{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,17]],"date-time":"2026-07-17T06:08:22Z","timestamp":1784268502219,"version":"3.55.0"},"reference-count":43,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,7,17]],"date-time":"2026-07-17T00:00:00Z","timestamp":1784246400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"\n Background<\/jats:title>\n \n Listeria monocytogenes<\/jats:italic>\n (\n L. monocytogenes<\/jats:italic>\n ) is a facultative intracellular bacterium and the etiological agent of listeriosis, an invasive infection that disproportionately affects neonates, older adults, pregnant women, and immunocompromised individuals. Among the major virulence determinants of\n L. monocytogenes<\/jats:italic>\n , listeriolysin O (LLO) and the pathogenicity island LIPI-4 have been implicated in intracellular survival and tissue tropism. Here, we examined whether LLO contributes to LIPI-4-associated pathogenicity. While both LIPI-4 and LLO are known to be important for\n L. monocytogenes<\/jats:italic>\n virulence, it remains to be determined whether LIPI-4 promotes infection by modulating LLO expression and secretion.\n <\/jats:p>\n <\/jats:sec>\n \n Methods<\/jats:title>\n \n Using the frozen raw chicken isolate LM928 as the parental strain, we constructed an\n hly<\/jats:italic>\n deletion mutant (LM928\u0394\n hly<\/jats:italic>\n ), an\n hly<\/jats:italic>\n \/LIPI-4 double-deletion mutant (LM928\u0394LIPI-4\/\n hly<\/jats:italic>\n ), and the corresponding complemented strains via homologous recombination. Genetic stability was evaluated by serial passaging followed by PCR confirmation. LLO expression was assessed by Western blotting, and bacterial growth kinetics and hemolytic activity were examined\n in vitro<\/jats:italic>\n . Adhesion, invasion, and intracellular replication were assessed in human cerebral microvascular endothelial cells (HCMEC\/D3) and placental trophoblast cells (HTR-8).\n In vivo<\/jats:italic>\n virulence was determined by quantifying bacterial burdens in the brain and spleen of infected mice.\n <\/jats:p>\n <\/jats:sec>\n \n Results<\/jats:title>\n \n All mutant and complemented strains remained genetically stable, and deletion of\n hly<\/jats:italic>\n or LIPI-4 had no measurable effect on bacterial growth\n in vitro<\/jats:italic>\n . LLO was not detected in the\n hly<\/jats:italic>\n deletion mutant or in the\n hly<\/jats:italic>\n \/LIPI-4 double-deletion mutant. In contrast, the LIPI-4 deletion mutant retained hemolytic activity, although at a reduced level compared with the wild-type strain. In cell infection assays, deletion of LIPI-4 resulted in reduced adhesion, invasion, and intracellular replication in both HCMEC\/D3 and HTR-8 cells, and these impairments were greater than those observed for the\n hly<\/jats:italic>\n mutant. Among all strains tested, the double-deletion mutant showed the most pronounced defects across the\n in vitro<\/jats:italic>\n assays. In a mouse infection model, deletion of either\n hly<\/jats:italic>\n or LIPI-4 reduced bacterial burdens in the brain and spleen, and the lowest burdens were observed in animals infected with the double-deletion strain.\n <\/jats:p>\n <\/jats:sec>\n \n Conclusion<\/jats:title>\n \n Our results indicate that\n hly<\/jats:italic>\n and LIPI-4 act together to promote\n L. monocytogenes<\/jats:italic>\n virulence, with LIPI-4 having a stronger impact on host cell interaction and intracellular persistence. These data support a coordinated role for LLO and LIPI-4 during systemic infection and provide insight into mechanisms that may contribute to tissue tropism and barrier crossing by\n L. monocytogenes<\/jats:italic>\n .\n <\/jats:p>\n <\/jats:sec>","DOI":"10.3389\/fmicb.2026.1853120","type":"journal-article","created":{"date-parts":[[2026,7,17]],"date-time":"2026-07-17T05:43:25Z","timestamp":1784267005000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Functional cooperation between LIPI-4 and listeriolysin O governs intracellular replication and systemic virulence of Listeria monocytogenes"],"prefix":"10.3389","volume":"17","author":[{"given":"Zhongke","family":"Yin","sequence":"first","affiliation":[{"name":"College of Animal Science and Technology, Shihezi University","place":["Shihezi, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhijie","family":"Luo","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Shihezi University","place":["Shihezi, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xun","family":"Ma","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Shihezi University","place":["Shihezi, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Caixia","family":"Liu","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Shihezi University","place":["Shihezi, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yatao","family":"Qi","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Shihezi University","place":["Shihezi, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lu","family":"Liu","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Shihezi University","place":["Shihezi, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruixuan","family":"Qian","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Shihezi University","place":["Shihezi, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jing","family":"Wang","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Shihezi University","place":["Shihezi, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guangdong","family":"Hu","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Shihezi University","place":["Shihezi, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,7,17]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"5574","DOI":"10.3390\/ijms26125574","article-title":"Metabolic rewiring of bacterial pathogens in response to antibiotic pressure\u2014a molecular perspective","volume":"26","author":"Acierno","year":"2025","journal-title":"Int. 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