Parasitol Res (1992) 78 :712-714
Parasitology Research 9 Springer-Verlag1992
Genetic variants of Giardia duodenMis differ in their metabolism* M.L. Hall, N.D. Costa, R.C.A. Thompson, A.J. Lymbery, B.P. Meloni, and R.G. Wales School of Veterinary Studies, Murdoch University,Murdoch,WesternAustralia, Australia 6150 Accepted July 21, 1992
There are numerous reports of differences between isolates of Giardia duodenalis, involving characteristics such as virulence, infectivity, antigenicity and susceptibility to drugs (reviewed in Thompson et al. 1990). In addition, there is evidence for variability in the degree of interspecies transmission by different isolates (reviewed in Woo and Paterson 1986). G. duodenalis isolates from Australia have been shown to be genetically heterogeneous and such heterogeneity occurs both within and between host species (Meloni et al. 1988, 1989). In contrast, electrophoretic studies on Swiss isolates of Giardia from several different hosts have demonstrated great genetic homogeneity (Stranden et al. 1990). Energy metabolism in Giardia has attracted a good deal of interest since the landmark studies on the end products of glucose metabolism reported by Lindmark (1980). The study undertaken by Lindmark (1980) and that of Weinbach et al. (1980) produced conflicting results on the nature of anaerobic respiration and details of oxygen consumption. Most of the metabolic studies have been confined to only one isolate, Portland 1 (American Type Culture Collection, catalogue number 30888), which was originally thought to have been of human origin but is now thought to have been isolated from a cat (Bertram etal. 1983; Meloni et al. 1988). Only Stranden and K6hler (1991) have reported results from metabolic studies in Swiss isolates from different hosts but of similar electrophoretic characteristics (again using Portland 1 as a reference) and they showed a great 9deal of similarity in the production of ethanol and acetate between these isolates. Using [1H]-nuclear magnetic resonance (NMR) spectroscopy to measure the metabolic end products of the Portland 1 isolate during culture, Edwards et al. (1989) reported not only that ethanol and acetate were produced by Giardia but also that alanine
* This work was supported by the National Health and Medical Research Council of Australia. The senior author (M.L.H.) is the recipient of a CommonwealthPostgraduate Research Award Correspondence to: N.D. Costa
increased to concentrations greater than those of either ethanol or acetate. Alanine production was found to follow the pattern of cell growth, whereas ethanol production occurred predominantly in the stationary stage following growth. Acetate appeared to be produced at a constant rate independent of cell growth. In a critical evaluation of the glucose metabolism in Giardia, Schofield et al. (1991) questioned the appropriateness of the glucose concentrations used and, furthermore, the use of glucose itself as a substrate for axenic culture of Giardia isolates. A sound understanding of the basic metabolism of Giardia is a prerequisite for providing a more rational basis for the development of new drugs. In the light of evidence of intraspecific genetic variation in Giardia and the recently demonstrated dangers of making broad generalisations about parasite metabolism from studies on particular laboratory isolates (Bryant and Flockhart 1986), we studied aspects of the energy metabolism of three isolates of G. duodenalis: Portland 1, selected as the reference isolate; BAH 12, a human isolate that is slow-growing relative to Portland 1 ; and BAC 1, another cat isolate whose growth rate is similar to that of Portland 1. Importantly, these isolates have previously been shown to differ not only in their growth but also in their isoenzyme and DNA profiles (Meloni et al. 1988, 1989). The aims of this study were therefore to culture Portland 1 and the two other Giardia isolates over the exponential growth phase to elucidate (1) their acetate, ethanol and alanine production and glucose utilisation and (2) whether variations in genotype and patterns of growth are associated with phenotypic variation in energy metabolism. Trophozoites were cultured in BIS-33 (Meloni and Thompson 1987) supplemented with newborn calf serum (medium 1), or in a serum-free medium (Wieder et al. 1983) in the presence and absence of glucose (media 2 and 3) to ascertain whether glucose is essential for cell growth, In the absence of glucose, osmoregutarlty was maintained by the addition of equal molarities of glutamate and aspartate.
