Breaking Boundaries in Malaria Research: Design of a Genetic Tool for High-Throughput Gametocidal Drug Screening

Malaria, a devastating illness remains a global health concern with an estimated 249 million malaria cases in 85 malaria-endemic countries around the world. Malaria elimination, challenged by drug resistance, requires strategic interventions that could be the implementation of antimalarials with selective actions on the different phases of the parasite life cycle. Of particular relevance is gametocytocidal drugs that could be used to prevent transmission of malaria infection to the mosquito


1.1• The current Malaria placement as a tropical disease
Malaria illness is one of the major concerns in the world, being an epidemic in 85 countries [1].The disease comprises two hosts: the human and the mosquito.The Life cycle in humans starts when the female mosquito Anopheles is infected with Plasmodium parasites and takes a blood meal in humans [2].Common antimalarials are losing efficacy due to rising resistance and do not tackle the form of transmission (gametocytes) [1].Currently, only two drugs from the 8 -8-aminoquinoline family (primaquine and tafenoquine) target gametocytes across all stages [3], [4], however, the treatment is very toxic for patients with glucose -6 -phosphate dehydrogenase deficiency (G6PD) [5], therefore there is a need to screen and discover novel drugs that not only affect asexual parasites but also gametocytes.Nevertheless, studying gametocytes pose challenges.Current in vitro production methods are labor-intensive [6].The standard approach involves subjecting parasites to stress conditions such as high parasitemia and nutrient starvation [7].Achieving a balance in parasitemia is crucial for generating healthy gametocytes and decreasing cell death [7].The assessment of parasitemia in gametocytes is subjective once it is user-dependent on a light microscope, and there is almost no human-friendly method to detect and quantify gametocytes [3], [8].Given these limitations, there is a significant need to screen and discover new drugs capable of targeting both asexual parasites (responsible for symptoms) and gametocytes (responsible for the disease transmission).

1.2• Unraveling the role of gametocytes: key players in Malaria's puzzle
Gametocytes are the form of transmission [9].The major key player in sexual commitment is the AP2 -G family [9], which promotes a cascade of genes to develop and commit the neutral forms into gametocytes [10].Gametocytes have five stages to fully develop, mature, and be functional [11].
To follow gametocytes, the Plasmodium falciparum sexual-specific protein 16 (pfs16) seems to play a major role with expression starting 35 hours post-merozoite invasion, with the signal expression being like the five stages of gametocytes [12].More recently, it was discovered that pfs16 promotes vector transmission of the disease by silencing the mosquito's immune system [13].

1.3• Fluorescence and luminescence
The fluorescence [19], [20], and luminescence [21] have been implemented as optical readouts of several biological markers in Plasmodium falciparum.Comparing fluorescence to luminescence, luminescence involves the emission of light by a substance without the need for an external light source whereas fluorescence specifically refers to the absorption and re-emission of light at a longer wavelength.Luminescence assays tend to be highly sensitive and stable, while fluorescence offers high brightness and spatial resolution, making it ideal for imaging applications.Marin-Mogollon investigators genetic-engineer a P. falciparum NF54 reporter line to be expressed in gametocytes, sporozoites, and liver stage through CRISPR -Cas9 for fluorescence and luminescence readings [22].Our goal is to develop a genetic-based strategy in a form of a plasmid further to be transfected into Plasmodium falciparum for identifying and quantifying gametocytes using fluorescence and/or luminescence.This genetic tool comprises the pfs16 gametocytes-promoter (for expression of a protein that exhibits fluorescence (EmiRFP670) and luminescence (Luciferase) solely at the gametocyte stage of the parasite).

2.1• In silico design
The genetically encoded EmiRFP670Luc@s16 tool is designed to be episomally expressed and/or integrated into the genome of P. falciparum, under the pfs16 promoter (specific to gametocytes) (Figure 2).In this tool, the N-terminal pfs16 promoter (specific of gametocytes) is in frame with an EmiRFP670 -enhanced red fluorescent protein, linked to luciferase through a linker (GGGATCCTGGCTAGCCAGTCGACCTGCAGGC).The sequence of the promoter pfs16 was obtained from the PlasmoDB database with the PF3D7_0406200 code, luciferase sequence was obtained from P. falciparum NF54-GFP-luc strain (MRA 1217, BEI Resources), and the fluorescent reporter protein EmiRFP670 was obtained from the addgene plasmid pLAMP1-emiRFP670 (#136570).The plasmid pDC2 -pfcamp -Snf7 -GFP -BSD -attP was used as the backbone plasmid and was kindly provided by David Fidock and Marcus Lee.The sequence of the cloning vectors pJET 1.2 and pNZY28 were supplied by ThermoFisher Scientific and NZYTech, respectively.The ApE -A Plasmid Editor v3.0.9 and SnapGene version 6.2.1 were used for the in silico design of EmiRFP670Luc@s16.

2.2• Engineering the EmiRFP670Luc16 cassete
Genomic DNA (gDNA) of P. falciparum 3D7 and NF54-GFP-luc strains was extracted using the NZY Tissue gDNA isolation kit (NZYTech).The pfs16 gene was amplified from P. falciparum 3D7 with primer P1 and P2, comprising the recognition sites for ApaI and AvrII, respectively.The P. falciparum NF54-GFPluc was used to amplify luciferase with primers P3 and P4, with restriction recognition sites for BglII and XhoI, respectively.The plasmid DNA (pDNA) from pLAMP1-emiRFP670 was extracted with the NZYMiniprep kit (NZYTech).The EmiRFP670 was amplified from the pLAMP1 -emiRFP670 pDNA with primers P5 and P6, with specific restriction sites for BshTI and AvrII, respectively.The pfs16 gene promoter was amplified with a Supreme NZYTaq II 2× Green Master Mix (NZYTech), while luciferase and EmiRFP670 were amplified with 2X Phusion Green Hot Start II High-Fidelity PCR Master Mix (ThermoFisher Scientific).The PCR programs and reaction conditions are depicted in Tables 1 and 2.     3, and restriction enzymes to find the positives recombinants.

