M1 protein triggers a phosphoinositide cascade for group A Streptococcus invasion of epithelial cells

Abstract

Invasion of nonphagocytic cells by bacteria provides a favorable niche for persistence and evasion of host defenses and antibiotics. M protein is a major virulence factor because it promotes high-frequency invasion of epithelial cells by group A Streptococcus (GAS) and also renders the bacterium resistant to phagocytosis. In this study, we investigated the role of M1 protein from serotype M1 strain 90-226 in regulating mammalian signal transduction and cytoskeletal rearrangement for bacterial entry. LY294002 and wortmannin, which are inhibitors of phosphatidylinositol 3-kinase (PI 3-K) blocked invasion of epithelial cells by GAS by 75 and 80%, respectively, but failed to inhibit invasion by Salmonella enterica serovar Typhimurium. Also, epithelial cells transiently transfected with dominant negative p85 and p110 genes, the regulatory and catalytic subunits of PI 3-K, respectively, were less able to be invaded by GAS. To separate the influence of other streptococcal virulence factors from M protein, Lactococcus lactis was engineered to express M1 protein on its surface. L. lactis(pLM1) invaded epithelial cells efficiently in vitro, and PI 3-K inhibitors blocked 90% of this invasion. Purified soluble M1 protein stimulated the formation of stress fibers and actin tuffs on epithelial cells. LY294002 and wortmannin inhibited these cellular changes. A phosphoinositide analogue also inhibited the invasion of epithelial cells by GAS. Therefore, M1 protein, either directly or via bound fibronectin, initiates signals that depend on the lipid kinase PI 3-K pathway, which paves the way for cytoskeletal rearrangement that internalize the bacterium.

FIG. 1.

Effect of different inhibitors on GAS, S. enterica serovar Typhimurium, and E. coli HB101(pVM101) invasion of HEp-2 cells. Confluent monolayers were preincubated with the inhibitors for 30 min, and the standard invasion assay was performed in the presence of the inhibitor. Values are mean and standard deviation for at least three experiments. The results are expressed as percent inhibition, which is calculated as follows: [(CFU_{no inhibitor} − CFU_inhibitor)/CFU_{no inhibitor}] × 100. Percent inhibition by staurosporine (a) and genistein (b) is shown. In the absence of inhibitor, 25% of the inoculum (1 × 10⁸ to 5 × 10⁸ CFU per well) was ingested by epithelial cells.

FIG. 2.

Effect of different inhibitors on GAS and S. enterica serovar Typhimurium invasion of HEp-2 cells. Monolayers were preincubated with inhibitors for 30 min, and the invasion was carried out in the presence of the inhibitor. Percent inhibition was calculated as described in the legend to fig. 1. Values are mean and standard deviation for at least three experiments. (a) Wortmannin, (b) LY294002, (c) Akt inhibitor ICIO. In the absence of inhibitor, 25% of the inoculum (1 × 10⁸ to 5 × 10⁸ CFU per well) was ingested by epithelial cells.

FIG. 3.

Inhibition of invasion of A549 cells by dominate negative and constitutive forms of PI 3-K. Data are expressed as the percent invasion of transiently transfected A549 cells relative to untransfected A549 cells with the LT1 transfection reagent. LT1 cells were exposed to the LT1 polyamine transfection reagent without plasmid DNA. The cells were transfected with pSG5 (vector only), pSG5-p85 (dominant negative p85), pSG5-p110 (dominant negative p110), or p110CAAX (constitutively active form of p110) (30). They were also transfected with a GFP fusion (pEGFP-p85) or vector only (pEGFPC2) (20).

FIG. 4.

M1 protein induction of localized cytoskeleton rearrangement and stress fibers in HEp-2 cells is dependent on the PI 3-K pathway. (a and b) Wortmannin inhibited the ingestion of streptococci but did not impede adherence to HEp-2 cells. Infected cells were fixed, and the extracellular bacteria were stained with rabbit anti-GAS antibody followed by anti-rabbit-FITC conjugate. The cells were permeabilized, and intracellular bacteria were again exposed to rabbit anti-GAS antibody and labeled with anti-rabbit Cy3. Arrows indicate the intracellular bacteria, which appear red. Extracellular bacteria are yellow. (a) Cells infected in the absence of wortmannin. (b) Cells infected in the presence of 50 nM wortmannin. (c to d) Cells preincubated with purified M protein. (c) Control untreated cells. (d) Cells treated with 400 ng of recombinant M1 protein per ml for 10 min. (e) Cells treated with wortmannin plus 400 ng of purified M1 protein per ml in MEM with Fn. Arrows indicate actin tufts of lamellipodia and stress fibers.

FIG. 5.

Effect of manumycin A, a farnesyl transferase I inhibitor, on GAS invasion of HEp-2 cells. Values are mean and standard deviation for least three experiments. The results are expressed as percent inhibition, which is calculated as described in the legend to Fig. 1.

FIG. 6.

Schematic illustration of signaling pathways postulated to be activated by M1 protein and PrtF of GAS. ◊, M protein; ○, PrtF; ▿, Fn.