Biology subjects

Lourido, S.

Publications and source records attributed to Lourido, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Proteome-wide crosslinking mass spectrometry reveals novel components of essential complexes in Toxoplasma

Protein-protein interactions underpin nearly all cellular processes, yet systematic definition of these networks remains limited outside a few model organisms. As a result, the architectures of essential complexes in many divergent lineages remain poorly characterized. Here we developed a high-coverage crosslinking mass spectrometry framework to map the proteome-wide interactome of the model apicomplexan parasite Toxoplasma gondii. From 29,624 crosslinked peptide pairs, we resolved a network of 2,859 protein-protein interactions that we integrated with structural modeling to resolve interaction interfaces. We identified and validated previously unrecognized components of essential protein complexes, including a structurally distinct ATP synthase subcomplex containing a highly divergent, apicomplexan-specific subunit essential for parasite fitness. Beyond revealing unexpected diversification of core mitochondrial machinery, these findings provide a general strategy to define the molecular architecture of divergent organisms and represent a foundational resource for hypothesis generation, structural inference, and discovery of lineage-specific vulnerabilities in pathogen biology.

microbiology↗

bioRxiv2025

Brain-immune interactions generate pathogen-specific sickness states

In nature, animals encounter diverse pathogens that trigger specific peripheral defense programs and elicit sickness behavior, a set of stereotyped physiological and behavioral changes thought to promote host fitness. Most studies to date have relied on one or a few mouse models of infection, limiting insights into pathogen-specific neuroimmune interactions that generate sickness. We hypothesized that different pathogens might elicit distinct sickness states by engaging different cell types and brain circuits. Using inflammatory models representing bacterial, viral, allergic, parasitic or colitis conditions, we assessed sickness across scales: organismal - behavior and physiology; cellular - brain-wide neural activity; and molecular - single-cell in situ transcriptomics in hypothalamus areas associated with social and homeostatic functions affected during sickness. Remarkably, immune challenges elicited unique repertoires of changes across all scales. Our findings reveal pathogen-specific sickness states encoded by the brain across scales, thereby broadening our understanding of how infections make us sick.

neuroscience↗

bioRxiv2025

Clustering of host N-glycans licenses Toxoplasma rhoptry discharge

Apicomplexan parasites must discharge the contents of specialized organelles called rhoptries into host cells to initiate the process of invasion. This process requires the prior recognition and binding of the host cell by proteins released from another set of parasite organelles, the micronemes. However, the host-parasite interactions required for rhoptry discharge are largely unknown. Here we performed a host-cell directed genome-wide screen for host factors required for rhoptry discharge from Toxoplasma gondii, the causative agent of toxoplasmosis. The screen identified host N-glycosylation and cholesterol biosynthesis as pathways required for normal rhoptry discharge. A trimeric microneme complex, MIC1/4/6, interfaces with both pathways by binding host N-glycans to cluster proteins in a process dependent on host plasma membrane cholesterol. The process can be inhibited by depletion of host cholesterol or competition with exogenous glycans. This clustering of host factors by MIC1/4/6 likely prepares the host membrane for rhoptry discharge, delineating a new step in the Toxoplasma invasion process.

microbiology↗

bioRxiv2025

Two distinct SWI/SNF complexes direct chromatin-linked transcriptional programs in Toxoplasma

Chromatin remodeling complexes dynamically modify DNA accessibility to mediate changes in gene expression during eukaryotic cell cycle progression, developmental transitions, and environmental adaptation. Higher eukaryotes have multiple remodeler subtypes based on the incorporation of different ATPases; however, the coordination and functional specificity of these diverse complexes is not well understood. Apicomplexan parasites such as Toxoplasma gondii have a limited set of chromatin remodelers offering a divergent setting in which to explore the function of homologous complexes. These parasites have selectively retained the Myb domain-containing proteins with homology to chromatin-associated regulators like SNF2H and SWI3. Here, a comprehensive analysis of the Myb protein family in Toxoplasma defines the composition of two SWI3 complexes defined by mutually exclusive ATPases with homology to the widely conserved BRG1 and BRM. Integrating transcriptomics with a custom chromatin-profiling strategy, we show that BRG1 is essential for the timely transcription of genes during mitosis and cytokinesis, while BRM ensures global transcriptional competency and fidelity throughout the cell cycle and developmental transitions. Our findings demonstrate that BRG1 and BRM perform distinct yet interdependent regulatory roles shaped by their chromatin context. This work uncovers ancestral principles of chromatin regulation and offers new insight into the functional diversification of SWI/SNF complexes across eukaryotes.

