Salmonella typhimurium Coordinately Regulates FliC Location and Reduces Dendritic Cell Activation and Antigen Presentation to CD4+ T cells

Robert C. Alaniz, Lisa A. Cummings, Molly A. Bergman, Sara L. Rassoulian-Barrett and Brad T. Cookson
J Immunol September 15, 2006, 177 (6) 3983-3993; DOI: https://doi.org/10.4049/jimmunol.177.6.3983

Abstract

During infection, Salmonella transitions from an extracellular-phase (STEX, growth outside host cells) to an intracellular-phase (STIN, growth inside host cells): changes in gene expression mediate survival in the phagosome and modifies LPS and outer membrane protein expression, including altered production of FliC, an Ag recognized by immune CD4+ T cells. Previously, we demonstrated that systemic STIN bacteria repress FliC below the activation threshold of FliC-specific T cells. In this study, we tested the hypothesis that changes in FliC compartmentalization and bacterial responses triggered during the transition from STEX to STIN combine to reduce the ability of APCs to present FliC to CD4+ T cells. Approximately 50% of the Salmonella-specific CD4+ T cells from Salmonella-immune mice were FliC specific and produced IFN-γ, demonstrating the potent immunogenicity of FliC. FliC expressed by STEX bacteria was efficiently presented by splenic APCs to FliC-specific CD4+ T cells in vitro. However, STIN bacteria, except when lysed, expressed FliC within a protected intracellular compartment and evaded stimulation of FliC-specific T cells. The combination of STIN-mediated responses that reduced FliC bioavailability were overcome by dendritic cells (DCs), which presented intracellular FliC within heat-killed bacteria; however, this ability was abrogated by live bacterial infection. Furthermore, STIN bacteria, unlike STEX, limited DC activation as measured by increased MHC class II, CD86, TNF-α, and IL-12 expression. These data indicate that STIN bacteria restrict FliC bioavailability by Ag compartmentalization, and together with STIN bacterial responses, limit DC maturation and cytokine production. Together, these mechanisms may restrain DC-mediated activation of FliC-specific CD4+ T cells.

The Gram-negative bacterium Salmonella enterica serovar Typhimurium (Salmonella typhimurium) replicates both outside and inside eukaryotic cells and causes gastroenteritis in humans and systemic infection in mice (1). After oral infection, Salmonella penetrates the intestinal epithelium via M cells to colonize the Peyer’s patches (PPs)4 (2). Salmonella then disseminates via the bloodstream to colonize the spleen and liver where it survives and replicates within the intracellular vacuoles of professional phagocytes (3). Salmonella modifies its gene expression during invasion, dissemination, and colonization, and these transitions occur in response to dynamic host environmental signals, such as Mg2+ (4), osmolarity (5), pH (6), O2 tension (7), bile salts (8), and cationic antimicrobial peptides (CAMPs) (9). Salmonella has therefore acquired an array of strategies to survive these varied host environments (10, 11).

The transition to an intracellular phase is a key bacterial virulence mechanism and is required for Salmonella survival in the host; bacteria unable to survive in phagocytes are avirulent in vivo (6). Salmonellae modify the expression of >40 genes, such as slyA (12) and genes controlled by the PmrA/PmrB and PhoP/PhoQ regulatory systems (4, 13), to promote intracellular bacterial survival in the phagosome (14). Wild-type (WT) bacteria grown in conditions simulating a eukaryotic phagosome (8 μM Mg2+) (4, 15) or mutant bacteria expressing excess PhoP (a global regulator of genes expressed in the phagosome) (6) in its active phosphorylated state (PhoPc) are models of intracellular-phase Salmonella (STIN). STIN bacteria specifically modify the bacterial membrane to promote survival (16). The most studied STIN outer membrane modifications occur in the LPS and include decreased O-Ag length (17), increased acylation of lipid A to a hepta-acylated form (PhoP-dependent) (18), and additions of aminoarabinose and phosphoethanolamine (PmrA dependent) (8, 13), and 2-hydroxymyristate (PhoP dependent) (17). These modifications reduce the inflammatory properties of lipid A (LPS) and increase STIN bacteria resistance to CAMPs and bile salts (8, 16). STIN bacteria also modify the proteins of the outer membrane through increased expression of Mig14, VirK, and PgtE that render STIN bacteria more resistant to CAMPs (19, 20). It is unclear what other advantages these modifications may have for bacterial survival and/or evasion.

FliC, the protein monomer of flagellin and the ligand for TLR5 (21, 22), is a major proinflammatory agent in vivo (23, 24). FliC promoter activity and transcription, regulated in a PhoP-dependent manner, are repressed in infected macrophages (14, 25), and STIN bacteria reduce FliC expression below a level required to stimulate TLR5 in vitro (26), putatively to limit recognition by innate host defenses. Although resistance to innate immunity is important for early Salmonella survival in vivo (16), bacterial mechanisms that inhibit recognition of Salmonella by adaptive immunity may also provide a considerable survival advantage.

Adaptive immunity to Salmonella infection relies not only on B cells (27, 28) but also on CD4+ T cells and the production of IFN-γ in vivo; mice lacking CD4+ T cells or those that fail to make IFN-γ do not survive challenge with avirulent Salmonella (29). FliC is one important Ag recognized by B and T cells from previously infected mice and humans (30, 31) and is a protective Ag after immunization (32, 33). However, after Salmonella infection and systemic colonization, adoptively transferred FliC-specific SM1-transgenic CD4+ T cells proliferate in the PPs, but not in the spleen (34), suggesting that FliC-specific CD4+ T cells may only provide protection in select compartments in vivo. The lack of FliC-specific T cell activation in the spleen is consistent with our recent data showing that APCs infected in vivo at a systemic site (spleen) fail to activate FliC-specific CD4+ T cells in vitro (25, 26). These results suggest that Salmonella modify FliC expression in different host tissues, and because FliC is not only a TLR5 ligand but also a target of CD4+ T cells and B cells, we hypothesized that multiple mechanisms are required to precisely regulate the bioavailability of FliC by Salmonella. We define the bioavailability of an Ag or ligand as expression in a biological context, at a level, or in a location that is accessible to innate and/or adaptive immune systems for acquisition, processing, or detection. Immunogenicity is a function of Ag bioavailability and is, therefore, bioavailable Ag for APC processing, and presentation is central to the initiation of host-adaptive T and B cell responses. Although the efficiency of T cell priming in vivo can be proportional to Ag dose (35), the effects of compartmentalization on Ag bioavailability and recognition by T cells have mostly been explored with model protein Ags expressed at supraphysiologic levels from constitutive promoters (36, 37, 38, 39, 40). Ag compartmentalization studies have not addressed a natural bacterial Ag, such as FliC, in the context of surface modifications of STIN bacteria that reduce the inflammatory properties of LPS and increase bacterial resistance to innate immunity. STIN modifications may further reduce the bioavailability of microbial Ags for APC presentation to T cells and may incompletely stimulate dendritic cells (DCs) resulting in suboptimal signals for efficient T cell activation (41), giving Salmonella a growth advantage in vivo.

