A Critical New Pathway for Toxin Secretion?
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《新英格兰医药杂志》
Vaccines that are composed of inactivated bacterial toxins work well for diseases such as diphtheria and tetanus, in which a single secreted toxin is the primary mediator of disease. Pathogens that rely on multiple secreted toxins, however, render vaccines impractical, partly owing to the fact that some toxins are directly injected into host cells and are therefore inaccessible to antibody. An alternative approach that is gaining momentum is to target the critical pathways required for toxin secretion.1 Five distinct protein secretion pathways have been known to contribute to the virulence of bacterial pathogens. Recent studies by the Mekalanos group (Pukatzki et al.2 and Mougous et al.3) have identified a sixth secretion pathway, present in both Vibrio cholerae and Pseudomonas aeruginosa, that is a potential target for vaccine development and for therapeutic intervention.
The classic diarrheal disease resulting from colonization of the small intestine by V. cholerae is mediated by and dependent on the secretion of cholera toxin. Some strains of V. cholerae, such as strain V52, lack the cholera toxin genes but still cause sporadic episodes of cholera-like and nondiarrheal diseases through poorly defined mechanisms. To gain further insight into the pathogenic mechanisms of strain V52, Pukatzki and colleagues2 established an infection system using amebae. Amebae have macrophage-like properties and can be used to model interactions between bacteria and macrophages.
With the use of a high-throughput screen, the investigators isolated V. cholerae mutants lacking the ability to kill amebae. Many of these organisms carried mutations in a cluster of genes that the investigators designated the virulence-associated secretion (VAS) genes, which together encode a new protein secretion system (designated "type VI") that is distinct from the previously described secretion systems known as types I through V (Figure 1).2 Four proteins are secreted by the VAS system into cell-culture supernatant fluid, and several of these proteins are essential for killing amebae and for VAS-dependent cytotoxic effects on mammalian macrophages.
Figure 1. Protein Secretion Pathways of Gram-Negative Bacteria.
Many pathogenic bacteria rely on specialized pathways to secrete proteins that are important for virulence. Each pathway has distinct properties that profoundly influence the manner in which the secreted products interact with host cells. Pore-forming toxins are commonly secreted into the extracellular milieu by the type I secretion pathway. Protein secretion by the type II pathway occurs in two steps: transport across the inner membrane, followed by transport across the outer membrane. Although the type I and II pathways facilitate protein secretion, they have no role in targeting toxins to host cells. However, the type III and type VI secretion pathways are involved in both toxin secretion and targeting to host cells. Each of these systems consists of a multiprotein complex that spans the bacterial-cell envelope and penetrates the host-cell plasma membrane, allowing the injection of toxins directly into the host cell while limiting antigen exposure to host defense mechanisms. The type V pathway represents a minor variation of the type II secretion pathway. Recent studies by Pukatzki et al.2 and Mougous et al.3 provide evidence of the existence of a type VI secretion pathway. Although mechanistic details are lacking, virulence factors may be secreted by the pathway directly into host cells.
Cell-free culture supernatant containing all the known VAS-secreted proteins lacked cytotoxic activity when mixed with macrophages — suggesting that physical contact between bacteria and host cells is necessary for the delivery of the VAS-related cytotoxin to host cells. This finding also suggests that the type VI secretion system injects cytotoxins directly into host cells.