microbiology↗

bioRxiv2025

5' untranslated regions tune Toxoplasma translation

Some of the longest 5' untranslated regions (UTRs) documented in eukaryotes belong to parasites of the phylum Apicomplexa. Translational regulation plays prominent roles in the development of these parasites, including the agents of toxoplasmosis (Toxoplasma gondii) and malaria. To understand the function of 5' UTRs in apicomplexan translation, we performed high-resolution ribosome profiling of T. gondii in human fibroblasts. We show that parasite translation efficiency (TE) is largely controlled by 5' UTR features and tuned by the number of upstream AUGs (uAUGs). Examination of ribosome occupancy reveals that, despite widespread assembly of parasite monosomes on uAUGs, ribosomes seldom translate uORFs. These determinants of translation are reaffirmed in a massively parallel reporter assay examining the effect of more than 30,000 synthetic 5' UTRs in T. gondii. A model trained on these results accurately predicted the TE of newly designed 5' UTRs. Together, this work defines the regulatory language of T. gondii translation, providing a framework to understand the evolution of exceptionally long 5' UTRs in apicomplexans.

molecular biology↗

bioRxiv2025

Alternative start codon selection shapes mitochondrial function during evolution, homeostasis, and disease

Mitochondrial endosymbiosis was a pivotal event in eukaryotic evolution, requiring core proteins to adapt to function both within the mitochondria and in the host cell. Here, we systematically profile the localization of protein isoforms generated by alternate start codon selection during translation. We identify hundreds of pairs of differentially-localized protein isoforms, many of which affect mitochondrial targeting and are essential for mitochondrial function. The emergence of dual-localized mitochondrial protein isoforms coincides with mitochondrial acquisition during early eukaryotic evolution. We further reveal that eukaryotes use diverse mechanisms--such as leaky ribosome scanning, alternative transcription, and paralog duplication--to maintain the production of dual-localized isoforms. Finally, we identify multiple isoforms that are specifically dysregulated by rare disease patient mutations and demonstrate how these mutations can help explain unique clinical presentations. Together, our findings illuminate the evolutionary and pathological relevance of alternative translation initiation, offering new insights into the molecular underpinnings of mitochondrial biology.

cell biology↗

bioRxiv2024

Metabolic Adaptability and Nutrient Scavenging in Toxoplasma gondii: Insights from Ingestion Pathway-Deficient Mutants

