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Babesia species are tick-borne intracellular parasites that infect the red blood cells of their mammalian host, leading to severe or fatal disease. Babesia spp. infect a wide range of mammalian species and cause a significant economic burden globally, predominantly through disease in cattle. Several Babesia spp. are increasingly being recognized as zoonotic pathogens of humans. Babesia spp. have complex life cycles involving multiple stages in the tick and the mammalian host. The parasite utilizes complex signaling pathways during replication, egress, and invasion in each of these stages. They must also rapidly respond to their environment when switching between the mammalian and tick stages. This review will focus on the signaling pathways and environmental stimuli that Babesia spp. utilize in the bloodstream and for transmission to the tick, with an emphasis on the role of phosphorylation- and calcium-based signaling during egress and invasion. The expanding availability of in vitro and in vivo culture systems, genomes, transcriptomes, and transgenic systems available for a range of Babesia spp. should encourage further biological and translational studies of these ubiquitous parasites., (© 2020 John Wiley & Sons Ltd.)

Malaria is one of the most life-threatening infectious diseases worldwide, caused by infection of humans with parasites of the genus Plasmodium. The complex life cycle of Plasmodium parasites is shared between two hosts, with infection of multiple cell types, and the parasite needs to adapt for survival and transmission through significantly different metabolic environments. Within the blood-stage alone, parasites encounter changing levels of key nutrients, including sugars, amino acids, and lipids, due to differences in host dietary nutrition, cellular tropism, and pathogenesis. In this review, we consider the mechanisms that the most lethal of malaria parasites, Plasmodium falciparum, uses to sense nutrient levels and elicit changes in gene expression during blood-stage infections. These changes are brought about by several metabolic intermediates and their corresponding sensor proteins. Sensing of distinct nutritional signals can drive P. falciparum to alter the key blood-stage processes of proliferation, antigenic variation, and transmission., (© 2020 John Wiley & Sons Ltd.)

The Plasmodium RhopH complex is a high molecular weight antigenic complex consisting of three subunits - RhopH1/clag, RhopH2 and RhopH3 - located in the rhoptry secretory organelles of the invasive merozoite. In Plasmodium falciparum RhopH1/clag is encoded by one of five clag genes. Two highly similar paralogous genes, clag 3.1 and clag 3.2, are mutually exclusively expressed. Here we show clonal switching from the clag 3.2 to the clag 3.1 paralogue in vitro. Chromatin immunoprecitation studies suggest that silencing of either clag 3 paralogue is associated with the enrichment of specific histone modifications associated with heterochromatin. We were able to disrupt the clag 3.2 gene, with a drug cassette inserted into the clag 3.2 locus being readily silenced in a position-dependent and sequence-independent manner. Activation of this drug cassette by drug selection results in parasites with the clag 3.1 locus silenced and lack full-length clag 3.1 or 3.2 transcripts. These clag 3-null parasites demonstrate a significant growth inhibition compared with wild-type parasites, providing the first genetic evidence for a role for these proteins in efficient parasite proliferation. Epigenetic regulation of these chromosomally proximal members of a multigene family provides a mechanism for both immune evasion and functional diversification., (© 2011 Blackwell Publishing Ltd.)

The pathogenesis of Plasmodium falciparum depends on efficient invasion into host erythrocytes. Parasite ligands encoded by multi-gene families interact with erythrocyte receptors. P. falciparum reticulocyte binding protein homologues (PfRhs) are expressed at the apical surface of invasive merozoites and have divergent ectodomains that are postulated to bind different erythrocyte receptors. Variant expression of these paralogues results in the use of alternative invasion pathways. Two PfRh proteins, PfRh2a and PfRh2b, are identical for 2700 N-terminal amino acids and differ only in a C-terminal 500 amino acid region, which includes a unique ectodomain, transmembrane domain and cytoplasmic domain. Despite their similarity, PfRh2b is required for a well-defined invasion pathway while PfRh2a is not required or sufficient for this pathway. Mapping the genomic region encoding these proteins revealed a recombinogenic locus with PfRh2a and PfRh2b in a head-to-head orientation. We have generated viable PfRh2a/2b chimeric parasites to identify the regions required for alternative invasion pathway utilization. We find that the differential ability to use these pathways is conferred by the cytoplasmic domains of PfRh2a and PfRh2b, not the ectodomain or transmembrane regions. Our results highlight the importance of the cytoplasmic domain for functional diversification of a major adhesive ligand family in malaria parasites.

