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The Vi capsular polysaccharide enables Salmonella enterica serovar typhi to evade microbe-guided neutrophil chemotaxis.

Wangdi T, Lee CY, Spees AM, Yu C, Kingsbury DD, Winter SE, Hastey CJ, Wilson RP, Heinrich V, Bäumler AJ - PLoS Pathog. (2014)

Bottom Line: Typhimurium) is associated with a localized gastroenteritis in humans.Typhi.Collectively, these data suggest that expression of the virulence-associated (Vi) capsular polysaccharide of S.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis, Davis, California, United States of America.

ABSTRACT
Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever, a disseminated infection, while the closely related pathogen S. enterica serovar Typhimurium (S. Typhimurium) is associated with a localized gastroenteritis in humans. Here we investigated whether both pathogens differ in the chemotactic response they induce in neutrophils using a single-cell experimental approach. Surprisingly, neutrophils extended chemotactic pseudopodia toward Escherichia coli and S. Typhimurium, but not toward S. Typhi. Bacterial-guided chemotaxis was dependent on the presence of complement component 5a (C5a) and C5a receptor (C5aR). Deletion of S. Typhi capsule biosynthesis genes markedly enhanced the chemotactic response of neutrophils in vitro. Furthermore, deletion of capsule biosynthesis genes heightened the association of S. Typhi with neutrophils in vivo through a C5aR-dependent mechanism. Collectively, these data suggest that expression of the virulence-associated (Vi) capsular polysaccharide of S. Typhi obstructs bacterial-guided neutrophil chemotaxis.

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The Vi capsular polysaccharide inhibits complement-dependent chemotaxis in vitro.(A and D) Neutrophil migration into the bottom compartment of a Boyden chamber containing the indicated attractants was measured in the presence or absence of the complement inhibitor Futhan. Bars represent averages ± standard error from at least 3 different donors. Vehicle; HBSS vehicle control, fMLP; N-formyl-L-methionyl-L-leucyl-L-phenylalanine; wt, wild type. (B) Schematic drawing of Vi capsular biosynthesis genes (black arrows) of S. Typhi. (C) Expression of the Vi capsular polysaccharide (Y-axis) was detected in cultures of the S. Typhi wild type (wt, left panel) and a non-capsulated S. Typhi strain (ΔtviB-vexE mutant, right panel) by flow cytometry using rabbit anti-Vi serum. (E) Fixation of C3b (Y-axis) on the surface of the S. Typhi wild type (wt, left panel) or a non-capsulated S. Typhi strain (ΔtviB-vexE mutant, right panel) that had been incubated for 30 minutes in 1% human serum was detected by flow cytometry using FITC-conjugated goat anti-human C3b antibody. (F) Quantification of C3b fixation detected by flow cytometry. A gate was set using a control in which bacteria were not stained with goat anti-human C3b antibody. Bars represent averages ± standard error from three independent experiments for each strain.
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ppat-1004306-g002: The Vi capsular polysaccharide inhibits complement-dependent chemotaxis in vitro.(A and D) Neutrophil migration into the bottom compartment of a Boyden chamber containing the indicated attractants was measured in the presence or absence of the complement inhibitor Futhan. Bars represent averages ± standard error from at least 3 different donors. Vehicle; HBSS vehicle control, fMLP; N-formyl-L-methionyl-L-leucyl-L-phenylalanine; wt, wild type. (B) Schematic drawing of Vi capsular biosynthesis genes (black arrows) of S. Typhi. (C) Expression of the Vi capsular polysaccharide (Y-axis) was detected in cultures of the S. Typhi wild type (wt, left panel) and a non-capsulated S. Typhi strain (ΔtviB-vexE mutant, right panel) by flow cytometry using rabbit anti-Vi serum. (E) Fixation of C3b (Y-axis) on the surface of the S. Typhi wild type (wt, left panel) or a non-capsulated S. Typhi strain (ΔtviB-vexE mutant, right panel) that had been incubated for 30 minutes in 1% human serum was detected by flow cytometry using FITC-conjugated goat anti-human C3b antibody. (F) Quantification of C3b fixation detected by flow cytometry. A gate was set using a control in which bacteria were not stained with goat anti-human C3b antibody. Bars represent averages ± standard error from three independent experiments for each strain.

Mentions: To further investigate the mechanism of bacterial-guided neutrophil chemotaxis we used a standard Boyden chamber assay [33]. In this assay, the migration of human neutrophils from the upper compartment of the Boyden chamber [33] into a bottom reservoir that contains human serum is measured in the presence or absence of chemoattractants. The presence in the bottom chamber of either S. Typhimurium, IL-8 or synthetic N-formyl peptide (N-formyl-L-methionyl-L-leucyl-L-phenylalanine, fMLP) elicited migration of significantly (P<0.01) larger numbers of human neutrophils into the bottom reservoir than the vehicle control (Fig. 2A). To investigate the contribution of complement we used Futhan, which specifically binds factor Bb in C3 convertase (i.e. surface bound C3bBb) and C5 convertase (i.e. surface bound C3bBbC3b), thereby inhibiting the production of C3a and C5a (Fig. S1) [34], [35]. Addition of the complement inhibitor Futhan blunted the S. Typhimurium-induced neutrophil migration into the bottom chamber (P<0.01) (Fig. 2A). In contrast, migration of human neutrophils elicited by IL-8 or N-formyl peptide was not significantly inhibited by the addition of Futhan (P>0.05). Collectively, these data suggested that neutrophil chemotaxis elicited by S. Typhimurium was mediated by a complement-dependent mechanism, which was distinct from the complement-independent mechanisms of neutrophil chemotaxis elicited by IL-8 or N-formyl peptide.


