Division of Biological Sciences, University of Missouri, Columbia, MO 65211-7400, USA1
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK2
Laboratory for Molecular Cell Biology, UCL, Gower Street, London WC1E 6BT, UK3
Author for correspondence: Robert H. Insall. Tel: +44 121 414 2507. Fax: +44 121 414 3982. e-mail: r.h.insall{at}bham.ac.uk
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ABSTRACT |
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Keywords: alcohol metabolism, methanol, xenobiotics, prodrug, isoniazid
Abbreviations: REMI, restriction enzyme mediated integration
The GenBank accession number for the sequence reported in this paper is AF090443.
a Ma. X. U. Garcia and C. Roberts contributed equally to this work.
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INTRODUCTION |
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These drug-resistance mutations have been essential in genetic mapping in Dictyostelium (Alexander et al., 1986 ; Welker & Deering, 1976
) and in integrating the genetic and physical maps of the Dictyostelium linkage groups (Loomis et al., 1995
). However, the structural gene encoded by acrA has remained unknown for the past 25 years. In this study, an insertional mutagenesis screen for the genes involved in methanol resistance has identified the Dictyostelium catalase A (catA) gene as acrA. We present genetic and biochemical evidence that a deficiency in CatA enzyme activity causes resistance to both acriflavine and methanol.
Catalases are ubiquitous, highly active enzymes which break down H2O2 to water and oxygen, and thus protect against cellular damage caused by peroxides. Dictyostelium has been shown to contain two catalases one, the product of the catA gene, is active in growing cells, whereas the other, encoded by catB, is expressed late in development (Garcia et al., 2000 ). CatA would therefore appear to be the standard metabolic enzyme, whereas CatB is predicted to have an as yet unknown role in development. Until the present work, no gene disruptants of catA had been isolated. However, a mutant which coincidentally expresses low levels of catA was found to be highly sensitive to exogenous H2O2, though normally resistant to UV light (Garcia et al., 2000
).
This work exposes the biochemical pathway underlying the toxic effects of methanol and provides an intriguing insight into the mechanism of acriflavine toxicity. Both pathways are centred on catalase.
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METHODS |
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Identification of mutations.
Genomic DNA was prepared from the acrA2 mutant XP210 as described by Sun & Devreotes (1991). This DNA was then used as a template for PCR reactions using a range of primer pairs covering the entire acrA gene. Initial sequencing identified a deletion at approximately 580 bp (numbered according to the GenBank entry no. AF090443). This was then precisely localized using a second PCR reaction, with primers from 141 to 157 bp and 1099 to 1083 bp and a sequencing primer from 704 to 690 bp (5'-GGCAGCTTCTTCAGC-3'). Sequences were aligned using MacVector (Oxford Molecular).
Assays for resistance to methanol and acriflavine.
Plates containing different concentrations of alcohols or acriflavine were prepared by adding appropriate additives to molten SM agar just before it began to set. Methanol plates were stored in separate boxes from others, as methanol vapour was found to cause significant toxicity to cells in surrounding plates. Set plates were spread with lawns of Klebsiella, then 10 µl drops containing approximately 104 Dictyostelium cells were spotted in the middle of each plate. After 5 d incubation at 22 °C, plates were examined and scored for cell growth.
Southern and Northern analyses.
Southern and Northern analyses were performed as described previously (Lee et al., 1996 ). The catA probe was prepared by excising the full-length catA cDNA as a 1·5 kb SalINotI fragment from plasmid FC-AH16 (Garcia et al., 2000
).
Catalase activity assays.
Vegetatively growing cells were harvested, washed with LPS buffer (20 mM KCl, 2·5 mM MgCl2, 40 mM potassium phosphate, pH 6·5, containing 0·5 mg streptomycin sulfate ml-1) and pelleted. The cell pellets were lysed in lysis buffer [10 mM potassium phosphate, pH 7·0, 0·1% Triton X-100, 1xprotease inhibitor cocktail (100x=20 mM AEBSF, 100 mg pepstatin A ml-1, 10 mg leupeptin ml-1)] and centrifuged for 3 min at 4 °C. Samples of 1·25 and 2·5 µl of the supernatants were then assayed for catalase activity by mixing with 10 mM H2O2 in 50 mM potassium phosphate, pH 7·0, and monitoring the degradation of H2O2 for 90 s (Garcia et al., 2000 ). Specific catalase activity was calculated as µmol H2O2 degraded min-1 (mg protein)-1. Catalase activities of the mutants were expressed as the percentage of the activities of the parental strains. Protein concentrations were determined using BCA reagent (Pierce).
