Escherichia coli O157:H7 Curli Fimbriae Promotes Biofilm Formation, Epithelial Cell Invasion, and Persistence in Cattle
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacteria, Plasmids, Primers, and Growth Conditions
2.2. Transposon Mutagenesis
2.3. Crystal Violet Biofilm Assay
2.4. Invasion Assay
2.5. Construction of csgD Promoter Mutants
2.6. Curli Isolation and Detection
2.7. Operon Sequences
2.8. Immunofluorescence Microscopy
2.9. Transmission Electron Microscopy
2.10. Cattle Challenge and O157 Enumeration
2.11. Statistical Analysis
3. Results
3.1. Tn5 Biofilm-Negative Insertions Map to LPS Synthesis or Curli Fimbriae Genes, but Only the Latter Class of Mutant Had Reduced Epithelial Cell Invasion
3.2. A Single Base Pair Change (A to T) in the csgD Promoter of O157 Strains Sakai and 43894, Conferred the Biofilm/Invasive-Positive Phenotype
3.3. Curli Fimbriae Are Expressed in O157 Strains 43895, 43894R, and SakaiR but Not in Wild Type Strains 43894, Sakai, or the Mutant csgB::Tn5
3.4. Deletion of csgB Resulted in the Same Phenotype as the csgB::Tn5
3.5. Curli Was Required for O157 Adherence to and Invasion of MAC-T Cells
3.6. The csgB::Tn5 Mutation Reduced Persistence of E. coli O157 Strain 43895 in Cattle
4. Discussion
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Strain or Plasmid | Description | Source or Reference |
---|---|---|
43895 | E. coli O157:H7 ATCC 43895, a clinical isolate, stx1+/stx2+, curli+ | ATCC * |
manC-Tn5 | Tn5 inserted in manC of 43895 | This work |
per-Tn5 | Tn5 inserted in per of 43895 | This work |
csgB-Tn5 | Tn5 inserted in csgB of 43895 | This work |
waaI-Tn5 | Tn5 inserted into waaI of 43895 | This work |
wzy-Tn5 | Tn5 inserted into wzy of 43895 | This work |
43895ΔcsgB | 43895 with csgB deletion | This work |
43895ΔcsgB(pcsgBA) | 43895ΔcsgB complemented csgB | This work |
43894 | E. coli O157:H7 ATCC 43894, a clinical isolate, stx1+/stx2+, curli- | ATCC |
43894R | 43894 mutant with A to T transversion in csgD promoter, curli+ | This work |
Sakai SakaiR | E. coli O157:H7, a clinical isolate, stx1+/stx2+, curli- Sakai mutant with A to T transversion in csgD promoter, curli+ | [16] This work |
FB38 | E. coli O157:H7, a bovine isolate, stx1+/stx2-, curli positive | Laboratory stock |
S17-1 λ pir | pro recA thi hsdR Hfr RP4-2 (Tc::Mu) (Km::Tn7, SmR, λ pir lysogen | [38] |
K-12 | E. coli MG1655 strain | Laboratory stock |
pKD4 | Template plasmid for mutagenesis (AmpR KanR) | [37] |
pKD3 | Template plasmid for mutagenesis (AmpR CmR) | [37] |
pKD46 | Red recombinase helper plasmid, RepA101(Ts), AmpR | [37] |
pACYC177 | Cloning vector, AmpR, KanR | ATCC |
pGP704.L | Suicide plasmid, mob+, sacBR+, pir-dependent oriR6K, AmpR | [39] |
pGP704csg1 | pGP704.L containing csgBA genes of 43895; allelic exchange plasmid | This work |
pcsgBA | csgBA and intergenic region of 43895 cloned into pACYC177 | This work |
Primer | * Sequence (5′–3′) |
---|---|
KAN-2 FP-1 | ACCTACAACAAAGCTCTCATCAACC |
KAN-2 RP-1 | GCAATGTAACATCAGAGATTTTGAG |
IBA-F | GTTTGGATCCAAACCCCGCTTTTTTTATTGATC (BamHI) |
IBA-R | GTTTCTGCAGTTAGTACTGATGAGCGGTCGCGT (PstI) |
csgDBign-F | GAGCCTGAAGAGATATCGTCCA |
csgDBign-R | GCGCACCCAGTATTGTTAAC |
csgB-LF | ATGAAAAACAAATTGTTATTTATGATGTTAACAATACTGGGTGTGTAGGCTGGAGCTGCTTCG |
csgB-LR | TTAACGTTGTGTCACGCGAATAGCCATTTGCGACTGTCTCTGCATATGAATATCCTCCTTA |
Dele-1F | AGAAGTACTGACAGATGTTGCACTGCTGTGTGTAGTAATAAATGTGTAGGCTGGAGCTGCTTCG |
Dele-1R | AACTTAATAAAACCTTAAGGTTAACATTTTAATATAACCAGTCATATGAATATCCTCCTTA |
DBC1bF | TATACCCGGGTTCTTGATCCTCCATGGCATAAAA (SmaI) |
DBC1bR | ATATAGATATCCTGCGTTACGATGGAAAGTATGTC(EcoRV) |
Strains a | Cell-Associated b (CFU × 107/Well) | Internalized c (CFU × 104/Well) |
---|---|---|
43895 | 8.78 ± 0.73 | 467 ± 190 |
ΔcsgB | * 0.71 ± 0.12 | ** 4.58 ± 0.89 |
ΔcsgB(pcsgBA) | 7.66 ± 0.67 | 389 ± 110 |
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Sheng, H.; Xue, Y.; Zhao, W.; Hovde, C.J.; Minnich, S.A. Escherichia coli O157:H7 Curli Fimbriae Promotes Biofilm Formation, Epithelial Cell Invasion, and Persistence in Cattle. Microorganisms 2020, 8, 580. https://doi.org/10.3390/microorganisms8040580
Sheng H, Xue Y, Zhao W, Hovde CJ, Minnich SA. Escherichia coli O157:H7 Curli Fimbriae Promotes Biofilm Formation, Epithelial Cell Invasion, and Persistence in Cattle. Microorganisms. 2020; 8(4):580. https://doi.org/10.3390/microorganisms8040580
Chicago/Turabian StyleSheng, Haiqing, Yansong Xue, Wei Zhao, Carolyn J. Hovde, and Scott A. Minnich. 2020. "Escherichia coli O157:H7 Curli Fimbriae Promotes Biofilm Formation, Epithelial Cell Invasion, and Persistence in Cattle" Microorganisms 8, no. 4: 580. https://doi.org/10.3390/microorganisms8040580