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Trypanosoma brucei brucei:Characterization of an ODC Null Bloodstream Form Mutant and the Action of Alpha-difluoromethylornithine
EXPERIMENTAL PARASITOLOGY ARTICLE NO.
88, 255–257 (1998)
PR974237
RESEARCH BRIEF Trypanosoma brucei brucei: Characterization of an ODC Null Bloodstream Form Mutant and the Action of Alpha-difluoromethylornithine
F. Li,* S.-b. Hua,† C. C. Wang,† and K. M. Gottesdiener*,1 *Department of Medicine, Columbia College of Physicians and Surgeons, 630 West 168th Street, New York, New York 10032, U.S.A.; and †Department of Pharmaceutical Chemistry, University of California San Francisco School of Pharmacy, 513 Parnassus Avenue, San Francisco, California 94143, U.S.A.
Li, F., Hua, S.-b., Wang, C. C., and Gottesdiener, K. M. 1998. Trypanosoma brucei brucei: Characterization of an ODC null bloodstream form mutant and the action of alpha-difluoromethylornithine. Experimental Parasitology 88, 255–257. Ornithine decarboxylase (ODC) is a key enzyme in the polyamine synthesis pathway in African trypanosomes. We report here the characterization of an ODC null bloodstream form Trypanosoma brucei brucei mutant, created by replacing the ODC gene with antibiotic resistant marker genes through transfection and homologous recombination. The null mutant expresses no ODC mRNA or protein and does not have ODC enzymatic activity. We tested the attenuation of the bloodstream form ODC2 mutants in mice, and showed that these mutants cannot multiply and are quickly cleared from the blood. We also tested the effect of DFMO on this ODC null mutant. q 1998 Academic Press Index Descriptors and Abbreviations: BS, bloodstream; DFMO, adifluoromethylornithine; ODC, ornithine decarboxylase (EC 4.1.1.17); WT, wild-type; VSG, variant surface glycoprotein. Key Words: Trypanosoma brucei; ornithine decarboxylase; DFMO; attenuation; gene knockout.
Trypanosoma brucei are pathogenic protozoan transmitted by the tsetse fly (Kuzoe 1991) that cause African sleeping sickness in humans or the nagana disease in livestock. Effective control of these diseases is still limited because of the lack of vaccines and the scant choices of antitrypanosome drugs (Kuzoe 1991; Wang 1995). We recently
1 To whom correspondence should be addressed at the present address: Merck Research Laboratories, P.O. Box 2000, RY 33-644, Rahway, NJ 07065. Fax: (908) 594-5405.
0014-4894/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
constructed an ODC2 procyclic trypanosome mutant (Li 1996). This procyclic mutant cell line could survive, but could not proliferate without exogenous polyamines. We hypothesized that an ODC2 bloodstream form cell line with similar characteristics could potentially function as an attenuated strain that would be useful in vaccine development, as it could express vaccine antigens without proliferation. Trypanosoma brucei brucei stock 427-60 (variant 118a) bloodstream form ODC null mutants were constructed using plasmid constructs and a knockout strategy similar to those previously described (Li 1996; Li and Gottesdiener 1996). Both Southern and Northern hybridization with the ODC coding region probe (Li 1996) demonstrated the absence of the ODC gene and gene expression in double knockout mutants (data not shown). The two single ODC knockout cell lines (containing a single ODC allele) had approximately half the ODC message of the wild-type cells, a result similar to that seen in the procyclic ODC single knockouts (Li et al. 1996), suggesting that neither BS nor procyclic trypanosomes can compensate for gene loss with increased steady state
TABLE I Testing Attenuation of ODC Null T. brucei brucei Bloodstream forms in Vivo
Trypanosomes T. T. T. T.
brucei brucei brucei brucei
Mice (No.) Gamma ODC irradiation Injected Survived
BS clone PH10 Null BS clone PH10 Null 427-60 (BS 118a) WT 427-60 (BS 118a) WT
None 460 Rad None 460 Rad
6 3 4 3
6 3 0 0
Note. BS, bloodstream; WT, wild-type.
