Biophysical properties of blood-stage Plasmodium falciparum malaria: from single-cell host-pathogen interactions to human protective polymorphisms

Malaria is a mosquito-borne infectious disease responsible for half a million deaths every year and long-term economic stagnation in many countries where it is endemic. All symptoms and pathology of malaria are caused by Plasmodium falciparum parasite, and are initiated when parasites invade human red blood cells, then mature and multiply inside them in approximately 48 hours. The invasion process is completed in less than a minute and is one of the most crucial, yet least understood, phases of malaria infection. It also represents a brief window in which the parasites are extracellular and hence exposed to the host immune system, therefore representing a potential target for vaccines and treatments. The work described in this thesis firstly includes the optimisation of a real-time live microscopy platform for recording parasite egress-invasion sequences under controlled conditions, and to investigate their morphology and kinetics. This set-up was employed to address the role of calcium in mediating successful invasion by observing the invasion process simultaneously in bright-field and fluorescence. Elevated calcium signal was found to be absent during the early steps of the process, implying that calcium does not trigger invasion, and an alternative invasion mechanism was suggested. To investigate whether parasite and host cell physical parameters were actively involved in invasion, adhesion forces between parasites and red cells were measured with optical tweezers, while the biophysical properties of the red blood cells such as bending modulus, tension, radius, and viscosity were assessed by analysing their plasma membrane fluctuations. In particular, cells from the Dantu blood group, a rare blood variant found mainly in East Africa that provides up to 70% protection against malaria, and from Beta-thalassaemia individuals, were studied. A general correlation between red blood cell membrane tension, invasion efficiency and dynamics was established, determining a protective tension threshold above which cells are less likely to be invaded. Finally, mature parasites have the ability to bind to the endothelium of peripheral blood vessels, causing impair flow that can lead to a range of fatal conditions. To study malaria cytoadherence to endothelial cells, a microfluidic device was designed to produce an in vitro physiologically relevant model of human circulation. Increasing cytoadhesion was experimentally associated with endothelial glycocalyx disruption as initial factor for malaria pathogenesis. Live imaging methods and techniques adopted in this study highlight mechanisms crucial for malaria infection, and represent an innovative and complementary study of this disease with respect to purely biological approaches. These findings show how changes in red blood cell biophysics can be linked to human evolutionary response against malaria with tangible effects on the population

The primate malarias

Front Matter -- 1. Evolution of the Primate Malarias -- 2. Historical Review -- 3. Ecology of the Hosts in Relation to the Transmission of Malaria -- 4. Life Cycle and the Phenomenon of Relapse -- 5. Plasmodium vivax -- 6. Plasmodium cynomolgi -- 7. Plasmodium eylesi -- 8. Plasmodium gonderi -- 9. Plasmodium hylobati -- 10. Plasmodium jefferyi -- 11. Plasmodium pitheci -- 12. Plasmodium schwetzi -- 13. Plasmodium simium -- 14. Plasmodium youngi -- 15. Plasmodium ovale -- 16. Plasmodium fieldi -- 17. Plasmodium simiovale -- 18. Plasmodium malariae -- 19. Plasmodium brasilianum -- 20. Plasmodium inui -- 21. Plasmodium rodhaini -- 22. Plasmodium falciparum -- 23. Plasmodium coatneyi -- 24. Plasmodium fragile -- 25. Plasmodium reichenowi -- 26. Plasmodium knowlesi -- 27. Plasmodium girardi -- 28. Plasmodium lemuris -- Author Index -- Subject IndexG. Robert Coatney, William E. Collins, McWilson Warren, Peter G. Contacos.The electronic version was produced in 2003 by James J. Sullivan, Gregory Noland, and Leanne Ward, Biology and Diagnostics Branch of the Division of Parasitic Diseases [CDC].Includes bibliographies

