{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,6]],"date-time":"2026-09-06T17:37:50Z","timestamp":1788716270031,"version":"build-2803163510"},"reference-count":59,"publisher":"American Society for Microbiology","issue":"10","license":[{"start":{"date-parts":[[2025,10,23]],"date-time":"2025-10-23T00:00:00Z","timestamp":1761177600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,10,23]],"date-time":"2025-10-23T00:00:00Z","timestamp":1761177600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"name":"National Institute of Wildlife Disease Control and Prevention"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["J Bacteriol"],"published-print":{"date-parts":[[2025,10,23]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      <jats:italic toggle=\"yes\">Mycobacterium avium<\/jats:italic>\n                      subsp.\n                      <jats:italic toggle=\"yes\">paratuberculosis<\/jats:italic>\n                      (MAP) is a causative agent of Johne\u2019s disease in ruminants and a\n                        potential zoonotic agent linked with Crohn\u2019s disease in humans.\n                        Despite the possible risk to public health, few studies have focused on the\n                        virulence of MAP against human macrophages. Therefore, a functional analysis\n                        of mycobacterial genes associated with virulence, especially the\n                        intracellular survival of MAP, was performed after infection of MAP CRISPR\n                        interference (CRISPRi) mutants in the human THP-1 macrophages. MAP mutants\n                        were targeted to four genes (\n                      <jats:italic toggle=\"yes\">mdh<\/jats:italic>\n                      ,\n                      <jats:italic toggle=\"yes\">pknG<\/jats:italic>\n                      ,\n                      <jats:italic toggle=\"yes\">MAP1981c<\/jats:italic>\n                      , and\n                      <jats:italic toggle=\"yes\">icl<\/jats:italic>\n                      ). The optimal\n                        concentration of anhydrotetracycline (ATc) was determined to be 5\n                        \u00b5g\/mL by measuring the survival of the cells and the downregulation\n                        of gene expression levels in the cells up to Day 3. The clump formation and\n                        intracellular survival of MAP were investigated using transmission electron\n                        microscopy and the colony-forming units, respectively. The clump formation\n                        of MAP mutants induced by CRISPRi was decreased in THP-1 macrophages at 24\n                        and 72 h post-infection. The survival rates of the MAP mutants significantly\n                        decreased with increasing ATc concentration and time course of infection in\n                            MAP\n                      <jats:italic toggle=\"yes\">-mdh<\/jats:italic>\n                      KD,\n                      <jats:italic toggle=\"yes\">MAP1981c<\/jats:italic>\n                      KD, and\n                            MAP\n                      <jats:italic toggle=\"yes\">-icl<\/jats:italic>\n                      KD. Conversely, the survival rate of THP-1\n                        macrophages increased with increasing ATc concentration. Our results suggest\n                        that these genes might be closely related to MAP virulence along with\n                        intracellular survival in THP-1 macrophages. These data can provide novel\n                        insights into the utilization of CRISPRi in further research on MAP\n                        virulence by exploring intracellular survival using mycobacterial genes\n                        related to the virulence of MAP during host infection.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      Johne\u2019s disease, caused by\n                      <jats:italic toggle=\"yes\">Mycobacterium avium<\/jats:italic>\n                      subsp.\n                      <jats:italic toggle=\"yes\">paratuberculosis<\/jats:italic>\n                      (MAP) is a worldwide issue in the\n                        dairy industry and has a possible connection to Crohn\u2019s disease (CD)\n                        in humans. Despite its potential contribution to the etiology of CD, there\n                        have been few studies focusing on the virulence of MAP against human\n                        macrophages. In the current study, we investigated MAP virulence along with\n                        intracellular survival in human THP-1 macrophages using functional analysis\n                        of MAP CRISPR interference (CRISPRi) mutants at the knockdown of genes\n                        associated with mycobacterial virulence. The identified potential genes\n                        represent novel candidate classes that could be necessary for MAP virulence\n                        by exploring intracellular survival during host infection and could provide\n                        novel insights for future studies on the utilization of CRISPRi.