title: Further characterization of a highly attenuated Yersinia pestis CO92 mutant deleted for the genes encoding Braun lipoprotein and plasminogen activator protease in murine alveolar and primary human macrophages.
abstract: We recently characterized the Δlpp Δpla double in-frame deletion mutant of Yersinia pestis CO92 molecularly, biologically, and immunologically. While Braun lipoprotein (Lpp) activates toll-like receptor-2 to initiate an inflammatory cascade, plasminogen activator (Pla) protease facilitates bacterial dissemination in the host. The Δlpp Δpla double mutant was highly attenuated in evoking bubonic and pneumonic plague, was rapidly cleared from mouse organs, and generated humoral and cell-mediated immune responses to provide subsequent protection to mice against a lethal challenge dose of wild-type (WT) CO92. Here, we further characterized the Δlpp Δpla double mutant in two murine macrophage cell lines as well as in primary human monocyte-derived macrophages to gauge its potential as a live-attenuated vaccine candidate. We first demonstrated that the Δpla single and the Δlpp Δpla double mutant were unable to survive efficiently in murine and human macrophages, unlike WT CO92. We observed that the levels of Pla and its associated protease activity were not affected in the Δlpp single mutant, and, likewise, deletion of the pla gene from WT CO92 did not alter Lpp levels. Further, our study revealed that both Lpp and Pla contributed to the intracellular survival of WT CO92 via different mechanisms. Importantly, the ability of the Δlpp Δpla double mutant to be phagocytized by macrophages, to stimulate production of tumor necrosis factor-α and interleukin-6, and to activate the nitric oxide killing pathways of the host cells remained unaltered when compared to the WT CO92-infected macrophages. Finally, macrophages infected with either the WT CO92 or the Δlpp Δpla double mutant were equally efficient in their uptake of zymosan particles as determined by flow cytometric analysis. Overall, our data indicated that although the Δlpp Δpla double mutant of Y. pestis CO92 was highly attenuated, it retained the ability to elicit innate and subsequent acquired immune responses in the host similar to that of WT CO92, which are highly desirable in a live-attenuated vaccine candidate.
authors.affiliation: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, 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, TX 77555, USA.
authors.forename: Bethany L
authors.initials: BL
authors.lastname: Tiner
authors.affiliation: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, USA.
authors.forename: Sadhana
authors.initials: S
authors.lastname: Chauhan
authors.affiliation: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, USA; Sealy Center for Vaccine Development, University of Texas Medical Branch, Galveston, TX 77555, USA; Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, TX 77555, USA; Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX 77555, USA; Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555, USA.
authors.forename: Vladimir L
authors.initials: VL
authors.lastname: Motin
authors.affiliation: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, USA.
authors.forename: Eric C
authors.initials: EC
authors.lastname: Fitts
authors.affiliation: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, USA.
authors.forename: Matthew B
authors.initials: MB
authors.lastname: Huante
authors.affiliation: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, USA; Sealy Center for Vaccine Development, University of Texas Medical Branch, Galveston, TX 77555, USA; Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX 77555, USA.
authors.forename: Janice J
authors.initials: JJ
authors.lastname: Endsley
authors.affiliation: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, USA.
authors.forename: Duraisamy
authors.initials: D
authors.lastname: Ponnusamy
authors.affiliation: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, USA; Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX 77555, USA. Electronic address: jisha@utmb.edu.
authors.forename: Jian
authors.initials: J
authors.lastname: Sha
authors.affiliation: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, USA; Sealy Center for Vaccine Development, University of Texas Medical Branch, Galveston, TX 77555, USA; Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, TX 77555, USA; Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX 77555, USA. Electronic address: achopra@utmb.edu.
authors.forename: Ashok K
authors.initials: AK
authors.lastname: Chopra
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D017353:Gene Deletion; D006801:Humans; D007113:Immunity, Innate; D008074:Lipoproteins / Q000172:deficiency*; D008264:Macrophages / Q000382:microbiology*; D016676:Macrophages, Alveolar / Q000382:microbiology*; D051379:Mice; D050296:Microbial Viability; D010447:Peptide Hydrolases / Q000172:deficiency*; D010931:Plague Vaccine; D010960:Plasminogen Activators / Q000172:deficiency*; D014613:Vaccines, Attenuated; D014774:Virulence; D015010:Yersinia pestis / Q000235:genetics / Q000254:growth & development*
chemical_list: D008074:Lipoproteins; D010931:Plague Vaccine; D014613:Vaccines, Attenuated; D010447:Peptide Hydrolases; D010960:Plasminogen Activators
keywords: Alveolar macrophages; Braun lipoprotein; Human monocyte-derived macrophages; Innate immunity; Intracellular survival; Plasminogen activator protease; Yersinia pestis