Biology subjects

Perez-Pulido, A. J.

Publications and source records attributed to Perez-Pulido, A. J..

10 recordsLinked to original sources

bioRxiv2026

Evolutionary expansion of the bacterial ompW gene family enables functional diversification during host adaptation

The outer membrane protein W (OmpW) family comprises minor porins essential for environmental adaptation and host interactions in Gram-negative bacteria. However, ompW is conventionally treated as a ubiquitous single-copy gene, masking its true evolutionary complexity. Through comprehensive phylogenetic analysis across thousands of reference genomes, we reveal that ompW is present in nearly half of Gram-negative lineages, exhibiting a patchy distribution governed by a birth-and-death model of evolution with recurrent duplications and horizontal transfer. Notably, copy number expansions concentrate in lineages associated with eukaryotic hosts, linking paralog accumulation to niche adaptation. Experimental validation in Acinetobacter baumannii reveals four distinct loci that maintain high structural conservation despite sequence divergence, yet undergo profound functional specialization spanning from baseline physiological maintenance to modulating host colonization and persistence in vitro and in vivo. These findings dismantle the single-copy paradigm, demonstrating that widespread gene family expansion and paralog diversification drive the phenotypic plasticity required for bacteria to colonize hostile host environments.

microbiology↗

bioRxiv2026

Scaling Functional Annotation Across Proteomes, Pangenomes and Metagenomes with Sma3s v3

High-throughput sequencing has generated protein datasets whose scale increasingly exceeds the practical limits of conventional functional annotation workflows. We present Sma3s v3, a scalable reimplementation of the Sma3s three-step annotation strategy, which combines transfer from highly similar homologs, orthology-based inference, and functional enrichment among homologous proteins. Sma3s v3 replaces BLAST-based searches with MMseqs2 and introduces parallel processing, reusable SQLite caches, taxonomic filtering, and traceable outputs that retain the evidence underlying each assignment. We evaluated the method on a Vibrio cholerae pangenome comprising 50,415 gene clusters from 11,295 quality-filtered genomes and on a metagenomic catalogue containing 843,935 proteins. After excluding non-informative assignments, Sma3s v3 annotated 30,662 pangenome clusters (60.8%), comparable to InterProScan (60.2%) and exceeding eggNOG-mapper (41.4%), while providing 5,747 annotations not recovered by either comparator. Gene Ontology comparisons showed broad semantic agreement between methods, with Sma3s v3 frequently contributing more non-redundant information in Molecular Function and Biological Process. Within the pangenome, annotation coverage reached 97.1% for core clusters and approximately 59% for accessory and unique clusters. Exact protein matches to non-Vibrio genera identified 1,838 candidate horizontally transferred clusters enriched in genetic mobility, antimicrobial resistance, and metal tolerance functions. In the metagenomic catalogue, Sma3s v3 annotated 728,014 proteins (86.3%), compared with 616,895 (73.1%) using InterProScan 2026, and recovered approximately 20,000 unique functional terms. These results establish Sma3s v3 as a scalable and interpretable tool for functional annotation and re-annotation of proteomes, pangenomes, and metagenomic protein catalogues.

bioinformatics↗

bioRxiv2025

Discovery of a stress-response integrative and conjugative element from Sphingopyxis granuli TFA broadly conserved across Sphingomonadales and Rhizobiales

Horizontal gene transfer is a pivotal element in the evolution of microbes, enabling them to acquire novel genes and phenotypes. Integrative and conjugative elements (ICEs) are a type of mobile genetic element that can integrate into the host genome and propagate during chromosome replication and cell division. The induction of ICE gene expression results in the excision of the ICE gene, the production of conserved conjugation machinery, a Type IV Secretion System, and the potential for DNA transfer to appropriate receptors. It has been observed that ICEs frequently contain cargo genes that do not typically align with the ICE life cycle. These genes often result in the manifestation of phenotypes that are of particular interest. The bacterium Sphingopyxis granuli strain TFA is being studied for its ability to degrade the contaminant tetralin present in crude oils. Genomic analysis identified eight possible integrative mobile elements in S. granuli TFA. Most of these regions exhibited a distribution pattern that was restricted to the species, and they lacked some functional modules that are characteristic of a complete ICE. This finding suggests the presence of degenerate structures or limited mobilization capacity. However, only two of the detected elements exhibited the capacity to retain all the modules necessary for transfer, integration, and maintenance. These elements also contained a cargo module that included genes associated with lipid metabolic pathways and resistance mechanisms. Among them, ICE3 was distinguished as the sole complete functional ICE that was also present in other species. Transcriptomic analysis under multiple stress conditions revealed differential and consistent activation of ICE3 genes, demonstrating their direct contribution to bacterial resilience and suggesting a key adaptive role in response to adverse environmental changes.

