Affinity purification of Plasmodium ookinetes from in vitro cultures using extracellular matrix gel
Graphical abstract
Introduction
Malaria is still a burden for many tropical and subtropical countries. It is caused by parasites of the genus Plasmodium which are transmitted to humans by Anopheles mosquitoes. After the mosquito takes a Plasmodium-infected blood meal, the parasite undergoes its sexual reproduction in the midgut lumen forming a motile zygote called the ookinete. The ookinete is responsible for establishing the infection in the mosquito by gliding out of the blood bolus and invading the midgut epithelium [1]. Once the ookinete traverses the midgut, it firmly attaches to the basal lamina and commences its differentiation into a sporozoites-producing oocyst. The basal lamina is a thin layer of extracellular matrix (ECM) that surrounds the midgut and is composed of type IV collagen, laminin, and entactin, among other proteoglycans [2]. The binding of the ookinete to the basal lamina is through several surface proteins, of which P25 and P21/P28 are the most abundant [[2], [3], [4], [5]].
Up to this day, the ookinete and the oocyst remain the most elusive to research. This is due to the difficulties of studying these stages in the mosquito, mainly because there is no practical way of extracting them but also because of the low number that survives in it. Hence the great interest to culture them in vitro and especially in axenic conditions, which eliminates the uncontrollable variables present in this complex interaction between the parasite, the blood, the mosquito midgut, and its microbiota [6,7]. Traditionally, the sexual stages are purified through centrifugation in density gradients [8], Nycodenz being the one that has performed the best in terms of cost, simplicity, and reduced toxicity [9,10]. The Nycodenz cushions can be coupled with DNA synthesis inhibitors to eliminate the asexual stages prior the ookinete culture [9,11,12], and an isotonic solution of NH4Cl can be used to selectively lyse the erythrocytes before the use of Nycodenz. However, NH4Cl causes modifications in the ookinete membrane [13], and the haemoglobin released from erythrocytes can induce oxidative stress in the parasite [14]. Another ookinete purification method is based on the detection of DNA content and cell sorting by flow cytometry, taking advantage of the tetraploid nature of ookinetes [15]. Finally, other methods are based on magnetic columns either solely, exploiting the magnetic properties of hemozoin [10] or with magnetic beads coated with anti-Pbs21 [16]. With these methods, a varying proportion of other parasite stages remain after purification since they also contain hemozoin, and macrogametes and zygotes also contain Pbs21 on the surface [10,16].
A couple of other methods, albeit not for purification, but rather as a way of culturing oocysts and testing ookinete invasion, involved the use of ECM components as a substrate for attachment and differentiation. In these studies, ookinete cultures were seeded as such [17] or after the erythrocytes were partially haemolysed and agglutinated [18]. Upon following days, the erythrocytes and other parasite stages were gradually eliminated with the medium changes, while the ookinetes and oocysts remained attached to the ECM. Here, we rake this experimental approach for purification purposes, extending it as a simple method for ookinete purification exploiting its adhesive properties without any previous enrichment or chemical treatment. The ookinetes are ready for further cultivation in the same container they were purified in, thus minimizing parasite manipulation. This method is particularly suitable for microscopy studies or oocyst culture. Notwithstanding, the parasites can be harvested from the ECM gel by digesting it with dispase II, a protease that cleaves type IV collagen.
Section snippets
Mice infections and ookinete culture
Plasmodium berghei ANKA strain clone 2.34, and the variant constitutively expressing the green fluorescent protein [19] (both kindly donated by Robert Sinden, Imperial College London), were kept as frozen stabilates. Parasites were maintained in BALB/c male mice of 6–8 weeks old up to the 8th passage by intraperitoneal inoculation. The blood was obtained from CO2 euthanized mice via cardiac puncture with a heparinised syringe (2.5 IU of saline-heparin per ml of blood). For ookinete culture,
Method development
As a proof of concept, the relationship between ookinete recovery and incubation time was determined in 4-chamber slides coated with ECM gel. As shown in Fig. 1A, a positive linear relationship was found. The recovery rate was of 100 ± 17.1 ookinetes per minute whilst the contaminant cells (CC) remained constant (slope = 4.6 ± 10.8 CC/min). The number of ookinetes and contaminant cells was measured between the washes to test whether this step causes ookinete loss during purification (Fig. 1B).
Discussion
In this study, a method for the gentle purification of ookinetes that relies on their invasive capabilities is presented. An in-depth analysis of this method shows that the purification outcome is positively influenced by incubation time, number of washes, ECM gel concentration, rocking during incubation, available surface, and initial amount of ookinetes seeded. However, the gain in ookinete yield and purity achieved when increasing the surface or splitting the culture into several containers
Funding
This project was possible thanks to the scholarship 487032/277845 from CONACyT granted to B.R.T. as part of the Programa de Maestría y Doctorado en Ciencias Bioquímicas at the Instituto de Biotecnología – UNAM.
Ethical standards
The Ethics, Biosafety, and Research Committees of our institution evaluated and approved the use of mice in this project.
Declaration of Competing Interest
The authors declare no competing interests.
Acknowledgments
We would like to thank Miguel Ángel Linares Aragón and Luis Giovanni Cassani López for technical support, and Gabriel Saucedo Eichelmann for revising the English. We also want to thank Mario Henry Rodríguez López and Wilhelm Ludwig Hansberg Torres for their helpful discussions and encouraging this project.
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