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Bacterial pathogenesis: The answer to virulence is in the pore
R264
Dispatch
Bacterial pathogenesis: The answer to virulence is in the pore Annick Gauthier and B. Brett Finlay
A wide variety of Gram-negative bacterial pathogens use a ‘type III’ protein secretion system to deliver bacterial virulence factors into host cells. Recent results suggest that Gram-positive pathogens may employ similar methods to deliver virulence factors into host cells. Address: Biotechnology Laboratory and Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3. E-mail: [email protected] Current Biology 2001, 11:R264–R267 0960-9822/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.
Delivery of virulence factors into host cells to interfere with and alter host processes is a crucial step in bacterial virulence. Pathogenic bacteria can be broadly divided into Gram-positive bacteria and Gram-negative bacteria, on the basis of their cell surface structures. Understanding protein delivery mechanisms has been pioneered in Gram-negative bacterial pathogens, with the identification of the so-called ‘type III’ secretion system in 1980 (reviewed in [1,2]). The type III secretion system delivers bacterial effectors directly from the cytoplasm of the bacterium into the host cell, thus crossing the bacterial inner membrane, peptidoglycan and the outer membrane, and the host plasma membrane. While the effectors are varied between pathogens, the greater than 20 type III apparatus components that have been identified are quite conserved across species. A type III secretion system has not been identified in Gram-positive pathogenic bacteria, nor until recently had any analagous system for delivering virulence factors into host cells been identified in such pathogens. This is not that surprising, considering the marked difference in cell-surface structure between Gram-negative and Grampositive bacteria — which do not have an outer membrane. New evidence now suggests that a system that is analogous to the type III system exists in Gram-positive pathogens; this is based on recently reported findings in Streptococcus pyogenes, the infectious agent of throat, soft tissue and systemic infections [3]. Gram-positive secretions
Many Gram-positive pathogens secrete A–B toxins, where ‘A’ is the enzymatically active component, and ‘B’ is the binding component which aids the internalization of the toxin into host cells [4]. These toxins, such as C2 toxin in Clostridium botulinum and diphtheria toxin from Corynebacterium diphtheriae, have an effect inside the host
cell, in these cases by ADP-ribosylating monomeric G actin and translation elongation factor 2, respectively. Similarly, anthrax toxin ‘protective antigen’ binds cells and mediates the entry of two other components to the cytoplasm via the endosomal pathway [5]. Gram-positive bacteria also secrete proteins that have an extracellular effect, such as the superantigens and proteases of S. pyogenes [6]. The question that arises is how secreted proteins are delivered into host cells, if the proteins are not A–B toxins but have an obvious requirement to reach an intracellular compartment. Group A and group C streptococcus have been known to secrete a protein with NADglycohydrolase activity for many years [7,8]. It seems evident that this protein could play a role in pathogenesis inside the host cell, but it is not understood how it could get to this site of action. Creating a pore
S. pyogenes streptolysin O is a member of a family of poreforming cholesterol-dependent cytolysins that includes Listeria monocytogenes listeriolysin O and Clostridium perfringens perfringolysin O (reviewed in [9]). This family of toxins shares a 30–60% similarity in amino acid sequence, with an invariant region at the carboxy-terminus (ECTGLAWEWWR) that is important for cytolytic activity. The cholesterol-dependent cytolysins are secreted as 47–60 kDa proteins by the general secretory pathway, with cleavable amino-terminal signal sequences. The secreted cytolysin monomers bind cholesterol in the host cell membrane and assemble into a large transmembrane pore of approximately 30 nm in diameter. While many of the cholesterol-dependent cytolysins have demonstrated cytolytic activity, it is thought that they may have a more subtle function as well [9]. An example of this is that while an slo mutant, defective in streptolysin O, still attached to host cells, it lost the ability to manipulate proinflammatory responses in keratinocytes [10]. Cytolysin-mediated translocation
By a stroke of good fortune, Madden et al. [3] identified a 52 kDa protein that could be detected in the cytosol of aCaT keratinocytes — by cross-reaction of antistreptolysin O antiserum to the 52 kDa band — only after infection by a streptolysin O-containing S. pyogenes. Amino-terminal sequencing of the 52 kDa band identified it as an S. pyogenes protein that is almost identical to the mature secreted form of group C streptococcal NAD-glycohydrolase [8]. Comparison of the sequence of the 52 kDa band to the S. pyogenes genome revealed that the region of identity was preceded by a stretch of 37 amino
Dispatch
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Table 1 Comparison of type III secretion and S. pyogenes cytolysin-mediated translocation.
