Expansion of a specific plasmodium falciparum PfMDR1 Haplotype in southeast Asia with increased substrate transport

Artemisinin-based combination therapies (ACTs) have been vital in reducing malaria mortality rates since the 2000s. Their efficacy, however, is threatened by the emergence and spread of artemisinin resistance in Southeast Asia. The Plasmodium falciparum multidrug resistance protein 1 (PfMDR1) transporter plays a central role in parasite resistance to ACT partner drugs through gene copy number variations (CNV) and/or single nucleotide polymorphisms (SNPs). Using genomic epidemiology, we show that multiple pfmdr1 copies encoding the N86 and 184F haplotype are prevalent across Southeast Asia. Applying genome editing tools on the Southeast Asian Dd2 strain and using a surrogate assay to measure transporter activity in infected red blood cells, we demonstrate that parasites harboring multicopy N86/184F PfMDR1 have a higher Fluo-4 transport capacity compared with those expressing the wild-type N86/Y184 haplotype. Multicopy N86/184F PfMDR1 is also associated with decreased parasite susceptibility to lumefantrine. These findings provide evidence of the geographic selection and expansion of specific multicopy PfMDR1 haplotypes associated with multidrug resistance in Southeast Asia.IMPORTANCE Global efforts to eliminate malaria depend on the continued success of artemisinin-based combination therapies (ACTs) that target Plasmodium asexual blood-stage parasites. Resistance to ACTs, however, has emerged, creating the need to define the underlying mechanisms. Mutations in the P. falciparum multidrug resistance protein 1 (PfMDR1) transporter constitute an important determinant of resistance. Applying gene editing tools combined with an analysis of a public database containing thousands of parasite genomes, we show geographic selection and expansion of a pfmdr1 gene amplification encoding the N86/184F haplotype in Southeast Asia. Parasites expressing this PfMDR1 variant possess a higher transport capacity that modulates their responses to antimalarials. These data could help tailor and optimize antimalarial drug usage in different regions where malaria is endemic by taking into account the regional prevalence of pfmdr1 polymorphisms.This work was funded by Portuguese National funds through the Foundation for Science and Technology (FCT) (project UIDB/50026/2020 and UIDP/50026/2020; fellowships PD/BD/127826/2016 to C.C., SFRH/BD/129769/2017 to M.S., SFRH/BD/145427/2019 to V.B., SFRH/BD/131540/2017 to R.S.P., and IF/00143/2015/CP1294/CT0001 to P.E.F. and contract funding to M.I.V. provided through DL 57/2016 [CRP]); by the projects NORTE-01-0145-FEDER-000013, NORTE-01-0145-FEDER-000023, and NORTE 01-0145-FEDER-028178, supported by Norte Portugal Regional Operational Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the Euro pean Regional Development Fund (ERDF); by the Institute Merieux through “Starting” Mérieux Research Grant 2016 to M.I.V.; by the ESCMID to P.E.F. and by the NIH R01 AI109023 and R37AI50234 to D.A.F.info:eu-repo/semantics/publishedVersio

Allele Summation of Diabetes Risk Genes Predicts Impaired Glucose Tolerance in Female and Obese Individuals

INTRODUCTION: Single nucleotide polymorphisms (SNPs) in approximately 40 genes have been associated with an increased risk for type 2 diabetes (T2D) in genome-wide association studies. It is not known whether a similar genetic impact on the risk of prediabetes (impaired glucose tolerance [IGT] or impaired fasting glycemia [IFG]) exists. METHODS: In our cohort of 1442 non-diabetic subjects of European origin (normal glucose tolerance [NGT] n = 1046, isolated IFG n = 142, isolated IGT n = 140, IFG+IGT n = 114), an impact on glucose homeostasis has been shown for 9 SNPs in previous studies in this specific cohort. We analyzed these SNPs (within or in the vicinity of the genes TCF7L2, KCNJ11, HHEX, SLC30A8, WFS1, KCNQ1, MTNR1B, FTO, PPARG) for association with prediabetes. RESULTS: The genetic risk load was significantly associated with the risk for IGT (p = 0.0006) in a model including gender, age, BMI and insulin sensitivity. To further evaluate potential confounding effects, we stratified the population on gender, BMI and insulin sensitivity. The association of the risk score with IGT was present in female participants (p = 0.008), but not in male participants. The risk score was significantly associated with IGT (p = 0.008) in subjects with a body mass index higher than 30 kg/m(2) but not in non-obese individuals. Furthermore, only in insulin resistant subjects a significant association between the genetic load and the risk for IGT (p = 0.01) was found. DISCUSSION: We found that T2D genetic risk alleles cause an increased risk for IGT. This effect was not present in male, lean and insulin sensitive subjects, suggesting a protective role of beneficial environmental factors on the genetic risk

Plasmodium falciparum Reticulocyte Binding-Like Homologue Protein 2 (PfRH2) Is a Key Adhesive Molecule Involved in Erythrocyte Invasion

