- Email: [email protected]
Folic acid antagonism of sulfa drug treatments
Research Update
TRENDS in Parasitology Vol.18 No.2 February 2002
49
Research News
Folic acid antagonism of sulfa drug treatments Ann M. Bayly and Ian G. Macreadie Folic acid antagonizes the sulfa drug treatment of malaria. This antagonism has been mimicked in a yeast model system, where the growth inhibition by sulfa drugs can be confounded. In addition, yeast studies show that the inhibition is independent of folic acid utilization, suggesting that the antagonism occurs by an unexpected mechanism.
Sulfa drugs are antifolates that have an important role in malaria chemotherapy. These antifolates compete with p-amino benzoate (PABA) leading to the depletion of dihydrofolic acid (DHF), which is essential for many reactions in all organisms (Fig. 1a). Many organisms, including animals, do not synthesize DHF, but instead take up folic acid (FA), which is converted to DHF. Therefore, exogenous FA would be expected to alleviate inhibition of sulfa drugs. Indeed, FA does inhibit the action of antifolates in vitro [1–3] and in vivo [4], but how FA achieves this action is not clear. In particular, there have been considerable discussions on whether FA utilization in Plasmodium falciparum affects sulfa drug resistance [5–7]. Recent studies in this field [8], where yeast was used as the experimental organism, help to understand the role of FA utilization in sulfa drug resistance. Strains that use FA had the same response to sulfa drugs as those that did not use FA. Some of the relevant results from these studies are summarized in Fig. 1. The parental yeast strain, YH1, has an intact pathway for DHF synthesis (FAS+). Two gene knockout mutants were made from YH1, EHY1 [which has a specific dihydropteroate synthase (DHPS) deficiency] and LCY1 [which is deficient in dihydrofolate synthase (DHFS)] [8]. DHPS and DHFS are the last two enzymes in DHF synthesis. Each knockout mutant is FAS− and dependent on exogenous DHF for growth (Fig. 1b). It was surprising that the majority of EHY1 and LCY1 cells could not use FA in place of DHF. However, from both strains, selected mutants (EHY1A and LCY1A) capable of using exogenous FA from the medium were isolated. http://parasites.trends.com
All four mutants were restored to FAS+, by transformation with the DHPS or DHFS gene lacking in the mutants, to enable a comparative study of FAS+ FA utilizers vs non-utilizers. The growth of these strains and the parent YH1 were then compared on various media (Fig. 1c). All strains exhibited the same growth on media without FA, indicating that endogenous folate synthesis provided the essential folate requirements. In the presence of the sulfa drug, sulfamethoxazole (SMX), growth was strongly inhibited when FA was absent. (a)
PABA+DHPPP Sulfa drug
DHPS DHP DHFS
FA
DHF Folate utilization cycle
(b)
DHF FASFA EA
(c)
E
LA
L
0 FAS+
SMX SMX+FA EA
E
Y
LA
L
TRENDS in Parasitology
Fig. 1. Effects of sulfamethoxazole (SMX) and folic acid (FA) on the growth of yeast. (a) Dihydrofolic acid (DHF) synthesis and utilization pathways. DHF could originate from cellular biosynthesis (black) or from exogenous FA (red). Sulfa drugs compete with p-amino benzoate (PABA) leading to loss of DHF. DHF is reduced and modified into several different folate cofactors that are recycled back to DHF. (b) FAS– strains EHY1A (EA), EHY1 (E), LCY1A (LA) and LCY1 (L) do not possess an intact pathway for DHF synthesis. (c) FAS+ derivatives of the mutant strains from (b) were transformed with plasmids p414.YFAS and p414. DHFS, in addition to the parental strain YH1 (Y). Yeast minimal media was used supplemented with uracil, tryptophan and leucine as required, plus DHF (10 µg ml–1), SMX (100 µg ml–1 or FA (10 µg ml–1), as indicated. Full details are in [8]. Abbreviations: DHFS, dihydrofolate synthase; DHP, dihydropteroate; DHPPP, DHP pyrophosphate; DHPS, dihydropteroate synthase; 0, no supplements added.
