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Purified flagellin activated the mitogen activated protein kinase (MAPK), stress-activated protein kinase (SAPK) and Ikappa B kinase (IKK) signaling pathways that lead to expression of the proinflammatory gene program in a temporal fashion nearly identical to that of infection of intestinal epithelial cells by Salmonella. Flagellin expression was required for Salmonella invasion of host cells and it activated NF-κB via toll-like receptor 5 (TLR5). Surprisingly, a number of cell lines found to be unresponsive to flagellin express TLR5 and expression of exogenous TLR5 in these cells induces NF-κB activity in response to flagellin challenge although not robustly. Conversely, overexpression of dominant-negative TLR5 alleles only partially blocks NF-κB activation by flagellin. These observations are consistent with the possibility of either a very stable TLR5 signaling complex, the existence of a low abundance flagellin co-receptor or required adapter, or both.

Since Salmonella sp. infection of intestinal epithelial cells in culture led to only roughly thirty percent infection but activation of NF-κB in nearly all of the cells, we anticipated that NF-κB activation was in response to host cell recognition of bacteria structural components or products produced by the bacteria and not by the invasion process. Invasion itself has been demonstrated not to be required for activation of the proinflamatory gene program as had previously been thought [16]. To investigate this possibility sterile-filtered S. dublin culture broth left either untreated or boiled for twenty minutes was used to challenge HT29 intestinal epithelial cells and NF-κB DNA binding activity was monitored by electromobility shift assays (EMSAs) of whole cell extracts (WCE) prepared forty-five minutes after exposure [3, 35]. Potent activation of NF-κB in response to the broth under both conditions was observed indicating the activating factor was heat-stable (AD, TT and JD, personal observations) and is not LPS since HT29 cells are not responsive to LPS [3, 35].

Protein factor in Salmonella culture broth leads to NF-κB activation. A, Salmonella dublin culture broth concentrated 100-fold was treated as indicated or infectious bacteria, as indicated was used to challenge HT29 cells. NF-κB DNA binding activity was assayed by EMSA from whole cell extracts prepared 45 min after treatment. Authenticity of the NF-κB DNA:protein complex was determined using p65(RelA)-specific and p50-specific antibody supershifts. B, Concentrated Salmonella dublin culture broth (IN) was chromatographed by gel permeation on a Superose 12 column. Eluted protein fractions were analyzed by fractionation on 10% SDS-PAGE and visualized by Coomassie blue (CB) staining. Molecular weight markers for chromatography and on the gels are indicated. Aliquots of each fraction as indicated was used to stimulate HT29 cells and resultant WCEs were analyzed by EMSA for NF-κB DNA binding activity. C, Concentrated Salmonella dublin culture broth (IN) was chromatographed by anion exchange chromatography on POROS HQ matrix. Proteins were eluted with an increasing NaCl gradient as indicated and analyzed on 10% SDS-PAGE and visualized by Coomassie blue (CB) staining. Input and aliquots of each fraction as indicated was used to stimulate HT29 cells and resultant WCEs were analyzed by EMSA for NF-κB DNA binding activity. Eluted material corresponding to protein bands B1-B6, a blank portion of the gel was isolated from a duplicate 10% SDS-PAGE gel as described in Experimental Procedures along with buffer samples from the beginning and end NaCl buffer gradient and used to stimulate HT29 cells and resultant WCEs were analyzed by EMSA for NF-κB DNA binding activity.

To determine if flagellin was indeed the factor that was responsible for triggering activation of NF-κB after exposure of intestinal epithelial cells to direct bacterial infection or to filtered culture broths of pathogenic Salmonella sp. we prepared infectious bacteria and boiled and filtered culture broths from the non-flagellated E. Coli DH5α, pathogenic S. dublin strain 2229, an isogenic S. dublin 2229 SopE- mutant, isogenic S. dublin 2229 SopB- mutant, isogenic S. dublin 2229 double SopE-/SopB- mutant (strain SE1SB2), S. typhimurium strain 1103, and isogenic S. typhimurium fliC::Tn10 insertion mutant (strain 86) and a S. typhimurium 1103 isogenic double mutant fliC-/fljB- and were used to challenge HT29 cells. Bacteria and culture broths were used to challenge HT29 intestinal epithelial cells and WCE extracts were prepared after forty-five minutes and analyzed for NF-κB DNA binding activity by EMSA. Salmonella strains could activate NF-κB, while Salmonella strains failing to produce flagellin (fliC and fliC-/fljB- mutants as indicated) also failed to activate NF-κB (Fig. 4A &4B). E. Coli DH5α is non-flagellated and does not produce flagellin failed to activate NF-κB. We also noticed through numerous experiments that S. dublin direct infections always activated NF-κB to a greater extent than S. typhimurium as observed in Fig. 4A while culture broths from both species activated NF-κB almost equally well (Fig. 4B). We believe this difference is due perhaps to S. dublin releasing more flagellin into the cell culture media than S. typhimurium during infection since purification of flagellin from both S. dublin and S. typhimurium and addition of equivalent amounts of chromatographically purified flagellin gave similar NF-κB activation profiles (TT & JD, unpublished observations).