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RESULTS |
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We first tested whether the disruption mutant was resistant to acriflavine. Wild-type (AX2) and catA mutant (IR29) cells were plated on SM agar plates containing increasing concentrations of methanol, ethanol or acriflavine. The results in Table 1 clearly show that the catA mutant indeed exhibits cross-resistance to acriflavine. This suggests, unexpectedly, that acriflavine must be oxidized by a catalase-dependent pathway for it to show significant toxicity to Dictyostelium.
In some organisms, in particular the mouse, catalases can play a major part in the breakdown of ethanol as well as methanol. There is only a very slight difference in the sensitivity of wild-type and catA mutant to ethanol (Table 1). This could imply either that the CatA catalase has very little activity against ethanol or that the products of ethanol oxidation are far less toxic to Dictyostelium than those from methanol.
Phenotypes of catA disruption mutants
None of the catA-deficient strains described in this work, in particular the disruptants IR29, IR40 and IR41, showed any obvious mutant phenotype during normal laboratory growth and development. Each strain grew at approximately wild-type rates and to wild-type cell densities. When starved, each strain developed at a normal rate, eventually producing apparently normal fruiting bodies. It therefore appears that catalase is not important for growth or development under normal laboratory conditions.
Previous work (Garcia et al., 2000 ) showed that strain X9 coincidentally expresses unusually low (
3%), but measurable, levels of catalase A (Madigan & Katz, 1989
). X9 cells were also found to be hypersensitive to H2O2. As shown in Fig. 3
, the catA null strain IR29 is extremely sensitive to H2O2 even more so than Garcia et al. (2000)
observed for X9 consistent with a more severe phenotype. At low concentrations of H2O2, which barely affect the parental strain, IR29 cells are nearly all killed (
90% at 0·1 mM;
99·9% at 0·3 mM). However, a significant but small number of IR29 cells survived treatment with 1 mM H2O2, which killed 99·9% of parental cells (Fig. 3
). This appears to be due to the stochastic nature of death by oxidative damage, rather than reversion, as the survivors are still sensitive to H2O2 when regrown (data not shown).
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A previously described acrA mutant contains a lesion in catA
As reported previously, sequencing of the catA gene in an acrA1 strain revealed no changes in the coding sequence. Together with the low level of expression, this suggests that the defect in acrA1 cells affects the regulation of expression (Garcia et al., 2000 ). We therefore isolated and sequenced various fragments of the catA gene from strain XP210, which contains the acrA2 allele. As shown in Fig. 5
, the sequencing data show that catA has a 13 bp deletion within the coding sequence, between positions 573 and 585 (numbered according to the GenBank entry for catA, accession no. AF090443). The effect of this mutation will be to introduce a frameshift into the coding sequence, in addition to the removal of 4 aa. The identification of this lesion independently confirms the connection between catA and the original acrA gene. Since all known mutations which confer methanol resistance have mapped to the same locus, this suggests that the catalase which is the product of catA is necessary and sufficient for cellular toxicity of methanol.
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DISCUSSION |
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Dictyostelium has two catalase genes, catA and catB, which are differentially expressed during growth and development. CatA enzyme activity is present in peroxisomes of growing cells and is specific to prestalk cells during late development while CatB enzyme activity is only expressed in prespore cells during late development (Garcia et al., 2000 ). Catalases are antioxidant enzymes that detoxify H2O2 in the cell by breaking it down to H2O and O2. The importance of this activity is emphasized by the high sensitivity of a mutant with diminished catA levels to treatment with exogenous H2O2 (Garcia et al., 2000
; Madigan & Katz, 1989
).