However, when FA was present, growth was restored to a level resembling that of growth in the absence of SMX. Similar results have been obtained when other sulfa drugs including dapsone and sulfanilamide were used in the place of SMX (data not shown). One conclusion drawn from these data is that, regardless of whether or not a strain can use exogenous FA (the main form found in the diet), there is no noticeable impact on growth or on sulfa drug resistance in the strains. Assuming that this statement also applies to Plasmodium, it would be expected that there is no difference in drug resistance owing to FA utilization phenotype of the strain. The observation that FA alleviates the inhibition of sulfa drugs is not new and has been observed in Plasmodium [6]. However, the theory that the mechanism might be a result of FA utilization now seem unlikely and this raises a major question about how FA exerts this effect. FA is not a PABA analogue and is unlikely to compete directly with sulfa drugs in the manner seen for PABA [9]. It is more likely that competition might occur between the DHP analogues that are produced when sulfa drugs, in place of PABA, are condensed with DHP pyrophosphate (DHPPP) [10–11]. In vitro, such DHP analogues can inhibit DHFS or serine hydroxymethyl transferase activity, and FA might alleviate growth inhibition at one or more of these inhibitory points. However, it has yet to be elucidated whether inhibition at these points occurs in vivo. Possible additional points for FA to alleviate inhibition might be in competition for folate membrane-carriers and folate-binding proteins that exist in animals, but have yet to be proven in yeast. We hope to address these questions, once again employing yeast as model for the study of antifolate resistance. References 1 Wang, P. et al. (1997) Sulfadoxine resistance in the human malaria parasite Plasmodium falciparum is determined by mutations in dihydropteroate synthetase and an additional factor associated with folate utilization. Mol. Microbiol. 23, 979–986
1471-4922/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S1471-4922(01)02202-4
50
Research Update
2 Watkins, W.M. et al. (1985) Antagonism of sulfadoxine and pyrimethamine antimalarial activity in vitro by p-aminobenzoic acid, p-aminobenzoylglutamic acid and folic acid. Mol. Biochem. Parasitol. 14, 55–61 3 Milhous, W.K. et al. (1985) In vitro activities of and mechanisms of resistance to antifol antimalarial drugs. Antimicrob. Agents Chemother. 27, 525–530 4 van Hensbroek, M.B. et al. (1995) Iron, but not folic acid, combined with effective antimalarial therapy promotes haematological recovery in African children after acute falciparum malaria. Trans. R. Soc. Trop. Med. Hyg. 89, 672–676 5 Sims, P. et al. (1998) The efficacy of antifolate antimalarial combinations in Africa. Parasitol. Today 14, 136–137
TRENDS in Parasitology Vol.18 No.2 February 2002
6 Sims, P. et al. (1999) Selection and synergy in Plasmodium falciparum. Parasitol. Today 15, 132–134 7 Watkins, W.M. et al. (1999) More on ‘The efficacy of antifolate antimalarial combinations in Africa’. Parasitol. Today 15, 131–132 8 Bayly, A.M. et al. (2001) Folic acid utilization related to sulfa drug resistance in Saccharomyces cerevisiae. FEMS Microbiol. Letts. 204, 389–390 9 Castelli, L.A. et al. (2001) Sulfa drug screening in yeast: fifteen sulfa drugs compete with p-aminobenzoate in Saccharomyces cerevisiae. FEMS Microbiol. Letts. 199, 181–184 10 Swedberg, G. et al. (1979) Characterization of a mutationally altered dihydropteroate synthase and its ability to form a sulfonamidecontaining dihydrofolate analog. J. Bacteriol. 137, 129–136
11 Roland, S. et al. (1979). The characteristics and significance of sulfonamides as substrates for Escherichia coli dihydropteroate synthase. J. Biol. Chem. 254, 10337–10345
Ann M. Bayly Ian G. Macreadie* Commonwealth Scientific and Industrial Research Organisation (CSIRO), Health Sciences and Nutrition, 343 Royal Parade Parkville 3052 and Royal Melbourne Institute of Technology (RMIT) University, Bundoora, Victoria, Australia. *e-mail: [email protected]
Meeting Report
Schistosomiasis immunology, epidemiology and diagnosis Andreas Ruppel, Malcolm W. Kennedy and John R. Kusel The seventh annual meeting on schistosomiasis organized by Andreas Ruppel was held 18–21 October 2001 at University of Heidelberg, Oberflockenbach, Germany.
