The DEAD-box RNA helicase PfDOZI imposes opposing actions on RNA metabolism in Plasmodium falciparum.

In malaria parasites, the regulation of mRNA translation, storage and degradation during development and life-stage transitions remains largely unknown. Here, we functionally characterized the DEAD-box RNA helicase PfDOZI in P. falciparum. Disruption of pfdozi enhanced asexual proliferation but reduced sexual commitment and impaired gametocyte development. By quantitative transcriptomics, we show that PfDOZI is involved in the regulation of invasion-related genes and sexual stage-specific genes during different developmental stages. PfDOZI predominantly participates in processing body-like mRNPs in schizonts but germ cell granule-like mRNPs in gametocytes to impose opposing actions of degradation and protection on different mRNA targets. We further show the formation of stress granule-like mRNPs during nutritional deprivation, highlighting an essential role of PfDOZI-associated mRNPs in stress response. We demonstrate that PfDOZI participates in distinct mRNPs to maintain mRNA homeostasis in response to life-stage transition and environmental changes by differentially executing post-transcriptional regulation on the target mRNAs.

Autochthonous Plasmodium vivax Infections, Florida, USA, 2023.

During May-July 2023, a cluster of 7 patients at local hospitals in Florida, USA, received a diagnosis of Plasmodium vivax malaria. Whole-genome sequencing of the organism from 4 patients and phylogenetic analysis with worldwide representative P. vivax genomes indicated probable single parasite introduction from Central/South America.

Porphyrin overdrive rewires cancer cell metabolism.

All cancer cells reprogram metabolism to support aberrant growth. Here, we report that cancer cells employ and depend on imbalanced and dynamic heme metabolic pathways, to accumulate heme intermediates, that is, porphyrins. We coined this essential metabolic rewiring "porphyrin overdrive" and determined that it is cancer-essential and cancer-specific. Among the major drivers are genes encoding mid-step enzymes governing the production of heme intermediates. CRISPR/Cas9 editing to engineer leukemia cell lines with impaired heme biosynthetic steps confirmed our whole-genome data analyses that porphyrin overdrive is linked to oncogenic states and cellular differentiation. Although porphyrin overdrive is absent in differentiated cells or somatic stem cells, it is present in patient-derived tumor progenitor cells, demonstrated by single-cell RNAseq, and in early embryogenesis. In conclusion, we identified a dependence of cancer cells on non-homeostatic heme metabolism, and we targeted this cancer metabolic vulnerability with a novel "bait-and-kill" strategy to eradicate malignant cells.

MalariaSED: a deep learning framework to decipher the regulatory contributions of noncoding variants in malaria parasites.

Malaria remains one of the deadliest infectious diseases. Transcriptional regulation effects of noncoding variants in this unusual genome of malaria parasites remain elusive. We developed a sequence-based, ab initio deep learning framework, MalariaSED, for predicting chromatin profiles in malaria parasites. The MalariaSED performance was validated by published ChIP-qPCR and TF motifs results. Applying MalariaSED to ~ 1.3 million variants shows that geographically differentiated noncoding variants are associated with parasite invasion and drug resistance. Further analysis reveals chromatin accessibility changes at Plasmodium falciparum rings are partly associated with artemisinin resistance. MalariaSED illuminates the potential functional roles of noncoding variants in malaria parasites.

A type II protein arginine methyltransferase regulates merozoite invasion in Plasmodium falciparum.

Protein arginine methyltransferases (PRMTs) regulate many important cellular processes, such as transcription and RNA processing in model organisms but their functions in human malaria parasites are not elucidated. Here, we characterize PfPRMT5 in Plasmodium falciparum, which catalyzes symmetric dimethylation of histone H3 at R2 (H3R2me2s) and R8, and histone H4 at R3 in vitro. PfPRMT5 disruption results in asexual stage growth defects primarily due to lower invasion efficiency of the merozoites. Transcriptomic analysis reveals down-regulation of many transcripts related to invasion upon PfPRMT5 disruption, in agreement with H3R2me2s being an active chromatin mark. Genome-wide chromatin profiling detects extensive H3R2me2s marking of genes of different cellular processes, including invasion-related genes in wildtype parasites and PfPRMT5 disruption leads to the depletion of H3R2me2s. Interactome studies identify the association of PfPRMT5 with invasion-related transcriptional regulators such as AP2-I, BDP1, and GCN5. Furthermore, PfPRMT5 is associated with the RNA splicing machinery, and PfPRMT5 disruption caused substantial anomalies in RNA splicing events, including those for invasion-related genes. In summary, PfPRMT5 is critical for regulating parasite invasion and RNA splicing in this early-branching eukaryote.