The obligate intracellular parasite Toxoplasma gondii replicates within a specialized compartment called the parasitophorous vacuole (PV). Recent work showed that despite living within a PV, Toxoplasma endocytoses proteins from the cytosol of infected host cells via a so-called ingestion pathway. The ingestion pathway is initiated by dense granule protein GRA14, which binds host ESCRT machinery to bud vesicles into the lumen of the PV. The protein-containing vesicles are internalized by the parasite and trafficked to the Plant Vacuole-like compartment (PLVAC), where cathepsin protease L (CPL) degrades the cargo and the chloroquine resistance transporter (CRT) exports the resulting peptides and amino acids to the parasite cytosol. However, although the ingestion pathway was proposed to be a conduit for nutrients, there is limited evidence for this hypothesis. We reasoned that if Toxoplasma uses the ingestion pathway to acquire nutrients, then parasites lacking GRA14, CPL, or CRT should rely more on biosynthetic pathways or alternative scavenging pathways. To explore this, we conducted a genome-wide CRISPR screen in wild-type (WT) parasites and {Delta}gra14, {Delta}cpl, and {Delta}crt mutants to identify genes that become more fitness conferring in ingestion-deficient parasites. Our screen revealed a significant overlap of genes that become more fitness conferring in the ingestion mutants compared to WT. Pathway analysis indicated that {Delta}cpl and {Delta}crt mutants relied more on pyrimidine biosynthesis, fatty acid biosynthesis, TCA cycle, and lysine degradation. Bulk metabolomic analysis showed reduced levels of glycolytic intermediates and amino acids in the ingestion mutants compared to WT, highlighting the pathways potential role in host resource scavenging. Interestingly, ingestion mutants showed an exacerbated growth defect when grown in amino acid-depleted media, suggesting a role for the Toxoplasma ingestion pathway during nutrient scarcity. ImportanceToxoplasma gondii is an obligate intracellular pathogen that infects virtually any nucleated cell in most warm-blooded animals. Infections are asymptomatic in most cases but people with weakened immunity can experience severe disease. For the parasite to replicate within the host, it must efficiently acquire essential nutrients, especially as it is unable to make several key metabolites. Understanding the mechanisms by which Toxoplasma scavenges nutrients from the host is crucial for identifying potential therapeutic targets. Our study highlights the function of the ingestion pathway in sustaining parasite metabolites and contributes to parasite replication under amino acid limiting conditions. This work advances our understanding of the metabolic adaptability of Toxoplasma.

microbiology↗

bioRxiv2024

Perforation of the host cell plasma membrane during Toxoplasma gondii invasion requires rhoptry exocytosis

Toxoplasma gondii is an obligate intracellular parasite, and the delivery of effector proteins from the parasite into the host cell during invasion is critical for invasion itself and for parasite virulence. The effector proteins are released from specialized apical secretory organelles known as rhoptries. While much has been learned recently about the structure and composition of the rhoptry exocytic machinery and the function of individual rhoptry effector proteins that are exocytosed, virtually nothing is known about how the released proteins are translocated across the host cell plasma membrane. Previous electrophysiology experiments reported an unanticipated observation that invasion by T. gondii is preceded by a transient increase in host cell plasma membrane conductance. Here, we confirm this electrophysiological observation and propose that the conductance transient represents a parasite-induced perforation in the host cell plasma membrane through which rhoptry proteins are delivered. As a first step towards testing this hypothesis, and to provide higher throughput than patch clamp electrophysiology, we developed an alternative assay to detect the perforation. This assay utilizes high-speed, multi-wavelength fluorescence imaging to enable simultaneous visualization of host cell perforation and parasite invasion. Using this assay, we interrogated a panel of mutant parasites conditionally depleted of key invasion-related proteins. Parasites lacking signaling proteins involved in triggering rhoptry secretion (e.g., CLAMP) or components of the rhoptry exocytic machinery (e.g., Nd9, RASP2) are defective in their ability to induce the perforation. These data are consistent with a model in which the perforating agents that disrupt host cell membrane integrity during invasion - and may thereby provide the conduit for delivery of rhoptry effector proteins - are stored within the rhoptries themselves and released upon contact with the host cell.

microbiology↗

bioRxiv2024

TgATG9 is required for autophagosome biogenesis and maintenance of chronic infection in Toxoplasma gondii

Toxoplasma gondii is a ubiquitous protozoan parasite that can reside long-term within hosts as intracellular tissue cysts comprised of chronic stage bradyzoites. To perturb chronic infection requires a better understanding of the cellular processes that mediate parasite persistence. Macroautophagy/autophagy is a catabolic and homeostatic pathway that is required for T. gondii chronic infection, although the molecular details of this process remain poorly understood. A key step in autophagy is the initial formation of the phagophore that sequesters cytoplasmic components and matures into a double-membraned autophagosome for delivery of the cargo to a cells digestive organelle for degradative recycling. While T. gondii appears to have a reduced repertoire of autophagy proteins, it possesses a putative phospholipid scramblase, TgATG9. Through structural modeling and complementation assays, we show herein that TgATG9 can partially rescue bulk autophagy in atg9{Delta} yeast. We demonstrated the importance of TgATG9 for proper autophagosome dynamics at the subcellular level using three-dimensional live cell lattice light sheet microscopy. Conditional knockdown of TgATG9 in T. gondii after bradyzoite differentiation resulted in markedly reduced parasite viability. Together, our findings provide insights into the molecular dynamics of autophagosome biogenesis within an early-branching eukaryote and pinpoint the indispensable role of autophagy in maintaining T. gondii chronic infection.

