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Regulation of the phosphorylation of human pharyngeal cell proteins by group A streptococcal surface dehydrogenase: signal transduction between streptococci and pharyngeal cells.

Pancholi V, Fischetti VA - J. Exp. Med. (1997)

Bottom Line: Intact streptococci and purified SDH induce a similar protein phosphorylation pattern with the de novo tyrosine phosphorylation of a 17-kD protein found in the membrane/particulate fraction of the pharyngeal cells.Treatment of pharyngeal cells with protein kinase inhibitors such as genistein and staurosporine significantly inhibited streptococcal invasion of pharyngeal cells.To identify the membrane receptor that elicits these signaling events, we found that SDH bound specifically to 30- and 32-kD membrane proteins in a direct ligand-binding assay.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, New York 10021, USA. panchov@rockvax.rockefeller.edu

ABSTRACT
Whether cell-to-cell communication results when group A streptococci interact with their target cells is unknown. Here, we report that upon contact with cultured human pharyngeal cells, both whole streptococci and purified streptococcal surface dehydrogenase (SDH) activate pharyngeal cell protein tyrosine kinase as well as protein kinase C, thus regulating the phosphorylation of cellular proteins. SDH, a major surface protein of group A streptococci, has both glyceraldehyde-3-phosphate dehydrogenase and ADP-ribosylating enzyme activities that may relate to early stages of streptococcal infection. Intact streptococci and purified SDH induce a similar protein phosphorylation pattern with the de novo tyrosine phosphorylation of a 17-kD protein found in the membrane/particulate fraction of the pharyngeal cells. However, this phosphorylation required the presence of cytosolic components. NH2-terminal amino acid sequence analysis identified the 17-kD protein as nuclear core histone H3. Both phosphotyrosine and phosphoserine-specific monoclonal antibodies reacted with the 17-kD protein by Western blot, suggesting that the binding of SDH to these pharyngeal cells elicits a novel signaling pathway that ultimately leads to activation of histone H3-specific kinases. Genistein-inhibitable phosphorylation of histone H3 indicates that tyrosine kinase plays a key role in this event. Treatment of pharyngeal cells with protein kinase inhibitors such as genistein and staurosporine significantly inhibited streptococcal invasion of pharyngeal cells. Therefore, these data indicated that streptococci/SDH-mediated phosphorylation plays a critical role in bacterial entry into the host cell. To identify the membrane receptor that elicits these signaling events, we found that SDH bound specifically to 30- and 32-kD membrane proteins in a direct ligand-binding assay. These findings clearly suggest that SDH plays an important role in cellular communication between streptococci and pharyngeal cells that may be important in host cell gene transcription, and hence in the pathogenesis of streptococcal infection.

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Streptococcal adherence to and invasion of  Detroit and FaDu human pharyngeal cells. Role of  protein tyrosine kinase and other protein kinases on the  streptococcal adherence and invasion was determined  by inhibiting these enzymes by specific kinase inhibitors, genistein, and staurosporine. Streptococcal adherence experiments with Detroit cells were carried out  for 3 h, while those with FaDu cells were carried out  for 1.25 h. The procedures after adhesion assay to determine streptococcal invasion for both the cell types  were identical. Number of colonized and invaded  streptococci were determined in terms of CFUs. In  each experiment, an average of the CFU counts from  4–6 individual wells of 24-well tissue culture plates was  calculated. Each bar represents the mean value of three  such experiments. Error bars indicate SD. Genistein or  staurosporine has no deleterious effect on the viability  of group A streptococci.
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Figure 5: Streptococcal adherence to and invasion of Detroit and FaDu human pharyngeal cells. Role of protein tyrosine kinase and other protein kinases on the streptococcal adherence and invasion was determined by inhibiting these enzymes by specific kinase inhibitors, genistein, and staurosporine. Streptococcal adherence experiments with Detroit cells were carried out for 3 h, while those with FaDu cells were carried out for 1.25 h. The procedures after adhesion assay to determine streptococcal invasion for both the cell types were identical. Number of colonized and invaded streptococci were determined in terms of CFUs. In each experiment, an average of the CFU counts from 4–6 individual wells of 24-well tissue culture plates was calculated. Each bar represents the mean value of three such experiments. Error bars indicate SD. Genistein or staurosporine has no deleterious effect on the viability of group A streptococci.

Mentions: To investigate whether the induction of tyrosine and serine/threonine phosphorylation of pharyngeal cellular proteins can influence the ability of group A streptococci to adhere and subsequently invade the host cell, these processes were studied in the presence and absence of protein kinase inhibitors (genistein or staurosporine) using both Detroit and FaDu cells. As shown in Fig. 5, streptococci adhered efficiently to Detroit pharyngeal cells (4.9 ± 0.5 × 106 CFU/ well), and this adherence was not significantly affected by treatment with the protein tyrosine kinase inhibitor, genistein, or a broad spectrum protein kinase inhibitor, staurosporine. However, in the presence of these inhibitors streptococcal invasion was significantly inhibited (Fig 5). Similar results were obtained when the adherence and invasion assays were performed using FaDu pharyngeal cells. The lower level of streptococcal adherence to and invasion of FaDu cells reflects the shorter incubation time used for the FaDu-based adherence assay as compared to Detroit cells (1.25 versus 3 h). Longer incubation of streptococci with FaDu cells resulted in detachment of the cells from the tissue culture plates. Both genistein and staurosporine inhibited streptococcal invasion of Detroit as well as FaDu pharyngeal cells by as much as 20-fold. These results indicate that SDH/streptococci-mediated induction of protein tyrosine kinase and protein kinase C are important for streptococcal invasion.


