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Amino acids 89–96 of Salmonella flagellin: a key site for its adjuvant effect independent of the TLR5 signaling pathway

Cellular & Molecular Immunology volume 14, pages 1023–1025 (2017)Cite this article

Campylobacter jejuni and Helicobacter pylori possess specific amino-acid changes in the TLR5 recognition site of flagellin, and these changes can abolish TLR5 recognition.9 To determine whether the alteration of aa 89–96 of flagellin could affect recognition by TLR5 and downstream signaling, Zhang et al replaced aa 89–96 of the S. typhimurium flagellin gene fliC with the corresponding flagellin (flaA) sequence from C. jejuni. The aa 89–96-mutant flagellin was obtained using a flagellated recombinant Salmonella strain (pTrc99a-fliC-89–96, flagellin with the native conformation) or expressed by Escherichia coli (pET30a-fliC-89–96). The pTrc99a-fliC-89–96 flagellin mutant induced a marked reduction in IL-8 secretion compared with that induced by pTrc99a-fliC-WT in HEK293-mTLR5 cells expressing the murine TLR5 under low stimulus concentrations. However, pET30a-fliC-89–96 flagellin was unable to induce IL-8 production in HEK293-mTLR5 cells, consistent with a previous report showing the loss of this ability in a mutant expressed by E. coli in which aa 89–96 of flagellin was deleted.10 Considering that E. coli-expressed flagellin may not retain all of the characteristics of native flagellin, it is reasonable to infer that pTrc99a-fliC-89–96 flagellin and pET30a-fliC-89–96 flagellin have different TLR5 activation abilities. These findings also suggest that other sites (in addition to aa 89–96) are responsible for TLR5 activation in flagellin, as confirmed by a recent report.11

Zhang et al. further evaluated the innate immune responses induced by flagellin mutants by measuring pro-inflammatory cytokines in vivo. The intraperitoneal injection of wild-type (WT) flagellin induced a robust secretion of cytokines, including IL-6 and tumor necrosis factor-α, in mice; in contrast, the aa 89–96 fliC→flaA mutant flagellin did not evoke substantial production of these cytokines. These observations suggest that the alteration of aa 89–96 (the TLR5-binding site) of flagellin could markedly decrease TLR5 activity in vivo. Flagellin can be recognized not only by cell surface TLR5 but also by cytosolic NAIP5/6. Thus, Zhang et al. assessed the ability of the flagellin mutant to activate NLRC4 signaling in murine peritoneal exudate cells. Notably, aa 89–96 fliC→flaA mutant flagellin induced significantly lower IL-1β and IL-18 secretion than did WT flagellin, indicating that 89–96 fliC→flaA mutant flagellin decreases NLRC4 signaling. The production of both IL-1β and IL-18 involves the processing of the proform by the inflammasome. However, the production of pro-IL-1β and pro-IL-18 can be influenced by TLR5 signaling.12 These observations may explain why reduced TLR5 activity was accompanied by decreased NLRC4 signaling in aa 89–96 fliC→flaA mutant flagellin.

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Acknowledgements

The authors (XK, ZP and XJ) were supported by the National Natural Science Foundation of China (31320103907, 31372415, 31730094), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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Authors and Affiliations

  1. Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, 225009, Jiangsu, China

    Xilong Kang, Zhiming Pan & Xinan Jiao

  2. Jiangsu Co-innovation Center for the Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, Jiangsu, China

    Xilong Kang, Zhiming Pan & Xinan Jiao

  3. Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agrifood Safety and Quality, MOA, Yangzhou University, Yangzhou, 225009, Jiangsu, China

    Xilong Kang, Zhiming Pan & Xinan Jiao

  4. Joint International Research Laboratory of Agriculture and Agri-product Safety of the Ministry of Education, Yangzhou, 225009, Jiangsu, China

    Xilong Kang, Zhiming Pan & Xinan Jiao

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Kang, X., Pan, Z. & Jiao, X. Amino acids 89–96 of Salmonella flagellin: a key site for its adjuvant effect independent of the TLR5 signaling pathway. Cell Mol Immunol 14, 1023–1025 (2017). https://doi.org/10.1038/cmi.2017.125

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