Characterization of NFDQ1 in Cryptosporidium parvum.
Cryptosporidium parvum
CRISPR/Cas9
NFDQ
Journal
Parasites & vectors
ISSN: 1756-3305
Titre abrégé: Parasit Vectors
Pays: England
ID NLM: 101462774
Informations de publication
Date de publication:
26 Oct 2024
26 Oct 2024
Historique:
received:
06
07
2024
accepted:
09
10
2024
medline:
27
10
2024
pubmed:
27
10
2024
entrez:
27
10
2024
Statut:
epublish
Résumé
Cryptosporidium spp. are important zoonotic parasites that can cause moderate to severe diarrhea in humans and animals. Among the three Cryptosporidium species infecting the intestines of calves, Cryptosporidium parvum has a broad host range and causes severe diarrhea in calves, while Cryptosporidium bovis and Cryptosporidium ryanae mainly infect calves without obvious clinical symptoms. Comparative genomic analysis revealed differences in the copy number of genes encoding the nonfinancial disclosure quality (NFDQ) secretory protein family among the three species, suggesting that this protein family may be associated with the host range or pathogenicity of Cryptosporidium spp. To understand the function of cgd8_10 encoded NFDQ1, tagged and knockout strains were constructed and characterized in this study. To determine the localization of NFDQ1, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology to tag the C-terminus of NFDQ1 with three hemagglutinin epitopes (3 × HA). The tagged strain was constructed, and the genomic insertion was confirmed by polymerase chain reaction (PCR). Immunofluorescence assays were performed to observe the localization of NFDQ1 both in extracellular sporozoites and at various intracellular developmental stages. Immunoelectron microscopy was used to study the ultrastructural localization of NFDQ1. Then, the ΔNFDQ1 strain was generated by CRISPR/Cas9 and the in vitro growth assay on HCT-8 cells was used to analyze of phenotypic changes after knockout NFDQ1 in parasites. The NFDQ1 tagging and knockout stains were successfully constructed by CRISPR/Cas9 technology and the insertions of transgenic strains were validated by PCR. The expression of NFDQ1 was validated in parasite by western blot. Immunofluorescence and immune-electron microscopy assay showed that NFDQ1 expressed in both asexual and sexual stages of C. parvum, where it was localized to the cytoplasm of the parasite. Upon ablation of NFDQ1, the ΔNFDQ1 strain showed an apparent growth retardation during sexual replication in vitro. NFDQ1 is a cytoplasmic protein without specific localization to secretory organelles, and it may participate in C. parvum growth during sexual reproduction. Future study should determine the role of NFDQ1 following C. parvum infection in vivo.
Sections du résumé
BACKGROUND
BACKGROUND
Cryptosporidium spp. are important zoonotic parasites that can cause moderate to severe diarrhea in humans and animals. Among the three Cryptosporidium species infecting the intestines of calves, Cryptosporidium parvum has a broad host range and causes severe diarrhea in calves, while Cryptosporidium bovis and Cryptosporidium ryanae mainly infect calves without obvious clinical symptoms. Comparative genomic analysis revealed differences in the copy number of genes encoding the nonfinancial disclosure quality (NFDQ) secretory protein family among the three species, suggesting that this protein family may be associated with the host range or pathogenicity of Cryptosporidium spp. To understand the function of cgd8_10 encoded NFDQ1, tagged and knockout strains were constructed and characterized in this study.
METHODS
METHODS
To determine the localization of NFDQ1, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology to tag the C-terminus of NFDQ1 with three hemagglutinin epitopes (3 × HA). The tagged strain was constructed, and the genomic insertion was confirmed by polymerase chain reaction (PCR). Immunofluorescence assays were performed to observe the localization of NFDQ1 both in extracellular sporozoites and at various intracellular developmental stages. Immunoelectron microscopy was used to study the ultrastructural localization of NFDQ1. Then, the ΔNFDQ1 strain was generated by CRISPR/Cas9 and the in vitro growth assay on HCT-8 cells was used to analyze of phenotypic changes after knockout NFDQ1 in parasites.
RESULTS
RESULTS
The NFDQ1 tagging and knockout stains were successfully constructed by CRISPR/Cas9 technology and the insertions of transgenic strains were validated by PCR. The expression of NFDQ1 was validated in parasite by western blot. Immunofluorescence and immune-electron microscopy assay showed that NFDQ1 expressed in both asexual and sexual stages of C. parvum, where it was localized to the cytoplasm of the parasite. Upon ablation of NFDQ1, the ΔNFDQ1 strain showed an apparent growth retardation during sexual replication in vitro.
CONCLUSIONS
CONCLUSIONS
NFDQ1 is a cytoplasmic protein without specific localization to secretory organelles, and it may participate in C. parvum growth during sexual reproduction. Future study should determine the role of NFDQ1 following C. parvum infection in vivo.
