Localization of the C3-Like ADP-Ribosyltransferase from Staphylococcus aureus during Bacterial Invasion of Mammalian Cells
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感染与免疫杂志 2006年第6期
Department of Microbial Pathogenesis, GBF—German Research Centre for Biotechnology, Braunschweig, Germany
Institut für Toxikologie der Medizinischen Hochschule Hannover, Hannover, Germany
Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Universitt Freiburg, Freiburg, Germany
ABSTRACT
The C3stau2 exoenzyme from Staphylococcus aureus is a C3-like ADP-ribosyltransferase which possesses no specific receptor-binding domain or translocation unit required for entry in target cells where its substrate is located. Here we show that C3stau2 can reach its target after invasion of staphylococci in eukaryotic cells without needing translocation.
TEXT
C3-like transferases are a family of enzymes with the capacity of exclusively ADP ribosylating RhoA, RhoB, and RhoC at asparagine 41 (2, 3, 7, 12). Members of the Rho subfamily of small GTPases are involved in multiple cellular functions, including cell morphology, cell adhesion, phagocytosis, endocytosis, apoptosis, and smooth-muscle contraction (4). Rho GTPases are key regulators of actin dynamics and function as molecular switches in various signal transduction pathways (1, 5, 6, 8). In vitro, the ADP ribosylation results in a functional inactivation of Rho reflected by disaggregation of the actin cytoskeleton of eukaryotic cells. Recently, we have characterized the C3stau2 exoenzyme, a new C3-like transferase produced by a clinical isolate of Staphylococcus aureus (13-15).
C3-like ADP-ribosyltransferases such as C3stau2 consist entirely of an enzyme domain, lacking a specific receptor-binding domain or a translocation unit that could be used to enter eukaryotic target cells to reach their intracellularly located substrates. The role in virulence of an exoenzyme that could not reach the intracellular target per se remains enigmatic and required further study. To explain this role, at least two possibilities are plausible: first, a translocation of bacterial exoenzymes through eukaryotic membranes, particularly in gram-positive bacteria, via bacterial pore-forming toxins as an equivalent to the type III secretion system of gram-negative bacteria (9). S. aureus produces the pore-forming alpha-toxin, and it remains to be investigated whether this toxin could facilitate the translocation of other exoproteins into target cells. The second possibility is that the C3-like exoenzymes do not need to be transported because they are released intracellularly. As staphylococci have the capacity to invade mammalian cells, we show here that S. aureus bacteria invade mammalian cells and, once intracellularly located, secrete the C3-like ADP-ribosyltransferase, which then reaches its target.
The S. aureus strain characterized in this study is a clinical isolate from a patient with postoperative infection and produces a C3-like ADP-ribosyltransferase designated as C3stau2 (14). Another clinical isolate, Sa59, which does not produce C3stau2, was included as a control. Wild-type or mutant Glu180Leu C3stau2 was expressed as recombinant glutathione S-transferase-fusion proteins in Escherichia coli, as previously described (13). Rabbit polyclonal anti-C3stau2 serum was prepared by immunizing a rabbit with 100 μg wild-type C3stau2 at days 0, 28, and 56, and immunoglobulin G was purified by affinity chromatography.
The human HeLa (ATCC CCL-2) epithelial cell line was cultured in Dulbecco's modified Eagle medium (Life Technologies) supplemented with 10% fetal calf serum (Life Technologies) and 5 mM glutamine (Life Technologies) at 37°C in an atmosphere containing 5% CO2. For invasion assays, cells were resuspended at a concentration of approximately 3 x 105 cells/ml in Dulbecco's modified Eagle medium in 24-well and 6-cm tissue plates (Nunc); for microscopic assays, approximately 2 x 105 cells/ml were seeded onto 12-mm-diameter glass coverslips placed on the bottom of the tissue culture plates, as previously described (10). Bacterial adherence and internalization were assessed as previously described (11). Electron microscopy analysis of the infected cells was performed as previously described (11).
