- Email: [email protected]
A novel approach to explore the role of plasminogen in bacterial pathogenesis
C O M M E N T
article 1~ accompanying our recent paper ~2,we present 'the best evidence so far' that the TVM membranes are an import system. The functions of tubules and ion channels in delivering exogenous nutrients to the parasite are major points of debate in malarial transport ~3. Our data suggest that, under physiological conditions, lowmolecular-weight nutrients can be transported through a tubular TVM pathway. Parasite-induced channels expressed early in development may underlie the first steps of solute transport into the infected RBC. When nutrient demand increases at the later trophozoite stage, the TVM pathway may increase the efficiency of delivering at least a subset of essential nutrients to the parasite. This may explain why this pathway is key (and appears) only at later stages of growth ~2. It is difficult to define in a single publication all of the substrates of a 'high-capacity' transport pathway
such as the TVM. Further studies are required to refine the specificity (glutamine vs. glutamate, etc.) of this pathway and its organization relative to channels in the infected RBC membrane as well as the parasite plasma and vacuolar membranes. Acknowledgements Our work is supported by NIH grants (M26670 and AI39071to K.H.,and AI26912 and AI01112to P.K.R.),the MacArthurFoundation (S.A.L.)and BurroughsWellc0meFund Awards (NewInvestigatorand New Initiatives in Malaria to K.H.). Kasturi Haldar and Sabine A. Lauer
Dept of Microbiology and Immunology, Stanford University, School of Medicine, Fairchild Science Building, Stanford, CA 94305-,5402, USA Pradipsinh K. Rathod
Dept of Biology, Institute for Biomolecular Studies, The Catholic University of America,
620 Michigan Avenue, NE Washington, DC 20064, USA References 1 Elmendorf, H.G. and Haldar, K. (1994) J. Cell Biol. 124, 4.49-462 2 Elmendorf, H.G. and Haldar, K. (1993) EMBO J. 12, 4763-4773 3 Elford, B.C. and Ferguson, D.J.P. (1993) Parasitol. Today 9, 80-81 4 Aikawa,M., Miller, LH. and Rabbege,J. (1975) Am. ]. Pathol. 79, 285-300 5 Aikawa, M. (1977) Bull. WHO 55, 139-156 6 Langreth, S.G. et al. (1978)J. Protozool. 25,443-452 7 Grellier, P. et al. (1991) J. Cell Biol. 112, 266-267 8 Pouvdle, B. et al. (1991) Nature 353, 73-75 9 Elmendorf, H.G. and Haldar, K. (1993) Parasitol. Today 9, 98-102 10 Lauer, S., Ghori, N. and Haldar, K. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9181-9185 11 Vogel,G. (1997) Science 276, 1031 12 Lauer, S. et al. (1997) Science 276, 1122-1125 13 Gero,A.M. and Kirk, K. (1994) Parasitol. Today 10, 395-399
A novel approach to explore the role of plasminogen "mbacterial pathogenesis Richard Lottenberg
p
lasminogen is a zymogen protein that is synthesized by the liver and is found in circulating blood and extracellular fluids. The mammalian plasminogen activators are proteases that convert plasminogen to the serine protease plasmin by cleavage of a single peptide bond. Specific protein inhibitors of plasminogen activators, as well as plasmin, prevent indiscriminate generation of proteolytic activity. Fibrinolysis The role of the plasminogen/plasmin system in homeostasis has been clearly demonstrated in the context of fibrinolysis 1. Breaks in the integrity of the vasculature elicit several responses, including the generation of insoluble fbrin from its precur-
sor, fibrinogen. If the fibrin does not remain localized, an intravascular blood clot, which also contains cellular elements, can form (a process known as thrombosis). Plasmin is responsible for fibrin degradation. The importance of an intact fibrinolytic mechanism is underscored by the identification of dysfunctional plasminogen or elevated levels of plasminogen activator inhibitors in patients experiencing a thrombotic R. Lottenberg is in the University of Florida College of Medicine, Dept of Medicine, Divn of Hematology/Oncology, Box 100277 HSC, Gainesville, FL 32610-0277, USA. tel: +1 352 392 3000,
fax" +1 352 392 8530, e-mail: [email protected]
Copyright © 1997 Elsevier Science Ltd. All rights reserved. 0966 842)(/97/$17.00
TRENDS
IN MICROBIOLOGY
466
~rOL.
5
NO.
