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Histoplasma capsulatum 100-kilodalton antigen: recombinant production, characterization, and evaluation of its possible application in the diagnosis of histoplasmosis


The goal of the present work was to develop a novel reagent with potential for histoplasmosis diagnosis. For this purpose, the genetic sequence of the 100 kDa protein of Histoplasma capsulatum (Hcp100) was cloned and expressed as a secretory protein in Pichia pastoris. After optimizing the culture conditions and purifying by immobilized metal ion affinity chromatography, the highest yield of Hcp100 reached approximately 1.3 mg/l with > 90% purity in shake flasks using basal salt medium supplemented with casamino acids after 72 h of methanol induction. To investigate its potential for diagnosis, its detection in urine samples using specific polyclonal antibodies as reagent was evaluated by dot blot in 6 patients with progressive disseminated histoplasmosis (PDH), of whom all had AIDS. Antigen was detected in urine from all 6 (100%) PDH patients. Urine samples from a pool of 20 healthy individuals did not react with the anti-Hcp100 antibodies. The dot blot assay performed in this study provides preliminary data of a simple technology that can be performed in medical institutions with limited resources to facilitate the rapid diagnosis of histoplasmosis, particularly the disseminated forms. Hence, use of these assays may provide a rapid diagnostic tool of PDH in endemic areas for histoplasmosis where PDH-related mortality is high, hastening treatment and improving patient survival. Finally, this novel antigen and its specific antibodies may provide an alternative diagnostic reagent to the largely unknown and poorly characterized polysaccharide antigens (HPA, galactomannan, histoplasmin) frequently used in the diagnostic tests.

Key Points

  • Few antigens are used as laboratory tools for the immunodiagnosis of histoplasmosis.

  • P. pastoris was an excellent system for recombinant Hcp100 expression.

  • Maximum expression levels of rHcp100 were achieved in BSM with 1% casamino acids.

  • Dot blot assays with anti-rHcp100 antisera can be successfully used for diagnosing PHD.

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  1. Adenis A, Nacher M, Hanf M, Basurko C, Dufour J, Huber F, Aznar C, Carme B, Coupple P (2014) Tuberculosis and histoplasmosis among human immunodeficiency virus-infected patients: a comparative study. Am J Trop Med Hyg 90:216–223.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. Almeida M d A, Pizzini CV, Damasceno LS, Muniz M d M, Almeida-Paes R, Peralta RH, Oliveira R d V, Vizzoni AG, de Andrade CL, Zancopé-Oliveira RM (2016) Validation of western blot for Histoplasma capsulatum antibody detection assay. BMC Infect Dis 16:87.

    CAS  Article  PubMed Central  Google Scholar 

  3. Bialek R, Feucht A, Aepinus C, Just-Nübling G, Robertson VJ, Knobloch J, Hohle R (2002) Evaluation of two nested PCR assays for detection of Histoplasma capsulatum DNA in human tissue. J Clin Microbiol 40:1644–1647

    CAS  Article  Google Scholar 

  4. Caceres DH, Scheel CM, Tobón AM, Ahiquist Cleveland A, Restrepo A, Brandt ME, Chiller T, Gómez BL (2014) Validation of an enzyme-linked immunosorbent assay that detects Histoplasma capsulatum antigenuria in Colombian patients with AIDS for diagnosis and follow-up during therapy. Clin Vaccine Immunol 21:1364–1368.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. Carvalho KC, Vallejo MC, Camargo ZP, Puccia R (2008) Use of recombinant gp43 isoforms expressed in Pichia pastoris for diagnosis of Paracoccidioidomycosis. Clin Vaccine Immunol 15:622–629.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. Colombo AL, Tobon A, Restrepo A, Queiroz-Telles F, Nucci M (2011) Epidemiology of endemic systemic fungal infections in Latin America. Med Mycol 49:785–798.

    Article  PubMed  Google Scholar 

  7. Colonna-Romano S, Porta A, Franco A, Kobayashi S, Maresca B (1998) Identification and isolation by DDRT-PCR of genes differentially expressed by Histoplasma capsulatum during macrophages infection. Microb Pathog 25:55–66

    CAS  Article  Google Scholar 

  8. Cunningham L, Cook A, Hanzlicek A, Harkin K, Wheat J, Goad C, Kirsch E (2015) Sensitivity and specificity of Histoplasma antigen detection by enzyme immunoassay. J Am Anim Hosp Assoc 51:306–310.

