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Host-adaptation of Francisella tularensis alters the bacterium's surface-carbohydrates to hinder effectors of innate and adaptive immunity.

Zarrella TM, Singh A, Bitsaktsis C, Rahman T, Sahay B, Feustel PJ, Gosselin EJ, Sellati TJ, Hazlett KR - PLoS ONE (2011)

Bottom Line: Lastly we show that upon host-adaptation, the human-virulent strain, F. tularensis SchuS4 also induces capsule production with the effect of reducing macrophage-activation and accelerating tularemia pathogenesis in mice.These capsules impede recognition of bacterial outer membrane constituents by antibody, complement, and Toll-Like Receptor 2.These changes in the host-pathogen interface have profound implications for pathogenesis and vaccine development.

View Article: PubMed Central - PubMed

Affiliation: Center for Immunology and Microbial Disease, Albany Medical College, Albany, New York, United States of America.

ABSTRACT

Background: The gram-negative bacterium Francisella tularensis survives in arthropods, fresh water amoeba, and mammals with both intracellular and extracellular phases and could reasonably be expected to express distinct phenotypes in these environments. The presence of a capsule on this bacterium has been controversial with some groups finding such a structure while other groups report that no capsule could be identified. Previously we reported in vitro culture conditions for this bacterium which, in contrast to typical methods, yielded a bacterial phenotype that mimics that of the bacterium's mammalian, extracellular phase.

Methods/findings: SDS-PAGE and carbohydrate analysis of differentially-cultivated F. tularensis LVS revealed that bacteria displaying the host-adapted phenotype produce both longer polymers of LPS O-antigen (OAg) and additional HMW carbohydrates/glycoproteins that are reduced/absent in non-host-adapted bacteria. Analysis of wildtype and OAg-mutant bacteria indicated that the induced changes in surface carbohydrates involved both OAg and non-OAg species. To assess the impact of these HMW carbohydrates on the access of outer membrane constituents to antibody we used differentially-cultivated bacteria in vitro to immunoprecipitate antibodies directed against outer membrane moieties. We observed that the surface-carbohydrates induced during host-adaptation shield many outer membrane antigens from binding by antibody. Similar assays with normal mouse serum indicate that the induced HMW carbohydrates also impede complement deposition. Using an in vitro macrophage infection assay, we find that the bacterial HMW carbohydrate impedes TLR2-dependent, pro-inflammatory cytokine production by macrophages. Lastly we show that upon host-adaptation, the human-virulent strain, F. tularensis SchuS4 also induces capsule production with the effect of reducing macrophage-activation and accelerating tularemia pathogenesis in mice.

Conclusion: F. tularensis undergoes host-adaptation which includes production of multiple capsular materials. These capsules impede recognition of bacterial outer membrane constituents by antibody, complement, and Toll-Like Receptor 2. These changes in the host-pathogen interface have profound implications for pathogenesis and vaccine development.

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Host-adapted F. tularensis produce more OAg capsule and bind less Ab directed against OM constituents.A) Intact bacteria (WT and wbtA), grown in MHB (M) or BHI (B), were incubated with the indicated sera/Ab, washed and probed for IgG heavy chain by western blot. The probed membranes were stripped and re-probed for total signals for the indicated proteins. B) Surface-bound Ab-to-FopA ratios from 5–8 independent experiments were quantified and expressed relative to that of the MHB-grown WT with same Ab. Asterisks indicate values that differed significantly (p≤0.05, T-test with Bonferonni correction). C) MHB- and MΦ- grown F. tularensis were incubated with Ab specific for the OAg-capsule and Tul4 and analyzed as in A above. Mean FopA-normalized, surface Ab values from 3 independent experiments are indicated along with standard deviations in parentheses.
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pone-0022335-g003: Host-adapted F. tularensis produce more OAg capsule and bind less Ab directed against OM constituents.A) Intact bacteria (WT and wbtA), grown in MHB (M) or BHI (B), were incubated with the indicated sera/Ab, washed and probed for IgG heavy chain by western blot. The probed membranes were stripped and re-probed for total signals for the indicated proteins. B) Surface-bound Ab-to-FopA ratios from 5–8 independent experiments were quantified and expressed relative to that of the MHB-grown WT with same Ab. Asterisks indicate values that differed significantly (p≤0.05, T-test with Bonferonni correction). C) MHB- and MΦ- grown F. tularensis were incubated with Ab specific for the OAg-capsule and Tul4 and analyzed as in A above. Mean FopA-normalized, surface Ab values from 3 independent experiments are indicated along with standard deviations in parentheses.

Mentions: To probe the surface of F. tularensis for OAg capsule and OM accessibility we incubated intact bacteria with various Ab and probed the washed bacteria for bound immunoglobulin heavy chain (HC) by western blot. Following development of the Ig HC signals, we re-probed the membranes for total FopA and quantified the data as surface Ab/total FopA and normalized the ratios to the corresponding MHB result. As shown in Fig. 3A and B, when F. tularensis was incubated with immune mouse serum (IMS) the amount of IgG bound to the surface of BHI-grown, WT F. tularensis was significantly reduced relative to that of its MHB-grown counterpart. The amount of IgG bound to the surface of MHB-grown, WT F. tularensis and the BHI-grown, OAg mutant was not significantly different (Fig 3B) and there were no significant differences in the amount of IgM bound to the surfaces of these three bacteria (Fig 3B).


