{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,18]],"date-time":"2026-10-18T02:46:27Z","timestamp":1792291587203,"version":"4.4.0"},"reference-count":43,"publisher":"American Society for Microbiology","issue":"8","license":[{"start":{"date-parts":[[2025,8,5]],"date-time":"2025-08-05T00:00:00Z","timestamp":1754352000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,8,5]],"date-time":"2025-08-05T00:00:00Z","timestamp":1754352000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32394013"],"award-info":[{"award-number":["32394013"]}],"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":["82172279"],"award-info":[{"award-number":["82172279"]}],"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":["82402641"],"award-info":[{"award-number":["82402641"]}],"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":["82070012"],"award-info":[{"award-number":["82070012"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Beijing High-level Public Health Project","award":["G2024-2-007"],"award-info":[{"award-number":["G2024-2-007"]}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2025,8,5]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Tuberculosis (TB), caused by\n                      <jats:italic toggle=\"yes\">Mycobacterium tuberculosis<\/jats:italic>\n                      (\n                      <jats:italic toggle=\"yes\">M.tb<\/jats:italic>\n                      ), remains a serious infectious disease posing significant global health challenges. A critical evolutionary feature of\n                      <jats:italic toggle=\"yes\">M.tb<\/jats:italic>\n                      is its genome encoding a set of eukaryotic-like secretory proteins, which facilitate intracellular survival by manipulating host immune responses. However, the specific eukaryotic-like secretory proteins that facilitate\n                      <jats:italic toggle=\"yes\">M.tb<\/jats:italic>\n                      intracellular survival and their regulatory mechanisms on host immunity remain uncharacterized. In this study, a mutant library comprising 137 potential eukaryotic-like secretory proteins was constructed using clustered regularly interspaced short palindromic repeats (CRISPR)-non-homologous end joining genome editing technology. Subsequently, macrophages were infected with the mutant library, and CRISPR sequencing enabled preliminary identification of virulence factors associated with bacterial intracellular persistence. To validate the screen, two genes (\n                      <jats:italic toggle=\"yes\">Rv0066c<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">Rv3139<\/jats:italic>\n                      ) exhibiting the most pronounced reduction in intracellular survival rates when mutated were selected for the construction of large-fragment knockout strains (\u0394\n                      <jats:italic toggle=\"yes\">Rv0066c<\/jats:italic>\n                      and \u0394\n                      <jats:italic toggle=\"yes\">Rv3139<\/jats:italic>\n                      ). Subsequent macrophage infection assays reconfirmed the impaired intracellular survival of these two mutants. RNA-seq analysis was conducted to characterize host gene expression profiles during \u0394\n                      <jats:italic toggle=\"yes\">Rv0066c<\/jats:italic>\n                      -infected macrophage interactions. RNA-seq analysis of macrophages infected with wild-type and \u0394\n                      <jats:italic toggle=\"yes\">Rv0066c<\/jats:italic>\n                      strains identified 138 differentially expressed genes, with 75 upregulated and 63 downregulated in \u0394\n                      <jats:italic toggle=\"yes\">Rv0066c<\/jats:italic>\n                      . Gene ontology clustering of these differentially expressed genes highlighted molecular functions related to chemokine binding, chemokine-mediated signaling pathways, Ras protein signal transduction, and calcineurin-mediated signaling. Collectively, this work established a potential eukaryotic-like secretory protein mutant library and identified two novel\n                      <jats:italic toggle=\"yes\">M.tb<\/jats:italic>\n                      effectors governing intracellular survival, providing potential new targets for anti-TB drug development.