12
tendency 2. In contrast, patients with inadequate concentrations of the major inhibitor of plasmin, ~t-2 antiplasmin, have a bleeding diathesis. Perhaps even more dramatic than the studies of individuals with congenital or acquired deficiencies are the profound effects observed from pharmacological manipulation of the plasminogerdplasmin system 3. The ability of thrombolytic agents, highly purified preparations of plasminogen activators, to facilitate rapid fibrin dissolution has improved the survival of patients suffering from coronary artery thrombosis. However, the degradation of fibrin at other sites by infusion of these drugs can lead to substantial bleeding. In summary, these clinical investigations have emphasized PII: S0966-842X(97)01171-2
DECEMBER
1997
C O M h , ~ E N T
that fibrinolysis is a tightly regulated process, but there is considerable potential in perturbing the plasminogen/plasmin system. Plasminogen activation in other biological processes
More recently, the role of the plasminogen/plasmin system in diverse physiological extracellular processes, such as ovulation, trophoblast implantation and embryogenesis, has started to be appreciated 4. Several approaches have also shown the importance of plasminogen activation in the growth and spread of malignant tumors s. The identification of mucous membrane abnormalities observed in rare individuals lacking functional plasminogen provides evidence for the participation of the system in wound healing 6. Each of these processes involves cell migration and/or tissue remodeling. A key feature at the cellular level is the assembly of plasminogen activators and plasmin(ogen) on the cell surface. Surface-bound plasmin provides localized proteolytic activity that is protected from physiological inhibition and promotes the degradation of extracellular matrix. Studies of transgenic mice
It has been possible to generate mice with deficiencies in endogenous plasminogen activators or plasminogen v,s. Homozygous deficiency of plasminogen (Pig -/-) predisposes to a spectrum of abnormalities, including thrombosis, gastrointestinal tract ulceration and defective wound healing 9. Mice deficient in both fibrinogen (Fib-t-) and plasminogen (Pig -t-) have abnormalities seen only with the Fib -~-mice, indicating that fibrin deposition contributes to the pathology in the various tissues. The availability of mice with deficiencies in the plasminogen/plasmin system provides opportunities for determining the role of plasminogen in a variety of pathological conditions. What is the role of plasminogen in bacterial pathogenesis?
It has been hypothesized that invasive bacteria can use plasmin to move through tissue planes ~°. Over 60 years ago, Tillett and Garner reported that pyogenic streptococci
TRIZNDS
produce a 'fibrinolysin 'H. Streptokinase was identified as the secreted plasminogen activator responsible for the clot lysis. Staphylokinase, a distinct plasminogen activator secreted by S t a p h y l o c o c c u s aureus, has similar activity. Despite the successful application of these bacterial products as thrombolytic agents in clinical practice, their roles in the infectious process have been difficult to delineate. Ten years ago it was observed that streptococci bind plasmin as an active protease and that the surface-bound enzyme is protected from inhibition by o~-2 antiplasmin 12. Subsequent investigations have indicated that streptococci secreting streptokinase can efficiently harness plasmin activity when simply grown in the presence of plasma ~3. Interestingly, a wide spectrum of Gram-positive and Gramnegative bacteria has also been found to interact with plasmin(ogen), thereby providing each of these microorganisms with the ability to capture surface-associated proteolytic activity ~°. The findings obtained from studies in vitro have recently been complemented by investigations of invasive pathogens in vivo. A plasmid-encoded product of Yersinia pestis possesses plasminogen activator activity. The tissueinvasive capability of an isogenic strain of Y. pestis lacking the expression of this protein is markedly compromised when the bacteria are introduced into mice by the subcutaneous route 14.The protein has also been identified as an adhesin ~s, so it remains to be shown that the plasminogen activator activity is exclusively responsible for the invasive phenotype. The recent study from Coleman et al. addressing plasminogen interactions with Borrelia burgdorferi provides the most compelling evidence that plasminogen activation contributes to a bacterial infection ~6. The investigators used Plg -~- mice to show that the blood meal of the tick provides plasminogen and plasminogen activator activity, which is necessary for dissemination of B. burgdorferi in the tick vector. Furthermore, they demonstrated a role for plasminogen in the establishment of bacteremia. The findings reported for Y. pestis and B. burgdorferi were made
IN MIC'ROBIOLOGY
467
VoL.
5
NO.
