Bacteroides fragilis adherence to Caco-2 cells

Abstract

The ability of ten Bacteroides fragilis strains isolated from intestinal and non-intestinal infections, normal flora and environment to adhere to human colon carcinoma cells, Caco-2, was examined. The adherence capacity varied among the strains tested from strongly adherent (76–100%) to non- or weakly adherent (0–25%). Negative staining with Indian ink showed that all the strains were capsulated, although strain 1032 (strongly adherent and originated from bacteremia) had the highest rate of capsulated cells in the culture. All strains studied presented an electron-dense layer and no fimbrial structures in their surface after PTA negative staining. The analysis of the strains with ruthenium red showed the presence of an acidic polysaccharide and also surface vesicles in all of them. The strain 1032 presented an aggregative adherence pattern toward Caco-2 cells monolayers. It could be seen trapped by elongated microvilli and surrounded by extracellular material in the scanning electron microscope. Treatment with sodium periodate (100 mM/1 h) reduced significantly its adherence capacity and also the expression of an electron-dense layer and of the capsule, detected with PTA and Indian ink staining, respectively. We suggest that the capsular polysaccharide might mediate the adherence of the B. fragilis to Caco-2 cells.

Introduction

Adherence of pathogenic microorganisms to host tissue is considered the first step in establishing infection in the host and it is an important virulence factor. Many mechanisms of attachment involving fimbriae, capsule and outer membrane components have been described for a variety of bacteria, including anaerobic pathogens such as Bacteroides fragilis [1], [2], [3], [4], [5], [6], [7], [8], [9], [10]. This species is a minor component of faeces and found as a major component of the mucosa-associated flora [11], [12], [13], [14]. The major virulence determinant identified for B. fragilis is the capsular polysaccharide, which has been widely studied and characterized as an abscess inductor and also as an adhesion factor to mesothelial cells [15], [16], [17], [18].

In vitro assays have been developed to study the adherence of pathogens to intestinal cells such as the use of the human colon carcinoma cells cultures (Caco-2 cells) which is also a useful tool for studying the intestinal epithelium differentiation because of its spontaneous capacity of differentiating and forming a confluent layer. This confluence begins after the 5th day of culture and this process is very similar to what happens in vivo, when immature intestinal cells become mature [19], [20], [21], [22], [23]. The aims of this work were to study the adherence capacity of different strains of B. fragilis to Caco-2 cells in different levels analysing if the differentiation of the cells interfers with the adhesion process. We also characterized the surface structures that may be involved in the adherence process through Indian ink, phosphotungstic acid and ruthenium red staining. We used the transmission and the scanning electron microscopy to observe the interactions beween bacteria and eukaryotic cells.