Role of the Brucella suis Lipopolysaccharide O Antigen in Phagosomal Genesis and in Inhibition of Phagosome-Lysosome Fusion in Murine Macrophages

Abstract

Brucella species are gram-negative, facultative intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment inside professional and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both cell types. However, the molecular mechanisms and the microbial factors involved are poorly understood. Smooth lipopolysaccharide (LPS) of Brucella has been reported to be an important virulence factor, although its precise role in pathogenesis is not yet clear. In this study, we show that the LPS O side chain is involved in inhibition of the early fusion between Brucella suis-containing phagosomes and lysosomes in murine macrophages. In contrast, the phagosomes containing rough mutants, which fail to express the O antigen, rapidly fuse with lysosomes. In addition, we show that rough mutants do not enter host cells by using lipid rafts, contrary to smooth strains. Thus, we propose that the LPS O chain might be a major factor that governs the early behavior of bacteria inside macrophages.Brucella species are gram-negative, facultative intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment inside professional (5,14,17,28) and nonprofessional (9, 25, 26) phagocytic cells. Usually, phagosomes mature gradually into phagolysosomes which are capable of degrading proteins by lysosomal enzymes. In the case of

Evidence of Brucella abortus OPS dictating uptake and restricting NF-κB activation in murine macrophages

“…A double antibody labeling procedure was employed to differentiate external bacteria (red) from internalized organisms (green) (data not shown). In agreement with previous reports [6,17,19], CA180 has greatly elevated binding capacity compared with S2308. The dynamics of internalization were determined according to the following criteria: i) fraction of cells containing internalized bacteria; ii) average bacterial burden per infected cell; and iii) fraction of cell-associated bacteria that was internalized (Fig.…”

“…Among those molecules, membrane cholesterol and ganglioside GM1 have been shown to be important in establishing Brucella entry and infection [24]. Methyl-β-cyclodextrin or filipin treatments of J774.A1 cells inhibited uptake of smooth Brucella in a dose dependent manner, while the treatments had limited effect on the uptake of rough Brucella [6]. In the current report, colocolization of Brucella with lipid rafts was visualized using fluorescent microscopy.…”

“…It has previously been reported that the uptake of rough organisms greatly exceeds that observed for smooth strains [6,17,19], and current experiments were conducted to reveal the dynamics of internalization. A double antibody labeling procedure was employed to differentiate external bacteria (red) from internalized organisms (green) (data not shown).…”

“…Invasion of HeLa cells by B. abortus requires small GTPase of the Rho subfamily [4] and requires energy input from the host cells [5]. It was also observed that Brucella invade macrophages through lipid raft microdomains [6]. In both epithelial cells and macrophages F-actin polymerization is involved in ingestion of Brucella, because inhibition of actin polymerization abolishes bacterial uptake [4,5,7].…”

“…Recent studies revealed that smooth Brucella invade phagocytes at lower number compared with rough organisms, and inhibit phagocyte apoptosis [17,18]. In contract, Brucella rough strains attach and invade macrophages at higher efficiency and activate macrophage [6,17,19,20]. However the mechanisms behind this have not been fully investigated.…”