713 When trophozoites were cultured in medium 1, four replicates were set up in 16-ml culture tubes for each time point and measurements were taken every 12 h for a total of 84 h. In serum-free medium (media 2 and 3), cultures were set up in duplicate for each time point and measurements were taken every 24 h. For all cultures, the inoculum contained approximately 3.2 x l0 s trophozoites, giving a final cell concentration of approximately 2.0 x 104 cells/ml. Culture tubes were incubated on a slight incline at 37~ C. Cell counts were done using a haemocytometer after the flask had been chilled in an ice bath for 30 rain to remove the trophozoites from the flask wall and the appropriate dilutions had been made. Trophozoites were then lysed by the addition of 2 ml 15% perchloric acid/10 ml medium, and the acidsoluble and -insoluble fractions were separated by centrifugation (2000 g for 5 rain). Neutralised extracts of the acid-soluble fractions were then analysed for ethanol and acetate using a modification of the method of Beutler (1984a, b) and for L-alanine by a modification of the method of Williamson (1984). Both the acid-soluble and the acid-insoluble fractions were assayed for glucose and glycogen (Keppler and Decker 1984). Glycogen content was recorded as the difference between the total glucose following treatment with amyloglycosidase and the original free glucose in the sample. Cell numbers of the Portland 1 and BAC I isolates increased exponentially until confluent monolayers of cells were present, whereas the BAH 12 isolate never formed confluent monolayers of cells. Linear regression analyses of log-transformed cell numbers as a function of time, i.e. from the initiation of cultures until peak cell numbers were reached, explained 91.2% of the variance in cell numbers for Portland 1, 87.7% of that for BAC i and 79.9% of that for BAH 12. Analyses of covariance indicated heterogeneity between slopes (F= 26.81, P < 0.001). At the 0.05 probability level, no difference was found in the slope of the growth curves generated for Portland 1 and BAC 1, but the slopes for both of these isolates differed from the slope of the growth curve for BAH 12. After 72 h of culture, the net ethanol production for the Portland 1, BAC 1, and BAH 12 isolates was 0.64_+0.16, 1.78___0.45 and 1.10+0.16 raM, respectively. The net acetate production was 1.46 + 0.14, 1.15 + 0.14 and 0.95 _ 0.10 mM, respectively, whereas the corresponding net alanine production was 10.96_+ 0.86, 12.89 + 0.80 and 2.72_+ 1.06 raM. The glucose equivalent of the combined end products producted by the Portland 1, BAC 1, and BAH 12 isolates was 6.53, 7.91 and 2.33 raM, respectively, and was extremely low in comparison with the concentration of glucose and glucoseequivalent glycogen. For quantification of the end products produced during growth and subsequent comparison of the end products and growth of the different isolates, the net end product formed was divided by the mean number of cells present over the 72 h of growth necessary for peak cell numbers to be achieved by the faster-growing isolates. The acetate, ethanol and alanine production per million cells for the three isolates is shown in Table 1. No significant difference in ethanol production was
Table 1. Mean concentration of end products produced by three isolates of Giardia duodenalis Isolate
Portland 1 BAC 1 BAH 12
End products (gmol/106 cells) Ethanol
Acetate
Alanine
0.93_+0.23 2.64_+0.67 17.04_+2.45"
2.10_+0.21 1.73_+0.22 14.75_+1.50"
15.78_+ 1.25 19.38_+ 1.2t 42.24_+16.50
Data represent mean values_+SEM for 4 replicates * Significantlydifferentfrom the other two values at P < 0.001 Table 2. End products produced by the mean number of cells present between inoculation and peak growth (48 h for Portland I and BAC 1, 96 h for BAH 12) in serum-freemedia in the presence and absence of glucose Isolate
Portland 1 BAC 1 BAH 12
End products (j.tmol/106cells) Presence of glucose Absence of glucose Ethanol
Acetate
Ethanol
Acetate
1.31 5.38 8.13
0 0.23 0.46
4.18 0 19.55
0.41 0.83 4.95
Data represent average values for duplicates
found between the Portland 1 and BAC 1 isolates, but both produced significantly less ethanol than did BAH 12. Similarly, no significant difference in acetate production was found between the Portland I and BAC 1 isolates, whereas both of these isolates produced significantly less (P
Parasitology Research 9 Springer-Verlag1992