Flagellin mutants fail to activate NF-κB. EMSAs assaying for NF-κB DNA binding activity in WCEs prepared 45 min from non-infected cells (UN) and after direct infection of HT29 cells with wild-type E. coli DH5α, wild-type Salmonella dublin or SopE- mutant, SopB- mutant, the SopE-/SopB- double mutant, wild-type Salmonella typhimurium strain 1103, the fliC- mutant (fliC::Tn10), the fliC-/fljB- double mutant as indicated at an MOI of 50. B, EMSAs assaying for NF-κB DNA binding activity in WCEs prepared 45 min after challenge of HT29 cells from non-infected cells (UN) or with sterile-filtered concentrated culture broths from wild-type and mutant bacteria as indicated.

The fliC-/fljB- double mutant Salmonella failed to invade HT29 cells compared to the wild-type Salmonella strain as determined by gentamycin protection/invasion assay (see Experimental Procedures). The flagellin fliC-/fljB- double mutant displayed a four orders of magnitude difference in its ability to invade HT29 cells (TT & JD, unpublished observations). To demonstrate this point further, we infected HT29 cells with either wild-type Salmonella or the fliC-/fljB- double mutant Salmonella (strain 134), both strains were transformed with the plasmid pFM10.1 that encodes GFP under the control of the Salmonella ssaH promoter and only functions once the bacteria has invaded the host cell [10, 34]. The wild-type Salmonella clearly was able to infect HT29 cells (GFP, Fig. 5B) while the flagellin mutant bacteria failed to invade HT29 cells as evidenced by the lack of GFP expression (Fig. 5B). To determine if flagellin is sufficient or that other bacterially produced proteins are required for invasion, we added either purified flagellin or sterile-filtered culture broths or a combination of both to HT29 cells that were challenged with the Salmonella fliC-/fljB- double mutant and assayed for invasion. Intestinal epithelial cells failed to be invaded using all tested combinations of purified flagellin and/or culture broths with the fliC-/fljB- double mutant strain (TT & JD, unpublished observations). To our knowledge there is no known direct connection between expression of flagellin genes and the effectiveness of the type III secretion system to deliver bacterially produced proteins such as SopE, SopE2 and SipA or other Sip or Sop proteins [7, 14, 15, 40, 41] that play important roles in initiating bacterial internalization. Furthermore, to evaluate the effectiveness of flagellin to stimulate p65 (RelA) nuclear localization in intestinal epithelial cells we challenged HT29 cells with purified flagellin and examined p65 (RelA) localization using indirect immunofluorescence and found p65 (RelA) nuclear localization in nearly every cell (Fig. 5B as indicated).

We wished to further examine the effect of purified flagellin and flagellin present on Salmonella on the temporal pattern of proinflammatory cytokine gene expression in intestinal epithelial cells in order to differentiate the effects of flagellin alone vs. flagellated Salmonella or non-flagellated Salmonella infection. HT29 cells were left untreated, stimulated with TNFα (10 ng/ml), or stimulated with flagellin (0.5 ug/ml), or infected with wild-type Salmonella typhimurium or the Salmonella fliC/fljB double mutant (at MOI of 50). After the indicated times after treatment or infection, HT29 cells were harvested in ice-cold PBS and the cell pellets lysed in Trizol and RNA was purified and used to prepare first-strand cDNA (see Experimental Procedures). Aliquots of the cDNA were used in semi-quantitative RT-PCR reactions using IL1α, IL-1β, IL-8, TNFα, MCP1 and β-actin gene specific primers (sequences available upon request) and the products were fractionated on ethidium bromide containing 1.2% agarose gels. Expression of the known NF-κB target genes IL-1β, IL-8, TNFα and MCP1 was increased in response to TNFα or purified flagellin exposure (Fig. 6C). Wild-type Salmonella infection also led to activation of these same genes although the expression of TNFα and MCP1 was transient in comparison and occurred immediately after infection. The Salmonella fliC-/fljB- double mutant failed to induce IL-1β, IL-8 and TNFα expression, however MCP1 expression was induced, although at lower levels than that induced by wild-type Salmonella, and also, the expression of MCP1 was not transient in nature and continued throughout the time course (9 h) (Fig. 6C). The expression level of β-actin served as an internal standard for comparison. Interestingly, IL-1α, which is not an NF-κB target gene was stimulated in response to HT29 cell challenge by all of the treatments. Obviously, the Salmonella fliC-/fljB- double mutant can activate other signaling pathways leading to IL-1α expression. We presently do not know what these signaling pathways are. 2b1af7f3a8

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