Erythrocyte invasion by Plasmodium merozoites is a complex, multistep process that is mediated by a number of parasite ligand-erythrocyte receptor interactions. One such family of parasite ligands includes the P. falciparum reticulocyte binding homologue (PfRH) proteins that are homologous with the P. vivax reticulocyte binding proteins and have been shown to play a role in erythrocyte invasion. There are five functional PfRH proteins of which only PfRH2a/2b have not yet been demonstrated to bind erythrocytes. In this study, we demonstrated that native PfRH2a/2b is processed near the N-terminus yielding fragments of 220 kDa and 80 kDa that exhibit differential erythrocyte binding specificities. The erythrocyte binding specificity of the 220 kDa processed fragment of native PfRH2a/2b was sialic acid-independent, trypsin resistant and chymotrypsin sensitive. This specific binding phenotype is consistent with previous studies that disrupted the PfRH2a/2b genes and demonstrated that PfRH2b is involved in a sialic acid independent, trypsin resistant, chymotrypsin sensitive invasion pathway. Interestingly, we found that the smaller 80 kDa PfRH2a/2b fragment is processed from the larger 220 kDa fragment and binds erythrocytes in a sialic acid dependent, trypsin resistant and chymotrypsin sensitive manner. Thus, the two processed fragments of PfRH2a/2b differed with respect to their dependence on sialic acids for erythrocyte binding. Further, we mapped the erythrocyte binding domain of PfRH2a/2b to a conserved 40 kDa N-terminal region (rPfRH240) in the ectodomain that is common to both PfRH2a and PfRH2b. We demonstrated that recombinant rPfRH240 bound human erythrocytes with the same specificity as the native 220 kDa processed protein. Moreover, antibodies generated against rPfRH240 blocked erythrocyte invasion by P. falciparum through a sialic acid independent pathway. PfRH2a/2b thus plays a key role in erythrocyte invasion and its conserved receptor-binding domain deserves attention as a promising candidate for inclusion in a blood-stage malaria vaccine

Interaction of Pattern Recognition Receptors with Mycobacterium Tuberculosis.

Tuberculosis (TB) is considered a major worldwide health problem with 10 million new cases diagnosed each year. Our understanding of TB immunology has become greater and more refined since the identification of Mycobacterium tuberculosis (MTB) as an etiologic agent and the recognition of new signaling pathways modulating infection. Understanding the mechanisms through which the cells of the immune system recognize MTB can be an important step in designing novel therapeutic approaches, as well as improving the limited success of current vaccination strategies. A great challenge in chronic disease is to understand the complexities, mechanisms, and consequences of host interactions with pathogens. Innate immune responses along with the involvement of distinct inflammatory mediators and cells play an important role in the host defense against the MTB. Several classes of pattern recognition receptors (PRRs) are involved in the recognition of MTB including Toll-Like Receptors (TLRs), C-type lectin receptors (CLRs) and Nod-like receptors (NLRs) linked to inflammasome activation. Among the TLR family, TLR1, TLR2, TLR4, and TLR9 and their down-stream signaling proteins play critical roles in the initiation of the immune response in the pathogenesis of TB. The inflammasome pathway is associated with the coordinated release of cytokines such as IL-1β and IL-18 which also play a role in the pathogenesis of TB. Understanding the cross-talk between these signaling pathways will impact on the design of novel therapeutic strategies and in the development of vaccines and immunotherapy regimes. Abnormalities in PRR signaling pathways regulated by TB will affect disease pathogenesis and need to be elucidated. In this review we provide an update on PRR signaling during M. tuberculosis infection and indicate how greater knowledge of these pathways may lead to new therapeutic opportunities

Deletion of individual Ku subunits in mice causes an NHEJ-independent phenotype potentially by altering apurinic/apyrimidinic site repair

Ku70 and Ku80 form a heterodimer called Ku that forms a holoenzyme with DNA dependent-protein kinase catalytic subunit (DNA-PKCS) to repair DNA double strand breaks (DSBs) through the nonhomologous end joining (NHEJ) pathway. As expected mutating these genes in mice caused a similar DSB repair-defective phenotype. However, ku70-/- cells and ku80 -/- cells also appeared to have a defect in base excision repair (BER). BER corrects base lesions, apurinic/apyrimidinic (AP) sites and single stand breaks (SSBs) utilizing a variety of proteins including glycosylases, AP endonuclease 1 (APE1) and DNA Polymerase β (Pol β). In addition, deleting Ku70 was not equivalent to deleting Ku80 in cells and mice. Therefore, we hypothesized that free Ku70 (not bound to Ku80) and/or free Ku80 (not bound to Ku70) possessed activity that influenced BER. To further test this hypothesis we performed two general sets of experiments. The first set showed that deleting either Ku70 or Ku80 caused an NHEJ-independent defect. We found ku80-/- mice had a shorter life span than dna-pkcs-/- mice demonstrating a phenotype that was greater than deleting the holoenzyme. We also found Ku70-deletion induced a p53 response that reduced the level of small mutations in the brain suggesting defective BER. We further confirmed that Ku80-deletion impaired BER via a mechanism that was not epistatic to Pol β. The second set of experiments showed that free Ku70 and free Ku80 could influence BER. We observed that deletion of either Ku70 or Ku80, but not both, increased sensitivity of cells to CRT0044876 (CRT), an agent that interferes with APE1. In addition, free Ku70 and free Ku80 bound to AP sites and in the case of Ku70 inhibited APE1 activity. These observations support a novel role for free Ku70 and free Ku80 in altering BER. © 2014 Choi et al

