- Email: [email protected]
Signs and portents: molecular signals and infectious diseases1
COMMENT Event
Signs and portents: molecular signals and infectious diseases Valerie A. Snewin and David W. Holden
T
he subject of this meeting – molecular signals and infectious diseases – was illustrated by wide-ranging presentations on bacteria, viruses and parasites, all of which have evolved clever tricks to interfere with host cell signalling, for example by mediating host cell invasion, preventing phagocytosis, permitting cellto-cell spread or modulating host immune responses. Two aspects of signalling emerged as common themes at the meeting: interference of host signalling pathways by Gram-negative bacterial proteins translocated into the host cell via type III secretion systems, and the role of glycosylphosphatidylinositol (GPI)\anchored parasite molecules as inducers of pathogenic processes. Actin recruitment Secreted products of several pathogenic bacteria are able to interact with members of the Rho family of small GTPases, including Rho, Rac and Cdc42. The functions of these GTPases were described by Alan Hall (MRC Laboratory of Molecular Cell Biology, London, UK). Members of this family control signal transduction pathways, linking membrane receptors to the dynamic actin cytoskeleton in a range of cell types, and regulate mitogen-activated protein kinase (MAP kinase) pathways. Hall reported that phagocytosis through macrophage immunoglobulin receptors (FcgR), involving pseudopod extension and membrane ruffling (type I pathway), is mediated by Cdc42 and Rac, whereas phagocytosis through the complement receptor CR3 (type II pathway) is mediated by Rho (Refs 1,2). Both pathways involve actin polymerization but they result in different biological responses; only the type I pathway
The 2nd Louis Pasteur Conference on Infectious Diseases, organized by J-L. Virelizier, R.R. Isberg, K. Joiner and S. Pellegrini, was held at the Pasteur Institute, Paris, France, 8–10 October 1998. V.A. Snewin* is in the Dept of Infectious Diseases and Microbiology, Imperial College School of Medicine, St Mary’s Campus, Norfolk Place, London, UK W2 1PG; D.W. Holden is in the Dept of Infectious Diseases and Microbiology, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London, UK W12 0NN. *tel: 144 171 594 3964, fax: 144 171 262 6299, e-mail: [email protected]
produces an inflammatory response. Salmonella exploits this system to enter host cells via membrane ruffling. This process involves a type III secretion system termed Inv/Spa. Secretion of SipA is dependent on this system and is required for effective entry of Salmonella into cultured cells. Jorge Galán (SUNY, Stony Brook, NY, USA) reported that SipA acts by binding actin in the presence of the actin bundling protein, plastin. Another secreted product of Inv/Spa is SopE, which interacts with Cdc42 and Rac1 (Ref. 3). Purified SopE can stimulate GDP/ GTP exchange in these GTPases; however, SopE is not present in all Salmonella isolates and, where it is absent, alternative mechanisms might exist for similar functions. The mechanism of invasion of epithelial cells by Shigella was reviewed by Philippe Sansonetti (Pasteur Institute, Paris, France). As Shigella makes contact with the cell surface, IpaA–D invasins are released by the Mxi–Spa type III secretion system. IpaA rapidly associates with the host protein vinculin and activates it to link actin to the plasma membrane4.