In this study, we assess mechanisms that limit the bioavailability of FliC, an important natural Ag of Salmonella recognized by CD4+ T cells. We demonstrate that Salmonella transitioning from an extracellular-phase (STEX) to STIN express FliC in a restricted bacterial compartment that reduces its bioavailability for efficient processing and presentation by APCs to FliC-specific T cells. The restricted expression of FliC occurs in the context of STIN bacteria that avoid fully activating DC maturation and production of TNF-α and IL-12, cytokines important for host defense to Salmonella infection. These observations reveal that reducing the bioavailability of FliC is a complex and multifaceted modification that occurs when Salmonella transitions to STIN growth.

Materials and Methods

Mice and immunizations

Six- to 8-wk-old female C3H/HeJ and C3H/HeN mice were obtained from The Jackson Laboratory and used at 6–14 wk of age. Consistent with our previously established model where immunization with viable attenuated S. typhimurium primes FliC-specific CD4+ T cell responses (25, 26, 31, 42, 43), susceptible C3H/HeJ mice were used for in vitro T cell stimulation assays and infected DC experiments (see below). To rigorously evaluate the ability of Salmonella surface modifications to reduce DC activation (43), we used DCs from C3H/HeN mice. Mice were immunized by oral infection with 109 S. typhimurium SL3261 (an aroA derivative of SL1344) by gavage with feeding needles (22 × 1½ with 1¼-mm ball, no. 7920; Popper & Sons). All mice were housed in specific pathogen-free conditions and cared for in accordance with University of Washington (Seattle, WA) Institutional Animal Care and Use Committee guidelines. Immune mice were used at 70–100 days postimmunization.

Generation of Salmonella FliC export mutants

The TnlacZ/in transposon mutagenesis technique described by Manoil and Bailey (44), was used to generate 31-aa in-frame insertion mutations in a FliC expressing plasmid (pTrcFliC). Plasmid DNA was isolated, and the insertion sites of ISlacZ/in were determined by DNA sequencing using the ABI BigDye terminator cycle sequencing ready reaction mix (PE Applied Biosystems) and the primer TnlacZ-II (5′ CGGGATCCCCCTGGATGG 3′). In-frame ISlacZ/in insertion derivatives were converted into 31-codon insertions by digesting with BamHI to remove all but 31 codons of the insertion sequence and religating the digested plasmid. The 31-codon insertion sequence (i31, where N, determined by the insertion site, is any nucleotide, and underlined base pairs indicate the BamHI site) is as follws: 5′ NCT GAC TCT TAT ACA CAA GTA GCG TCC TGG ACG GAA CCT TTC CCG TTT TCC ATC CAG GGG GAT CCA AGA TCT GAT CAA GAG ACA GNN NNN NNN. Plasmids carrying mutant alleles were transferred to flagellin-negative SL1344 (BC696) by electroporation. Total FliC expressed by each mutant was separated into intracellular (In-FliC) and extracellular (Ex-FliC) fractions: log-phase bacteria were incubated at 60°C for 20 min to depolymerize surface-attached flagella and centrifuged to separate extracellular monomers from intact bacteria. Plasmid-bearing mutants were selected for defective export phenotypes that limited FliC expression to a monomeric form located within an intracellular or extracellular compartment, as well as for WT FliC expression, normally expressed as a polymer on the surface of WT salmonellae. Western immunoblot analysis (described below) confirmed the FliC export phenotype of each strain. Motility was measured 18 h after inoculation to Luria-Bertani motility (0.4% agar) plates. Suicide vector pCVD442 (λpir-dependent replicon, AmpR, sacB gene conferring sucrose sensitivity) was used to replace the WT fliC allele on the chromosome as described previously (45). Briefly, plasmid pCVD442 carrying a mutant fliC allele was transferred to FljB-null SL1344 (BC687) by conjugation, and the resulting AmpR Salmonella were then grown in sucrose-containing medium to select for loss of sacB gene and plasmid DNA. Individual AmpS, sucroseR colonies were screened by PCR to identify the presence of the mutant allele, and the predicted motility and FliC export phenotype of each strain was confirmed as described above (see also Results). Three chromosomal mutants were chosen with unique FliC export phenotypes: FliCWT, where FliC is exported and polymerized on the bacterial surface as a filament (fliC D287::i31, with i31 in-frame insert following residue D287, BC737); FliCOUT, where FliC is only exported as a monomer (fliC N444::i31, BC739); and FliCIN, where FliC is retained intracellularly (fliC N444::i31-LR, BC762; contains i31 inserted at N444 followed by the last two residues of FliC at the C terminus). FliCWT, FliCOUT, and FliCIN all retained the IAk-restricted FliC339–350 epitope (42).

Bacterial Ags

All cultures were grown at 37°C in indicated medium. Bacterial Ags were prepared by heating bacterial cultures for 1 h at 65°C. STEX Ags were prepared from S. typhimurium strain ST14028 (ATCC) grown without aeration in TSB (BD Diagnostic Systems) containing 1% NaCl. STIN Ags were prepared from ST14028 pho-24 pmrA505 (BC497) grown in TSB with aeration at pH5.8 (STIN (PhoPc)) (25). Alternatively, STIN Ags were prepared by diluting overnight cultures of ST14028 grown with aeration in TSB 1/150 into N-minimal medium containing 8 μM Mg2+ and 34 mM glycerol (15) and incubating an additional 18–24 h with aeration (STIN (lowMg2+)). The FliC concentration of individual Ag preparations was determined by serial titration and Western blot analysis; dilutions with equivalent FliC and CFU concentrations were used as Ag. To generate bare bacterial Ag (bareST, in which FliC expression is confined to the intracellular compartment of intact bacteria), stationary-phase SL1344, grown under extracellular-phase conditions (see above), was heated to 65°C for 20 min and washed in PBS to depolymerize and remove surface-attached flagella; removal of extracellular FliC was confirmed by dot blot and Western blot analysis. Purified FliC monomers were prepared by removing flagella from logarithmic phase bacteria expressing only FliC (BC687) by mechanical shearing (46), followed by heating to depolymerize flagellar filaments. The resulting FliC monomers were passed through a Centricon YM-10 filtration unit (Millipore) to remove LPS, and were confirmed to be free of contaminating Ags as described previously (26). FliC also was purified from mutant strains after separation of whole bacteria by SDS-10% PAGE as described previously (31). Briefly, after electrophoretic separation, protein was eluted from appropriate gel sections, polyacrylamide was removed by filtration (Spin-X; Corning) and SDS-PAGE buffer replaced with PBS by diafiltration through Microcon YM-10 filtration units (Millipore). The concentration of gel-purified FliC protein was measured by ELISA: samples were coated to MaxiSorp 96-well plates (Nalge Nunc International) and probed with rabbit anti-FliC Ab (1/500 dilution, preabsorbed with FliC-null Salmonella before use; Accurate Chemical & Scientific), followed by HRP-labeled donkey anti-rabbit Ab (1/2000 dilution; Amersham Biosciences) and visualized using TMB-peroxidase substrate (Kirkegaard & Perry Laboratories).