bioinformatics↗

bioRxiv2024

The defensome of Acinetobacter baumannii reveals two genome groups according to their innate defense systems and phage profile

Phages are guilty of killing daily almost half of bacterial cells, while bacteria have developed defense mechanisms that number in the dozens. Individual defense systems are gained and lost by genomes of the same species, depending on their fitness advantage. Thus, some genomes have a certain combination of defense systems, while other genomes act as a reservoir for the rest of the systems, thus constituting the so-called pan-immune system of the species. Here we have analyzed thousands of genomes of the bacterium Acinetobacter baumannii, an opportunistic pathogen of humans of great clinical concern, and we have found 81 different defense systems heterogeneously distributed. By analyzing how these systems combine, we have found that more than half of the genomes lack the universal DNA-methylating restriction-modification systems (R-M) and harbor an alternative innate SspBCDE system that performs a DNA phosphorothioate modification. In addition, the adaptive CRISPR-Cas systems could act synergistically to the R-M systems, based on their frequency of co-appearance. The presence of one or the other innate system could modulate the evolution of the genomes of this species, causing them to present a different profile of phages integrated into the bacterial genome. We have also observed that the presence of many defense systems is associated with the presence of a higher number of prophages, which could be due to the fact that the prophage carries the system, or that the bacterium would not need these systems in environments where the phage is absent.

microbiology↗

bioRxiv2024

Hydroxyurea induces ER stress and cytoplasmic protein aggregation

The endoplasmic reticulum (ER) lumen provides the proper redox environment for disulfide bond formation, which is required for the appropriate folding of proteins that enter the secretory pathway and constitute membranes. Defective protein folding in the ER activates proteostatic mechanisms that are now beginning to be elucidated. Here, we show that hydroxyurea (HU) causes ER stress and triggers a transient perinuclear ER expansion, which leads to the clustering of nuclear pore complexes. This striking phenotype is mimicked by diamide (DIA), a specific thiol stress inductor, and prevented or rapidly reverted by dithiothreitol, a dithiol-reducing agent, suggesting that ER expansion is caused by disulfide stress. ER expansion induced by HU or DIA depends on glutathione (GSH), is Ire1-independent, and is associated with a unique transcriptome program that differs from the canonical unfolding protein response (UPR). The ER luminal expansion accumulates Hsp70 Bip1 chaperone, and it evolves parallel with the appearance of cytoplasmic protein aggregates containing heat stress proteins (HSPs), indicating that both HU and DIA are impinging on protein folding. Thus, our data reveal that HU induces disulfide stress that impinges on protein folding in the cytoplasm and ER.

cell biology↗

bioRxiv2023

A biological warfare between two bacterial viruses in a particular genomic region using a CRISPR-Cas system as a weapon

CRISPR-Cas systems are acquired immunity systems of bacteria and archaea that prevent infection by phages and other mobile genetic elements. It is currently known that this defense system has also been co-opted by viruses. These viruses could use CRISPR-Cas systems to compete against other rival viruses. We have discovered a virus in the bacterium Acinetobacter baumannii that incorporates a CRISPR-Cas system into an integration hotspot of the host genome. Once integrated, this could prevent the infection of the most frequent viruses in this bacterial species, especially one that competes with the CRISPR-Cas system itself for the same integration site. This latter virus is prevalent in strains of the species belonging to the so-called Global Clone 2, which causes the most frequent outbreaks worldwide. Knowledge of this new viral warfare using CRISPR-Cas systems, known to limit the entry of antibiotic resistance genes into bacteria, could be useful in the fight against the infections they cause. But it would also shed light on the way in which these defense systems expand in bacteria.

bioinformatics↗

bioRxiv2022

An outstanding bacterial membranome requires CRISPR-Cas systems to avoid the intervention of phages

Antimicrobial resistance is widely recognized as a serious global public health problem. To combat this threat, a thorough understanding of bacterial genomes is necessary. The current wide availability of bacterial genomes provides us with an in-depth understanding of the great variability of dispensable genes and their relationship with antimicrobials. Some of these accessory genes are those involved in CRISPR-Cas systems, which are acquired immunity systems that are present in part of bacterial genomes. They prevent viral infections through small DNA fragments called spacers. But the vast majority of these spacers have not yet been associated with the virus they recognize, and this has been named CRISPR dark matter. By analyzing the spacers of tens of thousands of genomes from six bacterial species highly resistant to antibiotics, we have been able to reduce the CRISPR dark matter from 80-90% to as low as 15% in some of the species. In addition, we have observed that, when a genome presents CRISPR-Cas systems, this is accompanied by particular collections of membrane proteins. Our results suggest that when a bacterium presents membrane proteins that make it compete better in its environment, and these proteins are in turn receptors for specific phages, it would be forced to acquire CRISPR-Cas immunity systems to avoid infection by these phages.