0966-842X/99/$ - see front matter © 1999 Elsevier Science. All rights reserved. TRENDS
IN
MICROBIOLOGY
62
This causes the formation of cellular projections that engulf the bacterium in a manner similar to that seen for Salmonella. However, whereas Salmonella entry requires Cdc42 and Rac, Shigella entry is dependent on Rho. In contrast, the type III secretion system of enteropathogenic Escherichia coli (EPEC) induces cytoskeletal changes that result in a pedestal-like structure on the epithelial cell surface underneath the adhering bacteria. The EPEC secretion system is encoded by a chromosomal pathogenicity island called the locus of enterocyte effacement (LEE), whose nucleotide sequence has recently been determined for both EPEC and enterohaemorrhagic E. coli (EHEC)5,6. Secreted Esp proteins and the bacterial outer membrane protein intimin are all encoded by the LEE (Mike Donnenberg, University of Maryland, Baltimore, MD, USA). EspB is translocated into the host cell cytoplasm and is essential for pedestal formation and attachment/effacing (A/E) activity. Expression of EspB following transfection of host cells leads to significant changes in actin staining, suggesting a direct involvement of this protein in the A/E process. A poster by Brendan Kenny (University of Bristol, UK) expanded on his recent startling finding that the intimin receptor is in fact a bacterial protein, termed translocated intimin receptor (Tir; previously Hp90)7. A central 55amino-acid domain in Tir that is required for interaction with intimin has now been defined8. Tir is translocated into the host cell, where, in the case of EPEC, it is phosphorylated. Phosphorylation is required for actin-nucleating activity. Could this strategy of injecting bacterial components that require host factors for activation be PII: S0966-842X(98)01445-0
VOL. 7 NO. 2 FEBRUARY 1999
COMMENT
a method of limiting bacterial infection to specific cell types? Interestingly, a non-LEE gene product, lymphocyte inhibitory factor (LIF), regulates lymphocyte activation and inhibits release of cytokines (M. Donnenberg). LIF is a large protein of 366 kDa and is only found in pathogenic E. coli. Gauging the importance of these nonA/E activities in vivo might require new infection models. Lysosome recruitment Whereas early interactions between bacteria and host cells often involve cytoskeletal protein rearrangements beneath adherent bacteria, Trypanosoma cruzi enters host cells by hijacking host cell organelles (Norma Andrews, Yale University, New Haven, CT, USA). T. cruzi can invade a broad range of mammalian cells by an actinindependent mechanism involving recruitment of lysosomes to the attachment site, where they fuse with the plasma membrane. Lysosome recruitment occurs through parasite signalling linked to the activity of a parasite enzyme, oligopeptidase B (Ref. 9). G-protein-coupled phospholipase C activation and inositol triphosphate formation leads to calcium release from intracellular stores. Increasing the calcium concentration leads to the formation of giant lysosomes through fusion events. Calcium-dependent lysosomal exocytosis can even be induced by the parasite in fibroblasts, which do not normally undergo exocytosis, and might represent a plasma membrane repair mechanism that is exploited by T. cruzi for cell entry. Anchors and toxins Other novel examples of pathogen regulation of host activities are emerging, including changes in host cell membrane fluidity and downregulation of the immune response during Leishmania infection of macrophages (Sam Turco, University of Kentucky, Lexington, KY, USA). Many parasite surface molecules attached via GPI anchors are known to play a role in parasite–host interactions. The GPI-anchored polysaccharide lipo-
TRENDS
IN