Genetic variants of Giardia duodenMis differ in their metabolism* M.L. Hall, N.D. Costa, R.C.A. Thompson, A.J. Lymbery, B.P. Meloni, and R.G. Wales School of Veterinary Studies, Murdoch University,Murdoch,WesternAustralia, Australia 6150 Accepted July 21, 1992
There are numerous reports of differences between isolates of Giardia duodenalis, involving characteristics such as virulence, infectivity, antigenicity and susceptibility to drugs (reviewed in Thompson et al. 1990). In addition, there is evidence for variability in the degree of interspecies transmission by different isolates (reviewed in Woo and Paterson 1986). G. duodenalis isolates from Australia have been shown to be genetically heterogeneous and such heterogeneity occurs both within and between host species (Meloni et al. 1988, 1989). In contrast, electrophoretic studies on Swiss isolates of Giardia from several different hosts have demonstrated great genetic homogeneity (Stranden et al. 1990). Energy metabolism in Giardia has attracted a good deal of interest since the landmark studies on the end products of glucose metabolism reported by Lindmark (1980). The study undertaken by Lindmark (1980) and that of Weinbach et al. (1980) produced conflicting results on the nature of anaerobic respiration and details of oxygen consumption. Most of the metabolic studies have been confined to only one isolate, Portland 1 (American Type Culture Collection, catalogue number 30888), which was originally thought to have been of human origin but is now thought to have been isolated from a cat (Bertram etal. 1983; Meloni et al. 1988). Only Stranden and K6hler (1991) have reported results from metabolic studies in Swiss isolates from different hosts but of similar electrophoretic characteristics (again using Portland 1 as a reference) and they showed a great 9deal of similarity in the production of ethanol and acetate between these isolates. Using [1H]-nuclear magnetic resonance (NMR) spectroscopy to measure the metabolic end products of the Portland 1 isolate during culture, Edwards et al. (1989) reported not only that ethanol and acetate were produced by Giardia but also that alanine
* This work was supported by the National Health and Medical Research Council of Australia. The senior author (M.L.H.) is the recipient of a CommonwealthPostgraduate Research Award Correspondence to: N.D. Costa
increased to concentrations greater than those of either ethanol or acetate. Alanine production was found to follow the pattern of cell growth, whereas ethanol production occurred predominantly in the stationary stage following growth. Acetate appeared to be produced at a constant rate independent of cell growth. In a critical evaluation of the glucose metabolism in Giardia, Schofield et al. (1991) questioned the appropriateness of the glucose concentrations used and, furthermore, the use of glucose itself as a substrate for axenic culture of Giardia isolates. A sound understanding of the basic metabolism of Giardia is a prerequisite for providing a more rational basis for the development of new drugs. In the light of evidence of intraspecific genetic variation in Giardia and the recently demonstrated dangers of making broad generalisations about parasite metabolism from studies on particular laboratory isolates (Bryant and Flockhart 1986), we studied aspects of the energy metabolism of three isolates of G. duodenalis: Portland 1, selected as the reference isolate; BAH 12, a human isolate that is slow-growing relative to Portland 1 ; and BAC 1, another cat isolate whose growth rate is similar to that of Portland 1. Importantly, these isolates have previously been shown to differ not only in their growth but also in their isoenzyme and DNA profiles (Meloni et al. 1988, 1989). The aims of this study were therefore to culture Portland 1 and the two other Giardia isolates over the exponential growth phase to elucidate (1) their acetate, ethanol and alanine production and glucose utilisation and (2) whether variations in genotype and patterns of growth are associated with phenotypic variation in energy metabolism. Trophozoites were cultured in BIS-33 (Meloni and Thompson 1987) supplemented with newborn calf serum (medium 1), or in a serum-free medium (Wieder et al. 1983) in the presence and absence of glucose (media 2 and 3) to ascertain whether glucose is essential for cell growth, In the absence of glucose, osmoregutarlty was maintained by the addition of equal molarities of glutamate and aspartate.
