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

Barquero-Calvo E, Conde-Alvarez R, Chacón-Díaz C, Quesada-Lobo L, Martirosyan A, Guzmán-Verri C, Iriarte M, Mancek-Keber M, Jerala R, Gorvel JP, Moriyón I, Moreno E, Chaves-Olarte E - PLoS ONE (2009)

Bottom Line: During evolution, innate immunity has been tuned to recognize pathogen-associated molecular patterns.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.

View Article: PubMed Central - PubMed

Affiliation: Programa de Investigación en Enfermedades Tropicales, Escuela de Medicina Veterinaria, Universidad Nacional, Heredia, Costa Rica.

ABSTRACT

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.

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Model of Brucella, Ochrobactrum and Escherichia lipid As and core oligosaccharide moieties. [18], [19], [45].The presence of three KDO and phosphorylated heptoses confers five negative charges to the Escherichia coli core oligosaccharide. The core oligosaccharide of Ochrobactrum LPS possesses galacturonic acid, in contrast to Brucella LPS core which does not possess this negatively charged acidic sugar. The differential sensitivity of Escherichia, Ochrobactrum and Brucella 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 [18]. Notice that upon saturation with PlxB, zeta potential became positive (1 mV) for Ochrobactrum LPS while remaining negative (−5 mV) for Brucella smooth LPS, suggesting hindered access to inner targets. Ochrobactrum and Brucella LPSs does not show significant differences in lipid A structure and acyl chain fluidity and display very similar structure [18], [19], [45]. It has been proposed that the quinovosamine sugar in Ochrobactrum and Brucella LPS links the O core oligosaccharide with the O-chain polysaccharide [21].
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pone-0005893-g010: Model of Brucella, Ochrobactrum and Escherichia lipid As and core oligosaccharide moieties. [18], [19], [45].The presence of three KDO and phosphorylated heptoses confers five negative charges to the Escherichia coli core oligosaccharide. The core oligosaccharide of Ochrobactrum LPS possesses galacturonic acid, in contrast to Brucella LPS core which does not possess this negatively charged acidic sugar. The differential sensitivity of Escherichia, Ochrobactrum and Brucella 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 [18]. Notice that upon saturation with PlxB, zeta potential became positive (1 mV) for Ochrobactrum LPS while remaining negative (−5 mV) for Brucella smooth LPS, suggesting hindered access to inner targets. Ochrobactrum and Brucella LPSs does not show significant differences in lipid A structure and acyl chain fluidity and display very similar structure [18], [19], [45]. It has been proposed that the quinovosamine sugar in Ochrobactrum and Brucella LPS links the O core oligosaccharide with the O-chain polysaccharide [21].

Mentions: Most biological effects of LPS depend on its interaction with the TLR4 co-receptor MD-2, an event that triggers a cascade of signals leading to the NF-κB-dependent activation of immune response genes. On the basis of the above-described results, we hypothesized that OaLPS would have a higher binding to MD-2 than BaLPS but lower than SeLPS, and that this would lead to an NF-κB activation between both types of LPSs. To test this possibility, we compared first the ability of OaLPS, BaLPS, and SeLPS to displace the hydrophobic probe bis-ANS from the binding site of human MD-2 (hMD2) [28]. We found that SeLPS was able to displace approximately 30% of bis-ANS from the hMD-2-binding site at concentrations from 1.25–5 µg/ml (Fig. 9A). Displacement by BaLPS, on the other hand, remained close to the background values obtained with an equal amount of water (Fig. 9A). At the highest concentration tested, OaLPS caused a displacement similar to that induced by SeLPS. However, at lower concentrations it approached the negligible values of BaLPS (Fig. 9A). The interaction with hMD-2 was also measured as the ability of the LPSs to block the recognition of hMD2 by anti-hMD-2 monoclonal antibody (Fig. 9B). SeLPS inhibited this antibody in a dose-dependent manner, with a 50% reduction in the signal at a concentration of 2 µg/ml. BaLPS did not reduce significantly the binding of the anti-hMD-2 antibody at any of the concentrations tested. Again, OaLPS showed an activity that laid between BaLPS and SeLPS since it blocked the antibody at concentrations higher than those of SeLPS (Fig. 9B). Finally, we assessed the activation of the terminal end of the signaling pathway by measuring the relative activity of a luciferase reporter under the control of the NF-κB promoter in HEK 293 cells transfected with expression vectors containing hMD-2, human TLR4 (hTLR4) and human CD14 (hCD14). As can be seen in Fig. 9C, 10 to 25 times higher concentrations of OaLPS were necessary to generate a relative luciferase activity similar to that induced by 1 µg/ml of SeLPS. Consistent with other observations, BaLPS induced a weaker luciferase activity (Fig. 9C).