phosphoglycan (LPG) acts as a ligand both for attachment to the sandfly midgut and for macrophage invasion, probably via the CR3 receptor10. LPG is essential for parasite survival in the macrophage. Upon infection, LPG is inserted into the macrophage lipid bilayer, which becomes stabilized, preventing activation of host protein kinase C. Consequently, the host cell oxidative burst, chemotaxis and interleukin 1 (IL-1) production are downregulated, facilitating parasite survival. Toxoplasma gondii can invade all nucleated mammalian cells, where it resides within a parasitophorous vacuole (Keith Joiner, Yale University). Soluble proteins are released into this vacuole from dense granules in the parasite. In contrast, the five major GPI-anchored parasite proteins are re-routed away from dense granules to the parasite surface11. Death from cerebral malaria is now thought to be caused by a plasmodial molecule known as ‘malaria toxin’, which induces a systemic inflammatory cascade dominated by cytokine excess. Parasite GPI anchor has been shown to be responsible for this effect through signal transduction leading to induction of tumour necrosis factor (TNF) and adhesin receptors on the endothelial microvilli12. The Plasmodium falciparum GPI anchor has been purified, and Louis Schofield (Royal Melbourne Hospital, Victoria, Australia) presented preliminary data showing that a monoclonal antibody raised against malaria parasite GPI is protective in a murine model of cerebral malaria and that there is a correlation between disease and the amount of this single effector found in the blood. In humans, the presence of anti-GPI circulating antibody appears to correlate with absence of disease, and there is potential for the use of ‘humanized’ anti-GPI monoclonal antibody as therapy in cerebral malaria. Gram-positive bacteria It is likely that microbial entry and replication have many as yet undiscovered effects on host cells,
MICROBIOLOGY
63
and we might need new methods to identify and measure such effects. Gram-positive bacteria include some of the most important human pathogens. However, with notable exceptions [Listeria signalling (Pascale Cossart, Pasteur Institute) and Mycobacterium cell wall lipid trafficking in macrophages (David Russell, Washington University, St Louis, MO, USA)], research into Gram-positive pathogen–host interactions was not well represented at the meeting. Promisingly, more sophisticated genetic tools are being developed for these microorganisms, as illustrated by a poster on Mycobacterium tuberculosis insertional mutagenesis (Luis Camacho, Pasteur Institute)13. Two posters discussed Staphylococcus aureus entry into host cells and induction of apoptosis (Bhanu Sinha, University Hospital, Geneva, Switzerland, and Bettina Schreiner, Institut Physiologie, Tübingen, Germany)14,15; however, we still know little about the importance of intracellular life to S. aureus. For many of the pathogens described at the meeting, only certain stages of infection are being investigated, a point highlighted by Stanley Falkow (Stanford University, Palo Alto, CA, USA). Although the meeting drew attention to complex and sophisticated biochemical signalling between pathogens and their hosts at these stages, it also served to remind us of our ignorance of how these and other mechanisms operate during the full course of infection in vivo. Acknowledgements V.A.S. thanks Professor Douglas Young and the Wellcome Trust for support, and Alice Dautry-Varsat, Genevieve Milon, Brendan Kenny, Howard Cooper, Brian Sheehan and Brian Robertson for helpful comments on the manuscript. We apologize for omissions resulting from space limitations. References 1 Caron, E. and Hall, A. (1998) Science 282, 1717–1721 2 Massol, P. et al. (1998) EMBO J. 17, 6219–6229 3 Hardt, W-D. et al. (1998) Cell 93, 815–826 4 Tran Van Nhieu, G., Ben-Ze’ev, A. and Sansonetti, P.J. (1997) EMBO J. 16, 2717–2729
VOL. 7 NO. 2 FEBRUARY 1999
COMMENT
5 Elliott, S.J. et al. (1998) Mol. Microbiol. 28, 1–4 6 Perna, N.T. et al. (1998) Infect. Immun. 66, 3810–3817 7 Kenny, B. et al. (1997) Cell 91, 511–520 8 Kenny, B. et al. Mol. Microbiol. (in press)
9 Caler, E.V. et al. (1998) EMBO J. 17, 4975–4986 10 Mengeling, B.J. and Turco, S.J. (1998) Curr. Opin. Struct. Biol. 8, 572–577 11 Karsten, V. et al. (1998) J. Cell Biol. 141, 1323–1333 12 Tachado, S.D. et al. (1997) Proc. Natl.