12
using animal models that represent natural hosts for these pathogens. Perhaps the difficulty in defining the role of plasminogen activation for other organisms, such as streptococci, has been the lack of an appropriate animal model. Group A streptococcus is a human pathogen, whereas certain group C streptococci appear specific for other mammalian hosts. Streptokinases from these streptococcal strains demonstrate plasminogen activator activity that is selective to the natural mammalian host iv. The ability to examine these bacteria relevantly in vivo undoubtedly requires effective interactions between the bacteria and the host plasminogen. As demonstrated by the intriguing findings published by Coleman et al. 16, the application of transgenic mouse technology may afford the best opportunity for furthering our understanding of the plasminogen-microorganism in~:eraction in the pathogenesis of invasive infections. References
1 Collen, D. (1980) Tbromb. Haemost. 43, 77-89 2 Lijnen,H.R. and Collen, D. (1989) Fibrinolysis 3, 67-77 3 Collen, D. (1997) Thromb. Haemost. 78, 742-746 4 Vassalli,J.D., Sappino, A.P. and Belir~,D. (1991) ]. Clin. Invest. 88, 1067-1072. 5 DanG,K. etal. (1994) Fibrinolysis 9, 189-203 6 Schuster,V. et al. (1997) Blood 90, 958-966 7 Carmeliet, P. et al. (1994) Nature 368, 419-424 8 Romer,J. etal. (1996) Nat. Med. 2, 287-294 9 Bugge,T.H. et al. (1996) Cell 87, 709-719 10 Boyle,M.D.P. and Lottenberg,R. (1997) Thromb. Haemost. 77, 1-10 11 Tillett, W.S. and Garner, R.L. (1933) J. Exp. Med. 58,485-502 12 Lottenberg,R., Broder, C.C. and Boyle,M.D.P. (1987) Infect. Immun. 55, 1914-1928 13 Lottenberg,R. et al. (1992)J. Infect. Dis. 166, 436-440 14 Sodeinde,O.A. et al. (1992)Science;!58, 1004-1007 15 Kienle,Z. et al. (•992)J. Gen. Microbiol. 138, 1670-1687 16 Coleman,J.L. et al. (1997) Cell 89, 1111-1119 17 McCoy,H.E., Broder, C.C. and Lottenberg, R. (1991)]. Infect. Dis. 164, 515-521
DECEMBER
1997
article 1~ accompanying our recent paper ~2,we present 'the best evidence so far' that the TVM membranes are an import system. The functions of tubules and ion channels in delivering exogenous nutrients to the parasite are major points of debate in malarial transport ~3. Our data suggest that, under physiological conditions, lowmolecular-weight nutrients can be transported through a tubular TVM pathway. Parasite-induced channels expressed early in development may underlie the first steps of solute transport into the infected RBC. When nutrient demand increases at the later trophozoite stage, the TVM pathway may increase the efficiency of delivering at least a subset of essential nutrients to the parasite. This may explain why this pathway is key (and appears) only at later stages of growth ~2. It is difficult to define in a single publication all of the substrates of a 'high-capacity' transport pathway
such as the TVM. Further studies are required to refine the specificity (glutamine vs. glutamate, etc.) of this pathway and its organization relative to channels in the infected RBC membrane as well as the parasite plasma and vacuolar membranes. Acknowledgements Our work is supported by NIH grants (M26670 and AI39071to K.H.,and AI26912 and AI01112to P.K.R.),the MacArthurFoundation (S.A.L.)and BurroughsWellc0meFund Awards (NewInvestigatorand New Initiatives in Malaria to K.H.). Kasturi Haldar and Sabine A. Lauer
Dept of Microbiology and Immunology, Stanford University, School of Medicine, Fairchild Science Building, Stanford, CA 94305-,5402, USA Pradipsinh K. Rathod
Dept of Biology, Institute for Biomolecular Studies, The Catholic University of America,
620 Michigan Avenue, NE Washington, DC 20064, USA References 1 Elmendorf, H.G. and Haldar, K. (1994) J. Cell Biol. 124, 4.49-462 2 Elmendorf, H.G. and Haldar, K. (1993) EMBO J. 12, 4763-4773 3 Elford, B.C. and Ferguson, D.J.P. (1993) Parasitol. Today 9, 80-81 4 Aikawa,M., Miller, LH. and Rabbege,J. (1975) Am. ]. Pathol. 79, 285-300 5 Aikawa, M. (1977) Bull. WHO 55, 139-156 6 Langreth, S.G. et al. (1978)J. Protozool. 25,443-452 7 Grellier, P. et al. (1991) J. Cell Biol. 112, 266-267 8 Pouvdle, B. et al. (1991) Nature 353, 73-75 9 Elmendorf, H.G. and Haldar, K. (1993) Parasitol. Today 9, 98-102 10 Lauer, S., Ghori, N. and Haldar, K. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9181-9185 11 Vogel,G. (1997) Science 276, 1031 12 Lauer, S. et al. (1997) Science 276, 1122-1125 13 Gero,A.M. and Kirk, K. (1994) Parasitol. Today 10, 395-399
