Braun Lipoprotein (Lpp) Contributes to Virulence of Yersiniae: Potential Role of Lpp in Inducing Bubonic and Pneumonic Plague

Abstract

Yersinia pestis evolved from Y. pseudotuberculosis to become the causative agent of bubonic and pneumonic plague. We identified a homolog of the Salmonella enterica serovar Typhimurium lipoprotein (lpp) gene in Yersinia species and prepared lpp gene deletion mutants of Y. pseudotuberculosis YPIII, Y. pestis KIM/D27 (pigmentation locus minus), and Y. pestis CO92 with reduced virulence. Mice injected via the intraperitoneal route with 5 ؋ 10 7 CFU of the ⌬lpp KIM/D27 mutant survived a month, even though this would have constituted a lethal dose for the parental KIM/D27 strain. Subsequently, these ⌬lpp KIM/D27-injected mice were solidly protected against an intranasally administered, highly virulent Y. pestis CO92 strain when it was given as five 50% lethal doses (LD 50 ). In a parallel study with the pneumonic plague mouse model, after 72 h postinfection, the lungs of animals infected with wild-type (WT) Y. pestis CO92 and given a subinhibitory dose of levofloxacin had acute inflammation, edema, and masses of bacteria, while the lung tissue appeared essentially normal in mice inoculated with the ⌬lpp mutant of CO92 and given the same dose of levofloxacin. Importantly, while WT Y. pestis CO92 could be detected in the bloodstreams and spleens of infected mice at 72 h postinfection, the ⌬lpp mutant of CO92 could not be detected in those organs. Furthermore, the levels of cytokines/chemokines detected in the sera were significantly lower in animals infected with the ⌬lpp mutant than in those infected with WT CO92. Additionally, the ⌬lpp mutant was more rapidly killed by macrophages than was the WT CO92 strain. These data provided evidence that the ⌬lpp mutants of yersiniae were significantly attenuated and could be useful tools in the development of new vaccines.

Characterization of an F1 Deletion Mutant of Yersinia pestis CO92, Pathogenic Role of F1 Antigen in Bubonic and Pneumonic Plague, and Evaluation of Sensitivity and Specificity of F1 Antigen Capture-Based Dipsticks

“…The WT Y. pestis CO92 strain is highly virulent and was first isolated in 1992 from a deceased pneumonic plague patient (14,26,40). It was acquired from the Centers for Disease Control and Prevention (CDC), Atlanta, GA. Y. pseudotuberculosis YPIII was purchased from the American Type Culture Collection (ATCC), Manassas, VA, and also was used as a negative control in this study in addition to the ⌬caf mutant.…”

“…For Western blot analysis, the bacterial cells were lysed in SDS-PAGE sample buffer and probed with the primary anti-F1 antigen antibodies (generated in the laboratory) at a dilution of 1:2,500, followed by horseradish peroxidase (HRP)-labeled, goat anti-mouse secondary antibodies (Southern Biotech, Birmingham, Alabama) at a dilution of 1:20,000. The blots were developed by using a SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL) (40). Similarly, the production of V antigen (LcrV), a component of the type III secretion system (T3SS) (19), in the WT and ⌬caf mutant of CO92 was analyzed by using antibodies to LcrV (generated in the laboratory) at a dilution of 1:2,500.…”

“…Female 5-to 6-week-old BALB/c mice were purchased from Jackson Laboratories (Bar Harbor, ME) and infected in the ABSL-3 facility either by the i.n. or s.c. route under an approved UTMB IACUC protocol to mimic pneumonic and bubonic plague, respectively (40). The animals were infected with either WT CO92 or its ⌬caf mutant and monitored for up to 30 to 35 days for mortality.…”

“…Samples from each flask were taken at 1-h intervals until the cultures reached their plateau phase. The OD 600 s at different time points were recorded and the CFU determined by plating, as we described previously (40).…”

Recent Advancement in the Development of Vaccines Against Y. pestis - A Potential Agent of Bioterrorism

“…Strain EV76 produces a robust T-cell response that contributes to protection against pneumonic plague in a murine model (Sha et al, 2008). Despite safety concerns and a high degree of immune variability among vaccine recipients, the NIIEG line of strain EV 76 is still in use today (Zilinskas, 2006).…”