Acad. Sci. U. S. A. 94, 4022–4027 13 Pelicic, V., Reyrat, J-M. and Gicquel, B. (1998) Mol. Microbiol. 28, 413–420 14 Wesson, C.A. et al. (1998) Infect. Immun. 66, 5238–5243 15 Menzies, B.E. and Kourteva, I. (1998) Infect. Immun. 66, 5994–5998
Letter CpG DNA: a novel immunomodulator
A
s detailed by Lipford et al. in their excellent review1, in recent years it has become clear that DNA serves as more than the genetic code. Our immune system has evolved the ability to distinguish microbial nucleic acids from our own on the basis of their differences in the frequency and methylation of CpG dinucleotides, in particular base contexts, termed ‘CpG motifs’. Remarkably, immune-stimulatory CpG (CpG-S) motifs serve both as powerful activators of innate immune defenses and as co-stimulators of antigen-specific acquired B- and T-cell immune responses. As CpG dinucleotides in the genomes of prokaryotes, viruses and retroviruses are generally not methylated, the ability to recognize the molecular pattern of unmethylated CpG motifs confers a more general method of detecting infections than do other pattern recognition receptors that detect, for example, endotoxins or high mannose proteins. The recognition of CpG DNA might therefore be an important trigger for protective immune responses against many pathogens. Of course, in their ongoing evolutionary battle to evade or subvert host defenses, pathogens have evolved myriad counter-strategies. In the case of the CpG defense, pathogens that must replicate their DNA inside host cells, such as viruses and retroviruses, appear to have evolved at least two counterstrategies. First, in all small DNA viruses and retroviruses, the genomic CpG levels are suppressed to just 6–20% of predicted levels based on random-base usage2,3. We have recently reported an even more intriguing strategy that has evolved in certain serotypes of adenovirus4. Adenovirus types 2 and 5 have selectively suppressed the levels of CpG-S motifs but have dramatically increased the levels of other types of CpG motifs. These motifs consist of
TRENDS
IN
various combinations of CGCG, CCG and CGG (Ref. 4). Thus, in addition to the CpG-S motifs, there are opposing neutralizing (CpG-N) motifs. This observation also provides an insight into the reason why vertebrate DNA is still nonstimulatory even when it is completely demethylated5; vertebrate DNA has an ~5-fold higher concentration of CpG-N compared with CpG-S motifs4. The recognition of the immune effects of these various sequence motifs adds an exciting new dimension to investigations into the evolution of microbial genomes. CpG-S motifs are now recognized as a major problem in gene therapy, particularly with respect to pulmonary delivery of nonviral vectors6,7. Immune stimulation by CpG-S motifs appears to be responsible for the occurrence of fever, myalgias and fatigue in cystic fibrosis patients who have inhaled plasmids encoding the cystic fibrosis transmembrane receptor together with lipids to enhance cell uptake7. Now that we understand something of the mechanism of CpG-S motifs and their susceptibility to inhibition by commonly used human drugs such as chloroquine, hydroxychloroquine and quinacrine8,9, and now that the ability of CpG-N motifs to block these immunostimulatory effects has been recognized, we have the opportunity to develop safer and potentially more-effective approaches to gene therapy. From the standpoint of the host response to CpG-S DNA, we have found that the optimal CpG motif for activating mouse cells, GACGTT, is surprisingly weak for activating human cells (A. Krieg et al., unpublished). Indeed, preliminary studies suggest that, to some degree at least, the CpG sequences recognized by the leukocytes of different species differ; that is to say, the CpG motifs are species specific. We have recently identified CpG
MICROBIOLOGY
64
motifs that are highly stimulatory for human and monkey peripheral blood mononuclear cells, and different motifs that are optimal for stimulating fish, cat and dog immune responses (A. Krieg et al., unpublished). Studies are under way to identify optimal CpG motifs for other species. Although I had earlier proposed that CpG DNA would turn out to be a cause of the autoimmune disease systemic lupus erythematosus10, it has now been demonstrated that CpG DNA not only fails to trigger lupus in normal or genetically predisposed mice but actually reduces disease severity11,12! The therapeutic potential of CpG DNA now appears to be extremely exciting. The role of CpG-S motifs in enhancing genetic vaccination has been reviewed previously in Trends Microbiol.13 As reviewed by Lipford et al.1, synthetic oligodeoxynucleotides (ODNs) containing CpG motifs are astonishingly strong adjuvants for inducing antigen-specific Th1-like T- and B-cell responses in vivo. This suggests that CpG-S ODNs could be used as additives to vaccines against infectious diseases, allergic diseases and cancer. The first human clinical trial using a CpG ODN for this purpose is planned for early 1999. In this trial, CpG ImmunoPharmaceuticals, Inc. (Wellesley, MA, USA) will evaluate the safety and efficacy of a human stimulatory CpG ODN for improving immune responses to Engerix B®, SmithKline Beecham’s vaccine against hepatitis B. In the near future, the clinical utility of this novel immunomodulator should become clear.