255
256 ODC message. Western analysis and enzymatic activity of the ODC deficient cell lines demonstrated that neither ODC protein nor ODC enzyme activity was present in the double knockout mutant cell lines (data not shown). We next studied attenuation of the double knockout cell lines by injection (1 3 107 tryps/mouse) into individual BALB/C mice, with or without prior irradiation (460 Rad), to inactivate the immune response prior to injection. As controls, wild-type trypanosomes 427-60 (118a, 1 3 105 tryps/mouse) were also injected into immunocompetent and irradiated mice. Trypanosomemia was monitored by counting trypanosomes from tail vein blood. The survival rate is shown in Table I. In nonirradiated mice, live ODC2 trypanosome cells could be seen in the blood on Days 2 and 3 after injection, but disappeared by Day 4. A similar result was seen after injection of the live ODC2 trypanosome cells into immune inactivated gamma-irradiated mice, and all animals (from both groups) remained trypanosome free for .2 weeks. In contrast, after wild-type trypanosome injection, in both irradiated and nonirradiated mice, trypanosomes were seen in the blood
LI ET AL.
from Day 2, reached a peak (3–5 3 108 cells/ml) on Days 4–6, and all animals died thereafter. Using the ODC null cell line PH10, we examined the effect of DFMO on the mutant cells by measuring in vitro growth curves at various putrescine concentrations, in the presence or absence of DFMO. We hypothesized that if ODC is the sole target for DFMO action, the growth curves of ODC2 cell PH10, at each particular putrescine concentration, would be unaffected by the absence or presence of DFMO in the media. In addition, we hypothesized that the growth of the wild type cell line in the presence of DFMO (making the wildtype trypanosomes effectively “ODC null”) in various concentrations of putrescine would be identical to the growth of ODC null knockout cell line under the same conditions. Figure 1 shows the experimental results on the growth of the ODC2 and the wild-type BS trypanosome cells at various putrescine concentrations and in the presence or absence of DFMO. These results indicated that the growth of ODC2 cells was dependent on the concentration of putrescine in the media, but was not affected by the absence or
FIG. 1. Action of DFMO on T. brucei brucei. (a) Double ODC gene knockout clone PH10 cultured in the absence of DFMO and at various putrescine concentrations. Days are indicated below (X axis), and cell number (the log10 value) is indicated on the left (Y axis). (b) Same as in (a), except 0.5 mM DFMO was added to the culturing medium. (c) Wild-type T. brucei 427-60 (118a) were cultured in the absence of DFMO and at various putrescine concentrations. (d) Wild-type T. brucei 427-60 (118a) were cultured in the presence of DFMO (0.5 mM) and at various putrescine concentrations.
