Biology subjects

Bernabeu, M.

Publications and source records attributed to Bernabeu, M..

13 recordsLinked to original sources

bioRxiv2026

Host retargeting predominated over gene transfer during early chromatophore integration in Paulinella micropora

The relative contributions of gene acquisition and protein retargeting to early organellogenesis remain debated. We addressed this question for the recently evolved chromatophore of Paulinella micropora, which provides a unique window into the initial stages of primary organelle integration. Using a stringent phylogenomic pipeline, we identified 282 P. micropora gene families whose origin can be attributed to horizontal gene transfer (HGT) with high confidence, including 115 for which we could confidently assess the donor lineages. In addition to the expected cyanobacterial signal from the chromatophore ancestor, we detected recurrent contributions from Gammaproteobacteria, Bacteroidota, Actinobacteriota, and Chlamydiota. Relative stem-length analyses indicate that these donors contributed in temporally structured periods (waves), with several non-cyanobacterial signals older than the median cyanobacterial signal and others clearly more recent. HGT-derived genes are enriched in transport and metabolic functions, which might have been relevant to host-symbiont interaction. However, based on computational predictions, the chromatophore-targeted proteome is dominated by vertically inherited host genes with only 36 of 287 predicted chromatophore-targeted nucleus-encoded proteins being of HGT origin, the majority of which are absent in Paulinella chromatophora, suggesting a recent origin in P. microporalineage. Thus, although repeated horizontal acquisition from multiple bacterial partners shaped the nuclear genome of P. micropora, early chromatophore integration appears to have been dominated by host-derived retargeting, with HGT providing a smaller but detectable complementary contribution. These results support a mixed model of organellogenesis in which targeting and gene transfer act together, but with host retargeting playing the dominant early role.

evolutionary biology↗

bioRxiv2025

Innate immune responses to Plasmodium falciparum disrupt the blood-brain barrier

Plasmodium falciparum accumulation at the blood-brain barrier (BBB) is a hallmark of cerebral malaria, a life-threatening complication. Conversely, the contribution of the immune response to vascular injury has long been debated. Here, we studied the role of innate immune cells as potential effectors of vascular damage using a human in vitro 3D-BBB model. Parasite-stimulated immune cells from malaria-naive donors increased adhesion to microvessels, at least partly through LFA-1. This caused barrier disruption and inflammatory activation of BBB cells. Secretion of TNF-, IFN-{gamma}, and granzyme B by monocytes, NK and {gamma}{delta} T cells correlated with vascular injury, and accumulation of immune cells was required for local barrier damage. Our computational analysis disentangled pathogenic mechanisms driven specifically by either P. falciparum parasite or immune cells, as well as shared pathways. These findings demonstrate how vascular-immune interactions may contribute to vascular injury in cerebral malaria and point towards the potential of immunomodulatory therapeutics.

immunology↗

bioRxiv2025

Febrile temperature enhances Plasmodium falciparum cytoadhesion by disrupting the endothelial glycocalyx

Fever, a universal host defense response in infection and inflammation, paradoxically contributes to neurological complications in malaria. Febrile temperatures are known to enhance parasite virulence protein expression, but direct effects on the human endothelium remain unknown. We found that a 1-hour exposure to 40 {degrees}C, representative of fever in children with cerebral malaria, increased adhesion of Plasmodium falciparum-infected red blood cells and neutrophils to 3D brain microvascular models displaying a wide wall shear stress gradient. Mechanistically, this brief hyperthermia triggered rapid endothelial glycocalyx shedding, exposing endothelial receptors for binding. This response was more pronounced in brain than in pulmonary microvessels, revealing a greater vulnerability of the cerebral vasculature to fever. Pharmacological inhibition of matrix metalloproteinase activity preserved glycocalyx integrity and abolished the temperature-induced increase in adhesion. These findings identify fever as a host-specific amplifier of malaria-associated microvascular pathology, highlighting the importance of antipyretic strategies to mitigate disease severity.

microbiology↗

bioRxiv2025

ETS-guided iPSC-endothelial models recapitulate malaria pathogenesis

The sequestration of the malaria parasite Plasmodium falciparum in the microvasculature is a major driver of severe malaria, but its pathogenic mechanisms still remain unknown. Advancements in induced pluripotent stem cell (iPSC) technologies offer unique opportunities to study parasite interactions with blood vessels in a well-defined host environment. However, endothelial iPSC-differentiation methods often result in cells with mixed epithelial identity. Here, we have generated an iPSC line with inducible and simultaneous expression of ETS transcription factors (ETV2, FLI1, ERG), which resulted in improved endothelial cell identity and strong barrier function. These cells display a high affinity to infected red blood cells. Exposure to parasite products caused significant endothelial metabolic changes and splicing alterations. Furthermore, it disrupted the iPSC-endothelial barrier, as a consequence of transcriptional downregulation of key barrier processes, and alteration of severe malaria biomarkers. Our novel iPSC-based approach represents a new in vitro platform to study the pathogenesis of vascular infections.