Exploring Molecular Diversity: There is Plenty of Room at Markush's

L'estratègia de les etapes inicials del descobriment de fàrmacs està normalment basada en un procés anomenat hit-to-lead que implica un extens estudi entorn de la síntesi de derivats d'una molècula original que prèviament hagi mostrat certa activitat biològica davant d'una diana concreta. Per tant, aquest procés comporta la síntesi de molts anàlegs que descriurien una subquimioteca, que generalment evidencia que aquests estudis estan molt focalitzats al voltant de l'espai químic del compost original. Així i tot, quan aquesta molècula és finalment patentada, es descriu un espai químic molt més vast per mitjà d'estructures Markush donant per suposat que alguns dels seus derivats puguin presentar també activitat biològica. Tot i això, la presència d'aquestes estructures no implica la síntesi comprovada de tota la biblioteca molecular sinó només una petita mostra de la mateixa. La nostra hipòtesi és que hi ha una gran part de l’espai químic d’aquestes biblioteques que està sense explorar i pot amagar possibles candidats que poden fins i tot superar l’activitat del hit original. A través d'aquest projecte, es proposa una alternativa que sosté que una selecció racional de poques molècules – basat en l'agrupament segons semblança molecular – pot representar de manera més significativa l'espai químic establert, oferint la possibilitat d'explorar regions desconegudes que podrien amagar més potencial biològic. Després de revisar els darrers fàrmacs aprovats per la FDA en el període del 2008 al 2020 i la base de dades de molècules bioactives de ChEMBL, s'ha dut a terme una exploració de l'ampli espai químic resultant de molècules petites amb propietats similars a les dels medicaments per definir nous espais accessibles que podrien ocultar activitat. Els resultats obtinguts de set casos d'estudis reals han demostrat que tant la selecció racional com l’aleatòria representen més significativament les biblioteques combinatòries declarades a les patents, que les molècules descrites fins ara. S'han realitzat dos estudis pràctics que implementen aquesta metodologia suggerida per descriure millor l'espai químic del fàrmac antipalúdic Tafenoquina i del Dacomitinib, un inhibidor de tirosina cinases de segona generació per al tractament del càncer de pulmó de cèl·lules no petites. L’exploració de l’espai químic d’aquestes dues famílies ha portat a la síntesi racional de set anàlegs antipalúdics i vuit inhibidors de cinases que han mostrat interessants activitats inhibidores. Aquests resultats demostren que l'aplicació de la quimioinformàtica per a la selecció de biblioteques pot millorar la capacitat d'inspeccionar millor els conjunts de dades químiques per identificar nous compostos precandidats i representar grans biblioteques per a posteriors campanyes de reposicionament.La estrategia de las etapas iniciales del descubrimiento de fármacos está normalmente basada en un proceso denominado hit-to-lead que implica un extenso estudio entorno a la síntesis de derivados de una molécula original que previamente haya expresado cierta actividad biológica frente a una diana concreta. Por ende, este proceso conlleva la síntesis de muchos análogos que describirían una sublibrería química, la cual generalmente evidencia que estos estudios están muy focalizados alrededor del espacio químico del compuesto original. Aún y así, cuando esta molécula es finalmente patentada, se describe un espacio químico mucho más vasto por medio de estructuras Markush teorizando que algunos de sus derivados puedan presentar también actividad biológica. Sin embargo, la presencia de estas estructuras no implica la síntesis comprobada de toda la biblioteca molecular sino solo una pequeña muestra de la misma. Nuestra hipótesis es que hay una gran parte del espacio químico de estas bibliotecas que está sin explorar y puede ocultar posibles candidatos que pueden hasta superar la actividad del hit original. A través de este proyecto, se propone una alternativa que sostiene que una selección racional de pocas moléculas – fundada en el agrupamiento según su similitud química – puede representar de manera más