Brucella Ornithine Lipids Figure 6. The OL-deficiency does not alter cytokine responses to B. abortus during the early stages of infection in mice. IL-6, IL-10, IL- 12p40 and TNF-a levels in the blood of mice intraperitoneally injected with 106 CFU of BAB-parental or BABDolsB (values normalized with respect to the values in mice inoculated with PBS). doi:10.1371/journal.pone.0016030.g006 charged groups which are limited to two 2-keto-3-doxyoctulosonic acid residues and the lipid A phosphates [39–43]. Moreover, it was proposed that the Brucella OLs could shield those negatively charged groups by virtue of their positively charged amino groups, as postulated for P. fluorescens [44]. Indeed, the results of this and previous works in other bacteria [45–47] support that Brucella OLs are in fact N-(acyloxyacyl)- ornithine OM structural elements with a free amino group that should be positively charged at neutral and acidic pH. Such an OL role would represent an advantage for a pathogen because the hydrophobic anchorage should make OLs more resistant than divalent cations to displacement by bacteri- cidal peptides and proteins. However, since OL deficiency did not increase the sensitivity of Brucella cells to polymyxins or the permeability to lysozyme (a cationic peptide), this hypothesis was disproved. Furthermore, any possible defect not detected by the in vitro methods seems not to be relevant in vivo, at least in cells and Figure 7. The OL-deficiency does not alter the ability of B. abortus to multiply in mouse cells. (A), bone-marrow derived macrophages; (B), RAW 264.7 macrophages; (C), Hela cells. Values are the mean 6 standard error of triplicate infections, and the results shown are representative of three independent experiments. doi:10.1371/journal.pone.0016030.g007 PLoS ONE | www.plosone.org 6 January 2011 | Volume 6 | Issue 1 | e16030