713 When trophozoites were cultured in medium 1, four replicates were set up in 16-ml culture tubes for each time point and measurements were taken every 12 h for a total of 84 h. In serum-free medium (media 2 and 3), cultures were set up in duplicate for each time point and measurements were taken every 24 h. For all cultures, the inoculum contained approximately 3.2 x l0 s trophozoites, giving a final cell concentration of approximately 2.0 x 104 cells/ml. Culture tubes were incubated on a slight incline at 37~ C. Cell counts were done using a haemocytometer after the flask had been chilled in an ice bath for 30 rain to remove the trophozoites from the flask wall and the appropriate dilutions had been made. Trophozoites were then lysed by the addition of 2 ml 15% perchloric acid/10 ml medium, and the acidsoluble and -insoluble fractions were separated by centrifugation (2000 g for 5 rain). Neutralised extracts of the acid-soluble fractions were then analysed for ethanol and acetate using a modification of the method of Beutler (1984a, b) and for L-alanine by a modification of the method of Williamson (1984). Both the acid-soluble and the acid-insoluble fractions were assayed for glucose and glycogen (Keppler and Decker 1984). Glycogen content was recorded as the difference between the total glucose following treatment with amyloglycosidase and the original free glucose in the sample. Cell numbers of the Portland 1 and BAC I isolates increased exponentially until confluent monolayers of cells were present, whereas the BAH 12 isolate never formed confluent monolayers of cells. Linear regression analyses of log-transformed cell numbers as a function of time, i.e. from the initiation of cultures until peak cell numbers were reached, explained 91.2% of the variance in cell numbers for Portland 1, 87.7% of that for BAC i and 79.9% of that for BAH 12. Analyses of covariance indicated heterogeneity between slopes (F= 26.81, P < 0.001). At the 0.05 probability level, no difference was found in the slope of the growth curves generated for Portland 1 and BAC 1, but the slopes for both of these isolates differed from the slope of the growth curve for BAH 12. After 72 h of culture, the net ethanol production for the Portland 1, BAC 1, and BAH 12 isolates was 0.64_+0.16, 1.78___0.45 and 1.10+0.16 raM, respectively. The net acetate production was 1.46 + 0.14, 1.15 + 0.14 and 0.95 _ 0.10 mM, respectively, whereas the corresponding net alanine production was 10.96_+ 0.86, 12.89 + 0.80 and 2.72_+ 1.06 raM. The glucose equivalent of the combined end products producted by the Portland 1, BAC 1, and BAH 12 isolates was 6.53, 7.91 and 2.33 raM, respectively, and was extremely low in comparison with the concentration of glucose and glucoseequivalent glycogen. For quantification of the end products produced during growth and subsequent comparison of the end products and growth of the different isolates, the net end product formed was divided by the mean number of cells present over the 72 h of growth necessary for peak cell numbers to be achieved by the faster-growing isolates. The acetate, ethanol and alanine production per million cells for the three isolates is shown in Table 1. No significant difference in ethanol production was
Table 1. Mean concentration of end products produced by three isolates of Giardia duodenalis Isolate
Portland 1 BAC 1 BAH 12
End products (gmol/106 cells) Ethanol
Acetate
Alanine
0.93_+0.23 2.64_+0.67 17.04_+2.45"
2.10_+0.21 1.73_+0.22 14.75_+1.50"
15.78_+ 1.25 19.38_+ 1.2t 42.24_+16.50
Data represent mean values_+SEM for 4 replicates * Significantlydifferentfrom the other two values at P < 0.001 Table 2. End products produced by the mean number of cells present between inoculation and peak growth (48 h for Portland I and BAC 1, 96 h for BAH 12) in serum-freemedia in the presence and absence of glucose Isolate
Portland 1 BAC 1 BAH 12
End products (j.tmol/106cells) Presence of glucose Absence of glucose Ethanol
Acetate
Ethanol
Acetate
1.31 5.38 8.13
0 0.23 0.46
4.18 0 19.55
0.41 0.83 4.95
Data represent average values for duplicates
found between the Portland 1 and BAC 1 isolates, but both produced significantly less ethanol than did BAH 12. Similarly, no significant difference in acetate production was found between the Portland I and BAC 1 isolates, whereas both of these isolates produced significantly less (P