Chemogenomic Profiling of a Plasmodium falciparum Transposon Mutant Library Reveals Shared Effects of Dihydroartemisinin and Bortezomib on Lipid Metabolism and Exported Proteins.

The antimalarial activity of the frontline drug artemisinin involves generation of reactive oxygen species (ROS) leading to oxidative damage of parasite proteins. To achieve homeostasis and maintain protein quality control in the overwhelmed parasite, the ubiquitin-proteasome system kicks in. Even though molecular markers for artemisinin resistance like pfkelch13 have been identified, the intricate network of mechanisms driving resistance remains to be elucidated. Here, we report a forward genetic screening strategy that enables a broader identification of genetic factors responsible for altering sensitivity to dihydroartemisinin (DHA) and a proteasome inhibitor, bortezomib (BTZ). Using a library of isogenic piggyBac mutants in P. falciparum, we defined phenotype-genotype associations influencing drug responses and highlighted shared mechanisms between the two processes, which mainly included proteasome-mediated degradation and the lipid metabolism genes. Additional transcriptomic analysis of a DHA/BTZ-sensitive piggyBac mutant showed it is possible to find differences between the two response mechanisms on the specific components for regulation of the exportome. Our results provide further insight into the molecular mechanisms of antimalarial drug resistance. IMPORTANCE Malaria control is seriously threatened by the emergence and spread of Plasmodium falciparum resistance to the leading antimalarial, artemisinin. The potent killing activity of artemisinin results from oxidative damage unleashed by free heme activation released by hemoglobin digestion. Although the ubiquitin-proteasome system is considered critical for parasite survival of this toxicity, the diverse genetic changes linked to artemisinin resistance are complex and, so far, have not included the ubiquitin-proteasome system. In this study, we use a systematic forward genetic approach by screening a library of P. falciparum random piggyBac mutants to decipher the genetic factors driving malaria parasite responses to the oxidative stress caused by antimalarial drugs. This study compares phenotype-genotype associations influencing dihydroartemisinin responses with the proteasome inhibitor bortezomib to delineate the role of ubiquitin-proteasome system. Our study highlights shared and unique pathways from the complex array of molecular processes critical for P. falciparum survival resulting from the oxidative damage of artemisinin.

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.

Large Mammals Have More Powerful Antibacterial Defenses Than Expected from Their Metabolic Rates.

AbstractTerrestrial mammals span seven orders of magnitude in body size, ranging from the <2-g Etruscan pygmy shrew (Suncus etruscus) to the >3,900-kg African elephant (Loxodonta africana). Although body size profoundly affects the behavior, physiology, ecology, and evolution of species, how investment in functional immune defenses changes with body size across species is unknown. Here, we (1) developed a novel 12-point dilution curve approach to describe and compare antibacterial capacity against three bacterial species among >160 terrestrial species of mammals and (2) tested published predictions about the scaling of immune defenses. Our study focused on the safety factor hypothesis, which predicts that broad, early-acting immune defenses should scale hypermetrically with body mass. However, our three statistical approaches demonstrated that antibacterial activity in sera across mammals exhibits isometry; killing capacity did not change with body size across species. Intriguingly, this result indicates that the serum of a large mammal is less hospitable to bacteria than would be predicted by its metabolic rates. In other words, if metabolic rates underlie the rates of physiological reactions as postulated by the metabolic theory of ecology, large species should have disproportionately lower antibacterial capacity than small species, but they do not. These results have direct implications for effectively modeling the evolution of immune defenses and identifying potential reservoir hosts of pathogens.

Epigenetic Potential and DNA Methylation in an Ongoing House Sparrow (Passer domesticus) Range Expansion.