significativa el espacio químico establecido, ofreciendo la posibilidad de explorar regiones desconocidas que podrían ocultar más potencial biológico. Después de revisar los últimos fármacos aprobados por la FDA en el período de 2008 a 2020 y la base de datos de moléculas bioactivas de ChEMBL, se ha llevado a cabo una exploración del amplio espacio químico resultante de moléculas pequeñas con propiedades similares a las de los medicamentos para definir nuevos espacios accesible que podrían ocultar actividad. Los resultados obtenidos de siete casos de estudios reales han demostrado que tanto la selección racional como la aleatoria representan más significativamente las bibliotecas combinatorias declaradas en las patentes que las moléculas descritas hasta la fecha. Se han desarrollado dos estudios prácticos que implementan esta metodología sugerida para describir mejor el espacio químico del fármaco antipalúdico Tafenoquina y Dacomitinib, un inhibidor de la tirosina quinasa de segunda generación para el tratamiento del cáncer de pulmón de células no pequeñas. La exploración del espacio químico de estas dos familias ha llevado a la síntesis racional de siete análogos antipalúdicos y ocho inhibidores de quinasas que han mostrado interesantes actividades inhibidoras. Estos resultados demuestran que la aplicación de la quimioinformática para la selección de bibliotecas puede mejorar la capacidad de inspeccionar mejor los conjuntos de datos químicos para identificar nuevos potenciales hits y representar grandes bibliotecas para fines de reposicionamiento.The early Drug Discovery strategy is commonly based on a hit-to-lead process which involves large research on the synthesis of derivatives of an original molecule that had previously shown biological activity against a specific biological target. Therefore, this process implies the synthesis of many analogs leading to the description of a chemical sub-library which generally leads to a highly focused study on the chemical space nearby the hit compound. However, when this drug is finally patented, a wider chemical space derived from a Markush structure is described, theorizing that some analogs within may present biological activity. Nevertheless, this claim involving the Markush structure does not imply the proven synthesis of all the chemical library but just a small population of it. We hypothesize that there is a great part of the chemical space of these libraries that is unexplored and can hide potential lead candidates which may even surpass the activity of the original hit. Through this project, an alternative is proposed claiming that a rational selection of a short sample of small molecules – founded on similarity-based clustering – can represent more significatively the stated chemical space offering the possibility to explore the unknown space that could hide more potential biological activity. After a review on the latest approved drugs by the FDA in the period from 2008 to 2020 and the ChEMBL database of bioactive molecules, an exploration of the resulting wide chemical space of small molecules with drug-like properties has been assessed in order to define accessible spots that might hide biological activity. The obtained results from seven real cases of study have proven that random and rationally selected molecules represent more significantly the combinatorial libraries stated in the patents rather than the reported molecules until date. Furthermore, two practical studies implementing our suggested methodology have been developed to better describe the chemical space of the antimalarial drug Tafenoquine and Dacomitinib, a second-generation tyrosine kinase inhibitor for non-small-cell lung cancer treatment. The assessment driven by a better chemical space exploration of these two families have led to the rational synthesis of seven antimalarial analogs and eight kinase inhibitors which have shown interesting inhibitory activities. Our results evince that the application of cheminformatics for library selection may improve the ability to better inspect chemical datasets in order to identify new potential hits and represent large libraries for further reprofiling purposes

