Influence of lactoferrin on the entry process of Escherichia coli HB101(pRI203) in HeLa cells

Abstract

Lactoferrin (Lf) is an iron-binding protein which plays an important role in the host defense systems of different mucosal surfaces including the intestinal mucosa. In the present research the role of apo-Lf and iron-saturated Lf in the invasion process of enteroinvasive bacteria, grown in iron stress or excess, was investigated. As enteroinvasive bacterium, Escherichia coli HB101 strain harboring a plasmid which contains the chromosomal inv gene from Yersinia pseudotuberculosis was utilized. The product of this gene (invasin) enables this microorganism to invade human epithelial cultured cells (HeLa). The results obtained showed that apo-Lf and iron-saturated Lf added at physiological concentration during the infection exerted a significant inhibition of adhesion (3.2 x 10(5) instead 3.4 x 10(6) adherent bacteria grown in iron excess; 1.6 x 10(3) instead of 2.3 x 10(4) adherent bacteria grown in iron-limited medium) and internalization (4.0 x 10(5) instead of 3.7 x 10(6) internalized bacteria grown in iron excess; 2.1 x 10(3) instead 2.8 x 10(4) internalized bacteria grown in iron-limited medium). It has also been demonstrated that in these experimental conditions Lf binds to HeLa cell membrane as well as to bacterial outer membrane. It is likely that this binding interfere with the early events of interaction between bacteria and eukaryotic cells. This inhibiting effect of Lf on the invasion efficiency of E. coli HB101 (pRI203) could be related to the cationic nature of the molecule, although other mechanisms cannot be ruled out.

Lactoferrin

“…Bacterial entry into host cells is mediated by the binding of bacterial invasin to a host integrin receptor. The effectiveness of apo-and iron-saturated bLf, and bLfcin, towards E. coli strain HB101 (pRI203) invasion is correlated with their ability to bind to both cultured cells and bacterial invasin [83,84]. Likewise, for Y. enterocolitica and Y. pseudotuberculosis, grown in conditions allowing maximal invasin synthesis, a 10-fold inhibition of invasion of cultured cells by bLfcin was observed [84].…”

“…Pre-incubation of apoand iron-saturated Lf with bacteria or with cultured cells, carried out at 4 °C to avoid internalisation, did not exert any anti-adhesive activity. Both hLf and bLf were shown, however, to inhibit E. coli HB101 (pRI203), Y. pseudotuberculosis and Y. enterocolitica adhesion through the ability of Lf to bind to both epithelial cells and invasins in a manner unrelated to its iron-binding capability [83,84]. Lf has also been shown to inhibit the adherence of enterotoxigenic E. coli (ETEC) to human epithelial cells and to intestinal mucosa of germfree mice [85], as well as the adhesion of three adhesive diarrheagenic E. coli strains (DAEC), enteroaggregative E. coli (EAEC) [86] and enteropathogenic E. coli (EPEC) [87].…”

“…The invasin, the inv gene product, promotes both bacterial binding and internalisation into mammalian cells [83]. Pre-incubation of apoand iron-saturated Lf with bacteria or with cultured cells, carried out at 4 °C to avoid internalisation, did not exert any anti-adhesive activity.…”

“…Moreover, a similar role also appears to apply to some parasites (see later). The fi rst demonstration of the inhibition of bacterial invasion by bLf involved a recombinant enteroinvasive E. coli strain HB101(pRI203) [83]. Bacterial entry into host cells is mediated by the binding of bacterial invasin to a host integrin receptor.…”

Lactoferrin

“…Studies have also demonstrated that LF can protect epithelial cells against microbial infection by inhibiting intracellular invasion by pathogenic bacteria, presumably by binding to surface bacterial and/or mammalian proteins and blocking their adhesion to host cells [57][58][59]. This mechanism appears to also account for most of the anti-viral activity described for LF.…”