title: Combinational deletion of three membrane protein-encoding genes highly attenuates yersinia pestis while retaining immunogenicity in a mouse model of pneumonic plague.
abstract: Previously, we showed that deletion of genes encoding Braun lipoprotein (Lpp) and MsbB attenuated Yersinia pestis CO92 in mouse and rat models of bubonic and pneumonic plague. While Lpp activates Toll-like receptor 2, the MsbB acyltransferase modifies lipopolysaccharide. Here, we deleted the ail gene (encoding the attachment-invasion locus) from wild-type (WT) strain CO92 or its lpp single and Δlpp ΔmsbB double mutants. While the Δail single mutant was minimally attenuated compared to the WT bacterium in a mouse model of pneumonic plague, the Δlpp Δail double mutant and the Δlpp ΔmsbB Δail triple mutant were increasingly attenuated, with the latter being unable to kill mice at a 50% lethal dose (LD50) equivalent to 6,800 LD50s of WT CO92. The mutant-infected animals developed balanced TH1- and TH2-based immune responses based on antibody isotyping. The triple mutant was cleared from mouse organs rapidly, with concurrent decreases in the production of various cytokines and histopathological lesions. When surviving animals infected with increasing doses of the triple mutant were subsequently challenged on day 24 with the bioluminescent WT CO92 strain (20 to 28 LD50s), 40 to 70% of the mice survived, with efficient clearing of the invading pathogen, as visualized in real time by in vivo imaging. The rapid clearance of the triple mutant, compared to that of WT CO92, from animals was related to the decreased adherence and invasion of human-derived HeLa and A549 alveolar epithelial cells and to its inability to survive intracellularly in these cells as well as in MH-S murine alveolar and primary human macrophages. An early burst of cytokine production in macrophages elicited by the triple mutant compared to WT CO92 and the mutant's sensitivity to the bactericidal effect of human serum would further augment bacterial clearance. Together, deletion of the ail gene from the Δlpp ΔmsbB double mutant severely attenuated Y. pestis CO92 to evoke pneumonic plague in a mouse model while retaining the required immunogenicity needed for subsequent protection against infection.
authors.affiliation: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, USA.
authors.forename: Bethany L
authors.initials: BL
authors.lastname: Tiner
authors.affiliation: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, USA Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, Texas, USA jisha@utmb.edu achopra@utmb.edu.
authors.forename: Jian
authors.initials: J
authors.lastname: Sha
authors.affiliation: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, USA.
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Kirtley
authors.affiliation: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, USA.
authors.forename: Tatiana E
authors.initials: TE
authors.lastname: Erova
authors.affiliation: Department of Pathology, University of Texas Medical Branch, Galveston, Texas, USA.
authors.forename: Vsevolod L
authors.initials: VL
authors.lastname: Popov
authors.affiliation: Department of Veterinary Sciences, University of Texas M. D. Anderson Cancer Center, Bastrop, Texas, USA.
authors.forename: Wallace B
authors.initials: WB
authors.lastname: Baze
authors.affiliation: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, USA.
authors.forename: Christina J
authors.initials: CJ
authors.lastname: van Lier
authors.affiliation: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, USA.
authors.forename: Duraisamy
authors.initials: D
authors.lastname: Ponnusamy
authors.affiliation: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, USA.
authors.forename: Jourdan A
authors.initials: JA
authors.lastname: Andersson
authors.affiliation: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, USA Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, Texas, USA Department of Pathology, University of Texas Medical Branch, Galveston, Texas, USA Center for Vaccine Development, University of Texas Medical Branch, Galveston, Texas, USA Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, Texas, USA.
authors.forename: Vladimir L
authors.initials: VL
authors.lastname: Motin
authors.affiliation: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, USA.
authors.forename: Sadhana
authors.initials: S
authors.lastname: Chauhan
authors.affiliation: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, USA Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, Texas, USA Center for Vaccine Development, University of Texas Medical Branch, Galveston, Texas, USA Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, Texas, USA jisha@utmb.edu achopra@utmb.edu.
authors.forename: Ashok K
authors.initials: AK
authors.lastname: Chopra
mesh_terms: D000217:Acyltransferases / Q000235:genetics*; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000907:Antibodies, Bacterial / Q000276:immunology; D001422:Bacterial Adhesion / Q000235:genetics / Q000276:immunology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D002460:Cell Line; D004195:Disease Models, Animal; D024881:Drug Resistance, Bacterial / Q000235:genetics; D005260:Female; D017353:Gene Deletion; D005839:Gentamicins / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D042541:Intracellular Space / Q000382:microbiology; D008074:Lipoproteins / Q000235:genetics*; D008264:Macrophages / Q000276:immunology / Q000382:microbiology; D051379:Mice; D010930:Plague / Q000276:immunology* / Q000473:pathology; D037521:Virulence Factors / Q000235:genetics*; D015010:Yersinia pestis / Q000235:genetics / Q000276:immunology / Q000472:pathogenicity*
chemical_list: C526543:Ail protein, Yersinia pestis; D000900:Anti-Bacterial Agents; D000907:Antibodies, Bacterial; D001425:Bacterial Outer Membrane Proteins; D005839:Gentamicins; D008074:Lipoproteins; D037521:Virulence Factors; D000217:Acyltransferases