Bacterial lysis

For restimulation of T cell lines, log-phase bacteria were disrupted by sonication on ice in four 1-min bursts at maximum setting using a probe sonicator. Unlysed cells were removed by centrifugation, and protein concentrations were determined using the bicinchoninic acid protein assay (Pierce). For all other experiments, stationary-phase cultures were boiled for 10 min in 0.2% SDS, treated with DNase (Invitrogen Life Technologies), and centrifuged to remove cellular debris. SDS was removed by buffer exchange using Microcon YM-10 filtration units before use as Ag.

Immunoblot analysis

Bacterial cultures were either spotted directly to nitrocellulose or transferred to nitrocellulose after separation by SDS-10% PAGE (∼107 CFU per sample). Membranes were probed with polyclonal anti-FliC (preabsorbed with ΔFliC/FljB Salmonella (BC379); Denka Seiken), or monoclonal anti-DnaK (StressGen), followed by goat anti-rabbit or anti-mouse IgG conjugated to HRP (Santa Cruz Biotechnology). Reactive HRP was detected by ECL (Amersham Biosciences).

T cell lines and clones

Salmonella-immune T cell lines, and FliC-specific T cell clone 3A7 were generated as described previously (26, 42). T cells were grown in RPMI 1640 supplemented with l-glutamine, 50 μΜ 2-ME, 10% FCS with antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin, and 50 μg/ml gentamicin) at 37°C in 7% CO2, and restimulated by coincubation with naive splenocyte APCs and Ag at 14- to 17-day intervals.

T cell stimulation assays

Irradiated 105 splenocyte APCs or 104 bone marrow-derived DC APCs (described below) and 104 T cells were combined in 96-well plates and assayed with serial dilutions of Ag in triplicate. Ag remained present throughout culture. 1 μCi of [3H]TdR (NEN) was added after 48 h and incorporation of 3H into newly synthesized DNA was measured after an additional 18–20 h using liquid scintillation spectrophotometry. All SEMs of triplicate samples were <10% of the mean, and for clarity of presentation, the error bars are not shown. Alternatively, IFN-γ secretion was measured from culture supernatant 72 h after stimulation with Ag and detected by sandwich ELISA (BD Pharmingen).

Infected DC APCs

DCs were derived from naive mice by in vitro culture of bone-marrow cells with 20 ng/ml GM-CSF (R&D Systems) for 7 days according to standard methods (47). DCs were infected with titrated CFU of stationary-phase bacteria in antibiotic-free medium for 15 min, washed, and incubated for an additional 2–3 h in medium containing 15 μg/ml gentamicin. After infection, DCs remained viable and were fixed with 0.2% paraformaldehyde/HBSS for 20 min, washed extensively, and resuspended in culture medium before use as APCs in T cell stimulation assays. Intracellular CFU were measured immediately before fixation by lysis of infected cells with 0.2% Triton X-100, and plating dilutions of cell lysate on to Luria-Bertani agar. Infected DCs retained full APC function for stimulating T cells (25).

Intracellular cytokine staining and flow cytometry

Production of cytokines by host cells in response to Salmonella Ags was detected by intracellular cytokine staining (ICS). Briefly, 4 × 106 splenocytes taken directly ex vivo from Salmonella-immune mice or 2.5 × 105 DCs cultured from naive mice were coincubated with titrations of HKAg, 25 μg/ml FliC monomer, 100 ng/ml LPS, or 12.5 μg/ml Ab to murine CD3 (clone 145.2C11) in single wells of 96-well U-bottom tissue culture plates. After overnight incubation at 37°C in 5% CO2, GolgiPlug (BD Biosciences) was added at 1/1000 dilution for the final 6 h (splenocyte) or 4 h (DC) of incubation, and cells were washed twice in cold PBS plus 0.5% (w/v) BSA (Sigma-Aldrich) (PBSA), and surface stained in the presence of Fc block (clone 2.4G2) with PE-labeled Abs to murine CD4 (L3T4, clone H129.19) or CD11c (clone HL3). Stained cells were fixed on ice with 2% paraformaldehyde and permeabilized with Perm/Wash Buffer followed by staining with APC-labeled Abs to murine IFN-γ (clone XMG1.2), TNF-α (clone MP6-XT22), or IL-12 (clone C15.6). Cells were resuspended in PBSA and stored at 4°C in the dark until analysis. For detection of DC maturation, DCs were surfaced stained with biotin-conjugated Abs to MHC-II (I-Ek, clone 14-4-4S) or to CD86 (clone B7-2) plus streptavidin-allophycocyanin after stimulation with Salmonella Ags as described for ICS. Unstained cells or cells stained with isotype control Ab were used as negative controls (data not shown). All flow cytometry reagents and Abs were purchased from BD Pharmingen unless otherwise noted. Data were acquired on a LSR 6 color analyzer (BD Biosciences) and analyzed using FlowJo Software (Tree Star).

Statistical analysis

Values of p < 0.05 were considered significant and determined using Student’s t test and the ANOVA test for multiple comparisons.

Results

FliC stimulates robust IFNγ secretion by CD4+ T cells recovered from Salmonella-immune mice

FliC-specific CD4+ T cells are not activated in systemic lymphoid organs after oral Salmonella infection (34), which is surprising for a pathogen capable of causing systemic infection (48). Further, the absence of FliC expression, during bacterial growth in the spleen (25) is in apparent conflict with FliC as a protective Ag (32, 33). To more thoroughly investigate the contribution of FliC-specific CD4+ T cells to the memory CD4+ T cell pool in immune mice, we quantified long-term FliC-specific memory CD4+ T cells from immune mice directly ex vivo, using intracellular cytokine staining (ICS). Long-term memory CD4+ T cells, specific for both Salmonella (Fig. 1⇓, first row) and FliC Ag (Fig. 1⇓, second row) were abundant in immune mice. In multiple experiments, FliC-specific CD4+ T cells represented ∼50% of the total Salmonella-specific CD4+ T cells, indicating the potent capacity of FliC Ag to prime T cells in vivo. Similar results from multiple murine haplotypes (H-2k, Fig. 1⇓; and H-2b, data not shown) firmly establish FliC as a prominent target Ag recognized by long-term memory CD4+ T cells from mice infected previously with Salmonella (31, 32).

