title: Promotion and Rescue of Intracellular Brucella neotomae Replication during Coinfection with Legionella pneumophila.
abstract: We established a newBrucella neotomaein vitromodel system for study of type IV secretion system-dependent (T4SS) pathogenesis in theBrucellagenus. Importantly,B. neotomaeis a rodent pathogen, and unlikeB. abortus,B. melitensis, andB. suis,B. neotomaehas not been observed to infect humans. It therefore can be handled more facilely using biosafety level 2 practices. More particularly, using a series of novel fluorescent protein andluxoperon reporter systems to differentially label pathogens and track intracellular replication, we confirmed T4SS-dependent intracellular growth ofB. neotomaein macrophage cell lines. Furthermore,B. neotomaeexhibited early endosomal (LAMP-1) and late endoplasmic reticulum (calreticulin)-associated phagosome maturation. These findings recapitulate prior observations for human-pathogenicBrucellaspp. In addition, during coinfection experiments withLegionella pneumophila, we found that defective intracellular replication of aB. neotomaeT4SSvirB4mutant was rescued and baseline levels of intracellular replication of wild-typeB. neotomaewere significantly stimulated by coinfection with wild-type but not T4SS mutantL. pneumophilaUsing confocal microscopy, it was determined that intracellular colocalization ofB. neotomaeandL. pneumophilawas required for rescue and that colocalization came at a cost toL. pneumophilafitness. These findings were not completely expected based on known temporal and qualitative differences in the intracellular life cycles of these two pathogens. Taken together, we have developed a new system for studyingin vitroBrucellapathogenesis and found a remarkable T4SS-dependent interplay betweenBrucellaandLegionelladuring macrophage coinfection.
authors.affiliation: Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Yoon-Suk
authors.initials: YS
authors.lastname: Kang
authors.affiliation: Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA jekirby@bidmc.harvard.edu.
authors.forename: James E
authors.initials: JE
authors.lastname: Kirby
mesh_terms: D000818:Animals; D002002:Brucella / Q000254:growth & development*; D002460:Cell Line; D060085:Coinfection; D006801:Humans; D016952:Legionella pneumophila / Q000254:growth & development*; D008264:Macrophages / Q000382:microbiology*; D051379:Mice; D056265:Microbial Interactions; D018613:Microscopy, Confocal; D010588:Phagosomes / Q000382:microbiology
keywords: Brucella; Legionella pneumophila; coinfection; fluorescent image analysis; intracellular pathogens; pathogenesis; phagosomes; reporter genes; type IV secretion system