A novel approach to explore the role of plasminogen "mbacterial pathogenesis Richard Lottenberg
p
lasminogen is a zymogen protein that is synthesized by the liver and is found in circulating blood and extracellular fluids. The mammalian plasminogen activators are proteases that convert plasminogen to the serine protease plasmin by cleavage of a single peptide bond. Specific protein inhibitors of plasminogen activators, as well as plasmin, prevent indiscriminate generation of proteolytic activity. Fibrinolysis The role of the plasminogen/plasmin system in homeostasis has been clearly demonstrated in the context of fibrinolysis 1. Breaks in the integrity of the vasculature elicit several responses, including the generation of insoluble fbrin from its precur-
sor, fibrinogen. If the fibrin does not remain localized, an intravascular blood clot, which also contains cellular elements, can form (a process known as thrombosis). Plasmin is responsible for fibrin degradation. The importance of an intact fibrinolytic mechanism is underscored by the identification of dysfunctional plasminogen or elevated levels of plasminogen activator inhibitors in patients experiencing a thrombotic R. Lottenberg is in the University of Florida College of Medicine, Dept of Medicine, Divn of Hematology/Oncology, Box 100277 HSC, Gainesville, FL 32610-0277, USA. tel: +1 352 392 3000,
fax" +1 352 392 8530, e-mail: [email protected]
Copyright © 1997 Elsevier Science Ltd. All rights reserved. 0966 842)(/97/$17.00
TRENDS
IN MICROBIOLOGY
466
~rOL.
5
NO.
12
tendency 2. In contrast, patients with inadequate concentrations of the major inhibitor of plasmin, ~t-2 antiplasmin, have a bleeding diathesis. Perhaps even more dramatic than the studies of individuals with congenital or acquired deficiencies are the profound effects observed from pharmacological manipulation of the plasminogerdplasmin system 3. The ability of thrombolytic agents, highly purified preparations of plasminogen activators, to facilitate rapid fibrin dissolution has improved the survival of patients suffering from coronary artery thrombosis. However, the degradation of fibrin at other sites by infusion of these drugs can lead to substantial bleeding. In summary, these clinical investigations have emphasized PII: S0966-842X(97)01171-2
DECEMBER
1997
C O M h , ~ E N T
that fibrinolysis is a tightly regulated process, but there is considerable potential in perturbing the plasminogen/plasmin system. Plasminogen activation in other biological processes
More recently, the role of the plasminogen/plasmin system in diverse physiological extracellular processes, such as ovulation, trophoblast implantation and embryogenesis, has started to be appreciated 4. Several approaches have also shown the importance of plasminogen activation in the growth and spread of malignant tumors s. The identification of mucous membrane abnormalities observed in rare individuals lacking functional plasminogen provides evidence for the participation of the system in wound healing 6. Each of these processes involves cell migration and/or tissue remodeling. A key feature at the cellular level is the assembly of plasminogen activators and plasmin(ogen) on the cell surface. Surface-bound plasmin provides localized proteolytic activity that is protected from physiological inhibition and promotes the degradation of extracellular matrix. Studies of transgenic mice
It has been possible to generate mice with deficiencies in endogenous plasminogen activators or plasminogen v,s. Homozygous deficiency of plasminogen (Pig -/-) predisposes to a spectrum of abnormalities, including thrombosis, gastrointestinal tract ulceration and defective wound healing 9. Mice deficient in both fibrinogen (Fib-t-) and plasminogen (Pig -t-) have abnormalities seen only with the Fib -~-mice, indicating that fibrin deposition contributes to the pathology in the various tissues. The availability of mice with deficiencies in the plasminogen/plasmin system provides opportunities for determining the role of plasminogen in a variety of pathological conditions. What is the role of plasminogen in bacterial pathogenesis?
