biostudies-literatureRoberts JRUniversity of MichiganNational Institutes of Health Office of Research Infrastructure ProgramsNational Institute of Allergy and Infectious Diseases Division of Microbiology and Infectious DiseasesNational Science Foundation Division of Graduate EducationNIAID NIH HHSNational Institutes of HealthNational Institute of General Medical SciencesNIH HHSArnold and Mabel Beckman FoundationNIGMS NIH HHSNational Science FoundationLife Sciences Institute169310https://www.ebi.ac.uk/biostudies/studies/S-EPMC12716638biostudies-literatureUnknown437(19)Legionella pneumophila is a pathogenic Gram-negative bacterium that causes Legionnaires' disease. The main virulence factor of L. pneumophila is the Dot/Icm Type IV Secretion System (T4SS), which translocates effector proteins into the cytoplasm of the host cell, allowing the bacterium to establish a replicative niche. The outer membrane core complex (OMCC), the T4SS machinery localized between the inner and outer membranes, is composed of at least nine proteins organized into various sub-complexes that include the dome, outer membrane cap (OMC), periplasmic ring (PR), and stalk. In this study we describe how two uncharacterized Dot/Icm T4SS components, Dis2 and Dis3, contribute to the structure of the T4SS, the ability of the T4SS to translocate effectors, and the pathogenicity of L. pneumophila. Using cryo-electron microscopy we show that OMCCs purified from a Δdis2 strain are only missing the density for Dis2, while OMCCs purified from the Δdis3 strain lack densities for Dis3 and DotF in the OMC. Despite missing these proteins, the OMC and PR of both mutant OMCCs remain structurally stable. Strains lacking dis2 and or dis3 efficiently replicate in human macrophages; however, they have subtle differences in translocation efficiency for four tested substrates. Combined these data indicate that Dis2 or Dis3 are not required for the stability or global organization of the OMCC, but each protein may contribute to the efficient translocation of specific effectors.Journal of molecular biologyThe Legionella pneumophila Dot/Icm Type IV Secretion System is Structurally and Functionally Resilient in Absence of Species-specific Proteins Dis2 and Dis3.PMC12716638S10 OD030275DGE 2241144R01 AI118932R21AI118932R21 AI164651F32AI1500271R24GM154186R24 GM154186Frick-Cheng AEChang LStyron HJDurie CLRoberts JROhi MDfalseThe Legionella pneumophila Dot/Icm Type IV Secretion System is Structurally and Functionally Resilient in Absence of Species-specific Proteins Dis2 and Dis3.Legionella pneumophila is a pathogenic Gram-negative bacterium that causes Legionnaires' disease. The main virulence factor of L. pneumophila is the Dot/Icm Type IV Secretion System (T4SS), which translocates effector proteins into the cytoplasm of the host cell, allowing the bacterium to establish a replicative niche. The outer membrane core complex (OMCC), the T4SS machinery localized between the inner and outer membranes, is composed of at least nine proteins organized into various sub-complexes that include the dome, outer membrane cap (OMC), periplasmic ring (PR), and stalk. In this study we describe how two uncharacterized Dot/Icm T4SS components, Dis2 and Dis3, contribute to the structure of the T4SS, the ability of the T4SS to translocate effectors, and the pathogenicity of L. pneumophila. Using cryo-electron microscopy we show that OMCCs purified from a Δdis2 strain are only missing the density for Dis2, while OMCCs purified from the Δdis3 strain lack densities for Dis3 and DotF in the OMC. Despite missing these proteins, the OMC and PR of both mutant OMCCs remain structurally stable. Strains lacking dis2 and or dis3 efficiently replicate in human macrophages; however, they have subtle differences in translocation efficiency for four tested substrates. Combined these data indicate that Dis2 or Dis3 are not required for the stability or global organization of the OMCC, but each protein may contribute to the efficient translocation of specific effectors.2025-01-01T00:00:00Z2025 Oct2026-06-06T05:25:47.48Z2026-05-26T03:12:23.066ZS-EPMC127166384058109210.1016/j.jmb.2025.169310