RESUMO
Immunity that controls parasitemia and inflammation during Plasmodium falciparum (Pf) malaria can be acquired with repeated infections. A limited understanding of this complex immune response impedes the development of vaccines and adjunctive therapies. We conducted a prospective systems biology study of children who differed in their ability to control parasitemia and fever following Pf infection. By integrating whole-blood transcriptomics, flow-cytometric analysis, and plasma cytokine and antibody profiles, we demonstrate that a pre-infection signature of B cell enrichment, upregulation of T helper type 1 (Th1) and Th2 cell-associated pathways, including interferon responses, and p53 activation associated with control of malarial fever and coordinated with Pf-specific immunoglobulin G (IgG) and Fc receptor activation to control parasitemia. Our hypothesis-generating approach identified host molecules that may contribute to differential clinical outcomes during Pf infection. As a proof of concept, we have shown that enhanced p53 expression in monocytes attenuated Plasmodium-induced inflammation and predicted protection from fever.
Assuntos
Linfócitos B/imunologia , Proteínas Sanguíneas/metabolismo , Inflamação/metabolismo , Malária Falciparum/metabolismo , Plasmodium falciparum/fisiologia , Células Th1/imunologia , Células Th2/imunologia , Proteína Supressora de Tumor p53/metabolismo , Adolescente , Adulto , Animais , Anticorpos Antiprotozoários/metabolismo , Criança , Pré-Escolar , Resistência à Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Lactente , Interferons/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estudos Prospectivos , Receptores Fc/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Adulto JovemRESUMO
Blocking Plasmodium, the causative agent of malaria, at the asymptomatic pre-erythrocytic stage would abrogate disease pathology and prevent transmission. However, the lack of well-defined features within vaccine-elicited antibody responses that correlate with protection represents a major roadblock to improving on current generation vaccines. We vaccinated mice (BALB/cJ and C57BL/6J) with Py circumsporozoite protein (CSP), the major surface antigen on the sporozoite, and evaluated vaccine-elicited humoral immunity and identified immunological factors associated with protection after mosquito bite challenge. Vaccination achieved 60% sterile protection and otherwise delayed blood stage patency in BALB/cJ mice. In contrast, all C57BL/6J mice were infected similar to controls. Protection was mediated by antibodies and could be passively transferred from immunized BALB/cJ mice into naïve C57BL/6J. Dissection of the underlying immunological features of protection revealed early deficits in antibody titers and polyclonal avidity in C57BL/6J mice. Additionally, PyCSP-vaccination in BALB/cJ induced a significantly higher proportion of antigen-specific B-cells and class-switched memory B-cell (MBCs) populations than in C57BL/6J mice. Strikingly, C57BL/6J mice also had markedly fewer CSP-specific germinal center experienced B cells and class-switched MBCs compared to BALB/cJ mice. Analysis of the IgG γ chain repertoires by next generation sequencing in PyCSP-specific memory B-cell repertoires also revealed higher somatic hypermutation rates in BALB/cJ mice than in C57BL/6J mice. These findings indicate that the development of protective antibody responses in BALB/cJ mice in response to vaccination with PyCSP was associated with increased germinal center activity and somatic mutation compared to C57BL/6J mice, highlighting the key role B cell maturation may have in the development of vaccine-elicited protective antibodies against CSP.
Assuntos
Vacinas Antimaláricas , Malária , Animais , Anticorpos Antiprotozoários , Formação de Anticorpos , Centro Germinativo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas de Protozoários/genéticaRESUMO
Terminal and benign diseases alike in adults, children, pregnant women, and others are successfully treated by pharmacological inhibitors that target human enzymes. Despite extensive global efforts to fight malaria, the disease continues to be a massive worldwide health burden, and new interventional strategies are needed. Current drugs and vector control strategies have contributed to the reduction in malaria deaths over the past 10 years, but progress toward eradication has waned in recent years. Resistance to antimalarial drugs is a substantial and growing problem. Moreover, targeting dormant forms of the malaria parasite Plasmodium vivax is only possible with two approved drugs, which are both contraindicated for individuals with glucose-6-phosphate dehydrogenase deficiency and in pregnant women. Plasmodium parasites are obligate intracellular parasites and thus have specific and absolute requirements of their hosts. Growing evidence has described these host necessities, paving the way for opportunities to pharmacologically target host factors to eliminate Plasmodium infection. Here, we describe progress in malaria research and adjacent fields and discuss key challenges that remain in implementing host-directed therapy against malaria.