Plasmodium falciparum is the most virulent of the Plasmodium species infective to humans. Different P. falciparum strains vary in their dependence on erythrocyte receptors for invasion and their ability to switch in their utilization of different receptor repertoires. Members of the reticulocyte-binding protein-like (RBL) family of invasion ligands are postulated to play a central role in defining ligand-receptor interactions, known as invasion pathways. Here we report the targeted gene disruption of PfRh2b and PfRh2a in W2mef, a parasite strain that is heavily dependent on sialic-acid receptors for invasion, and show that the PfRh2b ligand is functional in this parasite background. Like the parental line, parasites lacking either PfRh2a or PfR2b can switch to a sialic acid-independent invasion pathway. However, both of the switched lines exhibit a reduced efficiency for invasion into sialic acid-depleted cells, suggesting a role for both PfRh2b and PfRh2a in invasion via sialic acid-independent receptors. We also find a strong selective pressure for the reconstitution of PfRh2b expression at the expense of PfRh2a. Our results reveal the importance of genetic background in ligand-receptor usage by P. falciparum parasites, and suggest that the co-ordinate expression of PfRh2a, PfRh2b together mediate efficient sialic acid-independent erythrocyte invasion.

Epigenetic phenomena have been shown to play a role in the regulated expression of virulence genes in several pathogenic organisms, including the var gene family in Plasmodium falciparum. A better understanding of how P. falciparum can both maintain a single active var gene locus through many erythrocytic cycles and also achieve successive switching to different loci in order to evade the host immune system is greatly needed. Disruption of this tightly co-ordinated expression system presents an opportunity for increased clearance of the parasites by the immune system and, in turn, reduced mortality and morbidity. In the current issue of Molecular Microbiology, Lopez-Rubio and colleagues investigate the correlation of specific post-translational histone modifications with different transcriptional states of a single var gene, var2csa. Quantitative chromatin immunoprecipitation is used to demonstrate that different histone methylation marks are enriched at the 5' flanking and coding regions of active, poised or silenced var genes. They identify an increase of H3K4me2 and H3K4me3 in the 5' flanking region of an active var locus and expand on an earlier finding that H3K9me3 is enriched in the coding regions of silenced var genes. The authors also present evidence that H3K4me2 bookmarks the active var gene locus during later developmental stages for expression in the subsequent asexual cycle, hinting at a potential mechanism for transcriptional 'memory'. The stage is now set for work generating a complete catalogue of all histone modifications associated with var gene regulation as well as functional studies striving to uncover the precise mechanisms underlying these observations.

In recent years, the sequencing and annotation of complete genomes, together with the development of genetic and proteomic techniques to study previously intractable eukaryotic microbes, has revealed interesting new themes in the control of virulence gene expression. Families of variantly expressed genes are found adjacent to telomeres in the genomes of both pathogenic and non-pathogenic organisms. This subtelomeric DNA is normally heterochromatic and higher-order chromatin structure has now come to be recognized as an important factor controlling both the evolution and expression dynamics of these multigene families. In eukaryotic cells, higher-order chromatin structure plays a central role in many DNA processes including the control of chromosome integrity and recombination, DNA partitioning during cell division, and transcriptional control. DNA can be packaged in two distinct forms: euchromatin is relatively accessible to DNA binding proteins and generally contains active genes, while heterochromatin is densely packaged, relatively inaccessible and usually transcriptionally silent. These features of chromatin are epigenetically inherited from cell cycle to cell cycle. This review will focus on the epigenetic mechanisms used to control expression of virulence genes in medically important microbial pathogens. Examples of such control have now been reported in several evolutionarily distant species, revealing what may be a common strategy used to regulate many very different families of genes.

Ubiquitination is a post-translational modification implicated in a variety of cellular functions, including transcriptional regulation, protein degradation and membrane protein trafficking. Ubiquitin and the enzymes that act on it, although conserved and essential in eukaryotes, have not been well studied in parasites, despite sequencing of several parasite genomes. Several putative ubiquitin hydrolases have been identified in Plasmodium falciparum based on sequence homology alone, with no evidence of expression or function. Here we identify the first deubiquitinating enzyme in P. falciparum, PfUCH54, by its activity. We show that PfUCH54 also has deNeddylating activity, as assayed by a mammalian Nedd8-based probe. This activity is absent from mammalian homologues of PfUCH54. Given the importance of parasitic membrane protein trafficking as well as protein degradation in the virulence of this parasite, this family of enzymes may represent a target for pharmacological intervention with this disease.

A decline in our ability to successfully treat patients with malaria infections of the parasitic protozoan Plasmodium falciparum with cheap quinoline drugs has led to a huge escalation in morbidity and mortality in recent years. Many approaches have been taken, including classical genetics, reverse genetics and molecular epidemiology, to identify the molecular determinants underlying this resistance. The contribution of the P. falciparum multidrug resistance gene, pfmdr1, to antimalarial resistance has been a source of controversy for over a decade since it was first identified. In the current issue of Molecular Microbiology, Sidhu and colleagues use powerful reverse genetics to demonstrate the importance of commonly occurring alleles of pfmdr1 in conferring resistance to the second-line drugs quinine and sensitivity to the new alternatives mefloquine and artemisinin. They also elegantly highlight the importance of genetic background and epistasis between pfmdr1 and other potential modulators of drug resistance. Such molecular knowledge will facilitate surveillance/monitoring and aid the development of strategies for the reversal of resistance.

