Transcriptional plasticity of virulence genes provides malaria parasites with greater adaptive capacity for avoiding host immunity.

Chronic, asymptomatic malaria infections contribute substantially to disease transmission and likely represent the most significant impediment preventing malaria elimination and eradication. Plasmodium falciparum parasites evade antibody recognition through transcriptional switching between members of the var gene family, which encodes the major virulence factor and surface antigen on infected red blood cells. This process can extend infections for up to a year; however, infections have been documented to last for over a decade, constituting an unseen reservoir of parasites that undermine eradication and control efforts. How parasites remain immunologically "invisible" for such lengthy periods is entirely unknown. Here we show that in addition to the accepted paradigm of mono-allelic var gene expression, individual parasites can simultaneously express multiple var genes or enter a state in which little or no var gene expression is detectable. This unappreciated flexibility provides parasites with greater adaptive capacity than previously understood and challenges the dogma of mutually exclusive var gene expression. It also provides an explanation for the antigenically "invisible" parasites observed in chronic asymptomatic infections.

There and back again: malaria parasite single-cell transcriptomics comes full circle.

The Malaria Cell Atlas (MCA) is an ambitious, ongoing project to profile the intensity and heterogeneity of gene expression throughout the entire malaria parasite life cycle with single-cell resolution. Real et al. now complete the cycle by adding the transmission stages of the most virulent malaria parasite, Plasmodium falciparum, to this easy-to-use resource.

Disruption of the Plasmodium falciparum Life Cycle through Transcriptional Reprogramming by Inhibitors of Jumonji Demethylases.

Little is known about the role of the three Jumonji C (JmjC) enzymes in Plasmodium falciparum (Pf). Here, we show that JIB-04 and other established inhibitors of mammalian JmjC histone demethylases kill asexual blood stage parasites and are even more potent at blocking gametocyte development and gamete formation. In late stage parasites, JIB-04 increased levels of trimethylated lysine residues on histones, suggesting the inhibition of P. falciparum Jumonji demethylase activity. These epigenetic defects coincide with deregulation of invasion, cell motor, and sexual development gene programs, including gene targets coregulated by the PfAP2-I transcription factor and chromatin-binding factor, PfBDP1. Mechanistically, we demonstrate that PfJmj3 converts 2-oxoglutarate to succinate in an iron-dependent manner consistent with mammalian Jumonji enzymes, and this catalytic activity is inhibited by JIB-04 and other Jumonji inhibitors. Our pharmacological studies of Jumonji activity in the malaria parasite provide evidence that inhibition of these enzymatic activities is detrimental to the parasite.

Revisiting the initial steps of sexual development in the malaria parasite Plasmodium falciparum.

Human to vector transmission of malaria requires that some blood-stage parasites abandon asexual growth and convert into non-replicating sexual forms called gametocytes. The initial steps of gametocytogenesis remain largely uncharacterized. Here, we study this part of the malaria life cycle in Plasmodium falciparum using PfAP2-G, the master regulator of sexual conversion, as a marker of commitment. We demonstrate the existence of PfAP2-G-positive sexually committed parasite stages that precede the previously known committed schizont stage. We also found that sexual conversion can occur by two different routes: the previously described route in which PfAP2-G-expressing parasites complete a replicative cycle as committed forms before converting into gametocytes upon re-invasion, or a direct route with conversion within the same cycle as initial PfAP2-G expression. The latter route is linked to early PfAP2-G expression in ring stages. Reanalysis of published single-cell RNA-sequencing (RNA-seq) data confirmed the presence of both routes. Consistent with these results, using plaque assays we observed that, in contrast to the prevailing model, many schizonts produced mixed plaques containing both asexual parasites and gametocytes. Altogether, our results reveal unexpected features of the initial steps of sexual development and extend the current view of this part of the malaria life cycle.

Loss of USP28-mediated BRAF degradation drives resistance to RAF cancer therapies.

RAF kinase inhibitors are clinically active in patients with BRAF (V600E) mutant melanoma. However, rarely do tumors regress completely, with the majority of responses being short-lived. This is partially mediated through the loss of negative feedback loops after MAPK inhibition and reactivation of upstream signaling. Here, we demonstrate that the deubiquitinating enzyme USP28 functions through a feedback loop to destabilize RAF family members. Loss of USP28 stabilizes BRAF enhancing downstream MAPK activation and promotes resistance to RAF inhibitor therapy in culture and in vivo models. Importantly, we demonstrate that USP28 is deleted in a proportion of melanoma patients and may act as a biomarker for response to BRAF inhibitor therapy in patients. Furthermore, we identify Rigosertib as a possible therapeutic strategy for USP28-depleted tumors. Our results show that loss of USP28 enhances MAPK activity through the stabilization of RAF family members and is a key factor in BRAF inhibitor resistance.