AbstractDuring range expansions, organisms can use epigenetic mechanisms to adjust to conditions in novel areas by altering gene expression and enabling phenotypic plasticity. Here, we predicted that the number of CpG sites within the genome, one form of epigenetic potential, would be important for successful range expansions because DNA methylation can modulate gene expression and, consequently, plasticity. We asked how the number of CpG sites and DNA methylation varied across five locations in the ∼70-year-old Kenyan house sparrow (Passer domesticus) range expansion. We found that the number of CpG sites was highest toward the vanguard of the invasion and decreased toward the range core. Analysis suggests that this pattern may have been driven by selection, favoring birds with more CpG sites at the range edge. However, we cannot rule out other processes, including nonrandom gene flow. Additionally, DNA methylation did not change across the range expansion, nor was it more variable. We hypothesize that as new areas are colonized, epigenetic potential may be selectively advantageous early but eventually be replaced by less plastic and perhaps genetically canalized traits as populations adapt to local conditions. Although further work is needed on epigenetic potential, this form (CpG number) appears to be a promising mechanism to investigate as a driver of expansions via capacitated phenotypic plasticity in other natural and anthropogenic range expansions.

A unique class of Zn2+-binding serine-based PBPs underlies cephalosporin resistance and sporogenesis in Clostridioides difficile.

Treatment with ß-lactam antibiotics, particularly cephalosporins, is a major risk factor for Clostridioides difficile infection. These broad-spectrum antibiotics irreversibly inhibit penicillin-binding proteins (PBPs), which are serine-based enzymes that assemble the bacterial cell wall. However, C. difficile has four different PBPs (PBP1-3 and SpoVD) with various roles in growth and spore formation, and their specific links to ß-lactam resistance in this pathogen are underexplored. Here, we show that PBP2 (known to be essential for vegetative growth) is the primary bactericidal target for ß-lactams in C. difficile. PBP2 is insensitive to cephalosporin inhibition, and this appears to be the main basis for cephalosporin resistance in this organism. We determine crystal structures of C. difficile PBP2, alone and in complex with ß-lactams, revealing unique features including ligand-induced conformational changes and an active site Zn2+-binding motif that influences ß-lactam binding and protein stability. The Zn2+-binding motif is also present in C. difficile PBP3 and SpoVD (which are known to be essential for sporulation), as well as in other bacterial taxa including species living in extreme environments and the human gut. We speculate that this thiol-containing motif and its cognate Zn2+ might function as a redox sensor to regulate cell wall synthesis for survival in adverse or anaerobic environments.

Probing the distinct chemosensitivity of Plasmodium vivax liver stage parasites and demonstration of 8-aminoquinoline radical cure activity in vitro.

Improved control of Plasmodium vivax malaria can be achieved with the discovery of new antimalarials with radical cure efficacy, including prevention of relapse caused by hypnozoites residing in the liver of patients. We screened several compound libraries against P. vivax liver stages, including 1565 compounds against mature hypnozoites, resulting in one drug-like and several probe-like hits useful for investigating hypnozoite biology. Primaquine and tafenoquine, administered in combination with chloroquine, are currently the only FDA-approved antimalarials for radical cure, yet their activity against mature P. vivax hypnozoites has not yet been demonstrated in vitro. By developing an extended assay, we show both drugs are individually hypnozonticidal and made more potent when partnered with chloroquine, similar to clinically relevant combinations. Post-hoc analyses of screening data revealed excellent performance of ionophore controls and the high quality of single point assays, demonstrating a platform able to support screening of greater compound numbers. A comparison of P. vivax liver stage activity data with that of the P. cynomolgi blood, P. falciparum blood, and P. berghei liver stages reveals overlap in schizonticidal but not hypnozonticidal activity, indicating that the delivery of new radical curative agents killing P. vivax hypnozoites requires an independent and focused drug development test cascade.

A unique GCN5 histone acetyltransferase complex controls erythrocyte invasion and virulence in the malaria parasite Plasmodium falciparum.