257
ODC NULL BLOODSTREAM FORM T. brucei brucei
presence of DFMO, demonstrating, as expected, that DFMO did not affect other targets besides ODC (Figs. 1a and 1b). The wild-type trypanosomes continued log phase growth in the absence of DFMO, regardless of the amount of putrescine added to the medium (Fig. 1c). However, the wild-type growth curves in the presence of DFMO (and various amount of putrescine; Fig. 1d) are identical to those of the ODC null cells shown in Figs. 1a and 1b. Altogether, these results confirm the previous studies that suggest that ODC is the sole target for DFMO and that the mechanism of action of this drug is solely by inhibiting ODC, causing depletion of polyamines from the trypanosomes. As can be seen also, when cultured, the BS form ODC2 trypanosomes can survive only in the stationary phase for about 1 week in the absence of putrescine (Figs. 1a and 1b, 0 mM putrescine curves). This result is in stark contrast to the procyclic ODC2 mutants that survive for up to 7 weeks (Li 1996) without exogenous putrescine. Since the discovery that DFMO can cure acute infections, it has been established that endogenous synthesis of polyamines is essential for trypanosomes proliferation in mammalian blood. In this study we confirmed this conclusion. Although it has been speculated that DFMO might have other direct actions on the cell, our results indicate that this hypothesis is unlikely. An additional trait of the ODC null trypanosomes is attenuation: bloodstream form ODC2 mutants can be detected up to 72 h after injection into mice, before disappearing from the bloodstream. There was no obvious pathology in these ODC2 trypanosome inoculated mice. In contrast, injection of a much smaller number of WT trypanosomes into mice led to a peak of parasitemia after 4–6 days and death of the infected animals. These results suggest that the virulence of these ODC2 mutants is attenuated in the mammalian bloodstream, presumably because of the low levels of polyamines present. Alternately, the mutation might have affected the ability of
these trypanosomes to undergo antigenic variation, though this is less likely since the ODC2 cells also disappeared from the bloodstream of irradiated mice. These results suggest the potential utility of an attenuated ODC2 mutant strain of trypanosomes as a vaccine strain, presumably with expression of an appropriate antigen(s), if one or a subset of antigens could be identified. Considering the importance of polyamines for proliferation of all kinds of cells, this same approach might also be useful for other parasites that live in a polyaminedepleted environment.
REFERENCES Kuzoe, F. A. S. 1991. Perspectives in research on and control of African trypanosomiasis. Annals of Tropical Medicine and Parasitology 85, 33–41. Li, F., and Gottesdiener K. M. 1996. An efficient method for stable transfection of Trypanosoma brucei brucei blood-stream form. Nucleic Acids Research 24, 534–536. Li, F., Hua, S.-b. Wang, C. C., and Gottesdiener, K. M. 1996. Procyclic Trypanosoma brucei cell lines deficient in ornithine decarboxylase activity. Molecular and Biochemical Parasitology 78, 227–236. Wang, C. C. 1995. Molecular mechanisms and therapeutic approaches to the treatment of African Trypanosomiasis. Annual Review of Pharmacology and Toxicology 35, 93–127. Received 8 May 1997; accepted 12 September 1997
88, 255–257 (1998)
PR974237
RESEARCH BRIEF Trypanosoma brucei brucei: Characterization of an ODC Null Bloodstream Form Mutant and the Action of Alpha-difluoromethylornithine
F. Li,* S.-b. Hua,† C. C. Wang,† and K. M. Gottesdiener*,1 *Department of Medicine, Columbia College of Physicians and Surgeons, 630 West 168th Street, New York, New York 10032, U.S.A.; and †Department of Pharmaceutical Chemistry, University of California San Francisco School of Pharmacy, 513 Parnassus Avenue, San Francisco, California 94143, U.S.A.
Li, F., Hua, S.-b., Wang, C. C., and Gottesdiener, K. M. 1998. Trypanosoma brucei brucei: Characterization of an ODC null bloodstream form mutant and the action of alpha-difluoromethylornithine. Experimental Parasitology 88, 255–257. Ornithine decarboxylase (ODC) is a key enzyme in the polyamine synthesis pathway in African trypanosomes. We report here the characterization of an ODC null bloodstream form Trypanosoma brucei brucei mutant, created by replacing the ODC gene with antibiotic resistant marker genes through transfection and homologous recombination. The null mutant expresses no ODC mRNA or protein and does not have ODC enzymatic activity. We tested the attenuation of the bloodstream form ODC2 mutants in mice, and showed that these mutants cannot multiply and are quickly cleared from the blood. We also tested the effect of DFMO on this ODC null mutant. q 1998 Academic Press Index Descriptors and Abbreviations: BS, bloodstream; DFMO, adifluoromethylornithine; ODC, ornithine decarboxylase (EC 4.1.1.17); WT, wild-type; VSG, variant surface glycoprotein. Key Words: Trypanosoma brucei; ornithine decarboxylase; DFMO; attenuation; gene knockout.