First we investigated the invasion capacity of the C3stau2-producing S. aureus Hmi6 strain used in this study. The human epithelial HeLa cells and a mouse mammary epithelial cell line (Mm5MT) were used as hosts for S. aureus Hmi6 infection. Higher attachment (40% and 70%) and low invasion (2% and 3%) were observed for HeLa and mammary cells, respectively.
For a precise localization of the C3stau2 toxin during the infection process of S. aureus strain Hmi6, we used immunoelectron microscopy. As a control for unspecific label, noninfected cells were also processed and analyzed. These cells showed very low background label (Fig. 1A). Analysis by immunogold labeling of HeLa cells infected with the Hmi6 strain showed poor labeling of extracellular bacteria after 30 min of infection and no C3stau2 toxin associated with the host cells (Fig. 1B and C). After 1 h of infection, a large number of bacteria were attached (Fig. 1C) and some were localized intracellularly (Fig. 1D). More C3stau2 toxin was present on the surface of the intracellular (Fig. 1D) than in the extracellular bacteria, and almost no free toxin associated with the cellular membrane or in the cytosol was observed (Fig. 1B to D). After 2 h of infection, a large amount of gold particles was detected associated with all intracellular bacteria which were present in endosomes. Cellular membrane and cytoplasm continue to show almost no labeling (Fig. 2A and B).
A completely different pattern of labeling was observed in later stages of staphylococcal and mammalian cell interactions. After 3 h of infection staphylococci were observed free in the cytoplasm (Fig. 2C) or in degenerated endosomes (Fig. 2D). The C3stau2 toxin was associated with bacteria but was also observed for the first time in large amounts in secreted form in the host cytoplasm of infected cells (Fig. 2C and D). Quantification of the proportion of labeled toxin associated with bacteria or in the cytoplasm, assessed by examination of thin sections in the transmission electron microscopy, showed that the percentage of ADP-ribosyltransferase present in secreted form in the host cell cytoplasm increased steadily from 2 to 3 h after infection (from 7% at 2 h to 60% at 3 h). The host cells showed signs of injury after 4 h of infection (data not shown). S. aureus strain Sa59, which does not produce C3stau2 but has the capacity to invade eukaryotic cells and is not cytotoxic, showed no labeling in the same set of experiments (Fig. 2E). In all these experiments approximately 100 eukaryotic cells were examined, and a limited number of representative images were selected. These data indicate that (i) C3stau2 is produced by intracellular bacteria during infection and (ii) C3stau2 is released and localized in a secreted form in the cytoplasm of infected host cells. These results demonstrated that C3stau2 can reach its intracellular targets during the staphylococcal invasion process and therefore does not require receptor binding or a translocation unit. Thus, bacteria possessing C3stau2 transferases that modify Rho GTPases may modulate Rho-dependent cellular processes to the advantage of pathogen, thereby playing an important role in pathogenesis.
To establish the role of C3stau2 in virulence, we used the wild-type enzyme and a mutant toxin (C3stau2 Glu180Leu), which exhibited 10,000-fold-lower ADP ribosylating transferase activity. To facilitate the penetration of the toxin we used Influx pinocytic cell loading (IPCL) reagent to load either the native or the mutant toxin. HeLa were incubated with (i) medium alone and (ii) native or mutant toxin (0.5 μg/ml), (iii) IPCL reagent, or (iv) IPCL reagent plus native or mutant toxin (0.5 μg/ml). After 3 h the cells were fixed and permeabilized, and actin was stained with Texas Red-conjugated phalloidin. The IPCL reagent had no effect on HeLa cell morphology, and the cytoskeleton organization did not undergo significant changes (Fig. 3B) compared to control nonloaded cells (Fig. 3A). The cells loaded with 0.5 μg/ml of toxin for 10 min showed drastic morphological changes (Fig. 3D) in comparison to the control IPCL cells (Fig. 3B) and the nonloaded cells, which were incubated with 0.5 μg/ml of toxin alone for 3 h (Fig. 3C). The mutant toxin had little effect when incubated with the cells (Fig. 3E) and produced same disassembly of the actin stress fibers but no cytotoxicity when loaded into HeLa cells (Fig. 3F). Similar results were observed for other nonphagocytic cell lines such as skin epithelial cells (A431) and a mouse mammary epithelial-like cell line, Mm5MT (ATCC CRL-1637) (data not shown). These data indicate that the presence of C3Stau2 toxin, through its ADP ribosylation activity, results in disaggregation of the actin cytoskeleton and substantially contributes to virulence of internalized staphylococci. Our findings provide the first evidence showing how C3-like exoenzymes may reach their specific targets in vivo without a translocation unit and underline the importance of these enzymes in bacterial virulence.