Assuntos
Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Interações Hospedeiro-Parasita/efeitos dos fármacos , Malária/tratamento farmacológico , Terapia de Alvo Molecular , Humanos , Malária/parasitologiaRESUMO
Many of the gene regulatory processes of Plasmodium falciparum, the deadliest malaria parasite, remain poorly understood. To develop a comprehensive guide for exploring this organism's gene regulatory network, we generated a systems-level model of P. falciparum gene regulation using a well-validated, machine-learning approach for predicting interactions between transcription regulators and their targets. The resulting network accurately predicts expression levels of transcriptionally coherent gene regulatory programs in independent transcriptomic data sets from parasites collected by different research groups in diverse laboratory and field settings. Thus, our results indicate that our gene regulatory model has predictive power and utility as a hypothesis-generating tool for illuminating clinically relevant gene regulatory mechanisms within P. falciparum. Using the set of regulatory programs we identified, we also investigated correlates of artemisinin resistance based on gene expression coherence. We report that resistance is associated with incoherent expression across many regulatory programs, including those controlling genes associated with erythrocyte-host engagement. These results suggest that parasite populations with reduced artemisinin sensitivity are more transcriptionally heterogenous. This pattern is consistent with a model where the parasite utilizes bet-hedging strategies to diversify the population, rendering a subpopulation more able to navigate drug treatment.
Assuntos
Regulação da Expressão Gênica , Redes Reguladoras de Genes , Modelos Genéticos , Plasmodium falciparum/genética , Antimaláricos/farmacologia , Artemisininas/farmacologia , Resistência a Medicamentos/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Aprendizado de Máquina , Plasmodium falciparum/efeitos dos fármacos , Biologia de Sistemas , Transcrição GênicaRESUMO
Gliding motility and cell traversal by the Plasmodium ookinete and sporozoite invasive stages allow penetration of cellular barriers to establish infection of the mosquito vector and mammalian host, respectively. Motility and traversal are not observed in red cell infectious merozoites, and we have previously classified genes that are expressed in sporozoites but not merozoites (S genes) in order to identify proteins involved in these processes. The S4 gene has been described as criticaly involved in Cell Traversal for Ookinetes and Sporozoites (CelTOS), yet knockout parasites (s4/celtos¯) do not generate robust salivary gland sporozoite numbers, precluding a thorough analysis of S4/CelTOS function during host infection. We show here that a failure of oocysts to develop or survive in the midgut contributes to the poor mosquito infection by Plasmodium yoelii (Py) s4/celtos¯ rodent malaria parasites. We rescued this phenotype by expressing S4/CelTOS under the ookinete-specific circumsporozoite protein and thrombospondin-related anonymous protein-related protein (CTRP) promoter (S4/CelTOSCTRP ), generating robust numbers of salivary gland sporozoites lacking S4/CelTOS that were suitable for phenotypic analysis. Py S4/CelTOSCTRP sporozoites showed reduced infectivity in BALB/c mice when compared to wild-type sporozoites, although they appeared more infectious than sporozoites deficient in the related traversal protein PLP1/SPECT2 (Py plp1/spect2¯). Using in vitro assays, we substantiate the role of S4/CelTOS in sporozoite cell traversal, but also uncover a previously unappreciated role for this protein for sporozoite gliding motility.