Function Apparatus
Size of pore Effectors
Requires effector and pore-former in same bacterium Intermediates
Channel
Type III secretion
Cytolysin-mediated translocation
Delivers virulence factors into host cell
Delivers a virulence factor into host cell
More than 20 proteins: inner membrane, outer membrane, ATPase, channel/conduit forming, host cell pore-forming
One protein; potentially more? Cytolysin in host cell membrane General secretory pathway Sec system (6–7 proteins) form a complex that crosses bacterial cellular membrane.
≈2 nm
≈30 nm
Not amino-terminal processed Recognition signal may reside in amino-terminal amino acids or 5′ mRNA
Amino-terminal processed
Yes
Yes
No periplasmic or extracellular intermediates
Effector secreted via general secretory pathway into extracellular space then translocated
Continuous
Not continuous?
Contact-dependent
Usually
Translocation of SPN always requires adherence
Directional
Usually
Usually
acid residues characteristic of a signal sequence. This S. pyogenes protein was designated SPN, for S. pyogenes NAD-glycohydrolase. An spn mutant was constructed, which did not secrete SPN protein into the extracellular media or translocate SPN into HaCaT cell cytosol. While no NAD-glycohydrolase activity was detected in uninfected cells, high levels were seen in the cytosol of HaCaT cells infected with wild-type S. pyogenes, in a manner dependent on the presence of both SPN and streptolysin O [3]. The authors called this streptolysin O-dependent delivery of SPN from streptococcus into host cells ‘cytolysin-mediated translocation’.
note that, while there are similarities between these two systems, it would be premature to call the cytolysinmediated translocation a Gram-positive type III system. Table 1 shows a comparison of S. pyogenes cytolysinmediated translocation to the type III secretion system of Gram-negative pathogens. While the pore created in the host cell membrane by cytolysin-mediated translocation is much larger than that of the type III system, in both systems translocation is directional. Most of the effector proteins in both systems are translocated into the host cell and not to the extracellular environment ([3], reviewed in [2]).
In an elegant experiment, it was demonstrated by coinfecting HaCaT cells with an spn mutant and an slo mutant that SPN does not simply diffuse into host cells through the pores created by streptolysin O [3]. A diffusion process would only require the presence of both streptolysin O and SPN. SPN was not delivered in the co-infection experiment, and no NAD-glycohydrolase activity was detected in HaCaT cells, indicating that this is a coordinated process. Madden et al. [3] went on to show that the SPN activity in the host cell cytosol was not due to endocytosis or streptococcal invasion by using cytochalasin D, an inhibitor of actin polymerization and bacterial uptake. From additional experiments, they concluded that translocation is a polarized process in the presence of the cytolysin, where SPN is directed into the host cell.
The greatest similarity in the function of these systems seems to be delivery of virulence factors into host cells. However, there are many notable differences. Type III effectors do not have cleavable amino-terminal signal sequences (reviewed in [2]). So far, only one protein has been identified as part of the cytolysin-mediated translocation system — the cytolysin — while more than 20 proteins of the type III system have been identified, including proteins of the bacterial cytosol, inner membrane and outer membrane, proteins that form conduits to the host cell, and those that form a pore in the host cell membrane. The completion of the S. pyogenes genome (http://www.genome.ou.edu/strep.html) may provide clues to the existence of other cytolysin-mediated translocation components. While cytolysins are secreted by bacteria as water-soluble monomers, which then oligomerize and insert spontaneously into host cell membranes (reviewed in [9]), the host cell pore-forming type III proteins YopB and YopD from Yersinia enterocolitica
Gram-positive versus Gram-negative translocation
This cytolysin-mediated transport is reminiscent of Gramnegative type III secretion systems. It is important to
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Current Biology Vol 11 No 7
Figure 1
Cytolysin-mediated translocation Gram-positive bacterium Sec complex
Type III secretion system
Gram-negative bacterium Type III complex Inner membrane
Inner Membrane
Peptidoglycan / cell wall
Peptidoglycan / cell wall
Outer membrane
Protected protein channel?