Host-adaptation of Francisella tularensis alters the bacterium's surface-carbohydrates to hinder effectors of innate and adaptive immunity.

Zarrella TM, Singh A, Bitsaktsis C, Rahman T, Sahay B, Feustel PJ, Gosselin EJ, Sellati TJ, Hazlett KR - PLoS ONE (2011)

Host-adapted F. tularensis produce more OAg capsule and bind less Ab directed against OM constituents.A) Intact bacteria (WT and wbtA), grown in MHB (M) or BHI (B), were incubated with the indicated sera/Ab, washed and probed for IgG heavy chain by western blot. The probed membranes were stripped and re-probed for total signals for the indicated proteins. B) Surface-bound Ab-to-FopA ratios from 5–8 independent experiments were quantified and expressed relative to that of the MHB-grown WT with same Ab. Asterisks indicate values that differed significantly (p≤0.05, T-test with Bonferonni correction). C) MHB- and MΦ- grown F. tularensis were incubated with Ab specific for the OAg-capsule and Tul4 and analyzed as in A above. Mean FopA-normalized, surface Ab values from 3 independent experiments are indicated along with standard deviations in parentheses.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3142145&req=5

pone-0022335-g003: Host-adapted F. tularensis produce more OAg capsule and bind less Ab directed against OM constituents.A) Intact bacteria (WT and wbtA), grown in MHB (M) or BHI (B), were incubated with the indicated sera/Ab, washed and probed for IgG heavy chain by western blot. The probed membranes were stripped and re-probed for total signals for the indicated proteins. B) Surface-bound Ab-to-FopA ratios from 5–8 independent experiments were quantified and expressed relative to that of the MHB-grown WT with same Ab. Asterisks indicate values that differed significantly (p≤0.05, T-test with Bonferonni correction). C) MHB- and MΦ- grown F. tularensis were incubated with Ab specific for the OAg-capsule and Tul4 and analyzed as in A above. Mean FopA-normalized, surface Ab values from 3 independent experiments are indicated along with standard deviations in parentheses.
Mentions: To probe the surface of F. tularensis for OAg capsule and OM accessibility we incubated intact bacteria with various Ab and probed the washed bacteria for bound immunoglobulin heavy chain (HC) by western blot. Following development of the Ig HC signals, we re-probed the membranes for total FopA and quantified the data as surface Ab/total FopA and normalized the ratios to the corresponding MHB result. As shown in Fig. 3A and B, when F. tularensis was incubated with immune mouse serum (IMS) the amount of IgG bound to the surface of BHI-grown, WT F. tularensis was significantly reduced relative to that of its MHB-grown counterpart. The amount of IgG bound to the surface of MHB-grown, WT F. tularensis and the BHI-grown, OAg mutant was not significantly different (Fig 3B) and there were no significant differences in the amount of IgM bound to the surfaces of these three bacteria (Fig 3B).

Bottom Line: Lastly we show that upon host-adaptation, the human-virulent strain, F. tularensis SchuS4 also induces capsule production with the effect of reducing macrophage-activation and accelerating tularemia pathogenesis in mice.These capsules impede recognition of bacterial outer membrane constituents by antibody, complement, and Toll-Like Receptor 2.These changes in the host-pathogen interface have profound implications for pathogenesis and vaccine development.

View Article: PubMed Central - PubMed

Affiliation: Center for Immunology and Microbial Disease, Albany Medical College, Albany, New York, United States of America.

ABSTRACT

Background: The gram-negative bacterium Francisella tularensis survives in arthropods, fresh water amoeba, and mammals with both intracellular and extracellular phases and could reasonably be expected to express distinct phenotypes in these environments. The presence of a capsule on this bacterium has been controversial with some groups finding such a structure while other groups report that no capsule could be identified. Previously we reported in vitro culture conditions for this bacterium which, in contrast to typical methods, yielded a bacterial phenotype that mimics that of the bacterium's mammalian, extracellular phase.

Methods/findings: SDS-PAGE and carbohydrate analysis of differentially-cultivated F. tularensis LVS revealed that bacteria displaying the host-adapted phenotype produce both longer polymers of LPS O-antigen (OAg) and additional HMW carbohydrates/glycoproteins that are reduced/absent in non-host-adapted bacteria. Analysis of wildtype and OAg-mutant bacteria indicated that the induced changes in surface carbohydrates involved both OAg and non-OAg species. To assess the impact of these HMW carbohydrates on the access of outer membrane constituents to antibody we used differentially-cultivated bacteria in vitro to immunoprecipitate antibodies directed against outer membrane moieties. We observed that the surface-carbohydrates induced during host-adaptation shield many outer membrane antigens from binding by antibody. Similar assays with normal mouse serum indicate that the induced HMW carbohydrates also impede complement deposition. Using an in vitro macrophage infection assay, we find that the bacterial HMW carbohydrate impedes TLR2-dependent, pro-inflammatory cytokine production by macrophages. Lastly we show that upon host-adaptation, the human-virulent strain, F. tularensis SchuS4 also induces capsule production with the effect of reducing macrophage-activation and accelerating tularemia pathogenesis in mice.

Conclusion: F. tularensis undergoes host-adaptation which includes production of multiple capsular materials. These capsules impede recognition of bacterial outer membrane constituents by antibody, complement, and Toll-Like Receptor 2. These changes in the host-pathogen interface have profound implications for pathogenesis and vaccine development.

Show MeSH
Related in: MedlinePlus