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      Eukaryotic-like secretory proteins that subvert host immunity to enable intracellular persistence are a key evolutionary adaptation of\n                      <jats:italic toggle=\"yes\">Mycobacterium tuberculosis<\/jats:italic>\n                      (\n                      <jats:italic toggle=\"yes\">M.tb<\/jats:italic>\n                      ). In this study, we established a mutant library targeting 137 potential eukaryotic-like secretory proteins through clustered regularly interspaced short palindromic repeats (CRISPR)-non-homologous end joining genome editing technology. The library was subjected to macrophage infection assays, and CRISPR sequencing enabled identification of\n                      <jats:italic toggle=\"yes\">M.tb<\/jats:italic>\n                      persistence-associated virulence determinants. Validation screens highlighted two genes (\n                      <jats:italic toggle=\"yes\">Rv0066c<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">Rv3139<\/jats:italic>\n                      ) that displayed the most significant intracellular survival defects to generate large-fragment knockout strains (\u0394\n                      <jats:italic toggle=\"yes\">Rv0066c<\/jats:italic>\n                      and \u0394\n                      <jats:italic toggle=\"yes\">Rv3139<\/jats:italic>\n                      ). Macrophage infection experiments reconfirmed the compromised intracellular viability of both mutants. RNA-seq profiling of \u0394\n                      <jats:italic toggle=\"yes\">Rv0066c<\/jats:italic>\n                      -infected macrophages identified 138 differentially expressed genes, with functional enrichment in chemokine signaling, Ras protein signal transduction, and calcineurin-mediated signaling. To conclude, this study identified two novel\n                      <jats:italic toggle=\"yes\">M.tb<\/jats:italic>\n                      effectors contributing to intracellular survival as potential new targets for anti-TB drug development.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.00767-25","type":"journal-article","created":{"date-parts":[[2025,6,12]],"date-time":"2025-06-12T08:27:18Z","timestamp":1749716838000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":5,"title":["Identification of\n                    <i>Mycobacterium tuberculosis<\/i>\n                    intracellular survival-related virulence factors via CRISPR-based eukaryotic-like secretory protein mutant library screen"],"prefix":"10.1128","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3470-0948","authenticated-orcid":true,"given":"Weiyi","family":"Liu","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01espdw89","id-type":"ROR","asserted-by":"publisher"}],"name":"Beijing Key Laboratory for Drug Resistance Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yingchao","family":"Wang","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01espdw89","id-type":"ROR","asserted-by":"publisher"}],"name":"Beijing Key Laboratory for Drug Resistance Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiayuan","family":"Zhao","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01espdw89","id-type":"ROR","asserted-by":"publisher"}],"name":"Beijing Key Laboratory for Drug Resistance Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinyue","family":"Zhang","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01espdw89","id-type":"ROR","asserted-by":"publisher"}],"name":"Beijing Key Laboratory for Drug Resistance Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zihui","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongyan","family":"Jia","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01espdw89","id-type":"ROR","asserted-by":"publisher"}],"name":"Beijing Key Laboratory for Drug Resistance Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-2166-0331","authenticated-orcid":true,"given":"Chuanzhi","family":"Zhu","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01espdw89","id-type":"ROR","asserted-by":"publisher"}],"name":"Beijing Key Laboratory for Drug Resistance Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8267-2059","authenticated-orcid":true,"given":"Lanyue","family":"Zhang","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01espdw89","id-type":"ROR","asserted-by":"publisher"}],"name":"Beijing Key Laboratory for Drug Resistance Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6172-3630","authenticated-orcid":false,"given":"Liping","family":"Pan","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01espdw89","id-type":"ROR","asserted-by":"publisher"}],"name":"Beijing Key Laboratory for Drug Resistance Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0857-9793","authenticated-orcid":false,"given":"Zongde","family":"Zhang","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01espdw89","id-type":"ROR","asserted-by":"publisher"}],"name":"Beijing Key Laboratory for Drug