Murein Lipoprotein Is a Critical Outer Membrane Component Involved inSalmonella entericaSerovar Typhimurium Systemic Infection

Abstract

Lipopolysaccharide (LPS) and Braun (murein) lipoprotein (Lpp) are major components of the outer membrane of gram-negative enteric bacteria that function as potent stimulators of inflammatory and immune responses. In a previous paper, we provided evidence that two functional copies of the lipoprotein gene (lppAandlppB) located on the chromosome ofSalmonella entericaserovar Typhimurium contributed to bacterial virulence. In this study, we characterizedlppAandlppBsingle-knockout (SKO) mutants and compared them with anlppdouble-knockout (DKO) mutant using in vitro and in vivo models. Compared to thelppDKO mutant, which was nonmotile, the motility of thelppSKO mutants was significantly increased (73 to 77%), although the level of motility did not reach the level of wild-type (WT)S. entericaserovar Typhimurium. Likewise, the cytotoxicity was also significantly increased when T84 human intestinal epithelial cells and RAW264.7 murine macrophages were infected with thelppSKO mutants compared to the cytotoxicity when cells were infected with thelppDKO mutant. The level of interleukin-8 (IL-8) in polarized T84 cells infected with thelppBSKO mutant was significantly higher (two- to threefold higher), reaching the level in cells infected with WTS. entericaserovar Typhimurium, than the level in host cells infected with thelppASKO mutant. ThelppDKO mutant induced minimal levels of IL-8. Similarly, sera from mice infected with thelppBSKO mutant contained 4.5- to 10-fold-higher levels of tumor necrosis factor-α and IL-6; the levels of these cytokines were 1.7- to 3.0-fold greater in thelppASKO mutant-infected mice than in animals challenged with thelppDKO mutant. The increased cytokine levels observed with thelppBSKO mutant in mice correlated with greater tissue damage in the livers and spleens of these mice than in the organs of animals infected with thelppASKO andlppDKO mutants. Moreover, thelppBSKO mutant-infected mice had increased susceptibility to death. Since thelppDKO mutant retained intact LPS, we constructed anS. entericaserovar Typhimurium triple-knockout (TKO) mutant in which thelppAandlppBgenes were deleted from an existingmsbBmutant (msbBencodes an enzyme required for the acylation of lipid A). Compared to thelppDKO andmsbBSKO mutants, thelpp-msbBTKO mutant was unable to induce cytotoxicity and to produce cytokines and chemokines in vitro and in vivo. These studies provided the first evidence of the relative contributions of Lpp and lipid A acylation toSalmonellapathogenesis.