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

Barquero-Calvo E, Conde-Alvarez R, Chacón-Díaz C, Quesada-Lobo L, Martirosyan A, Guzmán-Verri C, Iriarte M, Mancek-Keber M, Jerala R, Gorvel JP, Moriyón I, Moreno E, Chaves-Olarte E - PLoS ONE (2009)

Model of Brucella, Ochrobactrum and Escherichia lipid As and core oligosaccharide moieties. [18], [19], [45].The presence of three KDO and phosphorylated heptoses confers five negative charges to the Escherichia coli core oligosaccharide. The core oligosaccharide of Ochrobactrum LPS possesses galacturonic acid, in contrast to Brucella LPS core which does not possess this negatively charged acidic sugar. The differential sensitivity of Escherichia, Ochrobactrum and Brucella 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 [18]. Notice that upon saturation with PlxB, zeta potential became positive (1 mV) for Ochrobactrum LPS while remaining negative (−5 mV) for Brucella smooth LPS, suggesting hindered access to inner targets. Ochrobactrum and Brucella LPSs does not show significant differences in lipid A structure and acyl chain fluidity and display very similar structure [18], [19], [45]. It has been proposed that the quinovosamine sugar in Ochrobactrum and Brucella LPS links the O core oligosaccharide with the O-chain polysaccharide [21].
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2691993&req=5

pone-0005893-g010: Model of Brucella, Ochrobactrum and Escherichia lipid As and core oligosaccharide moieties. [18], [19], [45].The presence of three KDO and phosphorylated heptoses confers five negative charges to the Escherichia coli core oligosaccharide. The core oligosaccharide of Ochrobactrum LPS possesses galacturonic acid, in contrast to Brucella LPS core which does not possess this negatively charged acidic sugar. The differential sensitivity of Escherichia, Ochrobactrum and Brucella 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 [18]. Notice that upon saturation with PlxB, zeta potential became positive (1 mV) for Ochrobactrum LPS while remaining negative (−5 mV) for Brucella smooth LPS, suggesting hindered access to inner targets. Ochrobactrum and Brucella LPSs does not show significant differences in lipid A structure and acyl chain fluidity and display very similar structure [18], [19], [45]. It has been proposed that the quinovosamine sugar in Ochrobactrum and Brucella LPS links the O core oligosaccharide with the O-chain polysaccharide [21].
Mentions: Most biological effects of LPS depend on its interaction with the TLR4 co-receptor MD-2, an event that triggers a cascade of signals leading to the NF-κB-dependent activation of immune response genes. On the basis of the above-described results, we hypothesized that OaLPS would have a higher binding to MD-2 than BaLPS but lower than SeLPS, and that this would lead to an NF-κB activation between both types of LPSs. To test this possibility, we compared first the ability of OaLPS, BaLPS, and SeLPS to displace the hydrophobic probe bis-ANS from the binding site of human MD-2 (hMD2) [28]. We found that SeLPS was able to displace approximately 30% of bis-ANS from the hMD-2-binding site at concentrations from 1.25–5 µg/ml (Fig. 9A). Displacement by BaLPS, on the other hand, remained close to the background values obtained with an equal amount of water (Fig. 9A). At the highest concentration tested, OaLPS caused a displacement similar to that induced by SeLPS. However, at lower concentrations it approached the negligible values of BaLPS (Fig. 9A). The interaction with hMD-2 was also measured as the ability of the LPSs to block the recognition of hMD2 by anti-hMD-2 monoclonal antibody (Fig. 9B). SeLPS inhibited this antibody in a dose-dependent manner, with a 50% reduction in the signal at a concentration of 2 µg/ml. BaLPS did not reduce significantly the binding of the anti-hMD-2 antibody at any of the concentrations tested. Again, OaLPS showed an activity that laid between BaLPS and SeLPS since it blocked the antibody at concentrations higher than those of SeLPS (Fig. 9B). Finally, we assessed the activation of the terminal end of the signaling pathway by measuring the relative activity of a luciferase reporter under the control of the NF-κB promoter in HEK 293 cells transfected with expression vectors containing hMD-2, human TLR4 (hTLR4) and human CD14 (hCD14). As can be seen in Fig. 9C, 10 to 25 times higher concentrations of OaLPS were necessary to generate a relative luciferase activity similar to that induced by 1 µg/ml of SeLPS. Consistent with other observations, BaLPS induced a weaker luciferase activity (Fig. 9C).

Bottom Line: During evolution, innate immunity has been tuned to recognize pathogen-associated molecular patterns.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.

View Article: PubMed Central - PubMed

Affiliation: Programa de Investigación en Enfermedades Tropicales, Escuela de Medicina Veterinaria, Universidad Nacional, Heredia, Costa Rica.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus