and the BABDolsB mutant in water:hexadecane was similar and widely different from that of an O-polysaccharide deficient B. abortus per mutant used as a reference (Figure 3 C). Similarly, Western-blot analysis of extracts of wild type BAB-parental and BABDolsB cell envelopes did not show differences in the reactivity of Omp 1, Omp2b, Omp31b, Omp25, Omp19, Omp16, and Omp10 (not shown). The absence of OLs does not influence proinflammatory responses to B. abortus or the ability to multiply intracellularly and generate chronic infections Since B. abortus induces a negligible proinflammatory response in mice at early times of infection [1], we examined whether the lack of OLs could alter this property by examining several markers. First, we tested the generation of fibrin D-dimer that would result from the activation of the blood clothing cascade caused by endotoxic microorganisms. For this, we infected Swiss Webster mice 106 CFU/mouse of BAB-parental or mutant BABDolsB (controls received 0.1 ml of 100 mM phosphate buffered saline (pH 7.2) [PBS]) and examined the serum 24 h later. For BAB-parental, the results confirmed previous reports [1] on the absence of any increase in fibrin D-dimers above the positive threshold (0.5 mg/ml). Similarly, infection with the OL- deficient mutant did not have a significant effect on fibrin D-dimer generation. Then, we determined the number of leukocytes in the peritoneal fluid and blood of mice injected intraperitoneally with 106 CFU of BAB-parental or BABDolsB, 105 CFU of S. typhimurium or 0.1 ml of PBS. Whereas the latter induced a leukocyte peritoneal recruitment and a progressive reduction in blood leukocyte numbers at later times, these linked effects were not observed in BAB-parental or BABDolsB mutant infected mice (Figure 4). Similarly, lymphocyte, neutrophil and monocyte numbers in blood and peritoneal fluid did not reveal significant differences in Brucella infected mice (Figure 5). We also measured TNF-a, IL-6, IL-10 and IL-12p40 in the blood of the same animals, and found that the normalized levels of these cytokines were similar in BAB-parental and BABDolsB infections (Figure 6). These levels were similar to and much lower than those reported for B. abortus 2308 and S. typhimurium, respectively [1]. Brucella Ornithine Lipids To complement the above-described studies, we tested the ability of the BABDolsB OL-deficient mutant to multiply in cells and to generate chronic infections. First, we infected bone marrow derived macrophages, RAW 264.7 macrophages and HeLa cells and monitored the intracellular survival of bacteria [36]. Figure 7 shows that the BABDolsB mutant retained the ability to multiply intracellularly in all these cells. Then, we inoculated BALB/c mice intraperitoneally with 56104 CFU/mouse of BABDolsB or of BAB- parental. Two, 6, and 12 weeks later, the bacteria in the spleens were counted, the identity of the isolates confirmed by PCR, and the spleen weights recorded. The results showed that the parental were practically identical throughout the experiment (average log10 CFU 6 SD values for BABDolsB and BAB-parental, respectively, were: week 2, 6.0960.24 and 6.2060.11; week 6, 6.6560.19 and 6.6960.32; and week 12: 5.8560.48 and 5.8060.46). Similarly, there were no splenomegaly differences (0.3560.05 and 0.4060.04; 0.5860.06 and 0.5760.12; 0.4660.09 and 0.4360.12). Consistent with the experiments in vitro, these observations suggest that the absence of OLs does not affect the OM stability in vivo and, accordingly, that OLs seem not to hamper the release of PAMP-bearing molecules and the establishment of chronic infections. Moreover, since BABDolsB did not promote a lower or higher proinflammatory response than BAB-parental, the results suggest that these lipids are neither detected by the pathogen recognition receptors nor antagonists in the recognition of other OM molecules during brucellosis. B. abortus OL do not stimulate cytokine secretion in murine macrophages To test whether Brucella OLs carry a PAMP, we used the BAB- parental and BABDolsB lipids, since the presence of PC in Brucella allows obtaining stable liposomes. As a positive control, we extracted the lipids of B. pertussis (containing OL but not PC) and generated B. pertussis OL-liposomes using the OL-free lipids of BABDolsB as carriers. After verification of the liposome composition (Figure 8 A), we stimulated RAW 264.7 macrophages. While the B. pertussis- BABDolsB liposomes notably stimulated TNF-a and IL-6 secretion, the BAB-parental or BABDolsB liposomes were inactive (Figure 8 B). Figure 3. B. abortus OL-deficient mutants do not show increased sensitivity to normal serum, complement activation activity or altered surface hydrophobicity. (A), survival of BAB-parental and BABDolsB after 90 min of incubation in non-immune serum (B. abortus per- and B. abortus BvrR- are mutants defective in the LPS O-polysaccharide or with an altered OM, respectively, that are sensitive to non-immune serum); (B), packed bacteria were incubated with normal rabbit serum and the complement remaining measured as the hemolytic activity using an erythrocyte- antibody system; (C), partition coefficients of of BAB-parental, BABDolsB, B. abortus per- in hexadecane and water. Data are the mean 6 standard error of triplicate measurements. doi:10.1371/journal.pone.0016030.g003 PLoS ONE | www.plosone.org 4 January 2011 | Volume 6 | Issue 1 | e16030