Assuntos
Plasmodium yoelii/fisiologia , Proteínas de Protozoários/metabolismo , Esporozoítos/metabolismo , Animais , Movimento Celular , Interações Hospedeiro-Parasita , Malária/parasitologia , Mosquitos Vetores , Plasmodium yoelii/genética , Proteínas de Protozoários/genéticaRESUMO
Histology-directed imaging mass spectrometry (IMS) is a spatially targeted IMS acquisition method informed by expert annotation that provides rapid molecular characterization of select tissue structures. The expert annotations are usually determined on digital whole slide images of histological stains where the staining preparation is incompatible with optimal IMS preparation, necessitating serial sections: one for annotation, one for IMS. Registration is then used to align staining annotations onto the IMS tissue section. Herein, we report a next-generation histology-directed platform implementing IMS-compatible autofluorescence (AF) microscopy taken prior to any staining or IMS. The platform enables two histology-directed workflows, one that improves the registration process between two separate tissue sections using automated, computational monomodal AF-to-AF microscopy image registration, and a registration-free approach that utilizes AF directly to identify ROIs and acquire IMS on the same section. The registration approach is fully automated and delivers state of the art accuracy in histology-directed workflows for transfer of annotations (â¼3-10 µm based on 4 organs from 2 species) while the direct AF approach is registration-free, allowing targeting of the finest structures visible by AF microscopy. We demonstrate the platform in biologically relevant case studies of liver stage malaria and human kidney disease with spatially targeted acquisition of sparsely distributed (composing less than one tenth of 1% of the tissue section area) malaria infected mouse hepatocytes and glomeruli in the human kidney case study.
Assuntos
Nefropatias/diagnóstico por imagem , Malária/diagnóstico por imagem , Imagem Óptica , Animais , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Eliminating malaria parasites during the asymptomatic but obligate liver stages (LSs) of infection would stop disease and subsequent transmission. Unfortunately, only a single licensed drug that targets all LSs, Primaquine, is available. Targeting host proteins might significantly expand the repertoire of prophylactic drugs against malaria. Here, we demonstrate that both Bcl-2 inhibitors and P53 agonists dramatically reduce LS burden in a mouse malaria model in vitro and in vivo by altering the activity of key hepatocyte factors on which the parasite relies. Bcl-2 inhibitors act primarily by inducing apoptosis in infected hepatocytes, whereas P53 agonists eliminate parasites in an apoptosis-independent fashion. In combination, Bcl-2 inhibitors and P53 agonists act synergistically to delay, and in some cases completely prevent, the onset of blood stage disease. Both families of drugs are highly effective at doses that do not cause substantial hepatocyte cell death in vitro or liver damage in vivo. P53 agonists and Bcl-2 inhibitors were also effective when administered to humanized mice infected with Plasmodium falciparum. Our data demonstrate that host-based prophylaxis could be developed into an effective intervention strategy that eliminates LS parasites before the onset of clinical disease and thus opens a new avenue to prevent malaria.
Assuntos
Antimaláricos/farmacologia , Estágios do Ciclo de Vida/efeitos dos fármacos , Fígado/parasitologia , Malária/parasitologia , Plasmodium/efeitos dos fármacos , Plasmodium/fisiologia , Profilaxia Pós-Exposição , Animais , Antimaláricos/administração & dosagem , Linhagem Celular , Modelos Animais de Doenças , Feminino , Imidazóis/administração & dosagem , Imidazóis/farmacologia , Indóis , Malária/tratamento farmacológico , Malária/metabolismo , Malária/prevenção & controle , Malária Falciparum/tratamento farmacológico , Malária Falciparum/metabolismo , Malária Falciparum/parasitologia , Malária Falciparum/prevenção & controle , Camundongos , Camundongos Transgênicos , Carga Parasitária , Piperazinas/administração & dosagem , Piperazinas/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pirróis/administração & dosagem , Pirróis/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismoRESUMO
After transmission by Anopheles mosquitoes, Plasmodium sporozoites travel to the liver, infect hepatocytes, and rapidly develop as intrahepatocytic liver stages (LS). Rodent models of malaria exhibit large differences in the magnitude of liver infection, both between parasite species and between strains of mice. This has been mainly attributed to differences in innate immune responses and parasite infectivity. Here, we report that BALB/cByJ mice are more susceptible to Plasmodium yoelii preerythrocytic infection than BALB/cJ mice. This difference occurs at the level of early hepatocyte infection, but expression levels of reported host factors that are involved in infection do not correlate with susceptibility. Interestingly, BALB/cByJ hepatocytes are more frequently polyploid; thus, their susceptibility converges on the previously observed preference of sporozoites to infect polyploid hepatocytes. Gene expression analysis demonstrates hepatocyte-specific differences in mRNA abundance for numerous genes between BALB/cByJ and BALB/cJ mice, some of which encode hepatocyte surface molecules. These data suggest that a yet-unknown receptor for sporozoite infection, present at elevated levels on BALB/cByJ hepatocytes and also polyploid hepatocytes, might facilitate Plasmodium liver infection.