microbiology↗

bioRxiv2024

Diverse ancestries reveal complex symbiotic interactions during eukaryogenesis

Summary paragraphThe origin of eukaryotes remains a central enigma in biology1. Ongoing debates agree on the pivotal role of a symbiosis between an alphaproteobacterium and an Asgard archaeon2,3. However, the nature, timing and contributions of other potential bacterial partners4-6 or the role of interactions with viruses7-9 remain contentious. To address these questions, we employed advanced phylogenomic approaches and comprehensive datasets spanning the known diversity of cellular life and viruses. Our analysis provided an updated reconstruction of the last eukaryotic common ancestor (LECA) proteome, in which we traced the phylogenetic origin of each protein family. We found compelling evidence for multiple waves of horizontal gene transfer from diverse bacterial donors, with some likely preceding the mitochondrial endosymbiosis. We inferred plausible traits of the major donors and their functional contributions to LECA. Our findings underscore the contribution of horizontal gene transfers in shaping the proteomes of pre-LECA ancestors and hint to a facilitating role of Nucleocytoviricota viruses. Altogether, our results suggest that ancient eukaryotes originated within complex microbial ecosystems through a succession of diverse associations that left a footprint of horizontally transferred genes.

evolutionary biology↗

bioRxiv2024

Pathogenic mechanisms of Plasmodium falciparum egress unveiled by a microvascular 3D blood-brain barrier model

Cerebral malaria is a severe neurovascular complication of Plasmodium falciparum infection, with high mortality rates even after treatment with effective antimalarials. Limitations in current experimental models have hindered our knowledge of the disease. We developed a 3D blood-brain barrier (BBB) model with enhanced barrier properties using primary brain endothelial cells, astrocytes and pericytes. Exposure to parasite egress products increased microvascular permeability, likely due to transcriptional downregulation of junctional and vascular development genes in endothelial cells. In addition, it increased the expression of ferroptosis markers, antigen presentation and type I interferon genes and upregulated the JAK-STAT pathway across all BBB cell types. Incubation with cytoadherent schizont-stage P. falciparum-infected erythrocytes induced a similar, but highly localized transcriptional shift, along with inter-endothelial gaps at sites of parasite egress, significantly increasing permeability. The co-administration of egress products with the JAK-STAT inhibitor Ruxolitinib prevented junctional disruption and BBB breakdown. These findings provide key insights into the parasite-mediated mechanisms driving brain microvascular pathogenesis in cerebral malaria and suggest potential avenues for adjunctive therapies.

microbiology↗

bioRxiv2024

Phylogeny-aware simulations suggest a low impact of unsampled lineages in the inference of gene flow during eukaryogenesis

Gene phylogenies are broadly used to analyse events of horizontal gene transfer, namely, their presence, the potential donor and acceptor lineages, and their relative timing. Recent phylogenomics analyses have reconstructed a relative chronology of gene acquisitions in the lineage leading to the eukaryotes, revealing waves of acquisition from different donors before and after the mitochondrial endosymbiosis. However, a recognised caveat is the potential biases introduced by the presence of incomplete taxon sampling resulting in so-called "ghost" lineages. Here, we assessed the robustness of the gene phylogeny-based branch length ratio method in the inference of the relative ordering of gene acquisition events during eukaryogenesis. We introduce a novel simulation framework that populates a known dated Tree of Life with plausible "ghost" lineages and simulates their gene transfers to the lineage leading to the last eukaryotic common ancestor. Our simulations suggest a generally low probability of misinterpreting the relative order of gene acquisitions from distinct ghost donors. However, we identify specific problematic phylogenetic placements where unsampled lineages are more likely to produce misleading results. Overall, our approach offers valuable guidance for the interpretation of future work on eukaryogenesis, and can be readily adapted to other evolutionary scenarios.

evolutionary biology↗

bioRxiv2024

Probabilistic modelling improves relative dating from gene phylogenies

O_LIEstablishing the timing of past evolutionary events is a fundamental task in the reconstruction of the history of life. State-of-the-art molecular dating methods generally involve the reconstruction of a species tree from conserved, vertically evolving genes, and the assumption of a molecular clock calibrated with the fossil record. Although this approach is extremely useful, its use is limited to speciation events and does not account for genes following different evolutionary paths. Recently, an alternative methodology for the relative dating of evolutionary events has been proposed that considers the distribution of branch lengths across sets of gene trees. C_LIO_LIHere, we validate this methodology by comparing the relative age estimates with a fossil-calibrated phylogeny and propose a model-based formalisation using a Bayesian framework. C_LIO_LIOur analyses revealed that the normalisation of the distances of interest with the branch lengths of a reference clade present across the set of gene trees results in narrower distributions, allowing the correct inference of the relative ordering of evolutionary events. C_LIO_LIWe show that distributions of normalised lengths can be modelled using gamma or lognormal distributions and demonstrate that inference of the posterior distribution of the mode allows accurate relative age estimation, as assessed by a strong correlation with the molecular clock-dated tree. C_LI

evolutionary biology↗

bioRxiv2024

Plasmodium falciparum disruption of pericyte angiopoietin-1 secretion contributes to barrier breakdown in a 3D brain microvessel model