Trypanosoma brucei are pathogenic protozoan transmitted by the tsetse fly (Kuzoe 1991) that cause African sleeping sickness in humans or the nagana disease in livestock. Effective control of these diseases is still limited because of the lack of vaccines and the scant choices of antitrypanosome drugs (Kuzoe 1991; Wang 1995). We recently
1 To whom correspondence should be addressed at the present address: Merck Research Laboratories, P.O. Box 2000, RY 33-644, Rahway, NJ 07065. Fax: (908) 594-5405.
0014-4894/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
constructed an ODC2 procyclic trypanosome mutant (Li 1996). This procyclic mutant cell line could survive, but could not proliferate without exogenous polyamines. We hypothesized that an ODC2 bloodstream form cell line with similar characteristics could potentially function as an attenuated strain that would be useful in vaccine development, as it could express vaccine antigens without proliferation. Trypanosoma brucei brucei stock 427-60 (variant 118a) bloodstream form ODC null mutants were constructed using plasmid constructs and a knockout strategy similar to those previously described (Li 1996; Li and Gottesdiener 1996). Both Southern and Northern hybridization with the ODC coding region probe (Li 1996) demonstrated the absence of the ODC gene and gene expression in double knockout mutants (data not shown). The two single ODC knockout cell lines (containing a single ODC allele) had approximately half the ODC message of the wild-type cells, a result similar to that seen in the procyclic ODC single knockouts (Li et al. 1996), suggesting that neither BS nor procyclic trypanosomes can compensate for gene loss with increased steady state
TABLE I Testing Attenuation of ODC Null T. brucei brucei Bloodstream forms in Vivo
Trypanosomes T. T. T. T.
brucei brucei brucei brucei
Mice (No.) Gamma ODC irradiation Injected Survived
BS clone PH10 Null BS clone PH10 Null 427-60 (BS 118a) WT 427-60 (BS 118a) WT
None 460 Rad None 460 Rad
6 3 4 3
6 3 0 0
Note. BS, bloodstream; WT, wild-type.
255
256 ODC message. Western analysis and enzymatic activity of the ODC deficient cell lines demonstrated that neither ODC protein nor ODC enzyme activity was present in the double knockout mutant cell lines (data not shown). We next studied attenuation of the double knockout cell lines by injection (1 3 107 tryps/mouse) into individual BALB/C mice, with or without prior irradiation (460 Rad), to inactivate the immune response prior to injection. As controls, wild-type trypanosomes 427-60 (118a, 1 3 105 tryps/mouse) were also injected into immunocompetent and irradiated mice. Trypanosomemia was monitored by counting trypanosomes from tail vein blood. The survival rate is shown in Table I. In nonirradiated mice, live ODC2 trypanosome cells could be seen in the blood on Days 2 and 3 after injection, but disappeared by Day 4. A similar result was seen after injection of the live ODC2 trypanosome cells into immune inactivated gamma-irradiated mice, and all animals (from both groups) remained trypanosome free for .2 weeks. In contrast, after wild-type trypanosome injection, in both irradiated and nonirradiated mice, trypanosomes were seen in the blood
LI ET AL.
from Day 2, reached a peak (3–5 3 108 cells/ml) on Days 4–6, and all animals died thereafter. Using the ODC null cell line PH10, we examined the effect of DFMO on the mutant cells by measuring in vitro growth curves at various putrescine concentrations, in the presence or absence of DFMO. We hypothesized that if ODC is the sole target for DFMO action, the growth curves of ODC2 cell PH10, at each particular putrescine concentration, would be unaffected by the absence or presence of DFMO in the media. In addition, we hypothesized that the growth of the wild type cell line in the presence of DFMO (making the wildtype trypanosomes effectively “ODC null”) in various concentrations of putrescine would be identical to the growth of ODC null knockout cell line under the same conditions. Figure 1 shows the experimental results on the growth of the ODC2 and the wild-type BS trypanosome cells at various putrescine concentrations and in the presence or absence of DFMO. These results indicated that the growth of ODC2 cells was dependent on the concentration of putrescine in the media, but was not affected by the absence or
FIG. 1. Action of DFMO on T. brucei brucei. (a) Double ODC gene knockout clone PH10 cultured in the absence of DFMO and at various putrescine concentrations. Days are indicated below (X axis), and cell number (the log10 value) is indicated on the left (Y axis). (b) Same as in (a), except 0.5 mM DFMO was added to the culturing medium. (c) Wild-type T. brucei 427-60 (118a) were cultured in the absence of DFMO and at various putrescine concentrations. (d) Wild-type T. brucei 427-60 (118a) were cultured in the presence of DFMO (0.5 mM) and at various putrescine concentrations.
