Antimalarial action of artesunate involves DNA damage mediated by reactive oxygen species.

Artemisinin-based combination therapy (ACT) is the recommended first-line treatment for Plasmodium falciparum malaria. It has been suggested that the cytotoxic effect of artemisinin is mediated by free radicals followed by the alkylation of P. falciparum proteins. The endoperoxide bridge, the active moiety of artemisinin derivatives, is cleaved in the presence of ferrous iron, generating reactive oxygen species (ROS) and other free radicals. However, the emergence of resistance to artemisinin in P. falciparum underscores the need for new insights into the molecular mechanisms of antimalarial activity of artemisinin. Here we show that artesunate (ART) induces DNA double-strand breaks in P. falciparum in a physiologically relevant dose- and time-dependent manner. DNA damage induced by ART was accompanied by an increase in the intracellular ROS level in the parasites. Mannitol, a ROS scavenger, reversed the cytotoxic effect of ART and reduced DNA damage, and modulation of glutathione (GSH) levels was found to impact ROS and DNA damage induced by ART. Accumulation of ROS, increased DNA damage, and the resulting antiparasite effect suggest a causal relationship between ROS, DNA damage, and parasite death. Finally, we also show that ART-induced ROS production involves a potential role for NADPH oxidase, an enzyme involved in the production of superoxide anions. Our results with P. falciparum provide novel insights into previously unknown molecular mechanisms underlying the antimalarial activity of artemisinin derivatives and may help in the design of next-generation antimalarial drugs against the most virulent Plasmodium species.

Role of cis-regulatory elements on the ring-specific hrp3 promoter in the human parasite Plasmodium falciparum.

Identification of promoter elements responsible for regulation of gene expression has been hampered by the AT richness of P. falciparum intergenic regions. Nested deletions of histidine-rich protein 3 (hrp3) promoter suggested the presence of a multipartite ring-specific element. Linker scanning (LS) of this ring-specific promoter showed that the alteration of several promoter regions decreased the luciferase activity compared to the wild-type configuration, indicating that these regions played a role in gene expression. No homology was observed by comparison of putative regulatory elements of other genes identified by bioinformatic analysis with the hrp3 enhancer, implying a different mechanism of gene regulation by the hrp3 promoter. LS and deletion analysis of the 5' untranslated region (UTR) of the hrp3 suggested that this region contains elements which interact with promoter elements to regulate gene expression. Analysis of the intron in the UTR region suggested that this region does not play a role in stage specificity in the hrp3 promoter. Together, our results indicate the presence of multiple mechanisms of gene regulation in the parasite.

Plasmodium falciparum: Preinitiation complex occupancy of active and inactive promoters during erythrocytic stage.

Over 80% of Plasmodium falciparum genes are developmentally regulated during the parasite's life cycle with most genes expressed in a "just in time" fashion. However, the molecular mechanisms of gene regulation are still poorly understood. Analysis of P. falciparum genome shows that the parasite appears to encode relatively few transcription factors homologous to those in other eukaryotes. We used Chromatin immunoprecipitation (ChIP) to study interaction of PfTBP and PfTFIIE with stage specific Plasmodium promoters. Our results indicate that PfTBP and PfTFIIE are bound to their cognate sequence in active and inactive erythrocytic-expressed promoters. In addition, TF occupancy appears to extend beyond the promoter regions, since PfTBP interaction with the coding and 3' end regions was also detected. No PfTBP or PfTFIIE interaction was detected on csp and pfs25 genes which are not active during the erythrocytic asexual stage. Furthermore, PfTBP and PfTFIIE binding did not appear to correlate with histone 3 and/or 4 acetylation, suggesting that histone acetylation may not be a prerequisite for PfTBP or PfTFIIE promoter interaction. Based on our observations we concluded that the PfTBP/PfTFIIE-containing preinitiation complex (PIC) would be preassembled on promoters of all erythrocytic-expressed genes in a fashion independent of histone acetylation, providing support for the "poised" model. Contrary to the classical model of eukaryotic gene regulation, PIC interaction with Plasmodium promoters occurred independent of transcriptional activity and to the notion that chromatin acetylation leads to PIC assembly.