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/jb.00244-25","type":"journal-article","created":{"date-parts":[[2025,9,12]],"date-time":"2025-09-12T15:47:55Z","timestamp":1757692075000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":5,"title":["Functional analysis of the intracellular survival of\n                    <i>Mycobacterium avium<\/i>\n                    subsp.\n                    <i>paratuberculosis<\/i>\n                    in THP-1 cells using CRISPR                    interference"],"prefix":"10.1128","volume":"207","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-8953-6413","authenticated-orcid":false,"given":"Jun Ho","family":"Lee","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04h9pn542","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Infectious Disease, College of Veterinary Medicine, Seoul National University","place":["Seoul, Republic of Korea"]},{"id":[{"id":"https:\/\/ror.org\/04h9pn542","id-type":"ROR","asserted-by":"publisher"}],"name":"BK21 FOUR Future Veterinary Medicine Leading Education and Research Center, College of Veterinary Medicine, Seoul National University","place":["Seoul, Republic of Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eun-Seo","family":"Lee","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04h9pn542","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Infectious Disease, College of Veterinary Medicine, Seoul National University","place":["Seoul, Republic of Korea"]},{"id":[{"id":"https:\/\/ror.org\/04h9pn542","id-type":"ROR","asserted-by":"publisher"}],"name":"BK21 FOUR Future Veterinary Medicine Leading Education and Research Center, College of Veterinary Medicine, Seoul National University","place":["Seoul, Republic of Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Su Min","family":"Kyung","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04h9pn542","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Infectious Disease, College of Veterinary Medicine, Seoul National University","place":["Seoul, Republic of Korea"]},{"id":[{"id":"https:\/\/ror.org\/04h9pn542","id-type":"ROR","asserted-by":"publisher"}],"name":"Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University","place":["Seoul, Republic of Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xi-Rui","family":"Xiang","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04h9pn542","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Infectious Disease, College of Veterinary Medicine, Seoul National University","place":["Seoul, Republic of Korea"]},{"id":[{"id":"https:\/\/ror.org\/04h9pn542","id-type":"ROR","asserted-by":"publisher"}],"name":"BK21 FOUR Future Veterinary Medicine Leading Education and Research Center, College of Veterinary Medicine, Seoul National University","place":["Seoul, Republic of Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hyun-Eui","family":"Park","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01wjejq96","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology, College of Medicine, Yonsei University Wonju","place":["Wonju, Republic of Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1782-5351","authenticated-orcid":true,"given":"Min-Kyoung","family":"Shin","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00saywf64","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology and Convergence Medical Science, College of Medicine, Gyeongsang National University","place":["Jinju, Republic of Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0548-0835","authenticated-orcid":true,"given":"Han Sang","family":"Yoo","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04h9pn542","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Infectious Disease, College of Veterinary Medicine, Seoul National University","place":["Seoul, Republic of Korea"]},{"id":[{"id":"https:\/\/ror.org\/04h9pn542","id-type":"ROR","asserted-by":"publisher"}],"name":"BK21 FOUR Future Veterinary Medicine Leading Education and Research Center, College of Veterinary Medicine, Seoul National University","place":["Seoul, Republic of Korea"]},{"id":[{"id":"https:\/\/ror.org\/04h9pn542","id-type":"ROR","asserted-by":"publisher"}],"name":"Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University","place":["Seoul, Republic of Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.20506\/rst.20.1.1275"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cvfa.2011.07.001"},{"key":"e_1_3_4_4_2","doi-asserted-by":"publisher","DOI":"10.5455\/OVJ.2022.v12.i4.16"},{"key":"e_1_3_4_5_2","doi-asserted-by":"publisher","DOI":"10.3748\/wjg.v20.i23.7403"},{"key":"e_1_3_4_6_2","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2015.00096"},{"key":"e_1_3_4_7_2","doi-asserted-by":"publisher","DOI":"10.21037\/tgh-23-16"},{"key":"e_1_3_4_8_2","doi-asserted-by":"publisher","DOI":"10.1007\/s10393-022-01602-x"},{"key":"e_1_3_4_9_2","doi-asserted-by":"publisher","DOI":"10.1080\/01652176.2001.9695105"},{"key":"e_1_3_4_10_2","doi-asserted-by":"publisher","DOI":"10.4236\/ns.2013.56088"},{"key":"e_1_3_4_11_2","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2180-2-2"},{"key":"e_1_3_4_12_2","doi-asserted-by":"publisher","DOI":"10.1079\/AHRR200134"},{"key":"e_1_3_4_13_2","doi-asserted-by":"publisher","DOI":"10.4142\/jvs.2008.9.1.1"},{"key":"e_1_3_4_14_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1472-765X.2006.01873.x"},{"key":"e_1_3_4_15_2","doi-asserted-by":"publisher","DOI":"10.1016\/s0165-2427(02)00065-x"},{"key":"e_1_3_4_16_2","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