Figure 1.
Flagella identification by transmission electron microscopy.
(A) C. sakazakii ATCC BAA-894 strain shows flagellar structures protruding from the bacteria after growth on TSA agar at 37°C. (B) Immunogold-labeling of flagella produced by C. sakazakii on TSA agar using anti-flagella antibody. The inset shows the micrograph of the whole bacteria using immunogold-labeling obtained by TEM. (C) Purified flagella. (D) Immunogold-labeling of purified flagella. Samples were negatively stained and electron micrographs were taken at a magnification of 19,000x.
Figure 2.
Biochemical analysis of flagella.
(A) Depolymerization of purified flagella in 12% SDS-PAGE gel. (B) Immunoblot assays with anti-flagella antibody recognizing the flagellin of ∼28-kDa. (C) Analysis of the protein band of ∼28-kDa by mass spectrometry. Molecular weight (MW).
Figure 3.
Multiple sequence alignment of flagellin from C. sakazakii ST1 ATCC BAA-894, C. sakazakii ST4 ATCC 29004, C. malonaticusT, C. muytjensiiT, C. turicensisT, and C. dublinensisT (Cronobacter_flagellin) with different known Gram-negative bacterial flagellins.
Sequences were aligned using the ClustalW progam. C. sakazakii, C turicensis, Enterobacter spp., Pectobacterium carotovorum subsp. Carotovorum, Citrobacter koseri ATCC BAA-895, Yersinia enterocolitica, Pantoea ananatis LMG 20103, and Yersinia pseudotuberculosis IP 32953.
Figure 4.
Flagella from C. sakazakii activate cytokine’s release.
Macrophage cells were exposed to flagella (1, 10, 25, 50 and 100 ng/ml) and flagellin (100 ng/ml Tx) for 24 h. (A) High values were observed in the IL-8 release due to the presence of different concentrations of purified flagella from Cronobacter species. (B) Induction of TNF-α in macrophage cells in response to different concentrations of purified flagella from Cronobacter species. (C) A non-significant release of IL-10 was observed when two different concentrations of purified flagella from Cronobacter species were incubated. In addition, cytokines release was also observed when the purified flagella of each Cronobacter species were dissociated in monomers by heat. Lipopolysaccharide 100 ng/ml (LPS), RPMI-1640 medium. Flagellin of Cronobacter (Tx). Flagella of EHEC strain O157:H7 and Flagella from S. enterica serovar Typhimurium (100 ng/ml). * and ** p<0.05.
Figure 5.
Anti-flagella antibodies block the secretion of pro-inflammatory cytokines.
Purified flagella (100 pg/ml) and flagellin (100 ng/ml) were pre-incubated with anti-flagella antibodies in LPS-free water before the addition to macrophage cells. The antibodies were tested undiluted and using different dilutions (1∶10, 1∶100, and 1∶200). (A) IL-8 release was inhibited when flagella (94, 88, 48 and 48%) and flagellin (96, 94, 52 and 51%) were incubated with different dilutions of anti-flagella. (B and C) TNF-α and IL-10 releases were blocked completely with the three dilutions and with the undiluted antibody. RPMI-1640 medium.
Figure 6.
Anti-human-TLR5-IgA antibody blocked the secretion of pro-inflammatory cytokines.
(A) An inhibition dependent on concentration of anti-hTLR5-IgA was observed in IL-8 release, in flagella (51, 55, and 59%) and flagellin (21, 52 and 53%) with 1, 10 and 20 µg of antibody, respectively. (B) TNF-α release was blocked between 75–97% for flagella and 13–58% for flagellin with 1 and 10 µg anti-human-IgA-TLR5, respectively. (C) IL-10 release was blocked 98% for flagella and 99% for flagellin with 1 and 10 µg anti-human-IgA-TLR5, respectively. RPMI-1640 medium.
Figure 7.
Transfection assay in HEK293-hTLR5 cells enables IL-8 secretion in response to C. sakazakii flagella and flagellin.
293-hTLR5 cells (HEK293 cells transfected with TLR5) secreted 197 pg/ml and 133 pg/ml of IL-8 after treatment with C. sakazakii (ST1) flagella and flagellin, respectively; and 196 pg/ml of IL-8 were released with FliC-Salmonella enterica serovar Typhimurium. 293-hTLR5 cells pre-incubated with anti-hTLR5-IgA antibodies at a concentration of 10 µg/ml showed a reduction of 76% in IL-8 release.