FIGURE 1.
FIGURE 1.

The flagellar subunit protein FliC stimulates robust IFN-γ secretion by CD4+ T cells recovered from Salmonella-immune mice. IFN-γ production by CD4+ T cells from the spleens of Salmonella-immune C3H/HeN mice (70–100 days postinfection) was measured by intracellular cytokine staining (ICS) directly ex vivo after overnight stimulation with intact FliC+ Salmonella, purified FliC, or anti-CD3 Ab (as a positive control). Data are presented as two-color density plots; the percentage of CD4+ cells expressing IFN-γ among total CD4+ cells is indicated. CD4+ T cell responses to total Salmonella Ag were comparable to anti-CD3 stimulation, indicative of the maximal memory CD4+ T cell response, and confirmed our ability to detect responses from the full CD4+ T cell long-term memory compartment. Data are representative of three independent experiments.

Intracellular FliC sequestration by intact Salmonella is not sufficient to reduce Ag bioavailability for presentation to CD4+ Tcells

Studies with Salmonella vaccine strains have suggested that the immunogenicity of model protein Ags is reduced upon expression within the intracellular compartment (36, 37, 49). However, the influence of compartmentalization on processing and presentation of a natural bacterial Ag, such as FliC has not been studied. Because flagellar FliC is surface exposed, we predicted that restricting its expression to a different bacterial compartment would change its bioavailability as an Ag for CD4+ T cells. Therefore, we generated mutant Salmonella strains that expressed FliC in different bacterial compartments (Fig. 2⇓A and Materials and Methods). FliCWT bacteria export and polymerize FliC on the surface; FliCIN retain FliC monomer exclusively inside the bacterial cell; FliCOUT export FliC as a monomer; ΔFliC produce no FliC (Fig. 2⇓B). Proliferation of the FliC-specific CD4+ T cell clone (3A7) that recognizes the FliC339–350 epitope in the context of H-2k (26, 42) was used to assess the ability of APCs to present FliC expressed by these bacterial strains. FliC Ag prepared from intact FliCWT and FliCOUT bacteria was readily presented to the FliC-specific T cell clone, whereas FliC from intact FliCIN bacteria failed to stimulate FliC-specific T cell proliferation (Fig. 2⇓C, left). Although these results appear to support the hypothesis that the intracellular location of FliC expressed by the FliCIN strain prevented APCs from presenting this Ag, lysed FliCIN bacteria failed to stimulate FliC-specific T cell proliferation (Fig. 2⇓C, right). To test whether FliC from the lysed FliCIN strain was somehow sequestered from presentation by insoluble bacterial fragments, intracellular FliC from each FliC mutant was purified and quantified (see Materials and Methods). Purified intracellular FliC from FliCIN had a dramatically reduced capacity to stimulate T cell proliferation, compared with purified FliC from the FliCWT and FliCOUT strains (Fig. 2⇓D). Because FliCIN bacteria expressed the stimulatory FliC339–350 epitope (42), we concluded that the altered Ag export properties by the FliCIN strain severely reduced FliC antigenicity by an unknown mechanism and thus precluded additional experiments with the FliCIN strain. Instead, we pursued an alternate strategy to test the hypothesis that Ag sequestered to an intracellular bacterial compartment reduces Ag bioavailability and prevents FliC presentation to T cells. To do this, we prepared WT Salmonella bacterial Ag with the surface-exposed FliC present (WTST) or removed ((bareST), containing only intracellular FliC) (Fig. 3⇓A and Materials and Methods). The location of FliC in intact bacteria, before (bareSTintact) or after lysis (bareSTlysed), was confirmed by immunoblot (Fig. 3⇓B). Both bareSTintact and bareSTlysed provided stimulatory Ag for FliC-specific T cell proliferation (Fig. 3⇓C) indicating that the FliC expressed inside intact Salmonella was available for presentation by APCs. Thus, confining FliC expression to the intracellular bacterial compartment is not singularly sufficient to reduce Ag bioavailability. Yet, as a facultative intracellular pathogen, Salmonella transitions from an extracellular-phase to an intracellular-phase in response to host environmental cues (10, 25). Our observations (Fig. 3⇓) used Salmonella grown in conditions promoting the extracellular phase (see Materials and Methods). Therefore, we tested how the intracellular phase affected FliC localization and bioavailability.

FIGURE 2.
FIGURE 2.

Mutant FliC expressed within an intracellular Salmonella compartment is nonimmunogenic. Salmonella expressing mutated fliC alleles encoding surface polymeric FliC (FliCWT), FliC that is retained intracellularly (FliCIN), or extracellular monomeric FliC (FliCOUT) were used to study the effect of compartmentalized FliC on T cell responses; FliC-null Salmonella (ΔFliC) are included as a negative control. FliC expression for all mutants was determined by motility (A) and Western blot analysis (B); FliC-specific T cell proliferation in response to intact (C, left) and lysed (C, right) bacteria was measured after confirming equivalent Ag levels by Western blot analysis (C, inset). FliC-specific T cell proliferation also was measured using SDS-PAGE purified FliC Ag from each Salmonella mutant (D). All SEMs of triplicate samples were <10% of the mean, and for clarity of presentation the error bars are not shown. ∗, p = 0.0006. Data are representative of two independent experiments.

FIGURE 3.
FIGURE 3.

Intracellular FliC expressed by intact Salmonella can be processed and presented efficiently to CD4+ T cells in vitro. A, Surface exposed FliC was removed from intact bacteria as described in Materials and Methods. B, FliC expression was confined to the intracellular compartment of the resulting “bare” bacteria (bareSTintact) as confirmed by immunoblot analysis: FliC was detectable by specific Ab only after bacterial lysis (bareSTlysed). C, Equivalent proliferation of a FliC-specfic T cell clone in response to APCs incubated with bareSTintact or bareSTlysed indicates that FliC from these intact bacteria can be efficiently processed and presented to CD4+ T cells. As a positive control, WTST (105 CFU per ml) stimulated proliferation (15 kcpm) from FliC-specific T cells. All SEMs of triplicate samples were <10% of the mean, and for clarity of presentation, the error bars are not shown. There was no significant difference in proliferation when T cells were stimulated with bareSTintact and bareSTlysed Ags. Data are representative of three experiments.