Protein channel Host plasma membrane
Host plasma membrane
Cytolysin
Pore-forming proteins
Host cell
Host cell
Current Biology
A model of cytolysin-mediated transport compared to Gram-negative pathogens type III secretion system. In cytolysin-mediated translocation, the effectors are secreted via the general secretory pathway into an as yet unidentified channel, which delivers the effectors to the large pore formed by the cytolysin for translocation into the host cell cytosol. For
type III secretion, the effectors are delivered through a continuous machinery through both bacterial membranes and host cell membranes. In enteropathogenic Escherichia coli, the secretion complex in the bacterium is formed by the Escs, the channel is formed by EspA, and the pore in the host cell membrane by EspB and EspD (reviewed in [1,2]).
are themselves substrates of the type III secretion system (reviewed in [2]).
The proof is still to come
The type III secretion system is thought to mediate onestep translocation of effectors across bacterial and host cell membranes in a continuous process [11]. On the surface, it appears that, since SPN has a cleavable amino-terminal signal sequence, it is secreted into the extracellular milieu by the general secretory pathway before it is translocated via streptolysin O [3]. However, a strong argument can be made that cytolysin-mediated translocation is a continuous process. If extracellular SPN is translocated through the streptolysin O pore, a mixed infection where one bacterium provides SPN and a separate bacterium supplies streptolysin O would yield SPN activity in the cytosol of host cells. As mentioned above, co-infection did not rescue translocation [3]. Similarly, in Yersinia enterocolitica it has been shown that effectors must be in the same bacterium as the one producing the pore-forming YopB and YopD in order to be delivered into host cells [12].
While Madden et al. [3] favour a model in which SPN is first secreted and then translocated via streptolysin O, they mention — and their data better support — a model for cytolysin-mediated translocation in which the effectors are translocated via a protected channel formed between the bacterium and the streptolysin O pore in the host cell (Figure 1). A needle-like complex has been visualized in many type III secretion systems, including Salmonella spp, Shigella spp and enteropathogenic Escherichia coli, and data suggest that this complex serves as a conduit for protein delivery (reviewed in [2]). The presence or absence of a protected protein channel in cytolysin-mediated translocation needs to be investigated. The new work reported by Madden et al. [3] is an exciting initial step towards understanding the delivery of virulence factors from Gram-positive bacteria into host cells. It remains to be shown if multiple proteins are involved in the cytolysin-mediated translocation process, as either
Dispatch
effectors or components of the translocation system. Considerable work is needed to determine if the effectors are translocated through a continuous channel. As additional studies progress, it will be interesting to determine how widespread this mechanism is in Gram-positive pathogens. Many lessons have been learned from the years of study of the Gram-negative type III secretion system, the most important being the complexity and intricacy of the virulence delivery systems. Acknowledgements We would like to thank Samantha Gruenheid and John H. Brumell for critical reading of this manuscript. Work in B.B.F.’s laboratory is supported by operating grants from the Medical Research Council of Canada and the Canadian Bacterial Disease Network. A.G. is supported by a doctoral research award from MRC, and B.B.F. is a Howard Hughes International Scholar and an MRC Scientist.