Arthur M. Krieg Dept of Internal Medicine, University of Iowa, and Veterans Administration Medical Center, Iowa City, IA 52242, USA CpG ImmunoPharmaceuticals, Inc. Wellesley, MA 02481, USA
VOL. 7 NO. 2 FEBRUARY 1999
Signs and portents: molecular signals and infectious diseases Valerie A. Snewin and David W. Holden
T
he subject of this meeting – molecular signals and infectious diseases – was illustrated by wide-ranging presentations on bacteria, viruses and parasites, all of which have evolved clever tricks to interfere with host cell signalling, for example by mediating host cell invasion, preventing phagocytosis, permitting cellto-cell spread or modulating host immune responses. Two aspects of signalling emerged as common themes at the meeting: interference of host signalling pathways by Gram-negative bacterial proteins translocated into the host cell via type III secretion systems, and the role of glycosylphosphatidylinositol (GPI)\anchored parasite molecules as inducers of pathogenic processes. Actin recruitment Secreted products of several pathogenic bacteria are able to interact with members of the Rho family of small GTPases, including Rho, Rac and Cdc42. The functions of these GTPases were described by Alan Hall (MRC Laboratory of Molecular Cell Biology, London, UK). Members of this family control signal transduction pathways, linking membrane receptors to the dynamic actin cytoskeleton in a range of cell types, and regulate mitogen-activated protein kinase (MAP kinase) pathways. Hall reported that phagocytosis through macrophage immunoglobulin receptors (FcgR), involving pseudopod extension and membrane ruffling (type I pathway), is mediated by Cdc42 and Rac, whereas phagocytosis through the complement receptor CR3 (type II pathway) is mediated by Rho (Refs 1,2). Both pathways involve actin polymerization but they result in different biological responses; only the type I pathway
The 2nd Louis Pasteur Conference on Infectious Diseases, organized by J-L. Virelizier, R.R. Isberg, K. Joiner and S. Pellegrini, was held at the Pasteur Institute, Paris, France, 8–10 October 1998. V.A. Snewin* is in the Dept of Infectious Diseases and Microbiology, Imperial College School of Medicine, St Mary’s Campus, Norfolk Place, London, UK W2 1PG; D.W. Holden is in the Dept of Infectious Diseases and Microbiology, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London, UK W12 0NN. *tel: 144 171 594 3964, fax: 144 171 262 6299, e-mail: [email protected]
produces an inflammatory response. Salmonella exploits this system to enter host cells via membrane ruffling. This process involves a type III secretion system termed Inv/Spa. Secretion of SipA is dependent on this system and is required for effective entry of Salmonella into cultured cells. Jorge Galán (SUNY, Stony Brook, NY, USA) reported that SipA acts by binding actin in the presence of the actin bundling protein, plastin. Another secreted product of Inv/Spa is SopE, which interacts with Cdc42 and Rac1 (Ref. 3). Purified SopE can stimulate GDP/ GTP exchange in these GTPases; however, SopE is not present in all Salmonella isolates and, where it is absent, alternative mechanisms might exist for similar functions. The mechanism of invasion of epithelial cells by Shigella was reviewed by Philippe Sansonetti (Pasteur Institute, Paris, France). As Shigella makes contact with the cell surface, IpaA–D invasins are released by the Mxi–Spa type III secretion system. IpaA rapidly associates with the host protein vinculin and activates it to link actin to the plasma membrane4.
