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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. [ABSTRACT FROM AUTHOR]

Drugs targeting multiple stages of the Plasmodium vivax life cycle are needed to reduce the health and economic burdens caused by malaria worldwide. N-myristoyltransferase (NMT) is an essential eukaryotic enzyme and a validated drug target for combating malaria. However, previous PvNMT inhibitors have failed due to their low selectivity over human NMTs. Herein, we apply a structure-guided hybridization approach combining chemical moieties of previously reported NMT inhibitors to develop the next generation of PvNMT inhibitors. A high-resolution crystal structure of PvNMT bound to a representative selective hybrid compound reveals a unique binding site architecture that includes a selective conformation of a key tyrosine residue. The hybridized compounds significantly decrease P. falciparum blood-stage parasite load and consistently exhibit dose-dependent inhibition of P. vivax liver stage schizonts and hypnozoites. Our data demonstrate that hybridized NMT inhibitors can be multistage antimalarials, targeting dormant and developing forms of liver and blood stage. Developing selective N-myristoyltransferase (NMT) inhibitors has been challenging. Here, the authors describe selective NMT inhibitors that can be used as multistage antimalarials, targeting dormant and developing forms of liver and blood stage. [ABSTRACT FROM AUTHOR]

The mechanistic target of rapamycin (mTOR) functions in two distinct complexes: mTORC1, and mTORC2. mTORC1 has been implicated in the pathogenesis of flaviviruses including dengue, where it contributes to the establishment of a pro-viral autophagic state. Activation of mTORC2 occurs upon infection with some viruses, but its functional role in viral pathogenesis remains poorly understood. In this study, we explore the consequences of a physical protein-protein interaction between dengue non-structural protein 5 (NS5) and host cell mTOR proteins during infection. Using shRNA to differentially target mTORC1 and mTORC2 complexes, we show that mTORC2 is required for optimal dengue replication. Furthermore, we show that mTORC2 is activated during viral replication, and that mTORC2 counteracts virus-induced apoptosis, promoting the survival of infected cells. This work reveals a novel mechanism by which the dengue flavivirus can promote cell survival to maximize viral replication. [ABSTRACT FROM AUTHOR]

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. Author summary: Identifying specific features of vaccine-elicited antibody responses that are associated with protection from malaria infection is a key step toward the development of a safe and effective vaccine. Here we compared antibody and B cell responses in two mouse strains that exhibited a differential ability to generate antibodies that protect from infection challenge. We found that protection was due to the presence of vaccine-elicited antibodies and could be transferred between strains, and that the ability of antibodies to neutralize the parasite was directly linked to the strength (affinity) with which it binds CSP. Thus, we sought to understand if there were differences in the two strains in the process of B cell maturation that leads to generation of high affinity, protective antibody responses after vaccination. Overall, our comparative analysis indicates that germinal center (GC) activity, a key process in B cell maturation, was significantly diminished in the non-protected strain. Further, we observed evidence of higher levels of somatic mutation, which is a result of germinal center activity, in protected mice. Thus, our results indicate that the ability to generate protective antibody responses was linked to enhanced B cell maturation in the protected strain, providing a key clue to the type of responses that should be generated by future vaccines. [ABSTRACT FROM AUTHOR]

Upon transmission to the human host, Plasmodium sporozoites exit the skin, are taken up by the blood stream, and then travel to the liver where they infect and significantly modify a single hepatocyte. Low infection rates within the liver have made proteomic studies of infected hepatocytes challenging, particularly in vivo , and existing studies have been largely unable to consider how protein and phosphoprotein differences are altered at different spatial locations within the heterogeneous liver. Using digital spatial profiling, we characterized changes in host signaling during Plasmodium yoelii infection in vivo without disrupting the liver tissue. Moreover, we measured alterations in protein expression around infected hepatocytes and identified a subset of CD163+ Kupffer cells that migrate towards infected cells during infection. These data offer the first insight into the heterogeneous microenvironment that surrounds the infected hepatocyte and provide insights into how the parasite may alter its milieu to influence its survival and modulate immunity. [ABSTRACT FROM AUTHOR]

Cellular decisions in human development, homeostasis, regeneration, and disease are coordinated in large part by signals that are spatially localized in tissues. These signals are often soluble, such that biomolecules produced by one cell diffuse to receiving cells. To recapitulate soluble factor patterning in vitro, several microscale strategies have been developed. However, these techniques often introduce new variables into cell culture experiments (e.g., fluid flow) or are limited in their ability to pattern diverse solutes in a user-defined manner. To address these challenges, we developed an adaptable method that facilitates spatial presentation of biomolecules across cells in traditional open cultures in vitro. This technique employs device inserts that are placed in standard culture wells, which support localized diffusive pattern transmission through microscale spaces between device features and adherent cells. Devices can be removed and cultures can be returned to standard media following patterning. We use this method to spatially control cell labeling with pattern features ranging in scale from several hundred microns to millimeters and with sequential application of multiple patterns. To better understand the method we investigate relationships between pattern fidelity, device geometry, and consumption and diffusion kinetics using finite element modeling. We then apply the method to spatially defining reporter cell heterogeneity by patterning a small molecule modulator of genetic recombination with the requisite sustained exposure. Finally, we demonstrate use of this method for patterning larger and more slowly diffusing particles by creating focal sites of gene delivery and infection with adenoviral, lentiviral, and Zika virus particles. Thus, our method leverages devices that interface with standard culture vessels to pattern diverse diffusible factors, geometries, exposure dynamics, and recipient cell types, making it well poised for adoption by researchers across various fields of biological research. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

