Nutrient sensing modulates malaria parasite virulence.

The lifestyle of intracellular pathogens, such as malaria parasites, is intimately connected to that of their host, primarily for nutrient supply. Nutrients act not only as primary sources of energy but also as regulators of gene expression, metabolism and growth, through various signalling networks that enable cells to sense and adapt to varying environmental conditions. Canonical nutrient-sensing pathways are presumed to be absent from the causative agent of malaria, Plasmodium, thus raising the question of whether these parasites can sense and cope with fluctuations in host nutrient levels. Here we show that Plasmodium blood-stage parasites actively respond to host dietary calorie alterations through rearrangement of their transcriptome accompanied by substantial adjustment of their multiplication rate. A kinome analysis combined with chemical and genetic approaches identified KIN as a critical regulator that mediates sensing of nutrients and controls a transcriptional response to the host nutritional status. KIN shares homology with SNF1/AMPKα, and yeast complementation studies suggest that it is part of a functionally conserved cellular energy-sensing pathway. Overall, these findings reveal a key parasite nutrient-sensing mechanism that is critical for modulating parasite replication and virulence.

GLUT1-mediated glucose uptake plays a crucial role during Plasmodium hepatic infection.

Intracellular pathogens have evolved mechanisms to ensure their survival and development inside their host cells. Here, we show that glucose is a pivotal modulator of hepatic infection by the rodent malaria parasite Plasmodium berghei and that glucose uptake via the GLUT1 transporter is specifically enhanced in P. berghei-infected cells. We further show that ATP levels of cells containing developing parasites are decreased, which is known to enhance membrane GLUT1 activity. In addition, GLUT1 molecules are translocated to the membrane of the hepatic cell, increasing glucose uptake at later stages of infection. Chemical inhibition of GLUT1 activity leads to a decrease in glucose uptake and the consequent impairment of hepatic infection, both in vitro and in vivo. Our results reveal that changes in GLUT1 conformation and cellular localization seem to be part of an adaptive host response to maintain adequate cellular nutrition and energy levels, ensuring host cell survival and supporting P. berghei hepatic development.

Simple, sensitive and quantitative bioluminescence assay for determination of malaria pre-patent period.

BACKGROUND: The first phase of malaria infection occurs in the liver and is clinically silent. Inside hepatocytes each Plasmodium sporozoite replicate into thousands of erythrocyte-infectious merozoites that when released into the blood stream result in clinical symptoms of the disease. The time between sporozoite inoculation and the appearance of parasites in the blood is defined as the pre-patent period, which is classically analysed by time-consuming and labor-intensive techniques, such as microscopy and PCR. METHODS: Luciferase-expressing Plasmodium berghei parasites were used to measure pre-patent period of malaria infection in rodents using a bioluminescence assay that requires only one microliter of blood collected from the tail-vein. The accuracy and sensitivity of this new method was compared with conventional microscopy and PCR based techniques, and its capacity to measure the impact of anti-malarial interventions against the liver evaluated. RESULTS: The described method is very sensitive allowing the detection of parasites during the first cycles of blood stage replication. It accurately translates differences in liver load due to inoculation of different sporozoite doses as well as a result of treatment with different primaquine regimens. CONCLUSIONS: A novel, simple, fast, and sensitive method to measure pre-patent period of malaria infection in rodents is described here. The sensitivity and accuracy of this new method is comparable to standard PCR and microscopy-based techniques, respectively.

Development of antibodies against the notch ligand Delta-Like-1 by phage display with activity against breast cancer cells.

Notch signalling is a well-established oncogenic pathway, and its ligand Delta-like 1 (DLL1) is overexpressed in estrogen receptor-positive (ER+) breast cancers and associated with poor patient prognosis. Hence, DLL1 has become an interesting therapeutic target for breast cancer. Here, the development of specific functional blocking anti-DLL1 antibodies with potential activity against ER+ breast cancer cells is reported. Human DLL1 proteins, containing the essential regions for binding to the Notch receptor and Notch signalling activation, were produced and used to select specific scFv antibody fragments by phage display. Fifteen unique scFvs were identified and reformatted into full IgGs. Characterization of these antibodies by ELISA, surface plasmon resonance and flow cytometry enabled selection of three specific anti-DLL1 IgGs, sharing identical VH regions, with nM affinities. Cellular assays on ER+ breast cancer MCF-7 cells showed that one of the IgGs (IgG-69) was able to partially impair DLL1-mediated activation of the Notch pathway, as determined by Notch reporter and RT-qPCR assays, and to attenuate cell growth. Treatment of MCF-7 cells with IgG-69 reduced mammosphere formation, suggesting that it decreases the breast cancer stem cell subpopulation. These results support the use of this strategy to develop and identify potential anti-DLL1 antibodies candidates against breast cancer.

