biostudies-literatureDaher WFondation pour la Recherche Médicale (Foundation for Medical Research in France)Agence Nationale de la Recherche (French National Research Agency)Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Santé du Canada)166https://www.ebi.ac.uk/biostudies/studies/S-EPMC11791044biostudies-literatureUnknown8(1)Type VII secretion systems participate in protein export, virulence, conjugation, and metabolic regulation. Five subtypes (ESX-1 to ESX-5) exist, each with specific roles and well-characterized secretion profiles in various mycobacterial species. Mycobacterium abscessus, encodes only ESX-3 and ESX-4. Here, single and double M. abscessus mutants lacking the main ATPases EccC3 and EccC4 were used to define ESX-3 and ESX-4 contributions to substrate secretion and virulence. Our results demonstrate that EsxG/H secretion depends entirely on ESX-3, whereas both ESX-3 and ESX-4 secrete EsxU/T. Furthermore, two newly identified PE/PPE substrates (MAB_0046/MAB_0047) require ESX-3 for secretion. Functional complementation restored secretion and revealed subpolar localization of these systems. MacropCommunications biologyDeletion of ESX-3 and ESX-4 secretion systems in Mycobacterium abscessus results in highly impaired pathogenicity.PMC1179104419-CE15-0012-01; SUNLIVEAntimicrobial Resistance 202309ARBEquipe FRM EQU202103012588Parmar SLe Moigne VTocheva EITasrini YSexton DLHerrmann JLDaher WBerdal VAguilera-Correa JJKremer LfalseDeletion of ESX-3 and ESX-4 secretion systems in Mycobacterium abscessus results in highly impaired pathogenicity.Type VII secretion systems participate in protein export, virulence, conjugation, and metabolic regulation. Five subtypes (ESX-1 to ESX-5) exist, each with specific roles and well-characterized secretion profiles in various mycobacterial species. Mycobacterium abscessus, encodes only ESX-3 and ESX-4. Here, single and double M. abscessus mutants lacking the main ATPases EccC3 and EccC4 were used to define ESX-3 and ESX-4 contributions to substrate secretion and virulence. Our results demonstrate that EsxG/H secretion depends entirely on ESX-3, whereas both ESX-3 and ESX-4 secrete EsxU/T. Furthermore, two newly identified PE/PPE substrates (MAB_0046/MAB_0047) require ESX-3 for secretion. Functional complementation restored secretion and revealed subpolar localization of these systems. Macrop2025-01-01T00:00:00Z2025 Feb2026-05-29T16:51:41.505Z2025-04-04T00:51:48.131ZS-EPMC117910443990063110.1038/s42003-025-07572-4