Assuntos
Suscetibilidade a Doenças , Endocitose , Hepatócitos/parasitologia , Malária/imunologia , Malária/parasitologia , Plasmodium yoelii/fisiologia , Animais , Feminino , Perfilação da Expressão Gênica , Camundongos Endogâmicos BALB CRESUMO
Plasmodium parasites infect hepatocytes of their mammalian hosts and undergo obligate liver stage development. The specific host cell attributes that are important for liver infection remain largely unknown. Several host signalling pathways are perturbed in infected hepatocytes, some of which are important in the generation of hepatocyte polyploidy. To test the functional consequence of polyploidy on liver infection, we infected hepatocytes with the rodent malaria parasite Plasmodium yoelii both in vitro and in vivo and examined the ploidy of infected and uninfected hepatocytes by flow cytometry. In both hepatoma cell lines and in the mouse liver, the fraction of polyploid cells was higher in the infected cell population than in the uninfected cell population. When the data were reanalysed by comparing the extent of Plasmodium infection within each ploidy subset, we found that infection rates were elevated in more highly polyploid cells and lower in diploid cells. Furthermore, we found that the parasite's preference for host cells with high ploidy is conserved among rodent malaria species and the human malaria parasite Plasmodium falciparum. This parasite preference for host cells of high ploidy cannot be explained by differences in hepatocyte size or DNA replication. We conclude that Plasmodium preferentially infects and develops in polyploid hepatocytes.
Assuntos
Hepatócitos/parasitologia , Fígado/patologia , Fígado/parasitologia , Malária/patologia , Malária/parasitologia , Plasmodium yoelii/crescimento & desenvolvimento , Poliploidia , Animais , Linhagem Celular , Citometria de Fluxo , Humanos , Malária/genética , CamundongosRESUMO
Forty percent of people worldwide are at risk of malaria infection, and despite control efforts it remains the most deadly parasitic disease. Unfortunately, rapid discovery and development of new interventions for malaria are hindered by the lack of small animal models that support the complex life cycles of the main parasite species infecting humans. Such tools must accommodate human parasite tropism for human tissue. Mouse models with human tissue developed to date have already enhanced our knowledge of human parasites, and are useful tools for assessing anti-parasitic interventions. Although these systems are imperfect, their continued refinement will likely broaden their utility. Some of the malaria parasite's interactions with human hepatocytes and human erythrocytes can already be modelled with available humanized mouse systems. However, interactions with other relevant human tissues such as the skin and immune system, as well as most transitions between life cycle stages in vivo will require refinement of existing humanized mouse models. Here, we review the recent successes achieved in modelling human malaria parasite biology in humanized mice, and discuss how these models have potential to become a valuable part of the toolbox used for understanding the biology of, and development of interventions to, malaria.
