title: High-throughput assay to phenotype Salmonella enterica Typhimurium association, invasion, and replication in macrophages.
abstract: Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock. Understanding the mechanisms underlying Salmonella-host interactions are important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.
authors.affiliation: Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University.
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
authors.affiliation: Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University.
authors.forename: Roberta
authors.initials: R
authors.lastname: Pugh
authors.affiliation: Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University.
authors.forename: Richard C
authors.initials: RC
authors.lastname: Laughlin
authors.affiliation: Department of Microbial and Molecular Pathogenesis, College of Medicine, Texas A&M University System Health Science Center.
authors.forename: Helene
authors.initials: H
authors.lastname: Andrews-Polymenis
authors.affiliation: University of California, Irvine.
authors.forename: Michael
authors.initials: M
authors.lastname: McClelland
authors.affiliation: Department of Medical Microbiology & Immunology, School of Medicine, University of California, Davis.
authors.forename: Andreas J
authors.initials: AJ
authors.lastname: Bäumler
authors.affiliation: Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University; GADAMS@cvm.tamu.edu.
authors.forename: L Garry
authors.initials: LG
authors.lastname: Adams
mesh_terms: D000818:Animals; D055786:Gene Knockout Techniques; D057166:High-Throughput Screening Assays / Q000379:methods*; D054884:Host-Pathogen Interactions; D008264:Macrophages / Q000382:microbiology*; D051379:Mice; D012481:Salmonella Infections, Animal / Q000382:microbiology; D012486:Salmonella typhimurium / Q000235:genetics / Q000254:growth & development / Q000502:physiology*