STIN naturally compartmentalizes FliC, which is protected from detection by specific Ab and unavailable for presentation by APCs to T cells

To determine whether FliC compartmentalization contributes to Ag regulation in the extracellular- to intracellular-phase transition, bacteria were selected to represent STEX (identical growth conditions as WTST in Fig. 3⇑) (7) or STIN (PhoPc and lowMg2+) (4, 15). FliC expression from intact or lysed STEX and STIN bacteria was compared by immunoblot analysis and revealed the presence of abundant surface FliC on intact STEX, but not on intact STIN (PhoPc and lowMg2+) (Fig. 4⇓A). However, FliC was detected after lysis of STEX and STIN (lowMg2+), indicating the presence of intracellular FliC in both strains (Fig. 4⇓B). These data demonstrate that the FliC Ag naturally compartmentalized by STIN (lowMg2+) was inaccessible to specific Ab (Fig. 4⇓A); an important finding given the fact protective immunity to virulent Salmonella infection requires humoral immunity (27, 28). As expected, STIN (PhoPc) did not express measurable FliC (26). The presence of detectable FliCIN in STIN (lowMg2+), in contrast with STIN (PhoPc), indicates that STIN (lowMg2+) bacteria represent Salmonella in the transition from STEX to STIN during the initial stages of infection (11), whereas STIN (PhoPc) bacteria uniquely recapitulate a more terminal intracellular-phase of Salmonella colonizing systemic sites (25, 26). Therefore, because STIN (PhoPc) bacteria did not express surface or intracellular FliC and do not stimulate FliC-specific CD4+ T cells (25), STIN (lowMg2+) bacteria were used for subsequent T cell studies.

FIGURE 4.
FIGURE 4.

Salmonella growth in low Mg2+ (loMg2+) naturally limits FliC expression to an intracellular bacterial compartment. Bacterial Ags were chosen to represent those Ags expressed in vivo by a facultatively intracellular pathogen (replicates both inside and outside of host cells). STEX = S. typhimurium ST14028 grown in conditions that simulate extracellular growth during penetration of the GI barrier (7 ); STIN (PhoPc) = ST14028 carrying the pho-24 allele that up-regulates the PhoP/PhoQ regulon, a global virulence system regulating ∼40 genes during intracellular growth in the phagosome (4 ,6 ); STIN (lowMg2+) = ST14028 grown in conditions (8 μM Mg2+) that stimulate transcription of genes required for intracellular survival (15 ). FliC expression was characterized by FliC-specific immunoblot analysis: equal numbers of intact bacteria were spotted directly to nitrocellulose (A) or transferred to nitrocellulose after separation by SDS-PAGE (B). FliC expressed by intact STEX bacteria was readily detected by specific Ab (A), whereas FliC expressed by STIN (lowMg2+) bacteria was detected only after bacterial lysis (B), indicating that FliC was naturally limited to an intracellular bacterial compartment. Total loaded protein was equivalent for all samples as determined by DnaK-specific Western blot analysis (C). Data are representative of two experiments.

Because STIN bacteria modify LPS and resist CAMPs (13, 17), and naturally compartmentalize FliC (Fig. 4⇑), we hypothesized that bacterial responses associated with the transition to STIN, and altered Ag sequestration, would combine to reduce FliC bioavailability and prevent APCs from presenting this Ag to T cells. FliC from intact STEX Ag was efficiently presented by APCs to FliC-specific CD4+ T cells (Fig. 5⇓A) as indicated by robust proliferation; in contrast, FliC from intact STIN (lowMg2+) Ag, which was exclusively intracellular (Fig. 4⇑), was not presented. However, when intracellular Ags were released by lysis of STIN (lowMg2+), FliC became available for presentation (Fig. 5⇓A), indicating the bacterial responses associated with the transition to STIN (lowMg2+) limited presentation of intracellular FliC.

FIGURE 5.
FIGURE 5.

Intact STIN bacteria limit presentation of FliC Ag to CD4+ T cells. A, STIN (lowMg2+) bacteria were tested for ability to stimulate FliC-specific T cell proliferation before and after bacterial lysis; intact STEX and FliC-null Salmonella (ΔFliC) are shown as positive and negative controls, respectively. The recovery of stimulatory activity after lysis indicates that the intracellular FliC Ag expressed by STIN bacteria is immunogenic, but that mechanical lysis of intact STIN is required for efficient processing and presentation of Ag to FliC-specific T cells. All SEMs of triplicate samples were <10% of the mean, and for clarity of presentation, the error bars are not shown. B, After in vivo priming with attenuated FliC+ Salmonella, CD4+ T cells were selectively amplified in vitro with bacterial Ags presented by naive splenocyte APCs to establish individual T cell lines. Each line is identified by the Ag used for the selective amplification. C, The proliferative response of CD4+ T cell lines to purified FliC confirms the presence of FliC-specific T cells only in lines TEX and TIN (lysed), demonstrating the ability of lysed, but not intact STIN bacteria to support propagation of FliC-specific T cells from previously infected mice. D, The equivalent responses of all T cell lines to ΔFliC Salmonella confirm that intact STIN bacteria provide Ag(s) capable of supporting the propagation of Salmonella-specific T cell populations in vitro and formally exclude the possibility that STIN bacteria are inhibitory for either Ag presentation or T cell proliferation. Data are representative of two independent experiments. ∗, p < 0.001.

