Characterization of the dual role of Plasmodium falciparum DNA methyltransferase in regulating transcription and translation.

DNA modifications are critical in fine-tuning the biological processes in model organisms. However, the presence of cytosine methylation (5mC) and the function of the putative DNA methyltransferase, PfDNMT2, in the human malaria pathogen, Plasmodium falciparum, remain controversial. Here, we revisited the 5mC in the parasite genome and the function of PfDNMT2. Low levels of genomic 5mC (0.1-0.2%) during asexual development were identified using a sensitive mass spectrometry procedure. Native PfDNMT2 displayed substantial DNA methylation activities, and disruption or overexpression of PfDNMT2 resulted in reduced or elevated genomic 5mC levels, respectively. PfDNMT2 disruption led to an increased proliferation phenotype, with the parasites having an extended schizont stage and producing a higher number of progenies. Consistent with PfDNMT2's interaction with an AP2 domain-containing transcription factor, transcriptomic analyses revealed that PfDNMT2 disruption led to a drastic alteration in the expression of many genes, some of which provided the molecular basis of enhanced proliferation after PfDNMT2 disruption. Furthermore, levels of tRNAAsp and its methylation rate at position C38, and the translation of a reporter containing an aspartate repeat were significantly reduced after PfDNMT2 disruption, while the levels of tRNAAsp and its C38 methylation were restored after complementation of PfDNMT2. Our study sheds new light on the dual function of PfDNMT2 during P. falciparum asexual development.

Onametostat, a PfPRMT5 inhibitor, exhibits antimalarial activity to Plasmodium falciparum.

Protein arginine methyltransferases (PRMTs) play critical roles in Plasmodium falciparum, a protozoan causing the deadliest form of malaria, making them potential targets for novel antimalarial drugs. Here, we screened 11 novel PRMT inhibitors against P. falciparum asexual growth and found that onametostat, an inhibitor for type II PRMTs, exhibited strong antimalarial activity with a half-maximal inhibitory concentration (IC50) value of 1.69 ± 0.04 µM. In vitro methyltransferase activities of purified PfPRMT5 were inhibited by onametostat, and a shift of IC50 to onametostat was found in the PfPRTM5 disruptant parasite line, indicating that PfPRTM5 is the primary target of onametostat. Consistent with the function of PfPRMT5 in mediating symmetric dimethylation of histone H3R2 (H3R2me2s) and in regulating invasion-related genes, onametostat treatment led to the reduction of H3R2me2s level in P. falciparum and caused the defects on the parasite's invasion of red blood cells. This study provides a starting point for identifying specific PRMT inhibitors with the potential to serve as novel antimalarial drugs.

Plasmodium falciparum GCN5 plays a key role in regulating artemisinin resistance-related stress responses.

Plasmodium falciparum causes the most severe malaria and is exposed to various environmental and physiological stresses in the human host. Given that GCN5 plays a critical role in regulating stress responses in model organisms, we aimed to elucidate PfGCN5's function in stress responses in P. falciparum. The protein level of PfGCN5 was substantially induced under three stress conditions [heat shock, low glucose starvation, and dihydroartemisinin, the active metabolite of artemisinin (ART)]. With a TetR-DOZI conditional knockdown (KD) system, we successfully down-regulated PfGCN5 to ~50% and found that KD parasites became more sensitive to all three stress conditions. Transcriptomic analysis via RNA-seq identified ~1,000 up- and down-regulated genes in the wild-type (WT) and KD parasites under these stress conditions. Importantly, DHA induced transcriptional alteration of many genes involved in many aspects of stress responses, which were heavily shared among the altered genes under heat shock and low glucose conditions, including ART-resistance-related genes such as K13 and coronin. Based on the expression pattern between WT and KD parasites under three stress conditions, ~300-400 genes were identified to be involved in PfGCN5-dependent, general, and stress-condition-specific responses with high levels of overlaps among three stress conditions. Notably, using ring-stage survival assay, we found that KD or inhibition of PfGCN5 could sensitize the ART-resistant parasites to the DHA treatment. All these indicate that PfGCN5 is pivotal in regulating general and ART-resistance-related stress responses in malaria parasites, implicating PfGCN5 as a potential target for malaria intervention.

