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

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In this study, we analyzed temporal gene expression patterns in human peripheral blood mononuclear cells (PBMCs) infected with the Francisella tularensis live vaccine strain from 1 to 24 h utilizing a whole human Affymetrix gene chip. We found that a considerable number of induced genes had similar expression patterns and functions as reported previously for gene expression profiling in patients with ulceroglandular tularemia. Among the six uniquely regulated genes reported for tularemia patients as being part of the alarm signal gene cluster, five, namely caspase 1, PSME2, TAP-1, GBP1, and GCH1, were induced in vitro. We also detected four out of the seven potential biomarkers reported in tularemia patients, namely TNFAIP6 at 4 h and STAT1, TNFSF10, and SECTM1 at 16 and 24 h. These observations underscore the value of using microarray expression profiling as an in vitro tool to identify potential biomarkers for human infection and disease. Our results indicate the potential involvement of several host pathways/processes in Francisella infection, notably those involved in calcium, zinc ion binding, PPAR signaling, and lipid metabolism, which further refines the current knowledge of F. tularensis infection and its effects on the human host. Ultimately, this study provides support for utilizing in vitro microarray gene expression profiling in human PBMCs to identify biomarkers of infection and predict in vivo immune responses to infectious agents.

To determine whether cytokines and T-cell subsets other than Th1 cells contribute to secondary immune responses against Francisella species, we investigated production of Th17-associated cytokines IL-17 and IL-22 in a recall response to Francisella tularensis. Peripheral blood mononuclear cells (PBMCs) from volunteers previously immunized with the F. tularensis live vaccine strain (LVS) were stimulated in vitro with bacterial lysates of LVS or a nonpathogenic type A B38 strain. Gene expression analysis by real-time PCR showed that IL-17 and IL-22 transcripts were induced in immune PBMCs at a significantly higher level than in cells from nonvaccinated volunteers stimulated with LVS or B38 antigens at 24 h. In addition, we detected both cell-associated and secreted IL-22 at 24 h after stimulation and IL-17 at 72 h post-stimulation. Intracellular IL-22 and IL-17 were observed in memory CD4+ cells and less in memory CD8+ cells. These findings suggest that Th17 responses in addition to the Th1 response may play an important role in adaptive immunity against Francisella.