Biology subjects

Moreira-Leite, F.

Publications and source records attributed to Moreira-Leite, F..

10 recordsLinked to original sources

bioRxiv2025

Discovery of a novel flagellar filament system underpinning Leishmania adhesion to surfaces

Adhesion to surfaces is a common strategy employed across biology, especially by pathogens. Within their sand fly vector, Leishmania parasites undergo multiple developmental stages, including the understudied haptomonad form, which adheres to the sand fly stomodeal valve via a highly modified flagellum. This adhesion, likely critical for efficient transmission, is mediated by a complex adhesion plaque from which filaments in the modified flagellum extend towards the cell body and likely connect to the flagellum attachment zone (FAZ), a cytoskeletal structure important for cell morphogenesis. However, the role of the FAZ in adhesion and its relationship with the Kinetoplastid-Insect Adhesion Proteins (KIAPs) and the filamentous structures of the plaque itself remains unclear. Here, to examine the role of the FAZ in adhesion, we generated FAZ2, FAZ5, and FAZ34 deletion mutants. Deletion of any of these FAZ proteins impaired parasite adhesion in vitro. Furthermore, we identified a novel and distinct set of extra-axonemal flagellar filaments important for adhesion and demonstrated that KIAP2 is an essential component of these filaments. Our findings underscore the importance of a robust connection from the cell body to the adhesion plaque for stable Leishmania adhesion via the highly modified flagellum.

cell biology↗

bioRxiv2024

Malaria parasites undergo a rapid and extensive metamorphosis after invasion of the host erythrocyte

Within the human host, the symptoms of malaria are caused by the replication of malaria parasites within erythrocytes. Growth inside the erythrocyte exposes the parasites to the normal surveillance of erythrocytes by the host organism, in particular the clearance of erythrocytes in the spleen. Here we show that the malaria parasite Plasmodium falciparum undergoes a rapid, multi-step metamorphosis that transforms the invasive merozoite into an amoeboid-shaped cell within minutes after invading erythrocytes. This transformation involves an increase in the parasite surface area and is mediated by factors already present in the merozoite, including the parasite phospholipid transfer protein PV6. Parasites lacking PV6 do not assume an amoeboid form and instead are spherical and have a smaller surface area than amoeboid forms. Furthermore, erythrocytes infected with parasites lacking PV6 undergo a higher loss of surface area upon infection by P. falciparum, which affects the traversal of infected erythrocytes through the spleen. This is the first evidence that after invasion, the parasite undergoes a rapid, complex metamorphosis within the host erythrocyte that promotes survival in the host.

microbiology↗

bioRxiv2024

Dramatic reduction in trypanosome motility occurs without large-scale changes to paraflagellar rod ultrastructure

Eukaryotic flagella - widely conserved structures involved in signalling, metabolism and motility - have a core microtubular axoneme that, in many organisms, is accompanied by prominent extra-axonemal structures. In kinetoplastids, including human parasites such as trypanosomes and Leishmania, a dense filamentous lattice called the paraflagellar rod (PFR) accompanies the axoneme for most of its length. While functional studies showed that the presence of the core PFR structure is required for normal motility, the evaluation of more subtle roles for the PFR in motility has been hampered by limited functional and localisation data, particularly on components not essential to form the core PFR, such as signalling and metabolism proteins. Here, we addressed these issues by using the genome-wide protein localisation database TrypTag to define a PFR proteome, which was used as a base for a subtler analysis of PFR structure and function. We combined the localisation of fluorescently tagged PFR proteins relative to other cellular components with electron microscopy data on the PFR ultrastructure to localise 81 proteins to specific subdomains of the PFR. Functional analysis of a subset of PFR proteins by gene deletion and RNAi demonstrated that a novel PFR component (PFC21) is required for correct assembly of the outer PFR domain. Importantly, in some single deletion mutants, cell motility was impaired without gross disruption to the core PFR ultrastructure. Thus, our study shows that the PFR has subtle, likely regulatory roles in motility unrelated to any physical constraints that the bulky PFR structure may impose on flagella function.

microbiology↗

bioRxiv2023

3D electron microscopy of the Leishmania mexicana cell cycle: Patterns of organelle duplication and segregation and their implications for parasite biology