Single-Cell Transcriptome Profiling of Protozoan and Metazoan Parasites.

Single-cell RNA sequencing (scRNAseq) technologies are changing the way we study populations of cells by allowing for an unbiased characterization of the composition of these populations. This Forum article highlights outstanding questions in parasitology that could benefit from scRNAseq and provides guiding thoughts for planning such experiments.

Single-cell RNA sequencing reveals a signature of sexual commitment in malaria parasites.

Pathogens have to balance transmission with persistence. For Plasmodium falciparum, the most widespread and virulent malaria parasite, persistence within its human host requires continuous asexual replication within red blood cells, while its mosquito-borne transmission depends on intra-erythrocytic differentiation into non-replicating sexual stages called gametocytes. Commitment to either fate is determined during the preceding cell cycle that begins with invasion by a single, asexually committed merozoite and ends, 48 hours later, with a schizont releasing newly formed merozoites, all committed to either continued asexual replication or differentiation into gametocytes. Sexual commitment requires the transcriptional activation of ap2-g (PF3D7_1222600), the master regulator of sexual development, from an epigenetically silenced state during asexual replication. AP2-G expression during this 'commitment cycle' prepares gene expression in nascent merozoites to initiate sexual development through a hitherto unknown mechanism. To maintain a persistent infection, the expression of ap2-g is limited to a sub-population of parasites (1-30%, depending on genetic background and growth conditions). As sexually committed schizonts comprise only a sub-population and are morphologically indistinguishable from their asexually committed counterparts, defining their characteristic gene expression has been difficult using traditional, bulk transcriptome profiling. Here we use highly parallel, single-cell RNA sequencing of malaria cultures undergoing sexual commitment to determine the transcriptional changes induced by AP2-G within this sub-population. By analysing more than 18,000 single parasite transcriptomes from a conditional AP2-G knockdown line and NF54 wild-type parasites at multiple stages of development, we show that sexually committed, AP2-G+ mature schizonts specifically upregulate additional regulators of gene expression, including other AP2 transcription factors, histone-modifying enzymes, and regulators of nucleosome positioning. These epigenetic regulators may act to facilitate the expression and/or repression of genes that are necessary for the initiation of gametocyte development in the subsequent cell cycle.

The functional readthrough extension of malate dehydrogenase reveals a modification of the genetic code.

Translational readthrough gives rise to C-terminally extended proteins, thereby providing the cell with new protein isoforms. These may have different properties from the parental proteins if the extensions contain functional domains. While for most genes amino acid incorporation at the stop codon is far lower than 0.1%, about 4% of malate dehydrogenase (MDH1) is physiologically extended by translational readthrough and the actual ratio of MDH1x (extended protein) to 'normal' MDH1 is dependent on the cell type. In human cells, arginine and tryptophan are co-encoded by the MDH1x UGA stop codon. Readthrough is controlled by the 7-nucleotide high-readthrough stop codon context without contribution of the subsequent 50 nucleotides encoding the extension. All vertebrate MDH1x is directed to peroxisomes via a hidden peroxisomal targeting signal (PTS) in the readthrough extension, which is more highly conserved than the extension of lactate dehydrogenase B. The hidden PTS of non-mammalian MDH1x evolved to be more efficient than the PTS of mammalian MDH1x. These results provide insight into the genetic and functional co-evolution of these dually localized dehydrogenases.

Identification of New Fungal Peroxisomal Matrix Proteins and Revision of the PTS1 Consensus.

The peroxisomal targeting signal type 1 (PTS1) is a seemingly simple peptide sequence at the C-terminal end of most peroxisomal matrix proteins. PTS1 can be described as a tripeptide with the consensus motif [S/A/C] [K/R/H] L. However, this description is neither necessary nor sufficient. It does not cover all cases of PTS1 proteins, and some proteins in accordance with this consensus do not target to the peroxisome. In order to find new PTS proteins in yeast and to arrive at a more complete description of the PTS1 consensus motif, we developed a machine learning approach that involves orthologue expansion of the set of known peroxisomal proteins. We performed a genome-wide in silico screen, characterised several PTS1-containing peptides and identified two new peroxisomal matrix proteins, which we named Pxp1 (Yel020c) and Pxp2 (Yjr111c). Based on these in silico and in vivo analyses, we revised the yeast PTS1 consensus which now includes all known PTS1 proteins.