ACKNOWLEDGMENTS
We thank E. Müller for her outstanding technical assistance in the electron microscopy studies and K. N. Timmis for his generous support and encouragement throughout this work.
This work has been supported by a grant from the Deutsche Forschungsgemeinschaft, Germany (grant SFB 388, project Ak6/10).
REFERENCES
1. Aktories, K., and J. T. Barbieri. 2005. Bacterial cytotoxins: targeting eukaryotic switches. Nat. Rev. Microbiol. 3:397-410.
2. Aktories, K., U. Weller, and G. S. Chhatwal. 1987. Clostridium botulinum type C produces a novel ADP-ribosyltransferase distinct from botulinum C2 toxin. FEBS Lett. 212:109-113.
3. Aktories, K., C. Wilde, and M. Vogelsgesang. 2004. Rho-modifying C3-like ADP-ribosyltransferases. Rev. Physiol. Biochem. Pharmacol. 152:1-22.
4. Bishop, A. L., and A. Hall. 2000. Rho GTPases and their effector proteins. Biochem. J. 348(Pt. 2):241-255.
5. Hall, A. 1998. Rho GTPases and the actin cytoskeleton. Science 279:509-514.
6. Inoue, S., M. Sugai, Y. Murooka, S. Y. Paik, Y. M. Hong, H. Ohgai, and H. Suginaka. 1991. Molecular cloning and sequencing of the epidermal cell differentiation inhibitor gene from Staphylococcus aureus. Biochem. Biophys. Res. Commun. 174:459-464.
7. Just, I., F. Hofmann, H. Genth, and R. Gerhard. 2001. Bacterial protein toxins inhibiting low-molecular-mass GTP-binding proteins. Int. J. Med. Microbiol. 291:243-250.
8. Kjoller, L., and A. Hall. 1999. Signaling to Rho GTPases. Exp. Cell Res. 253:166-179.
9. Madden, J. C., N. Ruiz, and M. Caparon. 2001. Cytolysin-mediated translocation (CMT): a functional equivalent of type III secretion in gram-positive bacteria. Cell 104:143-152.
10. Molinari, G., and G. S. Chhatwal. 1998. Invasion and survival of Streptococcus pyogenes in eukaryotic cells correlates with the source of the clinical isolates. J. Infect. Dis. 177:1600-1607.
11. Molinari, G., M. Rohde, S. R. Talay, G. S. Chhatwal, S. Beckert, and A. Podbielski. 2001. The role played by the group A streptococcal negative regulator Nra on bacterial interactions with epithelial cells. Mol. Microbiol. 40:99-114.
12. Wilde, C., and K. Aktories. 2001. The Rho-ADP-ribosylating C3 exoenzyme from Clostridium botulinum and related C3-like transferases. Toxicon 39:1647-1660.
13. Wilde, C., G. S. Chhatwal, and K. Aktories. 2002. C3stau, a new member of the family of C3-like ADP-ribosyltransferases. Trends Microbiol. 10:5-7.
14. Wilde, C., G. S. Chhatwal, G. Schmalzing, K. Aktories, and I. Just. 2001. A novel C3-like ADP-ribosyltransferase from Staphylococcus aureus modifying RhoE and Rnd3. J. Biol. Chem. 276:9537-9542.
