Brucella abortusRough Mutants Are Cytopathic for Macrophages in Culture

Abstract

Rough mutants of Brucella spp. are attenuated for survival in animal models. However, conflicting in vitro evidence has been obtained concerning the intracellular survival of rough mutants. Transposon-derived rough mutants isolated in our laboratory were previously shown to exhibit small but significant reductions in intracellular survival in a 12-h in vitro assay. Several recent publications report that rough mutants exhibited increased macrophage uptake relative to their smooth parental strains, and a reduction in numbers at the end of the assay has been interpreted as intracellular killing. In an effort to explore the role of O antigen in the interaction between Brucella abortus and macrophages, we have monitored the uptake of rough mutants and survival in vitro by using the murine macrophage cell line J774.A1. The results confirm a 10-to 20-foldincreased uptake of rough mutants over that of smooth organisms under standard conditions. Recovery of the rough mutants persisted up to 8 h postinfection, but at the point when intracellular replication of the smooth organisms was observed, the number of rough organisms recovered declined. Fluorescence microscopy revealed the intracellular multiplication of both smooth and rough organisms, and assays performed in the absence of antibiotic confirmed the replication of the rough organisms. Examination by phase-contrast microscopy revealed the lytic death of macrophages infected with the rough mutants, which was confirmed by the release of lactate dehydrogenase (LDH) from the cell cytoplasm. Thus, the decline in the number of rough organisms was the result of the lysis of macrophages and not from intracellular killing. The cytopathic effect is characterized as necrotic rather than apoptotic cell death based on early LDH release, annexin V and propidium iodide staining, morphological changes of infected cells and nuclei, and glycine protection. The cytopathic effect was observed with macrophages at multiplicities of infection (MOIs) of as low as 20 and was not observed with epithelial cells at MOIs of as high as 2000. These findings suggest a role for O antigen during the early stages of host-agent interaction that is essential in establishing an intracellular niche that maintains and supports persistent intracellular infection resulting in disease.Brucella spp. are facultative intracellular bacteria that cause brucellosis in a variety of animals and undulant fever in humans (3). Brucellosis is a worldwide zoonosis characterized by persistence of the organism in the reticuloendothelial system in secondary hosts and in the reproductive system in primary hosts. The ability of these organisms to survive in professional and nonprofessional phagocytic cells is the basis for disease (18). The gene products and mechanisms that are essential for the intracellular lifestyle are currently under investigation. Among these, the O antigen has been classified as a major virulence determinant of Brucella and is essential for survival of the classical species in the host. Although it is...

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

“…Bone marrow derived macrophages (BMDM) were prepared from C3H/HeJ (TLR4 d/d ) and C3H/HeOuJ (TLR4 +/+ ) mice as previously described [22]. Macrophages were seeded in 24-well or 6-well plates one day prior to infection with Brucella [19].…”

“…J774.A1 cells cultured on glass coverslips with a thickness of 0.17 mm (Fisher Scientific, Pittsburgh, PA) were infected with S2308 or CA180 at an MOI of 10 as described previously [19]. The infected cells were fixed with 3% (w/v) paraformaldehyde immediately after a 5-minute centrifugation, and following 5, 10, 20, and 30 minutes incubations.…”

“…For differential staining of intracellular and extracellular bacteria, fixed cells on coverslips were stained with a double antibody labeling procedure and observed as previously described [19]. At least 200 cells were detected to obtain each data.…”

“…The cells were washed with complete DMEM before infection with S2308 and CA180 at an MOI of 100. Uptake of smooth and rough Brucella by the treated cells was determined using the gentamicin protection assay [19].…”

“…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.…”