Exposure of Plasmodium sporozoites to the intracellular concentration of potassium enhances infectivity and reduces cell passage activity

https://doi.org/10.1016/j.molbiopara.2007.07.004Get rights and content

Abstract

Malaria sporozoites migrate through several cells prior to a productive invasion that involves the formation of a parasitophorous vacuole (PV) where sporozoites undergo transformation into Exo-erythorcytic forms (EEFs). The precise mechanism leading to sporozoite activation for invasion is unknown, but prior traversal of host cells is required. During cell migration sporozoites are exposed to large shifts in K+ concentration. We report here that incubation of sporozoites to the intracellular K+ concentration enhances 8–10 times the infectivity of Plasmodium berghei and 4–5 times the infectivity of Plasmodium yoelli sporozoites for a hepatocyte cell line, while simultaneously decreasing cell passage activity. The K+ enhancing effect was time and concentration dependent, and was significantly decreased by K+ channel inhibitors. Potassium-treated P. berghei sporozoites also showed enhanced numbers of EEFs in non-permissive cell lines. Treated sporozoites had reduced infectivity for mice, but infectivity was enhanced upon Kupffer cell depletion. Transcriptional analysis of K+ treated and control sporozoites revealed a high degree of correlation in their levels of gene expression, indicating that the observed phenotypic changes are not due to radical changes in gene transcription. Only seven genes were upregulated by more than two-fold in K+ treated sporozoites. The highest level was noted in PP2C, a phosphatase known to dephosphorylate the AKT potassium channel in plants.

Introduction

Plasmodium sporozoites traverse the cytosol of cells prior to transformation into exoerythrocytic (EEF) stages [1]. The ability of sporozoites to traverse host cells is required for the completion of the Plasmodium life cycle. When infected mosquitoes feed on the mammalian hosts, the sporozoites are deposited in a pool of blood formed by the rupture of skin capillaries [2]. As shown by real time studies, the parasites pass through skin cells prior to entering the blood or lymph [2], [3]. Once in the blood circulation, sporozoites are retained in the liver sinusoids by the interaction of the circumsporozoite protein (CS) with heparan sulfate proteoglycans (HSPGs) [4], [5]. Then the parasites glide onto the endothelium, and traverse Kupffer cells [6], to enter the liver parenchyma. Only after navigating rapidly through the cytoplasm of several hepatocytes [7], they turn on a program for a “productive” invasion that leads to the formation of a parasitophorous vacuole (PV) whereupon sporozoites transform into EEFs. The sporozoite “activation” is associated with the exocytosis of microneme products including TRAP (thrombospondin related anonymous protein), [8] an essential component of the molecular motor that drives the active invasion process [9], [10]. While sporozoites enter and exit the cytoplasm of host cells, the parasites are exposed to profound changes in K+ concentration. Here we explore the possibility that these shifts in [K+] trigger the profound phenotypic changes that sporozoites undergo when they travel from the skin to the liver.

Section snippets

Sporozoites

P. berghei and P. yoelii cycles were maintained by allowing Anopheles stephensi mosquitoes to feed on SW mice (Jackson Laboratory) that had been infected with sporozoites. Midgut infection level was assessed on day 12 for P. berghei and day 8 for P. yoelii by checking for the presence of the midgut oocyst stages. The infected salivary glands were dissected on days 18–20 post blood meal for P. berghei and on days 14–16 for P. yoelli in DMEM (Gibco) containing 2 mM l-glutamine and 4.5 g/l glucose

Results and discussion

P. berghei sporozoites were incubated for 35 min at room temperature in medium containing [142 mM] K+ (“intracellular” medium), or with the same medium but containing instead [142 mM] Na+ (“intracellular control” medium), or with “control” medium (DMEM containing 4.5 g/l glucose supplemented with 10% FCS). Following incubation in different media, the sporozoites were added to hepatoma cultures maintained in control medium. In this paper the sporozoites exposed to [142 mM] K+ are named “treated”. In

Acknowledgements

The authors thank Dr. Ruth Nussenzweig for valuable comments on the manuscript. We also thank Dr. Photini Sinnis and Dr. Alida Coppi for sharing the protocol and assistance with performing calcein green migration assay.

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