The VAS genes of V. cholerae are highly conserved in several gram-negative pathogens, including P. aeruginosa, a particularly insidious bacterium for patients with cystic fibrosis. Such patients are unusually susceptible to chronic colonization of the airways by the bacterium and, once colonized, remain so for life. Mougous and colleagues3 have recently reported that the VAS-like genes of P. aeruginosa also encode a functional type VI secretion system. More important, they provide data suggesting that the type VI secretion system contributes to the pathogenesis of chronic P. aeruginosa infection in patients with cystic fibrosis. First, expression of the P. aeruginosa VAS-like genes is coordinately regulated with other factors thought to contribute to the pathogenesis of chronic infections. Second, a protein secreted by the P. aeruginosa VAS system was present in sputum samples from patients with cystic fibrosis. Third, immunoreactivity to that protein was detected in serum from patients who had been colonized by P. aeruginosa for more than 10 years. Immunoreactivity to the same protein was absent or dramatically reduced in serum from patients who had been colonized by P. aeruginosa for less than 4 years. Although the sample was too small to yield significant results, this observation suggests that the expression of the VAS genes may increase over the course of chronic infections in patients with cystic fibrosis. Finally, another group had previously isolated P. aeruginosa mutants with attenuated pathogenicity in a rat model of chronic lung infection,4 and some of these organisms carried mutations that specifically disabled the type VI secretion pathway.
Specialized protein secretion systems are essential for the virulence of many bacterial pathogens. They have therefore become attractive therapeutic targets. The identification of the type VI secretion pathway is important for several reasons. Its discovery provides a target for therapeutic interventions aimed at disrupting protein secretion. This may be especially relevant for P. aeruginosa infections in the airways of patients with cystic fibrosis, in whom the elimination of the bacteria is all but impossible, despite aggressive antibiotic treatment. The mechanism by which P. aeruginosa persists in the airways of such patients is poorly understood. A deeper understanding of the type VI system may provide invaluable insight into the nature of persistence. Finally, the presence of VAS-like genes in Salmonella enterica, Yersinia pestis, and Escherichia coli O157:H7 suggests that the inhibition of type VI secretory activity may have wide use as a potential therapeutic approach.
No potential conflict of interest relevant to this article was reported.
Source Information
From the Department of Microbiology, University of Iowa, Iowa City.
References
Nordfelth R, Kauppi AM, Norberg HA, Wolf-Watz H, Elofsson M. Small-molecule inhibitors specifically targeting type III secretion. Infect Immun 2005;73:3104-3114.
Pukatzki S, Ma AT, Sturtevant D, et al. Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc Natl Acad Sci U S A 2006;103:1528-1533.
Mougous JD, Cuff ME, Raunser S, et al. A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus. Science 2006;312:1526-1530.
Potvin E, Lehoux DE, Kukavica-Ibrulj I, et al. In vivo functional genomics of Pseudomonas aeruginosa for high-throughput screening of new virulence factors and antibacterial targets. Environ Microbiol 2003;5:1294-1308.(Timothy L. Yahr, Ph.D.)
The classic diarrheal disease resulting from colonization of the small intestine by V. cholerae is mediated by and dependent on the secretion of cholera toxin. Some strains of V. cholerae, such as strain V52, lack the cholera toxin genes but still cause sporadic episodes of cholera-like and nondiarrheal diseases through poorly defined mechanisms. To gain further insight into the pathogenic mechanisms of strain V52, Pukatzki and colleagues2 established an infection system using amebae. Amebae have macrophage-like properties and can be used to model interactions between bacteria and macrophages.
With the use of a high-throughput screen, the investigators isolated V. cholerae mutants lacking the ability to kill amebae. Many of these organisms carried mutations in a cluster of genes that the investigators designated the virulence-associated secretion (VAS) genes, which together encode a new protein secretion system (designated "type VI") that is distinct from the previously described secretion systems known as types I through V (Figure 1).2 Four proteins are secreted by the VAS system into cell-culture supernatant fluid, and several of these proteins are essential for killing amebae and for VAS-dependent cytotoxic effects on mammalian macrophages.
Figure 1. Protein Secretion Pathways of Gram-Negative Bacteria.