microbiology↗

bioRxiv2024

The latent stage of Toxoplasma gondii is targeted by the immune response and host protective

Latency is a microbial strategy for persistence. For Toxoplasma gondii the ability of the bradyzoite stage to form long-lived cysts is critical for transmission, while their presence in neurons is considered important for immune evasion. Development of a mathematical model highlighted that immune pressure on bradyzoites should contribute to dynamics of cyst formation and reactivation. Experimental data demonstrated that a cyst-derived antigen was recognized by CD8+ T cells and that IFN-{gamma} signaling in neurons contributes to cyst control. In addition, modeling and the use of a parasite strain unable to form bradyzoites revealed that this stage was not required for long-term persistence, but the absence of cyst formation resulted in increased tachyzoite replication in the CNS with associated tissue damage and mortality. Thus, the latent form of T. gondii is under immune pressure, mitigates infection-induced damage, and promotes survival of host and parasite.

microbiology↗

bioRxiv2023

Translation initiation factor eIF1.2 promotes Toxoplasma stage conversion by regulating levels of key differentiation factors

AbstractsThe parasite Toxoplasma gondii persists in its hosts by converting from replicating tachyzoites to latent bradyzoites housed in tissue cysts. The molecular mechanisms that mediate T. gondii differentiation remain poorly understood. Through a mutagenesis screen, we identified translation initiation factor eIF1.2 as a critical factor for T. gondii differentiation. A F97L mutation in eIF1.2 or the genetic ablation of eIF1.2 ({Delta}eif1.2) markedly impeded bradyzoite cyst formation in vitro and in vivo. We demonstrated, at single-molecule level, that the eIF1.2 F97L mutation impacts the scanning process of the ribosome preinitiation complex on a model mRNA. RNA sequencing and ribosome profiling experiments unveiled that {Delta}eif1.2 parasites are defective in upregulating bradyzoite induction factors BFD1 and BFD2 during stress-induced differentiation. Forced expression of BFD1 or BFD2 significantly restored differentiation in {Delta}eif1.2 parasites. Together, our findings suggest that eIF1.2 functions by regulating the translation of key differentiation factors necessary to establish chronic toxoplasmosis.

microbiology↗

bioRxiv2023

SPARK regulates AGC kinases central to the Toxoplasma gondii asexual cycle

Apicomplexan parasites balance proliferation, persistence, and spread in their metazoan hosts. AGC kinases, such as PKG, PKA, and the PDK1 ortholog SPARK, integrate environmental signals to toggle parasites between replicative and motile life stages. Recent studies have cataloged pathways downstream of apicomplexan PKG and PKA; however, less is known about the global integration of AGC kinase signaling cascades. Here, conditional genetics coupled to unbiased proteomics demonstrates that SPARK complexes with an elongin-like protein to regulate the stability of PKA and PKG in the model apicomplexan Toxoplasma gondii. Defects attributed to SPARK depletion develop after PKG and PKA are down-regulated. Parasites lacking SPARK differentiate into the chronic form of infection, which may arise from reduced activity of a coccidian-specific PKA ortholog. This work delineates the signaling topology of AGC kinases that together control transitions within the asexual cycle of this important family of parasites.

