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Is the genetic variability of Cathepsin B important in the pathogenesis of Blastocystis spp.?

  • Nelly Raquel Gonzalez-Arenas
  • Guiehdani Villalobos
  • Gie Bele Vargas-Sanchez
  • Christian Alberto Avalos-Galarza
  • Laura Margarita Marquez-Valdelamar
  • Maria Elena Ramirez-Miranda
  • Angelica Olivo-Diaz
  • Mirza Romero-Valdovinos
  • Fernando Martinez-Hernandez
  • Pablo Maravilla
Original Paper
  • 22 Downloads

Abstract

The potential role of Blastocystis as a pathogen is controversial because it is found in both symptomatic and asymptomatic carriers. Since Cathepsin B has been identified as a main virulence factor that contributes to the pathogenesis of this parasite, the purpose of this study was to analyze the genetic polymorphisms of cathepsin B from Blastocystis from patients with irritable bowel syndrome and from asymptomatic carriers. DNA from fecal samples of both groups, which were previously genotyped by 18S sequencing, was used to amplify a fragment of the cathepsin B gene. Phylogenetic reconstructions were performed and some genetic population indexes were obtained. Amplicons of 27 samples (15 cases, 10 controls, and two commercial ATCC strains) were obtained and analyzed. Phylogenetic reconstructions using nucleotides or inferred amino acid sequences did not separate between cases or controls or among subtypes. Regarding the values of genetic variability, we found that the haplotype and nucleotide diversity indexes of cathepsin B from cases and controls were similar to the values of 18S from controls. By contrast, 18S from cases showed low variability, suggesting that the genetic variability of cathepsin B was not related to the symptomatology of Blastocystis carriers. However, since no polymorphisms related to cases or controls were found, it is logical to assume that the potential damage caused by Blastocystis in situ may be due to unclear mechanisms of Cathepsin B regulation and expression that should be studied in future studies.

Keywords

Blastocystis spp. cathepsin B Subtypes Irritable bowel syndrome Genetic polymorphism Pathogenesis 

Notes

Acknowledgments

The authors acknowledge Eduardo Lopez-Escamilla, Saul Cano-Colin, and William Arony Martinez-Flores for technical support.

Funding

This work was supported by Consejo Nacional de Ciencia y Tecnologia (CONACYT) grant CB-2011/168619. Nelly Raquel Gonzalez-Arenas is a doctoral student from Programa de Doctorado en Ciencias Biomedicas, Universidad Nacional Autonoma de Mexico (UNAM) and received fellowships 220301 and 25263 (CB-2011/168619) by CONACYT.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Informed consent

Informed consent was obtained from all participants included in the study.