The conference brought together scientists with a range of interests in basic, clinical and field research not only from Europe (Czech Republic, France, Germany, Italy, United Kingdom) but also from Oman, Egypt and, in particular, from China. This international group enabled extensive discussion on differences in epidemiology, diagnosis and immunopathology between Schistosoma japonicum and Schistosoma mansoni infections. A fruitful exchange between junior and senior scientists was also evident, in addition to discussions aimed at the development of joint research activities. Immunology and early infections
Michael Kirschfink (University of Heidelberg, Germany) presented an overview of immune evasion strategies (including examples from viruses, bacteria and parasites) and concentrated on evasion or manipulation of the complement system by these pathogens. In early schistosome infections (already at the skin stage), schistosomula (Sla) of S. mansoni induce a transitory thrombocytopaenia perhaps by inducing http://parasites.trends.com
platelet aggregation as a result of blood vessel rupture. In addition, adult schistosomes induce antibodies against host platelets, and the antigens which are responsible for this induction are present in Sla and adult worms, but not in the eggs of S. mansoni. These new insights into thrombocytopaenia could explain why blood clots do not form around adult worms (Ron Stanley, University of Wales, Bangor, UK). The early stages (cercariae and Sla) of S. japonicum activate the complement system not only by the alternative and classical pathways, but also by the mannose-binding lectin (MBL) pathway, and these parasite stages also bind C1q (a component of the complement system) directly to their surface (Wenshi Wang, University of Heidelberg). Cytotoxic complement activity was, however, limited, and serum even promoted the in vitro transformation of S. japonicum cercariae to Sla. The sensitivity of S. japonicum Sla to killing by macrophages was mediated to a large extent by nitric oxide (Xiao-Chun Long, Tongji Medical College, Wuhan, China). A stathmin-like protein was found in the pre- and post-acetabular glands of S. mansoni cercariae and also in the subtegumental layer of mechanically transformed schistosomula (Cristiana Valle, National Research Council, Rome, Italy). Stathmin displays microtubule-destabilizing
activity and this protein is only synthesized in the sporocyst stage just before or during cercarial shedding. Therefore, stathmins could be involved in the rapid changes in membrane formation during skin penetration. The development of S. mansoni in normal mice was compared with mice immunosuppressed with prednisolone three or 21 days after infection ∨ ∨ (Michal Giboda, Ceské Budejovice, Czech Republic). Early, but not late, prednisolone treatment of mice resulted in significantly reduced worm load and female fecundity when compared with non-treated controls. This result suggests a role for the immune system in worm development during the early phase of infection. Vaccination and pathogenesis
The principles of DNA vaccination were outlined by Volker Schirrmacher (German Cancer Research Center, Heidelberg, Germany) using lacZ as a model tumor-associated antigen, and then vaccinating mice with lacZ DNA plasmids or self-replicating RNA. Anti-tumor immunity was enhanced by immunomodulation, particularly by co-injection of cytokine-expressing constructs. The DNA vaccine approach was applied to schistosomes by incorporating the gene encoding the S. mansoni asparaginyl endopeptidase (Sm32) into a DNA vaccine (Katerina Chlichlia,
1471-4922/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S1471-4922(01)02206-1
TRENDS in Parasitology Vol.18 No.2 February 2002
49
Research News
Folic acid antagonism of sulfa drug treatments Ann M. Bayly and Ian G. Macreadie Folic acid antagonizes the sulfa drug treatment of malaria. This antagonism has been mimicked in a yeast model system, where the growth inhibition by sulfa drugs can be confounded. In addition, yeast studies show that the inhibition is independent of folic acid utilization, suggesting that the antagonism occurs by an unexpected mechanism.
