Deletion of the Braun Lipoprotein-Encoding Gene and Altering the Function of Lipopolysaccharide Attenuate the Plague Bacterium

Abstract

e Braun (murein) lipoprotein (Lpp) and lipopolysaccharide (LPS) are major components of the outer membranes of Enterobacteriaceae family members that are capable of triggering inflammatory immune responses by activating Toll-like receptors 2 and 4, respectively. Expanding on earlier studies that demonstrated a role played by Lpp in Yersinia pestis virulence in mouse models of bubonic and pneumonic plague, we characterized an msbB in-frame deletion mutant incapable of producing an acyltransferase that is responsible for the addition of lauric acid to the lipid A moiety of LPS, as well as a ⌬lpp ⌬msbB double mutant of the highly virulent Y. pestis CO92 strain. Although the ⌬msbB single mutant was minimally attenuated, the ⌬lpp single mutant and the ⌬lpp ⌬msbB double mutant were significantly more attenuated than the isogenic wild-type (WT) bacterium in bubonic and pneumonic animal models (mouse and rat) of plague. These data correlated with greatly reduced survivability of the aforementioned mutants in murine macrophages. Furthermore, the ⌬lpp ⌬msbB double mutant was grossly compromised in its ability to disseminate to distal organs in mice and in evoking cytokines/chemokines in infected animal tissues. Importantly, mice that survived challenge with the ⌬lpp ⌬msbB double mutant, but not the ⌬lpp or ⌬msbB single mutant, in a pneumonic plague model were significantly protected against a subsequent lethal WT CO92 rechallenge. These data were substantiated by the fact that the ⌬lpp ⌬msbB double mutant maintained an immunogenicity comparable to that of the WT strain and induced long-lasting T-cell responses against heat-killed WT CO92 antigens. Taken together, the data indicate that deletion of the msbB gene augmented the attenuation of the ⌬lpp mutant by crippling the spread of the double mutant to the peripheral organs of animals and by inducing cytokine/chemokine responses. Thus, the ⌬lpp ⌬msbB double mutant could provide a new live-attenuated background vaccine candidate strain, and this should be explored in the future.

Combinational Deletion of Three Membrane Protein-Encoding Genes Highly Attenuates Yersinia pestis while Retaining Immunogenicity in a Mouse Model of Pneumonic Plague

“…However, it is important to mention that while the ⌬lpp ⌬msbB double mutant was much more impaired in its ability to disseminate than the ⌬lpp single mutant, substantial numbers of the double mutant bacteria were still detected at the initial infection site (lungs) in some mice at 3 days postinfection (p.i.) (49). Similarly, the ⌬lpp ⌬msbB double mutant persisted in the spleen of mice by day 6 p.i.…”

“…Importantly, only animals initially challenged with the double mutant in a pneumonic plague model were significantly protected (55%) upon subsequent pneumonic infection with 10 LD 50 s of WT CO92 (49). The attenuated phenotype of the ⌬lpp ⌬msbB double mutant in mouse models correlated with its reduced survivability in murine RAW 264.7 macrophages (49). Furthermore, the ⌬lpp ⌬msbB double mutant evoked reduced levels of inflammatory cytokines compared to those induced by the WT bacterium in a pneumonic plague mouse model, which coincided with overall decreased dissemination of the mutant to the peripheral organs of mice (49).…”

“…The attenuated phenotype of the ⌬lpp ⌬msbB double mutant in mouse models correlated with its reduced survivability in murine RAW 264.7 macrophages (49). Furthermore, the ⌬lpp ⌬msbB double mutant evoked reduced levels of inflammatory cytokines compared to those induced by the WT bacterium in a pneumonic plague mouse model, which coincided with overall decreased dissemination of the mutant to the peripheral organs of mice (49). However, it is important to mention that while the ⌬lpp ⌬msbB double mutant was much more impaired in its ability to disseminate than the ⌬lpp single mutant, substantial numbers of the double mutant bacteria were still detected at the initial infection site (lungs) in some mice at 3 days postinfection (p.i.)…”

“…Both ⌬lpp single and ⌬lpp ⌬msbB double mutants exhibited significant attenuation (70 to 100%) compared to the wild-type (WT) bacterium in pneumonic and bubonic plague mouse models at a dose of 3 50% lethal doses (LD 50 s) (49). Importantly, only animals initially challenged with the double mutant in a pneumonic plague model were significantly protected (55%) upon subsequent pneumonic infection with 10 LD 50 s of WT CO92 (49). The attenuated phenotype of the ⌬lpp ⌬msbB double mutant in mouse models correlated with its reduced survivability in murine RAW 264.7 macrophages (49).…”

“…In our previous study, we investigated the effects of the deletion of the lpp and msbB genes on the pathogenesis of a highly virulent Y. pestis CO92 strain (49). Both ⌬lpp single and ⌬lpp ⌬msbB double mutants exhibited significant attenuation (70 to 100%) compared to the wild-type (WT) bacterium in pneumonic and bubonic plague mouse models at a dose of 3 50% lethal doses (LD 50 s) (49).…”