15. Wilde, C., I. Just, and K. Aktories. 2002. Structure-function analysis of the Rho-ADP-ribosylating exoenzyme C3stau2 from Staphylococcus aureus. Biochemistry 41:1539-1544.(Gabriella Molinari, Manfr)
Institut für Toxikologie der Medizinischen Hochschule Hannover, Hannover, Germany
Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Universitt Freiburg, Freiburg, Germany
ABSTRACT
The C3stau2 exoenzyme from Staphylococcus aureus is a C3-like ADP-ribosyltransferase which possesses no specific receptor-binding domain or translocation unit required for entry in target cells where its substrate is located. Here we show that C3stau2 can reach its target after invasion of staphylococci in eukaryotic cells without needing translocation.
TEXT
C3-like transferases are a family of enzymes with the capacity of exclusively ADP ribosylating RhoA, RhoB, and RhoC at asparagine 41 (2, 3, 7, 12). Members of the Rho subfamily of small GTPases are involved in multiple cellular functions, including cell morphology, cell adhesion, phagocytosis, endocytosis, apoptosis, and smooth-muscle contraction (4). Rho GTPases are key regulators of actin dynamics and function as molecular switches in various signal transduction pathways (1, 5, 6, 8). In vitro, the ADP ribosylation results in a functional inactivation of Rho reflected by disaggregation of the actin cytoskeleton of eukaryotic cells. Recently, we have characterized the C3stau2 exoenzyme, a new C3-like transferase produced by a clinical isolate of Staphylococcus aureus (13-15).
C3-like ADP-ribosyltransferases such as C3stau2 consist entirely of an enzyme domain, lacking a specific receptor-binding domain or a translocation unit that could be used to enter eukaryotic target cells to reach their intracellularly located substrates. The role in virulence of an exoenzyme that could not reach the intracellular target per se remains enigmatic and required further study. To explain this role, at least two possibilities are plausible: first, a translocation of bacterial exoenzymes through eukaryotic membranes, particularly in gram-positive bacteria, via bacterial pore-forming toxins as an equivalent to the type III secretion system of gram-negative bacteria (9). S. aureus produces the pore-forming alpha-toxin, and it remains to be investigated whether this toxin could facilitate the translocation of other exoproteins into target cells. The second possibility is that the C3-like exoenzymes do not need to be transported because they are released intracellularly. As staphylococci have the capacity to invade mammalian cells, we show here that S. aureus bacteria invade mammalian cells and, once intracellularly located, secrete the C3-like ADP-ribosyltransferase, which then reaches its target.
The S. aureus strain characterized in this study is a clinical isolate from a patient with postoperative infection and produces a C3-like ADP-ribosyltransferase designated as C3stau2 (14). Another clinical isolate, Sa59, which does not produce C3stau2, was included as a control. Wild-type or mutant Glu180Leu C3stau2 was expressed as recombinant glutathione S-transferase-fusion proteins in Escherichia coli, as previously described (13). Rabbit polyclonal anti-C3stau2 serum was prepared by immunizing a rabbit with 100 μg wild-type C3stau2 at days 0, 28, and 56, and immunoglobulin G was purified by affinity chromatography.
The human HeLa (ATCC CCL-2) epithelial cell line was cultured in Dulbecco's modified Eagle medium (Life Technologies) supplemented with 10% fetal calf serum (Life Technologies) and 5 mM glutamine (Life Technologies) at 37°C in an atmosphere containing 5% CO2. For invasion assays, cells were resuspended at a concentration of approximately 3 x 105 cells/ml in Dulbecco's modified Eagle medium in 24-well and 6-cm tissue plates (Nunc); for microscopic assays, approximately 2 x 105 cells/ml were seeded onto 12-mm-diameter glass coverslips placed on the bottom of the tissue culture plates, as previously described (10). Bacterial adherence and internalization were assessed as previously described (11). Electron microscopy analysis of the infected cells was performed as previously described (11).
First we investigated the invasion capacity of the C3stau2-producing S. aureus Hmi6 strain used in this study. The human epithelial HeLa cells and a mouse mammary epithelial cell line (Mm5MT) were used as hosts for S. aureus Hmi6 infection. Higher attachment (40% and 70%) and low invasion (2% and 3%) were observed for HeLa and mammary cells, respectively.