Operational Strategies for the Identification and Targeting of Hotspots of Malaria Transmission

Heterogeneous malaria exposure may result in distinct clusters of higher malaria burden, or hotspots, across space and time. Targeting control programs to these areas may be highly efficient, however, operationally attractive approaches for identifying hotspots are needed for any such program to be sustained by local malaria control programs. The principal aim of this project was to investigate the ability of convenient sampling to identify hotspots of malaria transmission in a low endemic transmission setting in the western Kenyan highlands: 1) The boundaries of hotspots, and associated uncertainties, was determined using a large community survey; 2) The value of convenience sampling to estimate transmission in the community was assessed using cross-­‐sectional surveys of 4964 children in 46 government primary schools and 3042 individuals in five rural-­‐health facilities; 3) The value of compound-­‐level screening of sentinel age groups that are likely to have patent level infections was determined and; 4) The potential use for convenience sampling in hotspot targeted approaches was assessed using spatial information on residences collected during the school and health-­‐facility surveys. The community-­‐based approach was able to detect 77% of the parasite infections in selected hotspots of malaria exposure using field-­‐based tools in sentinel age groups. Both convenience-­‐sampling approaches tested produced similar estimates of malaria transmission to the community when restricted to those residing in the same catchment areas and those testing positive for malaria were more likely to reside in a hotspot. The findings suggest that operationally attractive approaches provide reliable information on malaria transmission and may play an important role in targeted malaria control strategies. Future research on ascertaining what coverage of the hotspot is needed to see sustainable reductions in transmission would provide a threshold with which to gauge the utility of this strategy

How merozoite surface antigen-specific antibodies inhibit Plasmodium falciparum growth in vitro

In hyperendemic malarious regions adults develop protective immunity to Plasmodium falciparum infection. In order for this immunity to develop the host immune system must be able to recognise the parasite. One stage at which this occurs is prior to red blood cell invasion when the extracellular form of the parasite, the merozoite, presents the host immune system with a number of potential immunogens termed merozoite surface antigens. Antibodies to merozoite surface antigens are able to inhibit the growth and development of the parasite in vitro. This thesis explores the mechanisms by which merozoite surface antigen -specific antibodies exert this inhibition.The affinity, fine specificity and Fc- mediated effects of antibodies may affect their functional activity. Immortalised B cell lines producing merozoite surface antigen - specific human monoclonal antibodies were generated in order to investigate the effect of these factors on their growth inhibitory activity in vitro. The preliminary characterisation of these mAbs is described in chapter 3. However, sufficient quantities of these mAbs could not be generated for their functional activity to be investigated in vitro.Current dogma holds that the primary function of antibodies is to provide a molecular link between antigen recognition and pathogen destruction. However, all Abs have the ability to catalyse a reaction between singlet oxygen and water to generate hydrogen peroxide. This thesis explored the hypothesis that this antibody-catalysed water-oxidation pathway is responsible for the intraerythrocytic growth inhibition exerted by MSP-1-₁₉-specific Abs. An in vitro ACWO assay was developed to test this hypothesis and data suggest that ACWO may occur in infected RBCs associated with an anti- MSP-1-₁₉ monoclonal antibody.Antibodies specific to an intrinsically unstructured region from the C- terminal half of merozoite surface protein 3.3, designated MSP3.3C, are highly effective at inhibiting iii the in vitro growth of P. falciparum. This thesis explored the mechanisms responsible for this inhibition. This inhibition is caused by inhibition of the intraerythrocytic development of the parasite and not by inhibition of merozoite invasion. MSP3.3C specific Abs can access the intraerythrocytic parasite post invasion and completely arrest parasite development by inducing parasite death.The findings presented in this thesis expand current knowledge of the mechanisms by which MSA- specific Abs inhibit the growth of P. falciparum in vitro. This may prove informative both in terms of our understanding of naturally acquired antibody mediated immunity to P. falciparum asexual stages and in furthering effective vaccine design against this deadly pathogen

Apicomplexan F-actin is required for efficient nuclear entry during host cell invasion

