Neutrophils are required for the control of <i>O. anthropi</i> infections.

<p>(A) Neutrophils were depleted from twenty mice by injection of the anti-neutrophil RB6 antibody and ten additional mice were instead injected with PBS alone. One group of ten mice anti-neutrophil treated and the group of ten mice injected with PBS alone were then infected intraperitoneally with 10⁹ CFU/mouse of <i>O. anthropi</i>. The last group of ten mice treated with antibody anti-neutrophil was inoculated with PBS alone. The lethality of the bacteria was recorded at the indicated times. (B) <i>O. anthropi</i> was mixed with purified rat neutrophils at a ratio of 5±4 bacteria/cell. Control bacteria were not incubated with neutrophils. At the indicated times the viable CFU were determined and the percentage of bacterial replication was calculated. In “B” Values of p<0.01 (*) and p<0.001 (**) are indicated.</p

Genomic comparison of <i>Brucella</i> 2308 and <i>O. anthropi</i> LMG3331 ORFs involved in the synthesis of surface exposed or OM PAMPs.

1<p>Although acting in the O-PS biosynthetic pathway, these proteins are involved in the steps previous to O-sugar polymerization and, therefore, the corresponding sugars belong structurally to the core oligosaccharide.</p>2<p>These bacteria differ in O-PS sugar structure and have unrelated glycosyltransferases and O-PS export systems. Accordingly, only ORFs assigned to the core and lipid A are presented in the Table. Proteins known to be involved in ancillary pathways of <i>Brucella</i> core oligosaccharide (Pgm, ManBcore and ManCcore) are highly conserved in <i>O. anthropi</i>.</p>3<p>Only ORFs attributed in the annotations in databases to the lipid A modification systems <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005893#pone.0005893-Raetz1" target="_blank">[83]</a> are included.</p>4<p>This ORF, although not annoted in the <i>B.abortus</i> 2308 genome, was identified by using Oant_1613 as a query in the TBLASTN program (NCBI).</p>5<p>For lipoproteins, lipoprotein annotations were obtained from both genomes and verified with the lipoP (<a href="http://www.cbs.dtu.dk/services/LipoP/" target="_blank">http://www.cbs.dtu.dk/services/LipoP/</a>) in those cases in which there were discrepancies between the two annotations.</p

<i>O. anthropi</i> infection induces an intermediate blood leukocyte response.

<p>Groups of 6 mice were inoculated intraperitoneally with10⁶ CFU/ml of the indicated bacteria, bleed at different times and leukocytes counted in blood. (A) Total leukocytes number of mice infected with the indicated bacteria. (B) Relative blood leukocyte number of mice infected with <i>B. abortus</i>. (C) Relative blood leukocyte number of mice infected with <i>S. enterica</i>. (D) Relative blood leukocyte number of mice infected with <i>O. anthropi.</i> Values of <i>O. anthropi</i> in “A” were significantly different at p<0.01 (*) and p<0.001 (**) with respect to <i>B. abortus.</i> In “B, C and D” the standard error was less that 5% at all points.</p

<i>Oa</i>LPS induces cytokine responses that lay between <i>Ba</i>LPS. and <i>Se</i>LPS.

<p>(A, B and C) Groups of 6 mice were inoculated intraperitoneally with <i>Ba</i>LPS, <i>Ec</i>LPS or <i>Oa</i>LPS at the indicated concentrations. Mice were bled at 2 or 5 h and interleukins quantitated by ELISA. (D) Bone marrow macrophages from wild type TLR2−/− and TLR4−/− knockout mice were incubated with the indicated LPSs. After incubation TNF-α was quantified in the supernatant of cells at 24 h. The insert shows Raw 264.7 macrophages incubated with 25 µg of <i>Oa</i>LPS or <i>Ba</i>LPS and at the indicated times TNF-α quantified from cell culture supernatants. Values of p<0.05 (*) and p<0.005 (**) with respect to <i>Ba</i>LPS are indicated.</p

<i>O. anthropi</i> induces TNF-α that lays between <i>B. abortus</i> and <i>S. enterica</i>.

<p>(A) Murine Raw 264.7 macrophages were infected for 30 min with <i>O. anthropi</i> or <i>B. abortus</i> at a MOI of 500. Extracellular bacteria were killed by addition of gentamicin. At the indicated times samples from the culture media were taken and processed for TNF-α quantification. (B) Groups of 6 mice were inoculated intraperitoneally with <i>O. anthropi</i>. At the indicated times mice were bled and TNF-α levels were determined. The levels of TNF-α induction after 3 h of an equivalent inoculation of <i>B. abortus</i> or <i>S. enterica</i>. Value of p<0.001 (**) is indicated.</p

<i>Oa</i>LPS induces dendritic cell maturation that lay between <i>Ba</i>LPS. and <i>Se</i>LPS.

<p>(A) BMDC were incubated with 0.1 µg of <i>Ec</i>LPS, 10 µg of <i>Ba</i>LPS or 10 µg of <i>Oa</i>LPS in 1 ml culture and labeled with the anti-FK2 antibody for detection of DALIS at 24 h. The percentage of BMDC positive for DALIS is indicated. (B) BMDC incubated as in “A” were labeled with fluorescent anti-CD40, CD-80, CD-86 and MHC II. Values of median fluorescence correspond to four independent experiments. Values of p< 0.05 (*) and p<0.005 (**) with respect to <i>Ba</i>LPS are indicated.</p

Model of <i>Brucella, Ochrobactrum</i> and <i>Escherichia</i> lipid As and core oligosaccharide moieties. [18], [19], [45].

<p>The presence of three KDO and phosphorylated heptoses confers five negative charges to the <i>Escherichia coli</i> core oligosaccharide. The core oligosaccharide of <i>Ochrobactrum</i> LPS possesses galacturonic acid, in contrast to <i>Brucella</i> LPS core which does not possess this negatively charged acidic sugar. The differential sensitivity of <i>Escherichia</i>, <i>Ochrobactrum</i> and <i>Brucella</i> to cationic peptides expressed as sensitivity to polymyxin B (PlxB) units (U) is linked to the different LPSs properties such as uptake hydrophobic probes (NPN), PlxB binding, and LPS zeta potential (ζ) dependence after saturation with polymyxin B <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005893#pone.0005893-Velasco2" target="_blank">[18]</a>. Notice that upon saturation with PlxB, zeta potential became positive (1 mV) for <i>Ochrobactrum</i> LPS while remaining negative (−5 mV) for <i>Brucella</i> smooth LPS, suggesting hindered access to inner targets. <i>Ochrobactrum</i> and <i>Brucella</i> LPSs does not show significant differences in lipid A structure and acyl chain fluidity and display very similar structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005893#pone.0005893-Velasco2" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005893#pone.0005893-Moriyn3" target="_blank">[19]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005893#pone.0005893-Velasco3" target="_blank">[45]</a>. It has been proposed that the quinovosamine sugar in <i>Ochrobactrum</i> and <i>Brucella</i> LPS links the O core oligosaccharide with the O-chain polysaccharide <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005893#pone.0005893-Gonzlez1" target="_blank">[21]</a>.</p