Comparative chemical genomics in Babesia species identifies the alkaline phosphatase PhoD as a determinant of antiparasitic resistance.

Babesiosis is an emerging zoonosis and widely distributed veterinary infection caused by 100+ species of Babesia parasites. The diversity of Babesia parasites and the lack of specific drugs necessitate the discovery of broadly effective antibabesials. Here, we describe a comparative chemogenomics (CCG) pipeline for the identification of conserved targets. CCG relies on parallel in vitro evolution of resistance in independent populations of Babesia spp. (B. bovis and B. divergens). We identified a potent antibabesial, MMV019266, from the Malaria Box, and selected for resistance in two species of Babesia. After sequencing of multiple independently derived lines in the two species, we identified mutations in a membrane-bound metallodependent phosphatase (phoD). In both species, the mutations were found in the phoD-like phosphatase domain. Using reverse genetics, we validated that mutations in bdphoD confer resistance to MMV019266 in B. divergens. We have also demonstrated that BdPhoD localizes to the endomembrane system and partially with the apicoplast. Finally, conditional knockdown and constitutive overexpression of BdPhoD alter the sensitivity to MMV019266 in the parasite. Overexpression of BdPhoD results in increased sensitivity to the compound, while knockdown increases resistance, suggesting BdPhoD is a pro-susceptibility factor. Together, we have generated a robust pipeline for identification of resistance loci and identified BdPhoD as a resistance mechanism in Babesia species.

Extensive Shared Chemosensitivity between Malaria and Babesiosis Blood-Stage Parasites.

The apicomplexan parasites that cause malaria and babesiosis invade and proliferate within erythrocytes. To assess the potential for common antiparasitic treatments, we measured the sensitivities of multiple species of Plasmodium and Babesia parasites to the chemically diverse collection of antimalarial compounds in the Malaria Box library. We observed that these parasites share sensitivities to a large fraction of the same inhibitors and we identified compounds with strong babesiacidal activity.

The Plasmodium PHIST and RESA-Like Protein Families of Human and Rodent Malaria Parasites.

The phist gene family has members identified across the Plasmodium genus, defined by the presence of a domain of roughly 150 amino acids having conserved aromatic residues and an all alpha-helical structure. The family is highly amplified in P. falciparum, with 65 predicted genes in the genome of the 3D7 isolate. In contrast, in the rodent malaria parasite P. berghei 3 genes are identified, one of which is an apparent pseudogene. Transcripts of the P. berghei phist genes are predominant in schizonts, whereas in P. falciparum transcript profiles span different asexual blood stages and gametocytes. We pursued targeted disruption of P. berghei phist genes in order to characterize a simplistic model for the expanded phist gene repertoire in P. falciparum. Unsuccessful attempts to disrupt P. berghei PBANKA_114540 suggest that this phist gene is essential, while knockout of phist PBANKA_122900 shows an apparent normal progression and non-essential function throughout the life cycle. Epitope-tagging of P. falciparum and P. berghei phist genes confirmed protein export to the erythrocyte cytoplasm and localization with a punctate pattern. Three P. berghei PEXEL/HT-positive exported proteins exhibit at least partial co-localization, in support of a common vesicular compartment in the cytoplasm of erythrocytes infected with rodent malaria parasites.

Analysis of variant gene family expression by quantitative PCR.

Real-time polymerase chain reaction (PCR), or quantitative PCR (qPCR), is a rapid, sensitive, and specific method used for a broad variety of applications including quantitative gene expression analysis, DNA copy number measurement, characterization of gene and chromosomal deletions, and genotyping. Real-time reverse transcription (RT)-PCR has largely supplanted Northern blot and RNase protection assays, as two examples, as a means of quantifying transcript levels. The method utilizes small amounts of RNA and allows efficient screening of a large number of samples. Here, we describe the materials and methods required to perform real-time RT-PCR, including RNA purification, cDNA synthesis, and real-time PCR analysis of cDNA samples.

