Dissecting the molecular interaction between hepatocytes and Plasmodium liver parasites

Dissertation presented to obtain the Ph.D degree in Biology

Development of sporogonic cycle of Plasmodium vivax in experimentally infected Anopheles albimanus mosquitoes

The sporogonic cycle of Plasmodium vivax was established and maintained under laboratory conditions in two different strains of Anopheles albimanus mosquitoes using as a parasite source blood from human patients or from Aotus monkeys infected with the VCC-2 P.vivax colombian isolate. Both the Tecojate strain isolate from Guatemala and the Cartagena strain from the colombian Pacific coast were susceptible to infections with P.vivax. A higher percentage of Cartagena mosquitoes was infected per trial, however the Tecojate strain developed higher sporozoite loads. Intravenous inoculation of Aotus monkeys with sporozoites obtained from both anopheline strains resulted in successful blood infections. Animals infected with sporozoites from the Tecojate strain presented a patent period of 21-32 days whereas parasitemia appeared between days 19-53 in monkeys infected with sporozites from Cartagena strain.

The malaria circumsporozoite protein has two functional domains, each with distinct roles as sporozoites journey from mosquito to mammalian host.

Plasmodium sporozoites make a remarkable journey from the mosquito midgut to the mammalian liver. The sporozoite's major surface protein, circumsporozoite protein (CSP), is a multifunctional protein required for sporozoite development and likely mediates several steps of this journey. In this study, we show that CSP has two conformational states, an adhesive conformation in which the C-terminal cell-adhesive domain is exposed and a nonadhesive conformation in which the N terminus masks this domain. We demonstrate that the cell-adhesive domain functions in sporozoite development and hepatocyte invasion. Between these two events, the sporozoite must travel from the mosquito midgut to the mammalian liver, and N-terminal masking of the cell-adhesive domain maintains the sporozoite in a migratory state. In the mammalian host, proteolytic cleavage of CSP regulates the switch to an adhesive conformation, and the highly conserved region I plays a critical role in this process. If the CSP domain architecture is altered such that the cell-adhesive domain is constitutively exposed, the majority of sporozoites do not reach their target organs, and in the mammalian host, they initiate a blood stage infection directly from the inoculation site. These data provide structure-function information relevant to malaria vaccine development.

Sporozoite-mediated hepatocyte wounding limits Plasmodium parasite development via MyD88-mediated NF-kappa B activation and inducible NO synthase expression

Plasmodium sporozoites traverse several host cells before infecting hepatocytes. In the process, the plasma membranes of the cells are ruptured, resulting in the release of cytosolic factors into the microenvironment. This released endogenous material is highly stimulatory/immunogenic and can serve as a danger signal initiating distinct responses in various cells. Thus, our study aimed at characterizing the effect of cell material leakage during Plasmodium infection on cultured mouse primary hepatocytes and HepG2 cells. We observed that wounded cell-derived cytosolic factors activate NF-kappaB, a main regulator of host inflammatory responses, in cells bordering wounded cells, which are potential host cells for final parasite infection. This activation of NF-kappaB occurred shortly after infection and led to a reduction of infection load in a time-dependent manner in vitro and in vivo, an effect that could be reverted by addition of the specific NF-kappaB inhibitor BAY11-7082. Furthermore, no NF-kappaB activation was observed when Spect(-/-) parasites, which are devoid of hepatocyte traversing properties, were used. We provide further evidence that NF-kappaB activation causes the induction of inducible NO synthase expression in hepatocytes, and this is, in turn, responsible for a decrease in Plasmodium-infected hepatocytes. Furthermore, primary hepatocytes from MyD88(-/-) mice showed no NF-kappaB activation and inducible NO synthase expression upon infection, suggesting a role of the Toll/IL-1 receptor family members in sensing cytosolic factors. Indeed, lack of MyD88 significantly increased infection in vitro and in vivo. Thus, host cell wounding due to parasite migration induces inflammation which limits the extent of parasite infection

Effects of Plasmodium gallinaceum on hemolymph physiology of Aedes aegypti during parasite development

Insect disease vectors show diminished fecundity when infected with Plasmodium. This phenomenon has already been demonstrated in laboratory models such as Aedes aegypti, Anopheles gambiae and Anopheles stephensi. This study demonstrates several changes in physiological processes of A. aegypti occurring upon infection with Plasmodium gallinaceum, such as reduced ecdysteroid levels in hemolymph as well as altered expression patterns for genes involved in vitellogenesis, lipid transport and immune response. Furthermore, we could show that P. gallinaceum infected A. aegypti presented a reduction in reproductive fitness, accompanied by an activated innate immune response and increase in lipophorin expression, with the latter possibly representing a nutritional resource for Plasmodium sporozoites. (C) 2010 Elsevier Ltd. All rights reserved.