An enhancer-like region regulates hrp3 promoter stage-specific gene expression in the human malaria parasite Plasmodium falciparum.

The asexual blood stage of Plasmodium falciparum is comprised of morphologically distinct ring, trophozoite and schizont stages. Each of these developmental stages possesses a distinct pattern of gene expression. Regulation of P. falciparum gene expression is thought to occur, at least in part, at the promoter level. Previously, we have found that although the hrp3 mRNA is only seen in ring-stage parasites, deletion of a specific sequence in the 5' end of the promoter region decreased ring-stage expression of hrp3 and enabled detection of its transcripts in trophozoite-stage parasites. In order to investigate this stage specific regulation of gene expression, we employed a series of nested deletions of the 1.7-kb hrp3 promoter. Firefly luciferase gene was used as a reporter to evaluate the role of promoter sequences in gene regulation. Using this approach, we identified a ring-stage specific regulatory region on the hrp3 promoter located between -1.7 kb and -1.1 kb from the ATG initiation codon. Small 100-150 bp truncations on this enhancer-like region failed to uncover discrete regulatory sequences, suggesting the multipartite nature of this element. The data presented in this study demonstrate that stage specific promoter activity of the hrp3 gene in P. falciparum blood stage parasites is supported, at least in-part, by a small promoter region that can function in the absence of a larger chromosomal context.

An Atlas of Genetic Variation Linking Pathogen-Induced Cellular Traits to Human Disease.

Pathogens have been a strong driving force for natural selection. Therefore, understanding how human genetic differences impact infection-related cellular traits can mechanistically link genetic variation to disease susceptibility. Here we report the Hi-HOST Phenome Project (H2P2): a catalog of cellular genome-wide association studies (GWAS) comprising 79 infection-related phenotypes in response to 8 pathogens in 528 lymphoblastoid cell lines. Seventeen loci surpass genome-wide significance for infection-associated phenotypes ranging from pathogen replication to cytokine production. We combined H2P2 with clinical association data from patients to identify a SNP near CXCL10 as a risk factor for inflammatory bowel disease. A SNP in the transcriptional repressor ZBTB20 demonstrated pleiotropy, likely through suppression of multiple target genes, and was associated with viral hepatitis. These data are available on a web portal to facilitate interpreting human genome variation through the lens of cell biology and should serve as a rich resource for the research community.

Multifunctional Involvement of a C2H2 Zinc Finger Protein (PbZfp) in Malaria Transmission, Histone Modification, and Susceptibility to DNA Damage Response.

In sexually reproducing organisms, meiosis is an essential step responsible for generation of haploid gametes from diploid somatic cells. The quest for understanding regulatory mechanisms of meiotic recombination in Plasmodium led to identification of a gene encoding a protein that contains 11 copies of C2H2 zinc fingers (ZnF). Reverse genetic approaches were used to create Plasmodium berghei parasites either lacking expression of full-length Plasmodium berghei zinc finger protein (PbZfp) (knockout [KO]) or expressing PbZfp lacking C-terminal zinc finger region (truncated [Trunc]). Mice infected with KO parasites survived two times longer (P < 0.0001) than mice infected with wild-type (WT) parasites. In mosquito transmission experiments, the infectivity of KO and Trunc parasites was severely compromised (>95% oocyst reduction). KO parasites revealed a total lack of trimethylation of histone 3 at several lysine residues (K4, K27, and K36) without any effect on acetylation patterns (H3K9, H3K14, and H4K16). Reduced DNA damage and reduced expression of topoisomerase-like Spo11 in the KO parasites with normal Rad51 expression further suggest a functional role for PbZfp during genetic recombination that involves DNA double-strand break (DSB) formation followed by DNA repair. These finding raise the possibility of some convergent similarities of PbZfp functions to functions of mammalian PRDM9, also a C2H2 ZnF protein with histone 3 lysine 4 (H3K4) methyltransferase activity. These functions include the major role played by the latter in binding recombination hotspots in the genome during meiosis and trimethylation of the associated histones and subsequent chromatin recruitment of topoisomerase-like Spo11 to catalyze DNA DSB formation and DMC1/Rad51-mediated DNA repair and homologous recombination.IMPORTANCE Malaria parasites are haploid throughout their life cycle except for a brief time period when zygotes are produced as a result of fertilization between male and female gametes during transmission through the mosquito vector. The reciprocal recombination events that follow zygote formation ensure orderly segregation of homologous chromosomes during meiosis, creating genetic diversity among offspring. Studies presented in the current manuscript identify a novel C2H2 ZnF-containing protein exhibiting multifunctional roles in parasite virulence, mosquito transmission, and homologous recombination during meiosis. Understanding the transmission biology of malaria will result in the identification of novel targets for transmission-blocking intervention approaches.

