Biology subjects

Vega-Rodriguez, J.

Publications and source records attributed to Vega-Rodriguez, J..

9 recordsLinked to original sources

bioRxiv2025

Unlocking new understanding of Plasmodium sporozoite biology with expansion microscopy

Transmission of malaria relies on the formation of sporozoites in the mosquito midgut and their subsequent invasion of the salivary gland. Despite their importance, our understanding of the cell biology of sporozoite formation and salivary gland invasion is limited. Here, we apply a technique called Mosquito Tissue Ultrastructure Expansion Microscopy (MoTissU-ExM), which physically expands infected mosquito tissues while preserving both host and parasite ultrastructure. Using MoTissU-ExM we are able to observe a range of parasite structures and organelles including features previously seen only by electron microscopy as well as structure not observed before. We leverage MoTissU-ExM to investigate a number of cell biology events during sporozoite formation and salivary gland invasion. In particular we focus on the rhoptries, a secretory organelle important for host cell invasion. We establish a timeline for sporozoite rhoptry biogenesis, show that two rhoptries are used up during salivary gland invasion, and provide the first evidence that rhoptry pairs are specialized for different invasion events. Building on this new knowledge, we characterize the rhoptry protein RON11 and identify it as the first protein involved in sporozoite rhoptry biogenesis. Disruption of RON11 led to the production of sporozoites that specifically fail to invade the salivary gland epithelial cell, thereby blocking transmission of these parasites.

microbiology↗

bioRxiv2025

High-Resolution Proteomics Unveils Salivary Gland Disruption and Saliva-Hemolymph Protein Exchange in Plasmodium-Infected Mosquitoes

Plasmodium sporozoites, the stage that initiates a malaria infection, must invade the mosquito salivary glands (SGs) before transmitting to a vertebrate host. However, the effects of sporozoite invasion on salivary gland physiology and saliva composition remain largely unexplored. We examined the impact of Plasmodium infection on Anopheles gambiae salivary glands using high-resolution proteomics, gene expression, and morphological analysis. The data revealed differential expression of various proteins, including the enrichment of humoral proteins in infected salivary glands originating from the hemolymph. These proteins diffused into the SGs due to structural damage caused by the sporozoites during invasion. Conversely, saliva proteins diffused out into the circulation of infected mosquitoes. Moreover, infection altered saliva protein composition, as shown by proteomes from saliva collected from mosquitoes infected by P. berghei or P. falciparum, revealing a significant reduction of immune proteins compared to uninfected mosquitoes. This reduction is likely due to the association of these proteins with the surface of sporozoites within the mosquito salivary secretory cavities. The saliva protein profiles from mosquitoes infected with both Plasmodium species were remarkably similar, suggesting a conserved interaction between sporozoites and salivary glands. Our results provide a foundation for understanding the molecular interactions between Plasmodium sporozoites and mosquito salivary glands.

microbiology↗

bioRxiv2024

Mosquito Tissue Ultrastructure-Expansion Microscopy (MoTissU-ExM) enables ultrastructural and anatomical analysis of malaria parasites and their mosquito.

Study of malaria parasite cell biology is challenged by their small size, which can make visualisation of individual organelles difficult or impossible using conventional light microscopy. In recent years, the [fi]eld has attempted to overcome this challenge through the application of ultrastructure expansion microscopy (U-ExM), which physically expands a biological sample approximately 4.5-fold. To date, U-ExM has mostly been used to visualise blood-stage parasites and used exclusively on parasites in vitro. Here we develop Mosquito Tissue U-ExM (MoTissU-ExM), a method for preparing dissected mosquito salivary glands and midguts by U-ExM. MoTissU-ExM preserves both host and parasite ultrastructure, enabling visualisation of oocysts and sporozoites in situ. We validate that MoTissU-ExM samples expand as expected, provide a direct comparison of the same dissected tissues before and after MoTissU-ExM, and highlight some of the key host and parasite structures that can be visualised following MoTissU-ExM. Finally, we provide a point-by-point protocol for how to perform MoTissU-ExM, along with details on how best to image the expanded tissues, and how to troubleshoot common issues.

