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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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Transmission electron microscopy (TEM) of SDH-treated  and -untreated (Control) pharyngeal cells grown on Transwell membranes.  (A) TEM viewed at low magnification (3,250×). (B) TEM viewed at  higher magnification (6,600×) emphasizing both the nuclear morphology  and apical tight junction (arrow) of control and SDH-treated pharyngeal  cells. The SDH-treated panel shows the nucleus with marked chromatin  condensation (white arrows). (C) TEM of nonpolarized Detroit cells grown  on polystyrene plates treated similarly as in A. Original magnification,  6,600×. Nonapical tight junction (arrow) and chromatin condensation in  the nucleus of SDH-treated cells (white arrows) are indicated.
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Figure 4: Transmission electron microscopy (TEM) of SDH-treated and -untreated (Control) pharyngeal cells grown on Transwell membranes. (A) TEM viewed at low magnification (3,250×). (B) TEM viewed at higher magnification (6,600×) emphasizing both the nuclear morphology and apical tight junction (arrow) of control and SDH-treated pharyngeal cells. The SDH-treated panel shows the nucleus with marked chromatin condensation (white arrows). (C) TEM of nonpolarized Detroit cells grown on polystyrene plates treated similarly as in A. Original magnification, 6,600×. Nonapical tight junction (arrow) and chromatin condensation in the nucleus of SDH-treated cells (white arrows) are indicated.

Mentions: Protein phosphorylation in general has a significant impact on cell shape, size, and motility (27), and phosphorylation of histone proteins in particular has been shown to result in condensation of chromatin structure (21–23, 28–30). Therefore, we investigated whether we could observe these ultrastructural changes in SDH-treated Detroit cells. Results obtained by transmission electron microscopy of control untreated Detroit cells grown on the surface of Transwell membranes revealed a highly compact monolayer of cells with distinct tight junctions at their basolateral sides towards the apical region. In contrast, using the same parameters as the phosphorylation experiments, Detroit cells treated with SDH showed a clear disruption in this arrangement (Fig. 4 A) with a change in shape and size and a significant change in the nucleus-to-cytoplasmic size ratio. Furthermore, chromatin was found to be distinctly condensed within the nucleus when compared with control untreated cells (Fig. 4 B). Similar findings were also obtained with nonpolarized Detroit cells grown on polystyrene plates (Fig. 4 C). These findings further support the role of SDH-mediated induction of protein phosphorylation in cell morphology, and in particular that of histone H3 in nuclear condensation.


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)

Transmission electron microscopy (TEM) of SDH-treated  and -untreated (Control) pharyngeal cells grown on Transwell membranes.  (A) TEM viewed at low magnification (3,250×). (B) TEM viewed at  higher magnification (6,600×) emphasizing both the nuclear morphology  and apical tight junction (arrow) of control and SDH-treated pharyngeal  cells. The SDH-treated panel shows the nucleus with marked chromatin  condensation (white arrows). (C) TEM of nonpolarized Detroit cells grown  on polystyrene plates treated similarly as in A. Original magnification,  6,600×. Nonapical tight junction (arrow) and chromatin condensation in  the nucleus of SDH-treated cells (white arrows) are indicated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199133&req=5

Figure 4: Transmission electron microscopy (TEM) of SDH-treated and -untreated (Control) pharyngeal cells grown on Transwell membranes. (A) TEM viewed at low magnification (3,250×). (B) TEM viewed at higher magnification (6,600×) emphasizing both the nuclear morphology and apical tight junction (arrow) of control and SDH-treated pharyngeal cells. The SDH-treated panel shows the nucleus with marked chromatin condensation (white arrows). (C) TEM of nonpolarized Detroit cells grown on polystyrene plates treated similarly as in A. Original magnification, 6,600×. Nonapical tight junction (arrow) and chromatin condensation in the nucleus of SDH-treated cells (white arrows) are indicated.
Mentions: Protein phosphorylation in general has a significant impact on cell shape, size, and motility (27), and phosphorylation of histone proteins in particular has been shown to result in condensation of chromatin structure (21–23, 28–30). Therefore, we investigated whether we could observe these ultrastructural changes in SDH-treated Detroit cells. Results obtained by transmission electron microscopy of control untreated Detroit cells grown on the surface of Transwell membranes revealed a highly compact monolayer of cells with distinct tight junctions at their basolateral sides towards the apical region. In contrast, using the same parameters as the phosphorylation experiments, Detroit cells treated with SDH showed a clear disruption in this arrangement (Fig. 4 A) with a change in shape and size and a significant change in the nucleus-to-cytoplasmic size ratio. Furthermore, chromatin was found to be distinctly condensed within the nucleus when compared with control untreated cells (Fig. 4 B). Similar findings were also obtained with nonpolarized Detroit cells grown on polystyrene plates (Fig. 4 C). These findings further support the role of SDH-mediated induction of protein phosphorylation in cell morphology, and in particular that of histone H3 in nuclear condensation.

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