Biology subjects

Vazquez, R.

Publications and source records attributed to Vazquez, R..

10 recordsLinked to original sources

bioRxiv2026

An integrated RNA-centric imaging and omics approach reveals distinct properties and composition of neuronal RNA granules

RNA granules are essential regulators of post-transcriptional gene expression, enabling mRNA transport, localization, and local translation in neurons. The localized transcriptome is diverse; however, how different mRNAs are organized into granules for efficient localization and translation remains unknown. Here, we combine real-time endogenous single RNA imaging with protein and RNA proximity labeling to investigate two distinct endogenous neuronal mRNA granule populations, Actb and Arc, in stimulated primary hippocampal neurons. Using orthogonal RNA labeling systems in a dual knock-in mouse model, we show that Actb and Arc mRNAs are packaged into spatially segregated granules with distinct trafficking dynamics, localization kinetics, and responses to synaptic stimulation. Actb granules displayed rapid and sustained localization, whereas Arc granules showed delayed, transient recruitment, consistent with their respective roles in structural and activity-dependent plasticity. Proximity labeling reveals that these granules are distinct in their mRNA composition, despite sharing core RNA-binding proteins, suggesting that shared cis-regulatory elements within mRNA 3UTR regions drive selective co-packaging of mRNAs into unique granules. Together, these findings demonstrate that neuronal mRNAs are differentially sorted into molecularly and functionally distinct granules, providing a framework for understanding how precise spatio-temporal control of mRNA localization and translation is achieved across complex neuronal arbors.

neuroscience↗

bioRxiv2025

PhaLP 2.0: extending the community-oriented phage lysin database with a SUBLYME pipeline for metagenomic discovery

As biology becomes increasingly data-driven, so too does the field of phage lysins, enzymes that degrade bacterial cell walls and hold promise as alternatives to traditional antibiotics. Five years ago, we introduced PhaLP, a centralized resource for Phage Lytic Protein sequences and associated metadata to support global research efforts. Here, we present PhaLP 2.0, a significantly enhanced database designed to overcome key challenges in the computational study of lysins by integrating newly identified lysins obtained from thousands of metagenomes. To expand the known diversity of lysins beyond those from cultured phages, we developed SUBLYME, a protein embedding-based machine learning Software designed to Uncover and classify Bacteriophage Lysins in Metagenomic datasets. Using embeddings derived from the prior well-curated protein sequences of the original PhaLP database, we trained support vector machines to distinguish lysins from non-lysins in viromes and classify them as either endolysins or virion-associated lysins. The models achieved an average F1-score of 98% on held-out lysin clusters. SUBLYME enabled the discovery of 743,000 new lysin sequences from EnVhogDB, a virome-derived protein database, increasing the number of known lysin clusters by a factor of 40, from 1,000 to 40,000. PhaLP 2.0 entries were annotated by integrating Pfam functional predictions to the refined delineations obtained with SPAED, an algorithm that leverages the predicted aligned error matrix from AlphaFold predictions to identify domain boundaries. Both SUBLYME and the PhaLP 2.0 database are accessible online at https://github.com/Rousseau-Team/sublyme and http://phalp.ugent.be, respectively. Together, these advances establish PhaLP 2.0 as a comprehensive and scalable portal for the discovery, classification, and sequence analysis of phage lysins, paving the way for future antibacterial applications and evolutionary insights. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=59 SRC="FIGDIR/small/692814v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@181e26borg.highwire.dtl.DTLVardef@37e099org.highwire.dtl.DTLVardef@7a7fccorg.highwire.dtl.DTLVardef@5c42f1_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2025

Pro-inflammatory role of granzyme K producing bystander CD8+ T cells in acute myeloid leukemia

Acute myeloid leukemia (AML) is a heterogeneous group of blood malignancies with a 5-year survival rate below 30%, highlighting the urgent need for more effective therapeutic strategies. T cell-based immunotherapies have demonstrated remarkable success in solid tumors, yet the role of CD8+ T cells in AML remains unclear. In this study, we analyzed the composition, antigenic specificity, and function of CD8+ T cells in paired blood and bone marrow samples from AML patients. While we did not identify exhausted CD8+ T cells as seen in solid tumors, we observed a distinct population of functional CD69+ CD8+ T cells specifically enriched in the bone marrow. These cells primarily recognized non-tumor antigens, including epitopes derived from Epstein-Barr virus (EBV) and cytomegalovirus (CMV). Notably, this bystander CD8+ T cell population showed high expression of Granzyme K, a cytokine found in the bone marrow of AML patients. Granzyme K did not induce leukemic cell death but instead promoted the secretion of IL-8, a pro-inflammatory cytokine known to play a detrimental role in AML pathology. Rather than mounting an anti-tumor response, these CD8+ T cells contribute to a pro-inflammatory environment that may exacerbate AML progression and severity. These findings provide a rationale for exploring therapeutic strategies aimed at inhibiting pro-inflammatory CD8+ T cells and targeting Granzyme K activity in association with actual therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/669682v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1fc9798org.highwire.dtl.DTLVardef@4491b1org.highwire.dtl.DTLVardef@1b8feadorg.highwire.dtl.DTLVardef@58515d_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

