{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,23]],"date-time":"2026-04-23T09:31:41Z","timestamp":1776936701083,"version":"3.51.4"},"update-to":[{"DOI":"10.1371\/journal.pntd.0012777","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2025,1,24]],"date-time":"2025-01-24T00:00:00Z","timestamp":1737676800000}}],"reference-count":39,"publisher":"Public Library of Science (PLoS)","issue":"1","license":[{"start":{"date-parts":[[2025,1,13]],"date-time":"2025-01-13T00:00:00Z","timestamp":1736726400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32030109"],"award-info":[{"award-number":["32030109"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32273032"],"award-info":[{"award-number":["32273032"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Guangdong Major Project of Basic and Applied Basic Research","award":["2020B0301030007"],"award-info":[{"award-number":["2020B0301030007"]}]},{"DOI":"10.13039\/501100013313","name":"Overseas Expertise Introduction Project for Discipline Innovation","doi-asserted-by":"publisher","award":["D20008"],"award-info":[{"award-number":["D20008"]}],"id":[{"id":"10.13039\/501100013313","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Double First-class Discipline Promotion Project","award":["2023B10564003"],"award-info":[{"award-number":["2023B10564003"]}]}],"content-domain":{"domain":["www.plosntds.org"],"crossmark-restriction":false},"short-container-title":["PLoS Negl Trop Dis"],"abstract":"<jats:sec id=\"sec001\">\n<jats:title>Background<\/jats:title>\n<jats:p>The determinants of differences in host infectivity among <jats:italic>Cryptosporidium<\/jats:italic> species and subtypes are poorly understood. Results from recent comparative genomic studies suggest that gains and losses of multicopy subtelomeric genes encoding insulinase-like proteases (INS-19 and INS-20 in <jats:italic>Cryptosporidium parvum<\/jats:italic> and their orthologs in closely related species) may potentially contribute to these differences.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec002\">\n<jats:title>Methodology\/Principal findings<\/jats:title>\n<jats:p>In this study, we investigated the expression and biological function of the INS-19 and INS-20 of <jats:italic>C<\/jats:italic>. <jats:italic>parvum<\/jats:italic>. CRISPR\/Cas9 was used to endogenously tag both genes with the hemagglutinin epitope. Immunofluorescence analysis revealed that INS-19 and INS-20 are expressed at different developmental stages of the pathogen. Although knockout of either had no detectable effect on the <jats:italic>in vitro<\/jats:italic> growth of <jats:italic>C<\/jats:italic>. <jats:italic>parvum<\/jats:italic>, knockout of INS-20, deletion of its multiple domains, or mutation of the active motif in the functional domain reduced the intensity of <jats:italic>C<\/jats:italic>. <jats:italic>parvum<\/jats:italic> infection in IFN-\u03b3 knockout mice. Consistent with this, mice infected with the INS-20-deleted mutant had reduced intestinal damage and parasite burden.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec003\">\n<jats:title>Conclusions\/Significance<\/jats:title>\n<jats:p>These results suggest that INS-19 and INS-20 have stage-specific expression with distinct biological functions, and that the presence of the INS-20 in zoonotic <jats:italic>C<\/jats:italic>. <jats:italic>parvum<\/jats:italic> contributes to its infectivity and fitness in mice.<\/jats:p>\n<\/jats:sec>","DOI":"10.1371\/journal.pntd.0012777","type":"journal-article","created":{"date-parts":[[2025,1,13]],"date-time":"2025-01-13T19:05:57Z","timestamp":1736795157000},"page":"e0012777","update-policy":"https:\/\/doi.org\/10.1371\/journal.pntd.corrections_policy","source":"Crossref","is-referenced-by-count":4,"title":["Stage-specific expression and divergent functions of two insulinase-like proteases associated with host infectivity in Cryptosporidium"],"prefix":"10.1371","volume":"19","author":[{"given":"Yue","family":"Huang","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shifeng","family":"Pei","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xin","family":"Lv","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fuxian","family":"Yang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiaoqing","family":"Gong","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Na","family":"Li","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yaqiong","family":"Guo","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yaoyu","family":"Feng","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8532-2727","authenticated-orcid":true,"given":"Lihua","family":"Xiao","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2025,1,13]]},"reference":[{"issue":"9","key":"pntd.0012777.ref001","doi-asserted-by":"crossref","first-page":"909","DOI":"10.1016\/S1473-3099(17)30276-1","article-title":"Estimates