Identification of Candida glabrata genes involved in pH modulation and modification of the phagosomal environment in macrophages

notes: PMCID: PMC4006850types: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov'tCandida glabrata currently ranks as the second most frequent cause of invasive candidiasis. Our previous work has shown that C. glabrata is adapted to intracellular survival in macrophages and replicates within non-acidified late endosomal-stage phagosomes. In contrast, heat killed yeasts are found in acidified matured phagosomes. In the present study, we aimed at elucidating the processes leading to inhibition of phagosome acidification and maturation. We show that phagosomes containing viable C. glabrata cells do not fuse with pre-labeled lysosomes and possess low phagosomal hydrolase activity. Inhibition of acidification occurs independent of macrophage type (human/murine), differentiation (M1-/M2-type) or activation status (vitamin D3 stimulation). We observed no differential activation of macrophage MAPK or NFκB signaling cascades downstream of pattern recognition receptors after internalization of viable compared to heat killed yeasts, but Syk activation decayed faster in macrophages containing viable yeasts. Thus, delivery of viable yeasts to non-matured phagosomes is likely not triggered by initial recognition events via MAPK or NFκB signaling, but Syk activation may be involved. Although V-ATPase is abundant in C. glabrata phagosomes, the influence of this proton pump on intracellular survival is low since blocking V-ATPase activity with bafilomycin A1 has no influence on fungal viability. Active pH modulation is one possible fungal strategy to change phagosome pH. In fact, C. glabrata is able to alkalinize its extracellular environment, when growing on amino acids as the sole carbon source in vitro. By screening a C. glabrata mutant library we identified genes important for environmental alkalinization that were further tested for their impact on phagosome pH. We found that the lack of fungal mannosyltransferases resulted in severely reduced alkalinization in vitro and in the delivery of C. glabrata to acidified phagosomes. Therefore, protein mannosylation may play a key role in alterations of phagosomal properties caused by C. glabrata.Deutsche ForschungsgemeinschaftNational Institutes for HealthWellcome TrustBBSR

The Interaction between Early Life Epilepsy and Autistic-Like Behavioral Consequences: A Role for the Mammalian Target of Rapamycin (mTOR) Pathway

Early life seizures can result in chronic epilepsy, cognitive deficits and behavioral changes such as autism, and conversely epilepsy is common in autistic children. We hypothesized that during early brain development, seizures could alter regulators of synaptic development and underlie the interaction between epilepsy and autism. The mammalian Target of Rapamycin (mTOR) modulates protein translation and is dysregulated in Tuberous Sclerosis Complex, a disorder characterized by epilepsy and autism. We used a rodent model of acute hypoxia-induced neonatal seizures that results in long term increases in neuronal excitability, seizure susceptibility, and spontaneous seizures, to determine how seizures alter mTOR Complex 1 (mTORC1) signaling. We hypothesized that seizures occurring at a developmental stage coinciding with a critical period of synaptogenesis will activate mTORC1, contributing to epileptic networks and autistic-like behavior in later life. Here we show that in the rat, baseline mTORC1 activation peaks during the first three postnatal weeks, and induction of seizures at postnatal day 10 results in further transient activation of its downstream targets phospho-4E-BP1 (Thr37/46), phospho-p70S6K (Thr389) and phospho-S6 (Ser235/236), as well as rapid induction of activity-dependent upstream signaling molecules, including BDNF, phospho-Akt (Thr308) and phospho-ERK (Thr202/Tyr204). Furthermore, treatment with the mTORC1 inhibitor rapamycin immediately before and after seizures reversed early increases in glutamatergic neurotransmission and seizure susceptibility and attenuated later life epilepsy and autistic-like behavior. Together, these findings suggest that in the developing brain the mTORC1 signaling pathway is involved in epileptogenesis and altered social behavior, and that it may be a target for development of novel therapies that eliminate the progressive effects of neonatal seizures

mTOR: from growth signal integration to cancer, diabetes and ageing

In all eukaryotes, the target of rapamycin (TOR) signalling pathway couples energy and nutrient abundance to the execution of cell growth and division, owing to the ability of TOR protein kinase to simultaneously sense energy, nutrients and stress and, in metazoans, growth factors. Mammalian TOR complex 1 (mTORC1) and mTORC2 exert their actions by regulating other important kinases, such as S6 kinase (S6K) and Akt. In the past few years, a significant advance in our understanding of the regulation and functions of mTOR has revealed the crucial involvement of this signalling pathway in the onset and progression of diabetes, cancer and ageing.National Institutes of Health (U.S.)Howard Hughes Medical InstituteWhitehead Institute for Biomedical ResearchJane Coffin Childs Memorial Fund for Medical Research (Postdoctoral Fellowship)Human Frontier Science Program (Strasbourg, France