cell biology↗

bioRxiv2023

Host-Pathogen Interactions in the Plasmodium-Infected Mouse Liver at Spatial and Single-Cell Resolution

Upon infecting its vertebrate host, the malaria parasite initially invades the liver where it undergoes massive replication, whilst remaining clinically silent. The spatial coordination of factors regulating immune responses and metabolic zonation during malaria infection, in the true tissue context, remains unexplored. Here, we perform spatial transcriptomics in combination with single-nuclei RNA-sequencing (snRNA-seq) over multiple time points during liver infection to delineate transcriptional programs of host-pathogen interactions across P. berghei-infected liver tissues. Our data suggest changes in gene expression related to lipid metabolism in response to Plasmodium infection in the proximity of infected hepatocytes, such as the modulation of the expression of genes involved in peroxisome proliferator-activated receptor pathway signaling. The data further indicate the presence of inflammatory hotspots with distinct cell type compositions and differential liver inflammation programs along the lobular axis in the malaria-infected tissues. Furthermore, a significant upregulation of genes involved in inflammation is observed in liver tissues of control mice injected with mosquito salivary gland components, which is considerably delayed compared to P. berghei infected mice. Our study establishes a benchmark for investigating transcriptome changes during host-parasite interactions in tissues, it provides informative insights regarding in vivo study design linked to infection, and provides a useful tool for the discovery and validation of de novo intervention strategies aimed at malaria liver stage infection.

molecular biology↗

bioRxiv2023

Anopheles salivary apyrase regulates blood meal hemostasis and drives malaria parasite transmission

Mosquito salivary proteins play a crucial role in regulating hemostatic responses at the bite site during blood feeding. In this study, we investigate the function of Anopheles gambiae salivary apyrase (AgApyrase) in Plasmodium transmission. Our results demonstrate that salivary apyrase interacts with and activates tissue plasminogen activator, facilitating the conversion of plasminogen to plasmin, a human protein previously shown to be required for Plasmodium transmission. Microscopy imaging shows that mosquitoes ingest a substantial amount of apyrase during blood feeding which reduces coagulation in the blood meal by enhancing fibrin degradation and inhibiting platelet aggregation. Supplementation of Plasmodium infected blood with apyrase significantly enhanced Plasmodium infection in the mosquito midgut. In contrast, AgApyrase immunization inhibited Plasmodium mosquito infection and sporozoite transmission. This study highlights a pivotal role for mosquito salivary apyrase for regulation of hemostasis in the mosquito blood meal and for Plasmodium transmission to mosquitoes and to the mammal host, underscoring the potential for new strategies to prevent malaria transmission.

microbiology↗

bioRxiv2022

Single-cell transcriptomics to define Plasmodium falciparum stage-transition in the mosquito midgut

Malaria inflicts the highest rate of morbidity and mortality among the vector-borne diseases. The dramatic bottleneck of parasite numbers that occurs in the gut of the obligatory vector mosquito provides a promising target for novel control strategies. Using single-cell transcriptomics, we analyzed Plasmodium falciparum development in the mosquito gut, from unfertilized female gametes through the first 20 hours post blood feeding, including the zygote and ookinete stages. This study revealed the transcriptional trajectories of the ApiAP2 family of transcription factors, and of parasite stress genes in response to the harsh environment of the mosquito midgut. Further, employing structure-based functional predictions we found several upregulated genes predicted to encode intrinsically disordered proteins (IDPs), a category of proteins known for their importance in regulation of transcription, translation and protein-protein interactions. IDPs are known for their antigenic properties and may serve as suitable targets for antibody or peptide-based transmission suppression strategies.

