Daniel Y. Bargieri

Bio: Daniel Y. Bargieri is an academic researcher from University of São Paulo. The author has contributed to research in topics: Plasmodium vivax & Antigen. The author has an hindex of 18, co-authored 42 publications receiving 1304 citations. Previous affiliations of Daniel Y. Bargieri include Pasteur Institute & Federal University of São Paulo.

On the Cytoadhesion of Plasmodium vivax–Infected Erythrocytes

Abstract: Background Plasmodium falciparum and Plasmodium vivax are responsible for most of the global burden of malaria. Although the accentuated pathogenicity of P. falciparum occurs because of sequestration of the mature erythrocytic forms in the microvasculature, this phenomenon has not yet been noted in P. vivax. The increasing number of severe manifestations of P. vivax infections, similar to those observed for severe falciparum malaria, suggests that key pathogenic mechanisms (eg, cytoadherence) might be shared by the 2 parasites. Methods Mature P. vivax-infected erythrocytes (Pv-iEs) were isolated from blood samples collected from 34 infected patients. Pv-iEs enriched on Percoll gradients were used in cytoadhesion assays with human lung endothelial cells, Saimiri brain endothelial cells, and placental cryosections. Results Pv-iEs were able to cytoadhere under static and flow conditions to cells expressing endothelial receptors known to mediate the cytoadhesion of P. falciparum. Although Pv-iE cytoadhesion levels were 10-fold lower than those observed for P. falciparum-infected erythrocytes, the strength of the interaction was similar. Cytoadhesion of Pv-iEs was in part mediated by VIR proteins, encoded by P. vivax variant genes (vir), given that specific antisera inhibited the Pv-iE-endothelial cell interaction. Conclusions These observations prompt a modification of the current paradigms of the pathogenesis of malaria and clear the way to investigate the pathophysiology of P. vivax infections.

Apical membrane antigen 1 mediates apicomplexan parasite attachment but is dispensable for host cell invasion

Abstract: Apicomplexan parasites invade host cells by forming a ring-like junction with the cell surface and actively sliding through the junction inside an intracellular vacuole. Apical membrane antigen 1 is conserved in apicomplexans and a long-standing malaria vaccine candidate. It is considered to have multiple important roles during host cell penetration, primarily in structuring the junction by interacting with the rhoptry neck 2 protein and transducing the force generated by the parasite motor during internalization. Here, we generate Plasmodium sporozoites and merozoites and Toxoplasma tachyzoites lacking apical membrane antigen 1, and find that the latter two are impaired in host cell attachment but the three display normal host cell penetration through the junction. Therefore, apical membrane antigen 1, rather than an essential invasin, is a dispensable adhesin of apicomplexan zoites. These genetic data have implications on the use of apical membrane antigen 1 or the apical membrane antigen 1–rhoptry neck 2 interaction as targets of intervention strategies against malaria or other diseases caused by apicomplexans.

Host Cell Invasion by Apicomplexan Parasites: The Junction Conundrum

Abstract: Apicomplexans form a large phylum of parasitic protists, some of which cause severe diseases in humans. Most notorious is Plasmodium, the agent of malaria, which kills around a million people each year, mostly young children in Africa. Most successful is Toxoplasma, which parasitizes nearly a third of the human population, making those people at risk of life-threatening complications, primarily encephalitis or pneumonia, in case of immunosuppression. Other apicomplexans of human importance include Cryptosporidium, Isospora, and Sarcocystis, which are opportunistic pathogens that cause severe diarrhea often associated with AIDS. Several apicomplexan parasites cause heavy losses in livestock, particularly Theileria and Babesia in cattle and Eimeria in poultry. Most apicomplexans are obligate intracellular parasites. Their extracellular stages, called zoites, display several conserved features: they are elongated and polarized cells, their shape is maintained by a set of microtubules running longitudinally, and their anterior pole contains secretory vesicles, called micronemes and rhoptries, which secrete their content at the anterior tip of the parasite. Most zoites also share two unique properties among eukaryotic cells. They move on substrate by a gliding type of motility, i.e., without overt deformation of the cell shape, at speeds of several microns per second. They also typically invade host cells by forming a ring-like junction with the host cell membrane. Zoites slide through the junction into an invagination of the host cell surface that becomes the parasitophorous vacuole (PV) after pinching off from the host cell plasma membrane, in a process that takes less than a minute. Once inside the PV niche, the zoite can multiply into multiple new zoites that eventually egress the infected cell to infect new host cells. Much work has been performed since the late 1970s to understand the cellular and molecular bases of host cell invasion by apicomplexans, using various zoites as models. The overall invasion process encompasses several steps, including loose followed by intimate attachment, reorientation relative to the host cell surface, and organelle discharge with junction formation. The ultimate step, sliding through the junction inside the PV and called here internalization, is commonly viewed as powered by the zoite submembrane actin-myosin motor. The junction is thought to act as a traction point for the motor, to bridge the cortical cytoskeletons in the two cells, and to be made of parasite proteins conserved in the apicomplexan phylum. In this review, we confront these established notions with genetic data recently obtained in Plasmodium and Toxoplasma parasites. The Junction: From “Moving” to Stationary The first observation of a junction between an apicomplexan zoite and its host cell was made using Plasmodium merozoites and their target cells, erythrocytes [1]. Electron microscopy showed that the merozoite, after initial random binding, reorients so that its apical tip faces the erythrocyte surface, and then induces a circumferential zone of close apposition of the zoite and erythrocyte membranes over ∼250 nm and the thickening of the inner leaflet of the erythrocyte membrane [1]. This junctional area was described as “actively moving down the body of the merozoite,” since the poorly motile merozoite was not thought to be capable of actively moving inside the cell, and was thus termed “moving junction” [1]. Studies in the 1980s focused on the highly motile Eimeria sporozoites. They showed that several activities at the zoite surface were dependent on parasite actin, including the posterior translocation (capping) of various surface ligands and beads [2]. Videomicroscopic studies revealed that host cell invasion by Eimeria sporozoites was continuous with extracellular gliding [3]. This led to the proposal that the zoite actin-based system would power both gliding motility and host cell invasion by capping substrate-binding ligands or the junction, respectively, which implied that the zoite actively moved inside the host cell [3], [4]. After myosins were identified in Toxoplasma [5] and in Plasmodium [6], it was assumed that an actin-myosin motor powered the zoite motile processes. The role of the host cell during zoite invasion has been studied mainly with Toxoplasma tachyzoites, which can be made to invade host cells at high frequency and synchronicity. The host cell was initially described as displaying no detectable actin reorganization and playing no active role during tachyzoite invasion [7], [8]. More recent work found that Toxoplasma tachyzoites induced, specifically at the junction, host actin polymerization and recruitment of the Arp2/3 complex, an actin-nucleating factor, which is important for tachyzoite entry [9]. Videomicroscopic studies showed a stationary ring of host F-actin at the parasite constriction, in agreement with the junction acting as an anchor for zoite traction inside the cell. In addition to de novo actin polymerization at the junction, tachyzoite invasion also requires disorganization of the host cortical actin meshwork. This activity is in part dependent on Toxofilin [10], a Toxoplasma protein that sequesters actin monomers in vitro [11] and promotes actin turnover at the leading edge of the cell [10]. Localized actin disassembly might thus release G-actin necessary to feed actin reassembly at the junction, regulated by recruited Arp2/3 complex, to anchor the junction to the host cortical cytoskeleton.