Assuntos
Modelos Animais de Doenças , Interações Hospedeiro-Patógeno , Malária/patologia , Malária/parasitologia , Camundongos SCID , Plasmodium/imunologia , Plasmodium/fisiologia , Animais , Humanos , MasculinoRESUMO
Immunization with live-attenuated Plasmodium sporozoites completely protects against malaria infection. Genetic engineering offers a versatile platform to create live-attenuated sporozoite vaccine candidates. We previously generated a genetically attenuated parasite (GAP) by deleting the P52 and P36 genes in the NF54 wild-type (WT) strain of Plasmodium falciparum (Pf p52(-)/p36(-) GAP). Preclinical assessment of p52(-)/p36(-) GAP in a humanized mouse model indicated an early and severe liver stage growth defect. However, human exposure to >200 Pf p52(-)/p36(-) GAP-infected mosquito bites in a safety trial resulted in peripheral parasitemia in one of six volunteers, revealing that this GAP was incompletely attenuated. We have now created a triple gene deleted GAP by additionally removing the SAP1 gene (Pf p52(-)/p36(-)/sap1(-) GAP) and employed flippase (FLP)/flippase recognition target (FRT) recombination for drug selectable marker cassette removal. This next-generation GAP was indistinguishable from WT parasites in blood stage and mosquito stage development. Using an improved humanized mouse model transplanted with human hepatocytes and human red blood cells, we show that despite a high-dose sporozoite challenge, Pf p52(-)/p36(-)/sap1(-) GAP did not transition to blood stage infection and appeared to be completely attenuated. Thus, clinical testing of Pf p52(-)/p36(-)/sap1(-) GAP assessing safety, immunogenicity, and efficacy against sporozoite challenge is warranted.
Assuntos
Anopheles/parasitologia , Malária Falciparum/sangue , Plasmodium falciparum/fisiologia , Proteínas de Protozoários/genética , Animais , Modelos Animais de Doenças , Deleção de Genes , Técnicas de Inativação de Genes , Humanos , Malária Falciparum/parasitologia , Vacinas Atenuadas/genéticaRESUMO
Mutation and overexpression of receptor tyrosine kinases or the proteins they regulate serve as oncogenic drivers in diverse cancers. To better understand receptor tyrosine kinase signaling and its link to oncogenesis, we used protein microarrays to systematically and quantitatively measure interactions between virtually every SH2 or PTB domain encoded in the human genome and all known sites of tyrosine phosphorylation on 40 receptor tyrosine kinases and on most of the SH2 and PTB domain-containing adaptor proteins. We found that adaptor proteins, like RTKs, have many high affinity bindings sites for other adaptor proteins. In addition, proteins that drive cancer, including both receptors and adaptor proteins, tend to be much more highly interconnected via networks of SH2 and PTB domain-mediated interactions than nononcogenic proteins. Our results suggest that network topological properties such as connectivity can be used to prioritize new drug targets in this well-studied family of signaling proteins.
Assuntos
Carcinogênese/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosfotirosina/metabolismo , Células HEK293 , Humanos , Proteínas Oncogênicas/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de SinaisRESUMO
Evidence from clinical trials of malaria vaccine candidates suggests that both cell-mediated and humoral immunity to pre-erythrocytic parasite stages can provide protection against infection. Novel pre-erythrocytic antibody (Ab) targets could be key to improving vaccine formulations, which are currently based on targeting antigens such as the circumsporozoite protein (CSP). However, methods to assess the effects of sporozoite-specific Abs on pre-erythrocytic infection in vivo remain underdeveloped. Here, we combined passive transfer of monoclonal Abs (MAbs) or immune serum with a luciferase-expressing Plasmodium yoelii sporozoite challenge to assess Ab-mediated inhibition of liver infection in mice. Passive transfer of a P. yoelii CSP MAb showed inhibition of liver infection when mice were challenged with sporozoites either intravenously or by infectious mosquito bite. However, inhibition was most potent for the mosquito bite challenge, leading to a more significant reduction of liver-stage burden and even a lack of progression to blood-stage parasitemia. This suggests that Abs provide effective protection against a natural infection. Inhibition of liver infection was also achieved by passive transfer of immune serum from whole-parasite-immunized mice. Furthermore, we demonstrated that passive transfer of a MAb against P. falciparum CSP inhibited liver-stage infection in a humanized mouse/P. falciparum challenge model. Together, these models constitute unique and sensitive in vivo methods to assess serum-transferable protection against Plasmodium sporozoite challenge.