Regulation of the phosphorylation of human pharyngeal cell proteins by group A streptococcal surface dehydrogenase: signal transduction between streptococci and pharyngeal cells.

Pancholi V, Fischetti VA - J. Exp. Med. (1997)

Streptococcal adherence to and invasion of  Detroit and FaDu human pharyngeal cells. Role of  protein tyrosine kinase and other protein kinases on the  streptococcal adherence and invasion was determined  by inhibiting these enzymes by specific kinase inhibitors, genistein, and staurosporine. Streptococcal adherence experiments with Detroit cells were carried out  for 3 h, while those with FaDu cells were carried out  for 1.25 h. The procedures after adhesion assay to determine streptococcal invasion for both the cell types  were identical. Number of colonized and invaded  streptococci were determined in terms of CFUs. In  each experiment, an average of the CFU counts from  4–6 individual wells of 24-well tissue culture plates was  calculated. Each bar represents the mean value of three  such experiments. Error bars indicate SD. Genistein or  staurosporine has no deleterious effect on the viability  of group A streptococci.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199133&req=5

Figure 5: Streptococcal adherence to and invasion of Detroit and FaDu human pharyngeal cells. Role of protein tyrosine kinase and other protein kinases on the streptococcal adherence and invasion was determined by inhibiting these enzymes by specific kinase inhibitors, genistein, and staurosporine. Streptococcal adherence experiments with Detroit cells were carried out for 3 h, while those with FaDu cells were carried out for 1.25 h. The procedures after adhesion assay to determine streptococcal invasion for both the cell types were identical. Number of colonized and invaded streptococci were determined in terms of CFUs. In each experiment, an average of the CFU counts from 4–6 individual wells of 24-well tissue culture plates was calculated. Each bar represents the mean value of three such experiments. Error bars indicate SD. Genistein or staurosporine has no deleterious effect on the viability of group A streptococci.
Mentions: To investigate whether the induction of tyrosine and serine/threonine phosphorylation of pharyngeal cellular proteins can influence the ability of group A streptococci to adhere and subsequently invade the host cell, these processes were studied in the presence and absence of protein kinase inhibitors (genistein or staurosporine) using both Detroit and FaDu cells. As shown in Fig. 5, streptococci adhered efficiently to Detroit pharyngeal cells (4.9 ± 0.5 × 106 CFU/ well), and this adherence was not significantly affected by treatment with the protein tyrosine kinase inhibitor, genistein, or a broad spectrum protein kinase inhibitor, staurosporine. However, in the presence of these inhibitors streptococcal invasion was significantly inhibited (Fig 5). Similar results were obtained when the adherence and invasion assays were performed using FaDu pharyngeal cells. The lower level of streptococcal adherence to and invasion of FaDu cells reflects the shorter incubation time used for the FaDu-based adherence assay as compared to Detroit cells (1.25 versus 3 h). Longer incubation of streptococci with FaDu cells resulted in detachment of the cells from the tissue culture plates. Both genistein and staurosporine inhibited streptococcal invasion of Detroit as well as FaDu pharyngeal cells by as much as 20-fold. These results indicate that SDH/streptococci-mediated induction of protein tyrosine kinase and protein kinase C are important for streptococcal invasion.

Bottom Line: Intact streptococci and purified SDH induce a similar protein phosphorylation pattern with the de novo tyrosine phosphorylation of a 17-kD protein found in the membrane/particulate fraction of the pharyngeal cells.Treatment of pharyngeal cells with protein kinase inhibitors such as genistein and staurosporine significantly inhibited streptococcal invasion of pharyngeal cells.To identify the membrane receptor that elicits these signaling events, we found that SDH bound specifically to 30- and 32-kD membrane proteins in a direct ligand-binding assay.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, New York 10021, USA. panchov@rockvax.rockefeller.edu

ABSTRACT
Whether cell-to-cell communication results when group A streptococci interact with their target cells is unknown. Here, we report that upon contact with cultured human pharyngeal cells, both whole streptococci and purified streptococcal surface dehydrogenase (SDH) activate pharyngeal cell protein tyrosine kinase as well as protein kinase C, thus regulating the phosphorylation of cellular proteins. SDH, a major surface protein of group A streptococci, has both glyceraldehyde-3-phosphate dehydrogenase and ADP-ribosylating enzyme activities that may relate to early stages of streptococcal infection. Intact streptococci and purified SDH induce a similar protein phosphorylation pattern with the de novo tyrosine phosphorylation of a 17-kD protein found in the membrane/particulate fraction of the pharyngeal cells. However, this phosphorylation required the presence of cytosolic components. NH2-terminal amino acid sequence analysis identified the 17-kD protein as nuclear core histone H3. Both phosphotyrosine and phosphoserine-specific monoclonal antibodies reacted with the 17-kD protein by Western blot, suggesting that the binding of SDH to these pharyngeal cells elicits a novel signaling pathway that ultimately leads to activation of histone H3-specific kinases. Genistein-inhibitable phosphorylation of histone H3 indicates that tyrosine kinase plays a key role in this event. Treatment of pharyngeal cells with protein kinase inhibitors such as genistein and staurosporine significantly inhibited streptococcal invasion of pharyngeal cells. Therefore, these data indicated that streptococci/SDH-mediated phosphorylation plays a critical role in bacterial entry into the host cell. To identify the membrane receptor that elicits these signaling events, we found that SDH bound specifically to 30- and 32-kD membrane proteins in a direct ligand-binding assay. These findings clearly suggest that SDH plays an important role in cellular communication between streptococci and pharyngeal cells that may be important in host cell gene transcription, and hence in the pathogenesis of streptococcal infection.

Show MeSH
Related in: MedlinePlus