Plasmodium parasites have extensive needs from their host hepatocytes during the obligate liver stage of infection, yet there remains sparse knowledge of specific host regulators. Here we assess 34 host-targeted kinase inhibitors for their capacity to eliminate Plasmodium yoelii-infected hepatocytes. Using pre-existing activity profiles of each inhibitor, we generate a predictive computational model that identifies host kinases, which facilitate Plasmodium yoelii liver stage infection. We predict 47 kinases, including novel and previously described kinases that impact infection. The impact of a subset of kinases is experimentally validated, including Receptor Tyrosine Kinases, members of the MAP Kinase cascade, and WEE1. Our approach also predicts host-targeted kinase inhibitors of infection, including compounds already used in humans. Three of these compounds, VX-680, Roscovitine and Sunitinib, each eliminate >85% of infection. Our approach is well-suited to uncover key host determinants of infection in difficult model systems, including field-isolated parasites and/or emerging pathogens. [ABSTRACT FROM AUTHOR]

P lasmodium 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 P lasmodium 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 P lasmodium 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 P lasmodium falciparum. This parasite preference for host cells of high ploidy cannot be explained by differences in hepatocyte size or DNA replication. We conclude that P lasmodium preferentially infects and develops in polyploid hepatocytes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: Plasmodium parasites infect the liver and replicate inside hepatocytes before they invade erythrocytes and trigger clinical malaria. Analysis of host signaling pathways affected by liver-stage infection could provide critical insights into host–pathogen interactions and reveal targets for intervention. Using protein lysate microarrays, we found that Plasmodium yoelii rodent malaria parasites perturb hepatocyte regulatory pathways involved in cell survival, proliferation, and autophagy. Notably, the prodeath protein p53 was substantially decreased in infected hepatocytes, suggesting that it could be targeted by the parasite to foster survival. Indeed, mice that express increased levels of p53 showed reduced liver-stage parasite burden, whereas p53 knockout mice suffered increased liver-stage burden. Furthermore, boosting p53 levels with the use of the small molecule Nutlin-3 dramatically reduced liver-stage burden in vitro and in vivo. We conclude that perturbation of the hepatocyte p53 pathway critically impacts parasite survival. Thus, host pathways might constitute potential targets for host-based antimalarial prophylaxis. [ABSTRACT FROM AUTHOR]

Certain bacterial adhesins appear to promote a pathogen's extracellular lifestyle rather than its entry into host cells. However, little is known about the stimuli elicited upon such pathogen host-cell interactions. Here, we report that type IV pili (Tfp)-producing Neisseria gonorrhoeae (P+GC) induces an immediate recruitment of caveolin-1 (Cav1) in the host cell, which subsequently prevents bacterial internalization by triggering cytoskeletal rearrangements via downstream phosphotyrosine signaling. A broad and unbiased analysis of potential interaction partners for tyrosine-phosphorylated Cav1 revealed a direct interaction with the Rho-family guanine nucleotide exchange factor Vav2. Both Vav2 and its substrate, the small GTPase RhoA, were found to play a direct role in the Cav1-mediated prevention of bacterial uptake. Our findings, which have been extended to enteropathogenic Escherichia coli, highlight how Tfp-producing bacteria avoid host cell uptake. Further, our data establish a mechanistic link between Cav1 phosphorylation and pathogen-induced cytoskeleton reorganization and advance our understanding of caveolin function. [ABSTRACT FROM AUTHOR]

Both subunit and attenuated whole-sporozoite vaccination strategies against Plasmodium infection have shown promising initial results in malaria-naive westerners but less efficacy in malaria-exposed individuals in endemic areas. Here, we demonstrate proof of concept by using a rodent malaria model in which non-neutralizing antibodies (nNAbs) can directly interfere with protective anti-circumsporozoite protein (CSP) humoral responses. We characterize a monoclonal antibody, RAM1, against Plasmodium yoelii sporozoite major surface antigen CSP. Unlike the canonical Py CSP repeat domain binding and neutralizing antibody (NAb) 2F6, RAM1 does not inhibit sporozoite traversal or entry of hepatocytes in vitro or infection in vivo. Although 2F6 and RAM1 bind non-overlapping regions of the CSP-repeat domain, pre-treatment with RAM1 abrogates the capacity of NAb to block sporozoite traversal and invasion in vitro. Importantly, RAM1 reduces the efficacy of the polyclonal humoral response against Py CSP in vivo. Collectively, our data provide a proof of concept that nNAbs can alter the efficacy of malaria vaccination. [Display omitted] • We isolate a non-neutralizing antibody from Py CSP-immunized BALB/cJ mice • A non-neutralizing Ab, RAM1, abrogates the blocking capacity of an αCSP NAb • A non-neutralizing Ab, RAM1, reduces the vaccine efficacy in mice in vivo • Further exploration for the role of pre-existing nNAbs in the field is warranted Plasmodium CSP is a complex protein with multiple unique domains and repetitive motifs. Vijayan et al. isolate a non-neutralizing CSP major repeat binding antibody that interferes with the protective efficacy of NAbs. This implicates direct antibody interference as a mechanism by which pre-existing nNAbs may interfere with vaccine-elicited NAbs. [ABSTRACT FROM AUTHOR]