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antiprotozoários/imunologia , Soros Imunes/imunologia , Malária/prevenção & controle , Plasmodium falciparum/imunologia , Plasmodium yoelii/imunologia , Animais , Modelos Animais de Doenças , Feminino , Humanos , Imunização Passiva , Fígado/imunologia , Fígado/parasitologia , Malária/imunologia , Malária/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCIDRESUMO
The molecular pathways that regulate megakaryocyte production have historically been identified through multiple candidate gene approaches. Several transcription factors critical for generating megakaryocytes were identified by promoter analysis of megakaryocyte-specific genes, and their biological roles then verified by gene knockout studies; for example, GATA-1, NF-E2, and RUNX1 were identified in this way. In contrast, other transcription factors important for megakaryopoiesis were discovered through a systems approach; for example, c-Myb was found to be critical for the erythroid versus megakaryocyte lineage decision by genome-wide loss-of-function studies. The regulation of the levels of these transcription factors is, for the most part, cell intrinsic, although that assumption has recently been challenged. Epigenetics also impacts megakaryocyte gene expression, mediated by histone acetylation and methylation. Several cytokines have been identified to regulate megakaryocyte survival, proliferation, and differentiation, most prominent of which is thrombopoietin. Upon binding to its receptor, the product of the c-Mpl proto-oncogene, thrombopoietin induces a conformational change that activates a number of secondary messengers that promote cell survival, proliferation, and differentiation, and down-modulate receptor signaling. Among the best studied are the signal transducers and activators of transcription (STAT) proteins; phosphoinositol-3-kinase; mitogen-activated protein kinases; the phosphatases PTEN, SHP1, SHP2, and SHIP1; and the suppressors of cytokine signaling (SOCS) proteins. Additional signals activated by these secondary mediators include mammalian target of rapamycin; ß(beta)-catenin; the G proteins Rac1, Rho, and CDC42; several transcription factors, including hypoxia-inducible factor 1α(alpha), the homeobox-containing proteins HOXB4 and HOXA9, and a number of signaling mediators that are reduced, including glycogen synthase kinase 3α(alpha) and the FOXO3 family of forkhead proteins. More recently, systematic interrogation of several aspects of megakaryocyte formation have been conducted, employing genomics, proteomics, and chromatin immunoprecipitation (ChIP) analyses, among others, and have yielded many previously unappreciated signaling mechanisms that regulate megakaryocyte lineage determination, proliferation, and differentiation. This chapter focuses on these pathways in normal and neoplastic megakaryopoiesis, and suggests areas that are ripe for further study.
Assuntos
Megacariócitos/fisiologia , Biologia de Sistemas , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem da Célula/genética , Citocinas/genética , Regulação da Expressão Gênica no Desenvolvimento , Estudos de Associação Genética , Humanos , Megacariócitos/citologia , Proto-Oncogene Mas , Trombopoese/genéticaRESUMO
Phosphosignaling networks control cellular processes. We built kinase-mediated regulatory networks elicited by thrombin stimulation of brain endothelial cells using two computational strategies: Temporal Pathway Synthesizer (TPS), which uses phosphoproetiomics data as input, and Temporally REsolved KInase Network Generation (TREKING), which uses kinase inhibitor screens. TPS and TREKING predicted overlapping barrier-regulatory kinases connected with unique network topology. Each strategy effectively describes regulatory signaling networks and is broadly applicable across biological systems.