The opportunistic pathogen Toxoplasma gondii is an obligate intracellular parasite part of the phylum Apicomplexa, able to infect all warm-blooded animals including humans. Invasion by apicomplexan parasites such as Plasmodium falciparum and Toxoplasma gondii to host cells requires the establishment and crossing through of a small ring-like junctional structure serving as an interface and stabiliser between the parasite and host cell plasma membrane. During the invasion process, the host cell possibly resist invasion to some degree, exerting force on the parasite’s entry point as de novo actin polymerisation has been characterised in this location (Gonzalez et al., 2009). Additionally, the parasite is required to generate force via an actomyosin motor to achieve host cell membrane penetration successfully, leading to mechanical deformation when the parasite is squeezing through the junctional ring. This actomyosin motor depends on a protein complex termed the glideosome, that pulls actin to achieve forward motility. Actin plays a key role in the parasite’s biology with important functions not only during invasion but also during replication, apicoplast maintenance and egress. Until recently, the lack of reliable F-actin sensors hampered the characterisation of actin dynamics during these processes. With the use of nanobodies with the potential to recognise actin (Periz et al., 2017), a complex actin behaviour was uncovered allowing the assessment of in vivo dynamics through the parasite’s lytic cycle. The uncovered flow of F-actin presented new opportunities to address debate over stablished hypothesis on parasite’s actin and to extend the initial roles attributed to actin including the establishment of cytoplasmic actin pool through the parasite’s life. Additionally, these F-actin dynamics were shown to be affected by traditional actin modulating drugs, as well as interference with actin binding factors resulting in abrogation of these dynamics and phenotypes associated with motility. Additionally in this thesis, it is suggested that F-actin’s role in invasion goes beyond powering the glideosome via force traction, but to facilitate nucleus passage and deformation. Real time and super resolution microscopy highlighted that during invasion events, the junction ring can oppose nucleus passage as parasites deficient of core components of the acto-myosin system have been shown to be incapable of withstand pressure exerted at the junction ring, leading to blebbing and collapse of the invading parasite (Bichet et al., 2016). Although some of these parasites are able to complete invasion, the dynamics are visibly affected suggesting more systems are at play during invasion. The literature shows that other eukaryotic systems deploy nucleus protection and displacement mechanisms to facilitate migration through tight spaces by the concerted action of actomyosin complexes and cytoskeletal structures (Petrie et al., 2012; Petrie and Yamada, 2015; McGregor, Hsia and Lammerding, 2016). This thesis proposes that the F-actin machinery facilitates nucleus passage through the junctional ring, offering a model fort the dual contribution of F-actin forces by constricting and pushing/pulling the nucleus during host cell invasion by these apicomplexan parasites, sharing similar mechanism with those of larger eukaryotes

Ultrasensitive detection of toxocara canis excretory-secretory antigens by a nanobody electrochemical magnetosensor assay.

peer reviewedHuman Toxocariasis (HT) is a zoonotic disease caused by the migration of the larval stage of the roundworm Toxocara canis in the human host. Despite of being the most cosmopolitan helminthiasis worldwide, its diagnosis is elusive. Currently, the detection of specific immunoglobulins IgG against the Toxocara Excretory-Secretory Antigens (TES), combined with clinical and epidemiological criteria is the only strategy to diagnose HT. Cross-reactivity with other parasites and the inability to distinguish between past and active infections are the main limitations of this approach. Here, we present a sensitive and specific novel strategy to detect and quantify TES, aiming to identify active cases of HT. High specificity is achieved by making use of nanobodies (Nbs), recombinant single variable domain antibodies obtained from camelids, that due to their small molecular size (15kDa) can recognize hidden epitopes not accessible to conventional antibodies. High sensitivity is attained by the design of an electrochemical magnetosensor with an amperometric readout with all components of the assay mixed in one single step. Through this strategy, 10-fold higher sensitivity than a conventional sandwich ELISA was achieved. The assay reached a limit of detection of 2 and15 pg/ml in PBST20 0.05% or serum, spiked with TES, respectively. These limits of detection are sufficient to detect clinically relevant toxocaral infections. Furthermore, our nanobodies showed no cross-reactivity with antigens from Ascaris lumbricoides or Ascaris suum. This is to our knowledge, the most sensitive method to detect and quantify TES so far, and has great potential to significantly improve diagnosis of HT. Moreover, the characteristics of our electrochemical assay are promising for the development of point of care diagnostic systems using nanobodies as a versatile and innovative alternative to antibodies. The next step will be the validation of the assay in clinical and epidemiological contexts