References 1. Hueck CJ: Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev 1998, 62:379-433. 2. Cornelis GR, Van Gijsegem F: Assembly and function of type III secretory systems. Annu Rev Microbiol 2000, 54:735-774. 3. Madden JC, Ruiz N, Caparon M: Cytolysin-mediated translocation (cytolysin-mediated translocation): a functional equivalent of type III secretion in Gram-positive bacteria. Cell 2001, 104:143-152. 4. Schmitt CK, Meysick KC, O’Brien AD: Bacterial toxins: friends or foes? Emerg Infect Dis 1999, 5:224-234. 5. Duesbery NS, Vande Woude GF: Anthrax toxins. Cell Mol Life Sci 1999, 55:1599-1609. 6. Cunningham MW: Pathogenesis of group A streptococcal infections. Clin Microbiol Rev 2000, 13:470-511. 7. Carlson AS, Kellner A, Bernheimer AW, Freeman EB: A streptococcal enzyme that acts specifically upon diphosphopyridine nucleotide: chracterization of the enzyme and its separation from streptolysin O. J Exp Med 1957, 106:15-26. 8. Gerlach D, Ozegowski JH, Gunther E, Vettermann S, Kohler W: Purification and some properties of streptococcal NADglycohydrolase. FEMS Microbiol Lett 1996, 136:71-78. 9. Billington SJ, Jost BH, Songer JG: Thiol-activated cytolysins: structure, function and role in pathogenesis. FEMS Microbiol Lett 2000, 182:197-205. 10. Ruiz N, Wang B, Pentland A, Caparon M: Streptolysin O and adherence synergistically modulate proinflammatory responses of keratinocytes to group A streptococci. Mol Microbiol 1998, 27:337-346. 11. Lee VT, Anderson DM, Schneewind O: Targeting of Yersinia Yop proteins into the cytosol of HeLa cells: one-step translocation of YopE across bacterial and eukaryotic membranes is dependent on SycE chaperone. Mol Microbiol 1998, 28:593-601. 12. Sory MP, Boland A, Lambermont I, Cornelis GR: Identification of the YopE and YopH domains required for secretion and internalization into the cytosol of macrophages, using the cyaA gene fusion approach. Proc Natl Acad Sci USA 1995, 92:11998-12002.
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Dispatch
Bacterial pathogenesis: The answer to virulence is in the pore Annick Gauthier and B. Brett Finlay
A wide variety of Gram-negative bacterial pathogens use a ‘type III’ protein secretion system to deliver bacterial virulence factors into host cells. Recent results suggest that Gram-positive pathogens may employ similar methods to deliver virulence factors into host cells. Address: Biotechnology Laboratory and Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3. E-mail: [email protected] Current Biology 2001, 11:R264–R267 0960-9822/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.
Delivery of virulence factors into host cells to interfere with and alter host processes is a crucial step in bacterial virulence. Pathogenic bacteria can be broadly divided into Gram-positive bacteria and Gram-negative bacteria, on the basis of their cell surface structures. Understanding protein delivery mechanisms has been pioneered in Gram-negative bacterial pathogens, with the identification of the so-called ‘type III’ secretion system in 1980 (reviewed in [1,2]). The type III secretion system delivers bacterial effectors directly from the cytoplasm of the bacterium into the host cell, thus crossing the bacterial inner membrane, peptidoglycan and the outer membrane, and the host plasma membrane. While the effectors are varied between pathogens, the greater than 20 type III apparatus components that have been identified are quite conserved across species. A type III secretion system has not been identified in Gram-positive pathogenic bacteria, nor until recently had any analagous system for delivering virulence factors into host cells been identified in such pathogens. This is not that surprising, considering the marked difference in cell-surface structure between Gram-negative and Grampositive bacteria — which do not have an outer membrane. New evidence now suggests that a system that is analogous to the type III system exists in Gram-positive pathogens; this is based on recently reported findings in Streptococcus pyogenes, the infectious agent of throat, soft tissue and systemic infections [3]. Gram-positive secretions
Many Gram-positive pathogens secrete A–B toxins, where ‘A’ is the enzymatically active component, and ‘B’ is the binding component which aids the internalization of the toxin into host cells [4]. These toxins, such as C2 toxin in Clostridium botulinum and diphtheria toxin from Corynebacterium diphtheriae, have an effect inside the host