RESUMO
Kinases are key players in endothelial barrier regulation, yet their temporal function and regulatory phosphosignaling networks are incompletely understood. We developed a novel methodology, Temporally REsolved KInase Network Generation (TREKING), which combines a 28-kinase inhibitor screen with machine learning and network reconstruction to build time-resolved, functional phosphosignaling networks. We demonstrated the utility of TREKING for identifying pathways mediating barrier integrity after activation by thrombin with or without TNF preconditioning in brain endothelial cells. TREKING predicted over 100 kinases involved in barrier regulation and discerned complex condition-specific pathways. For instance, the MAPK-activated protein kinase 2 (MAPKAPK2/MK2) had early barrier-weakening activity in both inflammatory conditions but late barrier-strengthening activity exclusively with thrombin alone. Using temporal Western blotting, we confirmed that MAPKAPK2/MK2 was differentially phosphorylated under the two inflammatory conditions. We further showed with lentivirus-mediated knockdown of MAPK14/p38α and drug targeting the MAPK14/p38α-MAPKAPK2/MK2 complex that a MAP3K20/ZAK-MAPK14/p38α axis controlled the late activation of MAPKAPK2/MK2 in the thrombin-alone condition. Beyond the MAPKAPK2/MK2 switch, TREKING predicts extensive interconnected networks that control endothelial barrier dynamics.
Assuntos
Células Endoteliais , Proteína Quinase 14 Ativada por Mitógeno , Células Endoteliais/metabolismo , Trombina/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Inibidores de Proteínas QuinasesRESUMO
The Leishmania life cycle alternates between promastigotes, found in the sandfly, and amastigotes, found in mammals. When an infected sandfly bites a host, promastigotes are engulfed by phagocytes (i.e., neutrophils, dendritic cells, and macrophages) to establish infection. When these phagocytes die or break down, amastigotes must be re-internalized to survive within the acidic phagolysosome and establish disease. To define host kinase regulators of Leishmania promastigote and amastigote uptake and survival within macrophages, we performed an image-based kinase regression screen using a panel of 38 kinase inhibitors with unique yet overlapping kinase targets. We also targeted inert beads to complement receptor 3 (CR3) or Fcγ receptors (FcR) as controls by coating them with complement/C3bi or IgG respectively. Through this approach, we identified several putative host kinases that regulate receptor-mediated phagocytosis and/or the uptake of L. amazonensis. Findings included kinases previously implicated in Leishmania uptake (such as Src family kinases (SFK), Abl family kinases (ABL1/c-Abl, ABL2/Arg), and spleen tyrosine kinase (SYK)), but we also uncovered many novel kinases. Our methods also predicted host kinases necessary for promastigotes to convert to amastigotes or for amastigotes to survive within macrophages. Overall, our results suggest that the concerted action of multiple interconnected networks of host kinases are needed over the course of Leishmania infection, and that the kinases required for the parasite's life cycle may differ substantially depending on which receptors are bound and the life cycle stage that is internalized. In addition, using our screen, we identified kinases that appear to preferentially regulate the uptake of parasites over beads, indicating that the methods required for Leishmania to be internalized by macrophages may differ significantly from generalized phagocytic mechanisms. Our findings are intended to be used as a hypothesis generation resource for the broader scientific community studying the roles of kinases in host-pathogen interactions.
RESUMO
N-myristoyltransferase (NMT) is a promising antimalarial drug target. Despite biochemical similarities between Plasmodium vivax and human NMTs, our recent research demonstrated that high selectivity is achievable. Herein, we report PvNMT-inhibiting compounds aimed at identifying novel mechanisms of selectivity. Various functional groups are appended to a pyrazole moiety in the inhibitor to target a pocket formed beneath the peptide binding cleft. The inhibitor core group polarity, lipophilicity, and size are also varied to probe the water structure near a channel. Selectivity index values range from 0.8 to 125.3. Cocrystal structures of two selective compounds, determined at 1.97 and 2.43 Å, show that extensions bind the targeted pocket but with different stabilities. A bulky naphthalene moiety introduced into the core binds next to instead of displacing protein-bound waters, causing a shift in the inhibitor position and expanding the binding site. Our structure-activity data provide a conceptual foundation for guiding future inhibitor optimizations.