0966-842X/99/$ - see front matter © 1999 Elsevier Science. All rights reserved. TRENDS
IN
MICROBIOLOGY
62
This causes the formation of cellular projections that engulf the bacterium in a manner similar to that seen for Salmonella. However, whereas Salmonella entry requires Cdc42 and Rac, Shigella entry is dependent on Rho. In contrast, the type III secretion system of enteropathogenic Escherichia coli (EPEC) induces cytoskeletal changes that result in a pedestal-like structure on the epithelial cell surface underneath the adhering bacteria. The EPEC secretion system is encoded by a chromosomal pathogenicity island called the locus of enterocyte effacement (LEE), whose nucleotide sequence has recently been determined for both EPEC and enterohaemorrhagic E. coli (EHEC)5,6. Secreted Esp proteins and the bacterial outer membrane protein intimin are all encoded by the LEE (Mike Donnenberg, University of Maryland, Baltimore, MD, USA). EspB is translocated into the host cell cytoplasm and is essential for pedestal formation and attachment/effacing (A/E) activity. Expression of EspB following transfection of host cells leads to significant changes in actin staining, suggesting a direct involvement of this protein in the A/E process. A poster by Brendan Kenny (University of Bristol, UK) expanded on his recent startling finding that the intimin receptor is in fact a bacterial protein, termed translocated intimin receptor (Tir; previously Hp90)7. A central 55amino-acid domain in Tir that is required for interaction with intimin has now been defined8. Tir is translocated into the host cell, where, in the case of EPEC, it is phosphorylated. Phosphorylation is required for actin-nucleating activity. Could this strategy of injecting bacterial components that require host factors for activation be PII: S0966-842X(98)01445-0
VOL. 7 NO. 2 FEBRUARY 1999
COMMENT
a method of limiting bacterial infection to specific cell types? Interestingly, a non-LEE gene product, lymphocyte inhibitory factor (LIF), regulates lymphocyte activation and inhibits release of cytokines (M. Donnenberg). LIF is a large protein of 366 kDa and is only found in pathogenic E. coli. Gauging the importance of these nonA/E activities in vivo might require new infection models. Lysosome recruitment Whereas early interactions between bacteria and host cells often involve cytoskeletal protein rearrangements beneath adherent bacteria, Trypanosoma cruzi enters host cells by hijacking host cell organelles (Norma Andrews, Yale University, New Haven, CT, USA). T. cruzi can invade a broad range of mammalian cells by an actinindependent mechanism involving recruitment of lysosomes to the attachment site, where they fuse with the plasma membrane. Lysosome recruitment occurs through parasite signalling linked to the activity of a parasite enzyme, oligopeptidase B (Ref. 9). G-protein-coupled phospholipase C activation and inositol triphosphate formation leads to calcium release from intracellular stores. Increasing the calcium concentration leads to the formation of giant lysosomes through fusion events. Calcium-dependent lysosomal exocytosis can even be induced by the parasite in fibroblasts, which do not normally undergo exocytosis, and might represent a plasma membrane repair mechanism that is exploited by T. cruzi for cell entry. Anchors and toxins Other novel examples of pathogen regulation of host activities are emerging, including changes in host cell membrane fluidity and downregulation of the immune response during Leishmania infection of macrophages (Sam Turco, University of Kentucky, Lexington, KY, USA). Many parasite surface molecules attached via GPI anchors are known to play a role in parasite–host interactions. The GPI-anchored polysaccharide lipo-
TRENDS
IN