Sulfa drugs are antifolates that have an important role in malaria chemotherapy. These antifolates compete with p-amino benzoate (PABA) leading to the depletion of dihydrofolic acid (DHF), which is essential for many reactions in all organisms (Fig. 1a). Many organisms, including animals, do not synthesize DHF, but instead take up folic acid (FA), which is converted to DHF. Therefore, exogenous FA would be expected to alleviate inhibition of sulfa drugs. Indeed, FA does inhibit the action of antifolates in vitro [1–3] and in vivo [4], but how FA achieves this action is not clear. In particular, there have been considerable discussions on whether FA utilization in Plasmodium falciparum affects sulfa drug resistance [5–7]. Recent studies in this field [8], where yeast was used as the experimental organism, help to understand the role of FA utilization in sulfa drug resistance. Strains that use FA had the same response to sulfa drugs as those that did not use FA. Some of the relevant results from these studies are summarized in Fig. 1. The parental yeast strain, YH1, has an intact pathway for DHF synthesis (FAS+). Two gene knockout mutants were made from YH1, EHY1 [which has a specific dihydropteroate synthase (DHPS) deficiency] and LCY1 [which is deficient in dihydrofolate synthase (DHFS)] [8]. DHPS and DHFS are the last two enzymes in DHF synthesis. Each knockout mutant is FAS− and dependent on exogenous DHF for growth (Fig. 1b). It was surprising that the majority of EHY1 and LCY1 cells could not use FA in place of DHF. However, from both strains, selected mutants (EHY1A and LCY1A) capable of using exogenous FA from the medium were isolated. http://parasites.trends.com
All four mutants were restored to FAS+, by transformation with the DHPS or DHFS gene lacking in the mutants, to enable a comparative study of FAS+ FA utilizers vs non-utilizers. The growth of these strains and the parent YH1 were then compared on various media (Fig. 1c). All strains exhibited the same growth on media without FA, indicating that endogenous folate synthesis provided the essential folate requirements. In the presence of the sulfa drug, sulfamethoxazole (SMX), growth was strongly inhibited when FA was absent. (a)
PABA+DHPPP Sulfa drug
DHPS DHP DHFS
FA
DHF Folate utilization cycle
(b)
DHF FASFA EA
(c)
E
LA
L
0 FAS+
SMX SMX+FA EA
E
Y
LA
L
TRENDS in Parasitology
Fig. 1. Effects of sulfamethoxazole (SMX) and folic acid (FA) on the growth of yeast. (a) Dihydrofolic acid (DHF) synthesis and utilization pathways. DHF could originate from cellular biosynthesis (black) or from exogenous FA (red). Sulfa drugs compete with p-amino benzoate (PABA) leading to loss of DHF. DHF is reduced and modified into several different folate cofactors that are recycled back to DHF. (b) FAS– strains EHY1A (EA), EHY1 (E), LCY1A (LA) and LCY1 (L) do not possess an intact pathway for DHF synthesis. (c) FAS+ derivatives of the mutant strains from (b) were transformed with plasmids p414.YFAS and p414. DHFS, in addition to the parental strain YH1 (Y). Yeast minimal media was used supplemented with uracil, tryptophan and leucine as required, plus DHF (10 µg ml–1), SMX (100 µg ml–1 or FA (10 µg ml–1), as indicated. Full details are in [8]. Abbreviations: DHFS, dihydrofolate synthase; DHP, dihydropteroate; DHPPP, DHP pyrophosphate; DHPS, dihydropteroate synthase; 0, no supplements added.