Assuntos
Aciltransferases , Antimaláricos , Inibidores Enzimáticos , Plasmodium vivax , Pirazóis , Pirazóis/química , Pirazóis/farmacologia , Pirazóis/síntese química , Plasmodium vivax/enzimologia , Plasmodium vivax/efeitos dos fármacos , Aciltransferases/antagonistas & inibidores , Aciltransferases/metabolismo , Aciltransferases/química , Relação Estrutura-Atividade , Antimaláricos/química , Antimaláricos/farmacologia , Antimaláricos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/síntese química , Cristalografia por Raios X , Humanos , Modelos Moleculares , Sítios de LigaçãoRESUMO
The Leishmania life cycle alternates between promastigotes, found in the sandfly, and amastigotes, found in mammals. When an infected sandfly bites a host, promastigotes are engulfed by phagocytes (i.e., neutrophils, dendritic cells, and macrophages) to establish infection. When these phagocytes die or break down, amastigotes must be re-internalized to survive within the acidic phagolysosome and establish disease. To define host kinase regulators of Leishmania promastigote and amastigote uptake and survival within macrophages, we performed an image-based kinase regression screen using a panel of 38 kinase inhibitors with unique yet overlapping kinase targets. We also targeted inert beads to complement receptor 3 (CR3) or Fcγ receptors (FcR) as controls by coating them with complement/C3bi or IgG respectively. Through this approach, we identified several putative host kinases that regulate receptor-mediated phagocytosis and/or the uptake of L. amazonensis. Findings included kinases previously implicated in Leishmania uptake (such as Src family kinases (SFK), Abl family kinases (ABL1/c-Abl, ABL2/Arg), and spleen tyrosine kinase (SYK)), but we also uncovered many novel kinases. Our methods also predicted host kinases necessary for promastigotes to convert to amastigotes or for amastigotes to survive within macrophages. Overall, our results suggest that the concerted action of multiple interconnected networks of host kinases are needed over the course of Leishmania infection, and that the kinases required for the parasite's life cycle may differ substantially depending on which receptors are bound and the life cycle stage that is internalized. In addition, using our screen, we identified kinases that appear to preferentially regulate the uptake of parasites over beads, indicating that the methods required for Leishmania to be internalized by macrophages may differ significantly from generalized phagocytic mechanisms. Our findings are intended to be used as a hypothesis generation resource for the broader scientific community studying the roles of kinases in host-pathogen interactions.
RESUMO
Introduction: Dengue is an arboviral disease causing severe illness in over 500,000 people each year. Currently, there is no way to constrain dengue in the clinic. Host kinase regulators of dengue virus (DENV) infection have the potential to be disrupted by existing therapeutics to prevent infection and/or disease progression. Methods: To evaluate kinase regulation of DENV infection, we performed kinase regression (KiR), a machine learning approach that predicts kinase regulators of infection using existing drug-target information and a small drug screen. We infected hepatocytes with DENV in vitro in the presence of a panel of 38 kinase inhibitors then quantified the effect of each inhibitor on infection rate. We employed elastic net regularization on these data to obtain predictions of which of 291 kinases are regulating DENV infection. Results: Thirty-six kinases were predicted to have a functional role. Intriguingly, seven of the predicted kinases - EPH receptor A4 (EPHA4), EPH receptor B3 (EPHB3), EPH receptor B4 (EPHB4), erb-b2 receptor tyrosine kinase 2 (ERBB2), fibroblast growth factor receptor 2 (FGFR2), Insulin like growth factor 1 receptor (IGF1R), and ret proto-oncogene (RET) - belong to the receptor tyrosine kinase (RTK) family, which are already therapeutic targets in the clinic. We demonstrate that predicted RTKs are expressed at higher levels in DENV infected cells. Knockdown of EPHB4, ERBB2, FGFR2, or IGF1R reduces DENV infection in hepatocytes. Finally, we observe differential temporal induction of ERBB2 and IGF1R following DENV infection, highlighting their unique roles in regulating DENV. Discussion: Collectively, our findings underscore the significance of multiple RTKs in DENV infection and advocate further exploration of RTK-oriented interventions against dengue.