References

  1. Abdulla MH, O’Brien T, Mackey ZB, Sajid M, Grab DJ, McKerrow JH (2008) RNA interference of Trypanosoma brucei Cathepsin B and L affects disease progression in a mouse model. PLoS Negl Trop Dis 2:e298CrossRefGoogle Scholar
  2. Ajjampur SSR, Tan KSW (2016) Pathogenic mechanisms in Blastocystis spp. — interpreting results from in vitro and in vivo studies. Parasitol Int 65:772–779CrossRefGoogle Scholar
  3. Alexander J, Graham H, Jeremy C (1998) Leishmania mexicana cysteine proteinase-deficient mutants have attenuated virulence for mice and potentiate a Th1 response. J Immunol 161:6794–6801PubMedGoogle Scholar
  4. Alfellani MA, Taner-Mulla D, Jacob AS, Imeede CA, Yoshikawa H, Stensvold CR, Clark CG (2013) Genetic diversity of Blastocystis in livestock and zoo animals. Protist 164:497–509CrossRefGoogle Scholar
  5. Artimo P, Jonnalagedda M, Arnold K, Baratin D, Csardi G, de Castro E, Duvaud S, Flegel V, Fortier A, Gasteiger E, Grosdidier A, Hernandez C, Ioannidis V, Kuznetsov D, Liechti R, Moretti S, Mostaguir K, Redaschi N, Rossier G, Xenarios I, Stockinger H (2012) ExPASy: SIB bioinformatics resource portal. Nucleic Acids Res 40:W597–W603CrossRefGoogle Scholar
  6. Chan VJ, Paul M, McKerrow JH, Sakanari JA (1999) Expression and alteration of the S2 subsite of the Leishmania major cathepsin B-like cysteine protease. Biochem J 15:113–117CrossRefGoogle Scholar
  7. Cirioni O, Giacometti A, Drenaggi D, Ancarani F (1999) Prevalence and clinical relevance of Blastocystis hominis in diverse patient cohorts. Eur J Epidemiol 15:389–393CrossRefGoogle Scholar
  8. Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797CrossRefGoogle Scholar
  9. El Safadi D, Gaayeb L, Meloni D, Cian A, Poirier P, Wawrzyniak I, Delbac F, Dabboussi F, Delhaes L, Seck M et al (2014) Children of Senegal River Basin show the highest prevalence of Blastocystis sp. ever observed worldwide. BMC Infect. Dis 14:1–11CrossRefGoogle Scholar
  10. Faust DM, Guillen N (2012) Virulence and virulence factors in Entamoeba histolytica, the agent of human amoebiasis. Microbes Infect 14:1428–1441CrossRefGoogle Scholar
  11. Finn RD, Coggill P, Eberhardt RY, Eddy SR, Mistry J, Mitchell AL, Potter SC, Punta M, Qureshi M, Sangrador-Vegas A, Salazar GA, Tate J, Bateman A (2016) The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res 44:D279–D285CrossRefGoogle Scholar
  12. Gerbaba TK, Gedamu L (2013) Cathepsin B gene disruption induced Leishmania donovani proteome remodeling implies Cathepsin B role in secretome regulation. PLoS One 8:e79951CrossRefGoogle Scholar
  13. Giacometti A, Cirioni O, Fortuna F, Scalise G (1999) Irritable bowel syndrome in patients with Blastocystis hominis infection. Eur J Clin Microbiol Infect Dis 18:436–439CrossRefGoogle Scholar
  14. Giacometti A, Cirioni O, Antonicelli L, D’Amato G, Silvestri C, Del Prete MS, Scalise G (2003) Prevalence of intestinal parasites among individuals with allergic skin diseases. J Parasitol 89:490–492CrossRefGoogle Scholar
  15. Glantz SA (1992) Primer of biostatistics: the program v 3.01. McGraw-Hill, ColumbusGoogle Scholar
  16. Hall T (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98Google Scholar
  17. Hartl D and Clark AG (1997) Principles of population genetics (Sinauer Associates)Google Scholar
  18. Hedrick P (2010) Genetics of populations (Jones and Bartlett)Google Scholar
  19. Jimenez-Gonzalez DE, Martinez-Flores WA, Reyes-Gordillo J, Ramirez-Miranda ME, Arroyo-Escalante S, Romero-Valdovinos M, Stark D, Souza-Saldivar V, Martinez-Hernandez F, Flisser A, Olivo-Diaz A, Maravilla P (2012) Blastocystis infection is associated with irritable bowel syndrome in a mexican patient population. Parasitol Res 110:1269–1275CrossRefGoogle Scholar
  20. Krogsgaard LR, Engsbro AL, Stensvold CR, Nielsen HV, Bytzer P (2015) The prevalence of intestinal parasites is not greater among individuals with irritable bowel syndrome: a population-based case-control study. Clin Gastroenterol Hepatol 13:507–513.e2CrossRefGoogle Scholar
  21. Librado P, Rozas J (2009) DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25:1451–1452CrossRefGoogle Scholar
  22. Mirza H, Tan KSW (2009) Blastocystis exhibits inter- and intra-subtype variation in cysteine protease activity. Parasitol Res 104:355–361CrossRefGoogle Scholar
