Arginine metabolism has a pivotal function for the encystation of Giardia duodenalis

TL;DR

Giardia parasites use arginine metabolism for energy, and a specific enzyme variant in human-specific parasites reduces their ability to form infectious cysts.

Contribution

The study identifies amino acid changes in ADI that affect Giardia's encystation efficiency, linking enzyme function to parasite life cycle progression.

Findings

ADI from human-specific Giardia AII has ~5-fold lower arginine affinity than zoonotic genotypes.

ADI is essential for efficient encystation, and its reduced activity in AII correlates with lower cyst formation.

Arginine is required for encystation, and ADI mediates this dependency.

Abstract

Arginine metabolism plays a key role in the energy metabolism of the intestinal parasite Giardia duodenalis, an amitochondrial protozoan that infects humans and animals and causes significant morbidity. Despite that an arginine deiminase (ADI) has been implicated in virulence, it remains unknown if ADI allele variants from the different genetic G. duodenalis subgroups (assemblages) differ in function. Here, the hypothesis was tested that sequence variation detected between G. duodenalis ADI alleles from the two G. duodenalis assemblage types found in humans affects functional parameters of the enzyme with potential consequences in life cycle progression. The ADI enzyme’s affinity for arginine was ~ 5fold reduced in sub-assemblage AII isolates, a human specific assemblage, in comparison to zoonotic sub-assemblage AI and B isolates. We identified the two amino acid residues responsible for the lower substrate affinity of ADIAII variant. By combining genetic ADI-knockout mutants, biochemical assays of substrate affinity, and cellular analyses of life-cycle progression, we demonstrate that ADI is required for efficient parasite encystation and that the lower substrate affinity in ADIAII correlates with reduced encystation efficiency. We further demonstrate that arginine is required for efficient encystation, and use an ADI knockout strain to confirm that ADI mediates this arginine dependence. Thus, we suggest that ADI is a quantitative trait that affects life cycle progression of G. duodenalis with putative clinical and epidemiological relevance.

In the human pathogenic parasite Giardia duodenalis, arginine deiminase (ADI) mediates the first step in the arginine dihydrolase pathway (ADH), metabolizing arginine to provide chemical energy in form of ATP. The bacterial-derived ADH pathway had been inherited by horizontal gene transfer, and ADI has been proposed as a virulence factor. Combining biochemical and genetic approaches with ADI knock-out mutants, we show here that arginine and its metabolizing enzyme ADI are essential for efficient life cycle progression (encystation) to form infectious cysts. Furthermore, we show a drastically impaired arginine substrate affinity for the human-specific G. duodenalis genotype AII in comparison to the zoonotic genotypes AI and B and identified the molecular entities responsible for this altered substrate affinity. This lower substrate affinity also correlated with lower cyst formation in vitro by the AII genotype.

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