Phosphorylation of IcsA by cAMP‐dependent protein kinase and its effect on intercellular spread of Shigella flexneri

Abstract

Shigella flexneri, a Gram-negative bacillus belonging to the family Enterobacteriaceae, causes bacillary dysentery in humans by invading colonic epithelial cells. Processes by which epithelial cells, which are not professional phagocytes, may limit the spread of the invading microorganisms are poorly understood. This paper shows that IcsA (VirG), a 120 kDa bacterial outer membrane protein responsible for intracellular and cell-to-cell spread through polymerization of actin, is a major substrate for phosphorylation by cyclic-dependent protein kinases. Site-directed mutagenesis of a sequence encoding phosphorylation consensus motif SSRRASS, located at residues 754-760, almost completely abolished the ability of this protein to be phosphorylated by protein kinase A. Such mutants expressed a 'super lcs' phenotype, characterized by an increased capacity to spread from cell-to-cell during the first three hours of infection in the HeLa cell infection assay. These data suggest that host-cell phosphorylation of key virulence proteins located on the bacterial surface may represent a significant host defence mechanism during the invasion process.

Cleavage of Shigella surface protein VirG occurs at a specific site, but the secretion is not essential for intracellular spreading

“…Indeed, d'Hauteville and Sansonetti (8) showed that phosphorylation of IcsA (VirG) by cyclic AMP-dependent protein kinase was abolished when either or both of the Arg residues located at positions 758 and 759 were altered. These mutants expressed a ''super Ics'' phenotype, characterized by an increased capacity to spread from one cell to another during the first 3 h of HeLa cell infection, as well as an increased rate of spreading in the plaque assay or the Serény test (8).…”

“…It should be mentioned that the Arg-Arg site of in vitro VirG cleavage determined in this study coincided with a sequence encoding a phosphorylation consensus motif, Ser-Ser-ArgArg-Ala-Ser-Ser, located at residues 756 through 762 (8). Indeed, d'Hauteville and Sansonetti (8) showed that phosphorylation of IcsA (VirG) by cyclic AMP-dependent protein kinase was abolished when either or both of the Arg residues located at positions 758 and 759 were altered.…”

“…These mutants expressed a ''super Ics'' phenotype, characterized by an increased capacity to spread from one cell to another during the first 3 h of HeLa cell infection, as well as an increased rate of spreading in the plaque assay or the Serény test (8). Although we did not quantatively assay the initial intracellular spreading rate of YSH6000 virG202 mutant, the earlier reaction in the Serény test would be indicative of the super Ics phenotype as described by d'Hauteville and Sansonetti (8). Because the phosphorylation consensus motif found in the VirG sequence contains the specific cleavage site for VirG secretion, and because alteration of the Arg-Arg bond results in blockage of both phosphorylation (8) and secretion of VirG (this study), the super Ics phenotype might result from the inability to cleave VirG protein.…”

Unipolar localization and ATPase activity of IcsA, a Shigella flexneri protein involved in intracellular movement

“…In infection of semiconfluent HeLa cells, the icsA mutant (i) does not diffuse within the cell cytoplasm but rather forms microcolonies near the cell nucleus, (ii) does not elicit the accumulation of polymerized actin on the surface of the bacteria that is observed with the wild-type strain, and (iii) does not spread into adjacent cells (7,20,29,48,49). Upon infection of macaque monkeys, the icsA mutant causes markedly less mucosal destruction than the wild-type strain (40) Labelling of bacteria grown in vitro and bacteria infecting HeLa cells with IcsA antiserum.…”

“…We have previously constructed shigella mutants that are unable to spread within and between cells (7,20,40). Each of * Corresponding author.…”