The Apicomplexan parasite responsible for the most virulent form of malaria, Plasmodium falciparum, invades human erythrocytes through multiple ligand-receptor interactions. Some strains of P. falciparum are sensitive to neuraminidase treatment of the host erythrocyte and these parasites have been termed sialic acid-dependent as they utilize receptors containing sialic acid. In contrast, other strains can efficiently invade neuraminidase-treated erythrocytes and hence are sialic acid-independent. The molecular interactions that allow P. falciparum to differentially utilize receptors for merozoite invasion are not understood. The P. falciparum reticulocyte-binding protein homologue (PfRh or PfRBL) family have been implicated in the invasion process but their exact role is unknown. PfRh1, a member of this protein family, appears to be expressed in all parasite lines analysed but there are marked differences in the level of expression between different strains. We have used targeted gene disruption of the PfRh1 gene in P. falciparum to show that the encoded protein is required for sialic acid-dependent invasion of human erythrocytes. The DeltaPfRh1 parasites are able to invade normally; however, they utilize a pattern of ligand-receptor interactions that are more neuraminidase-resistant. Current data suggest a strategy based on the differential function of specific PfRh proteins has evolved to allow P. falciparum parasites to utilize alternative receptors on the erythrocyte surface for evasion of receptor polymorphisms and the host immune system.

The declining efficacy of chloroquine and pyrimethamine/sulphadoxine in the treatment of human malaria has led to the use of newer antimalarials such as mefloquine and artemisinin. Sequence polymorphisms in the pfmdr1 gene, the gene encoding the plasmodial homologue of mammalian multidrug resistance transporters, have previously been linked to resistance to chloroquine in some, but not all, studies. In this study, we have used a genetic cross between the strains HB3 and 3D7 to study inheritance of sensitivity to the structurally unrelated drugs mefloquine and artemisinin, and to several other antimalarials. We find a complete allelic association between the HB3-like pfmdr1 allele and increased sensitivity to these drugs in the progeny. Different pfmdr1 sequence polymorphisms in other unrelated lines were also associated with increased sensitivity to these drugs. Our results indicate that the pfmdr1 gene is an important determinant of susceptibility to antimalarials, which has major implications for the future development of resistance.

The efficacy of sulphadoxine/pyrimethamine (S/P) in treatment of uncomplicated falciparum malaria in Africa is increasingly compromised by development of resistance. The occurrence of mutations associated with the active site sequence in the Plasmodium falciparum genes coding for dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) is associated with in vitro resistance to pyrimethamine and sulphadoxine. This study investigates the occurrence of these mutations in infected blood samples taken from Tanzanian children before treatment with S/P and their relationship to parasite breakthrough by day 7. The results show that alleles of DHPS (436-alanine, 437-alanine and 540-lysine) were significantly reduced in prevalence on day 7 after S/P treatment. In this area, a DHPS with 436-serine, 437-glycine and 540-glutamate appears to play a major role in resistance to S/P in vivo. Evidence for the influence of mutations in the DHFR gene in this investigation is not clear, probably because of the high prevalence of 'resistance-related' mutations at day 0 in the local parasite population. For apparently the same reason, it was not possible to show a statistical association between S/P resistance and the presence of particular polymorphisms in the DHFR and DHPS genes before treatment.

Recently the efficacy of sulfadoxine/pyrimethamine (S/P) in treatment of uncomplicated falciparum malaria in Tanzania has been seriously compromised by the development of resistance. The occurrence of active site mutations in the Plasmodium falciparum gene sequence coding for dihydrofolate reductase (DHFR) is known to confer resistance to pyrimethamine. This study investigates the occurrence of these mutations in infected blood samples taken from Tanzanian children before treatment with S/P and their relationship to parasite breakthrough by day 7. The results confirm the occurrence of one or more DHFR mutations in all the samples, but no relationship was found with the presence of parasites in the blood at day 7. The results suggest that alterations in the coding region for dihydropteroate synthetase (DHPS), the enzyme target for sulfadoxine, should be studied in order to predict resistance to the S/P combination. It has been proposed earlier that sulfadoxine could itself act on DHFR, because of a false dihydrofolate produced by drug metabolism through DHPS and dihydrofolate synthase. The results of this treatment study suggest that such a possibility is unlikely.