Abstract
Escherichia coli Nissle1917 (EcN) is a non-pathogenic probiotic strain widely used to maintain gut health, treat gastrointestinal disorders, and modulate the gut microbiome due to its anti-inflammatory and competitive exclusion effects against pathogenic bacteria. Heparin, abundant on intestinal mucosal surfaces, is a highly sulfated glycosaminoglycan primarily produced by mast cells. Currently, the interaction between EcN surface protein and heparin has remained elusive. In this study, the flagellin FliC responsible for EcN's movement was separated and characterized as a heparin binding protein by mass spectrometry (MS) analysis. The recombinant FliC protein, expressed by plasmid pET28a( +)-fliC, was further prepared to confirm the interaction between FliC and heparin. The results showed that heparin-Sepharose's ability to bind FliC was 48-fold higher than its ability to bind the negative control, bovine serum albumin (BSA). Neither the knockout of gene fliC nor the addition of heparin affects the growth of EcN, but both significantly inhibit the swimming of EcN. Adding 10 mg/ml heparin reduced the swimming diameter of the wild type and the complemented strain to 29–41% of the original, but that did not affect the swimming ability of the knockout strains. These results demonstrate that heparin interacts with EcN flagellin FliC and inhibits bacteria swimming. Exploring this interaction could improve our understanding of the relationship between hosts and microorganisms and provide a potential basis for disease treatment.
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The data that support the findings of this study are available from the corresponding author upon reasonable request.
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This work was supported by the National Natural Science Foundation of China (31670120).
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Funding was provided by the National Natural Science Foundation of China (31670120).
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G.Y. and L.Y. conducted the investigations and prepared the initial draft. X.Z. contributed to the conceptualization, supervision, and reviewed and edited the manuscript. All authors participated in reviewing the manuscript.
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203_2023_3622_MOESM1_ESM.eps
Supplementary file1 (EPS 1744 KB) Fig. S1 SDS-PAGE analysis of purified proteins obtained from the heparin-Sepharose column. Lane 1, protein marker. The molecular weights from top to bottom are 97.4 kDa, 66.2 kDa, 42.7 kDa, 31 kDa, and 14.4 kDa, respectively; lane 2, bacterial surface protein extract; lane 3, the protein washed out during the column washing process; lane 4, the heparin-binding protein eluted from the column.
203_2023_3622_MOESM2_ESM.eps
Supplementary file2 (EPS 2280 KB) Fig. S2 SDS-PAGE analysis of the purified recombinant FliC obtained from the nickel-NTA column. Lane 1, the cell extract obtained from the induced culture; Lane 2, the cell extract from the uninduced culture; Lane 3, the protein marker. The molecular weights from top to bottom are 97.4 kDa, 66.2 kDa, 42.7 kDa, 31 kDa, and 14.4 kDa, respectively; lane 4, the purified recombinant FliC
203_2023_3622_MOESM3_ESM.eps
Supplementary file3 (EPS 270836 KB) Fig. S3 Schematic representation of fliC deletion and complement. a The schematic representation of fliC deletion using RED homologous recombination. b Analysis of fliC deletion. The DNA fragments obtained from PCR with primer pairs 0019/0445 and 0020/0446 were analyzed by agarose electrophoresis. Lane 1, DNA marker; lane 2, PCR analysis of wild type EcN strain, showing a 2,999 bp DNA band; lane 3, PCR validation of the fliC deficient strain, showing two bands at 905 bp and 1,809 bp, respectively. c The schematic representation of fliC complement. The FliC-expressing plasmid pAH162-fliC was inserted into the phi80 site of the ∆fliC strain. d Analysis of fliC complement. After PCR with primer pairs 0028/0078 and 0029/0079, the DNA fragments were analyzed by agarose electrophoresis. Lane 1, DNA marker; lane 2, PCR analysis of wild type EcN strain, showing a 546 bp DNA band; lane 3, PCR validation of the fliC complement strain, showing two bands at 409 bp and 732 bp, respectively
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Yang, G., Yang, L. & Zhou, X. Inhibition of bacterial swimming by heparin binding of flagellin FliC from Escherichia coli strain Nissle 1917. Arch Microbiol 205, 286 (2023). https://doi.org/10.1007/s00203-023-03622-9
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DOI: https://doi.org/10.1007/s00203-023-03622-9