Naturally induced humoral response against Plasmodium vivax reticulocyte binding protein 2P1.

BACKGROUND: Plasmodium vivax is the most prevalent malaria parasite in many countries. A better understanding of human immunity to this parasite can provide new insights for vaccine development. Plasmodium vivax Reticulocyte Binding Proteins (RBPs) are key parasite proteins that interact with human proteins during erythrocyte invasion and are targets of the human immune response. The aim of this study is to characterize the human antibody response to RBP2P1, the most recently described member of the RBP family. METHODS: The levels of total IgG and IgM against RBP2P1 were measured using plasmas from 68 P. vivax malaria patients and 525 villagers in a malarious village of western Thailand. The latter group comprises asymptomatic carriers and healthy uninfected individuals. Subsets of plasma samples were evaluated for anti-RBP2P1 IgG subtypes and complement-fixing activity. RESULTS: As age increased, it was found that the level of anti-RBP2P1 IgG increased while the level of IgM decreased. The main anti-RBP2P1 IgG subtypes were IgG1 and IgG3. The IgG3-seropositive rate was higher in asymptomatic carriers than in patients. The higher level of IgG3 was correlated with higher in vitro RBP2P1-mediated complement fixing activity. CONCLUSIONS: In natural infection, the primary IgG response to RBP2P1 was IgG1 and IgG3. The predominance of these cytophilic subtypes and the elevated level of IgG3 correlating with complement fixing activity, suggest a possible role of anti-RBP2P1 antibodies in immunity against P. vivax.

The Blood Stage Antigen RBP2-P1 of Plasmodium vivax Binds Reticulocytes and Is a Target of Naturally Acquired Immunity.

The interactions between Plasmodium parasites and human erythrocytes are prime targets of blood stage malaria vaccine development. The reticulocyte binding protein 2-P1 (RBP2-P1) of Plasmodium vivax, a member of the reticulocyte binding protein family, has recently been shown to be highly antigenic in several settings endemic for malaria. Yet, its functional characteristics and the relevance of its antibody response in human malaria have not been examined. In this study, the potential function of RBP2-P1 as an invasion ligand of P. vivax was evaluated. The protein was found to be expressed in schizonts, be localized at the apical end of the merozoite, and preferentially bind reticulocytes over normocytes. Human antibodies to this protein also exhibit erythrocyte binding inhibition at physiologically relevant concentrations. Furthermore, RBP2-P1 antibodies are associated with lower parasitemia and tend to be higher in asymptomatic carriers than in patients. This study provides evidence supporting a role of RBP2-P1 as an invasion ligand and its consideration as a vaccine target.

Efficient synchronization of Plasmodium knowlesi in vitro cultures using guanidine hydrochloride.

BACKGROUND: Long-term in vitro culture of blood stage Plasmodium parasites invariably leads to asynchronous parasite development. The most often used technique to synchronize Plasmodium falciparum culture is sorbitol treatment, which differentially induces osmotic lysis of trophozoite- and schizont-infected red blood cells due to presence of the new permeation pathways in the membranes of these cells. However, sorbitol treatment does not work well when used to synchronize the culture-adapted Plasmodium knowlesi A1-H.1 line. METHODS: A number of common solutes were tested in lieu of sorbitol for synchronization of P. knowlesi A1-H.1 ring stage. RESULTS: Guanidine hydrochloride was found to selectively lyse trophozoite- and schizont-infected red blood cells, yielding highly synchronous and viable rings. CONCLUSIONS: A method for synchronization of P. knowlesi in human red blood cells was developed. Requiring only common laboratory reagents, this method is simple and should be applicable to most laboratory settings.

Gene Models, Expression Repertoire, and Immune Response of Plasmodium vivax Reticulocyte Binding Proteins.