Disruption of the vascular protective angiopoietin-Tie axis is common in cerebral malaria (CM) patients, who display elevated angiopoietin-2 (Ang-2) and reduced angiopoietin-1 (Ang-1) blood concentrations. The role of pericytes in CM pathogenesis remains unexplored, despite being a major source of brain Ang-1 secretion and evidence of pericyte damage observed in CM postmortem samples. Here we engineered a human 3D microfluidics-based brain microvessel model containing the minimal cellular components to replicate the angiopoietin-Tie axis, human primary brain microvascular endothelial cells and pericytes. This model replicated pericyte vessel coverage and ultrastructural interactions present in the brain microvasculature. When exposed to P. falciparum-iRBC egress products, 3D brain microvessels presented decreased Ang-1 secretion, increased vascular permeability, and minor ultrastructural changes in pericyte morphology. Notably, P. falciparum-mediated barrier disruption was partially reversed after pre-treatment with recombinant Ang-1 and the Tie-2 activator, AKB-9778. Our approach suggests a novel mechanistic role of pericytes in CM pathogenesis and highlights the potential of therapeutics that target the angiopoietin-Tie axis to rapidly counteract vascular dysfunction caused by P. falciparum. The paper explainedO_ST_ABSProblemC_ST_ABSCerebral malaria (CM) is a severe complication of Plasmodium falciparum infection, resulting in the majority of [~]600000 malarial deaths annually. Despite anti-malarial drug administration upon hospitalization, fatality rates still range from 15-25% and many survivors suffer long term neurological disabilities. A common dysregulated vascular pathway identified in CM patients is the angiopoietin-Tie axis. Treatments that restore this vascular homeostatic pathway appear as a potential avenue for adjunctive therapies in experimental rodent CM models. Nevertheless, the use of rodent CM models for therapeutic discovery is not ideal, given that P. falciparum pathogenesis is species-specific. Therefore, the development of novel and advanced human 3D microvascular models offers new avenues to study disease pathogenesis and explore potential adjunctive CM treatments. ResultsIn this study, we generate a 3D human brain microvasculature model that reproduces in vivo interactions between two key cell types necessary to reproduce the protective angiopoietin-Tie axis: human brain endothelial cells and pericytes. Addition of P. falciparum-infected red blood cell (iRBC) egress products causes vascular disruption and hampers the release of the vascular protective factor, angiopoietin-1, from brain pericytes. 18-hour pre-treatment of Ang-1 for 18h prevents iRBC egress product-induced vascular disruption. A short pre-treatment of the microvessels with AKB-9778, a downstream pharmaceutical inducer of angiopoietin-Tie axis activity currently in phase II clinical trial for diabetic retinopathy, partially restores vascular integrity. Our study highlights the role of pericytes in CM and the therapeutic potential of interventions that restore the angiopoietin-Tie2 axis as adjunctive CM treatments. ImpactOur study demonstrates the potential of bioengineered vascular models to recapitulate dysregulated pathways previously characterized in malaria patients, and in providing a physiologically-relevant platform to test adjunctive therapies. The use of the 3D brain microvascular model has enhanced our understanding of the mechanisms behind CM pathogenesis, uncovering a previously unappreciated effect of P. falciparum on brain pericytes, linking angiopoietin-Tie axis dysregulation and microvasculature disruption. These findings pave the way for the identification of novel, fast-acting therapeutics, such as AKB-9778, to restore vascular integrity in CM patients.

microbiology↗

bioRxiv2024

Broadly inhibitory antibodies against severe malaria virulence proteins

Plasmodium falciparum pathology is driven by the accumulation of parasite-infected erythrocytes in microvessels. This process is mediated by the parasites polymorphic erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. A subset of PfEMP1 variants that bind human endothelial protein C receptor (EPCR) through their CIDR1 domains is responsible for severe malaria pathogenesis. A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP1 variants. Here, we describe two broadly reactive and binding-inhibitory human monoclonal antibodies against CIDR1. The antibodies isolated from two different individuals exhibited a similar and consistent EPCR-binding inhibition of 34 CIDR1 domains, representing five of the six subclasses of CIDR1. Both antibodies inhibited EPCR binding of both recombinant full-length and native PfEMP1 proteins as well as parasite sequestration in bioengineered 3D brain microvessels under physiologically relevant flow conditions. Structural analyses of the two antibodies in complex with two different CIDR1 antigen variants reveal similar binding mechanisms that depend on interactions with three highly conserved amino acid residues of the EPCR-binding site in CIDR1. These broadly reactive antibodies likely represent a common mechanism of acquired immunity to severe malaria and offer novel insights for the design of a vaccine or treatment targeting severe malaria.

immunology↗

bioRxiv2023

Targeting plasmid-encoded proteins that contain immunoglobulin-like domains to combat antimicrobial resistance.