The unicellular parasite Leishmania has a precisely defined cell architecture that is inherited by each subsequent generation, requiring a highly coordinated pattern of duplication and segregation of organelles and cytoskeletal structures. A framework of nuclear division and morphological changes is known from light microscopy, yet this has limited resolution and the intrinsic organisation of organelles within the cell body and their manner of duplication and inheritance is unknown. Using volume electron microscopy approaches, we have produced three-dimensional reconstructions of different promastigote cell cycle stages to give a spatial and quantitative overview of organelle positioning, division and inheritance. The first morphological indications seen in our dataset that a new cell cycle had begun were the assembly of a new flagellum, the duplication of the contractile vacuole and the increase in volume of the nucleus and kinetoplast. We showed that the progression of the cytokinesis furrow created a specific pattern of membrane indentations and sub-pellicular microtubule organisation indicates that is likely a preferred site of new microtubule insertion. The daughter cells retained these indentations in their cell body for a period post-abscission. By comparing cultured and sand fly derived promastigotes, we found an increase in the number and overall volume of lipid droplets in the promastigotes from the sand fly, reflecting a change in their metabolism to ensure transmissibility to the mammalian host. Our insights into the cell cycle mechanics of Leishmania will be invaluable for future molecular cell biology analyses of these important parasites.

cell biology↗

bioRxiv2023

Discovery of essential kinetoplastid-insect adhesion proteins and their function in Leishmania-sand fly interactions

Leishmania species, members of the kinetoplastid parasites, cause leishmaniasis, a neglected tropical disease, in millions of people worldwide1. Leishmania has a complex life cycle with multiple developmental forms, as it cycles between a sand fly vector and a mammalian host; understanding their life cycle is critical to understanding disease spread2. One of the key life cycle stages is the haptomonad form, which is attached to the insect through its flagellum. This adhesion, which is conserved across kinetoplastid parasites, is implicated to have an important function within their life cycles and hence on disease transmission3-5. Here, we discovered kinetoplastid-insect adhesion proteins (KIAPs), which are localised in the attached haptomonad flagellum. Deletion of these KIAPs impaired cell adhesion in vitro and prevented Leishmania from colonising the stomodeal valve in the sand fly, without affecting cell growth. This result will provide important insights for a comprehensive understanding of the Leishmania life cycle.

microbiology↗

bioRxiv2023

Atypical flagella assembly and haploid genome coiling during male gamete formation in Plasmodium

Plasmodium spp. sexual reproduction occurs within the Anopheles mosquito and is essential for gametogenesis and onwards transmission to mammalian hosts. Upon activation, the male P. berghei gametocyte undergoes three rounds of inter-nuclear mitosis and assembles eight basal bodies and axonemes around the nucleus prior to ex-flagellation, resulting in 8 flagellated male gametes in 12-15 minutes. However there is little understanding of the 3D organisation of this rapid process of male gametogenesis. In this study we used serial block face scanning electron microscopy (SBF-SEM) and cellular electron tomography (ssET) of P. berghei microgametocytes to examine the 3D architecture of key structures during male gamete formation. Our data has revealed an exquisite organisation of axonemes coiling around the nucleus in opposite directions forming a central axoneme band in microgametocytes. Furthermore, we discovered that the nucleus of microgametes is tightly coiled around the axoneme in an exquisitely complex structure whose formation starts before microgamete emergence during ex-flagellation. Our discoveries of the detailed 3D organisation of the flagellated microgamete and the haploid genome highlights some of the atypical mechanisms of axoneme assembly and haploid genome organisation during male gamete formation in the malaria parasite Plasmodium spp.

cell biology↗

bioRxiv2022

Formation and three-dimensional architecture of Leishmania adhesion in the sand fly vector

Attachment to a substrate to maintain position in a specific ecological niche is a common strategy across biology, especially for eukaryotic parasites. During development in the sand fly vector, the eukaryotic parasite Leishmania adheres to the stomodeal valve, as the specialised haptomonad form. Dissection of haptomonad adhesion is a critical step for understanding parasite transmission. Nevertheless, haptomonad studies are limited, as this is a technically challenging life cycle form to investigate. Here, we have combined three-dimensional electron microscopy approaches, including serial block face scanning electron microscopy (SBFSEM) and serial tomography to dissect the organisation and architecture of haptomonads in the sand fly. We showed that the attachment plaque contains distinct structural elements. Using time-lapse light microscopy, we identified five stages of haptomonad differentiation, and showed that calcium is necessary for haptomonad adhesion to the surface. This study provides the structural and regulatory foundations of the haptomonad form, which are critical for a holistic understanding of Leishmania transmission.

microbiology↗

bioRxiv2021

Cellular electron tomography of the apical complex in the apicomplexan parasite Eimeria tenella shows a highly organised gateway for regulated secretion.

