Use of Fimbrial Rod for F18ab Fimbriae+ STEC Colonization to Host Cells

Summary

Here we present a protocol to study the function of fimbriae in bacterial colonization.

Abstract

Type 1 fimbriae are important virulence determinants of some Gram-negative pathogens, which promote bacterial colonization. The fimbrial rod is primarily composed of multiple copies of the major fimbrial subunit FimA. FimH adhesin, however, is present as a fibrillar tip structure that drive bacteria binding to host cellular mannose containing receptor. Here, we provide protocols to evaluate and compare the function of type 1 fimbrial subunits in F18ab fimbriae⁺ Shiga toxin-producing Escherichia coli (STEC). We found that both FimA and FimH are required for bacterial adhesion, invasion, and biofilm formation. Deleting fimA gene showed much more reduction in bacterial adhesion and invasion to porcine intestinal columnar epithelial cells IPEC-J2, than that of fimH mutant. Biofilm formation was significantly reduced in both mutants with an equal level. In addition, qPCR demonstrated that either fimA or fimH deletion down-regulated the bacterial flagella and F18 fimbriae genes expression, while up-regulated adhesin was involved in diffuse adherence-I (AIDA-I) gene expression, suggesting the co-regulation of cell surface-localized adhesins in F18ab fimbriae⁺ STEC.

Introduction

Bacterial fimbriae mediated adhesion facilitates bacterial attachment to a target cell surface and establishes an initial infection. Type 1 fimbriae are widely distributed among Escherichia coli (E. coli) and promote bacterial attachment to mammalian cells by binding to the mannose-containing receptor^1,2,3. In contrast to pathogenic strains, 85% of tested commensal E. coli strains of human origin do not express type 1 fimbriae⁴, which indicates its critical roles in disease infection. Type 1 fimbriae are also important virule....

Protocol

1. Cell culture

1. Maintain IPEC-J2 cells in a 25 cm² flask containing 5 mL of antibiotic-free F12-RPMI1640 (1:1) mixed media supplemented with 10% fetal bovine serum (FBS) at 37 °C, in a 5% CO₂ incubator.
2. One day before the adhesion assay, use 1 mL of 0.05% trypsin-EDTA solution to trypsinize IPEC-J2 cells for 3 min. Gently remove the trypsin-EDTA solution before cells start shedding from the flask. Add 3 mL of growth media and suspend the cells.
3. Use 10 µL of .......

Discussion

The methods provided here help to efficiently determine the function of fimbriae in bacterial colonization. Interestingly, in this study, deletion of fimA showed 15% less adhesion than fimH mutant, suggesting that tip adhesin may not be the only factor required for F18ab fimbriae⁺ STEC adhesion and that fimbrial rod subunit, FimA, works in bacterial attachment as well (Figure 1A). A recent study proposed that FimA modulated mechanical properties of the fimbrial s.......

Materials

Name	Company	Catalog Number	Comments
96-well microplate	Corning	3599	adhesion and invasion assay
96-well microplate(Round bottom)	Corning	3799	biofilm formation
crystal violet	Sinopharm Chemical Reagent	71012314	Biofilm staining
dextrose	Sangon Biotech	A610219	Culture broth
Ex Taq	TaKaRa	RR01A	PCR
F12 medium	Gibco	11765062	Cell culture
FeSO4	Sangon Biotech	A501386	Culture broth
K2HPO4	Sinopharm Chemical Reagent	20032116	Culture broth
KH2PO4	Sinopharm Chemical Reagent	10017608	Culture broth
L-Arabinose	Sangon Biotech	A610071	λ-Red recombination
MgSO4	Sinopharm Chemical Reagent	20025117	Culture broth
NaCl	Sinopharm Chemical Reagent	10019308	Culture broth
(NH4)2SO4	Sinopharm Chemical Reagent	10002917	Culture broth
Micro spectrophotometer	Thermo Fisher	Nano Drop one	Nucleic acid concentration detection
New-born calf serum	Gibco	16010159	Cell culture
Peptone	Sangon Biotech	A505247	Culture broth
PrimeScript RT reagent Kit with gDNA Eraser	TaKaRa	RR047	qPCR
Real-Time PCR	Applied Biosystems	7500 system	qPCR
RPMI1640 medium	Gibco	11875500	Cell culture
Spectrophotometer	Eppendorf	BioSpectrometer	Absorbance detection
Spectrophotometer (96-well microplate)	BioTek	Epoch	Absorbance detection
SYBR Premix Ex Taq II	TaKaRa	RR820	qPCR
Tabletop centrifuge	Thermo Fisher	Micro 17(R)	Centrifugation
thiamine hydrochloride	Sangon Biotech	A500986	Culture broth
Triton X-100	Sangon Biotech	A110694	adhesion and invasion assay
TRIzol	Invitrogen	15596018	RNA isolation
Tryptone	Oxoid	LP0042	Culture broth
Yeast extract	Oxoid	LP0021	Culture broth