Malaria parasites induce changes in the permeability of the infected erythrocyte membrane to numerous solutes, including toxic compounds. In Plasmodium falciparum, this is mainly mediated by PSAC, a broad-selectivity channel that requires the product of parasite clag3 genes for its activity. The two paralogous clag3 genes, clag3.1 and clag3.2, can be silenced by epigenetic mechanisms and show mutually exclusive expression. Here we show that resistance to the antibiotic blasticidin S (BSD) is associated with switches in the expression of these genes that result in altered solute uptake. Low concentrations of the drug selected parasites that switched from clag3.2 to clag3.1 expression, implying that expression of one or the other clag3 gene confers different transport efficiency to PSAC for some solutes. Selection with higher BSD concentrations resulted in simultaneous silencing of both clag3 genes, which severely compromises PSAC formation as demonstrated by blocked uptake of other PSAC substrates. Changes in the expression of clag3 genes were not accompanied by large genetic rearrangements or mutations at the clag3 loci or elsewhere in the genome. These results demonstrate that malaria parasites can become resistant to toxic compounds such as drugs by epigenetic switches in the expression of genes necessary for the formation of solute channels.

Background. Many genes of the malaria parasite Plasmodium falciparum show clonally variant expression regulated at the epigenetic level. These genes participate in fundamental host-parasite interactions and contribute to adaptive processes. However, little is known about their expression patterns during human infections. A peculiar case of clonally variant genes are the 2 nearly identical clag3 genes, clag3.1 and clag3.2, which mediate nutrient uptake and are linked to resistance to some toxic compounds.Methods. We developed a procedure to characterize the expression of clag3 genes in naturally infected patients and in experimentally infected human volunteers.Results. We provide the first description of clag3 expression during human infections, which revealed mutually exclusive expression and identified the gene predominantly expressed. Adaptation to culture conditions or selection with a toxic compound resulted in isolate-dependent changes in clag3 expression. We also found that clag3 expression patterns were reset during transmission stages.Conclusions. Different environment conditions select for parasites with different clag3 expression patterns, implying functional differences between the proteins encoded. The epigenetic memory is likely erased before parasites start infection of a new human host. Altogether, our findings support the idea that clonally variant genes facilitate the adaptation of parasite populations to changing conditions through bet-hedging strategies.

The product of the Plasmodium falciparum genes clag3.1 and clag3.2 plays a fundamental role in malaria parasite biology by determining solute transport into infected erythrocytes. Expression of the two clag3 genes is mutually exclusive, such that a single parasite expresses only one of the two genes at a time. Here we investigated the properties and mechanisms of clag3 mutual exclusion using transgenic parasite lines with extra copies of clag3 promoters located either in stable episomes or integrated in the parasite genome. We found that the additional clag3 promoters in these transgenic lines are silenced by default, but under strong selective pressure parasites with more than one clag3 promoter simultaneously active are observed, demonstrating that clag3 mutual exclusion is strongly favored but it is not strict. We show that silencing of clag3 genes is associated with the repressive histone mark H3K9me3 even in parasites with unusual clag3 expression patterns, and we provide direct evidence for heterochromatin spreading in P. falciparum. We also found that expression of a neighbor ncRNA correlates with clag3.1 expression. Altogether, our results reveal a scenario where fitness costs and non-deterministic molecular processes that favor mutual exclusion shape the expression patterns of this important gene family.

During the intraerythrocytic asexual cycle malaria parasites acquire nutrients and other solutes through a broad selectivity channel localized at the membrane of the infected erythrocyte termed the plasmodial surface anion channel (PSAC). The protein product of the Plasmodium falciparum clonally variant clag3.1 and clag3.2 genes determines PSAC activity. Switches in the expression of clag3 genes, which are regulated by epigenetic mechanisms, are associated with changes in PSAC-dependent permeability that can result in resistance to compounds toxic for the parasite, such as blasticidin S. Here, we investigated whether other antimalarial drugs require CLAG3 to reach their intracellular target and consequently are prone to parasite resistance by epigenetic mechanisms. We found that the bis-thiazolium salts T3 (also known as albitiazolium) and T16 require the product of clag3 genes to enter infected erythrocytes. P. falciparum populations can develop resistance to these compounds via the selection of parasites with dramatically reduced expression of both genes. However, other compounds previously demonstrated or predicted to enter infected erythrocytes through transport pathways absent from noninfected erythrocytes, such as fosmidomycin, doxycycline, azithromycin, lumefantrine, or pentamidine, do not require expression of clag3 genes for their antimalarial activity. This suggests that they use alternative CLAG3-independent routes to access parasites. Our results demonstrate that P. falciparum can develop resistance to diverse antimalarial compounds by epigenetic changes in the expression of clag3 genes. This is of concern for drug development efforts because drug resistance by epigenetic mechanisms can arise quickly, even during the course of a single infection.

Background: Type 2 diabetes mellitus (T2DM) is a highly prevalent disease associated with an increased risk of comorbidities, premature death, and health costs. Prediabetes is a stage of glucose alteration previous to T2DM, that can be reversed. The aim of the study is to develop and evaluate a low-intensity, multifaceted, digital intervention to prevent T2DM. The intervention comprises: (1) the use of mobile health technology to send tailored text messages promoting lifestyle changes to people at risk of T2DM and (2) the provision of online education to primary healthcare physicians and nurses about management of prediabetes. Methods: In stages 1–4 we will design, develop and pilot-test the intervention. In Stage 5 we will conduct a phase II, six-month, three-arm, cluster randomized, clinical trial with 42 primary care professionals and 420 patients at risk of T2DM. Patients will be allocated to a control group (usual care), intervention A (patient messaging intervention), or intervention B (patient messaging intervention plus online education to their primary healthcare professionals). The primary outcome will be glycated haemoglobin. All the procedures obtained ethical approval in June 2021 (CEI-IB Ref No: IB4495/21PI). Discussion: Digital health interventions can effectively prevent T2DM and reduce important T2DM risk factors such as overweight or hypertension. In Spain, this type of intervention is understudied. Moreover, there is controversy regarding the type of digital health interventions that are more effective. Findings from this study may contribute to address T2DM prevention, through a low-cost and easily implementable intervention.

type 2 diabetes mellitus (T2DM) based on: i) the use of a system comprising mobile health (mHealth) technology integrated with electronic health records to send tailored text messages (SMSs) promoting lifestyle changes in people at risk of T2DM, and, ii) the provision of online education to primary healthcare workers about prediabetes management. Design: The PREDIABETEXT project is a controlled, parallel, three-arm, cluster randomized trial and involve five phases. Methods: In phases 1-4 we will develop and pilot-test the different components of the intervention (In phase 1 we will develop the brief text messages targeted to patients; In phase 2 we will adapt our existing technology system to deliver the messages; In phase 3 we will develop an educational intervention targeted to Primary Care workers, and; In phase 4 we will pilot-test and optimise both interventions). In phase 5 we will conduct a phase II, six-month, three-arm, cluster randomised, clinical trial with 42 primary care workers and 420 patients at risk of T2DM (HbA1c from 6% to 6.4% or fasting plasma glucose 110-125mg/dl, or both). Patients will be allocated to a control (usual care) group, intervention A (patient messaging intervention), or intervention B (patient messaging intervention plus online education to their primary healthcare workers). The primary outcome will be HbA1c. Secondary outcomes will include additional clinical, physiological, behavioural and psychological outcomes. Discussion: Recent trials suggest that digital health interventions can effectively prevent T2DM and reduce important T2DM risk factors such as overweight or hypertension. In Spain this type of interventions is understudied.

No abstract