microbiology↗

bioRxiv2022

Combining transgenesis with paratransgenesis to fight malaria

Malaria is among the deadliest infectious diseases and Plasmodium, the causative agent, needs to complete a complex development cycle in its vector mosquito for transmission to occur. Two promising strategies to curb transmission are transgenesis, consisting of genetically engineering mosquitoes to express anti-malarial effector molecules and paratransgenesis, consisting of introducing into the mosquito, commensal bacteria engineered to express anti-malarial effector molecules. Although both approaches restrict parasite development in the mosquito, it is not known how their effectiveness compares. Here we provide an in-depth assessment of transgenesis and paratransgenesis and evaluate the combination of the two approaches. Using the Q-system to drive gene expression, we engineered mosquitoes to produce and secrete two effectors - scorpine and the MP2 peptide - into the mosquito gut and salivary glands. We also engineered Serratia, a commensal bacterium capable to spread through mosquito populations, to secrete the same two effectors into the mosquito gut. Whereas both mosquito-based and bacteria-based approaches strongly reduced the oocyst and sporozoite intensity, a substantially stronger reduction of P. falciparum development was achieved when transgenesis and paratransgenesis were combined. Most importantly, transmission of P. berghei from infected to naive mice was maximally inhibited by the combination of the two approaches. Combining these two strategies promise to become a powerful approach to combat malaria. SignificanceMalaria kills hundreds of thousand persons yearly. Clearly, new approaches are needed to fight this disease. Two promising approaches are based on the concept of genetically modifying the mosquito to make it a poor vector for the parasite: 1) transgenesis (engineering the mosquito to deliver anti-malarial compounds) and 2) paratransgenesis (engineering mosquito symbiotic bacteria to deliver anti-malarial compounds). The key questions addressed by this manuscript are: which of the two is the most promising approach? And because transgenesis and paratransgenesis are not mutually exclusive, would the combination of both be the most effective strategy? Our results argue for the combination of the two, showing the additive impact that these two strategies may have in controlling malaria transmission in the field.

microbiology↗

bioRxiv2020

A high affinity human monoclonal antibody against Pfs230 binds multiple parasite stages and blocks oocyst formation in mosquitoes

Malaria elimination requires tools that interrupt parasite transmission. Here, we characterized B cell receptor responses among Malian adults vaccinated against the first domain of the cysteine-rich 230kDa gamete surface protein Pfs2301-3 to neutralize sexual stage P. falciparum parasites and halt their further spread. We generated nine Pfs230 human monoclonal antibodies (mAbs). One mAb potently blocked transmission to mosquitoes in a complement-dependent manner and reacted strongly to gamete surface while eight mAbs showed only low or no blocking activity. This study provides a rational basis to improve malaria vaccines and develop therapeutic antibodies for malaria elimination.

immunology↗

bioRxiv2018

Generation of Transgenic Human Malaria Parasites With Strong Fluorescence in the Transmission Stages

Malaria parasites have a complex life cycle that includes specialized stages for transmission between their mosquito and human hosts. These stages are an understudied part of the lifecycle yet targeting them is an essential component of the effort to shrink the malaria map. The human parasite Plasmodium falciparum is responsible for the majority of deaths due to malaria. Our goal was to generate transgenic P. falciparum lines that could complete the lifecycle and produce fluorescent transmission stages for more in-depth and high-throughput studies. Using zinc-finger nuclease technology to engineer a marker-free integration site, we generated three transgenic P. falciparum lines in which tdtomato or gfp were stably integrated into the genome. Expression was driven by either stage-specific peg4 and csp promoters or the constitutive ef1a promoter. Phenotypic characterization of these lines demonstrates that they complete the life cycle with high infection rates and give rise to fluorescent mosquito stages. The transmission stages are sufficiently bright for intra-vital imaging, flow cytometry and scalable screening of chemical inhibitors and potentially inhibitory antibodies.

microbiology↗