Methanol itself is essentially non-toxic but gets oxidized to formaldehyde and then to formic acid (Fig. 6) which are both highly toxic and reactive metabolites (Kruse, 1992
). In mammals, most of the toxicity of methanol is attributed to formic acid, which is a potent inhibitor of cytochrome oxidase, thereby causing histotoxic hypoxia and metabolic acidosis (Liesivuori & Savolainen, 1991
; Tephly, 1991
). Indeed, administering formic acid alone can mimic methanol toxicity in animal models (Martin-Amat et al., 1978
). Thus, the pathways that regulate the relative rates of formic acid generation and formic acid oxidation to CO2 greatly determine the toxic effects of methanol in different species. Our work shows that this is also the case for Dictyostelium.
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The next metabolic step is the rapid oxidation of formaldehyde to formic acid, which is catalysed primarily by formaldehyde dehydrogenase in all species studied (Liesivuori & Savolainen, 1991 ), although there is evidence that catalase can also catalyse this reaction (Tephly, 1991
; van Dijken et al., 1982
; Veenhuis et al., 1983
). Formaldehyde itself is potentially toxic, but because there are multiple pathways for its oxidation, it does not accumulate and directly exert a toxic effect in mammals (Kavet & Nauss, 1990
).
The identification of the Dictyostelium catA gene as the acrA locus indicates that CatA enzyme activity is overwhelmingly the major enzyme in the metabolism of methanol in this organism. Despite many years of screening, no methanol-resistant mutants have been found outside the acrA locus and deletion of catA allows cells to survive about fivefold higher levels of methanol than wild-type, so it is unlikely that any other enzymes are contributing significantly to methanol breakdown. Thus, the lack of CatA activity in the methanol-resistant mutants enables them to survive the presence of methanol by preventing the formation of formaldehyde and/or formic acid (Fig. 6)
While the importance of catalase peroxidase activity in methanol metabolism has been previously recognized in other systems, the present study is the first to demonstrate a link between catalase activity and acriflavine resistance. Genes associated with acriflavine resistance have been identified in different organisms. Molecular cloning of these genes revealed that many of them encode multidrug resistance pumps that transport acriflavine out of the cell (De Rossi et al., 1998 ; Masaoka et al., 2000
; Nakaune et al., 1998
; Pereira et al., 1998
). Inactivating mutations in these transporter genes lead to acriflavine sensitivity (Ma et al., 1994
). Conversely, mutations that lead to their overexpression or to an increased efflux activity lead to acriflavine resistance (Andrade et al., 2000
; Klyachko & Neyfakh, 1998
; Nakaune et al., 1998
). The observation that mutations in Dictyostelium catA lead to acriflavine resistance represents a novel mechanism for resistance to this drug. It is possible that the peroxidase activity of CatA is involved in a pathway which modifies acriflavine to produce a cytotoxic form. Such a role for catalase has been demonstrated for the antituberculosis drug isoniazid, an antibiotic used against Mycobacterium tuberculosis infections. Recent work has indicated that isoniazid is a prodrug that has to be activated in vivo by the mycobacterial catalase-peroxidase KatG to form a reactive intermediate that can interact with its cellular targets (Johnsson & Schultz, 1994
; Rozwarski et al., 1998
). Loss-of-function mutations in KatG are thus sufficient to confer isoniazid resistance and are the most common mechanism for resistance in clinical isolates (Miesel et al., 1998
; Zhang et al., 1992
).
We predict that the other loci involved in acriflavine resistance, acrB and acrC, also encode enzymes and that together with catA they are responsible for the metabolism of acriflavine to its final, biologically active form. It will be interesting to test this hypothesis by isolating REMI mutations in acrB and acrC and cloning their genes. This should allow the complete metabolic pathway to be mapped out. It is also worth noting that acrA allows a strong negative selection, which is sufficiently powerful to allow isolation of mutants without the use of mutagens and measurement of basal mutation levels during normal growth (Loomis, 1987 ). We therefore anticipate that the molecular identification of the gene will allow far more versatile genetic selections than are currently available. For example, an extrachromosomal plasmid containing an antibiotic selection marker and catA could be selected positively with antibiotic and negatively by growth on methanol. Such experimental tools will become invaluable as the sequencing projects reveal the full complexity of the Dictyostelium genome.
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ACKNOWLEDGEMENTS |
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Received 17 August 2001;
revised 25 September 2001;
accepted 2 October 2001.
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