Collaborative, cross-disciplinary research may rapidly advance the fight against drug resistance. This Editorial discusses the state of research on drug resistance in the fields of cancer, infectious disease, and agriculture. Reaching across the aisle for a more cross-collaborative approach may lead to exciting breakthroughs toward tackling the challenges of drug resistance in each field. [ABSTRACT FROM AUTHOR]

Summary Plasmodium parasites are highly selective when infecting hepatocytes and induce many changes within the host cell upon infection. While several host cell factors have been identified that are important for liver infection, our understanding of what facilitates the maintenance of infection remains incomplete. Here, we describe a role for phosphorylated ribosomal protein S6 (Ser235/236) (p-RPS6) in Plasmodium yoelii -infected hepatocytes. Blocking RPS6 phosphorylation prior to infection decreases the number of liver stage parasites within 24 h. Infected hepatocytes exhibit elevated levels of p-RPS6 while simultaneously abrogating the induction of phosphorylation of RPS6 in response to insulin stimulation. This is in contrast with the regulation of p-RPS6 by Toxoplasma gondii, which elevates levels of p-RPS6 after infection but does not alter the response to insulin. Our data support a model in which RPS6 phosphorylation is uncoupled from canonical regulators in Plasmodium- infected hepatocytes and is relied on by the parasite to maintain infection. Graphical Abstract Highlights • p-RPS6 is elevated in hepatocyte populations susceptible to Plasmodium (Py) • Inhibiting RPS6 phosphorylation prior to infection decreases Py infection • In Py- infected hepatocytes, canonical Akt/ RPS6 signaling is disrupted • Response to insulin is abrogated in Py- infected hepatocytes After mosquito-to-human transmission, Plasmodium parasites infect hepatocytes. Glennon et al. demonstrate that infected cells exhibit elevated levels of ribosomal protein S6 phosphorylation, and this phosphorylation appears uncoupled from canonical regulators. This work raises the possibility that Plasmodium -infected hepatocytes are governed by non-canonical, re-wired signal transduction cascades. [ABSTRACT FROM AUTHOR]

After transmission by Anopheles mosquitoes, malaria parasite sporozoites target the liver, where they infect hepatocytes and multiply thousands of times. The release of new parasites into the blood stream then initiates symptomatic red blood cell infection. Although successful replication within hepatocytes is critical for host infection, little is known about parasite-hepatocyte interactions that ensure parasite survival and development. In this issue of EMBO Reports, the Mota group describes a beneficial role of the host ER stress pathway for Plasmodium survival in infected hepatocytes . They demonstrate that proteins and transcripts that act in the unfolded protein response ( UPR) are elevated in hepatocytes in response to infection. Reversing these perturbations by eliminating the splicing of XBP1 or knockdown of CREBH is detrimental to parasite development. These findings are of significant interest in light of recent findings that elucidate other aspects of liver-stage parasite-hepatocyte interactions and raise new, intriguing questions for the field (Fig 1). [ABSTRACT FROM AUTHOR]

A letter to the editor is presented regarding a study on the crucial role played by hepatocyte growth factor (HGF) receptor in liver-stage infection.

Receptor tyrosine kinases (RTKs) process extracellular cues by activating a broad array of signaling proteins. Paradoxically, they often use the same proteins to elicit diverse and even opposing phenotypic responses. Binary, ‘on–off’ wiring diagrams are therefore inadequate to explain their differences. Here, we show that when six diverse RTKs are placed in the same cellular background, they activate many of the same proteins, but to different quantitative degrees. Additionally, we find that the relative phosphorylation levels of upstream signaling proteins can be accurately predicted using linear models that rely on combinations of receptor-docking affinities and that the docking sites for phosphoinositide 3-kinase (PI3K) and Shc1 provide much of the predictive information. In contrast, we find that the phosphorylation levels of downstream proteins cannot be predicted using linear models. Taken together, these results show that information processing by RTKs can be segmented into discrete upstream and downstream steps, suggesting that the challenging task of constructing mathematical models of RTK signaling can be parsed into separate and more manageable layers. [ABSTRACT FROM AUTHOR]