Asexual blood stage | Growth of asexual blood stages is normal in the presence of pABA in diet of mice. In milk-fed mice adcs- parasite propagation was severely impaired, whereas WT parasites displayed normal growth. Upon pABA supplementation via the drinking water, adcs- growth was fully restored, and mutant parasites grew indistinguishable from WT in milk-fed mice.
Continued pABA restriction led to the retraction of adcs- parasitemia from day 12 onward, which was immediately reverted upon addition of pABA on day 18. During more extended periods of pABA deficiency, adcs- parasitemia dropped below the detection threshold, followed by alternating periods of re-emergence and retraction, indicative of latent malaria infection. |
Additional remarks phenotype | Mutant/mutation
The mutant lacks expression of ACDS and expresses GFP under the constitutive hsp70 promoter. In addition the selectable marker cassette has been removed by negative selection (see below).
Protein (function)
Malaria parasites replicate at high rates in the mammalian bloodstream. Consequently, they require large quantities of folate and folate precursors, which they scavenge from the serum or synthesize de novo. In contrast, the mammalian host strictly depends on the dietary uptake of preformed folates. Folates promote essential functions during one-carbon transfer reactions and consist of three major moieties: para-aminobenzoate (pABA), pterin, and glutamate.It is believed that chorismate, which is produced via the shikimate pathway, is converted to pABA in a two-step reaction. A potential aminodeoxychorismate synthase (ADCS, PBANKA_0823300) and an aminodeoxychorismate lyase (ADCL, PBANKA_1322100) are predicted by Plasmodium genome sequences.
From the paper: 'We show that the parasite depends on de novo folate synthesis only when dietary intake of pABA by the mammalian host is restricted and that only pABA, rather than fully formed folate, is taken up efficiently. This adaptation, which readily adjusts infection to highly variable pABA levels in the mammalian diet, is specific to blood stages and may have evolved to avoid folate competition between the parasite and its host.'
Phenotype
Growth of asexual blood stages is normal in the presence of pABA in diet of mice. In milk-fed mice adcs- parasite propagation was severely impaired, whereas WT parasites displayed normal growth. Upon pABA supplementation via the drinking water, adcs- growth was fully restored, and mutant parasites grew indistinguishable from WT in milk-fed mice.
Continued pABA restriction led to the retraction of adcs- parasitemia from day 12 onward, which was immediately reverted upon addition of pABA on day 18. During more extended periods of pABA deficiency, adcs- parasitemia dropped below the detection threshold, followed by alternating periods of re-emergence and retraction, indicative of latent malaria infection.
Additional information
Transgenic parasite lines expressing the endogenous acds and adcl genes (RMgm-4596, RMgm-4597) fused to mCherry-3xMyc were generated and analyzed by live fluorescence microscopy. Co-localization with a cytosolic GFP marker revealed that both enzymes localize to the parasite cytoplasm. ADCS expression was restricted to blood stage trophozoites and liver stages, but remained below the detection limit in rings, in schizonts, and throughout mosquito infection. This finding indicates distinct roles for ADCS in the mammalian host only. In contrast, ADCL was ubiquitously expressed during life cycle progression.
'We also compared blood infection of transgenic parasites lacking the second pABA synthesis enzyme, ADCL, in mice receiving different diets. adcl- parasites (RMgm-4595) also depend on nutritional pABA, demonstrating that both enzymes, ADCS and ADCL, promote pABA biogenesis during blood infection. The ubiquitous expression of ADCL suggests additional functions, especially during mosquito stages. A previous study demonstrated a substantial secondary aminotransferase activity of Plasmodium falciparum ADCL, which was suggested as contributing to the detoxification of exogenous D-amino acids'
Other mutants |