    Article  PubMed  Google Scholar 

  9. Dantas KC, de Freitas RS, Moreira AP, Silva MV, Benard G, Vasconcellos C, Criado PR (2013) The use of nested polymerase chain reaction (nested PCR) for the early diagnosis of Histoplasma capsulatum infection in serum and whole blood of HIV-positive patients. An Bras Dermatol 88:141–143

    Article  Google Scholar 

  10. Dantas KC, de Freitas RS, García RS, da Silva MV, Muricy EC, Kohara VS, Vicentini AP (2014) Importance of the association of molecular and immunological diagnosis in immunocompetent patient with Histoplasma capsulatum and Cryptococcus neoformans infection: a case report. J Venom Anim Toxins Incl Trop Dis 20:36.

    Article  PubMed  PubMed Central  Google Scholar 

  11. de Freitas RS, Carvalho-Vivi KO, Zamboni IM, Assis CM, Costa Martins JE, Vicentini Moreira AP (2009) The importance of serological assays in diagnosis acute pulmonary histoplasmosis. J Venom Anim Toxins Incl Trop Dis 15:278–288

    Article  Google Scholar 

  12. de Freitas RS, Kamikawa CM, Vicentini AP (2018) Fast protocol for the production of Histoplasma capsulatum antigens for antibody detection in the immunodiagnosis of histoplasmosis. Rev Iberoam Micol 35:27–31.

    Article  PubMed  Google Scholar 

  13. EasySelectTM Pichia Expression Kit. User manual. Invitrogen-Life Technologies. 2010;(25-0172):56–8

  14. Falci DR, Hoffmann ER, Paskulin DD, Pasqualotto AC (2017) Progressive disseminated histoplasmosis: a systematic review on the performance of non-culture-based diagnostic tests. Braz J Infect Dis 21:7–11.

    Article  PubMed  Google Scholar 

  15. Gajurel K, Dhakal R, Deresinski S (2018) Diagnosis and treatment of histoplasmosis in solid organ transplant patients. Curr Opin Infect Dis 31:301–308.

    Article  PubMed  Google Scholar 

  16. González-González AE, Taylor ML, Curiel-Quesada E (2012) Relevant aspects of the Hcp100 molecular marker of Histoplasma capsulatum and its potential therapeutic use in histoplasmosis. Rev Iberoam Micol 29:115–119.

    Article  PubMed  Google Scholar 

  17. Guedes HL, Guimaraes AJ, Muniz M d M, Pizzini CV, Hamilton AJ, Peralta JM, Deepe GS Jr, Zancopé-Oliveira RM (2003) PCR assay for identification of Histoplasma capsulatum based on the nucleotide sequence of the M antigen. J Clin Microbiol 41:535–539

    CAS  Article  Google Scholar 

  18. Guimaraes AJ, Nosanchuk JD, Zancopé-Oliveira RM (2006) Diagnosis of histoplasmosis. Braz J Microbiol 37:1–13

    Article  Google Scholar 

  19. Kaplan W, Littlejohn T (2001) Swiss-PDB viewer (deep view). Briefings in bioinformatics. Brief Bioinform 2:195–197

    CAS  Article  Google Scholar 

  20. Kauffman CA (2007) Histoplasmosis: a clinical and laboratory update. Clin Microbiol Rev 20:115–132

    CAS  Article  Google Scholar 

  21. Lindsley MD, Holland HL, Bragg SL, Hurst SF, Wannemuehler KA, Morrison CJ (2007) Production and evaluation of reagents for detection of Histoplasma capsulatum antigenuria by enzyme immunoassay. Clin Vaccine Immunol 14:700–709

    CAS  Article  Google Scholar 

  22. Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast 22:249–270

    CAS  Article  Google Scholar 

  23. Miller R, Assi M, AST Infectious Diseases Community of Practice (2013) Endemic fungal infections in solid organ transplantation. Am J Transplant 13:250–261.

    CAS  Article  PubMed  Google Scholar 

  24. Nacher M, Adenis A, Mc Donald S, Do Socorro Mendonca Gomes M, Singh S, Lopes Lima I, Malcher Leite R, Hermelijn S, Wongsokarijo M, Van Eer M, Marques Da Silva S, Mesquita Da Costa M, Silva M, Calvacante M, do Menino Jesus Silva Leitao T, Gomez BL, Restrepo A, Tobon A, Canteros CE, Aznar C, Blanchet D, Vantilcke V, Vautrin C, Boukhari R, Chiller T, Scheel C, Ahlquist A, Roy M, Lortholary O, Carme B, Crouppié P, Vreden S (2013) Disseminated histoplasmosis in HIV-infected patients in South America: a neglected killer continues on its rampage. PLoS Negl Trop Dis 7:e2319.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Negroni R, Iovannitti C, Arechavala AL, Carnovale S, Euguchi K (1998) Preparation and study of an exoantigen of Histoplasma capsulatum for serological reactions. Rev Iberoam Micol 15:282–285