The apical complex of apicomplexan parasites is essential for host cell invasion and intracellular survival and as the site of regulated exocytosis from specialised secretory organelles called rhoptries and micronemes. Despite its importance, there is little data on the three-dimensional organisation and quantification of these organelles within the apical complex or how they are trafficked to this specialised region of plasma membrane for exocytosis. In coccidian apicomplexans there is an additional tubulin-containing hollow barrel structure, the conoid, which provides a structural gateway for this specialised secretion. Using a combination of cellular electron tomography and serial block face-scanning electron microscopy (SBF-SEM) we have reconstructed the entire apical end of Eimeria tenella sporozoites. We discovered that conoid fibre number varied, but there was a fixed spacing between fibres, leading to conoids of different sizes. Associated apical structures varied in size to accommodate a larger or smaller conoid diameter. However, the number of subpellicular microtubules on the apical polar ring surrounding the conoid did not vary, suggesting a control of apical complex size. We quantified the number and location of rhoptries and micronemes within cells and show a highly organised gateway for trafficking and docking of rhoptries, micronemes and vesicles within the conoid around a set of intra-conoidal microtubules. Finally, we provide ultrastructural evidence for fusion of rhoptries directly through the parasite plasma membrane early in infection and the presence of a pore in the parasitophorous vacuole membrane, providing a structural explanation for how rhoptry proteins (ROPs) may be trafficked between the parasite and the host cytoplasm. SignificanceApicomplexan parasites cause a wide range of human and animal diseases. The apical complex is essential for motility, host cell invasion and intracellular survival within a specialised vacuole called the parasitophorous vacuole. We know that molecules important for all of these processes are secreted from the apical complex via a set of secretory organelles and there is even evidence that some parasite molecules can enter the host cell from the parasitohorous vacuole, but there is little understanding of exactly how this occurs. Here we have used three dimensional electron microscopy to reconstruct the entire apical end of the parasite and whole individual parasites. Our results provide important insights into the structural organisation and mechanisms for delivery of parasite molecules via this important area of the cell.

microbiology↗

bioRxiv2020

Role for the flagellum attachment zone in the resolution of cell membrane morphogenesis during Leishmania cell division

The shape and form of the flagellated eukaryotic parasite Leishmania is sculpted to its ecological niches and needs to be transmitted to each generation with great fidelity. The shape of the Leishmania cell is defined by the sub-pellicular microtubule array and the positioning of the nucleus, kinetoplast and the flagellum within this array. The flagellum emerges from the anterior end of the cell body through an invagination of the cell body membrane called the flagellar pocket. Within the flagellar pocket the flagellum is laterally attached to the side of the flagellar pocket by a cytoskeletal structure called the flagellum attachment zone (FAZ). During the cell cycle single copy organelles duplicate with a new flagellum assembling alongside the old flagellum and these are then segregated between the two daughter cells by cytokinesis, which initiates at the anterior cell tip. Here, we have investigated the role of the FAZ in the morphogenetic resolution of the anterior cell tip during cell division. We have deleted the FAZ filament protein, FAZ2 and investigated its function using light and electron microscopy and infection studies. The loss of FAZ2 caused a disruption in membrane organisation at the anterior cell tip, resulting in cells that late in division were connected to each other by a membranous bridge structure between their flagella. These changes had a great impact in vivo with the FAZ2 null mutant unable to develop and proliferate in sand flies and causing a reduced parasite burden in mice. Our study provides a deeper understanding of membrane-cytoskeletal interactions that define the shape and form of an individual cell and the remodelling of that form during cell division. Author summaryLeishmania are parasites that cause leishmaniasis in humans with symptoms ranging from mild cutaneous lesions to severe visceral disease. The life cycle of these parasites alternates between the human host and the sand fly vector, with distinct forms in both. These different forms have different cell shapes that are adapted for survival in these different environments. Leishmania parasites have an elongated cell shape with a flagellum extending from one end and this shape is due to a protein skeleton beneath the cell membrane. This skeleton is made up of different units one of which is called the flagellum attachment zone (FAZ), that connects the flagellum to the cell body. We have found that one of the proteins in the FAZ called FAZ2 is important for generating the shape of the cell at the point where the flagellum exits the cell. When we deleted FAZ2 we found that the cell membrane at the tip of the was distorted resulting in unusual connections between the flagella of different cells. We found that the disruption to the cell shape reduces the ability of the parasite to infect mice and develop in the sand fly, which shows the importance of the parasite shape.

cell biology↗

bioRxiv2018

Basalin: an evolutionary unconstrained protein revealed via a conserved role in basal plate function

Most motile flagella have an axoneme that contains nine outer microtubule doublets and a central pair (CP) of microtubules. The CP is thought to coordinate the flagellar beat and defects in CP projections are associated with loss of motility and human disease. In most cilia, the CP nucleate near a basal plate at the distal end of the transition zone (TZ). Here, we show that the trypanosome TZ protein basalin is essential for building the basal plate, and its loss is associated with inefficient recruitment of CP assembly factors to the TZ, loss of the CP and flagellum paralysis. Guided by synteny, we identified highly divergent basalin orthologs in the genomes of related Leishmania species. Basalins are predicted to be highly unstructured, suggesting that they may act as hubs facilitating many protein-protein interactions. This raises the general concept that proteins involved in cytoskeletal functions and apparently appearing organism-specific, may have highly divergent and cryptic orthologs in other species.

cell biology↗