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This study explores host-targeted therapies by focusing on deubiquitinating enzymes (DUBs), key regulators of the ubiquitin-proteasome system (UPS) that mediate host-pathogen interactions. Using\n                      <jats:italic toggle=\"yes\">Salmonella<\/jats:italic>\n                      -infected macrophages, we screened a UPS-targeted compound library and identified several compounds that enhanced bacterial clearance without affecting host cell viability. Among these, the dual USP25\/USP28 inhibitor AZ-1 emerged as one of the top candidates. Transcriptomic profiling revealed infection-induced upregulation of DUBs, particularly USP25, USP46, and OTUD7B. USP25 knockdown significantly reduced intracellular\n                      <jats:italic toggle=\"yes\">Salmonella<\/jats:italic>\n                      , confirming its role as a critical host factor. AZ-1 also exhibited broad-spectrum intracellular activity against multidrug-resistant\n                      <jats:italic toggle=\"yes\">Pseudomonas aeruginosa<\/jats:italic>\n                      ,\n                      <jats:italic toggle=\"yes\">Klebsiella pneumoniae<\/jats:italic>\n                      , and\n                      <jats:italic toggle=\"yes\">Acinetobacter baumannii<\/jats:italic>\n                      .\n                      <jats:italic toggle=\"yes\">In vivo<\/jats:italic>\n                      , AZ-1 reduced fecal bacterial loads, clinical scores, and infection-induced weight loss, though it did not extend survival. AZ-1 had no direct antibacterial activity in axenic culture, indicating a host-targeting mechanism. Transcriptomic and signaling analyses revealed AZ-1 suppressed key immune pathways, including nuclear factor-kappa B (NF-\u03baB) signaling. These findings establish DUBs as promising targets for host-directed therapies and support further development of UPS-targeted agents to combat antimicrobial resistance.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Antibiotic-resistant infections, particularly those caused by intracellular pathogens, represent an urgent public health threat due to their ability to evade immune responses and resist conventional antibiotics. This study identifies the ubiquitin-proteasome system, specifically deubiquitinating enzymes, as viable targets for host-directed therapy. We demonstrate that the USP25\/USP28 inhibitor AZ-1 enhances intracellular bacterial clearance without compromising host cell viability and is effective against several multidrug-resistant gram-negative pathogens. Knockdown of USP25 alone also reduced intracellular\n                        <jats:italic toggle=\"yes\">Salmonella<\/jats:italic>\n                        , stressing out its proposed role in bacterial persistence. AZ-1 improved early infection outcomes\n                        <jats:italic toggle=\"yes\">in vivo<\/jats:italic>\n                        but was insufficient as monotherapy. These findings support a novel therapeutic approach that targets host pathways to enhance bacterial clearance, offering a promising adjunct to traditional antibiotics in the fight against antimicrobial resistance.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/mbio.00312-25","type":"journal-article","created":{"date-parts":[[2025,9,3]],"date-time":"2025-09-03T13:01:22Z","timestamp":1756904482000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":2,"title":["Targeting deubiquitinating enzymes and ubiquitin pathway modulators to enhance host defense against bacterial infections"],"prefix":"10.1128","volume":"16","author":[{"given":"John","family":"Santelices","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02y3ad647","id-type":"ROR","asserted-by":"publisher"}],"name":"Microbiology and Cell Science Department, Institute of Food and 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