cell, in these cases by ADP-ribosylating monomeric G actin and translation elongation factor 2, respectively. Similarly, anthrax toxin ‘protective antigen’ binds cells and mediates the entry of two other components to the cytoplasm via the endosomal pathway [5]. Gram-positive bacteria also secrete proteins that have an extracellular effect, such as the superantigens and proteases of S. pyogenes [6]. The question that arises is how secreted proteins are delivered into host cells, if the proteins are not A–B toxins but have an obvious requirement to reach an intracellular compartment. Group A and group C streptococcus have been known to secrete a protein with NADglycohydrolase activity for many years [7,8]. It seems evident that this protein could play a role in pathogenesis inside the host cell, but it is not understood how it could get to this site of action. Creating a pore
S. pyogenes streptolysin O is a member of a family of poreforming cholesterol-dependent cytolysins that includes Listeria monocytogenes listeriolysin O and Clostridium perfringens perfringolysin O (reviewed in [9]). This family of toxins shares a 30–60% similarity in amino acid sequence, with an invariant region at the carboxy-terminus (ECTGLAWEWWR) that is important for cytolytic activity. The cholesterol-dependent cytolysins are secreted as 47–60 kDa proteins by the general secretory pathway, with cleavable amino-terminal signal sequences. The secreted cytolysin monomers bind cholesterol in the host cell membrane and assemble into a large transmembrane pore of approximately 30 nm in diameter. While many of the cholesterol-dependent cytolysins have demonstrated cytolytic activity, it is thought that they may have a more subtle function as well [9]. An example of this is that while an slo mutant, defective in streptolysin O, still attached to host cells, it lost the ability to manipulate proinflammatory responses in keratinocytes [10]. Cytolysin-mediated translocation
By a stroke of good fortune, Madden et al. [3] identified a 52 kDa protein that could be detected in the cytosol of aCaT keratinocytes — by cross-reaction of antistreptolysin O antiserum to the 52 kDa band — only after infection by a streptolysin O-containing S. pyogenes. Amino-terminal sequencing of the 52 kDa band identified it as an S. pyogenes protein that is almost identical to the mature secreted form of group C streptococcal NAD-glycohydrolase [8]. Comparison of the sequence of the 52 kDa band to the S. pyogenes genome revealed that the region of identity was preceded by a stretch of 37 amino
Dispatch
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Table 1 Comparison of type III secretion and S. pyogenes cytolysin-mediated translocation.
Function Apparatus
Size of pore Effectors
Requires effector and pore-former in same bacterium Intermediates
Channel
Type III secretion
Cytolysin-mediated translocation
Delivers virulence factors into host cell
Delivers a virulence factor into host cell
More than 20 proteins: inner membrane, outer membrane, ATPase, channel/conduit forming, host cell pore-forming
One protein; potentially more? Cytolysin in host cell membrane General secretory pathway Sec system (6–7 proteins) form a complex that crosses bacterial cellular membrane.
≈2 nm
≈30 nm
Not amino-terminal processed Recognition signal may reside in amino-terminal amino acids or 5′ mRNA
Amino-terminal processed
Yes
Yes
No periplasmic or extracellular intermediates
Effector secreted via general secretory pathway into extracellular space then translocated
Continuous
Not continuous?
Contact-dependent
Usually
Translocation of SPN always requires adherence
Directional
Usually
Usually
acid residues characteristic of a signal sequence. This S. pyogenes protein was designated SPN, for S. pyogenes NAD-glycohydrolase. An spn mutant was constructed, which did not secrete SPN protein into the extracellular media or translocate SPN into HaCaT cell cytosol. While no NAD-glycohydrolase activity was detected in uninfected cells, high levels were seen in the cytosol of HaCaT cells infected with wild-type S. pyogenes, in a manner dependent on the presence of both SPN and streptolysin O [3]. The authors called this streptolysin O-dependent delivery of SPN from streptococcus into host cells ‘cytolysin-mediated translocation’.
note that, while there are similarities between these two systems, it would be premature to call the cytolysinmediated translocation a Gram-positive type III system. Table 1 shows a comparison of S. pyogenes cytolysinmediated translocation to the type III secretion system of Gram-negative pathogens. While the pore created in the host cell membrane by cytolysin-mediated translocation is much larger than that of the type III system, in both systems translocation is directional. Most of the effector proteins in both systems are translocated into the host cell and not to the extracellular environment ([3], reviewed in [2]).
