Consequences of in vitro hyperoxia on Plasmodium falciparum tolerance to artemisinin

Abstract

Plasmodium falciparum is the major causative agent of malaria in man and is responsible for over 400,000 deaths per year, predominantly in Sub-Saharan Africa. P. falciparum exists in many forms within both the human host and mosquito vector. In clinical malaria the parasite resides within the red blood cells of the human host and is transmitted from one host to another through the blood feeding bite of the female Anopheles mosquito. Malaria, as a disease, is completely curable, and many countries have eradicated malaria, including Australia in 1981. However, malaria remains endemic in most poor countries. Overall, both case numbers and mortality rates are decreasing each year, primarily due to improved preventative measures, and importantly the use of artemisinin combination therapies (ACT) employed globally. The chink in the armour of chemotherapeutics currently available for the treatment of malaria is parasite drug resistance. Artemisinin was believed to be the exception, as stable in vitro generated artemisinin resistant parasites proved difficult to obtain. However, in 2007, clinical parasite delayed clearance times were observed for the artemisinin derivative, artesunate, used as a monotherapy in Western Cambodia, the epicentre of anti-malarial drug resistance origins. Standard laboratory drug sensitivity tests did not detect differences in parasite sensitivity between those from patients with or without delayed parasite clearance times. The use of artemisinin derivatives as a combination therapy was advocated, but underlying artemisinin tolerance, or partial resistance originating in Western Cambodia, has resulted in the acquisition of partner drug resistance and ultimately failure of standard 3-day ACT treatment regimens. A single nucleotide polymorphism in a kelch-13 (K13) propeller domain (PF3D7_1343700) was identified through escalating artemisinin drug exposure and recovery in vitro, correlating with extended in vivo parasite clearance times, providing a reliable molecular marker of artemisinin tolerance. This singular study, resulting in the parasite strain F32-ART5, is the only in vitro artemisinin drug resistance study to acquire a K13 mutation (M476I), which in itself was intriguing. Based on the validated scientific foundation that in vitro culturing conditions for P. falciparum can have experimental consequences, the culturing conditions involved in the generation of F32-ART5, and that of several other studies, were reviewed. A significant difference in oxygen levels between the K13 mutant parasite F32-ART5 culturing conditions and other studies was identified. F32-ART5 was generated under hyperoxic conditions (21%O2), known to cause oxidative stress in P. falciparum, whilst other studies employed lower O2 levels of 1-5%. As well as K13 mutations, a parasite “genetic background” with enhanced antioxidative stress, repair and survival signatures is associated with extended parasite clearance times and acquisition of K13 mutations in founder populations of P. falciparum in Western Cambodia. The coincidence of a K13 mutation acquired in hyperoxic or stressed conditions (F32-ART5), and the relationship between anti-oxidative stress capabilities with K13 mutations in vivo, appeared to be not so coincidental when viewed together. Based on these two observations, it was hypothesised that hyperoxia would cause enhanced tolerance to artemisinin-based drugs, such as dihydroartemisinin. To test this hypothesis, a recently developed in vitro assay was employed, which identifies dihydroartemisinin tolerance of K13 mutant parasites and is referred to as the ring stage survival assay, or RSA. In this study, the effect of hyperoxic exposure on twelve P. falciparum strains comprising those with naturally acquired K13 mutations, genetically engineered K13 parasite strains, K13 wild type clinical isolates and laboratory strains, were evaluated for dihydroartemisinin tolerance and growth characteristics in response to hyperoxic stimuli. To effectively monitor the impact of hyperoxia on parasite strain tolerance to dihydroartemisinin, a robust and highly reproducible protocol for the ring stage survival assay was established, incorporating a highly structured in vitro culturing programme. The 12 parasite strains were tested for the effect of hyperoxia on growth, as well as dihydroartemisinin tolerance. Parasite growth effects in hyperoxic conditions were deemed to be “genetic background” associated, with little or no impact of the presence or not of K13 mutations. Dihydroartemisinin tolerance was demonstrated to be K13 dependant, noticeably reduced after hyperoxic exposure, and the degree of parasite survival modulated by the parasite genetic background. The effect of hyperoxic exposure on dihydroartemisinin tolerance was observed to be parasite stage specific, namely between 0-20hr post red blood cell invasion and associated with the growth cycle prior to drug challenge. This study has demonstrated the effect parasite “genetic background”, in combination with K13 mutations on the ability of P. falciparum to modulate tolerance to dihydroartemisinin and other endoperoxide drugs. Without due consideration of in vitro culturing parameters used for P. falciparum, and comprehensive review of why only one study obtained a K13 mutation in vitro, this tolerance modulatory effect associated with the parasite genetic background, that is observed under oxidative stress conditions, may not have been identified. In conclusion, it is proposed that P. falciparum with K13 mutations and an associated “genetic background” are responsive to environmental stimulus and have a fitness advantage when responding to changes in environmental background. In stress conditions this genetic background maintains growth and a base level of DHA tolerance, whilst under non-stressed conditions, it has an even greater advantage due to elevated dihydroartemisinin tolerance. The transmission from stressed to non-stressed conditions could therefore select for the K13 mutations in both conditions.