For a precise localization of the C3stau2 toxin during the infection process of S. aureus strain Hmi6, we used immunoelectron microscopy. As a control for unspecific label, noninfected cells were also processed and analyzed. These cells showed very low background label (Fig. 1A). Analysis by immunogold labeling of HeLa cells infected with the Hmi6 strain showed poor labeling of extracellular bacteria after 30 min of infection and no C3stau2 toxin associated with the host cells (Fig. 1B and C). After 1 h of infection, a large number of bacteria were attached (Fig. 1C) and some were localized intracellularly (Fig. 1D). More C3stau2 toxin was present on the surface of the intracellular (Fig. 1D) than in the extracellular bacteria, and almost no free toxin associated with the cellular membrane or in the cytosol was observed (Fig. 1B to D). After 2 h of infection, a large amount of gold particles was detected associated with all intracellular bacteria which were present in endosomes. Cellular membrane and cytoplasm continue to show almost no labeling (Fig. 2A and B).
A completely different pattern of labeling was observed in later stages of staphylococcal and mammalian cell interactions. After 3 h of infection staphylococci were observed free in the cytoplasm (Fig. 2C) or in degenerated endosomes (Fig. 2D). The C3stau2 toxin was associated with bacteria but was also observed for the first time in large amounts in secreted form in the host cytoplasm of infected cells (Fig. 2C and D). Quantification of the proportion of labeled toxin associated with bacteria or in the cytoplasm, assessed by examination of thin sections in the transmission electron microscopy, showed that the percentage of ADP-ribosyltransferase present in secreted form in the host cell cytoplasm increased steadily from 2 to 3 h after infection (from 7% at 2 h to 60% at 3 h). The host cells showed signs of injury after 4 h of infection (data not shown). S. aureus strain Sa59, which does not produce C3stau2 but has the capacity to invade eukaryotic cells and is not cytotoxic, showed no labeling in the same set of experiments (Fig. 2E). In all these experiments approximately 100 eukaryotic cells were examined, and a limited number of representative images were selected. These data indicate that (i) C3stau2 is produced by intracellular bacteria during infection and (ii) C3stau2 is released and localized in a secreted form in the cytoplasm of infected host cells. These results demonstrated that C3stau2 can reach its intracellular targets during the staphylococcal invasion process and therefore does not require receptor binding or a translocation unit. Thus, bacteria possessing C3stau2 transferases that modify Rho GTPases may modulate Rho-dependent cellular processes to the advantage of pathogen, thereby playing an important role in pathogenesis.
To establish the role of C3stau2 in virulence, we used the wild-type enzyme and a mutant toxin (C3stau2 Glu180Leu), which exhibited 10,000-fold-lower ADP ribosylating transferase activity. To facilitate the penetration of the toxin we used Influx pinocytic cell loading (IPCL) reagent to load either the native or the mutant toxin. HeLa were incubated with (i) medium alone and (ii) native or mutant toxin (0.5 μg/ml), (iii) IPCL reagent, or (iv) IPCL reagent plus native or mutant toxin (0.5 μg/ml). After 3 h the cells were fixed and permeabilized, and actin was stained with Texas Red-conjugated phalloidin. The IPCL reagent had no effect on HeLa cell morphology, and the cytoskeleton organization did not undergo significant changes (Fig. 3B) compared to control nonloaded cells (Fig. 3A). The cells loaded with 0.5 μg/ml of toxin for 10 min showed drastic morphological changes (Fig. 3D) in comparison to the control IPCL cells (Fig. 3B) and the nonloaded cells, which were incubated with 0.5 μg/ml of toxin alone for 3 h (Fig. 3C). The mutant toxin had little effect when incubated with the cells (Fig. 3E) and produced same disassembly of the actin stress fibers but no cytotoxicity when loaded into HeLa cells (Fig. 3F). Similar results were observed for other nonphagocytic cell lines such as skin epithelial cells (A431) and a mouse mammary epithelial-like cell line, Mm5MT (ATCC CRL-1637) (data not shown). These data indicate that the presence of C3Stau2 toxin, through its ADP ribosylation activity, results in disaggregation of the actin cytoskeleton and substantially contributes to virulence of internalized staphylococci. Our findings provide the first evidence showing how C3-like exoenzymes may reach their specific targets in vivo without a translocation unit and underline the importance of these enzymes in bacterial virulence.