microbiology↗

bioRxiv2023

Functional profiling of the Toxoplasma genome during acute mouse infection

Within a host, pathogens encounter a diverse and changing landscape of cell types, nutrients, and immune responses. Examining host-pathogen interactions in animal models can therefore reveal aspects of infection absent from cell culture. We use CRISPR-based screens to functionally profile the entire genome of the model apicomplexan parasite Toxoplasma gondii during mouse infection. Barcoded gRNAs were used to track mutant parasite lineages, enabling detection of bottlenecks and mapping of population structures. We uncovered over 300 genes that modulate parasite fitness in mice with previously unknown roles in infection. These candidates span multiple axes of host-parasite interaction, including determinants of tropism, host organelle remodeling, and metabolic rewiring. We mechanistically characterized three novel candidates, including GTP cyclohydrolase I, against which a small-molecule inhibitor could be repurposed as an antiparasitic compound. This compound exhibited antiparasitic activity against T. gondii and Plasmodium falciparum, the most lethal agent of malaria. Taken together, we present the first complete survey of an apicomplexan genome during infection of an animal host, and point to novel interfaces of host-parasite interaction that may offer new avenues for treatment.

microbiology↗

bioRxiv2023

Analysis of CDPK1 targets identifies a trafficking adaptor complex that regulates microneme exocytosis in Toxoplasma

Apicomplexan parasites use Ca2+-regulated exocytosis to secrete essential virulence factors from specialized organelles called micronemes. Ca2+-dependent protein kinases (CDPKs) are required for microneme exocytosis; however, the molecular events that regulate trafficking and fusion of micronemes with the plasma membrane remain unresolved. Here, we combine sub-minute resolution phosphoproteomics and bio-orthogonal labeling of kinase substrates in Toxoplasma gondii to identify 163 proteins phosphorylated in a CDPK1-dependent manner. In addition to known regulators of secretion, we identify uncharacterized targets with predicted functions across signaling, gene expression, trafficking, metabolism, and ion homeostasis. One of the CDPK1 targets is a putative HOOK activating adaptor. In other eukaryotes, HOOK homologs form the FHF complex with FTS and FHIP to activate dynein-mediated trafficking of endosomes along microtubules. We show the FHF complex is partially conserved in T. gondii, consisting of HOOK, an FTS homolog, and two parasite-specific proteins (TGGT1_306920 and TGGT1_316650). CDPK1 kinase activity and HOOK are required for the rapid apical trafficking of micronemes as parasites initiate motility. Moreover, parasites lacking HOOK or FTS display impaired microneme protein secretion, leading to a block in the invasion of host cells. Taken together, our work provides a comprehensive catalog of CDPK1 targets and reveals how vesicular trafficking has been tuned to support a parasitic lifestyle.

microbiology↗

bioRxiv2022

A conserved complex of microneme proteins mediates rhoptry discharge in Toxoplasma

Apicomplexan parasites discharge specialized organelles called rhoptries upon host cell contact to mediate invasion. The events that drive rhoptry discharge are poorly understood, yet essential to sustain the apicomplexan parasitic life cycle. Rhoptry discharge appears to depend on proteins secreted from another set of organelles called micronemes, which in Toxoplasma gondii includes MIC8 and the microneme-associated CRMP complex. Here, we examine the function of the microneme protein CLAMP, uncovering its essential role in rhoptry discharge. CLAMP forms a distinct complex with two other microneme proteins, the invasion-associated SPATR, and a previously uncharacterized protein we name CLAMP-linked invasion protein (CLIP). CLAMP-deficiency does not impact parasite adhesion or microneme protein secretion; however, knockdown of any member of the CLAMP complex affects rhoptry discharge. Phylogenetic analysis suggests orthologs of the essential complex components, CLAMP and CLIP, are ubiquitous across apicomplexans. Nevertheless, SPATR, which appears to act as an accessory factor in Toxoplasma, is essential during Plasmodium falciparum blood stages. Our results reveal a new protein complex that mediates rhoptry discharge following host-cell contact.