Highly dynamic host actin reorganization around developing Plasmodium inside hepatocytes

Plasmodium sporozoites are transmitted by Anopheles mosquitoes and infect hepatocytes, where a single sporozoite replicates into thousands of merozoites inside a parasitophorous vacuole. The nature of the Plasmodium-host cell interface, as well as the interactions occurring between these two organisms, remains largely unknown. Here we show that highly dynamic hepatocyte actin reorganization events occur around developing Plasmodium berghei parasites inside human hepatoma cells. Actin reorganization is most prominent between 10 to 16 hours post infection and depends on the actin severing and capping protein, gelsolin. Live cell imaging studies also suggest that the hepatocyte cytoskeleton may contribute to parasite elimination during Plasmodium development in the liver.

Genetically attenuated, P36p-deficient malarial sporozoites induce protective immunity and apoptosis of infected liver cells.

Immunization with Plasmodium sporozoites that have been attenuated by gamma-irradiation or specific genetic modification can induce protective immunity against subsequent malaria infection. The mechanism of protection is only known for radiation-attenuated sporozoites, involving cell-mediated and humoral immune responses invoked by infected hepatocytes cells that contain long-lived, partially developed parasites. Here we analyzed sporozoites of Plasmodium berghei that are deficient in P36p (p36p(-)), a member of the P48/45 family of surface proteins. P36p plays no role in the ability of sporozoites to infect and traverse hepatocytes, but p36p(-) sporozoites abort during development within the hepatocyte. Immunization with p36p(-) sporozoites results in a protective immunity against subsequent challenge with infectious wild-type sporozoites, another example of a specifically genetically attenuated sporozoite (GAS) conferring protective immunity. Comparison of biological characteristics of p36p(-) sporozoites with radiation-attenuated sporozoites demonstrates that liver cells infected with p36p(-) sporozoites disappear rapidly as a result of apoptosis of host cells that may potentiate the immune response. Such knowledge of the biological characteristics of GAS and their evoked immune responses are essential for further investigation of the utility of an optimized GAS-based malaria vaccine.

Live and let die: manipulation of host hepatocytes by exoerythrocytic Plasmodium parasites

The generation of rodent Plasmodium strains expressing fluorescent proteins in all life cycle stages has had a big impact on malaria research. With this tool in hand, for the first time it was possible to follow in real time by in vivo microscopy the infection route of Plasmodium sporozoites transmitted to the mammalian host by Anopheles mosquitoes. Recently, this work has been extended to the analysis of both hepatocyte infection by Plasmodium sporozoites, as well as liver merozoite transport into blood vessels. The stunning results of these studies have considerably changed our understanding of hepatocyte invasion and parasite liberation. Here, we describe the most important findings of the last years and in addition, we elaborate on the molecular events during the intracellular development of Plasmodium exoerythrocytic forms that give rise to erythrocyte infecting merozoites.

A new thrombospondin-related anonymous protein homologue in Neospora caninum (NcMIC2-like1)

Neospora caninum is an Apicomplexan protozoan that has the dog as a definitive host and cattle (among other animals) as intermediate hosts. It causes encephalopathy in dogs and abortion in cows, with significant loss in worldwide livestock. As any Apicomplexan, the parasite invades the cells using proteins contained in the phylum-specific organelles, like the micronemes, rhoptries and dense granules. The aim of this study was the characterization of a homologue (denominated NcMIC2-like1) of N. caninum thrombospondin-related anonymous protein (NcMIC2), a micronemal protein previously shown to be involved in the attachment and connection with the intracellular motor responsible for the active process of invasion. A polyclonal antiserum raised against the recombinant NcMIC2-like1 functional core (thrombospondin and integrin domains) recognized the native form of NcMIC2-like1, inhibited the in vitro invasion process and localized NcMIC2-like1 at the apical complex of the parasite by confocal immunofluorescence, indicating its micronemal localization. The new molecule, NcMIC2-like1, has features that differentiates it from NcMIC2 in a substantial way to be considered a homologue dagger.

the liver endoplasmic reticulum in malaria liver infection

Malaria, a disease caused by Plasmodium, represents a major health problem with a still disconcertingly high mortality rate (655 000 malaria deaths were estimated by the World Health Organization in 2012), mainly in Africa [1]. After a bite by an infected Anopheles mosquito occurs, Plasmodium sporozoites reach their target organ, the liver, within minutes. After traversing several hepatocytes, the parasite invades a final one and establishes a parasitophorous vacuole, where it replicates exponentially generating thousands of infective merozoites, the red blood cell infectious forms that are released in the blood stream. The liver stage is the first obligatory phase of malaria infection and, although no symptoms are associated with it, it is absolutely crucial to the establishment of a successful infection.(...)

