The Two Murein Lipoproteins ofSalmonella entericaSerovar Typhimurium Contribute to the Virulence of the Organism

Abstract

Septic shock due to Salmonella and other gram-negative enteric pathogens is a leading cause of death worldwide. The role of lipopolysaccharide in sepsis is well studied; however, the contribution of other bacterial outer membrane components, such as Braun (murein) lipoprotein (Lpp), is not well defined. The genome of Salmonella enterica serovar Typhimurium harbors two copies of the lipoprotein (lpp) gene. We constructed a serovar Typhimurium strain with deletions in both copies of the lpp gene (lpp1 and lpp2) by marker exchange mutagenesis. The integrity of the cell membrane and the secretion of the effector proteins through the type III secretion system were not affected in the lpp double-knockout mutant. Subsequently, the virulence potential of this mutant was examined in a cell culture system using T84 intestinal epithelial and RAW264.7 macrophage cell lines and a mouse model of salmonellosis. The lpp double-knockout mutant was defective in invading and inducing cytotoxic effects in T84 and RAW264.7 cells, although binding of the mutant to the host cell was not affected when compared to the wild-type (WT) serovar Typhimurium. The motility of the mutant was impaired, despite the finding that the number of flagella was similar in the lpp double knockout mutant and the WT serovar Typhimurium. Deletion in the lpp genes did not affect the intracellular survival and replication of Salmonella in macrophages and T84 cells. Induction of the proinflammatory cytokines tumor necrosis factor alpha and interleukin-8 (IL-8) was significantly reduced in macrophages and T84 cells infected with the lpp double-knockout mutant. The levels of IL-8 remained unaffected in T84 cells when infected with either live or heat-killed WT and lpp mutant, indicating that invasion was not required for IL-8 production and that Toll-like receptor 2 signaling might be affected in the Lpp double-knockout mutant. These effects of the Lpp protein could be restored by complementation of the isogenic mutant. The lpp double-knockout mutant was avirulent in mice, and animals infected with this mutant were protected from a lethal challenge dose of WT serovar Typhimurium. The severe combined immunodeficient mice, on the other hand, were susceptible to infection by the lpp double-knockout mutant. The serovar Typhimurium mutants from which only one of the lpp (lpp1 or lpp2) genes was deleted were also avirulent in mice. Taken together, our data indicated that Lpp specifically contributed to the virulence of the organism.

A unique Coxiella burnetii lipoprotein involved in metal binding (LimB)

“…One potential set of virulence factors that has received relatively little attention is the pathogen's considerable assemblage of lipoproteins. Lipoproteins have long been recognized as virulence determinants in a variety of bacterial pathogens, and a number of them have been developed into vaccine immunogens (Fortney et al, 2000;Hayashi & Wu, 1990;Schwan et al, 1995;Sha et al, 2004). Lipoproteins are anchored to the inner or outer membrane through a covalently linked lipid moiety (Babu et al, 2006;Hantke & Braun, 1973;Inouye et al, 1977).…”

Peptidoglycan-associated lipoprotein ofAggregatibacter actinomycetemcomitansinduces apoptosis and production of proinflammatory cytokines via TLR2 in murine macrophages RAW 264.7in vitro

“…Bacterial lipoproteins are established bacterial virulence factors [11–13,24,26,36] that function as inflammatory stimulants in infection and disease progression. Our study is the first to show pro-inflammatory effects of AaPAL, an outer membrane lipoprotein of a major periodontal pathogen, A. actinomycetemcomitans .…”

“…However, current interest concerning Gram-negative infections is increasingly focused on bacterial outer membrane proteins (OMPs), especially lipoproteins [10]. In fact, recent studies have suggested that bacterial lipoproteins are major players in inflammatory reactions caused by pathogenic species, such as Shigella flexneri, Brucella abortus , and Salmonella enterica [11–13]. Regarding A. actinomycetemcomitans , 23 different lipoproteins are detectable among the proteins secreted by A. actinomyctemcomitans biofilm [14], but their bioactivity is poorly understood [15–17].…”

Immunological responses against Salmonella enterica serovar Typhimurium Braun lipoprotein and lipid A mutant strains in Swiss-Webster mice: Potential use as live-attenuated vaccines

“…Maturation of Lpp requires modification of the lipid moiety, which is catalyzed by enzymes, specifically glycerol transferase, O-acyl transferase, signal peptidase II, and N-acyl transferase [8]. Two functional copies of the lpp gene (designated as lppA and lppB) exist on the chromosome of S. Typhimurium 14028 located in tandem and separated by 82 bp [9]. Deletion of both copies of the lpp gene results in a S. Typhimurium mutant that is minimally invasive to epithelial cells, non-motile, and severely impaired in its ability to induce cytotoxicity in murine macrophages (RAW 264.7 cells) and T84 human colonic epithelial cells, possibly due to the reduced production of proinflammatory cytokines and chemokines (e.g., tumor necrosis factor-alpha [TNF-α] and interleukin [IL]-8) [9].…”

“…Two functional copies of the lpp gene (designated as lppA and lppB) exist on the chromosome of S. Typhimurium 14028 located in tandem and separated by 82 bp [9]. Deletion of both copies of the lpp gene results in a S. Typhimurium mutant that is minimally invasive to epithelial cells, non-motile, and severely impaired in its ability to induce cytotoxicity in murine macrophages (RAW 264.7 cells) and T84 human colonic epithelial cells, possibly due to the reduced production of proinflammatory cytokines and chemokines (e.g., tumor necrosis factor-alpha [TNF-α] and interleukin [IL]-8) [9]. The lpp (lppAB) double knockout (DKO) mutant was avirulent in mice following oral and intraperitoneal (i.p.)…”