RESUMEN
BACKGROUND: Plasmodium falciparum parasites export more than 400 proteins into the cytosol of their host erythrocytes. These exported proteins catalyse the formation of knobs on the erythrocyte plasma membrane and an overall increase in erythrocyte rigidity, presumably by modulating the endogenous erythrocyte cytoskeleton. In uninfected erythrocytes, Band 4.1 (4.1R) plays a key role in regulating erythrocyte shape by interacting with multiple proteins through the three lobes of its cloverleaf-shaped N-terminal domain. In P. falciparum-infected erythrocytes, the C-lobe of 4.1R interacts with the P. falciparum protein mature parasite-infected erythrocyte surface antigen (MESA), but it is not currently known whether other P. falciparum proteins bind to other lobes of the 4.1R N-terminal domain. METHODS: In order to identify novel 4.1R interacting proteins, a yeast two-hybrid screen was performed with a fragment of 4.1R containing both the N- and α-lobes. Positive interactions were confirmed and investigated using site-directed mutagenesis, and antibodies were raised against the interacting partner to characterise it's expression and distribution in P. falciparum infected erythrocytes. RESULTS: Yeast two-hybrid screening identified a positive interaction between the 4.1R N- and α-lobes and PF3D7_0402000. PF3D7_0402000 is a member of a large family of exported proteins that share a domain of unknown function, the PHIST domain. Domain mapping and site-directed mutagenesis established that it is the PHIST domain of PF3D7_0402000 that interacts with 4.1R. Native PF3D7_0402000 is localized at the parasitophorous vacuole membrane (PVM), and colocalizes with a subpopulation of 4.1R. DISCUSSION: The function of the majority of P. falciparum exported proteins, including most members of the PHIST family, is unknown, and in only a handful of cases has a direct interaction between P. falciparum-exported proteins and components of the erythrocyte cytoskeleton been established. The interaction between 4.1R and PF3D7_0402000, and localization of PF3D7_0402000 with a sub-population of 4.1R at the PVM could indicate a role in modulating PVM structure. Further investigation into the mechanisms for 4.1R recruitment is needed. CONCLUSION: PF3D7_0402000 was identified as a new binding partner for the major erythrocyte cytoskeletal protein, 4.1R. This interaction is consistent with a growing body of literature that suggests the PHIST family members function by interacting directly with erythrocyte proteins.
Asunto(s)
Membrana Eritrocítica/metabolismo , Interacciones Huésped-Parásitos , Plasmodium falciparum/fisiología , Proteínas Protozoarias/metabolismo , Análisis Mutacional de ADN , Humanos , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Proteínas Protozoarias/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Outbreaks of viral hemorrhagic fever caused by filoviruses have become more prevalent in recent years, with outbreaks of Ebola virus (EBOV), Sudan virus (SUDV), and Marburg virus (MARV) all occurring in 2022 and 2023. While licensed vaccines are now available for EBOV, vaccine candidates for SUDV and MARV are all in preclinical or early clinical development phases. During the recent outbreak of SUDV virus disease, the Biomedical Advanced Research and Development Authority (BARDA), as part of the Administration for Strategic Preparedness and Response within the U.S. Department of Health and Human Services, implemented key actions with our existing partners to advance preparedness and enable rapid response to the outbreak, while also aligning with global partners involved in the implementation of clinical trials in an outbreak setting. Beyond pre-existing plans prior to the outbreak, BARDA worked with product sponsors to expedite manufacturing of vaccine doses that could be utilized in clinical trials. While the SUDV outbreak has since ended, a new outbreak of MARV disease has emerged. It remains critical that we continue to advance a portfolio of vaccines against SUDV and MARV while also expediting manufacturing activities ahead of, or in parallel if needed, outbreaks.
RESUMEN
On March 12, 2021, the Biomedical Advanced Research and Development Authority (BARDA) sponsored a virtual market research workshop, "Partnering on Vaccines to Counter Multi-Drug Resistant Threats," to discuss the threat of antimicrobial resistance in the context of BARDA's mission space and the challenges encountered during the development of vaccines for specific antimicrobial resistant bacteria. The workshop convened representatives with expertise in vaccine development from government, academia, and industry. This report summarizes the presentations and subsequent discussions from the workshop and highlights existing challenges to advance the development of vaccine candidates for antimicrobial resistant pathogens, including Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus.