Identifiants
pubmed: 39462401
doi: 10.1186/s13071-024-06532-x
pii: 10.1186/s13071-024-06532-x
doi:
Substances chimiques
Protozoan Proteins
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
439Subventions
Organisme : National Natural Science Foundation of China
ID : U21A20258
Organisme : 111 Center
ID : D20008
Organisme : Double First-Class Discipline Promotion Project
ID : 2023B10564003
Organisme : National Key Research and Development Program of China
ID : 2022YFD1802100
Organisme : Basic and Applied Basic Research Foundation of Guangdong Province
ID : 2023A1515012862
Informations de copyright
© 2024. The Author(s).
Références
Striepen B. Parasitic infections: time to tackle cryptosporidiosis. Nature. 2013;503:189–91.
doi: 10.1038/503189a
pubmed: 24236315
Dong S, Yang Y, Wang Y, Yang D, Yang Y, Shi Y, et al. Prevalence of Cryptosporidium infection in the global population: a systematic review and meta-analysis. Acta Parasitol. 2020;65:882–9.
doi: 10.2478/s11686-020-00230-1
pubmed: 32514837
Wang RJ, Li JQ, Chen YC, Zhang LX, Xiao LH. Widespread occurrence of Cryptosporidium infections in patients with HIV/AIDS: epidemiology, clinical feature, diagnosis, and therapy. Acta Trop. 2018;187:257–63.
doi: 10.1016/j.actatropica.2018.08.018
pubmed: 30118699
Gilbert IH, Vinayak S, Striepen B, Manjunatha UH, Khalil IA, Van Voorhis WC, et al. Safe and effective treatments are needed for cryptosporidiosis, a truly neglected tropical disease. BMJ Global Health. 2023;8:e012540.
doi: 10.1136/bmjgh-2023-012540
pubmed: 37541693
pmcid: 10407372
Caravedo MA, White AC Jr. Treatment of cryptosporidiosis: nitazoxanide yes, but we can do better. Expert Rev Anti Infect Ther. 2023;21:167–73.
doi: 10.1080/14787210.2023.2160704
pubmed: 36533398
Ryan UM, Feng Y, Fayer R, Xiao L. Taxonomy and molecular epidemiology of Cryptosporidium and Giardia—a 50 year perspective (1971–2021). Int J Parasitol. 2021;51:1099–119.
doi: 10.1016/j.ijpara.2021.08.007
pubmed: 34715087
Prediger J, Ježková J, Holubová N, Sak B, Konečný R, Rost M, et al. Cryptosporidium sciurinum n. sp. (Apicomplexa: Cryptosporidiidae) in Eurasian Red Squirrels (Sciurus vulgaris). Microorganisms. 2021;9:2050.
doi: 10.3390/microorganisms9102050
pubmed: 34683369
pmcid: 8537388
Huang J, Chen M, He Y, Chen H, Huang M, Li N, et al. Cryptosporidium equi n. sp. (Apicomplexa: Cryptosporidiidae): biological and genetic characterisations. Int J Parasitol. 2023;53:545–54.
doi: 10.1016/j.ijpara.2023.02.008
pubmed: 37150475
Tůmová L, Ježková J, Prediger J, Holubová N, Sak B, Konečný R, et al. Cryptosporidium mortiferum n. sp. (Apicomplexa: Cryptosporidiidae), the species causing lethal cryptosporidiosis in Eurasian red squirrels (Sciurus vulgaris). Parasit Vectors. 2023;16:235.
doi: 10.1186/s13071-023-05844-8
pubmed: 37454101
pmcid: 10349434
Yang X, Guo Y, Xiao L, Feng Y. Molecular epidemiology of human cryptosporidiosis in low- and middle-income countries. Clin Microbiol Rev. 2021;34:e00087.
doi: 10.1128/CMR.00087-19
pubmed: 33627442
pmcid: 8549823
Guo Y, Ryan U, Feng Y, Xiao L. Association of common zoonotic pathogens with concentrated animal feeding operations. Front Microbiol. 2021;12:810142.
doi: 10.3389/fmicb.2021.810142
pubmed: 35082774
Feng Y, Ryan UM, Xiao L. Genetic diversity and population structure of Cryptosporidium. Trends Parasitol. 2018;34:997–1011.
doi: 10.1016/j.pt.2018.07.009
pubmed: 30108020
Guo Y, Ryan U, Feng Y, Xiao L. Emergence of zoonotic Cryptosporidium parvum in China. Trends Parasitol. 2022;38:335–43.
doi: 10.1016/j.pt.2021.12.002
pubmed: 34972653
Xiao L. Molecular epidemiology of cryptosporidiosis: An update. Exp Parasitol. 2010;124:80–9.
doi: 10.1016/j.exppara.2009.03.018
pubmed: 19358845
Fayer R, Santín M, Trout JM. Prevalence of Cryptosporidium species and genotypes in mature dairy cattle on farms in eastern United States compared with younger cattle from the same locations. Vet Parasitol. 2007;145:260–6.
