Antigen detection in enteropathogenic Escherichia coli using secretory immunoglobulin A antibodies isolated from human breast milk.

Enteropathogenic Escherichia coli (EPEC) produces a characteristic attaching and effacing (A/E) lesion in the small intestines of infected children. The immune response to EPEC infection remains poorly characterized. The molecular targets that elicit protective immunity against EPEC disease are unknown. In this study protein antigens from EPEC were identified using secretory immunoglobulin A (sIgA) antibodies isolated from milk from Mexican women by Western blot analysis. Purified sIgA antibodies, which inhibit the adherence of EPEC to cells, reacted to many EPEC proteins, the most prominent of which were intimin (a 94-kDa outer membrane protein) and two unknown proteins with apparent molecular masses of 80 and 70 kDa. A culture supernatant protein of 110 kDa also reacted strongly with the sIgA antibodies. The molecular size of this protein and its reactivity with specific anti-EspC antiserum suggest that it is EPEC-secreted protein C (EspC). These EPEC surface protein antigens were consistently recognized by all the different sIgA samples obtained from 15 women. Screening of clinical isolates of various O serogroups from cases of severe infantile diarrhea revealed that all EPEC strains able to produce the A/E lesion showed expression of intimin and the 80- and 70-kDa proteins. Such proteins reacted strongly with the purified sIgA pool. Moreover, nonvirulent E. coli strains were unable to generate a sIgA response. The immunogenic capacities of the 80- and 70-kDa proteins as virulence antigens have not been previously reported. The strong sIgA response to intimin and the 80- and 70-kDa proteins obtained in this study indicates that such antigens stimulate intestinal immune responses and may elicit protective immunity against EPEC disease.

Phosphorylation of myosin light chain at distinct sites and its association with the cytoskeleton during enteropathogenic Escherichia coli infection.

Myosin light chain, the most prominent host cell phosphoprotein during adhesion of enteropathogenic Escherichia coli to cultured HEp-2 cells, was shown to be distributed between cytosolic and cytoskeletal cell fractions; its association with the cytoskeletal fraction increased with time of enteropathogenic E. coli incubation. Phosphopeptide mapping indicated cytosolic and cytoskeletal myosin light chain phosphorylation at different sites by protein kinase C and myosin light chain kinase.

Multisite phosphorylation of the 80 kDa (MARCKS) protein kinase C substrate in C3H/10T1/2 fibroblasts. Quantitative analysis of individual sites by solid-phase microsequencing.

A synthetic peptide, KKKKRFSFKKSFKLSGFSFKK, containing the phosphorylation sites of the acidic 80-87 kDa protein kinase C substrate was used to identify phosphopeptides in enzyme digests of this protein from mouse fibroblast C3H/10T1/2 cells. Stimulation of phosphorylation occurred, in vivo, with TPA at Ser7, Ser11 and Ser18, and, with two less potent phorbol esters, at Ser7 and Ser18. Okadaic acid effected a net phosphorylation of Ser7 and/or Ser11. Solid-phase sequencing showed that, in vitro, the order of initial rate of phosphorylation was Ser11 greater than Ser7 greater than Ser18, while Ser18 was preferentially phosphorylated when either Ser7 or Ser11 was occupied. No significant phosphorylation of Ser15 was detected.

Intestinal epithelial cell protein phosphorylation in enteropathogenic Escherichia coli diarrhoea.

The ability of enteropathogenic Escherichia coli (EPEC) to cause diarrhoea in man is associated with the formation of characteristic histopathological lesions in small-intestine enterocytes, with gross cytoskeletal damage and loss of brush-border microvilli. Investigation of enterocyte protein phosphorylation in response to EPEC infection showed that the major phosphorylated protein, identified by immunoprecipitation, is myosin light-chain--an important cytoskeletal protein known to affect actin organisation in non-muscle cells. High enterocyte concentrations of actin and myosin were observed at sites of bacterial infection. Our findings indicate that enterocyte cytoskeletal changes in response to EPEC may be directly triggered by bacterial adherence through signal transduction pathways that stimulate protein kinase activity.

Purification of a 20 kDa phosphoprotein from epithelial cells and identification as a myosin light chain. Phosphorylation induced by enteropathogenic Escherichia coli and phorbol ester.

Previous studies on the mechanism of enteropathogenic Escherichia coli (EPEC) infection have revealed an increase in the phosphorylation state of a number of proteins in human laryngeal HEp-2 cells. The most prominent was an acidic phosphoprotein(s) of Mr 20-21 kDa. The present study reports: (a) a simple method for purification of phosphorylated 20 kDa protein; (b) identification of the 20 kDa phosphoprotein as myosin light chain; and (c) that the phorbol ester, TPA, also increased the phosphorylation of the 20 kDa myosin light chain. In contrast to the effects of EPEC, TPA stimulation resulted in the dissociation of myosin from the cytoskeleton to the cytosol.

Protein phosphorylation by protein kinase C in HEp-2 cells infected with enteropathogenic Escherichia coli.

Infection of HEp-2 monolayers with enteropathogenic Escherichia coli 2036-80 (O119) stimulated phosphorylation of several target cell proteins, the most prominent of which had apparent molecular weights of 21,000 and 29,000. Proteins of the same size were phosphorylated in response to known activators of the calcium-phospholipid-dependent protein kinase C. Screening of clinical isolates of various O serogroups revealed that all strains able to form the characteristic attaching and effacing lesion of enteropathogenic E. coli showed elevated phosphorylation of 21,000- and 29,000-dalton protein species.