{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,10]],"date-time":"2026-02-10T13:02:10Z","timestamp":1770728530109,"version":"3.49.0"},"reference-count":57,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2023,5,22]],"date-time":"2023-05-22T00:00:00Z","timestamp":1684713600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001509","name":"Royal Society Te Ap\u0101rangi","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100001509","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100017625","name":"Massey Foundation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100017625","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell. Infect. Microbiol."],"abstract":"<jats:p>Cryptosporidiosis is a worldwide diarrheal disease caused by the protozoan <jats:italic>Cryptosporidium<\/jats:italic>. The primary symptom is diarrhea, but patients may exhibit different symptoms based on the species of the <jats:italic>Cryptosporidium<\/jats:italic> parasite they are infected with. Furthermore, some genotypes within species are more transmissible and apparently virulent than others. The mechanisms underpinning these differences are not understood, and an effective <jats:italic>in vitro<\/jats:italic> system for <jats:italic>Cryptosporidium<\/jats:italic> culture would help advance our understanding of these differences. Using COLO-680N cells, we employed flow cytometry and microscopy along with the <jats:italic>C. parvum<\/jats:italic>-specific antibody Sporo-Glo\u2122 to characterize infected cells 48\u00a0h following an infection with <jats:italic>C. parvum<\/jats:italic> or <jats:italic>C. hominis<\/jats:italic>. The <jats:italic>Cryptosporidium parvum<\/jats:italic>-infected cells showed higher levels of signal using Sporo-Glo\u2122 than <jats:italic>C. hominis<\/jats:italic>-infected cells, which was likely because Sporo-Glo\u2122 was generated against <jats:italic>C. parvum<\/jats:italic>. We found a subset of cells from infected cultures that expressed a novel, dose-dependent auto-fluorescent signal that was detectable across a range of wavelengths. The population of cells that expressed this signal increased proportionately to the multiplicity of infection. The spectral cytometry results confirmed that the signature of this subset of host cells closely matched that of oocysts present in the infectious ecosystem, pointing to a parasitic origin. Present in both <jats:italic>C. parvum<\/jats:italic> and <jats:italic>C. hominis<\/jats:italic> cultures, we named this Sig M, and due to its distinct profile in cells from both infections, it could be a better marker for assessing <jats:italic>Cryptosporidium<\/jats:italic> infection in COLO-680N cells than Sporo-Glo\u2122. We also noted Sig M\u2019s impact on Sporo-Glo\u2122 detection as Sporo-Glo\u2122 uses fluoroscein\u2013isothiocynate, which is detected where Sig M also fluoresces. Lastly, we used NanoString nCounter<jats:sup>\u00ae<\/jats:sup> analysis to investigate the transcriptomic landscape for the two <jats:italic>Cryptosporidium<\/jats:italic> species, assessing the gene expression of 144 host and parasite genes. Despite the host gene expression being at high levels, the levels of putative intracellular <jats:italic>Cryptosporidium<\/jats:italic> gene expression were low, with no significant difference from controls, which could be, in part, explained by the abundance of uninfected cells present as determined by both Sporo-Glo\u2122 and Sig M analyses. This study shows for the first time that a natural auto-fluorescent signal, Sig M, linked to <jats:italic>Cryptosporidium<\/jats:italic> infection can be detected in infected host cells without any fluorescent labeling strategies and that the COLO-680N cell line and spectral cytometry could be useful tools to advance the understanding of <jats:italic>Cryptosporidium<\/jats:italic> infectivity.<\/jats:p>","DOI":"10.3389\/fcimb.2023.1178576","type":"journal-article","created":{"date-parts":[[2023,5,22]],"date-time":"2023-05-22T04:43:43Z","timestamp":1684730623000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":5,"title":["A novel, stain-free, natural auto-fluorescent signal, Sig M, identified from cytometric and transcriptomic analysis of infectivity of Cryptosporidium hominis and Cryptosporidium parvum"],"prefix":"10.3389","volume":"13","author":[{"given":"Paul","family":"Ogbuigwe","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Joanna M.","family":"Roberts","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Matthew A.","family":"Knox","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Axel","family":"Heiser","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anthony","family":"Pita","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Neville A.","family":"Haack","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Juan\u00a0Carlos","family":"Garcia-Ramirez","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Niluka","family":"Velathanthiri","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Patrick\u00a0J.","family":"Biggs","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nigel P.","family":"French","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David T. 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