A Natural Protective Mechanism Against Malaria

Malaria is an infectious disease of humans and other animals including birds, reptiles and most mammals. It is transmitted via the inoculation of Plasmodium sporozoites into the skin through the bite of an infected female Anopheles mosquito. Although every year, around 700.000 lives are perished, mainly children under the age of 3-5 years old, to Plasmodium infection this deadly parasite has a relatively low efficiency of transmission from mosquitoes into humans.(...)

T cell responses to repeat and non-repeat regions of the circumsporozoite protein detected in volunteers immunized with Plasmodium falciparum sporozoites

The design of malarial vaccine based on the circumsporozoite (CS) protein, a majuor surface antigen of the sporozoite stage of the malaria parasite, requires the identification of T and B cell epitopes for inclusion in recombinant or synthetic vaccine candidates. We have investigated the specificity and function of a series of T cell clones, derived from volunteers immunized with Plasmodium falciparum sporozoites in an effort to identify relevant epitopes in the immune response to the pre-erythrocytic stages of the parasite. CD4+ T cell clones were obtained wich specifically recognized a repetitive epitope located in the 5'repeat region of the CS protein. This epitope, when conjugated to the 3'repeat region in a synthetic MAPs construct, induced high titers of antisporozoite antibodies in C57B1 mice. A second T cell epitope, which mapped to aa 326-345 of the carboxy terminal, was recognized by lytic, as well as non-lytic, CD4+ T cells derived from the sporozoite-immunized volunteers. The demonstration of CD4+ CTL in the volunteers, and the recent studies inthe rodent model (Renia et al., 1991; Tsuji et al., 1990), suggested that CS-specific CD4+ T cells, in addition to their indirect role as helper cells in the induction of antibody and CD8 + effector cells, may also play a direct role in protection against sporozoite challenge by targeting EEF within the liver.

Anti-Plasmodium Activity of Angiotensin II and Related Synthetic Peptides

Plasmodium species are the causative agents of malaria, the most devastating insect-borne parasite of human populations. Finding and developing new drugs for malaria treatment and prevention is the goal of much research. Angiotensins I and II (ang I and ang II) and six synthetic related peptides designated Vaniceres 1-6 (VC1-VC6) were assayed in vivo and in vitro for their effects on the development of the avian parasite, Plasmodium gallinaceum. Ang II and VC5 injected into the thoraces of the insects reduced mean intensities of infection in the mosquito salivary glands by 88% and 76%, respectively. Although the mechanism(s) of action is not completely understood, we have demonstrated that these peptides disrupt selectively the P. gallinaceum cell membrane. Additionally, incubation in vitro of sporozoites with VC5 reduced the infectivity of the parasites to their vertebrate host. VC5 has no observable agonist effects on vertebrates, and this makes it a promising drug for malaria prevention and chemotherapy.

Susceptibility of two karyotypic forms of Anopheles aconitus (Diptera: Culicidae) to Plasmodium falciparum and P. vivax

Four laboratory-raised colonies of two karyotypic forms of Anopheles aconitus, i.e., Form B (Chiang Mai and Phet Buri strains) and C (Chiang Mai and Mae Hong Son strains), were experimentally infected with Plasmodium falciparum and P. vivax using an artificial membrane feeding technique and dissected eight and 12 days after feeding for oocyst and sporozoite rates, respectively. The results revealed that An. aconitus Form B and C were susceptible to P. falciparum and P. vivax, i.e., Form B (Chiang Mai and Phet Buri strains/P. falciparum and P. vivax) and Form C (Chiang Mai and Mae Hong Son strains/P. vivax). Comparative statistical analyses of the oocyst rates, average number of oocysts per infected midgut and sporozoite rates among all strains of An. aconitus Form B and C to the ingroup control vectors, An. minimus A and C, exhibited mostly no significant differences, confirming the high potential vector of the two Plasmodium species. The sporozoite-like crystals found in the median lobe of the salivary glands, which could be a misleading factor in the identification of true sporozoites in salivary glands were found in both An. aconitus Form B and C.