Effects of Opsonization and Gamma Interferon on Growth ofBrucella melitensis16M in Mouse Peritoneal Macrophages In Vitro

Abstract

Entry of opsonized pathogens into phagocytes may benefit or, paradoxically, harm the host. Opsonization may trigger antimicrobial mechanisms such as reactive oxygen or nitric oxide (NO) production but may also provide a safe haven for intracellular replication. Brucellae are natural intramacrophage pathogens of rodents, ruminants, dogs, marine mammals, and humans. We evaluated the role of opsonins in Brucellamacrophage interactions by challenging cultured murine peritoneal macrophages with Brucella melitensis 16M treated with complement-and/or antibody-rich serum. Mouse serum rich in antibody against Brucella lipopolysaccharide (LPS) (aLPS) and human complement-rich serum (HCS) each enhanced the macrophage uptake of brucellae. Combinations of suboptimal levels of aLPS (0.01%) and HCS (2%) synergistically enhanced uptake. The intracellular fate of ingested bacteria was evaluated with an optimal concentration of gentamicin (2 g/ml) to control extracellular growth but not kill intracellular bacteria. Bacteria opsonized with aLPS and/or HCS grew equally well inside macrophages in the absence of gamma interferon (IFN-␥). Macrophage activation with IFN-␥ inhibited replication of both opsonized and nonopsonized brucellae but was less effective in inhibiting replication of nonopsonized bacteria. IFN-␥ treatment of macrophages with opsonized or nonopsonized bacteria enhanced NO production, which was blocked by N G -monomethyl L-arginine (MMLA), an NO synthesis inhibitor. MMLA also partially blocked IFN-␥-mediated bacterial growth inhibition. These studies suggest that primary murine macrophages have limited ability to control infection with B. melitensis, even when activated by IFN-␥ in the presence of highly opsonic concentrations of antibody and complement. Additional cellular immune responses, e.g., those mediated by cytotoxic T cells, may play more important roles in the control of murine brucellosis.Brucella spp., short, nonmotile, nonsporulating, nonencapsulated, gram-negative aerobic rods, are important facultative intracellular pathogens of humans and livestock. Brucella melitensis usually infects sheep, goats, and camels and is the most pathogenic species for humans (1). Like other facultative intracellular pathogenic bacteria (e.g., Francisella tularensis, Listeria monocytogenes, Mycobacterium spp., and Legionella pneumophila), clearance of Brucella infection relies on both cellmediated immunity (1,3,7,12,20,21,27,30,38) and humoral responses (10,22,29,35). The interplay of these two arms of the immune response, however, is not well understood.Successful infection of the host by Brucella reflects the ability of the bacterium to establish itself in an intracellular environment favorable for its replication. The presence of complement or antibody in the extracellular fluid favors killing of some Brucella strains (35,38). Opsonization by these humoral factors also enhances uptake by phagocytic cells that shelter the bacteria. The intracellular fate of brucellae may depend on the bacterial species or the kind of ph...

Real-Time Monitoring of IntracellularStaphylococcus aureusReplication

“…Indeed, when concentrations of gentamicin of 100 g ml Ϫ1 or 50 g ml Ϫ1 , which are routinely used in traditional intracellular replication assays, were added to monolayers infected with Lux ϩ S. aureus, no bioluminescence was observed, indicating that bacterial replication had been completely abolished (data not shown). This observation concurs with recent reports that have demonstrated that gentamicin is capable of entering macrophages and killing intracellular bacteria (10,11,15,35). Hamrick et al (15) demonstrated that even at concentrations of 5 g ml Ϫ1 gentamicin was able to affect intracellular bacterial viability.…”

Vaccination with the RecombinantBrucellaOuter Membrane Protein 31 or a Derived 27-Amino-Acid Synthetic Peptide Elicits a CD4+T Helper 1 Response That Protects againstBrucella melitensisInfection

“…The importance of IFN-␥ in resolution of Brucella infection is supported by many studies (20,49). Indeed, BALB/c or C57BL/6 IFN-␥ knockout mice died approximately 6 weeks after infection with B. abortus strain 2308 (36).…”

BrucellaLumazine Synthase Elicits a Mixed Th1-Th2 Immune Response and Reduces Infection in Mice Challenged withBrucella abortus544 Independently of the Adjuvant Formulation Used

“…Each mouse was injected at days 0 and 15. Sera were obtained at 15,30,45, and 60 days after the first immunization. Mice used as the positive control group in the protection experiments were subcutaneously immunized with 8 ϫ 10 8 heat-killed B. melitensis H38S bacteria in 200 l of IFA.…”

“…In particular, Th1 immune responses characterized by production of gamma interferon (IFN-␥) are associated with protective immunity to Brucella (15,24,38). These responses are best stimulated by live vaccines or potentially by multiple injections of appropriate protective antigens in the presence of adjuvants which favor cell-mediated immune mechanisms.…”