doi: 10.1016/j.vetpar.2006.12.009
pubmed: 17287086
Rieux A, Paraud C, Pors I, Chartier C. Molecular characterization of Cryptosporidium isolates from beef calves under one month of age over three successive years in one herd in western France. Vet Parasitol. 2014;202:171–9.
doi: 10.1016/j.vetpar.2014.03.004
pubmed: 24685023
pmcid: 7116914
Ralston B, Thompson RC, Pethick D, McAllister TA, Olson ME. Cryptosporidium andersoni in Western Australian feedlot cattle. Aust Vet J. 2010;88:458–60.
doi: 10.1111/j.1751-0813.2010.00631.x
pubmed: 20973292
Xu Z, Li N, Guo Y, Feng Y, Xiao L. Comparative genomic analysis of three intestinal species reveals reductions in secreted pathogenesis determinants in bovine-specific and non-pathogenic Cryptosporidium species. Microb Genom. 2020;6:e000379.
pubmed: 32416746
pmcid: 7371110
Guo Y, Tang K, Rowe LA, Li N, Roellig DM, Knipe K, et al. Comparative genomic analysis reveals occurrence of genetic recombination in virulent Cryptosporidium hominis subtypes and telomeric gene duplications in Cryptosporidium parvum. BMC Genom. 2015;16:320.
doi: 10.1186/s12864-015-1517-1
Mazurie AJ, Alves JM, Ozaki LS, Zhou S, Schwartz DC, Buck GA. Comparative genomics of cryptosporidium. Int J Genom. 2013;2013:832756.
Liu S, Roellig DM, Guo Y, Li N, Frace MA, Tang K, et al. Evolution of mitosome metabolism and invasion-related proteins in Cryptosporidium. BMC Genom. 2016;17:1006.
doi: 10.1186/s12864-016-3343-5
He X, Huang W, Sun L, Hou T, Wan Z, Li N, et al. A productive immunocompetent mouse model of cryptosporidiosis with long oocyst shedding duration for immunological studies. J Infect. 2022;84:710–21.
doi: 10.1016/j.jinf.2022.02.019
pubmed: 35192895
Wang T, Guo Y, Roellig DM, Li N, Santin M, Lombard J, et al. Sympatric recombination in zoonotic Cryptosporidium leads to emergence of populations with modified host preference. Mol Biol Evol. 2022;39:msac150.
doi: 10.1093/molbev/msac150
pubmed: 35776423
pmcid: 9317183
Jia R, Huang W, Huang N, Yu Z, Li N, Xiao L, et al. High infectivity and unique genomic sequence characteristics of Cryptosporidium parvum in China. PLoS Negl Trop Dis. 2022;16:e0010714.
doi: 10.1371/journal.pntd.0010714
pubmed: 35994488
pmcid: 9436107
Xu R, Feng Y, Xiao L, Sibley LD. Insulinase-like protease 1 contributes to macrogamont formation in Cryptosporidium parvum. mBio. 2021;12:e03405-20.
doi: 10.1128/mBio.03405-20
pubmed: 33688009
pmcid: 8092296
Vinayak S, Pawlowic MC, Sateriale A, Brooks CF, Studstill CJ, Bar-Peled Y, et al. Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum. Nature. 2015;523:477–80.
doi: 10.1038/nature14651
pubmed: 26176919
pmcid: 4640681
Santín M. Clinical and subclinical infections with Cryptosporidium in animals. N Z Vet J. 2013;61:1–10.
doi: 10.1080/00480169.2012.731681
pubmed: 23134088
Santín M, Trout JM, Fayer R. A longitudinal study of cryptosporidiosis in dairy cattle from birth to 2 years of age. Vet Parasitol. 2008;155:15–23.
doi: 10.1016/j.vetpar.2008.04.018
pubmed: 18565677
Guérin A, Strelau KM, Barylyuk K, Wallbank BA, Berry L, Crook OM, et al. Cryptosporidium uses multiple distinct secretory organelles to interact with and modify its host cell. Cell Host Microbe. 2023;31:650–64.
doi: 10.1016/j.chom.2023.03.001
pubmed: 36958336
Dumaine JE, Sateriale A, Gibson AR, Reddy AG, Gullicksrud JA, Hunter EN, et al. The enteric pathogen Cryptosporidium parvum exports proteins into the cytosol of the infected host cell. Elife. 2021;10:e70451.
doi: 10.7554/eLife.70451
pubmed: 34866573
pmcid: 8687662
Guérin A, Roy NH, Kugler EM, Berry L, Burkhardt JK, Shin JB, et al. Cryptosporidium rhoptry effector protein ROP1 injected during invasion targets the host cytoskeletal modulator LMO7. Cell Host Microbe. 2021;29:1407-1420.e1405.
doi: 10.1016/j.chom.2021.07.002
pubmed: 34348092
pmcid: 8475647