microbiology↗

bioRxiv2022

CDPK2A and CDPK1 form a signaling module upstream of Toxoplasma motility

The transition between parasite replication and dissemination is regulated in apicomplexan parasites by fluctuations in cytosolic calcium concentrations, effectuated by calcium-dependent protein kinases (CDPKs). We examined the role of CDPK2A in the lytic cycle of Toxoplasma, analyzing its role in the regulation of cellular processes associated with parasite motility. We used chemical-genetic approaches and conditional depletion to determine that CDPK2A contributes to the initiation of parasite motility through microneme discharge. We demonstrate that the N-terminal extension of CDPK2A is necessary for the proteins function. Conditional depletion revealed an epistatic interaction between CDPK2A and CDPK1, suggesting that the two kinases work together to mediate motility in response to certain stimuli. This signaling module appears distinct from that of CDPK3 and PKG, which also controls egress. CDPK2A is revealed as an important regulator of the Toxoplasma kinetic phase, linked to other kinases that govern this critical transition. Our work uncovers extensive interconnectedness between the signaling pathways that govern parasite motility. IMPORTANCEThis work uncovers interactions between various signaling pathways that govern Toxoplasma gondii egress. Specifically, we compare the function of three canonical calcium dependent protein kinases (CDPKs) using chemical-genetic and conditional-depletion approaches. We describe the function of a previously uncharacterized CDPK, CDPK2A, in the Toxoplasma lytic cycle, demonstrating it contributes to parasite fitness through regulation of microneme discharge, gliding motility, and egress from infected host cells. Comparison of analog-sensitive (AS) kinase alleles and conditionally-depleted alleles uncovered epistasis between CDPK2A and CDPK1 implying a partial functional redundancy. Understanding the topology of signaling pathways underlying key events in the parasite life cycle can aid in efforts targeting parasite kinases for anti-parasitic therapies.

microbiology↗

bioRxiv2022

De novo mapping of the apicomplexan Ca2+-responsive proteome

Apicomplexan parasites cause persistent mortality and morbidity worldwide through diseases including malaria, toxoplasmosis, and cryptosporidiosis. Ca2+ signaling pathways have been repurposed in these eukaryotic pathogens to regulate parasite-specific cellular processes governing the transition between the replicative and lytic phases of the infectious cycle. Despite the presence of conserved Ca2+-responsive proteins, little is known about how specific signaling elements interact to impact pathogenesis. We mapped the Ca2+-responsive proteome of the model apicomplexan T. gondii via time-resolved phosphoproteomics and thermal proteome profiling. The waves of phosphoregulation following PKG activation and stimulated Ca2+ release corroborate known physiological changes but identify specific proteins operating in these pathways. Thermal profiling of parasite extracts identified many expected Ca2+-responsive proteins, such as parasite Ca2+-dependent protein kinases. Our approach also identified numerous Ca2+-responsive proteins that are not predicted to bind Ca2+, yet are critical components of the parasite signaling network. We characterized protein phosphatase 1 (PP1) as a Ca2+-responsive enzyme that relocalized to the parasite apex upon Ca2+ store release. Conditional depletion of PP1 revealed that the phosphatase regulates Ca2+ uptake to promote parasite motility. PP1 may thus be partly responsible for Ca2+-regulated serine/threonine phosphatase activity in apicomplexan parasites.

microbiology↗

bioRxiv2022

A positive feedback loop controls Toxoplasma chronic differentiation

Successful infection strategies must balance pathogen amplification and persistence. In Toxoplasma gondii, this is accomplished through differentiation into dedicated cyst-forming chronic stages that avoid clearance by the host immune system. The transcription factor BFD1 is both necessary and sufficient for stage conversion; however, its regulation is not understood. We examine five factors transcriptionally activated by BFD1. One of these is a cytosolic RNA-binding protein of the CCCH-type zinc finger family, which we name BFD2. Parasites lacking BFD2 fail to induce BFD1 and are consequently unable to fully differentiate in culture or in mice. BFD2 interacts with the BFD1 transcript in a stress-dependent manner. Deletion of BFD2 reduces BFD1 protein levels, but not mRNA abundance. The reciprocal effects on BFD2 transcription and BFD1 translation outline a positive feedback loop that enforces commitment to differentiation. BFD2 helps explain how parasites commit to the chronic gene-expression program and elucidates how the balance between proliferation and persistence is achieved over the course of infection.

microbiology↗