Expansion of experimental genetics approaches for Plasmodium berghei with versatile transfection vectors

Abstract

Experimental reverse genetic approaches have proven powerful in the study of the biology of the malaria parasite. The murine malaria model parasite Plasmodium berghei is the genetically most amendable Plasmodium species and allows full access to the entire life cycle in vivo. Here, we describe a next-generation, highly versatile transfection vector set that facilitates advancing experimental genetic strategies towards a genome-wide scale. Through 36 consecutive cloning and 17 subcloning steps an optimized vector set was generated from the standard transfection plasmid. These targeting vectors, collectively referred to as the Berghei Adaptable Transfection (pBAT) plasmids, contain key elements that permit recycling of the drug-selectable cassette, robust green fluorescent labelling of recombinant parasites, carboxy-terminal tagging of target proteins with a red fluorescent–epitope tag fusion, and expression of heterologous genes. The vectors were further optimized for small size, versatile restriction endonuclease recognition sites and potential exchange of individual vector elements. We show that stable integration into a transgene expression site, an intergenic locus at a synteny breakpoint on P. berghei chromosome 6, is phenotypically silent and generated a bright green fluorescent parasite line for imaging applications. We provide an example, P. berghei actin 2, for targeted gene deletion and illustrate that the positive selection marker can be recycled, thereby permitting multiple rounds of genetic manipulations. We propose that the vectors described herein will greatly facilitate functional assignment to predicted and orphan Plasmodium gene models by multiple experimental genetics approaches.

Introduction

Experimental reverse genetics in the rodent malaria model parasite Plasmodium berghei is an established approach to study gene function [1]. It has consistently generated novel insights into Plasmodium biology and is the most straightforward and efficient in any species of Plasmodium [2]. Crucially, the complete life cycle can be studied in vivo, allowing for a truly systematic and eventually genome-wide scale endeavour.

The majority of transfection plasmids that have been used in the past were all based on the original P. berghei transfection vectors [3]. The standard pBSKS-based selection vector (b3D) contains a drug-selectable cassette comprising a pyrimethamine-insensitive Toxoplasma gondii dihydrofolate reductase-thymidilate synthetase (Tgdhfr-ts) mutant gene under the control of 2.3 kb and 0.5 kb of the 5′ and 3′ untranslated regions (UTR) of the P. berghei DHFR-TS orthologue [4]. An alternative pUC19-based vector containing the human dihydrofolate reductase (hDHFR) gene that confers additional resistance to the antifolate W99210 is used [5]. More recent vector improvements include recycling of the drug-selectable cassette using negative selection with 5-fluorocytosine [6] and a cassette expressing green fluorescent protein (GFP) under the control of the constitutive P. berghei elongation factor 1α (PbEF1α) promoter. Parasites modified with the latter have been isolated using fluorescence-assisted cell sorting (FACS) and are now widely used as a reference strain (GFPcon, [7]).

For the purpose of transgenic integration of expression cassettes without affecting parasite life cycle progression, we wanted to explore an intergenic locus at a synteny breakpoint [8]. Thus far, only perceived ‘dispensable’ loci have been used; such as the gamete surface antigen 230p [9], the ribosomal RNA C- and D-units [10], and, in Plasmodium yoelii, the S1 locus [11]. Though no phenotypic assays have yet detected an effect of these gene disruptions, this may change with the development of new analytical tools. Therefore, a site for stable integration should ideally be devoid of genes and negative for transcription or expression data.

The existing transfection plasmids presented a number of additional limitations. First, the exchange of vector elements, such as the drug-selectable marker, a fluorescent protein, epitope tags, and regulatory elements, is complicated by the lack of any strategic restriction endonuclease (REase) recognition sites, requiring extensive vector redesign. Moreover, a plethora of unnecessary REase recognition sites within the vector elements greatly restricted the number of cloning sites. Hence, in our vector design we removed all unwanted REase recognition sites within the various vector elements, while introducing strategic sites enabling straightforward exchange of these elements.

The pyrimethamine-sensitive, green fluorescent GFPcon reference line [7] has proven to be a very useful and powerful tool in the study of P. berghei. However, the PbEF1α promoter driving GFP expression shows low activity in motile, extracellular stages, like ookinetes, male microgametes, and salivary gland sporozoites, making these stages more difficult to trace with fluorescence microscopy. Furthermore, the GFP expression cassette has been introduced by disrupting gamete surface antigen 230p. Therefore, we designed the vectors to include a GFP expression cassette that enables more intense fluorescence in all life cycle stages. By incorporating such a cassette in the targeting vector, the use of a green fluorescent recipient strain is no longer necessary. We also aimed at generating a novel pyrimethamine-sensitive reference strain, termed Bergreen, which provides stronger GFP expression and absence of any gene disruptions.

Finally, we significantly reduced sizes of the plasmid backbone and the 5′PbDHFR-TS sequence used as the promoter for the drug-selectable cassette, to accommodate all vector elements in a vector of standard size.

Thus to further improve tools available for experimental genetic approaches in P. berghei, we designed a set of optimized, versatile P. berghei adaptable transfection (pBAT) vectors containing recyclable drug-selectable and GFP-expression cassettes. Integration of these two elements in a single vector facilitates generation of green fluorescent mutant P. berghei parasites.