Affinity purification of Plasmodium ookinetes from in vitro cultures using extracellular matrix gel

https://doi.org/10.1016/j.parint.2020.102242Get rights and content

Highlights

  • In vitro Plasmodium ookinetes can be easily purified using an extracellular matrix gel.

  • Ookinete selective binding allows their purification by rinsing the contaminant cells.

  • This method is gentler to the ookinetes than the most common purification method.

  • Ookinetes are ready for oocyst culture after purification with purities above 85%.

  • Micro-cultures and micro-purifications can be performed without mice sacrifice.

Abstract

Malaria transmission depends on the parasites' successful invasion of the mosquito. This is achieved by the ookinete, a motile zygote that forms in the blood bolus after the mosquito takes an infectious blood meal. The ookinete invades the midgut epithelium and strongly attaches to the basal lamina, differentiating into an oocyst that produces the vertebrate-invasive sporozoites. Despite their importance, the ookinete and the oocyst are the least studied stages of the parasite. Much of what we know about the ookinete comes from in vitro experiments, which are hindered by the concomitant contamination with blood cells and other parasite stages. Although methods to purify them exist, they vary in terms of yield, costs, and difficulty to perform. A method for ookinete purification taking advantage of their adhesive properties was herein developed. The method consists of covering any culture-suitable surface with extracellular matrix gel, after which the ookinete culture is incubated on the gel to allow for ookinete attachment. The contaminant cells are then simply washed away. This procedure results in purer and less stressed ookinete preparations, which, by the nature of the method, are ready for oocyst production. Furthermore, it allows for micro-purifications using only 1 μl of blood, opening the possibility to make axenic ookinete cultures without sacrificing mice

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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      Plasmodium berghei ANKA strain, clone 2.34 that constitutively expresses the green fluorescent protein (GFP) (kindly provided by R. Sinden, Imperial College, UK) (Franke-Fayard et al., 2004), was used throughout the experiments. Parasite cultures were prepared as described in Recio-Tótoro et al. (2021) (Recio-Tótoro et al., 2021) to enrich ookinetes preparation. The ookinetes were counted in a Neubauer chamber, centrifuged at 1500×g, and resuspended in uninfected mouse blood to a final density of 1.2 × 106 ookinetes/ml.

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