These data support the notion that the reduced bioavailability of an Ag encapsulated inside intact bacteria could delay activation of Ag-specific T cells (36, 38). Therefore, we examined the ability of intact STIN (lowMg2+) (Fig. 4⇑) to supply stimulatory Ag for FliC-specific CD4+ T cell expansion over several rounds of restimulation during a long-term culture in vitro (25). In these conditions, APCs have several days to acquire and present FliC inside STIN (lowMg2+) bacteria. Salmonella immune splenocytes, containing FliC-specific and non-FliC-specific T cells (Fig. 1⇑ and Ref. 25), were selectively restimulated in vitro (see Materials and Methods) with intact STEX, intact STIN (lowMg2+), or lysed STIN (lowMg2+) bacteria to generate the polyclonal Salmonella-specific T cell lines TEX, TIN (intact), and TIN (lysed), respectively (Fig. 5⇑B). Proliferative responses to purified FliC were used to reveal whether or not FliC-specific CD4+ T cells were present in each line. TIN (intact) failed to proliferate in the presence of purified FliC (Fig. 5⇑C). This indicated that intact STIN (lowMg2+) bacteria contain intracellular FliC (Fig. 4⇑) that is not bioavailable to APCs for presentation to FliC-specific T cells present in the original input immune splenocytes (Fig. 1⇑). However, FliC-specific proliferation by lines TEX and TIN (lysed) (Fig. 5⇑C) indicated that the STEX and lysed STIN (lowMg2+) bacterial Ags stimulated the expansion of FliC-specific T cells. This confirmed that unlike intact STIN (lowMg2+) bacteria, the FliC expressed by STEX and lysed STIN (lowMg2+) bacteria was capable of supporting FliC-specific T cell proliferation (Fig. 5A⇑) and long-term expansion (Fig. 5⇑C). Importantly, all three lines (TEX, TIN (lysed), and TIN (intact)) responded to Ag prepared from FliC-null Salmonella (Fig. 5⇑D). This confirmed that STIN (lowMg2+) bacteria could propagate Salmonella-specific T cells with non-FliC specificities, and that STIN (lowMg2+) bacteria were neither cytotoxic nor inhibitory for either APCs or T cells. These data (Figs. 4⇑ and 5⇑) support the idea that bacterial responses triggered by phagosomal growth conditions (26), as found with intact STIN (lowMg2+) bacteria, make compartmentalized FliC unavailable for APC presentation to CD4+ T cells.

DCs infected with STIN fail to activate FliC-specific CD4+ T cells

DCs possess superior Ag presentation abilities and are the major APCs for initiating pathogen-specific T cell responses in vivo (41, 50). Therefore, we hypothesized that DCs could overcome the reduced bioavailability of FliC expressed by STIN (lowMg2+) bacteria and present FliC to T cells. Indeed, DCs pulsed with STEX, intact STIN (lowMg2+), or lysed STIN (lowMg2+) bacteria stimulated nearly equivalent FliC-specific T cell proliferation (Fig. 6⇓A), in contrast with splenocyte APCs (Fig. 5⇑). However, when DCs were infected with STIN (lowMg2+) bacteria in vitro (Fig. 6⇓, B and C), their ability to present FliC to T cells was abrogated. DCs infected with STIN (lowMg2+) required ∼1000-fold more bacteria, compared with DCs infected with STEX to stimulate FliC-specific CD4+ T cell proliferation (Fig. 6⇓B) and IFN-γ secretion (Fig. 6⇓C). These observations demonstrate that infected DCs are competent to activate Salmonella-specific T cells (25) and support our previous findings that APCs, infected in vitro and in vivo with STIN bacteria, only stimulate Salmonella-specific T cells responding to non-FliC Ags. These data establish that DCs fail to present intracellularly compartmentalized FliC Ag expressed by live STIN (lowMg2+) bacteria.

FIGURE 6.
FIGURE 6.

After infection with STIN bacteria, DCs fail to efficiently process and present FliC Ag. A, Bone marrow-derived DCs processed and presented FliC equally well from exogenously added intact or lysed STIN bacterial Ag; intact STEX, and ΔFliC bacteria are included as positive and negative controls, respectively. There was no significant difference in proliferation when T cells were stimulated with DCs presenting intact or lysed STIN bacterial Ag. B and C, After in vitro infection with STEX, STIN (lowMg2+) or FliC-null (ΔFliC) Salmonella (see Materials and Methods), DCs were unable to efficiently stimulate FliC-specific T cell responses after STIN (lowMg2+) infection as measured by proliferation (B) or IFN-γ secretion (C) (see Materials and Methods). DCs infected with STIN (lowMg2+) required ∼1000-fold more bacteria than DCs infected with STEX to stimulate FliC-specific CD4+ T cell proliferation (B) and IFN-γ secretion (C). All SEMs of triplicate samples were <10% of the mean, and for clarity of presentation, the error bars are not shown. ∗, DCs presenting STIN differs significantly from STEX for T cell stimulation, p < 0.002. Data are representative of two independent experiments.

STIN restrict robust DC maturation and cytokine secretion in vitro

Microbes that succeed in limiting effective DC maturation or proinflammatory cytokine secretion may achieve an advantage in vivo because suboptimal APC signals serve to limit T cell activation (41). We therefore tested the hypothesis that STIN (lowMg2+) bacteria, which compartmentalize FliC and reduce its bioavailability (Figs. 3–6⇑⇑⇑⇑), would fail to promote efficient DC maturation and proinflammatory cytokine production. Bone marrow-derived DCs exhibited an immature phenotype in vitro characterized by low MHC-II (Fig. 7⇓A) and CD86 (Fig. 7⇓B) expression. At low multiplicity of infection (MOI), representing the initial bacterial–host cell interaction early after infection in vivo (2, 51, 52), STIN (lowMg2+ and PhoPc) bacteria failed to stimulate efficient DC maturation, compared with STEX, which stimulated robust DC maturation as measured by increased MHC-II and CD86 expression (Fig. 7⇓, A and B). At higher MOI, both STEX and STIN (lowMg2+ and PhoPc) bacteria induced maximal DC maturation (Fig. 7⇓, A and B). Consistent with the reduced ability of STIN bacteria to promote DC maturation (Fig. 7⇓), reduced proinflammatory cytokine production by DCs in response to STIN (lowMg2+ and PhoPc) bacteria also was observed: STIN (lowMg2+ and PhoPc) bacteria induced ∼10-fold less TNF-α (Fig. 8⇓A) and IL-12 (Fig. 8⇓B), compared with STEX at low MOIs. These data demonstrate that STIN bacteria stimulate suboptimal DC expression of MHC-II, CD86, TNF-α, and IL-12 and support the notion that, in the microenvironment of a T cell–DC interaction, independent bacterial responses may combine to produce significant limitations for T cell activation. In this model (Fig. 9⇓), we hypothesize STIN bacteria restrict FliC bioavailability through Ag compartmentalization and membrane remodeling and limit DC maturation and cytokine production. During early Salmonella infection when bacterial burden is initially low, these mechanisms may facilitate evasion of DC-mediated activation of FliC-specific CD4+ T cells in vivo.

FIGURE 7.
FIGURE 7.

STIN bacteria avoid inducing efficient DC maturation in vitro. DC maturation marker expression was measured after stimulation with intact STEX, STIN (PhoPc), or STIN (lowMg2+) bacteria as described in the Materials and Methods. Representative samples after stimulation are presented as histograms; percentages of high MHC-II (A) or CD86 (B) expression of gated CD11c+ cells are indicated. Unstimulated DCs (Unstim), representing immature DCs, are included as a negative control; LPS (100 ng/ml) stimulation is included as a positive control. Data are representative of two independent experiments.

FIGURE 8.
FIGURE 8.