  23. Nourrisson C, Wawrzyniak I, Cian A, Livrelli V, Viscogliosi E, Delbac F, Poirier P (2016) On Blastocystis secreted cysteine proteases: a legumain-activated cathepsin B increases paracellular permeability of intestinal Caco-2 cell monolayers. Parasitology 143:1713–1722CrossRefGoogle Scholar
  24. Pandey PK, Verma P, Marathe N, Shetty S, Bavdekar A, Patole MS, Stensvold CR, Shouche YS (2015) Prevalence and subtype analysis of Blastocystis in healthy Indian individuals. Infect Genet Evol 31:296–299CrossRefGoogle Scholar
  25. Parkar U, Traub RJ, Vitali S, Elliot A, Levecke B, Robertson I, Geurden T, Steele J, Drake B, Thompson RCA (2010) Molecular characterization of Blastocystis isolates from zoo animals and their animal-keepers. Vet Parasitol 169:8–17CrossRefGoogle Scholar
  26. Petersen AM, Stensvold CR, Mirsepasi H, Engberg J, Friis-Møller A, Porsbo LJ, Hammerum AM, Nordgaard-Lassen I, Nielsen HV, Krogfelt KA (2013) Active ulcerative colitis associated with low prevalence of Blastocystis and Dientamoeba fragilis infection. Scand. J. Gastroenterol 48:638–639CrossRefGoogle Scholar
  27. Piranshahi AR, Tavalla M, Khademvatan S (2018) Genomic analysis of Blastocystis hominis isolates in patients with HIV-positive using locus SSU-rDNA. J. Parasit. Dis 42:28–33CrossRefGoogle Scholar
  28. Poirier P, Wawrzyniak I, Albert A, El Alaoui H, Delbac F, Livrelli V et al (2011) Development and evaluation of a real-time PCR assay for detection and quantification of Blastocystis parasites in human stool samples: prospective study of patients with hematological malignancies. J Clin Microbiol 49:975–983CrossRefGoogle Scholar
  29. Poirier P, Wawrzyniak I, Vivarès CP, Delbac F, El Alaoui H (2012) New Insights into Blastocystis spp.: a potential link with irritable bowel syndrome. PLoS Pathog 8:e1002545CrossRefGoogle Scholar
  30. Poirier P, Meloni D, Nourrisson C, Wawrzyniak I, Viscogliosi E, Livrelli V, Delbac F (2014) Molecular subtyping of Blastocystis spp. using a new rDNA marker from the mitochondria-like organelle genome. Parasitology 141:670–681CrossRefGoogle Scholar
  31. Reed SL, Keene WE, McKerrow JH (1989) Thiol proteinase expression and pathogenicity of Entamoeba histolytica. J Clin Microbiol 27:2772–2777PubMedPubMedCentralGoogle Scholar
  32. Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA and Huelsenbeck JP (2012) MrBayes 3.2: Efficient bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61, 539–542.CrossRefGoogle Scholar
  33. Sajid M, McKerrow JH (2002) Cysteine proteases of parasitic organisms. Mol. Biochem. Parasitol 120:1–21CrossRefGoogle Scholar
  34. Sanchez-Aguillon F, Lopez-Escamilla E, Velez-Perez F, Martinez-Flores WA, Rodriguez-Zulueta P, Martinez-Ocaña J et al (2013) Parasitic infections in a Mexican HIV/AIDS cohort. J Infect Dev Ctries 7:763–766Google Scholar
  35. Santin M, Gómez-Muñoz MT, Solano-Aguilar G, Fayer R (2011) Development of a new PCR protocol to detect and subtype Blastocystis spp. from humans and animals. Parasitol. Res 109:205–212CrossRefGoogle Scholar
  36. Scanlan PD (2012) Blastocystis: past pitfalls and future perspectives. Trends Parasitol 28:327–334CrossRefGoogle Scholar
  37. Scanlan PD, Stensvold CR, Rajilić-Stojanović M, Heilig HGHJ, De Vos WM, O’Toole PW, Cotter PD (2014) The microbial eukaryote Blastocystis is a prevalent and diverse member of the healthy human gut microbiota. FEMS Microbiol Ecol 90:326–330CrossRefGoogle Scholar
  38. Stensvold CR, Van der Giezen M (2018) Associations between gut microbiota and common luminal intestinal parasites. Trends Parasitol 34:369–377CrossRefGoogle Scholar
  39. Stensvold CR, Nielsen HV, Mølbak K, Smith HV (2009) Pursuing the clinical significance of Blastocystis – diagnostic limitations. Trends Parasitol 25:23–29CrossRefGoogle Scholar
  40. Surangsrirat S, Thamrongwittawatpong L, Piyaniran W, Naaglor T, Khoprasert C, Taamasri P, Mungthin M, Leelayoova S (2006) Assessment of the association between Blastocystis infection and irritable bowel syndrome. J Med Assoc Thai 93:S119–S124Google Scholar
  41. Tan KSW (2008) New insights on classification, identification and clinical relevance of Blastocystis spp. Clin Microbiol Rev 21:639–665CrossRefGoogle Scholar