Members of the Plasmodium vivax reticulocyte binding protein (PvRBP) family are believed to mediate specific invasion of reticulocytes by P. vivax. In this study, we performed molecular characterization of genes encoding members of this protein family. Through cDNA sequencing, we constructed full-length gene models and verified genes that are protein coding and those that are pseudogenes. We also used quantitative PCR to measure their in vivo transcript abundances in clinical P. vivax isolates. Like genes encoding related invasion ligands of P. falciparum, Pvrbp expression levels vary broadly across different parasite isolates. Through antibody measurements, we found that host immune pressure may be the driving force behind the distinctly high diversity of one of the family members, PvRBP2c. Mild yet significant negative correlation was found between parasitemia and the PvRBP2b antibody level, suggesting that antibodies to the protein may interfere with invasion.

Plasmodium falciparum GCN5 plays a key role in regulating artemisinin resistance-related stress responses.

Plasmodium falciparum causes the most severe malaria and is exposed to various environmental and physiological stresses in the human host. Given that GCN5 plays a critical role in regulating stress responses in model organisms, we aimed to elucidate PfGCN5's function in stress responses in P. falciparum . The protein level of PfGCN5 was substantially induced under three stress conditions (heat shock, low glucose starvation, and dihydroartemisinin, the active metabolite of artemisinin (ART)). With a TetR-DOZI conditional knockdown (KD) system, we successfully down-regulated PfGCN5 to ∼50% and found that KD parasites became more sensitive to all three stress conditions. Transcriptomic analysis via RNA-seq identified ∼1,000 up-and down-regulated genes in the wildtype (WT) and KD parasites under these stress conditions. Importantly, DHA induced transcriptional alteration of many genes involved in many aspects of stress responses, which were heavily shared among the altered genes under heat shock and low glucose conditions, including ART-resistance-related genes such as K13 and coronin . Based on the expression pattern between WT and KD parasites under three stress conditions, ∼300-400 genes were identified to be involved in PfGCN5-dependent, general and stress-condition-specific responses with high levels of overlaps among three stress conditions. Notably, using ring-stage survival assay (RSA), we found that KD or inhibition of PfGCN5 could sensitize the ART-resistant parasites to the DHA treatment. All these indicate that PfGCN5 is pivotal in regulating general and ART-resistance-related stress responses in malaria parasites, implicating PfGCN5 as a potential target for malaria intervention. IMPORTANCE: Malaria leads to about half a million deaths annually and these casualties were majorly caused by the infection of Plasmodium falciparum . This parasite strives to survive by defending against a variety of stress conditions, such as malaria cyclical fever (heat shock), starvation due to low blood sugar (glucose) levels (hypoglycemia), and drug treatment. Previous studies have revealed that P. falciparum has developed unique stress responses to different stresses including ART treatment, and ART-resistant parasites harbor elevated stress responses. In this study, we provide critical evidence on the role of PfGCN5, a histone modifier, and a chromatin coactivator, in regulating general and stress-specific responses in malaria parasites, indicating that PfGCN5 can be used as a potential target for anti-malaria intervention.

SLC35B2 expression is associated with a poor prognosis of invasive ductal breast carcinoma.

BACKGROUND: Breast cancer is the most common malignancy in women worldwide, including Thailand, and is a major cause of mortality and morbidity, despite advances in diagnosis and treatment. Novel gene expression in breast cancer is a focus in searches for prognostic biomarkers and new therapeutic targets. MATERIALS AND METHODS: The mRNA expression of novel B4GALT4, SLC35B2, and WDHD1 genes in breast cancer were examined in invasive ductal breast carcinoma (IDC) patients using quantitative real-time reverse transcription polymerase chain reaction (QRT-PCR). RESULTS: Among these genes, increased expression of SLC35B2 mRNA was significantly associated with TNM stage III+IV of IDC (p<0.001). Hence, up-regulation of SLC35B2 may serve as a prognostic biomarker for poor prognosis, and is also a potential therapeutic target in breast cancer.