phosphoglycan (LPG) acts as a ligand both for attachment to the sandfly midgut and for macrophage invasion, probably via the CR3 receptor10. LPG is essential for parasite survival in the macrophage. Upon infection, LPG is inserted into the macrophage lipid bilayer, which becomes stabilized, preventing activation of host protein kinase C. Consequently, the host cell oxidative burst, chemotaxis and interleukin 1 (IL-1) production are downregulated, facilitating parasite survival. Toxoplasma gondii can invade all nucleated mammalian cells, where it resides within a parasitophorous vacuole (Keith Joiner, Yale University). Soluble proteins are released into this vacuole from dense granules in the parasite. In contrast, the five major GPI-anchored parasite proteins are re-routed away from dense granules to the parasite surface11. Death from cerebral malaria is now thought to be caused by a plasmodial molecule known as ‘malaria toxin’, which induces a systemic inflammatory cascade dominated by cytokine excess. Parasite GPI anchor has been shown to be responsible for this effect through signal transduction leading to induction of tumour necrosis factor (TNF) and adhesin receptors on the endothelial microvilli12. The Plasmodium falciparum GPI anchor has been purified, and Louis Schofield (Royal Melbourne Hospital, Victoria, Australia) presented preliminary data showing that a monoclonal antibody raised against malaria parasite GPI is protective in a murine model of cerebral malaria and that there is a correlation between disease and the amount of this single effector found in the blood. In humans, the presence of anti-GPI circulating antibody appears to correlate with absence of disease, and there is potential for the use of ‘humanized’ anti-GPI monoclonal antibody as therapy in cerebral malaria. Gram-positive bacteria It is likely that microbial entry and replication have many as yet undiscovered effects on host cells,
MICROBIOLOGY
63
and we might need new methods to identify and measure such effects. Gram-positive bacteria include some of the most important human pathogens. However, with notable exceptions [Listeria signalling (Pascale Cossart, Pasteur Institute) and Mycobacterium cell wall lipid trafficking in macrophages (David Russell, Washington University, St Louis, MO, USA)], research into Gram-positive pathogen–host interactions was not well represented at the meeting. Promisingly, more sophisticated genetic tools are being developed for these microorganisms, as illustrated by a poster on Mycobacterium tuberculosis insertional mutagenesis (Luis Camacho, Pasteur Institute)13. Two posters discussed Staphylococcus aureus entry into host cells and induction of apoptosis (Bhanu Sinha, University Hospital, Geneva, Switzerland, and Bettina Schreiner, Institut Physiologie, Tübingen, Germany)14,15; however, we still know little about the importance of intracellular life to S. aureus. For many of the pathogens described at the meeting, only certain stages of infection are being investigated, a point highlighted by Stanley Falkow (Stanford University, Palo Alto, CA, USA). Although the meeting drew attention to complex and sophisticated biochemical signalling between pathogens and their hosts at these stages, it also served to remind us of our ignorance of how these and other mechanisms operate during the full course of infection in vivo. Acknowledgements V.A.S. thanks Professor Douglas Young and the Wellcome Trust for support, and Alice Dautry-Varsat, Genevieve Milon, Brendan Kenny, Howard Cooper, Brian Sheehan and Brian Robertson for helpful comments on the manuscript. We apologize for omissions resulting from space limitations. References 1 Caron, E. and Hall, A. (1998) Science 282, 1717–1721 2 Massol, P. et al. (1998) EMBO J. 17, 6219–6229 3 Hardt, W-D. et al. (1998) Cell 93, 815–826 4 Tran Van Nhieu, G., Ben-Ze’ev, A. and Sansonetti, P.J. (1997) EMBO J. 16, 2717–2729
VOL. 7 NO. 2 FEBRUARY 1999
COMMENT
5 Elliott, S.J. et al. (1998) Mol. Microbiol. 28, 1–4 6 Perna, N.T. et al. (1998) Infect. Immun. 66, 3810–3817 7 Kenny, B. et al. (1997) Cell 91, 511–520 8 Kenny, B. et al. Mol. Microbiol. (in press)
9 Caler, E.V. et al. (1998) EMBO J. 17, 4975–4986 10 Mengeling, B.J. and Turco, S.J. (1998) Curr. Opin. Struct. Biol. 8, 572–577 11 Karsten, V. et al. (1998) J. Cell Biol. 141, 1323–1333 12 Tachado, S.D. et al. (1997) Proc. Natl.