Global Mapping of the Inc-Human Interactome Reveals that Retromer Restricts Chlamydia Infection.

Chlamydia trachomatis is a leading cause of genital and ocular infections for which no vaccine exists. Upon entry into host cells, C. trachomatis resides within a membrane-bound compartment­the inclusion­and secretes inclusion membrane proteins (Incs) that are thought to modulate the host-bacterium interface. To expand our understanding of Inc function(s), we subjected putative C. trachomatis Incs to affinity purification-mass spectroscopy (AP-MS). We identified Inc-human interactions for 38/58 Incs with enrichment in host processes consistent with Chlamydia's intracellular life cycle. There is significant overlap between Inc targets and viral proteins, suggesting common pathogenic mechanisms among obligate intracellular microbes. IncE binds to sorting nexins (SNXs) 5/6, components of the retromer, which relocalizes SNX5/6 to the inclusion membrane and augments inclusion membrane tubulation. Depletion of retromer components enhances progeny production, revealing that retromer restricts Chlamydia infection. This study demonstrates the value of proteomics in unveiling host-pathogen interactions in genetically challenging microbes.

Opposing roles for two molecular forms of replication protein A in Rad51-Rad54-mediated DNA recombination in Plasmodium falciparum.

UNLABELLED: The bacterial RecA protein and its eukaryotic homologue Rad51 play a central role in the homologous DNA strand exchange reaction during recombination and DNA repair. Previously, our lab has shown that PfRad51, the Plasmodium falciparum homologue of Rad51, exhibited ATPase activity and promoted DNA strand exchange in vitro. In this study, we evaluated the catalytic functions of PfRad51 in the presence of putative interacting partners, especially P. falciparum homologues of Rad54 and replication protein A. PfRad54 accelerated PfRad51-mediated pairing between single-stranded DNA (ssDNA) and its homologous linear double-stranded DNA (dsDNA) in the presence of 0.5 mM CaCl2. We also present evidence that recombinant PfRPA1L protein serves the function of the bacterial homologue single-stranded binding protein (SSB) in initiating homologous pairing and strand exchange activity. More importantly, the function of PfRPA1L was negatively regulated in a dose-dependent manner by PfRPA1S, another RPA homologue in P. falciparum. Finally, we present in vivo evidence through comet assays for methyl methane sulfonate-induced DNA damage in malaria parasites and accompanying upregulation of PfRad51, PfRad54, PfRPA1L, and PfRPA1S at the level of transcript and protein needed to repair DNA damage. This study provides new insights into the role of putative Rad51-interacting proteins involved in homologous recombination and emphasizes the physiological role of DNA damage repair during the growth of parasites. IMPORTANCE: Homologous recombination plays a major role in chromosomal rearrangement, and Rad51 protein, aided by several other proteins, plays a central role in DNA strand exchange reaction during recombination and DNA repair. This study reports on the characterization of the role of P. falciparum Rad51 in homologous strand exchange and DNA repair and evaluates the functional contribution of PfRad54 and PfRPA1 proteins. Data presented here provide mechanistic insights into DNA recombination and DNA damage repair mechanisms in this parasite. The importance of these research findings in future work will be to investigate if Rad51-dependent mechanisms are involved in chromosomal rearrangements during antigenic variation in P. falciparum. A prominent determinant of antigenic variation, the extraordinary ability of the parasite to rapidly change its surface molecules, is associated with var genes, and antigenic variation presents a major challenge to vaccine development.

Novel nanosomes for gene delivery to Plasmodium falciparum-infected red blood cells.

