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

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 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 results^22,23. 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.