Asunto(s)
Antibacterianos , Vacunas , Antibacterianos/uso terapéutico , Bacterias , Farmacorresistencia Bacteriana , Escherichia coli , Pruebas de Sensibilidad Microbiana , Investigación , Staphylococcus aureusRESUMEN
Sudan ebolavirus (SUDV) is one of four members of the Ebolavirus genus known to cause Ebola Virus Disease (EVD) in humans, which is characterized by hemorrhagic fever and a high case fatality rate. While licensed therapeutics and vaccines are available in limited number to treat infections of Zaire ebolavirus, there are currently no effective licensed vaccines or therapeutics for SUDV. A well-characterized animal model of this disease is needed for the further development and testing of vaccines and therapeutics. In this study, twelve cynomolgus macaques (Macaca fascicularis) were challenged intramuscularly with 1000 PFUs of SUDV and were followed under continuous telemetric surveillance. Clinical observations, body weights, temperature, viremia, hematology, clinical chemistry, and coagulation were analyzed at timepoints throughout the study. Death from SUDV disease occurred between five and ten days after challenge at the point that each animal met the criteria for euthanasia. All animals were observed to exhibit clinical signs and lesions similar to those observed in human cases which included: viremia, fever, dehydration, reduced physical activity, macular skin rash, systemic inflammation, coagulopathy, lymphoid depletion, renal tubular necrosis, hepatocellular degeneration and necrosis. The results from this study will facilitate the future preclinical development and evaluation of vaccines and therapeutics for SUDV.
RESUMEN
Lipid raft microdomains, a component of detergent resistant membranes (DRMs), are routinely exploited by pathogens during host-cell entry. Multiple membrane-surface proteins mediate Plasmodium ookinete invasion of the Anopheles midgut, a critical step in the parasite life cycle that is successfully targeted by transmission-blocking vaccines (TBV). Given that lipid rafts are a common feature of host-pathogen interactions, we hypothesized that they promote the partitioning of midgut surface proteins and thus facilitate ookinete invasion. In support of this hypothesis, we found that five of the characterized Anopheles TBV candidates, including the leading Anopheles TBV candidate, AgAPN1, are present in Anopheles gambiae DRMs. Therefore, to extend the repertoire of putative midgut ligands that can be targeted by TBVs, we analyzed midgut DRMs by tandem mass spectrometry. We identified 1452 proteins including several markers of DRMs. Since glycosylphosphotidyl inositol (GPI)-anchored proteins partition to DRMs, we characterized the GPI subproteome of An. gambiae midgut brush-border microvilli and found that 96.9% of the proteins identified in the GPI-anchored fractions were also present in DRMs. Our study vastly expands the number of candidate malarial TBV targets for subsequent analysis by the broader community and provides an inferred role for midgut plasmalemma microdomains in ookinete cell invasion.
Asunto(s)
Anopheles/metabolismo , Sistema Digestivo/metabolismo , Proteínas Ligadas a GPI/metabolismo , Interacciones Huésped-Parásitos , Proteínas de Insectos/metabolismo , Microdominios de Membrana/metabolismo , Plasmodium/fisiología , Animales , Anopheles/parasitología , Colesterol/metabolismo , Sistema Digestivo/citología , Epitelio/metabolismo , Femenino , Proteínas Ligadas a GPI/aislamiento & purificación , Glicosilación , Inmunoglobulinas/química , Proteínas de Insectos/aislamiento & purificación , Microvellosidades/metabolismo , Modelos Biológicos , Estructura Terciaria de ProteínaRESUMEN
SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) play a central role in regulating and facilitating vesicular traffic in eukaryotic cells. While SNAREs have been well characterized in other eukaryotes, little is known about their role in the unique protein trafficking pathways in Plasmodium falciparum. We have identified seven Qa-SNAREs in the P. falciparum genome and confirmed the gene structure of all seven, which in one case differs from the predicted structure in the database. Based on comprehensive sequence alignments we made predictions for the intracellular locations of all seven P. falciparum Qa-SNAREs, and confirmed the predicted location for one Qa-SNARE, PfStx1, which is most closely related to other eukaryotic plasma membrane Qa-SNAREs such as syntaxin 1. This is the first identified trafficking component localized proximal to the P. falciparum plasma membrane.
