Lactoferrin downregulates pro-inflammatory cytokines upexpressed in intestinal epithelial cells infected with invasive or noninvasive Escherichia coli strains.

Intestinal epithelial cells are able to differentially interact with commensal or pathogenic microorganisms, triggering a physiological or destructive inflammation, respectively. To mimic commensal-enteroinvasive bacteria-host cell interaction, we infected Caco-2 cells with noninvasive Escherichia coli HB101 and with recombinant invasive E. coli HB101(pRI203). Using DNA microarray mRNA profiling and ELISA assays, we studied the expression of several cytokine and cytokine-related genes in infected Caco-2 cells in the absence or presence of bovine lactoferrin (bLf). Infection of Caco-2 cells with the noninvasive strain induced a slight increase in the expression of interleukin 8 (IL-8), whereas infection with invasive E. coli HB101(pRI203) induced a significant increase in the expression of IL-8 as well as other pro-inflammatory cytokines. The addition of bLf, in native- or holo-form, did not influence expression of cytokine genes by uninfected Caco-2 cells, but it decreased expression of IL-8 by cells infected with E.coli HB101. Moreover, except for IL-8, bLfs dramatically downregulated pro-inflammatory cytokines upexpressed by Caco-2 cells infected with the invasive strain. Although IL-8 was decreased by bLfs, it remained upregulated, suggesting that it could be a signal of persistence of intracellular bacteria. The bLf ability to reduce expression of some pro-inflammatory cytokines, which appears independent of its iron saturation, might represent an important natural mechanism in regulating epithelial cell responses to pathogenic bacteria and in limiting cell damage and the spread of infections.

Ala160 and His116 residues are involved in activity and specificity of apyrase, an ATP-hydrolysing enzyme produced by enteroinvasive Escherichia coli.

The virulence plasmid-carried apy (phoN2) gene of Shigella and related enteroinvasive Escherichia coli (EIEC) encodes apyrase, an ATP-diphosphohydrolase belonging to class A of the non-specific acid phosphatases (A-NSAPs). Apyrase and A-NSAPs share three domains of conserved amino acids (domains D1-D3) containing residues forming the putative active site of apyrase. In spite of their similarity, apyrase and A-NSAPs show different substrate specificity, apyrase being able to hydrolyse nucleotide tri- and diphosphates, but not monophosphates, as well as p-nitrophenyl phosphate (pNPP), while A-NSAPs are also active towards monophosphates and pNPP. In this paper, to get further insights into the structure-function relationship of apyrase, a random and site-directed mutagenesis of the apy gene of EIEC strain HN280 was conducted. Results indicate that amino acids located within the D2 and D3 conserved domains (Ser157 and Arg192, respectively) as well as residues located in the N-terminal (Ser97) and C-terminal (Glu233) domains are required for enzyme activity. Surprisingly, Ala160, located near the D2 domain and considered to be important for enzyme specificity, is required for enzyme activity, as its substitution with Thr led to the inactivation of enzyme activity. Furthermore, residue His116 is involved in apyrase specificity, since the H116L apyrase mutant shows substrate specificity resembling that of A-NSAPs.