The Vi capsular polysaccharide enables Salmonella enterica serovar typhi to evade microbe-guided neutrophil chemotaxis.

Wangdi T, Lee CY, Spees AM, Yu C, Kingsbury DD, Winter SE, Hastey CJ, Wilson RP, Heinrich V, Bäumler AJ - PLoS Pathog. (2014)

The Vi capsular polysaccharide inhibits complement-dependent chemotaxis in vitro.(A and D) Neutrophil migration into the bottom compartment of a Boyden chamber containing the indicated attractants was measured in the presence or absence of the complement inhibitor Futhan. Bars represent averages ± standard error from at least 3 different donors. Vehicle; HBSS vehicle control, fMLP; N-formyl-L-methionyl-L-leucyl-L-phenylalanine; wt, wild type. (B) Schematic drawing of Vi capsular biosynthesis genes (black arrows) of S. Typhi. (C) Expression of the Vi capsular polysaccharide (Y-axis) was detected in cultures of the S. Typhi wild type (wt, left panel) and a non-capsulated S. Typhi strain (ΔtviB-vexE mutant, right panel) by flow cytometry using rabbit anti-Vi serum. (E) Fixation of C3b (Y-axis) on the surface of the S. Typhi wild type (wt, left panel) or a non-capsulated S. Typhi strain (ΔtviB-vexE mutant, right panel) that had been incubated for 30 minutes in 1% human serum was detected by flow cytometry using FITC-conjugated goat anti-human C3b antibody. (F) Quantification of C3b fixation detected by flow cytometry. A gate was set using a control in which bacteria were not stained with goat anti-human C3b antibody. Bars represent averages ± standard error from three independent experiments for each strain.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4125291&req=5

ppat-1004306-g002: The Vi capsular polysaccharide inhibits complement-dependent chemotaxis in vitro.(A and D) Neutrophil migration into the bottom compartment of a Boyden chamber containing the indicated attractants was measured in the presence or absence of the complement inhibitor Futhan. Bars represent averages ± standard error from at least 3 different donors. Vehicle; HBSS vehicle control, fMLP; N-formyl-L-methionyl-L-leucyl-L-phenylalanine; wt, wild type. (B) Schematic drawing of Vi capsular biosynthesis genes (black arrows) of S. Typhi. (C) Expression of the Vi capsular polysaccharide (Y-axis) was detected in cultures of the S. Typhi wild type (wt, left panel) and a non-capsulated S. Typhi strain (ΔtviB-vexE mutant, right panel) by flow cytometry using rabbit anti-Vi serum. (E) Fixation of C3b (Y-axis) on the surface of the S. Typhi wild type (wt, left panel) or a non-capsulated S. Typhi strain (ΔtviB-vexE mutant, right panel) that had been incubated for 30 minutes in 1% human serum was detected by flow cytometry using FITC-conjugated goat anti-human C3b antibody. (F) Quantification of C3b fixation detected by flow cytometry. A gate was set using a control in which bacteria were not stained with goat anti-human C3b antibody. Bars represent averages ± standard error from three independent experiments for each strain.
Mentions: To further investigate the mechanism of bacterial-guided neutrophil chemotaxis we used a standard Boyden chamber assay [33]. In this assay, the migration of human neutrophils from the upper compartment of the Boyden chamber [33] into a bottom reservoir that contains human serum is measured in the presence or absence of chemoattractants. The presence in the bottom chamber of either S. Typhimurium, IL-8 or synthetic N-formyl peptide (N-formyl-L-methionyl-L-leucyl-L-phenylalanine, fMLP) elicited migration of significantly (P<0.01) larger numbers of human neutrophils into the bottom reservoir than the vehicle control (Fig. 2A). To investigate the contribution of complement we used Futhan, which specifically binds factor Bb in C3 convertase (i.e. surface bound C3bBb) and C5 convertase (i.e. surface bound C3bBbC3b), thereby inhibiting the production of C3a and C5a (Fig. S1) [34], [35]. Addition of the complement inhibitor Futhan blunted the S. Typhimurium-induced neutrophil migration into the bottom chamber (P<0.01) (Fig. 2A). In contrast, migration of human neutrophils elicited by IL-8 or N-formyl peptide was not significantly inhibited by the addition of Futhan (P>0.05). Collectively, these data suggested that neutrophil chemotaxis elicited by S. Typhimurium was mediated by a complement-dependent mechanism, which was distinct from the complement-independent mechanisms of neutrophil chemotaxis elicited by IL-8 or N-formyl peptide.

Bottom Line: Typhimurium) is associated with a localized gastroenteritis in humans.Typhi.Collectively, these data suggest that expression of the virulence-associated (Vi) capsular polysaccharide of S.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis, Davis, California, United States of America.

ABSTRACT
Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever, a disseminated infection, while the closely related pathogen S. enterica serovar Typhimurium (S. Typhimurium) is associated with a localized gastroenteritis in humans. Here we investigated whether both pathogens differ in the chemotactic response they induce in neutrophils using a single-cell experimental approach. Surprisingly, neutrophils extended chemotactic pseudopodia toward Escherichia coli and S. Typhimurium, but not toward S. Typhi. Bacterial-guided chemotaxis was dependent on the presence of complement component 5a (C5a) and C5a receptor (C5aR). Deletion of S. Typhi capsule biosynthesis genes markedly enhanced the chemotactic response of neutrophils in vitro. Furthermore, deletion of capsule biosynthesis genes heightened the association of S. Typhi with neutrophils in vivo through a C5aR-dependent mechanism. Collectively, these data suggest that expression of the virulence-associated (Vi) capsular polysaccharide of S. Typhi obstructs bacterial-guided neutrophil chemotaxis.

Show MeSH
Related in: MedlinePlus