of global, regional, and national morbidity, mortality, and aetiologies of diarrhoeal diseases: a systematic analysis for the Global Burden of Disease Study 2015","volume":"17","author":"C Troeger","year":"2017","journal-title":"The Lancet infectious diseases"},{"issue":"25","key":"pntd.0012777.ref002","doi-asserted-by":"crossref","first-page":"568","DOI":"10.15585\/mmwr.mm6825a3","article-title":"Cryptosporidiosis Outbreaks\u2014United States, 2009\u20132017","volume":"68","author":"R Gharpure","year":"2019","journal-title":"MMWR Morb Mortal Wkly Rep"},{"issue":"1","key":"pntd.0012777.ref003","first-page":"223","article-title":"Cryptosporidium and Giardia in ruminants","volume":"36","author":"M Santin","year":"2020","journal-title":"Veterinary Clinics of North America: Food Animal Practice"},{"issue":"13\u201314","key":"pntd.0012777.ref004","doi-asserted-by":"crossref","first-page":"1099","DOI":"10.1016\/j.ijpara.2021.08.007","article-title":"Taxonomy and molecular epidemiology of Cryptosporidium and Giardia\u2013a 50 year perspective (1971\u20132021)","volume":"51","author":"UM Ryan","year":"2021","journal-title":"International journal for parasitology"},{"issue":"11","key":"pntd.0012777.ref005","doi-asserted-by":"crossref","first-page":"997","DOI":"10.1016\/j.pt.2018.07.009","article-title":"Genetic diversity and population structure of Cryptosporidium","volume":"34","author":"Y Feng","year":"2018","journal-title":"Trends in parasitology"},{"issue":"6","key":"pntd.0012777.ref006","doi-asserted-by":"crossref","first-page":"433","DOI":"10.1111\/j.1550-7408.2002.tb00224.x","article-title":"Cryptosporidium hominis n. sp. (Apicomplexa: Cryptosporidiidae) from Homo sapiens","volume":"49","author":"UM Morgan-Ryan","year":"2002","journal-title":"J Eukaryot Microbiol"},{"issue":"1","key":"pntd.0012777.ref007","first-page":"1","article-title":"Comparative analysis reveals conservation in genome organization among intestinal Cryptosporidium species and sequence divergence in potential secreted pathogenesis determinants among major human-infecting species","volume":"20","author":"Z Xu","year":"2019","journal-title":"BMC genomics"},{"issue":"4","key":"pntd.0012777.ref008","doi-asserted-by":"crossref","first-page":"650","DOI":"10.1016\/j.chom.2023.03.001","article-title":"Cryptosporidium uses multiple distinct secretory organelles to interact with and modify its host cell","volume":"31","author":"A Guerin","year":"2023","journal-title":"Cell host & microbe"},{"issue":"14","key":"pntd.0012777.ref009","article-title":"Characterization of Dense Granule Metalloproteinase INS-16 in Cryptosporidium parvum","volume":"23","author":"H Cui","year":"2022","journal-title":"Int J Mol Sci"},{"issue":"2","key":"pntd.0012777.ref010","first-page":"e03405","article-title":"Insulinase-like protease 1 contributes to macrogamont formation in Cryptosporidium parvum","volume":"12","author":"R Xu","year":"2021","journal-title":"Mbio"},{"key":"pntd.0012777.ref011","doi-asserted-by":"crossref","first-page":"354","DOI":"10.3389\/fmicb.2019.00354","article-title":"Characterization of a species-specific insulinase-like protease in Cryptosporidium parvum","volume":"10","author":"S Zhang","year":"2019","journal-title":"Frontiers in Microbiology"},{"issue":"4","key":"pntd.0012777.ref012","doi-asserted-by":"crossref","first-page":"861","DOI":"10.3390\/microorganisms9040861","article-title":"Comparative study of two insulinlike proteases in Cryptosporidium parvum","volume":"9","author":"W He","year":"2021","journal-title":"Microorganisms"},{"issue":"10","key":"pntd.0012777.ref013","doi-asserted-by":"crossref","first-page":"452","DOI":"10.3390\/microorganisms7100452","article-title":"Characterization of INS-15, a metalloprotease potentially involved in the invasion of Cryptosporidium parvum","volume":"7","author":"R Xu","year":"2019","journal-title":"Microorganisms"},{"issue":"1","key":"pntd.0012777.ref014","doi-asserted-by":"crossref","first-page":"320","DOI":"10.1186\/s12864-015-1517-1","article-title":"Comparative genomic analysis reveals occurrence of genetic recombination in virulent Cryptosporidium hominis subtypes and telomeric gene duplications in Cryptosporidium parvum","volume":"16","author":"Y Guo","year":"2015","journal-title":"BMC genomics"},{"issue":"1","key":"pntd.0012777.ref015","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1101\/gr.275325.121","article-title":"Long-read assembly and comparative evidence-based reanalysis of Cryptosporidium genome sequences reveal expanded transporter repertoire and duplication of entire chromosome ends including subtelomeric regions","volume":"32","author":"RP Baptista","year":"2022","journal-title":"Genome Res"},{"key":"pntd.0012777.ref016","article-title":"Comparative genomics analysis reveals sequence characteristics potentially related to host preference in Cryptosporidium xiaoi","author":"J Li","year":"2024","journal-title":"Int J Parasitol"},{"issue":"9","key":"pntd.0012777.ref017","doi-asserted-by":"crossref","first-page":"366","DOI":"10.3390\/microorganisms7090366","article-title":"Divergent Copies of a Cryptosporidium parvum-Specific Subtelomeric Gene","volume":"7","author":"S Zhang","year":"2019","journal-title":"Microorganisms"},{"issue":"26","key":"pntd.0012777.ref018","doi-asserted-by":"crossref","DOI":"10.1128\/MRA.00363-21","article-title":"Draft genome assemblies of two Cryptosporidium hominis isolates from New Zealand","volume":"10","author":"M Knox","year":"2021","journal-title":"Microbiology Resource Announcements"},{"issue":"1","key":"pntd.0012777.ref019","doi-asserted-by":"crossref","first-page":"16324","DOI":"10.1038\/srep16324","article-title":"Revisiting the reference genomes of human pathogenic Cryptosporidium species: reannotation of C. parvum Iowa and a new C. hominis reference","volume":"5","author":"JP Isaza","year":"2015","journal-title":"Scientific reports"},{"key":"pntd.0012777.ref020","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s12864-015-1805-9","article-title":"Generation of whole genome sequences of new Cryptosporidium hominis and Cryptosporidium parvum isolates directly from stool samples","volume":"16","author":"SJ Hadfield","year":"2015","journal-title":"BMC genomics"},{"issue":"5","key":"pntd.0012777.ref021","doi-asserted-by":"crossref","first-page":"281","DOI":"10.1016\/j.ijpara.2016.12.002","article-title":"Comparative genomic analysis of the IId subtype family of Cryptosporidium parvum","volume":"47","author":"Y Feng","year":"2017","journal-title":"International Journal for Parasitology"},{"key":"pntd.0012777.ref022","author":"W Huang","year":"2023","journal-title":"Unique genomic characteristics underlie the infectivity of divergent Cryptosporidium hominis subtypes to animals"},{"issue":"5","key":"pntd.0012777.ref023","first-page":"e1606","article-title":"Diarrhoea outbreak caused by coinfections of Cryptosporidium parvum subtype IIdA20G1 and rotavirus in pre-weaned dairy calves","volume":"69","author":"N Li","year":"2022","journal-title":"Transboundary and Emerging Diseases"},{"issue":"8","key":"pntd.0012777.ref024","doi-asserted-by":"crossref","first-page":"e0010714","DOI":"10.1371\/journal.pntd.0010714","article-title":"High infectivity and unique genomic sequence characteristics of Cryptosporidium parvum in China","volume":"16","author":"R Jia","year":"2022","journal-title":"PLoS Negl Trop Dis"},{"issue":"1","key":"pntd.0012777.ref025","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1016\/j.chom.2019.05.007","article-title":"A stem-cell-derived platform enables complete Cryptosporidium development in vitro and genetic tractability","volume":"26","author":"G Wilke","year":"2019","journal-title":"Cell host & microbe"},{"issue":"7561","key":"pntd.0012777.ref026","doi-asserted-by":"crossref","first-page":"477","DOI":"10.1038\/nature14651","article-title":"Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum","volume":"523","author":"S Vinayak","year":"2015","journal-title":"Nature"},{"issue":"2","key":"pntd.0012777.ref027","doi-asserted-by":"crossref","DOI":"10.1073\/pnas.2007807118","article-title":"The intestinal parasite Cryptosporidium is controlled by an enterocyte intrinsic inflammasome that depends on NLRP6","volume":"118","author":"A Sateriale","year":"2021","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"2","key":"pntd.0012777.ref028","doi-asserted-by":"crossref","first-page":"314","DOI":"10.2307\/3282084","article-title":"Isolation of Cryptosporidium oocysts and sporozoites using discontinuous sucrose and isopycnic Percoll gradients","volume":"73","author":"MJ Arrowood","year":"1987","journal-title":"J Parasitol"},{"issue":"2","key":"pntd.0012777.ref029","doi-asserted-by":"crossref","first-page":"641","DOI":"10.1128\/JCM.02962-14","article-title":"Isolation and enrichment of Cryptosporidium DNA and verification of DNA purity for whole-genome sequencing","volume":"53","author":"Y Guo","year":"2015","journal-title":"Journal of clinical microbiology"},{"key":"pntd.0012777.ref030","author":"JT Robinson","year":"2011","journal-title":"Integrative genomics viewer"},{"issue":"17","key":"pntd.0012777.ref031","doi-asserted-by":"crossref","first-page":"5845","DOI":"10.1128\/AEM.01699-15","article-title":"Development and Evaluation of Three Real-Time PCR Assays for Genotyping and Source Tracking Cryptosporidium spp. in Water","volume":"81","author":"N Li","year":"2015","journal-title":"Appl Environ Microbiol"},{"key":"pntd.0012777.ref032","doi-asserted-by":"crossref","first-page":"201","DOI":"10.1016\/j.ins.2014.05.037","article-title":"Transmission protocol for secure big data in two-hop wireless networks with cooperative jamming","volume":"281","author":"Y Shen","year":"2014","journal-title":"Information Sciences"},{"issue":"7113","key":"pntd.0012777.ref033","doi-asserted-by":"crossref","first-page":"870","DOI":"10.1038\/nature05143","article-title":"Structures of human insulin-degrading enzyme reveal a new substrate recognition mechanism","volume":"443","author":"Y Shen","year":"2006","journal-title":"Nature"},{"issue":"31","key":"pntd.0012777.ref034","doi-asserted-by":"crossref","first-page":"3644","DOI":"10.2174\/138161209789271799","article-title":"Insulin-degrading enzyme: structure-function relationship and its possible roles in health and disease","volume":"15","author":"A Fernandez-Gamba","year":"2009","journal-title":"Curr Pharm Des"},{"key":"pntd.0012777.ref035","doi-asserted-by":"crossref","first-page":"651512","DOI":"10.3389\/fmicb.2021.651512","article-title":"Preliminary characterization of two small insulinase-like proteases in Cryptosporidium parvum","volume":"12","author":"R Xu","year":"2021","journal-title":"Frontiers in Microbiology"},{"issue":"1","key":"pntd.0012777.ref036","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1006\/prep.2000.1217","article-title":"Functional human insulin-degrading enzyme can be expressed in bacteria","volume":"19","author":"V Chesneau","year":"2000","journal-title":"Protein expression and purification"},{"key":"pntd.0012777.ref037","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1186\/1743-422X-3-7","article-title":"Mutational analysis of the potential catalytic residues of the VV G1L metalloproteinase","volume":"3","author":"KM Honeychurch","year":"2006","journal-title":"Virol J"},{"issue":"1","key":"pntd.0012777.ref038","doi-asserted-by":"crossref","first-page":"100054","DOI":"10.1016\/j.vetvac.2024.100054","article-title":"The first commercially approved efficacious Cryptosporidium vaccine protecting New-Born calves from severe diarrhea","volume":"3","author":"M Timmermans","year":"2024","journal-title":"Veterinary Vaccine"},{"issue":"1","key":"pntd.0012777.ref039","doi-asserted-by":"crossref","first-page":"1175","DOI":"10.1038\/s42003-024-06885-0","article-title":"Variant surface protein GP60 contributes to host infectivity of Cryptosporidium parvum","volume":"7","author":"M Li","year":"2024","journal-title":"Communications Biology"}],"updated-by":[{"DOI":"10.1371\/journal.pntd.0012777","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2025,1,24]],"date-time":"2025-01-24T00:00:00Z","timestamp":1737676800000}}],"container-title":["PLOS Neglected Tropical Diseases"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pntd.0012777","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,1,24]],"date-time":"2025-01-24T19:22:45Z","timestamp":1737746565000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pntd.0012777"}},"subtitle":[],"editor":[{"given":"Susan","family":"Madison-Antenucci","sequence":"first","affiliation":[],"role":[{"role":"editor","vocabulary":"crossref"}]}],"short-title":[],"issued":{"date-parts":[[2025,1,13]]},"references-count":39,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2025,1,13]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pntd.0012777","relation":{"new_version":[{"id-type":"doi","id":"10.1371\/journal.pntd.0012777","asserted-by":"object"}]},"ISSN":["1935-2735"],"issn-type":[{"value":"1935-2735","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,1,13]]}}}