Sexual stage adhesion proteins form multi-protein complexes in the malaria parasite Plasmodium falciparum.

The sexual phase of the malaria parasite Plasmodium falciparum is accompanied by the coordinated expression of stage-specific adhesive proteins. Among these are six secreted proteins with multiple adhesion domains, termed P. falciparum LCCL domain-containing protein (PfCCp) proteins, which are expressed in the parasitophorous vacuole of the differentiating gametocytes and which are later associated with macrogametes. Although the majority of the PfCCp proteins are implicated in parasite development in the mosquito vector, their functions remain unknown. In the present study we investigated the molecular interactions between the PfCCp proteins during gametocyte development and emergence. Using five different gene-disruptant parasite lines, we show that the lack of one PfCCp protein leads to the loss of other PfCCp family members. Co-immunoprecipitation assays on gametocyte lysates revealed formation of complexes involving all PfCCp proteins, and affinity chromatography co-elution binding assays with recombinant PfCCp domains further indicated direct binding between distinct adhesion domains. PfCCp-coated latex beads bind to newly formed macrogametes but not to gametocytes or older macrogametes 6 or 24 h post-activation. In view of these data, we propose that the PfCCp proteins form multi-protein complexes that are exposed during gametogenesis, thereby mediating cell contacts of macrogametes.

Analysis of mutant Plasmodium berghei parasites lacking expression of multiple PbCCp genes.

Plasmodium encodes a family of six secreted multi-domain adhesive proteins, termed PCCps, which are released from gametocytes during emergence within the mosquito midgut. The expression and cellular localization of PCCp proteins predict a role either in gametocyte development or within the mosquito midgut during the transition from gametes into the ookinete stage. However, mutant parasites lacking expression of any single PCCp protein show a phenotype at the oocyst stage with a failure of oocyst maturation and sporozoite formation. In this study we investigated the stage-specific transcription of the PCCp genes of the rodent malaria parasite, Plasmodium berghei, and analyzed their promoter activities. Transcript expression analysis by quantitative real time RT-PCR showed that as in the human malaria parasite, Plasmodium falciparum, all PbCCp genes are predominantly transcribed in the gametocyte stage with a low level of transcription in the oocyst stage. Transgenic P. berghei parasites that contain the reporter protein GFP driven by the promoter regions of PbCCps showed pronounced GFP expression exclusively in gametocytes, in agreement with the RT-PCR data. To determine whether functional redundancies of different PCCp family members could explain the lack of a phenotype in gametocytes or gametes in single knockout mutant parasites, double gene null mutant P. berghei parasites were generated lacking either PCCp1 and PCCp3, or PCCp1 and PCCp4. The phenotype of these double knockout mutants was similar to that observed for single gene knockout mutants and manifest at the oocyst rather than the gametocyte or other stages within the mosquito midgut lumen.

The Plasmodium TRAP/MIC2 family member, TRAP-Like Protein (TLP), is involved in tissue traversal by sporozoites.

In the apicomplexan protozoans motility and cell invasion are mediated by the TRAP/MIC2 family of transmembrane proteins, members of which link extracellular adhesion to the intracellular actomyosin motor complex. Here we characterize a new member of the TRAP/MIC2 family, named TRAP-Like Protein (TLP), that is highly conserved within the Plasmodium genus. Similar to the Plasmodium sporozoite protein, TRAP, and the ookinete protein, CTRP, TLP possesses an extracellular domain architecture that is comprised of von Willebrand factor A (vWA) and thrombospondin type 1 (TSP1) domains, plus a short cytoplasmic domain. Comparison of the vWA domain of TLP genes from multiple Plasmodium falciparum isolates showed relative low sequence diversity, suggesting that the protein is not under selective pressures of the host immune system. Analysis of transcript levels by quantitative reverse transcription polymerase chain reaction (RT-PCR) showed that TLP is predominantly expressed in salivary gland sporozoites of P. falciparum and P. berghei. Targeted disruption of P. berghei TLP resulted in a decreased capacity for cell traversal by sporozoites, and reduced infectivity of sporozoites in vivo, whereas in vitro sporozoite motility and hepatocyte invasion were unaffected. These results indicate a role of TLP in cell traversal by sporozoites.