In an elegant experiment, it was demonstrated by coinfecting HaCaT cells with an spn mutant and an slo mutant that SPN does not simply diffuse into host cells through the pores created by streptolysin O [3]. A diffusion process would only require the presence of both streptolysin O and SPN. SPN was not delivered in the co-infection experiment, and no NAD-glycohydrolase activity was detected in HaCaT cells, indicating that this is a coordinated process. Madden et al. [3] went on to show that the SPN activity in the host cell cytosol was not due to endocytosis or streptococcal invasion by using cytochalasin D, an inhibitor of actin polymerization and bacterial uptake. From additional experiments, they concluded that translocation is a polarized process in the presence of the cytolysin, where SPN is directed into the host cell.
The greatest similarity in the function of these systems seems to be delivery of virulence factors into host cells. However, there are many notable differences. Type III effectors do not have cleavable amino-terminal signal sequences (reviewed in [2]). So far, only one protein has been identified as part of the cytolysin-mediated translocation system — the cytolysin — while more than 20 proteins of the type III system have been identified, including proteins of the bacterial cytosol, inner membrane and outer membrane, proteins that form conduits to the host cell, and those that form a pore in the host cell membrane. The completion of the S. pyogenes genome (http://www.genome.ou.edu/strep.html) may provide clues to the existence of other cytolysin-mediated translocation components. While cytolysins are secreted by bacteria as water-soluble monomers, which then oligomerize and insert spontaneously into host cell membranes (reviewed in [9]), the host cell pore-forming type III proteins YopB and YopD from Yersinia enterocolitica
Gram-positive versus Gram-negative translocation
This cytolysin-mediated transport is reminiscent of Gramnegative type III secretion systems. It is important to
R266
Current Biology Vol 11 No 7
Figure 1
Cytolysin-mediated translocation Gram-positive bacterium Sec complex
Type III secretion system
Gram-negative bacterium Type III complex Inner membrane
Inner Membrane
Peptidoglycan / cell wall
Peptidoglycan / cell wall
Outer membrane
Protected protein channel?
Protein channel Host plasma membrane
Host plasma membrane
Cytolysin
Pore-forming proteins
Host cell
Host cell
Current Biology
A model of cytolysin-mediated transport compared to Gram-negative pathogens type III secretion system. In cytolysin-mediated translocation, the effectors are secreted via the general secretory pathway into an as yet unidentified channel, which delivers the effectors to the large pore formed by the cytolysin for translocation into the host cell cytosol. For
type III secretion, the effectors are delivered through a continuous machinery through both bacterial membranes and host cell membranes. In enteropathogenic Escherichia coli, the secretion complex in the bacterium is formed by the Escs, the channel is formed by EspA, and the pore in the host cell membrane by EspB and EspD (reviewed in [1,2]).
are themselves substrates of the type III secretion system (reviewed in [2]).
The proof is still to come
The type III secretion system is thought to mediate onestep translocation of effectors across bacterial and host cell membranes in a continuous process [11]. On the surface, it appears that, since SPN has a cleavable amino-terminal signal sequence, it is secreted into the extracellular milieu by the general secretory pathway before it is translocated via streptolysin O [3]. However, a strong argument can be made that cytolysin-mediated translocation is a continuous process. If extracellular SPN is translocated through the streptolysin O pore, a mixed infection where one bacterium provides SPN and a separate bacterium supplies streptolysin O would yield SPN activity in the cytosol of host cells. As mentioned above, co-infection did not rescue translocation [3]. Similarly, in Yersinia enterocolitica it has been shown that effectors must be in the same bacterium as the one producing the pore-forming YopB and YopD in order to be delivered into host cells [12].