However, when FA was present, growth was restored to a level resembling that of growth in the absence of SMX. Similar results have been obtained when other sulfa drugs including dapsone and sulfanilamide were used in the place of SMX (data not shown). One conclusion drawn from these data is that, regardless of whether or not a strain can use exogenous FA (the main form found in the diet), there is no noticeable impact on growth or on sulfa drug resistance in the strains. Assuming that this statement also applies to Plasmodium, it would be expected that there is no difference in drug resistance owing to FA utilization phenotype of the strain. The observation that FA alleviates the inhibition of sulfa drugs is not new and has been observed in Plasmodium [6]. However, the theory that the mechanism might be a result of FA utilization now seem unlikely and this raises a major question about how FA exerts this effect. FA is not a PABA analogue and is unlikely to compete directly with sulfa drugs in the manner seen for PABA [9]. It is more likely that competition might occur between the DHP analogues that are produced when sulfa drugs, in place of PABA, are condensed with DHP pyrophosphate (DHPPP) [10–11]. In vitro, such DHP analogues can inhibit DHFS or serine hydroxymethyl transferase activity, and FA might alleviate growth inhibition at one or more of these inhibitory points. However, it has yet to be elucidated whether inhibition at these points occurs in vivo. Possible additional points for FA to alleviate inhibition might be in competition for folate membrane-carriers and folate-binding proteins that exist in animals, but have yet to be proven in yeast. We hope to address these questions, once again employing yeast as model for the study of antifolate resistance. References 1 Wang, P. et al. (1997) Sulfadoxine resistance in the human malaria parasite Plasmodium falciparum is determined by mutations in dihydropteroate synthetase and an additional factor associated with folate utilization. Mol. Microbiol. 23, 979–986
1471-4922/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S1471-4922(01)02202-4
50
Research Update
2 Watkins, W.M. et al. (1985) Antagonism of sulfadoxine and pyrimethamine antimalarial activity in vitro by p-aminobenzoic acid, p-aminobenzoylglutamic acid and folic acid. Mol. Biochem. Parasitol. 14, 55–61 3 Milhous, W.K. et al. (1985) In vitro activities of and mechanisms of resistance to antifol antimalarial drugs. Antimicrob. Agents Chemother. 27, 525–530 4 van Hensbroek, M.B. et al. (1995) Iron, but not folic acid, combined with effective antimalarial therapy promotes haematological recovery in African children after acute falciparum malaria. Trans. R. Soc. Trop. Med. Hyg. 89, 672–676 5 Sims, P. et al. (1998) The efficacy of antifolate antimalarial combinations in Africa. Parasitol. Today 14, 136–137
TRENDS in Parasitology Vol.18 No.2 February 2002
6 Sims, P. et al. (1999) Selection and synergy in Plasmodium falciparum. Parasitol. Today 15, 132–134 7 Watkins, W.M. et al. (1999) More on ‘The efficacy of antifolate antimalarial combinations in Africa’. Parasitol. Today 15, 131–132 8 Bayly, A.M. et al. (2001) Folic acid utilization related to sulfa drug resistance in Saccharomyces cerevisiae. FEMS Microbiol. Letts. 204, 389–390 9 Castelli, L.A. et al. (2001) Sulfa drug screening in yeast: fifteen sulfa drugs compete with p-aminobenzoate in Saccharomyces cerevisiae. FEMS Microbiol. Letts. 199, 181–184 10 Swedberg, G. et al. (1979) Characterization of a mutationally altered dihydropteroate synthase and its ability to form a sulfonamidecontaining dihydrofolate analog. J. Bacteriol. 137, 129–136
11 Roland, S. et al. (1979). The characteristics and significance of sulfonamides as substrates for Escherichia coli dihydropteroate synthase. J. Biol. Chem. 254, 10337–10345
Ann M. Bayly Ian G. Macreadie* Commonwealth Scientific and Industrial Research Organisation (CSIRO), Health Sciences and Nutrition, 343 Royal Parade Parkville 3052 and Royal Melbourne Institute of Technology (RMIT) University, Bundoora, Victoria, Australia. *e-mail: [email protected]
Meeting Report
Schistosomiasis immunology, epidemiology and diagnosis Andreas Ruppel, Malcolm W. Kennedy and John R. Kusel The seventh annual meeting on schistosomiasis organized by Andreas Ruppel was held 18–21 October 2001 at University of Heidelberg, Oberflockenbach, Germany.