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Proteínas Protozoarias/genética , Proteínas Qa-SNARE/genética , Vías Secretoras/genética , Secuencia de Aminoácidos , Animales , Membrana Celular/química , ADN Protozoario/química , ADN Protozoario/genética , Datos de Secuencia Molecular , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas Qa-SNARE/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Novel strategies to directly thwart malaria transmission are needed to maintain the gains achieved by current control measures. Transmission-blocking interventions (TBIs), namely vaccines and drugs targeting parasite or mosquito molecules required for vector-stage parasite development, have been recognized as promising approaches for preventing malaria transmission. However, the number of TBI targets is limited and their degree of conservation among the major vector-parasite systems causing human disease is unclear. Therefore, discovery and characterization of novel proteins involved in vector-stage parasite development of Plasmodium falciparum and Plasmodium vivax is paramount. We mined the recent Anopheles gambiae midgut lipid raft proteome for putative mosquito-derived TBI targets and characterized a secreted glycoconjugate of unknown function, AgSGU. We analyzed molecular variation in this protein among a range of anopheline mosquitoes, determined its transcriptomic and proteomic profiles, and conducted both standard and direct membrane feeding assays with P. falciparum (lab/field) and P. vivax (field) in An. gambiae and Anopheles dirus. We observed that α-AgSGU antibodies significantly reduced midgut infection intensity for both lab and field isolates of P. falciparum in An. gambiae and An. dirus. However, no transmission-reducing effects were noted when comparable concentrations of antibodies were included in P. vivax-infected blood meals. Although antibodies against AgSGU exhibit transmission-reducing activity, the high antibody titer required for achieving 80% reduction in oocyst intensity precludes its consideration as a malaria mosquito-based TBI candidate. However, our results suggest that P. falciparum and P. vivax ookinetes use a different repertoire of midgut surface glycoproteins for invasion and that α-AgSGU antibodies, as well as antibodies to other mosquito-midgut microvillar surface proteins, may prove useful as tools for interrogating Plasmodium-mosquito interactions.
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Anopheles/metabolismo , Anopheles/parasitología , Proteínas de Insectos/metabolismo , Malaria Falciparum/transmisión , Malaria Vivax/transmisión , Plasmodium falciparum/patogenicidad , Plasmodium vivax/patogenicidad , Secuencia de Aminoácidos , Animales , Anopheles/genética , Evolución Molecular , Tracto Gastrointestinal/metabolismo , Expresión Génica , Variación Genética , Proteínas de Insectos/química , Proteínas de Insectos/genética , Datos de Secuencia Molecular , Selección Genética , Alineación de SecuenciaRESUMEN
Streptococcus pneumoniae cause considerable morbidity and mortality, with persistent neurological sequelae, particularly in young children and the elderly. It is widely assumed that carriage occurs through direct mucosal colonization from the environment whereas meningitis results from invasion from the blood. However, the results of published studies can be interpreted that pneumococci may enter the brain directly from the nasal cavity by axonal transport through olfactory nerves. This hypothesis is based on findings that (i) teichoic acid of the pneumococcal cell wall interact with gangliosides (GLS), (ii) the interaction of GLS with cholera toxin leads to axonal transport through the olfactory nerves into the brain, and (iii) viruses enter the brain through axonal transport into olfactory nerves. After nasal inoculation, we observe high numbers of pneumococci in nasal washes and the olfactory nerves and epithelium. Significant numbers of pneumococci also infected the olfactory bulbs, brain, and the trigeminal ganglia. The absence of bacteremia in this model makes it unlikely that the bacteria entered the brain from the blood stream. Recovery of colony-forming units from the brain, lungs, olfactory nerves, and epithelium and nasal washes was inhibited by incubating pneumococci with GLS before nasal inoculation. These findings, confirmed by PCR and immunohistochemistry, support a GLS-mediated process of infection and are consistent with pneumococci reaching the brain through retrograde axonal transport.