ACKNOWLEDGMENTS
We thank E. Müller for her outstanding technical assistance in the electron microscopy studies and K. N. Timmis for his generous support and encouragement throughout this work.
This work has been supported by a grant from the Deutsche Forschungsgemeinschaft, Germany (grant SFB 388, project Ak6/10).
REFERENCES
1. Aktories, K., and J. T. Barbieri. 2005. Bacterial cytotoxins: targeting eukaryotic switches. Nat. Rev. Microbiol. 3:397-410.
2. Aktories, K., U. Weller, and G. S. Chhatwal. 1987. Clostridium botulinum type C produces a novel ADP-ribosyltransferase distinct from botulinum C2 toxin. FEBS Lett. 212:109-113.
3. Aktories, K., C. Wilde, and M. Vogelsgesang. 2004. Rho-modifying C3-like ADP-ribosyltransferases. Rev. Physiol. Biochem. Pharmacol. 152:1-22.
4. Bishop, A. L., and A. Hall. 2000. Rho GTPases and their effector proteins. Biochem. J. 348(Pt. 2):241-255.
5. Hall, A. 1998. Rho GTPases and the actin cytoskeleton. Science 279:509-514.
6. Inoue, S., M. Sugai, Y. Murooka, S. Y. Paik, Y. M. Hong, H. Ohgai, and H. Suginaka. 1991. Molecular cloning and sequencing of the epidermal cell differentiation inhibitor gene from Staphylococcus aureus. Biochem. Biophys. Res. Commun. 174:459-464.
7. Just, I., F. Hofmann, H. Genth, and R. Gerhard. 2001. Bacterial protein toxins inhibiting low-molecular-mass GTP-binding proteins. Int. J. Med. Microbiol. 291:243-250.
8. Kjoller, L., and A. Hall. 1999. Signaling to Rho GTPases. Exp. Cell Res. 253:166-179.
9. Madden, J. C., N. Ruiz, and M. Caparon. 2001. Cytolysin-mediated translocation (CMT): a functional equivalent of type III secretion in gram-positive bacteria. Cell 104:143-152.
10. Molinari, G., and G. S. Chhatwal. 1998. Invasion and survival of Streptococcus pyogenes in eukaryotic cells correlates with the source of the clinical isolates. J. Infect. Dis. 177:1600-1607.
11. Molinari, G., M. Rohde, S. R. Talay, G. S. Chhatwal, S. Beckert, and A. Podbielski. 2001. The role played by the group A streptococcal negative regulator Nra on bacterial interactions with epithelial cells. Mol. Microbiol. 40:99-114.
12. Wilde, C., and K. Aktories. 2001. The Rho-ADP-ribosylating C3 exoenzyme from Clostridium botulinum and related C3-like transferases. Toxicon 39:1647-1660.
13. Wilde, C., G. S. Chhatwal, and K. Aktories. 2002. C3stau, a new member of the family of C3-like ADP-ribosyltransferases. Trends Microbiol. 10:5-7.
14. Wilde, C., G. S. Chhatwal, G. Schmalzing, K. Aktories, and I. Just. 2001. A novel C3-like ADP-ribosyltransferase from Staphylococcus aureus modifying RhoE and Rnd3. J. Biol. Chem. 276:9537-9542.
15. Wilde, C., I. Just, and K. Aktories. 2002. Structure-function analysis of the Rho-ADP-ribosylating exoenzyme C3stau2 from Staphylococcus aureus. Biochemistry 41:1539-1544.(Gabriella Molinari, Manfr)