Development of Dl1.72, a Novel Anti-DLL1 Antibody with Anti-Tumor Efficacy against Estrogen Receptor-Positive Breast Cancer.

The Notch-signaling ligand DLL1 has emerged as an important player and promising therapeutic target in breast cancer (BC). DLL1-induced Notch activation promotes tumor cell proliferation, survival, migration, angiogenesis and BC stem cell maintenance. In BC, DLL1 overexpression is associated with poor prognosis, particularly in estrogen receptor-positive (ER+) subtypes. Directed therapy in early and advanced BC has dramatically changed the natural course of ER+ BC; however, relapse is a major clinical issue, and new therapeutic strategies are needed. Here, we report the development and characterization of a novel monoclonal antibody specific to DLL1. Using phage display technology, we selected an anti-DLL1 antibody fragment, which was converted into a full human IgG1 (Dl1.72). The Dl1.72 antibody exhibited DLL1 specificity and affinity in the low nanomolar range and significantly impaired DLL1-Notch signaling and expression of Notch target genes in ER+ BC cells. Functionally, in vitro treatment with Dl1.72 reduced MCF-7 cell proliferation, migration, mammosphere formation and endothelial tube formation. In vivo, Dl1.72 significantly inhibited tumor growth, reducing both tumor cell proliferation and liver metastases in a xenograft mouse model, without apparent toxicity. These findings suggest that anti-DLL1 Dl1.72 could be an attractive agent against ER+ BC, warranting further preclinical investigation.

Specific Eco-evolutionary Contexts in the Mouse Gut Reveal Escherichia coli Metabolic Versatility.

Members of the gut microbiota are thought to experience strong competition for nutrients. However, how such competition shapes their evolutionary dynamics and depends on intra- and interspecies interactions is poorly understood. Here, we test the hypothesis that Escherichia coli evolution in the mouse gut is more predictable across hosts in the absence of interspecies competition than in the presence of other microbial species. In support, we observed that lrp, a gene encoding a global regulator of amino acid metabolism, was repeatedly selected in germ-free mice 2 weeks after mono-colonization by this bacterium. We established that this specific genetic adaptation increased E. coli's ability to compete for amino acids, and analysis of gut metabolites identified serine and threonine as the metabolites preferentially consumed by E. coli in the mono-colonized mouse gut. Preference for serine consumption was further supported by testing a set of mutants that showed loss of advantage of an lrp mutant impaired in serine metabolism in vitro and in vivo. Remarkably, the presence of a single additional member of the microbiota, Blautia coccoides, was sufficient to alter the gut metabolome and, consequently, the evolutionary path of E. coli. In this environment, the fitness advantage of the lrp mutant bacteria is lost, and mutations in genes involved in anaerobic respiration were selected instead, recapitulating the eco-evolutionary context from mice with a complex microbiota. Together, these results highlight the metabolic plasticity and evolutionary versatility of E. coli, tailored to the specific ecology it experiences in the gut.

The Notch ligand DLL1 exerts carcinogenic features in human breast cancer cells.

CONCLUSIONS: These findings provide further evidence that DLL1 exerts carcinogenic effects in BC cells. The dissimilar effects of DLL1 downregulation observed amongst MCF-7, BT474, and MDA-MB-231 cells is likely due to their distinctive genetic and biologic characteristics, suggesting that DLL1 contributes to BC through various mechanisms.

Host AMPK Is a Modulator of Plasmodium Liver Infection.

Manipulation of the master regulator of energy homeostasis AMP-activated protein kinase (AMPK) activity is a strategy used by many intracellular pathogens for successful replication. Infection by most pathogens leads to an activation of host AMPK activity due to the energetic demands placed on the infected cell. Here, we demonstrate that the opposite is observed in cells infected with rodent malaria parasites. Indeed, AMPK activity upon the infection of hepatic cells is suppressed and dispensable for successful infection. By contrast, an overactive AMPK is deleterious to intracellular growth and replication of different Plasmodium spp., including the human malaria parasite, P. falciparum. The negative impact of host AMPK activity on infection was further confirmed in mice under conditions that activate its function. Overall, this work establishes the role of host AMPK signaling as a suppressive pathway of Plasmodium hepatic infection and as a potential target for host-based antimalarial interventions.