STIN bacteria avoid stimulating robust cytokine secretion by DCs in vitro. The production of TNF-α (A) or IL-12 (B) from DCs was measured by ICS after stimulation with intact STEX, STIN (PhoPc), or STIN (lowMg2+) bacteria as described in Materials and Methods. Representative samples after stimulation are presented as density plots; the percentage of cytokine positive CD11c+ DCs is indicated. Unstimulated DCs (Unstim) are included as a negative control; LPS (100 ng/ml) stimulation is included as a positive control. Data are representative of two independent experiments.

FIGURE 9.
FIGURE 9.

Intracellular-phase Salmonella regulates FliC location and coordinately reduces DC activation and Ag presentation. Salmonella, a facultatively intracellular pathogen, replicates both inside and outside of host cells. STEX bacteria provide abundant, accessible FliC and potent inflammatory stimuli for DCs: DCs that acquire STEX (during invasion or directly from gut lumen) efficiently present FliC to Ag-specific CD4+ T cells (Tc) and induce robust T cell expansion and effector function (IFN-γ production) due to increased expression of TNF-α, IL-12, and MHC-II/peptide+CD86 complexes (left). We hypothesize that when small numbers of STEX bacteria penetrate the intestinal mucosa (52 ) and transition to STIN bacteria, a process critical for survival inside phagocytes (77 ), STIN restrict access of FliC to DCs in conjunction with outer membrane modifications that reduce their proinflammatory signature (right); a phenomenon that may be relevant in vivo. Although FliC from STEX bacteria is fully bioavailable, APCs may have access to either STEX or STIN bacteria after natural oral infection because the transition to STIN may initially begin in the intestine before entering a host APC (78 ,79 ,80 ). Although FliC may be available from other bacterial sources, DCs that encounter and acquire STIN do not efficiently present FliC or provide signals (TNF-α, IL-12, and MHC-II:peptide:+CD86) to induce T cell activation (proliferation and IFN-γ). These observations augment our previous models of FliC regulation (25 ,80 ) by establishing an additional level of Ag regulation: coordinate Ag compartmentalization, Ag repression, and surface modifications all have deleterious effects on the ability of CD4+ T cells to respond to Salmonella-infected APCs, and thus could have ramifications for FliC-specific T cell priming and/or recognition of infected host cells. PRR, microbial pattern recognition receptor.

Discussion

Protective CD4+ T cell-mediated immunity against infection with Salmonella requires the recognition of bacterial protein Ags processed and presented by APCs on MHC-II molecules. However, salmonellae use multiple strategies to escape detection by host innate and adaptive immunity (1). In this study, we establish that memory FliC-specific CD4+ T cells are an abundant fraction of the total Salmonella-specific CD4+ T cells in Salmonella immune mice. This observation is consistent with previous studies showing that immunization with FliC, an important Ag also recognized by B cells (27), induces protective immunity (32, 33). In addition, we demonstrate that, unlike STEX, STIN bacteria reduce the bioavailability of FliC by restricting FliC expression exclusively within the bacterial cell. However, FliC expression inside bacteria, per se, is not sufficient to reduce Ag bioavailability for presentation to T cells and FliC must be encapsulated inside STIN bacteria. In this microbial context, FliC is protected from detection by specific Ab and unavailable for presentation by APCs to FliC-specific CD4+ T cells. Furthermore, STIN bacteria avoid efficiently stimulating DC maturation and proinflammatory cytokine production, intrinsic functions for DCs as the preeminent initiators of T cell-mediated host defense in vivo (41), which compounds the influences of compartmentalized FliC in limiting activation of FliC-specific T cells. Taken together, our results directly address natural mechanisms of Salmonella immunopathogenesis impacted by altering Ag bioavailability. Through a combination of Ag compartmentalization (Fig. 4⇑) and molecular modifications of the bacterial cell (Figs. 7⇑ and 8⇑), we illuminate novel mechanisms that Salmonella may use in vivo to reduce their recognition by both the innate and adaptive arms of host immunity (Fig. 9⇑). These data support the concept that Ag availability for T cell activation is not solely dependent on Ag quantity (25), but also relies on the biological context of Ag expression (35, 36, 37, 38, 39, 40).

This study augments our previous findings demonstrating systemic STIN bacteria repress FliC expression to a level that fails to activate FliC-specific CD4+ T cells to proliferate or produce IFN-γ (25). Consistent with this observation, transferred FliC-specific SM1 Tg T cells are activated in the PP, but not in the spleen, after oral infection (34). Together, these data support the notion that Salmonella reduces FliC expression at systemic sites to evade detection by FliC-specific CD4+ T cells and suggest that FliC Ag is only available in the gut for T cell priming in vivo. These results also suggest that, compared with STIN (lowMg2+), STIN (PhoPc) bacteria may represent the more terminal phase(s) of systemic STIN bacteria found in vivo as FliC protein is undetectable in this strain after lysis (Fig. 4⇑). In contrast to STIN (PhoPc) bacteria, STIN (lowMg2+) bacteria may represent salmonellae at an early transitional phase: they contain detectable FliC Ag that is not surface exposed and is expressed exclusively inside bacteria. Furthermore, we establish that STIN (lowMg2+) bacteria restrict FliC bioavailability and provide suboptimal activation stimuli to DCs, a phenomenon that may be compounded in vivo by uptake of Salmonella by PP DCs that are predisposed to suppress inflammation (53). Together, these mechanisms may serve to limit the magnitude or the quality of FliC-specific CD4+ T cell priming in vivo and permit intracellular Salmonella to avoid immune detection, and after penetrating the GI barrier, to colonize systemic sites (3, 25) and/or establish chronic infection (54).

