Metabolic Changes in Giardia intestinalis during Differentiation

Abstract

The oxygen uptake rate and metronidazole (MTZ) sensitivity in Giardia spp. cysts is greatly reduced from that in trophozoites. Thus, this project was undertaken to assess when in the encystation process these phenomena occur. Oxygen uptake rates approximately doubled (from approximately 4.9 to 8.3 microM O2 min(-1) 10(-6) cells) during the first 5 hr into encystation. This increase was followed by a marked decrease to 2.3 microM O2 min(-1) 10(-6) by 12 hr. By 50 hr into encystation, oxygen uptake was 0.7 microM O2 min(-1) 10(-6) cells. Glucose stimulated oxygen uptake by 89% in trophozoites but did not demonstrably stimulate oxygen uptake in cells after 12 hr into encystment. Deoxy-D-glucose uptake dropped by more than an order of magnitude in encysting cells compared to nonencysting cells. In contrast, aspartate uptake remained relatively constant regardless of whether cells were encysting or not. This suggests that there is a change in the parasite's ability to transport glucose during cyst formation; a similar change in the parasite's ability to transport aspartate was not observed after 40 hr into encystation. MTZ inhibited oxygen uptake by 77% in trophozoites, but there was no detectable inhibition of oxygen uptake 8 hr after trophozoites were transferred to encystation medium. We propose that this resistance to MTZ may be due to a change in metabolic flux away from the pyruvate ferredoxin oxidoreductase pathway. Oxygen uptake by noninduced cysts increased exponentially during the 30 min following the induction of excystation. Likewise, MTZ sensitivity returned within 15 min after the induction of excystation, and by 30 min into excystation full sensitivity had returned.

Menadione kills trophozoites and cysts of Giardia intestinalis

“…Although metronidazole is one of the most potent antigiardial agents currently in use (Breccia et al, 1982;Ellis et al, 1993b) its toxicity towards G. intestinalis cysts is somewhat limited (Paget et al, 1993(Paget et al, , 1998. Therefore, a comparison between the anti-giardial efficacies of the nitroimidazole (metronidazole) and menadione against both developmental stages of the organism was conducted.…”

“…Organisms were counted on a Fuchs-Rosenthal haemocytometer slide; typically this gave cell numbers of 2610 6 ml 21 after 2 days growth in stoppered tubes. Viability of trophozoites was assessed by motility and of cysts by their capacity for excystation (Bingham & Meyer, 1979;Paget et al, 1998).…”

Stage-Specific Expression and Targeting of Cyst Wall Protein–Green Fluorescent Protein Chimeras inGiardia

“…Several genes that are up-regulated during Giardia encystation have been identified recently by biochemical approaches, molecular genetics, or metabolic studies (Ellis et al, 1996;Que et al, 1996;Steimle et al, 1997;Bulik et al, 1998;Paget et al, 1998;Van Keulen et al, 1998;Knodler et al, 1999). Among these, cyst wall protein (CWP) 1 and CWP2 constitute major components of an extracellular cyst wall (Lujan et al, 1995b;Mowatt et al, 1995), the function of which has not been determined.…”

“…Formation of environmentally resistant cysts is a key step in Giardia development as in other eukaryotic pathogens (Eichinger, 1997;Taratuto and Venturiello, 1997;Dubey et al, 1998) that has not been well characterized on a molecular level. Therefore, a better understanding of the mechanisms that govern stage-specific regulation of genes and the synthesis, transport, and assembly of the cyst wall is needed.Several genes that are up-regulated during Giardia encystation have been identified recently by biochemical approaches, molecular genetics, or metabolic studies (Ellis et al, 1996;Que et al, 1996;Steimle et al, 1997;Bulik et al, 1998;Paget et al, 1998;Van Keulen et al, 1998;Knodler et al, 1999). Among these, cyst wall protein (CWP) 1 and CWP2 constitute major components of an extracellular cyst wall (Lujan et al, 1995b;Mowatt et al, 1995), the function of which has not been determined.…”

Síntesis de las enzimas de piruvato, oxidorreductasa de ferredoxina y deshidrogenasa E de alcohol durante el desenquistamiento (excystacion) de Giardia intestinalis

“…However, little is known about this stage, especially about the molecular processes involved in its regulation. Paget et al (1998) (8) demonstrated that when excystation is initiated, an increase in oxygen uptake occurs; this increases exponentially during the following 30 minutes and is linked to an increase in metabolic rate. Are the associated enzymes synthesized de novo during excystation or are these enzymes carried over from the trophozoite stage?…”