The Differential Interaction of Brucella and Ochrobactrum with Innate Immunity Reveals Traits Related to the Evolution of Stealthy Pathogens

International audienceBACKGROUND: During evolution, innate immunity has been tuned to recognize pathogen-associated molecular patterns. However, some alpha-Proteobacteria are stealthy intracellular pathogens not readily detected by this system. Brucella members follow this strategy and are highly virulent, but other Brucellaceae like Ochrobactrum are rhizosphere inhabitants and only opportunistic pathogens. To gain insight into the emergence of the stealthy strategy, we compared these two phylogenetically close but biologically divergent bacteria. METHODOLOGY/PRINCIPAL FINDINGS: In contrast to Brucella abortus, Ochrobactrum anthropi did not replicate within professional and non-professional phagocytes and, whereas neutrophils had a limited action on B. abortus, they were essential to control O. anthropi infections. O. anthropi triggered proinflammatory responses markedly lower than Salmonella enterica but higher than B. abortus. In macrophages and dendritic cells, the corresponding lipopolysaccharides reproduced these grades of activation, and binding of O. anthropi lipopolysaccharide to the TLR4 co-receptor MD-2 and NF-kappaB induction laid between those of B. abortus and enteric bacteria lipopolysaccharides. These differences correlate with reported variations in lipopolysaccharide core sugars, sensitivity to bactericidal peptides and outer membrane permeability. CONCLUSIONS/SIGNIFICANCE: The results suggest that Brucellaceae ancestors carried molecules not readily recognized by innate immunity, so that non-drastic variations led to the emergence of stealthy intracellular parasites. They also suggest that some critical envelope properties, like selective permeability, are profoundly altered upon modification of pathogen-associated molecular patterns, and that this represents a further adaptation to the host. It is proposed that this adaptive trend is relevant in other intracellular alpha-Proteobacteria like Bartonella, Rickettsia, Anaplasma, Ehrlichia and Wolbachia

Free thiol group of MD-2 as the target for inhibition of the lipopolysaccharide-induced cell activation

MD-2 is a part of the Toll-like 4 signaling complex with an indispensable role in activation of the lipopolysaccharide (LPS) signaling pathway and thus a suitable target for the therapeutic inhibition of TLR4 signaling. Elucidation of MD-2 structure provides a foundation for rational design of inhibitors that bind to MD-2 and inhibit LPS signaling. Since the hydrophobic binding pocket of MD-2 provides little specificity for inhibitors, we have investigated targeting the solvent-accessible cysteine residue within the hydrophobic binding pocket of MD-2. Compounds with affinity for the hydrophobic pocket that contain a thiol-reactive group, which mediates covalent bond formation with the free cysteine residue of MD-2, were tested. Fluorescent compounds 2-(4'-(iodoacetamido)anilino)naphthalene-6-sulfonic acid and N-pyrene maleimide formed a covalent bond with MD-2 through Cys(133) and inhibited LPS signaling. Cell activation was also inhibited by thiol-reactive compounds JTT-705 originally targeted against cholesterol ester transfer protein and antirheumatic compound auranofin. Oral intake of JTT-705 significantly inhibited endotoxin-triggered tumor necrosis factor alpha production in mice. The thiol group of MD-2 also represents the target of environmental or endogenous thiol-reactive compounds that are produced in inflammation

Free Thiol Group of MD-2 as the Target for Inhibition of the Lipopolysaccharide-induced Cell Activation*

MD-2 is a part of the Toll-like 4 signaling complex with an indispensable role in activation of the lipopolysaccharide (LPS) signaling pathway and thus a suitable target for the therapeutic inhibition of TLR4 signaling. Elucidation of MD-2 structure provides a foundation for rational design of inhibitors that bind to MD-2 and inhibit LPS signaling. Since the hydrophobic binding pocket of MD-2 provides little specificity for inhibitors, we have investigated targeting the solvent-accessible cysteine residue within the hydrophobic binding pocket of MD-2. Compounds with affinity for the hydrophobic pocket that contain a thiol-reactive group, which mediates covalent bond formation with the free cysteine residue of MD-2, were tested. Fluorescent compounds 2-(4′-(iodoacetamido)anilino)naphthalene-6-sulfonic acid and N-pyrene maleimide formed a covalent bond with MD-2 through Cys133 and inhibited LPS signaling. Cell activation was also inhibited by thiol-reactive compounds JTT-705 originally targeted against cholesterol ester transfer protein and antirheumatic compound auranofin. Oral intake of JTT-705 significantly inhibited endotoxin-triggered tumor necrosis factor α production in mice. The thiol group of MD-2 also represents the target of environmental or endogenous thiol-reactive compounds that are produced in inflammation

The differential interaction of Brucella and Ochrobactrum with innate immunity reveals traits related to the evolution of stealthy pathogens

Background: During evolution, innate immunity has been tuned to recognize pathogen-associated molecular patterns. However, some alpha-Proteobacteria are stealthy intracellular pathogens not readily detected by this system. Brucella members follow this strategy and are highly virulent, but other Brucellaceae like Ochrobactrum are rhizosphere inhabitants and only opportunistic pathogens. To gain insight into the emergence of the stealthy strategy, we compared these two phylogenetically close but biologically divergent bacteria. Methodology/principal Findings: In contrast to Brucella abortus, Ochrobactrum anthropi did not replicate within professional and non-professional phagocytes and, whereas neutrophils had a limited action on B. abortus, they were essential to control O. anthropi infections. O. anthropi triggered proinflammatory responses markedly lower than Salmonella enterica but higher than B. abortus. In macrophages and dendritic cells, the corresponding lipopolysaccharides reproduced these grades of activation, and binding of O. anthropi lipopolysaccharide to the TLR4 co-receptor MD-2 and NF-kappaB induction laid between those of B. abortus and enteric bacteria lipopolysaccharides. These differences correlate with reported variations in lipopolysaccharide core sugars, sensitivity to bactericidal peptides and outer membrane permeability. Conclusions/significance: The results suggest that Brucellaceae ancestors carried molecules not readily recognized by innate immunity, so that non-drastic variations led to the emergence of stealthy intracellular parasites. They also suggest that some critical envelope properties, like selective permeability, are profoundly altered upon modification of pathogen-associated molecular patterns, and that this represents a further adaptation to the host. It is proposed that this adaptive trend is relevant in other intracellular alpha-Proteobacteria like Bartonella, Rickettsia, Anaplasma, Ehrlichia and Wolbachia

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

<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

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>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