Effect of the antimicrobial peptide gomesin against different life stages of Plasmodium spp.

While seeking strategies for interfering with Plasmodium development in vertebrate/invertebrate hosts, we tested the activity of gomesin, an antimicrobial peptide isolated from the hemocytes of the spider Acanthoscurria gomesiana. Gomesin was tested against asexual, sexual and pre-sporogonic forms of Plasmodium falciparum and Plasmodium berghei parasites. The peptide inhibited the in vitro growth of intraerythrocytic forms of P. falciparum. When gomesin was added to in vitro culture of P. berghei mature gametocytes, it significantly inhibited the exflagellation of male gametes and the formation of ookinetes. In vivo, the peptide reduced the number of oocysts of both Plasmodium species in Anopheles stephensi mosquitoes, and did not appear to affect the mosquitoes. These properties make gomesin an excellent candidate as a transmission blocking agent for the genetic engineering of mosquitoes.

Using bacteria to express and display anti-Plasmodium molecules in the mosquito midgut.

Bacteria capable of colonizing mosquito midguts are attractive vehicles for delivering anti-malaria molecules. We genetically engineered Escherichia coli to display two anti-Plasmodium effector molecules, SM1 and phospholipase-A(2), on their outer membrane. Both molecules significantly inhibited Plasmodium berghei development when engineered bacteria were fed to mosquitoes 24h prior to an infective bloodmeal (SM1=41%, PLA2=23%). Furthermore, prevalence and numbers of engineered bacteria increased dramatically following a bloodmeal. However, E. coli survived poorly in mosquitoes. Therefore, Enterobacter agglomerans was isolated from mosquitoes and selected for midgut survival by multiple passages through mosquitoes. After four passages, E. agglomerans survivorship increased from 2 days to 2 weeks. Since E. agglomerans is non-pathogenic and widespread, it is an excellent candidate for paratransgenic control strategies.

Mosquito transgenesis: what is the fitness cost?

The generation of transgenic mosquitoes with a minimal fitness load is a prerequisite for the success of strategies for controlling mosquito-borne diseases using transgenic insects. It is important to assemble as much information as possible on this subject because realistic estimates of transgene fitness costs are essential for modeling and planning release strategies. Transgenic mosquitoes must have minimal fitness costs, because such costs would reduce the effectiveness of the genetic drive mechanisms that are used to introduce the transgenes into field mosquito populations. Several factors affect fitness of transgenic mosquitoes, including the potential negative effect of transgene products and insertional mutagenesis. Studies to assess fitness of transgenic mosquitoes in the field (as opposed to the laboratory) are still needed.

Towards genetic manipulation of wild mosquito populations to combat malaria: advances and challenges.

Malaria kills millions of people every year, yet there has been little progress in controlling this disease. For transmission to occur, the malaria parasite has to complete a complex developmental cycle in the mosquito. The mosquito is therefore a potential weak link in malaria transmission, and generating mosquito populations that are refractory to the parasite is a potential means of controlling the disease. There has been considerable progress over the last decade towards developing the tools for creating a refractory mosquito. Accomplishments include germline transformation of several important mosquito vectors, the completed genomes of the mosquito Anopheles gambiae and the malaria parasite Plasmodium falciparum, and the identification of promoters and effector genes that confer resistance in the mosquito. These tools have provided researchers with the ability to engineer a refractory mosquito vector, but there are fundamental gaps in our knowledge of how to transfer this technology safely and effectively into field populations. This review considers strategies for interfering with Plasmodium development in the mosquito, together with issues related to the transfer of laboratory-acquired knowledge to the field, such as minimization of transgene fitness load to the mosquito, driving genes through populations, avoiding the selection of resistant strains, and how to produce and release populations of males only.