  42. Turkeltaub JA, McCarty TR, Hotez PJ (2015) The intestinal protozoa: emerging impact on global health and development. Curr. Opin. Gastroenterol 31:38–44CrossRefGoogle Scholar
  43. Valença-Barbosa C, de Jesus-Batista R, Pereira IR, d’Avila Levy CM, Werneck de Macedo H, Carneiro-Santos HL (2017) Distribution of Blastocystis subtypes isolated from humans from an urban community in Rio de Janeiro, Brazil. Parasit. Vectors 10(1):518–524CrossRefGoogle Scholar
  44. Vargas-Sanchez GB, Romero-Valdovinos M, Ramirez-Guerrero C, Vargas-Hernandez I, Ramirez-Miranda ME, Martinez-Ocaña J, Valadez A, Ximenez C, Lopez-Escamilla E, Hernandez-Campos ME et al (2015) Blastocystis isolates from patients with irritable bowel syndrome and from asymptomatic carriers exhibit similar parasitological loads, but significantly different generation times and genetic variability across multiple subtypes. Plos One 10:e0124006CrossRefGoogle Scholar
  45. Velasco J, González F, Díaz T, Peña-Guillén J, María A (2011) Profiles of enteropathogens in asymptomatic children from indigenous communities of Mérida, Venezuela. J Infect Dev Ctries 5:278–285PubMedGoogle Scholar
  46. Villegas-Gómez I, Martínez-Hernández F, Urrea-Quezada A, González-Díaz M, Durazo M, Hernández J, Orozco-Mosqueda GE, Villalobos G, Maravilla P, Valenzuela O (2016) Comparison of the genetic variability of Blastocystis subtypes between human carriers from two contrasting climatic regions of México. Infect. Genet. Evol 44:334–340CrossRefGoogle Scholar
  47. Wawrzyniak I, Texier C, Poirier P, Viscogliosi E, Tan KSW, Delbac F, El Alaoui H (2012) Characterization of two cysteine proteases secreted by Blastocystis ST7, a human intestinal parasite. Parasitol. Int 61:437–442CrossRefGoogle Scholar
  48. Wu Z, Mirza H, Tan KSW (2014) Intra-subtype variation in enteroadhesion accounts for differences in epithelial barrier disruption and is associated with metronidazole resistance in Blastocystis Subtype-7. PLoS Negl. Trop. Dis 8:e2885CrossRefGoogle Scholar
  49. Yaicharoen R, Sripochang S, Sermsart B, Pidetcha P (2005) Prevalence of Blastocystis hominis infection in asymptomatic individuals from Bangkok, Thailand. Southeast Asian J. Trop. Med. Public Health 36:17–20PubMedGoogle Scholar
  50. Yakoob J, Jafri W, Khan R, Islam M, Beg MA, Zaman V (2004) Iritable bowel syndrome: in search of an etiology: role of Blastocystis hominis. Am J Trop Med Hyg 74:383–385CrossRefGoogle Scholar
  51. Yoshikawa H, Abe N, Iwasawa M, Takahashi Y, Nagano I, Wu Z, Takahashi Y (2000) Genomic analysis of Blastocystis hominis strains isolated from two long-term health care facilities. J. Clin. Microbiol 38:1324–1330PubMedPubMedCentralGoogle Scholar
  52. Yoshikawa H, Wu Z, Nagano I, Takahashi Y (2003) Molecular comparative studies among Blatocystis isolates obtained from humans and animals. J. Parasitol 89:585–594CrossRefGoogle Scholar
  53. Yoshikawa H, Dogruman-AI F, Turk S, Kustimur S, Balaban N, Sultan N (2011) Evaluation of DNA extraction kits for molecular diagnosis of human Blastocystis subtypes from fecal samples. Parasitol. Res 109:1045–1050CrossRefGoogle Scholar
  54. Yu Z, Morrizon M (2004) Improved extraction of PCR-quality community DNA from digesta and fecal samples. Biotechniques 36:808–812CrossRefGoogle Scholar
  55. Zierdt CH (1991) Blastocystis hominis-past and future. Clin. Microbiol. Rev 4:61–79CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Nelly Raquel Gonzalez-Arenas
    • 1
  • Guiehdani Villalobos
    • 2
  • Gie Bele Vargas-Sanchez
    • 1
  • Christian Alberto Avalos-Galarza
    • 1
  • Laura Margarita Marquez-Valdelamar
    • 3
  • Maria Elena Ramirez-Miranda
    • 1
  • Angelica Olivo-Diaz
    • 1
  • Mirza Romero-Valdovinos
    • 1
  • Fernando Martinez-Hernandez
    • 1
  • Pablo Maravilla
    • 1
  1. 1.Hospital General “Dr. Manuel Gea Gonzalez”Mexico CityMexico
  2. 2.Departamento de Ecología Evolutiva, Instituto de EcologiaUniversidad Nacional Autonoma de MexicoMexico CityMexico
  3. 3.Departamento de Secuenciacion, Instituto de BiologiaUniversidad Nacional Autonoma de MexicoMexico CityMexico

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