257
ODC NULL BLOODSTREAM FORM T. brucei brucei
presence of DFMO, demonstrating, as expected, that DFMO did not affect other targets besides ODC (Figs. 1a and 1b). The wild-type trypanosomes continued log phase growth in the absence of DFMO, regardless of the amount of putrescine added to the medium (Fig. 1c). However, the wild-type growth curves in the presence of DFMO (and various amount of putrescine; Fig. 1d) are identical to those of the ODC null cells shown in Figs. 1a and 1b. Altogether, these results confirm the previous studies that suggest that ODC is the sole target for DFMO and that the mechanism of action of this drug is solely by inhibiting ODC, causing depletion of polyamines from the trypanosomes. As can be seen also, when cultured, the BS form ODC2 trypanosomes can survive only in the stationary phase for about 1 week in the absence of putrescine (Figs. 1a and 1b, 0 mM putrescine curves). This result is in stark contrast to the procyclic ODC2 mutants that survive for up to 7 weeks (Li 1996) without exogenous putrescine. Since the discovery that DFMO can cure acute infections, it has been established that endogenous synthesis of polyamines is essential for trypanosomes proliferation in mammalian blood. In this study we confirmed this conclusion. Although it has been speculated that DFMO might have other direct actions on the cell, our results indicate that this hypothesis is unlikely. An additional trait of the ODC null trypanosomes is attenuation: bloodstream form ODC2 mutants can be detected up to 72 h after injection into mice, before disappearing from the bloodstream. There was no obvious pathology in these ODC2 trypanosome inoculated mice. In contrast, injection of a much smaller number of WT trypanosomes into mice led to a peak of parasitemia after 4–6 days and death of the infected animals. These results suggest that the virulence of these ODC2 mutants is attenuated in the mammalian bloodstream, presumably because of the low levels of polyamines present. Alternately, the mutation might have affected the ability of
these trypanosomes to undergo antigenic variation, though this is less likely since the ODC2 cells also disappeared from the bloodstream of irradiated mice. These results suggest the potential utility of an attenuated ODC2 mutant strain of trypanosomes as a vaccine strain, presumably with expression of an appropriate antigen(s), if one or a subset of antigens could be identified. Considering the importance of polyamines for proliferation of all kinds of cells, this same approach might also be useful for other parasites that live in a polyaminedepleted environment.
REFERENCES Kuzoe, F. A. S. 1991. Perspectives in research on and control of African trypanosomiasis. Annals of Tropical Medicine and Parasitology 85, 33–41. Li, F., and Gottesdiener K. M. 1996. An efficient method for stable transfection of Trypanosoma brucei brucei blood-stream form. Nucleic Acids Research 24, 534–536. Li, F., Hua, S.-b. Wang, C. C., and Gottesdiener, K. M. 1996. Procyclic Trypanosoma brucei cell lines deficient in ornithine decarboxylase activity. Molecular and Biochemical Parasitology 78, 227–236. Wang, C. C. 1995. Molecular mechanisms and therapeutic approaches to the treatment of African Trypanosomiasis. Annual Review of Pharmacology and Toxicology 35, 93–127. Received 8 May 1997; accepted 12 September 1997