2022.994421"},{"key":"e_1_3_4_17_2","doi-asserted-by":"publisher","DOI":"10.3389\/fgene.2021.735147"},{"key":"e_1_3_4_18_2","doi-asserted-by":"publisher","DOI":"10.1186\/s13567-022-01089-y"},{"key":"e_1_3_4_19_2","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2019.02874"},{"key":"e_1_3_4_20_2","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2023.1042638"},{"key":"e_1_3_4_21_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.chembiol.2018.09.009"},{"key":"e_1_3_4_22_2","doi-asserted-by":"publisher","DOI":"10.1002\/prot.24743"},{"key":"e_1_3_4_23_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cyto.2018.01.022"},{"key":"e_1_3_4_24_2","doi-asserted-by":"publisher","DOI":"10.1155\/2018\/1450828"},{"key":"e_1_3_4_25_2","doi-asserted-by":"publisher","DOI":"10.15252\/embr.202052175"},{"key":"e_1_3_4_26_2","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2018.00206"},{"key":"e_1_3_4_27_2","doi-asserted-by":"publisher","unstructured":"Facciuolo A Gonzalez-Cano P Napper S Griebel PJ Mutharia LM. 2016. Marked differences in mucosal immune responses induced in ileal versus jejunal Peyer\u2019s patches to Mycobacterium avium subsp:e0158747. doi:10.1371\/journal.pone.0158747","DOI":"10.1371\/journal.pone.0158747"},{"key":"e_1_3_4_28_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-020-75799-8"},{"key":"e_1_3_4_29_2","doi-asserted-by":"publisher","DOI":"10.1128\/JB.181.23.7161-7167.1999"},{"key":"e_1_3_4_30_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.biochi.2014.12.016"},{"key":"e_1_3_4_31_2","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2023.1149419"},{"key":"e_1_3_4_32_2","doi-asserted-by":"publisher","DOI":"10.2174\/0929866529666221006121831"},{"key":"e_1_3_4_33_2","doi-asserted-by":"publisher","DOI":"10.1016\/B978-0-12-818140-9.00017-9"},{"key":"e_1_3_4_34_2","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2021.635227"},{"key":"e_1_3_4_35_2","doi-asserted-by":"publisher","DOI":"10.1016\/bs.pmbts.2021.01.007"},{"key":"e_1_3_4_36_2","doi-asserted-by":"publisher","DOI":"10.1134\/S0026893322060139"},{"key":"e_1_3_4_37_2","doi-asserted-by":"publisher","DOI":"10.1038\/nmicrobiol.2016.274"},{"key":"e_1_3_4_38_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.tube.2022.102292"},{"key":"e_1_3_4_39_2","doi-asserted-by":"publisher","DOI":"10.1128\/spectrum.00631-23"},{"key":"e_1_3_4_40_2","doi-asserted-by":"publisher","DOI":"10.3390\/antibiotics13060483"},{"key":"e_1_3_4_41_2","doi-asserted-by":"publisher","DOI":"10.1079\/9781789243413.0000"},{"key":"e_1_3_4_42_2","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2024.1327969"},{"key":"e_1_3_4_43_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0308911"},{"key":"e_1_3_4_44_2","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2021.633510"},{"key":"e_1_3_4_45_2","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dkab437"},{"key":"e_1_3_4_46_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkw625"},{"key":"e_1_3_4_47_2","doi-asserted-by":"publisher","DOI":"10.1128\/mbio.03683-21"},{"key":"e_1_3_4_48_2","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2023.1089911"},{"key":"e_1_3_4_49_2","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2016.01562"},{"key":"e_1_3_4_50_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbc.2022.101859"},{"key":"e_1_3_4_51_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.drudis.2017.04.012"},{"key":"e_1_3_4_52_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.vaccine.2011.04.090"},{"key":"e_1_3_4_53_2","doi-asserted-by":"publisher","DOI":"10.1099\/mic.0.033084-0"},{"key":"e_1_3_4_54_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.molmed.2015.07.006"},{"key":"e_1_3_4_55_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-0716-1460-0_16"},{"key":"e_1_3_4_56_2","doi-asserted-by":"publisher","DOI":"10.3168\/jds.2021-20305"},{"key":"e_1_3_4_57_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.tube.2021.102079"},{"key":"e_1_3_4_58_2","doi-asserted-by":"publisher","DOI":"10.1186\/s11658-024-00565-x"},{"key":"e_1_3_4_59_2","doi-asserted-by":"publisher","DOI":"10.1385\/1-59259-220-1:1"},{"key":"e_1_3_4_60_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.fm.2020.103611"}],"container-title":["Journal of Bacteriology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/jb.00244-25","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/jb.00244-25","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,23]],"date-time":"2025-10-23T13:21:28Z","timestamp":1761225688000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.asm.org\/doi\/10.1128\/jb.00244-25"}},"subtitle":[],"editor":[{"given":"Michael J.","family":"Federle","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2025,10,23]]},"references-count":59,"journal-issue":{"issue":"10","published-print":{"date-parts":[[2025,10,23]]}},"alternative-id":["10.1128\/jb.00244-25"],"URL":"https:\/\/doi.org\/10.1128\/jb.00244-25","relation":{},"ISSN":["0021-9193","1098-5530"],"issn-type":[{"value":"0021-9193","type":"print"},{"value":"1098-5530","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,10,23]]},"assertion":[{"value":"2025-06-17","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-08-11","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-09-12","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e00244-25"}}