Malaria threatens millions of people annually and is a burden to human health and economic development. Unfortunately in terms of disease control, no effective vaccines are available and the efficacy of treatment is limited by drug resistance. Genetic manipulation in Plasmodium falciparum is hampered due to the absence of robust methods for genetic analyses. Electroporation-based transfection methods have allowed the study of gene function in P. falciparum, with low efficiency. A lipid nanoparticle was developed that allowed nuclear targeting of pDNA with increased efficiency in reporter assay, compared to traditional electroporation method. This method has for the first time, facilitated transfection using both circular and linear DNA in P. falciparum thereby serving as an alternative to electroporation with an increase in transfection efficiency. Availability of a robust method for functional genomic studies in these organisms may be a catalyst for discovery of novel targets for developing drugs and vaccines.

Detection and species identification of malaria parasites by isothermal tHDA amplification directly from human blood without sample preparation.

We report the clinical and analytical performance of an isothermal thermophilic helicase-dependent amplification assay for blood Plasmodium parasite detection and species-level identification. The assay amplifies the 18S rRNA gene fragment of all Plasmodium species and uses a species-specific probe and a pan-malarial probe to definitively identify Plasmodium falciparum from other infectious Plasmodium species. Amplicon-probe hybridization products are detected with a disposable dipstick enclosed in a cassette. With a pan-malarial-positive and P. falciparum-negative result, an additional test is performed to detect if the pan-malarial-positive band was the result of the presence of Plasmodium vivax. The assay uses only 2 µL of human whole blood directly for a 50-µL amplification reaction, without any pre-amplification processing. The clinical performance of the assay was validated using 88 samples from New York patients suspected of malaria or babesiosis. The overall sensitivity of the assay was 96.6% (95% CI, 87.3% to 99.4%), and the specificity was 100% (95% CI, 85.4% to 100%), compared with gold standard microscopy and a laboratory-developed molecular assay, respectively. The analytical sensitivity was 50 copies of DNA per assay or 200 parasites per microliter of blood, and the assay can detect samples with parasitemia levels <1%. This novel molecular diagnostic assay requires minimal laboratory instrumentation and uses un-processed blood as input; it can be readily performed in the field.

Comparative analysis of stage specific gene regulation of apicomplexan parasites: Plasmodium falciparum and Toxoplasma gondii.

Apicomplexans comprise some of the most life threatening parasites infecting human and livestock and includes Plasmodium and Toxoplasma, the causative agents of malaria and toxoplasmosis respectively, in humans as well as Neospora caninum (abortion in livestock, neosporosis in dogs), Cryptosporidium (Diarrheal cryptosporidiosis and opportunistic infections in AIDS patients) and Eimeria (poultry coccidiosis). These parasites are characterized by a complex life cycle usually alternating between sexual and asexual cycles in different hosts. The need to adapt to different host environments demands a tight regulation of gene expression during parasite development. Therefore, the understanding of parasite biology will facilitate the control of the infection and the disease. In this review we emphasize the progress made so far in gene regulation in two medically important parasites, namely Plasmodium falciparum and Toxoplasma gondii, as well as other less known apicomplexan. The genome of both Plasmodium and Toxoplasma has been sequenced and since then there has been a significant progress in understanding the molecular mechanisms that control stage specific gene expression in the two parasites. In addition, the information gained in each of the parasite can be used in studying mechanisms that are still elusive in the other apicomplexans that are not readily available. Additionally, they can serve as model system for other disease causing Apicomplexan parasites.

Plasmodium falciparum: hrp3 promoter region is associated with stage-specificity and episomal recombination.

The asexual blood stage of Plasmodium falciparum in the human host is comprised of morphologically distinct ring, trophozoite and schizont stages, each of which possesses a distinct pattern of gene expression. Episomal promoter recombination has been recently reported in malaria parasites. We aim to investigate the nature of this process, and its relationship with promoter activity by employing a series of nested deletions of the ring-specific hrp3 promoter. Our results showed a discrete promoter region that is preferentially used for recombination. The P. falciparum hrp3 mRNA is only seen in ring-stage parasites but deletion of the recombination region was associated with decreased ring-stage expression and concurrent detection of transcripts in trophozoite-stage parasites. Our results describe a ring-stage specific regulatory region possibly involved in episomal promoter recombination, suggesting that common sequences might mediate both processes.