In addition to regulating FliC bioavailability, STIN bacteria also use mechanisms capable of directly inhibiting Ag presentation to T cells. Others have shown, compared with WT STEX bacteria, STIN (PhoPc) bacteria inefficiently provide OVA to macrophages for presentation to Ag-specific MHC-II-restricted T cells, whereas the use of DCs restored Ag presentation efficiency (55). Similarly, heat-inactivated STIN (PhoPc) bacteria fail to inhibit Ag presentation, suggesting PhoP-regulated gene products may be responsible for decreased Ag presentation when infecting with live STIN (PhoPc) (56). Although Ag compartmentalization or bacterial surface modifications were not directly examined, these studies support our findings: DCs, but not splenic APCs, present FliC from STIN to T cells, and live infection with STIN abrogates DC presentation of FliC Ag (Fig. 5⇑ and 6⇑). Our observations correlate with data showing STIN (lowMg2+) salmonellae actively interfere with DC Ag presentation, phagosomal maturation, and MHC-II expression through the production of type III secretion system effector proteins encoded on Salmonella-pathogenicity island 2 (SPI-2) (57). Recent studies show OVA-pulsed DCs infected with WT Salmonella have lower MHC-II expression and stimulate decreased OVA-specific T cell activation, in contrast with DCs infected with mutant strains lacking the SPI-2 effectors SseB/C and SifA (58, 59). These results required infection of DCs with live Salmonella, consistent with our observations (Figs. 5⇑ and 6⇑), because heat-inactivated bacteria were not inhibitory (58). Importantly, in our studies using infected DC APCs, STIN (lowMg2+) bacteria stimulated nearly equivalent levels of proliferation from non-FliC-specific T cells as did STEX (25), indicating Ag presentation of native Salmonella Ags occurs despite any bacterial mediated SPI-2 effector-dependent inhibition of APC function that may occur. Notably, the use of plasmids for constitutive heterologous Ag expression at supraphysiologic levels (56, 58, 59) differs substantially from the highly evolved regulation of a natural Ag, such as FliC. In addition, we examined Ag presentation after infection with numbers of bacteria representing those that penetrate the gut (46) and in the absence of Ab opsonization (58, 60), which is only likely to play a role in vivo after the activation of Salmonella-specific B cells. Our system reflects FliC expression under physiologic conditions and reveals how the bacterial transition to STIN restricts the bioavailability of FliC for presentation to T cells. We propose that, during infection in vivo, the combination of inhibitory SPI-2 effector proteins (58), and regulated FliC abundance (25) and location (Fig. 4⇑), couple with global surface modifications (26) to reduce FliC bioavailability and limit activation of FliC-specific CD4+ T cells (Fig. 9⇑).

In contrast with acute infections caused by bacteria capable of overwhelming host defenses, chronic or persistent infections, such as those caused by salmonellae, result from pathogens using a number of strategies in vivo to limit host innate and adaptive recognition (1). Salmonella limits innate immune recognition of LPS and flagellin (FliC) by TLR4 and TLR5, respectively, through surface modifications after the transition to STIN (25, 26, 61). The importance of immune recognition of LPS is illustrated by the fact C3H/HeJ mice (nonfunctional TLR4) are susceptible to Salmonella infection (62) and have delayed adaptive immune responses (43), compared with WT C3H/HeN mice. LPS-specific modifications by STIN bacteria increases their resistance to innate CAMPs (8); similar CAMP resistance mediated by LPS modifications occurs in Pseudomonas aeruginosa isolates from chronic cystic fibrosis patients (63). In addition, chronic human pathogens in ε Proteobacteria, represented by Helicobacter pylori and Campylobacter jejuni, express flagella that are functional but do not stimulate TLR5 (64). Helicobacter pylori uses evasion of TLR5 recognition to achieve a survival advantage in vivo (65). Human adapted Salmonella typhi establish a persistent infection in vivo, and expression of the Vi capsular Ag reduces host IL-8 production and PMN recruitment to the intestinal mucosa (66). These findings illustrate that microbes use multiple mechanisms to reduce innate-mediated recognition and inflammation and that regulating FliC (flagellin) bioavailability is another important method of evasion.

Targeting DC function is likely a beneficial strategy for many microbes because DCs exist to acquire Ag and activate T cells. Consistent with the notion that major modifications associated with STIN bacteria occur at the outer membrane (16, 26), DCs stimulated with STIN bacteria fail to mature (Fig. 7⇑) or produce TNF-α and IL-12 (Fig. 8⇑), two critical cytokines that promote protective immunity to intracellular bacteria (67). DCs are also susceptible to inhibition by Type III secretion of SPI-2 effectors (57). Bordetella bronchiseptica establishes chronic pulmonary infection in mice and uses Type III secretion of adenylate cyclase toxin to drive DCs into semimature/toleragenic state (68). Mycobacterium tuberculosis modifies the phagosome of APCs to reduce bacterial degradation and inhibit Ag processing (69). Furthermore, malaria-causing Plasmodium species render DCs inhibitory for T cell responses (70), refractory to TLR stimulation, and deficient for proinflammatory cytokine (TNF-α, IL-12) production (71). These observations illustrate how pathogens modulate host defenses by inhibiting the functions of DCs, the pivotal cellular link between innate and adaptive immunity (41).

Similar to the Salmonella regulation of FliC, other successfully persistent pathogens lower the bioavailability of important T cell Ags. The protective T cell Ag, ESAT-6, made during early growth by M. tuberculosis is later reduced, promoting evasion of Ag-specific T cells and the establishment of chronic infection (72). Plasmodium species rarely elicit sterilizing host immunity; partially through the expression of polymorphic Ags, but mainly through phenotypic phase variation (sporozoite and merozoite stages) as a mechanism to remodel their antigenic mosaic and to evade T cell recognition (73). Furthermore, Leishmania promastigotes rapidly change to an amastigote stage during infection, replicate in macrophages, and reduce and sequester from presentation Ags recognized by CD4+ T cells, e.g., LACK (74, 75). These observations suggest multiple microbes, including Salmonella, use related evasion strategies, including regulation of Ag bioavailability, to remain below the detection threshold of host surveillance: mechanisms that promote survival and persistence in vivo.

We speculate mechanisms, partly accomplished through STIN surface modifications, that decrease Salmonella bioavailability for innate and adaptive immune recognition are responsible for the continuous shedding of S. typhimurium from healthy humans after resolution of clinical disease. Our study also supports the notion that surface modifications may play a role during transitional phases of S. typhi infection by altering the molecular composition of surface structures, or physically masking their antigenic properties, or inhibiting their detection by innate and adaptive immune responses (76).

Acknowledgments

We thank the members of the Cookson laboratory for their support and critical review of the manuscript and for providing a stimulating and collegial environment for scientific discovery. We especially thank Matthew L. Johnson for maintenance and experimentation with T cell lines and clones.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was supported by National Institutes of Health Grant AI47242.

  • ↵2 Current address: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02112.

  • ↵3 Address correspondence and reprint requests to: Dr. Brad T. Cookson, Department of Laboratory Medicine and Microbiology, University of Washington Medical Center, Box 357110, 1959 Northeast Pacific Street, Seattle, WA 98195. E-mail address: cookson{at}u.washington.edu

  • ↵4 Abbreviations used in this paper: PP, Peyer’s patch; CAMP, cationic antimicrobial peptide; STIN, intracellular-phase Salmonella; STEX, extracellular-phase Salmonella; DC, dendritic cell; ICS, intracellular cytokine staining; MHC class II, MHC-II; MOI, multiplicity of infection; SPI-2, Salmonella-pathogenicity island 2.

  • Received November 29, 2005.
  • Accepted June 26, 2006.

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