The histone acetyltransferase GCN5-associated SAGA complex is evolutionarily conserved from yeast to human and functions as a general transcription co-activator in global gene regulation. In this study, we identified a divergent GCN5 complex in Plasmodium falciparum, which contains two plant homeodomain (PHD) proteins (PfPHD1 and PfPHD2) and a plant apetela2 (AP2)-domain transcription factor (PfAP2-LT). To dissect the functions of the PfGCN5 complex, we generated parasite lines with either the bromodomain in PfGCN5 or the PHD domain in PfPHD1 deleted. The two deletion mutants closely phenocopied each other, exhibiting significantly reduced merozoite invasion of erythrocytes and elevated sexual conversion. These domain deletions caused dramatic decreases not only in histone H3K9 acetylation but also in H3K4 trimethylation, indicating synergistic crosstalk between the two euchromatin marks. Domain deletion in either PfGCN5 or PfPHD1 profoundly disturbed the global transcription pattern, causing altered expression of more than 60% of the genes. At the schizont stage, these domain deletions were linked to specific down-regulation of merozoite genes involved in erythrocyte invasion, many of which contain the AP2-LT binding motif and are also regulated by AP2-I and BDP1, suggesting targeted recruitment of the PfGCN5 complex to the invasion genes by these specific factors. Conversely, at the ring stage, PfGCN5 or PfPHD1 domain deletions disrupted the mutually exclusive expression pattern of the entire var gene family, which encodes the virulent factor PfEMP1. Correlation analysis between the chromatin state and alteration of gene expression demonstrated that up- and down-regulated genes in these mutants are highly correlated with the silent and active chromatin states in the wild-type parasite, respectively. Collectively, the PfGCN5 complex represents a novel HAT complex with a unique subunit composition including an AP2 transcription factor, which signifies a new paradigm for targeting the co-activator complex to regulate general and parasite-specific cellular processes in this low-branching parasitic protist.

The apicoplast link to fever-survival and artemisinin-resistance in the malaria parasite.

The emergence and spread of Plasmodium falciparum parasites resistant to front-line antimalarial artemisinin-combination therapies (ACT) threatens to erase the considerable gains against the disease of the last decade. Here, we develop a large-scale phenotypic screening pipeline and use it to carry out a large-scale forward-genetic phenotype screen in P. falciparum to identify genes allowing parasites to survive febrile temperatures. Screening identifies more than 200 P. falciparum mutants with differential responses to increased temperature. These mutants are more likely to be sensitive to artemisinin derivatives as well as to heightened oxidative stress. Major processes critical for P. falciparum tolerance to febrile temperatures and artemisinin include highly essential, conserved pathways associated with protein-folding, heat shock and proteasome-mediated degradation, and unexpectedly, isoprenoid biosynthesis, which originated from the ancestral genome of the parasite's algal endosymbiont-derived plastid, the apicoplast. Apicoplast-targeted genes in general are upregulated in response to heat shock, as are other Plasmodium genes with orthologs in plant and algal genomes. Plasmodium falciparum parasites appear to exploit their innate febrile-response mechanisms to mediate resistance to artemisinin. Both responses depend on endosymbiont-derived genes in the parasite's genome, suggesting a link to the evolutionary origins of Plasmodium parasites in free-living ancestors.

Light pollution affects West Nile virus exposure risk across Florida.

Emerging infectious diseases (EIDs) present global health threats, and their emergences are often linked to anthropogenic change. Artificial light at night (ALAN) is one form of anthropogenic change that spans beyond urban boundaries and may be relevant to EIDs through its influence on the behaviour and physiology of hosts and/or vectors. Although West Nile virus (WNV) emergence has been described as peri-urban, we hypothesized that exposure risk could also be influenced by ALAN in particular, which is testable by comparing the effects of ALAN on prevalence while controlling for other aspects of urbanization. By modelling WNV exposure among sentinel chickens in Florida, we found strong support for a nonlinear relationship between ALAN and WNV exposure risk in chickens with peak WNV risk occurring at low ALAN levels. Although our goal was not to discern how ALAN affected WNV relative to other factors, effects of ALAN on WNV exposure were stronger than other known drivers of risk (i.e. impervious surface, human population density). Ambient temperature in the month prior to sampling, but no other considered variables, strongly influenced WNV risk. These results indicate that ALAN may contribute to spatio-temporal changes in WNV risk, justifying future investigations of ALAN on other vector-borne parasites.

The human malaria parasite genome is configured into thousands of coexpressed linear regulatory units.