The conference brought together scientists with a range of interests in basic, clinical and field research not only from Europe (Czech Republic, France, Germany, Italy, United Kingdom) but also from Oman, Egypt and, in particular, from China. This international group enabled extensive discussion on differences in epidemiology, diagnosis and immunopathology between Schistosoma japonicum and Schistosoma mansoni infections. A fruitful exchange between junior and senior scientists was also evident, in addition to discussions aimed at the development of joint research activities. Immunology and early infections
Michael Kirschfink (University of Heidelberg, Germany) presented an overview of immune evasion strategies (including examples from viruses, bacteria and parasites) and concentrated on evasion or manipulation of the complement system by these pathogens. In early schistosome infections (already at the skin stage), schistosomula (Sla) of S. mansoni induce a transitory thrombocytopaenia perhaps by inducing http://parasites.trends.com
platelet aggregation as a result of blood vessel rupture. In addition, adult schistosomes induce antibodies against host platelets, and the antigens which are responsible for this induction are present in Sla and adult worms, but not in the eggs of S. mansoni. These new insights into thrombocytopaenia could explain why blood clots do not form around adult worms (Ron Stanley, University of Wales, Bangor, UK). The early stages (cercariae and Sla) of S. japonicum activate the complement system not only by the alternative and classical pathways, but also by the mannose-binding lectin (MBL) pathway, and these parasite stages also bind C1q (a component of the complement system) directly to their surface (Wenshi Wang, University of Heidelberg). Cytotoxic complement activity was, however, limited, and serum even promoted the in vitro transformation of S. japonicum cercariae to Sla. The sensitivity of S. japonicum Sla to killing by macrophages was mediated to a large extent by nitric oxide (Xiao-Chun Long, Tongji Medical College, Wuhan, China). A stathmin-like protein was found in the pre- and post-acetabular glands of S. mansoni cercariae and also in the subtegumental layer of mechanically transformed schistosomula (Cristiana Valle, National Research Council, Rome, Italy). Stathmin displays microtubule-destabilizing
activity and this protein is only synthesized in the sporocyst stage just before or during cercarial shedding. Therefore, stathmins could be involved in the rapid changes in membrane formation during skin penetration. The development of S. mansoni in normal mice was compared with mice immunosuppressed with prednisolone three or 21 days after infection ∨ ∨ (Michal Giboda, Ceské Budejovice, Czech Republic). Early, but not late, prednisolone treatment of mice resulted in significantly reduced worm load and female fecundity when compared with non-treated controls. This result suggests a role for the immune system in worm development during the early phase of infection. Vaccination and pathogenesis
The principles of DNA vaccination were outlined by Volker Schirrmacher (German Cancer Research Center, Heidelberg, Germany) using lacZ as a model tumor-associated antigen, and then vaccinating mice with lacZ DNA plasmids or self-replicating RNA. Anti-tumor immunity was enhanced by immunomodulation, particularly by co-injection of cytokine-expressing constructs. The DNA vaccine approach was applied to schistosomes by incorporating the gene encoding the S. mansoni asparaginyl endopeptidase (Sm32) into a DNA vaccine (Katerina Chlichlia,
1471-4922/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S1471-4922(01)02206-1