SPAED: Harnessing AlphaFold Output for Accurate Segmentation of Phage Endolysin Domains

SummarySPAED is an accessible tool for the accurate segmentation of protein domains that applies hierarchical clustering to the predicted aligned error (PAE) matrix obtained from AlphaFold predictions. It leverages information contained in the PAE matrix to better identify domain-linker boundaries and detect disordered regions. On a dataset of 376 bacteriophage endolysins (proteins that degrade the bacterial cell wall), SPAED achieves a mean intersect-over-union score of 96% and a domain-boundary-distance score of 89% compared to 94% and 70%, respectively, for the state-of-the-art tool Chainsaw. Availability and ImplementationSPAED is available on the web at http://spaed.ca and available for download at https://github.com/Rousseau-Team/spaed. ContactElsa Rousseau - elsa.rousseau@ift.ulaval.ca, Roberto Vazquez - rvazqf@gmail.com

bioinformatics↗

bioRxiv2025

Diversity, structure-function relationships and evolution of cell wall-binding domains of staphylococcal phage endolysins

Endolysins, encoded by phages, lyse bacterial hosts at the end of the replication cycle by degrading peptidoglycan. Consequently, they have evolved in response to host cell wall structures, leading to complex modular architectures, particularly in Gram-positive bacteria. These architectures feature diverse enzymatically active domains (EADs) and cell wall-binding domains (CBDs). This study investigates the structure-function relationships of CBDs in staphylococcal phage endolysins, exploring their evolutionary origins and the extent to which binding specificity can be predicted from sequence data. A set of 182 staphylococcal endolysin sequences was analyzed, revealing predominantly three-domain architectures, occasionally disrupted by species-specific mobile genetic elements. Most CBDs exhibited an SH3-like fold, classified into two major subfamilies: SH3b_P (including the well-characterized SH3_5 family) and SH3b_T. The composition of endolysin domains correlated with specific CBD families, suggesting co-evolution of CBDs and compatible EADs to ensure functional synergy. To assess binding properties, 24 CBDs were fused to eGFP and tested against a panel of staphylococci, revealing diverse specificity profiles. However, no clear correlation emerged between binding specificity, phylogenetic subgroups, or bacterial hosts. This suggests that minor structural modifications significantly impact function and that CBD specificity is not a major selective pressure in the staphylococcal bacteria-phage interface.

molecular biology↗

bioRxiv2024

Chemoreceptor family in plant-associated bacteria responds preferentially to the plant signal molecule glycerol 3-phosphate

Plant pathogens and plant-associated bacteria contain about twice as many chemoreceptors as the bacterial average, indicating that chemotaxis is particularly important for bacteria-plant interactions. However, information on the corresponding chemoreceptors is limited. In this study, we identified the chemoreceptor PacP from the phytopathogen Pectobacterium atrosepticum, which exclusively recognized C3 phosphorylated compounds at its sCache ligand binding domain, mediating chemoattraction. Using a motif of PacP amino acid residues involved in ligand binding, we identified a chemoreceptor family, termed sCache_PC3, that was specific for C3 phosphorylated compounds. Isothermal titration calorimetry studies revealed that family members preferentially bound glycerol 3-phosphate, a key plant signaling molecule. Additionally, family members recognized glycerol 2-phosphate and glycolysis intermediates glyceraldehyde 3-phosphate, dihydroxyacetone phosphate and 3-phosphoglycerate. This study presents the first evidence of chemoreceptors that bind phosphorylated compounds. We show that the sCache_PC3 family has evolved from an ancestral sCache domain that respond primarily to Krebs cycle intermediates. Members of the sCache_PC3 family were mainly found in bacteria that interact with plants, including many important plant pathogens such as Brenneria, Dickeya, Musicola, Pectobacterium, and Herbaspirillum. Glycerol 3-phosphate is a signal molecule that is excreted by plants in response to stress and infection. Chemotaxis towards this molecule may thus be a means for bacteria to localize stressed plants and move to infection sites. This study lays the groundwork for investigating the functional importance of chemotaxis to phosphorylated C3 compounds in plant-bacteria interactions and virulence. Significance statementThe bacterial lifestyle has shaped the evolution of signal transduction systems, and the number and type of chemoreceptors varies greatly between bacteria occupying various ecological niches. Our understanding of the relationship between lifestyle and chemoreceptor function is limited and the discovery of a chemoreceptor family in plant-associated bacteria that primarily responds to an important plant signal molecule is a significant advancement, allowing for further studies to determine its physiological relevance. The lack of knowledge about signals recognized by bacterial receptors is currently a major challenge in microbiology. This study illustrates the potential of combining experimental ligand screening with computational ligand prediction to identify signals recognized by uncharacterized receptors.