The human malaria parasite Plasmodium falciparum thrives in radically different host environments in mosquitoes and humans, with only a limited set of transcription factors. The nature of regulatory elements or their target genes in the P. falciparum genome remains elusive. Here, we found that this eukaryotic parasite uses an efficient way to maximally use genetic and epigenetic regulation to form regulatory units (RUs) during blood infections. Genes located in the same RU tend to have the same pattern of expression over time and are associated with open chromatin along regulatory elements. To precisely define and quantify these RUs, a novel hidden Markov model was developed to capture the regulatory structure in a genome-wide fashion by integrating expression and epigenetic evidence. We successfully identified thousands of RUs and cross-validated with previous findings. We found more genes involved in red blood cell (RBC) invasion located in the same RU as the PfAP2-I (AP2-I) transcription factor, demonstrating that AP2-I is responsible for regulating RBC invasion. Our study has provided a regulatory mechanism for a compact eukaryotic genome and offers new insights into the in vivo transcriptional regulation of the P. falciparum intraerythrocytic stage.

False Alarms in Consumer Genomics Add to Public Fear and Potential Health Care Burden.

We have entered an era of direct-to-consumer (DTC) genomics. Patients have relayed many success stories of DTC genomics about finding causal mutations of genetic diseases before showing any symptoms and taking precautions. However, consumers may also take unnecessary medical actions based on false alarms of "pathogenic alleles". The severity of this problem is not well known. Using publicly available data, we compared DTC microarray genotyping data with deep-sequencing data of 5 individuals and manually checked each inconsistently reported single nucleotide variants (SNVs). We estimated that, on average, a person would have ~5 "pathogenic" alleles reported due to wrongly reported genotypes if using a 23andMe genotyping microarray. We also found that the number of wrongly classified "pathogenic" alleles per person is at least as significant as those due to wrongly reported genotypes. We show that the scale of the false alarm problem could be large enough that the medical costs will become a burden to public health.

Lineage-Specific Expansion of Plasmodium falciparum Parasites With pfhrp2 Deletion in the Greater Mekong Subregion.

Deletion of the pfhrp2 gene in Plasmodium falciparum can lead to false-negative rapid diagnostic test (RDT) results, constituting a major challenge for evidence-based malaria treatment. Here we analyzed the whole genome sequences of 138 P. falciparum clinical samples collected from the China-Myanmar boarder for pfhrp2 and pfhrp3 gene deletions. We found pfhrp2 and pfhrp3 deletions in 9.4% and 3.6% of samples, respectively, with no samples harboring deletions of both genes. The pfhrp2 deletions showed 2 distinct breakpoints, representing 2 different chromosomal deletion events. A phylogenetic analysis performed using genome-wide single-nucleotide polymorphisms revealed that the 2 pfhrp2 breakpoint groups as well as all the pfhrp3-negative parasites formed separate clades, suggesting they might have resulted from clonal expansion of pfhrp2- and pfhrp3-negative parasites. These findings highlight the need for urgent surveys to determine the prevalence of pfhrp2-negative parasites causing false-negative RDT results and a plan for switching of RDTs pending the survey results.

An adaptable soft-mold embossing process for fabricating optically-accessible, microfeature-based culture systems and application toward liver stage antimalarial compound testing.

Advanced cell culture methods for modeling organ-level structure have been demonstrated to replicate in vivo conditions more accurately than traditional in vitro cell culture. Given that the liver is particularly important to human health, several advanced culture methods have been developed to experiment with liver disease states, including infection with Plasmodium parasites, the causative agent of malaria. These models have demonstrated that intrahepatic parasites require functionally stable hepatocytes to thrive and robust characterization of the parasite populations' response to investigational therapies is dependent on high-content and high-resolution imaging (HC/RI). We previously reported abiotic confinement extends the functional longevity of primary hepatocytes in a microfluidic platform and set out to instill confinement in a microtiter plate platform while maintaining optical accessibility for HC/RI; with an end-goal of producing an improved P. vivax liver stage culture model. We developed a novel fabrication process in which a PDMS soft mold embosses hepatocyte-confining microfeatures into polystyrene, resulting in microfeature-based hepatocyte confinement (µHEP) slides and plates. Our process was optimized to form both microfeatures and culture wells in a single embossing step, resulting in a 100 µm-thick bottom ideal for HC/RI, and was found inexpensively amendable to microfeature design changes. Microfeatures improved intrahepatic parasite infection rates and µHEP systems were used to reconfirm the activity of reference antimalarials in phenotypic dose-response assays. RNAseq of hepatocytes in µHEP systems demonstrated microfeatures sustain hepatic differentiation and function, suggesting broader utility for preclinical hepatic assays; while our tailorable embossing process could be repurposed for developing additional organ models.