Interactions betweenBrucella melitensisand Human Phagocytes: Bacterial Surface O-Polysaccharide Inhibits Phagocytosis, Bacterial Killing, and Subsequent Host Cell Apoptosis

Abstract

Brucellae are gram-negative intracellular pathogens which can survive and multiply within the phagocytic cells of their hosts and are resistant to the bactericidal action of serum. Brucella melitensis is considered the principal cause of human brucellosis (40,41) and is more virulent than B. abortus (42). These species may occur as either smooth or rough variants depending on the expression of O-polysaccharides (OPS) as a component of the bacterial outer membrane lipopolysaccharide (LPS). In rough strains expression of OPS is limited or absent and attenuation in virulence is generally observed (1,4,19,26,31,35). Our previous studies demonstrated that smooth B. melitensis and B. abortus strains bind fewer complement components on their surface than their corresponding roughmutant organisms (13) obtained by disruption or deletion of the wboA gene (13,26,37,40). However, OPS-deficient strains derived from smooth, virulent B. melitensis 16M are as resistant as the wild type to the bactericidal action of nonimmune human serum (HS) and bind less complement than OPS-deficient strains derived from B. abortus 2308 (13). Since rough mutants of B. melitensis are protected from extracellular complement-mediated killing, we decided to investigate whether the attenuation in virulence previously observed in these strains was associated with differences in their interaction with macrophages.The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a self-fluorescing protein that requires no substrates and emits bright green fluorescence at 509 nm (3). We took advantage of the properties of the GFP to study the interaction of fluorescent rough and smooth B. melitensis strains with human mononuclear phagocytes and to evaluate the importance of the presence of OPS in the pathogenesis of brucellosis. We introduced plasmid pBBR1MCS-6Y (29) expressing GFP into 16M, OPS-deficient ⌬wboA B. melitensis strain WRR51, and WRR51 complemented with wboA and examined interactions of the fluorescent bacteria with human and murine macrophages by fluorescent and electron microscopy, flow cytometry, and release of lactate dehydrogenase (LDH). We found that infection of mononuclear phagocytes with WRR51 was followed by host cell apoptosis and bacterial death. In contrast, infection with either 16M or wboA-complemented WRR51 led to intracellular bacterial replication but not host cell apoptosis. Moreover, infection with the latter

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.…”

“…With OPS, smooth Brucella S2308 invade macrophages via lipid rafts, which requires PI3-kinase and TLR4 signal and directs intracellular trafficking through a unique pathway to the replication niche [16]. Without OPS, rough Brucella mutant CA180 invade macrophages through a different pathway and are either destroyed as a result of macrophage activation or released prematurely as a result of cell death [17,19].…”

“…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).…”

“…Brucella LPS has been recognized as an important virulence factor involved in bacterial adhesion and internalization [16]. 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].…”

The Role of Apoptosis and Autophagy in Bovine Abortions Associated with Brucella Spp

“…Experimental studies using macrophage cell cultures and human macrophages showed that Brucella spp., an intracellular bacterium, triggered apoptotic cell death [26,27]. The genes regulating apoptosis are known as caspases [28].…”