microbiology↗

bioRxiv2024

The new SH3_T domain increases the structural and functional variability among SH3b-like CBDs from staphylococcal phage endolysins

Endolysins, proteins encoded by phages to lyse their hosts and release their progeny, have evolved to adapt to the structural features of each host. The endolysins from Staphylococcus-infecting phages typically feature complex architectures with two enzymatically active domains (EADs) and one cell wall-binding domain (CBD) belonging to the bacterial SH3 (SH3b) superfamily. This study focuses on three SH3b-like CBDs from exemplary staphylococcal phage endolysins (LysRODI, LysC1C, and LysIPLA5) that were structurally and functionally characterized. While RODI_CBD and C1C_CBD were assigned to the well-known SH3_5 family, a new family, SH3b_T, was identified using the CBD from LysIPLA5 as a model. GFP-fused CBDs were created to assess their differential binding to a collection of staphylococcal strains. IPLA5_CBD showed enhanced binding to Staphylococcus epidermidis, while RODI_CBD and C1C_CBD exhibited distinct binding profiles, with RODI_CBD targeting Staphylococcus aureus specifically and C1C_CBD displaying broad binding. Sequence comparisons suggested that a few differences in key amino acids could be responsible for the latter binding difference. The CBDs modulated the activity spectrum of synthetic EAD-CBD combinations in accordance with the previous binding profiles, but in a manner that was also dependent on the EAD present in the fusion protein. These results serve as a context for the diversity and versatility of SH3b domains in staphylococcal endolysins, providing insights on how (i) the CBDs from this superfamily have diverged to adapt to diverse bacterial ligands in spite of sharing a common fold; and (ii) the evolution of specificity relies on the EAD-CBD combination rather than solely the CBD. IMPORTANCEClinical management of bacterial infections is nowadays compromised by the rise in antimicrobial resistance. The development of new antimicrobial therapies with diverse modes of action is therefore of pivotal importance to complement the current standard of care. Phage endolysins are a new class of antibacterial agents based on rapid peptidoglycan degradation. The natural reservoir of phage endolysins offers a practically infinite diversity. This works reveals a broadly spread but still unknown phage endolysin domain targeting staphylococci while providing structural-functional insights that are paramount to understand the evolution of endolysins and how they can be applied as an antimicrobial.

molecular biology↗

bioRxiv2023

HOMOCYSTEINE POTENTIATES AMYLOID β-INDUCED CEREBRAL ENDOTHELIAL CELL APOPTOSIS, BLOOD BRAIN BARRIER DYSFUNCTION AND ANGIOGENIC IMPAIRMENT

Cerebrovascular dysfunction has been implicated as a major contributor to Alzheimers Disease (AD) pathology, with cerebral endothelial cell (cEC) stress promoting ischemia, cerebral-blood flow impairments and blood-brain barrier (BBB) permeability. Recent evidence suggests that cardiovascular (CV)/cerebrovascular risk factors, including hyperhomocysteinemia (Hhcy), exacerbate AD pathology and risk. Yet, the underlying molecular mechanisms for this interaction remain unclear. Our lab has demonstrated that amyloid beta 40 (A{beta}40) species, and particularly A{beta}40-E22Q (vasculotropic Dutch mutant), promote death receptor 4 and 5 (DR4/DR5)-mediated apoptosis in human cECs, barrier permeability and angiogenic impairment. Previous studies show that Hhcy also induces EC dysfunction, but it remains unknown whether A{beta} and homocysteine function through common molecular mechanisms. We tested the hypotheses that Hhcy exacerbates A{beta}-induced cEC DR4/5-mediated apoptosis, barrier dysfunction, and angiogenesis defects. This study was the first to demonstrate that Hhcy specifically potentiates A{beta}40-E22Q-mediated activation of the DR4/5-mediated extrinsic apoptotic pathway in cECs, including DR4/5 expression, caspase 8/9/3 activation, cytochrome-c release and DNA fragmentation. Additionally, we revealed that Hhcy intensifies the deregulation of the same cEC junction proteins mediated by A{beta}, precipitating BBB permeability. Furthermore, Hhcy and A{beta}40-E22Q, impairing VEGF-A/VEGFR2 signaling and VEGFR2 endosomal trafficking, additively decrease cEC angiogenic capabilities. Overall, these results show that the presence of the CV risk factor Hhcy exacerbates A{beta}-induced cEC apoptosis, barrier dysfunction, and angiogenic impairment. This study reveals specific mechanisms through which amyloidosis and Hhcy jointly operate to produce brain EC dysfunction and death, highlighting new potential molecular targets against vascular pathology in comorbid AD/CAA and Hhcy conditions.