While Madden et al. [3] favour a model in which SPN is first secreted and then translocated via streptolysin O, they mention — and their data better support — a model for cytolysin-mediated translocation in which the effectors are translocated via a protected channel formed between the bacterium and the streptolysin O pore in the host cell (Figure 1). A needle-like complex has been visualized in many type III secretion systems, including Salmonella spp, Shigella spp and enteropathogenic Escherichia coli, and data suggest that this complex serves as a conduit for protein delivery (reviewed in [2]). The presence or absence of a protected protein channel in cytolysin-mediated translocation needs to be investigated. The new work reported by Madden et al. [3] is an exciting initial step towards understanding the delivery of virulence factors from Gram-positive bacteria into host cells. It remains to be shown if multiple proteins are involved in the cytolysin-mediated translocation process, as either
Dispatch
effectors or components of the translocation system. Considerable work is needed to determine if the effectors are translocated through a continuous channel. As additional studies progress, it will be interesting to determine how widespread this mechanism is in Gram-positive pathogens. Many lessons have been learned from the years of study of the Gram-negative type III secretion system, the most important being the complexity and intricacy of the virulence delivery systems. Acknowledgements We would like to thank Samantha Gruenheid and John H. Brumell for critical reading of this manuscript. Work in B.B.F.’s laboratory is supported by operating grants from the Medical Research Council of Canada and the Canadian Bacterial Disease Network. A.G. is supported by a doctoral research award from MRC, and B.B.F. is a Howard Hughes International Scholar and an MRC Scientist.
References 1. Hueck CJ: Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev 1998, 62:379-433. 2. Cornelis GR, Van Gijsegem F: Assembly and function of type III secretory systems. Annu Rev Microbiol 2000, 54:735-774. 3. Madden JC, Ruiz N, Caparon M: Cytolysin-mediated translocation (cytolysin-mediated translocation): a functional equivalent of type III secretion in Gram-positive bacteria. Cell 2001, 104:143-152. 4. Schmitt CK, Meysick KC, O’Brien AD: Bacterial toxins: friends or foes? Emerg Infect Dis 1999, 5:224-234. 5. Duesbery NS, Vande Woude GF: Anthrax toxins. Cell Mol Life Sci 1999, 55:1599-1609. 6. Cunningham MW: Pathogenesis of group A streptococcal infections. Clin Microbiol Rev 2000, 13:470-511. 7. Carlson AS, Kellner A, Bernheimer AW, Freeman EB: A streptococcal enzyme that acts specifically upon diphosphopyridine nucleotide: chracterization of the enzyme and its separation from streptolysin O. J Exp Med 1957, 106:15-26. 8. Gerlach D, Ozegowski JH, Gunther E, Vettermann S, Kohler W: Purification and some properties of streptococcal NADglycohydrolase. FEMS Microbiol Lett 1996, 136:71-78. 9. Billington SJ, Jost BH, Songer JG: Thiol-activated cytolysins: structure, function and role in pathogenesis. FEMS Microbiol Lett 2000, 182:197-205. 10. Ruiz N, Wang B, Pentland A, Caparon M: Streptolysin O and adherence synergistically modulate proinflammatory responses of keratinocytes to group A streptococci. Mol Microbiol 1998, 27:337-346. 11. Lee VT, Anderson DM, Schneewind O: Targeting of Yersinia Yop proteins into the cytosol of HeLa cells: one-step translocation of YopE across bacterial and eukaryotic membranes is dependent on SycE chaperone. Mol Microbiol 1998, 28:593-601. 12. Sory MP, Boland A, Lambermont I, Cornelis GR: Identification of the YopE and YopH domains required for secretion and internalization into the cytosol of macrophages, using the cyaA gene fusion approach. Proc Natl Acad Sci USA 1995, 92:11998-12002.
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