Resistance Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.molimm.2020.08.005"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1038\/nature12799"},{"key":"e_1_3_4_4_2","doi-asserted-by":"publisher","DOI":"10.1093\/abbs\/gmx080"},{"key":"e_1_3_4_5_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.abq0132"},{"key":"e_1_3_4_6_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cellimm.2018.10.009"},{"key":"e_1_3_4_7_2","doi-asserted-by":"publisher","DOI":"10.1111\/febs.16369"},{"key":"e_1_3_4_8_2","doi-asserted-by":"publisher","DOI":"10.1007\/s00294-016-0664-5"},{"key":"e_1_3_4_9_2","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms9922"},{"key":"e_1_3_4_10_2","doi-asserted-by":"publisher","DOI":"10.1038\/srep34827"},{"key":"e_1_3_4_11_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1120251109"},{"key":"e_1_3_4_12_2","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.02364-19"},{"key":"e_1_3_4_13_2","doi-asserted-by":"publisher","DOI":"10.1128\/mSystems.00070-19"},{"key":"e_1_3_4_14_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/28.1.27"},{"key":"e_1_3_4_15_2","doi-asserted-by":"publisher","DOI":"10.1084\/jem.20181776"},{"key":"e_1_3_4_16_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41423-020-0502-z"},{"key":"e_1_3_4_17_2","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.02133-16"},{"key":"e_1_3_4_18_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.aav5912"},{"key":"e_1_3_4_19_2","doi-asserted-by":"publisher","DOI":"10.1111\/mmi.12981"},{"key":"e_1_3_4_20_2","doi-asserted-by":"publisher","DOI":"10.3892\/mmr.2013.1687"},{"key":"e_1_3_4_21_2","doi-asserted-by":"publisher","DOI":"10.1107\/S1744309111008189"},{"key":"e_1_3_4_22_2","doi-asserted-by":"publisher","DOI":"10.1080\/22221751.2020.1861913"},{"key":"e_1_3_4_23_2","doi-asserted-by":"publisher","DOI":"10.1099\/00221287-147-8-2307"},{"key":"e_1_3_4_24_2","doi-asserted-by":"publisher","DOI":"10.1021\/acsmedchemlett.5b00373"},{"key":"e_1_3_4_25_2","doi-asserted-by":"publisher","DOI":"10.2147\/DDDT.S111443"},{"key":"e_1_3_4_26_2","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2018.00171"},{"key":"e_1_3_4_27_2","doi-asserted-by":"publisher","DOI":"10.1091\/mbc.e04-04-0329"},{"key":"e_1_3_4_28_2","doi-asserted-by":"publisher","DOI":"10.1002\/pmic.201100043"},{"key":"e_1_3_4_29_2","doi-asserted-by":"publisher","DOI":"10.1101\/cshperspect.a021121"},{"key":"e_1_3_4_30_2","doi-asserted-by":"publisher","DOI":"10.1038\/31159"},{"key":"e_1_3_4_31_2","doi-asserted-by":"publisher","DOI":"10.1021\/bi400037w"},{"key":"e_1_3_4_32_2","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2091-6-20"},{"key":"e_1_3_4_33_2","doi-asserted-by":"publisher","DOI":"10.3390\/pathogens12091109"},{"key":"e_1_3_4_34_2","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.169.9.5181"},{"key":"e_1_3_4_35_2","doi-asserted-by":"publisher","DOI":"10.1101\/cshperspect.a022459"},{"key":"e_1_3_4_36_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0018367"},{"key":"e_1_3_4_37_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M114.582502"},{"key":"e_1_3_4_38_2","doi-asserted-by":"publisher","DOI":"10.1128\/JB.00502-13"},{"key":"e_1_3_4_39_2","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2024.1402024"},{"key":"e_1_3_4_40_2","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.102980"},{"key":"e_1_3_4_41_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-024-07605-8"},{"key":"e_1_3_4_42_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.chembiol.2012.11.008"},{"key":"e_1_3_4_43_2","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2017.016"},{"key":"e_1_3_4_44_2","doi-asserted-by":"publisher","DOI":"10.1186\/s13059-014-0554-4"}],"container-title":["Microbiology Spectrum"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/spectrum.00767-25","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/spectrum.00767-25","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,8,5]],"date-time":"2025-08-05T13:05:41Z","timestamp":1754399141000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.asm.org\/doi\/10.1128\/spectrum.00767-25"}},"subtitle":[],"editor":[{"given":"Xinchun","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2025,8,5]]},"references-count":43,"journal-issue":{"issue":"8","published-print":{"date-parts":[[2025,8,5]]}},"alternative-id":["10.1128\/spectrum.00767-25"],"URL":"https:\/\/doi.org\/10.1128\/spectrum.00767-25","relation":{},"ISSN":["2165-0497"],"issn-type":[{"value":"2165-0497","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,8,5]]},"assertion":[{"value":"2025-03-17","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-05-17","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-06-12","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e00767-25"}}