bioinformatics↗

bioRxiv2021

Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes

The requirement for Cas nucleases to recognize a specific PAM is a major restriction for genome editing. SpCas9 variants SpG and SpRY, recognizing NGN and NRN PAM, respectively, have contributed to increase the number of editable genomic sites in cell cultures and plants. However, their use has not been demonstrated in animals. We have characterized and optimized the activity of SpG and SpRY in zebrafish and C. elegans. Delivered as mRNA-gRNA or ribonucleoprotein (RNP) complexes, SpG and SpRY were able to induce mutations in vivo, albeit at a lower rate than SpCas9 in equivalent formulations. This lower activity was overcome by optimizing mRNA-gRNA or RNP concentration, leading to efficient mutagenesis at regions inaccessible to SpCas9. We also found that the CRISPRscan algorithm can predict SpG and SpRY activity in vivo. Finally, we applied SpG and SpRY to generate knock-ins by homology-directed repair. Altogether, our results expand the CRISPR-Cas targeting genomic landscape in animals.

genetics↗

bioRxiv2021

L-arginine ameliorates defective autophagy in GM2 gangliosidoses by mTOR modulation

Tay-Sachs and Sandhoff diseases (GM2 gangliosidosis) are autosomal recessive disorders of lysosomal function that cause fatal and progressive neurodegeneration in infants and young children. Impaired hydrolysis catalysed by {beta}-hexosaminidase A (HexA) leads to the accumulation of its specific substrate, GM2 ganglioside, in neuronal lysosomes. Despite the development of a florid storage phenotype, the role of autophagy and its regulation by the mammalian target of rapamycin (mTOR) has yet to be explored in the neuropathogenesis. Accordingly, we investigated the effects on autophagy and lysosomal integrity using skin fibroblasts obtained from patients with Tay-Sachs and Sandhoff diseases. Pathological autophagosomes, with enhanced expression of the p62/SQSTM1 protein, suggested impaired autophagic flux, an abnormality confirmed by electron microscopy and biochemical studies revealing the accelerated release of mature cathepsins and HexA into the cytosol, indicating increased lysosomal permeability. GM2 fibroblasts showed inappropriately diminished mTOR signalling with reduced basal mTOR activity. Accordingly, provision of a positive nutrient signal by L-arginine supplementation partially restored mTOR activity and ameliorated the cytopathological abnormalities - and immediately suggests an avenue for therapeutic exploration in this cruel disease. We also contend that the expression of autophagy/lysosome/mTOR-associated molecules may prove useful peripheral biomarkers for facile monitoring of systemic treatment of GM2 gangliosidosis and neurodegenerative disorders that affect the lysosomal function and disrupt autophagy

cell biology↗

bioRxiv2020

Serial co-expression analysis of host factors from SARS-CoV viruses highly converges with former high-throughput screenings and proposes key regulators and co-option of cellular pathways

The current genomics era is bringing an unprecedented growth in the amount of gene expression data, only comparable to the exponential growth of sequences in databases during the last decades. This data now allows the design of secondary analyses that take advantage of this information to create new knowledge through specific computational approaches. One of these feasible analyses is the evaluation of the expression level for a gene through a series of different conditions or cell types. Based on this idea, we have developed ASACO, Automatic and Serial Analysis of CO-expression, which performs expression profiles for a given gene along hundreds of normalized and heterogeneous transcriptomics experiments and discover other genes that show either a similar or an inverse behavior. It might help to discover co-regulated genes, and even common transcriptional regulators in any biological model, including human diseases or microbial infections. The present SARS-CoV-2 pandemic is an opportunity to test this novel approach due to the wealth of data that is being generated, which could be used for validating results. In addition, new cell mechanisms identified could become new therapeutic targets. Thus, we have identified 35 host factors in the literature putatively involved in the infectious cycle of SARS-CoV and/or SARS-CoV-2 and searched for genes tightly co-expressed with them. We have found around 1900 co-expressed genes whose assigned functions are strongly related to viral cycles. Moreover, this set of genes heavily overlap with those identified by former laboratory high-throughput screenings (with p-value near 0). Some of these genes aim to cellular structures such as the stress granules, which could be essential for the virus replication and thereby could constitute potential targets in the current fight against the virus. Additionally, our results reveal a series of common transcription regulators, involved in immune and inflammatory responses, that might be key virus targets to induce the coordinated expression of SARS-CoV-2 host factors. All of this proves that ASACO can discover gene co-regulation networks with potential for proposing new genes, pathways and regulators participating in particular biological systems. HighlightsO_LIASACO identifies regulatory associations of genes using public transcriptomics data. C_LIO_LIASACO highlights new cell functions likely involved in the infection of coronavirus. C_LIO_LIComparison with high-throughput screenings validates candidates proposed by ASACO. C_LIO_LIGenes co-expressed with hosts genes used by SARS-CoV-2 are related to stress granules. C_LI

bioinformatics↗