A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes

Abstract: National Institute of General Medical Sciences (U.S.) (Center for Integrative Synthetic Biology Grant P50GM098792)

Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing

Abstract: methods for the large-scale analysis of genetic variation in Plasmodium falciparum by deep sequencing of parasite DNA obtained from the blood of patients with malaria, either directly or after short-term culture Analysis of 86,158 exonic single nucleotide polymorphisms that passed genotyping quality control in 227 samples from Africa, Asia and Oceania provides genomewide estimates of allele frequency distribution, population structure and linkage disequilibrium By comparing the genetic diversity of individual infections with that of the local parasite population, we derive a metric of within-host diversity that is related to the level of inbreeding in the population An open-access web application has been established for the exploration of regional differences in allele frequency and of highly differentiated loci in the P falciparum genome The genetic diversity and evolutionary plasticity of P falciparum are major obstacles for malaria elimination New forms of resistance against antimalarial drugs are continually emerging 1,2 , and new forms of antigenic variation are a critical point of vulnerability for future malaria vaccines Effective tools are needed to detect evolutionary changes in the parasite population and to monitor the spread of genetic variants that affect malaria control Here we describe the use of deep sequencing to analyse P falciparum diversity, using blood samples from patients with malaria The P falciparum genome has several unusual features that greatly complicate sequence analysis, such as extreme AT bias, large tracts of nonunique sequence and several large families of intensely polymorphic genes 3 Our aim was therefore not to determine the entire genome sequence of individual field samples—which would be prohibitively expensive with current technologies—but to define an initial set of single nucleotide polymorphisms (SNPs) distributed across the P falciparum genome, whose genotype can be ascertained with confidence in parasitized blood samples by deep sequencing An additional complication in the analysis of P falciparum genome variation is that the billions of haploid parasites that infect a single individual can be a complex mixture of genetic types Previous studies 4–8 have largely focused on laboratory-adapted parasite clones, but the within-host diversity of natural infections is of fundamental biological interest Parasites in the blood replicate asexually, but when they are taken up in the blood meal of an Anopheles mosquito they undergo sexual mating If the parasites in the blood are of diverse genetic types, this process of sexual mating can generate novel recombinant forms Deep sequencing provides new ways of investigating within-host diversity and the role of sexual recombination in parasite evolution

Flagellin as an Adjuvant: Cellular Mechanisms and Potential

Abstract: Flagellin is a potent activator of a broad range of cell types involved in innate and adaptive immunity. An increasing number of studies have demonstrated the effectiveness of flagellin as an adjuvant, as well as its ability to promote cytokine production by a range of innate cell types, trigger a generalized recruitment of T and B lymphocytes to secondary lymphoid sites, and activate TLR5+CD11c+ cells and T lymphocytes in a manner that is distinct from cognate Ag recognition. The plasticity of flagellin has allowed for the generation of a range of flagellin–Ag fusion proteins that have proven to be effective vaccines in animal models. This review summarizes the state of our current understanding of the adjuvant effect of flagellin and addresses important areas of current and future research interest.