    CAS  PubMed  Google Scholar 

  26. Oladele RO, Avanlowo OO, Richardson MD, Denning DW (2018) Histoplasmosis in Africa: an emerging or a neglected disease? PLoS Negl Trop Dis 12:e0006046.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Porta A, Colonna-Romano S, Callebaut I, Franco A, Marzullo L, Kobayashi GS, Maresca B (1999) An homologue of the human 100-kDa protein (p100) is differentially expressed by Histoplasma capsulatum during infection of murine macrophages. Biochem Biophys Res Commun 254:605–613

    CAS  Article  Google Scholar 

  28. Riviére S, Denis B, Bougnoux M-E, Lanternier F, Lecuit M, Lortholary O (2012) Serum Aspergillus galactomannan for the management of disseminated histoplasmosis in AIDS. Am J Trop Med Hyg 87:303–305.

    Article  PubMed  PubMed Central  Google Scholar 

  29. Sinha J, Plantz BA, Inan M, Meagher MM (2005) Cause of proteolytic degradation of secreted recombinant proteins produced in methylotrophic yeast Pichia pastoris: case study with recombinant ovine interferon-tau. Biotechnol Bioeng 89:103–112

    Article  Google Scholar 

  30. Srinivasan A, Kleiman MB, Debelenko L, Stokes DC, De Vincenzo J, Wheat JL (2009) False-negative Histoplasma antigen in acute pulmonary histoplasmosis: the value of urinary concentration by ultrafiltration and heat denaturation of serum proteins in detection of Histoplasma antigen. Pediatr Infect Dis 28:447–449.

    Article  Google Scholar 

  31. Theel ES, Harring JA, Dababneh AS, Rollins LO, Bestrom JE, Jespersen DJ (2015) Reevaluation of commercial reagents for detection of Histoplasma capsulatum antigen in urine. J Clin Microbiol 53:1198–1203.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. Wheat LJ, Garringer T, Brizendine E, Connolly P (2002) Diagnosis of histoplasmosis by antigen detection based upon experience at the histoplasmosis reference laboratory. Diagn Microbiol Infect Dis 43:29–37

    CAS  Article  Google Scholar 

  33. Wheat JL, Azar MM, Bahr NC, Spec A, Relich RF, Hage C (2016) Histoplasmosis. Infect Dis Clin N Am 30:207–227.

    Article  Google Scholar 

  34. WHO (2017) Accessed 7th July 2018

  35. Wu M, Shen Q, Yang Y, Zhang S, Qu W, Chen J, Sun H, Chen S (2013) Disruption of YPS1 and PEP4 genes reduces proteolytic degradation of secreted HAS-PTH in Pichia pastoris GS115. J Ind Microbiol Biotechnol 40:589–599.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. Xiao A, Zhou X, Zhou L, Zhang Y (2006) Improvement of cell viability and hirudin production by ascorbic acid in Pichia pastoris fermentation. Appl Microbiol Biotechnol 72:837–844

    CAS  Article  Google Scholar 

  37. Yang J, Yan R, Roy A, Xu D, Poisson J, Zhang Y (2015) The I-TASSER Suite: protein structure and function prediction. Nat Methods 12:7–8.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. Zhang Y, Liu R, Wu X (2007) The proteolytic systems and heterologous proteins degradation in the methylotrophic yeast Pichia pastoris. Ann Microbiol 57:553

    CAS  Article  Google Scholar 

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This work was supported by the University of Buenos Aires (Grant UBACyT 20020130200222BA). DGM, ADN, and MLC are staff members of CONICET. MAT thanks the doctoral scholarship to CONICET.

Author information




All authors contributed to the study conception and design as well as to material preparation, data collection, and analysis. The first draft of the manuscript was written by MLC and ADN and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to María L. Cuestas.

Ethics declarations

This study was approved by the institutional review committee “Dr Vicente Federico Del Giúdice” at Hospital Nacional Alejandro Posadas, Buenos Aires, Argentina (Ref. 260 EMnP0S0/19). Informed consents were obtained from all participants. The procedures involving animals followed the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Comité Institucional para el Cuidado y Uso de Animales de Laboratorio (CICUAL-FFyB, Protocol number 1018-19).

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The authors declare that they have no conflict of interest.

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Toscanini, M.A., Maglio, D.G., Capece, P. et al. Histoplasma capsulatum 100-kilodalton antigen: recombinant production, characterization, and evaluation of its possible application in the diagnosis of histoplasmosis. Appl Microbiol Biotechnol 104, 5861–5872 (2020).

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  • Histoplasma capsulatum
  • Recombinant Hcp100
  • Pichia pastoris
  • Antibodies anti-rHcp100
  • Diagnosis
  • Casamino acids