Antimicrobial resistance (AMR) poses a significant threat to human health. Although vaccines have been developed to combat AMR, it has proven challenging to associate specific vaccine antigens with AMR. Bacterial plasmids play a crucial role in the transmission of AMR. Our recent research has identified a group of bacterial plasmids (specifically, IncHI plasmids) that encode large molecular mass proteins containing bacterial immunoglobulin-like domains. These proteins are found on the external surface of the bacterial cells, such as in the flagella or conjugative pili. In this study, we show that these proteins are antigenic and can protect mice from infection caused by an AMR Salmonella strain harboring one of these plasmids. Furthermore, we successfully generated nanobodies targeting these proteins, that were shown to interfere with the conjugative transfer of IncHI plasmids. Considering that these proteins are also encoded in other groups of plasmids, such as IncA/C and IncP2, targeting them could be a valuable strategy in combating AMR infections caused by bacteria harboring different groups of AMR plasmids. Since the selected antigens are directly linked to AMR itself, the protective effect extends beyond specific microorganisms to include all those carrying the corresponding resistance plasmids.

microbiology↗

bioRxiv2022

Pulsed stimulated Brillouin microscopy enables high-sensitivity mechanical imaging of live and fragile biological specimens

Brillouin microscopy is an emerging optical elastography technique capable of assessing mechanical properties of biological samples in a 3D, all-optical and hence non-contact fashion. The typically weak Brillouin scattering signal can be substantially enhanced via a stimulated photon-phonon process, which improves the signal-to-background ratio (SBR) as well as provides higher spectral resolution. However, current implementations of stimulated Brillouin spectroscopy (SBS) require high pump powers, which prohibit applications in many areas of biology, especially when studying photosensitive samples, or when live-imaging in 3D and/or over extended time periods. Here, we present a pulsed SBS scheme that takes full advantage of the non-linearity of the pump-probe interaction in SBS. In particular, we show that through quasi-pulsing and diligent optimization of signal detection parameters, the required pump laser power can be decreased ~20-fold without affecting the signal levels or spectral precision. Moreover, we devise a custom analysis approach that facilitates the analysis of complex, multi-peaked Brillouin spectra in order to harness the high spectral resolution of SBS for the specific identification of biomechanical components inside the point-spread function of the microscope. We then demonstrate the low-phototoxicity and high-specificity of our pulsed SBS approach by imaging sensitive single cells, zebrafish larvae, and mouse embryos as well as adult C. elegans with sub-cellular detail. Furthermore, our method permits observing the mechanics of organoids and C. elegans embryos over time. We expect that the substantially lower photo-burden and improved SBR of pulsed SBS will facilitate studying biomechanics in 3D at high spatio-temporal resolution in living biological specimens in a non-invasive manner, opening up exciting new possibilities for the field of mechanobiology.

bioengineering↗

bioRxiv2021

Roles of proteins containing immunoglobulin-like domains in the conjugation of bacterial plasmids.

Horizontal transfer of bacterial plasmids generates genetic variability and contributes to the dissemination of the genes that enable bacterial cells to develop antimicrobial resistance (AMR). Several aspects of the conjugative process have long been known, namely, those related to the proteins that participate in the establishment of cell-to-cell contact and to the enzymatic processes associated with the processing of plasmid DNA and its transfer to the recipient cell. In this work, we describe the role of newly identified proteins that influence the conjugation of several plasmids. Genes encoding high-molecular-weight proteins that contain one or several immunoglobulin-like domains (Big) are located in the transfer regions of several plasmids that usually harbor AMR determinants. These Big proteins are exported to the external medium and target two extracellular organelles: the flagella and the conjugative pili. The plasmid-encoded Big proteins facilitate conjugation by reducing cell motility and facilitating cell-to-cell contact by binding both to the flagella and to the conjugative pilus. They use the same export machinery as that used by the conjugative pilus components. In the examples characterized in this paper, these proteins influence conjugation at environmental temperatures (i.e., 25{degrees}C). This suggests that they may play relevant roles in the dissemination of plasmids in natural environments. Taking into account that they interact with outer surface organelles, they could be targeted to control the dissemination of different bacterial plasmids carrying AMR determinants.

microbiology↗