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

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

Published: September 24, 2020

doi:

Mingxu Zhou*^1,2, Qiangde Duan*^1,3, Yang Yang³, Guoqiang Zhu³

¹College of Veterinary Medicine,Yangzhou University, ²Institute of Veterinary Immunology & Engineering,Jiangsu Academy of Agricultural Sciences, ³Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses

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 receptor¹^,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 virulence factors for extra-intestinal pathogens, such as uropathogenic E. coli (UPEC) and neonatal meningitis-causing E. coli (NMEC)²^,⁵^,⁶.

Infections caused by F18 fimbriae⁺ (including two variants: ab and ac) Shiga toxin-producing E. coli (STEC) strains are associated with porcine edema disease (ED) and post-weaning diarrhea (PWD)⁷. Porcine F18 fimbriae⁺ STEC attaches to intestinal epithelial receptors by a variety of surface adhesins, including F18 fimbriae, flagella, E. coli common pilus (ECP) and the adhesin involved in diffuse adherence (AIDA-I)⁸^,⁹^,¹⁰^,¹¹. Previously, we had investigated the function of type 1 fimbriae in F18ac fimbriae⁺ ETEC, which demonstrated that type 1 fimbriae facilitate bacterial biofilm formation and adhesion to host cells¹². However, as the pathogenesis of F18ab and F18ac fimbriae⁺ STEC are not totally the same⁷, the role of type 1 fimbriae in F18ab fimbriae⁺ STEC remains unclear. The fimbrial rod is primarily composed of multiple copies of the major fimbrial subunit FimA, and FimH adhesin is assembled into a fibrillar tip structure that drive bacteria binding to host cellular mannose containing receptor¹³. Using λ-Red recombination¹⁴, we had successfully knocked out fimA/fimH gene from a F18ab fimbriae⁺ STEC strain F107/86 (wild-type, O139:H1, Stx2e⁺), and constructed complement strains for this study¹⁵.

Here, we describe a protocol to study the function of bacterial fimbriae in colonization. Bacteria adhesion assay and invasion assay are major methods to investigate the bacteria fimbrial binding performance. It is complicated and costly to perform an animal challenge model or isolate the primary cell line for further infection assays¹⁶. Usually, neither of these results are stable with good repeatability since the individual differences are present between the tested animal. In this study, IPEC-J2 cells are used. These are porcine intestinal columnar epithelial cells that have been isolated from a neonatal piglet’s mid-jejunum¹⁷. It is a stable in vitro cell model for examining the interactions of various animal and human pathogens, including Salmonella enterica and pathogenic E. coli, with intestinal epithelial cells¹⁸, helping explain the role of fimbriae in intestinal infection conveniently and quickly. Otherwise, IPEC-1 cells are another widely used porcine intestinal epithelial cell line, in which case the composition of cellular receptors are different from IPEC-J2¹⁹. For the study of mammary pathogenic bacteria, it is better to use mammary epithelial cell line MAC-T²⁰. Hence, for different bacterial pathogenic conditions, choice of a suitable cell line which mimic in vivo environments is important.

In addition, the biofilm is another essential characteristic for bacterial survival during colonization²¹. In the previous works, silver and congo red were used to stain the biofilm formation in the glass tubes that visually showed the results²²^,²³. However, the difference of biofilm formation ability between varying strains cannot be measured. Here, we also present a protocol for the quantification of bacterial biofilm formation in vitro, which could easily evaluate the ability of fimbriae in biofilm formation.

The methods proposed in this study utilize a fast and simple in vitro way to determine the function of bacterial fimbriae during the bacteria infection process, which can be widely adapted to other researches in the study of virulence factor in bacterial pathogenic mechanism.

Protocol

1. Cell culture 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. 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. Use 10 µL of …

Representative Results

FimA is more important than FimH in F18ab fimbriae+ STEC adhesion and invasion to IPEC-J2 cells. Compared to WT strain, deleting fimA reduced F18ab fimbriae+ STEC adhesion to IPEC-J2 cells by approximately 86% (p < 0.01), while deleting fimH reduced STEC adhesion by approximately 71% (p < 0.01) (Figure 1A). Blocking the adhesin FimH of WT strain by co-incubating with 4% D-mannose showed an equal adhesion ability with the &#916…

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…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This study was supported by grants from the National Natural Science Foundation of China (No. 31672579).

Materials

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

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