Enteropathogenic Escherichia coli (EPEC) are a class of diarrheagenic organisms that induce a characteristic attaching and effacing lesion in enterocytes and various cultured cell lines. Infection of cultured HEp-2 cells by EPEC isolates 2036-80 (serotype 0119) and E2348-69 (serotype 0127) resulted in significant elevation of intracellular free calcium levels, determined quantitatively with the fluorescent calcium indicator dye 2-{[2-bis (carboxymethyl)amino-5-methylphenoxyjmethyl}-6-methoxy-8-bis(carboxymethyl)aminoquinoline. This effect, which was not observed on infection with non-lesion-forming E. coli strains, was inhibited by dantrolene, a drug that prevents calcium mobilization from intracellular stores. Moreover, activated protein kinase C in infected cells was dissociated from cell membranes by a process that was inhibited by cyclosporin A, suggesting involvement of the calcium-dependent protease calpain. A qualitative method for observing intracellular calcium fluxes by fluorescence microscopy with the recently described fluorescein-based indicator fluo-3 was used to screen a collection of well-characterized E. coli isolates from patients with infantile enteritis. Increased localized calcium-dependent fluo-3 fluorescence was observed only in HEp-2 cells infected with known lesion-forming EPEC strains. We propose that enhancement of intracellular free calcium levels in enterocytes infected with EPEC would result in formation of the characteristic lesion by calcium-dependent activation of actin-depolymerizing proteins, with eventual loss of absorptive capacity.

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A protein toxin of approximately 120,000 Da secreted by nonhemolytic enteroaggregative Escherichia coli strains cross-reacted in Western blots (immunoblots) with antibodies raised against the C-terminal region ofE. coli hemolysin. Treatment of HEp-2 cells with enteroaggregative E. coli or culture supernatants caused elevation of intracellular calcium and stimulated calcium-dependent protein phosphorylation.

The complete primary structure of inhibitor-2, a specific inhibitor of protein phosphatase-I , has been determined. The protein consists of a single polypeptide chain of 203 residues, and has a relative molecular mass of 22 835 Da. This molecular mass is significantly lower than earlier estimates based on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The threonyl residue phosphorylated by glycogen synthase kinase-3 is located at position 72. The molecule is very hydrophilic, lacks cysteine residues and the single tryptophanyl and phenylalanyl residues are at positions 46 and 139, respectively. The N-terminal alanyl residue is N-acetylated. Digestion with Straphylococcus aureus V8 proteinase, trypsin, or cleavage with cyanogen bromide, destroyed the biological activity of inhibitor-2, demonstrating that many large fragments (e. g. 1 -49, 49 -92, 67 ~ 101, 108 -134, 142 -182 and 163 -197) are inactive. Digestion with clostripain generated a peptide comprising residues 25 -114 which retained 2% of the inhibitory potency of the parent molecule. There is no sequence homology between inhibitor-2 and inhibitor-I .In 1976, Huang and Glinsmdnn [l] identified two proteins in rabbit skeletal muscle, termed inhibitor-I (1-1) and inhibitor-2 (I-2), that were specific inhibitors of a protein phosphatase, subsequently termed protein phosphatase-1 (reviewed in [2, 31). 1-1 was only an inhibitor after it had been phosphorylated by cyclic-AMP-dependent protein kinase, whereas the activity of 1-2 was unaffected by this protein kinasc [I].1-2 can interact with protein phosphatase-I in two distinct ways. Firstly, at extremely low concentrations (Kd z 0.1 nM), it combines with the catalytic (C) subunit of protein phosphatase-I to produce an inactive 1-2 . C complex [4, 51, termed protein phosphatase-lI [6]. This form has also been termed the Mg-ATP-dependent protein phosphatase [7] because its activation requires preincubation with Mg-ATP and a protein kinase, termed factor FA [8] or glycogen synthase kinase-3 [9, 101. Activation of protein phosphatase-lI is triggered by the phosphorylation of a specific threonyl residue on 1-2 [4, 5, 11 -131 and is the first example of a protein phosphatase that is activated by a protein kinase. Secondly, at higher concentrations ( K d E S nM), 1-2 inactivates protein phosphatase-I by a different mechanism that is not reversed by preincubation with Mg-ATP and glycogen synthase kinase-3, and presumably results from the binding of 1-2 at a separate site on the enzyme [S].1-2 is also a substrate for other protein kinases, such as casein kinase-I1 [I41 and the insulin receptor [15]. Phosphorylation by casein kinase-11, which occurs on seryl residues, does not activate protein phosphatase-lI directly, but enhances the rate of phosphorylation of 1-2 and activation of protein phosphatase-1, by glycogen synthase kinase-3 [I 41. Phosphorylation by the insulin receptor occurs on a tyrosyl residue(s), but the functional significance of this modification is unknown.1-1 and 1-2 have been purified...

Infection of HEp-2 monolayers with enteropathogenic Escherichia coli 2036-80 (0119) 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 0 serogroups revealed that all strains able to form the characteristic attaching and effacing lesion of enteropathogenic E. coli showed elevated phosphorylation of 21,000and 29,000-dalton protein species.