Many pathogenic bacteria rely on specialized pathways to secrete proteins that are important for virulence. Each pathway has distinct properties that profoundly influence the manner in which the secreted products interact with host cells. Pore-forming toxins are commonly secreted into the extracellular milieu by the type I secretion pathway. Protein secretion by the type II pathway occurs in two steps: transport across the inner membrane, followed by transport across the outer membrane. Although the type I and II pathways facilitate protein secretion, they have no role in targeting toxins to host cells. However, the type III and type VI secretion pathways are involved in both toxin secretion and targeting to host cells. Each of these systems consists of a multiprotein complex that spans the bacterial-cell envelope and penetrates the host-cell plasma membrane, allowing the injection of toxins directly into the host cell while limiting antigen exposure to host defense mechanisms. The type V pathway represents a minor variation of the type II secretion pathway. Recent studies by Pukatzki et al.2 and Mougous et al.3 provide evidence of the existence of a type VI secretion pathway. Although mechanistic details are lacking, virulence factors may be secreted by the pathway directly into host cells.
Cell-free culture supernatant containing all the known VAS-secreted proteins lacked cytotoxic activity when mixed with macrophages — suggesting that physical contact between bacteria and host cells is necessary for the delivery of the VAS-related cytotoxin to host cells. This finding also suggests that the type VI secretion system injects cytotoxins directly into host cells.
The VAS genes of V. cholerae are highly conserved in several gram-negative pathogens, including P. aeruginosa, a particularly insidious bacterium for patients with cystic fibrosis. Such patients are unusually susceptible to chronic colonization of the airways by the bacterium and, once colonized, remain so for life. Mougous and colleagues3 have recently reported that the VAS-like genes of P. aeruginosa also encode a functional type VI secretion system. More important, they provide data suggesting that the type VI secretion system contributes to the pathogenesis of chronic P. aeruginosa infection in patients with cystic fibrosis. First, expression of the P. aeruginosa VAS-like genes is coordinately regulated with other factors thought to contribute to the pathogenesis of chronic infections. Second, a protein secreted by the P. aeruginosa VAS system was present in sputum samples from patients with cystic fibrosis. Third, immunoreactivity to that protein was detected in serum from patients who had been colonized by P. aeruginosa for more than 10 years. Immunoreactivity to the same protein was absent or dramatically reduced in serum from patients who had been colonized by P. aeruginosa for less than 4 years. Although the sample was too small to yield significant results, this observation suggests that the expression of the VAS genes may increase over the course of chronic infections in patients with cystic fibrosis. Finally, another group had previously isolated P. aeruginosa mutants with attenuated pathogenicity in a rat model of chronic lung infection,4 and some of these organisms carried mutations that specifically disabled the type VI secretion pathway.
Specialized protein secretion systems are essential for the virulence of many bacterial pathogens. They have therefore become attractive therapeutic targets. The identification of the type VI secretion pathway is important for several reasons. Its discovery provides a target for therapeutic interventions aimed at disrupting protein secretion. This may be especially relevant for P. aeruginosa infections in the airways of patients with cystic fibrosis, in whom the elimination of the bacteria is all but impossible, despite aggressive antibiotic treatment. The mechanism by which P. aeruginosa persists in the airways of such patients is poorly understood. A deeper understanding of the type VI system may provide invaluable insight into the nature of persistence. Finally, the presence of VAS-like genes in Salmonella enterica, Yersinia pestis, and Escherichia coli O157:H7 suggests that the inhibition of type VI secretory activity may have wide use as a potential therapeutic approach.
No potential conflict of interest relevant to this article was reported.
Source Information
From the Department of Microbiology, University of Iowa, Iowa City.
References
Nordfelth R, Kauppi AM, Norberg HA, Wolf-Watz H, Elofsson M. Small-molecule inhibitors specifically targeting type III secretion. Infect Immun 2005;73:3104-3114.
Pukatzki S, Ma AT, Sturtevant D, et al. Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc Natl Acad Sci U S A 2006;103:1528-1533.
Mougous JD, Cuff ME, Raunser S, et al. A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus. Science 2006;312:1526-1530.
Potvin E, Lehoux DE, Kukavica-Ibrulj I, et al. In vivo functional genomics of Pseudomonas aeruginosa for high-throughput screening of new virulence factors and antibacterial targets. Environ Microbiol 2003;5:1294-1308.(Timothy L. Yahr, Ph.D.)