neuroscience↗

bioRxiv2022

Monomodular Pseudomonas aeruginosa phage JG004 lysozyme (Pae87) contains a bacterial surface-active antimicrobial peptide-like region and a possible substrate-binding subdomain

Phage lysins are a source of novel antimicrobials to tackle the bacterial antibiotic resistance crisis. The engineering of phage lysins is being explored as a game-changing technological strategy for introducing a more precise approach in the way we apply antimicrobial therapy. Such engineering efforts will benefit from a better understanding of lysin structure and function. In this work, the antimicrobial activity of the endolysin from Pseudomonas aeruginosa phage JG004, termed Pae87, has been characterized. This lysin had been previously identified as an antimicrobial agent candidate, able to interact with the Gram-negative surface and disrupt it. Further evidence is hereby provided on this matter, based on a structural and biochemical study. A high-resolution crystal structure of Pae87 complexed with a peptidoglycan fragment showed a separate substrate-binding region within the catalytic domain, 18 [A] away from the catalytic site and located at the opposite side of the lysin molecule. This substrate binding region was conserved among phylogenetically related lysins lacking an additional cell wall binding domain, but not among those containing such a module. Two glutamic acids were identified as relevant for the peptidoglycan degradation activity, although Pae87 antimicrobial activity was seemingly unrelated to it. In contrast, an antimicrobial peptide-like region within Pae87 C-terminus, named P87, was found to be able to actively disturb the outer membrane and have antibacterial activity by itself. Therefore, we propose an antimicrobial mechanism for Pae87 in which the P87 peptide plays the role of binding to the outer membrane and disrupting the cell wall function, either with or without the participation of Pae87 catalytic activity. SynopsisThe structure of the monomodular Pseudomonas aeruginosa bacteriophage JG004 lysin Pae87 is presented and investigated in relationship with its function repurposed as an antimicrobial agent. The structure with its peptidoglycan ligand revealed a possible cell wall binding region. A C-terminal antimicrobial peptide-like region is shown to be important for disrupting the bacterial cell wall.

molecular biology↗

bioRxiv2021

Sequence-function relationships in phage-encoded bacterial cell wall lytic enzymes and their implications for phage-derived products design

Phage (endo)lysins are thought to be a viable alternative to usual antibiotic chemotherapy to fight resistant bacterial infections. However, a landscape view of lysins structure and properties regarding their function, with an applied focus, is somewhat lacking. Current literature suggests that specific features typical of lysins from phages infecting Gram-negative bacteria (G-) (higher net charge, amphipathic helices) are responsible for an improved interaction with G- envelope. Such antimicrobial peptide (AMP)-like elements are also of interest for antimicrobial molecules design. Thus, this study aims to provide an updated view on the primary structural landscape of phage lysins to clarify the evolutionary importance of several sequence-predicted properties, particularly for the interaction with the G- surface. A database of 2,182 lysin sequences was compiled, containing relevant information such as domain architectures, data on the phages host bacteria and sequence-predicted physicochemical properties. Based on such classifiers, an investigation on the differential appearance of certain features was conducted. Such analyses revealed different lysin architectural variants that are preferably found in phages infecting certain bacterial hosts. Particularly, some physicochemical properties (higher net charge, hydrophobicity, hydrophobic moment and aliphatic index) were associated to G- phage lysins, appearing specifically at their C-terminal end. Evidences on the remarkable genetic specialization of lysins regarding the features of the bacterial hosts have been provided, specifically supporting the nowadays common hypothesis that lysins from G- usually contain AMP-like regions. IMPORTANCEPhage-encoded lytic enzymes, also called lysins, are one of the most promising alternatives to common antibiotics. The lysins potential as novel antimicrobials to tackle antibiotic-resistant bacteria not only arises from features such as a lower chance to provoke resistance, but also from their versatility as synthetic biology parts. Functional modules derived from lysins are currently being used for the design of novel antimicrobials with desired properties. This study provides a view of the lysins diversity landscape by examining a set of phage lysin genes. This way, we have uncovered the fundamental differences between the lysins from phages that infect bacteria with different superficial architectures, and, thus, also the reach of their specialization regarding cell wall structures. These results provide clarity and evidences to sustain some of the common hypothesis in current literature, as well as make available an updated and characterized database of lysins sequences for further developments.

microbiology↗