3.1• Selection of genes, their amplification, and subsequent vector cloning
The EmiRFP670Luc@s16 genetic tool aims at the identification and quantification of gametocytes in P. falciparum.To achieve this, we selected the psf16 promoter for the tool to be specific to gametocytes.The psf16 gene is only expressed upon sexual commitment and is similarly expressed following 35 hours post-invasion until the rest of the gametocytes' cycle (around 15 days) [13].This allows the identification of all gametocyte stages, including the early stage I, whose morphology resembles the one of asexual mature parasites [8], [23].The pfs16 was designed to be in frame with the EmiRFP670 protein linked to luciferase.These reporter proteins were selected to allow the detection and quantification of the gametocytes.EmiRFP670, derived from Rhodopseudomonas palustris, presents an enhanced signal, when compared to other red fluorescent proteins, due to a increase on photostability, and brightness [24].Specifically, this one displays an excitation peak of around 642 nm and an emission of around 670 nm [24].The detection of this protein can be performed by any fluorescence reading equipment such as through flow cytometry, fluorimeter detection, and fluorescent microscopy, the latest also allowing localization studies [22], [25], [26].
To improve the readout capacities, we also include a protein that emits luminescence, in this case, we chose luciferase.Luciferase is more sensitive (present low signal-to-noise ratio) and allows the test of frozen samples [9].
The PCR amplification of each gene was confirmed through visualization of agarose gel electrophoresis under ultraviolet light with the expected amplicon sizes enunciated in Table 2.

| VOL [03] | [2024]
JUS -JOURNAL UMINHO SCIENCE Afterward, each gene was cloned into an intermediate commercial plasmid (pJET and pNZY28) to facilitate the final cloning through restriction-ligation reactions (Figure 3A).Positive recombinants were verified with restriction enzymes (Figure 3B) before being outsourced for Sanger Sequencing.The pDC2 -pfcamp -Snf7 -GFP -BSD -attP plasmid was used as the backbone plasmid for our vector construct, EmiRFP670Luc@s16 cassette.The pDC2 plasmid was selected for its capacity to enable the expression of fluorescent and luminescent proteins under the control of a gametocidal promoter (pfs16).Additionally, this plasmid contains the attP sequence, facilitating genome integration through recombination of the attPsite in the plasmid with agenomic attB site, facilitated by the bxb1 integrase system [17].Furthermore, this plasmid presents a promoter (pfcamp), followed by two genes (snf7 and gfp) with sites for restriction enzymes (ApaI and AvrII, BshTI and AvrII, and BglII and XhoI).Thus, these restriction enzymes were used to allow the replacement of the original genes (pfcamp, snf7, and gfp) by our genes of interest (pfs16, emiRFP670,

• CONCLUSION
In this article, we report EmiRFP670Luc@s16 as a genetic tool to detect and quantify gametocytes through the expression of a red fluorescent protein and luciferase.By using pfs16, a specific sexual promoter, the EmiRFP670Luc@s16 cassette will target the 5 P. falciparum stages of gametocytes with almost constant expression levels throughout their lifecycle.The EmiRFP670Luc@s16 was designed to express the EmiRFP670, which allows live cell-imaging and quantification by optical readouts, including fluorescence microscopy, fluorimeter and flow cytometry, and luciferase for the sensitive detection of frozen samples through luminescence.The herein described a genetic tool is designed to allow both epissomal and genomic integration expression, through parasite selection with BSD.Genomic integration of this tool, as previously mentioned, can occur through the bxb1 integrase system, however this requires an attB-engineered parasite strain.The epissomal plasmid is more versatile as it does not require the time-consuming genome integration of recombinants, nor specific strains.We are confident that the vector construction we have developed represents a significant advancement in the field.This innovative tool enables rapid and precise optical readout of gametocytes, making it ideal for high-throughput screening of gametocidal drugs.Additionally, it offers the opportunity to assess the function of proteins involved in gametocyte development.By revolutionizing the screening process for anti-gametocyte drugs, this genetically engineered tool also has the potential to identify key players in gametocyte development, thus informing the discovery of new transmission blocking antimalarial drugs.

Figure 1 -
Figure 1 -Engineering a Plasmodium falciparum NF54 transgenic line to detect and quantify Gametocytes for highthroughput gametocidal drug screening by fluorescence and luminescence through episomal expression.

Figure 2 -
Figure 2 -Gametocytes under the pfs16 promotor, and blasticidin-S deaminase (bsd) selection cassette to select the parasites for episomal expression.
After amplification, the three fragments were inserted into cloning vectors; pfs16 was introduced into the pNZY28 commercial plasmid from the NZY-A PCR cloning kit (NZYTech), while luciferase and EmiRFP670 were inserted into pJET1.2commercial plasmid from the CloneJET PCR Cloning kit (ThermoFisher Scientific) as shown in Figure3.These constructs were then transformed into NZYStar competent cells (NZYTech) through the heat shock method.Positive recombinants were screened through restriction enzymes, followed by Sanger sequencing (STAB VIDA, Lda).

Figure 3 -
Figure 3 -An overview of the strategy for amplifying and ligating the fragments (pfs16, EmiRFP670, and Luciferase), which were then transformed into the pJET and pNZY28 commercial plasmids.

Table 2 -
Primers used for pfs16, EmiRFP670, and luciferase amplifications with the respective PCR programs, restriction recognition sites, and amplicon size.