnik V. , Macek P. , Frangez R. . ( 2007; ). Ostreolysin affects rat aorta ring tension and endothelial cell viability in vitro . . Toxicon 49:, 1211–1213. [CrossRef] [PubMed]
    [Google Scholar]
  38. Rubartelli A. , Sitia R. . ( 1997; ). Secretion of mammalian proteins that lack a signal sequence. . In Unusual Secretory Pathways: from Bacteria to Man, pp. 87–104. Edited by Kuchler K. et al. . Austin, TX:: R. G. Landes;.[CrossRef]
    [Google Scholar]
  39. Rutqvist L. . ( 1965; ). Studies on Aspergillus fumigatus; toxin production by different strains and serological comparison of the strains. . Acta Vet Scand 6:, 224–233.[PubMed]
    [Google Scholar]
  40. Schmechel D. , Simpson J. P. , Beezhold D. H. , Lewis D. M. . ( 2006; ). The development of species-specific immunodiagnostics for Stachybotrys chartarum: the role of cross-reactivity. . J Immunol Methods 309:, 150–159. [CrossRef] [PubMed]
    [Google Scholar]
  41. Seeger W. , Obernitz R. , Thomas M. , Walmrath D. , Suttorn N. , Holland I. B. , Grimminger F. , Eberspächer B. , Hugo F. , Bhakdi S. . ( 1991; ). Lung vascular injury after administration of viable hemolysin-forming Escherichia coli in isolated rabbit lungs. . Am Rev Respir Dis 143:, 797–805.[PubMed] [CrossRef]
    [Google Scholar]
  42. Segal B. H. , DeCarlo E. S. , Kwon-Chung K. J. , Malech H. L. , Gallin J. I. , Holland S. M. . ( 1998; ). Aspergillus nidulans infection in chronic granulomatous disease. . Medicine (Baltimore) 77:, 345–354. [CrossRef] [PubMed]
    [Google Scholar]
  43. Sukroongreung S. , Thakerngpol K. . ( 1985; ). Abnormal form of Aspergillus terreus isolated from mycotic abscesses. . Mycopathologia 91:, 47–52. [CrossRef] [PubMed]
    [Google Scholar]
  44. Tiwari S. , Singh S. M. , Jain S. . ( 1995; ). Chronic bilateral suppurative otitis media caused by Aspergillus terreus . . Mycoses 38:, 297–300. [CrossRef] [PubMed]
    [Google Scholar]
  45. Van Emon J. M. , Reed A. W. , Yike I. , Vesper S. J. . ( 2003; ). ELISA measurement of stachylysin in serum to quantify human exposures to the indoor mold Stachybotrys chartarum . . J Occup Environ Med 45:, 582–591. [CrossRef] [PubMed]
    [Google Scholar]
  46. Verghese S. , Palani R. , Thirunavakarasu N. , Chellamma T. , Pathipata P. . ( 2008; ). Peritonitis due to Aspergillus terreus in a patient undergoing continuous ambulatory peritoneal dialysis. . Mycoses 51:, 174–176. [CrossRef] [PubMed]
    [Google Scholar]
  47. Vesper S. J. , Vesper M. J. . ( 2004; ). Possible role of fungal hemolysins in sick building syndrome. . Adv Appl Microbiol 55:, 191–213. [CrossRef] [PubMed]
    [Google Scholar]
  48. Wadhwani K. , Srivastava A. K. . ( 1984; ). Fungi from otitis media of agricultural field workers. . Mycopathologia 88:, 155–159. [CrossRef] [PubMed]
    [Google Scholar]
  49. Wartenberg D. , Lapp K. , Jacobsen I. D. , Dahse H. M. , Kniemeyer O. , Heinekamp T. , Brakhage A. A. . ( 2011; ). Secretome analysis of Aspergillus fumigatus reveals Asp-hemolysin as a major secreted protein. . Int J Med Microbiol 301:, 602–611. [CrossRef] [PubMed]
    [Google Scholar]
  50. Xiang X. , Roghi C. , Morris N. R. . ( 1995; ). Characterization and localization of the cytoplasmic dynein heavy chain in Aspergillus nidulans . . Proc Natl Acad Sci U S A 92:, 9890–9894. [CrossRef] [PubMed]
    [Google Scholar]
  51. Žužek M. C. , Macek P. , Sepcić K. , Cestnik V. , Frangez R. . ( 2006; ). Toxic and lethal effects of ostreolysin, a cytolytic protein from edible oyster mushroom (Pleurotus ostreatus), in rodents. . Toxicon 48:, 264–271. [CrossRef] [PubMed]
    [Google Scholar]
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