Acad. Sci. U. S. A. 94, 4022–4027 13 Pelicic, V., Reyrat, J-M. and Gicquel, B. (1998) Mol. Microbiol. 28, 413–420 14 Wesson, C.A. et al. (1998) Infect. Immun. 66, 5238–5243 15 Menzies, B.E. and Kourteva, I. (1998) Infect. Immun. 66, 5994–5998
Letter CpG DNA: a novel immunomodulator
A
s detailed by Lipford et al. in their excellent review1, in recent years it has become clear that DNA serves as more than the genetic code. Our immune system has evolved the ability to distinguish microbial nucleic acids from our own on the basis of their differences in the frequency and methylation of CpG dinucleotides, in particular base contexts, termed ‘CpG motifs’. Remarkably, immune-stimulatory CpG (CpG-S) motifs serve both as powerful activators of innate immune defenses and as co-stimulators of antigen-specific acquired B- and T-cell immune responses. As CpG dinucleotides in the genomes of prokaryotes, viruses and retroviruses are generally not methylated, the ability to recognize the molecular pattern of unmethylated CpG motifs confers a more general method of detecting infections than do other pattern recognition receptors that detect, for example, endotoxins or high mannose proteins. The recognition of CpG DNA might therefore be an important trigger for protective immune responses against many pathogens. Of course, in their ongoing evolutionary battle to evade or subvert host defenses, pathogens have evolved myriad counter-strategies. In the case of the CpG defense, pathogens that must replicate their DNA inside host cells, such as viruses and retroviruses, appear to have evolved at least two counterstrategies. First, in all small DNA viruses and retroviruses, the genomic CpG levels are suppressed to just 6–20% of predicted levels based on random-base usage2,3. We have recently reported an even more intriguing strategy that has evolved in certain serotypes of adenovirus4. Adenovirus types 2 and 5 have selectively suppressed the levels of CpG-S motifs but have dramatically increased the levels of other types of CpG motifs. These motifs consist of
TRENDS
IN
various combinations of CGCG, CCG and CGG (Ref. 4). Thus, in addition to the CpG-S motifs, there are opposing neutralizing (CpG-N) motifs. This observation also provides an insight into the reason why vertebrate DNA is still nonstimulatory even when it is completely demethylated5; vertebrate DNA has an ~5-fold higher concentration of CpG-N compared with CpG-S motifs4. The recognition of the immune effects of these various sequence motifs adds an exciting new dimension to investigations into the evolution of microbial genomes. CpG-S motifs are now recognized as a major problem in gene therapy, particularly with respect to pulmonary delivery of nonviral vectors6,7. Immune stimulation by CpG-S motifs appears to be responsible for the occurrence of fever, myalgias and fatigue in cystic fibrosis patients who have inhaled plasmids encoding the cystic fibrosis transmembrane receptor together with lipids to enhance cell uptake7. Now that we understand something of the mechanism of CpG-S motifs and their susceptibility to inhibition by commonly used human drugs such as chloroquine, hydroxychloroquine and quinacrine8,9, and now that the ability of CpG-N motifs to block these immunostimulatory effects has been recognized, we have the opportunity to develop safer and potentially more-effective approaches to gene therapy. From the standpoint of the host response to CpG-S DNA, we have found that the optimal CpG motif for activating mouse cells, GACGTT, is surprisingly weak for activating human cells (A. Krieg et al., unpublished). Indeed, preliminary studies suggest that, to some degree at least, the CpG sequences recognized by the leukocytes of different species differ; that is to say, the CpG motifs are species specific. We have recently identified CpG
MICROBIOLOGY
64
motifs that are highly stimulatory for human and monkey peripheral blood mononuclear cells, and different motifs that are optimal for stimulating fish, cat and dog immune responses (A. Krieg et al., unpublished). Studies are under way to identify optimal CpG motifs for other species. Although I had earlier proposed that CpG DNA would turn out to be a cause of the autoimmune disease systemic lupus erythematosus10, it has now been demonstrated that CpG DNA not only fails to trigger lupus in normal or genetically predisposed mice but actually reduces disease severity11,12! The therapeutic potential of CpG DNA now appears to be extremely exciting. The role of CpG-S motifs in enhancing genetic vaccination has been reviewed previously in Trends Microbiol.13 As reviewed by Lipford et al.1, synthetic oligodeoxynucleotides (ODNs) containing CpG motifs are astonishingly strong adjuvants for inducing antigen-specific Th1-like T- and B-cell responses in vivo. This suggests that CpG-S ODNs could be used as additives to vaccines against infectious diseases, allergic diseases and cancer. The first human clinical trial using a CpG ODN for this purpose is planned for early 1999. In this trial, CpG ImmunoPharmaceuticals, Inc. (Wellesley, MA, USA) will evaluate the safety and efficacy of a human stimulatory CpG ODN for improving immune responses to Engerix B®, SmithKline Beecham’s vaccine against hepatitis B. In the near future, the clinical utility of this novel immunomodulator should become clear.
Arthur M. Krieg Dept of Internal Medicine, University of Iowa, and Veterans Administration Medical Center, Iowa City, IA 52242, USA CpG ImmunoPharmaceuticals, Inc. Wellesley, MA 02481, USA
VOL. 7 NO. 2 FEBRUARY 1999