It has been hypothesized that invasive bacteria can use plasmin to move through tissue planes ~°. Over 60 years ago, Tillett and Garner reported that pyogenic streptococci
TRIZNDS
produce a 'fibrinolysin 'H. Streptokinase was identified as the secreted plasminogen activator responsible for the clot lysis. Staphylokinase, a distinct plasminogen activator secreted by S t a p h y l o c o c c u s aureus, has similar activity. Despite the successful application of these bacterial products as thrombolytic agents in clinical practice, their roles in the infectious process have been difficult to delineate. Ten years ago it was observed that streptococci bind plasmin as an active protease and that the surface-bound enzyme is protected from inhibition by o~-2 antiplasmin 12. Subsequent investigations have indicated that streptococci secreting streptokinase can efficiently harness plasmin activity when simply grown in the presence of plasma ~3. Interestingly, a wide spectrum of Gram-positive and Gramnegative bacteria has also been found to interact with plasmin(ogen), thereby providing each of these microorganisms with the ability to capture surface-associated proteolytic activity ~°. The findings obtained from studies in vitro have recently been complemented by investigations of invasive pathogens in vivo. A plasmid-encoded product of Yersinia pestis possesses plasminogen activator activity. The tissueinvasive capability of an isogenic strain of Y. pestis lacking the expression of this protein is markedly compromised when the bacteria are introduced into mice by the subcutaneous route 14.The protein has also been identified as an adhesin ~s, so it remains to be shown that the plasminogen activator activity is exclusively responsible for the invasive phenotype. The recent study from Coleman et al. addressing plasminogen interactions with Borrelia burgdorferi provides the most compelling evidence that plasminogen activation contributes to a bacterial infection ~6. The investigators used Plg -~- mice to show that the blood meal of the tick provides plasminogen and plasminogen activator activity, which is necessary for dissemination of B. burgdorferi in the tick vector. Furthermore, they demonstrated a role for plasminogen in the establishment of bacteremia. The findings reported for Y. pestis and B. burgdorferi were made
IN MIC'ROBIOLOGY
467
VoL.
5
NO.
12
using animal models that represent natural hosts for these pathogens. Perhaps the difficulty in defining the role of plasminogen activation for other organisms, such as streptococci, has been the lack of an appropriate animal model. Group A streptococcus is a human pathogen, whereas certain group C streptococci appear specific for other mammalian hosts. Streptokinases from these streptococcal strains demonstrate plasminogen activator activity that is selective to the natural mammalian host iv. The ability to examine these bacteria relevantly in vivo undoubtedly requires effective interactions between the bacteria and the host plasminogen. As demonstrated by the intriguing findings published by Coleman et al. 16, the application of transgenic mouse technology may afford the best opportunity for furthering our understanding of the plasminogen-microorganism in~:eraction in the pathogenesis of invasive infections. References
1 Collen, D. (1980) Tbromb. Haemost. 43, 77-89 2 Lijnen,H.R. and Collen, D. (1989) Fibrinolysis 3, 67-77 3 Collen, D. (1997) Thromb. Haemost. 78, 742-746 4 Vassalli,J.D., Sappino, A.P. and Belir~,D. (1991) ]. Clin. Invest. 88, 1067-1072. 5 DanG,K. etal. (1994) Fibrinolysis 9, 189-203 6 Schuster,V. et al. (1997) Blood 90, 958-966 7 Carmeliet, P. et al. (1994) Nature 368, 419-424 8 Romer,J. etal. (1996) Nat. Med. 2, 287-294 9 Bugge,T.H. et al. (1996) Cell 87, 709-719 10 Boyle,M.D.P. and Lottenberg,R. (1997) Thromb. Haemost. 77, 1-10 11 Tillett, W.S. and Garner, R.L. (1933) J. Exp. Med. 58,485-502 12 Lottenberg,R., Broder, C.C. and Boyle,M.D.P. (1987) Infect. Immun. 55, 1914-1928 13 Lottenberg,R. et al. (1992)J. Infect. Dis. 166, 436-440 14 Sodeinde,O.A. et al. (1992)Science;!58, 1004-1007 15 Kienle,Z. et al. (•992)J. Gen. Microbiol. 138, 1670-1687 16 Coleman,J.L. et al. (1997) Cell 89, 1111-1119 17 McCoy,H.E., Broder, C.C. and Lottenberg, R. (1991)]. Infect. Dis. 164, 515-521
DECEMBER
1997