CD4+ T cells and toll-like receptors recognize Salmonella antigens expressed in bacterial surface organelles - PubMed (original) (raw)

CD4+ T cells and toll-like receptors recognize Salmonella antigens expressed in bacterial surface organelles

Molly A Bergman et al. Infect Immun. 2005 Mar.

Abstract

A better understanding of immunity to infection is revealed from the characteristics of microbial ligands recognized by host immune responses. Murine infection with the intracellular bacterium Salmonella generates CD4+ T cells that specifically recognize Salmonella proteins expressed in bacterial surface organelles such as flagella and membrane vesicles. These natural Salmonella antigens are also ligands for Toll-like receptors (TLRs) or avidly associated with TLR ligands such as lipopolysaccharide (LPS). PhoP/PhoQ, a regulon controlling Salmonella virulence and remodeling of LPS to resist innate immunity, coordinately represses production of surface-exposed antigens recognized by CD4+ T cells and TLRs. These data suggest that genetically coordinated surface modifications may provide a growth advantage for Salmonella in host tissues by limiting both innate and adaptive immune recognition.

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Figures

FIG. 1.

FIG. 1.

_Salmonella_-specific CD4+ T cells from orally immunized mice recognize multiple bacterial antigens. _Salmonella_-specific CD4+ T cells derived from immune mice (line MAR.D) were assayed for proliferative responses to SDS-PAGE-fractionated bacteria (see Materials and Methods). Responses of CD4+ T-cell clones 7.4.8 and F4 are also shown. Molecular weights (MW; 103) of protein standards are shown at the top; arrows indicate responses to fraction 10 and FliC (approximately 52,000). The data shown are representative of four experiments.

FIG. 2.

FIG. 2.

CD4+ T-cell clone F4 recognizes antigen expressed in bacterial MVs. Concentrated S. enterica serovar Typhimurium culture supernatant was (i) separated by size exclusion chromatography, and fractions were used as stimulatory antigen in proliferation assays with _Salmonella_-specific CD4+ T-cell clone F4 (molecular weight standards: fluorescein-labeled nitrobenzoxadiazole [NBD] phosphatidylcholine vesicles [% fluorescence intensity], thyroglobulin, and albumin [A280]) (A) or (ii) examined by electron microscopy (B). Bar = 100 nm. The data shown are representative of three experiments.

FIG. 3.

FIG. 3.

The unique composition of MVs includes surface-exposed outer membrane proteins. Concentrated MVs were compared to whole bacteria (WB) by SDS-PAGE with Coomassie staining (A) and Western analysis for the presence of cytoplasmic-inner membrane (SecA) and outer membrane (OmpA) marker proteins (isolated subcellular bacterial fractions containing cytoplasm and periplasm [CYT/PP] or inner-outer membrane [IM/OM] proteins are provided for comparison) (B) or treated with or without proteinase K (PK) in the presence or absence of detergent and used as the stimulatory antigen in F4 proliferation assays (C). (D) Surface exposure of F4 stimulatory antigen was confirmed by conversion from a 10-kDa to a 5-kDa species after proteinase K treatment of intact bacteria.

FIG. 4.

FIG. 4.

Bacterial surface organelles contain natural antigens recognized by CD4+ T cells. CD4+ T-cell clones, derived from _Salmonella_-infected mice in the absence of the dominant FliC antigen, recognize either antigens in MVs (n = 12 clones) (A) or the alternate flagellin molecule FljB (n = 12 clones) (B). Proliferative responses of representative T-cell clones were measured in the presence of isolated MVs (102 dilution), purified FljB protein (micrograms per milliliter), or FljB+ FliC− or FljB− FliC− salmonellae (CFU per milliliter).

FIG. 5.

FIG. 5.

Bacterial surface organelles contain natural antigens recognized by TLRs. CHO cells transiently transfected with expression vectors for TLR2 (A) or TLR5 (B), together with an NF-κB luciferase reporter, were used to assess the ability of MVs and purified FljB to serve as TLR ligands. The data shown are representative of three experiments.

FIG. 6.

FIG. 6.

PhoP/PhoQ negatively regulates the expression of natural Salmonella antigens recognized by CD4+ T cells and TLRs. Wild-type (WT) bacteria were compared with PhoP− and PhoP-activated (PhoPc, PhoP*) strains for the ability to stimulate FljB-specific clone D3D6 (A), FliC-specific clone 3A7 (B), and MV-specific T-cell clone F4 (C) or TLR5-dependent NF-κB activation in CHO cells (D). Representative data from multiple experiments are shown.

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