Biology subjects

de Souza, R. F.

Publications and source records attributed to de Souza, R. F..

13 recordsLinked to original sources

bioRxiv2026

An RNase Toxin Hijacks Elongation Factor-Tu to Cleave the Ribosomal Sarcin-Ricin Loop

Ribosome-targeting toxins typically recognize and attack their substrates directly. Here, we provide the functional characterization of SRLase1, a widespread member of a recently identified RNase superfamily, and show that it cleaves the sarcin-ricin loop (SRL) of 23S rRNA. Unlike previously described ribosome-targeting toxins, SRLase1 requires the host elongation factor Tu (EF-Tu) for activity. EF-Tu normally engages the ribosome as part of the aminoacyl-tRNA delivery cycle, but SRLase1 instead exploits this factor through a mechanism incompatible with canonical ternary-complex formation. This host-factor-assisted mechanism reveals a distinct strategy for translation inhibition, in which a toxin repurposes a conserved component of the elongation machinery to access a vulnerable ribosomal target. Homologs of SRLase1 are widespread across bacterial phyla and are frequently encoded in conflict-associated genomic loci. Together, these findings define a widespread family of SRL-targeting toxins, expand the mechanistic repertoire of ribosomal antibacterial toxins, and demonstrate how conserved components of the host translation machinery can be co-opted to promote toxin activity.

microbiology↗

bioRxiv2026

A structure-guided classification framework reveals the diversity and catalytic architecture of BECR ribonuclease

Microorganisms across all domains of life engage in molecular conflict, deploying toxins to inhibit competitors or respond to biological threats. Among these, ribonuclease toxins are particularly widespread and diverse. A substantial fraction is associated with the BECR fold, a compact /{beta} architecture that supports RNase activity despite extensive divergence. Although several canonical members are well characterized, many BECR-fold proteins remain difficult to identify because of low sequence similarity, variation in catalytic residues, and structural elaborations that obscure evolutionary relationships. The growing availability of high-confidence protein structure predictions provides an opportunity to reassess this deeply divergent protein landscape. Here, we integrate iterative profile-HMM searches, profile-similarity networks, structural analyses, active-site mapping, and genomic context to examine BECR proteins across the tree of life. Our analysis resolves an expanded BECR-fold landscape comprising canonical BECR and BECR-like superfamilies, refines the organization of canonical BECR proteins and identifies previously unrecognized families. We further validate BECR-Tox2 as a toxin neutralized by a cognate immunity protein and show that its homologs occur in both Menshen-like anti-phage systems and polymorphic toxin loci. Together, these findings expand and clarify the BECR-fold landscape and provide a framework for identifying and interpreting highly divergent proteins of this fold.

microbiology↗

bioRxiv2026

ppigFinder: an integrated desktop application for bacterial genome annotation and AlphaFold 3 based protein protein interaction screening

Motivation. AlphaFold-based structure prediction has transformed structural biology by enabling accurate protein modelling and providing a powerful framework for inferring protein-protein interactions (PPIs). However, discovering candidate PPIs directly from genome sequences remains a fragmented and largely trial-and-error process, typically requiring separate tools for open reading frame (ORF) prediction, functional annotation, candidate selection, iterative testing of potential partners, manual preparation of individual structural-prediction jobs, and downstream interpretation of confidence metrics. Results. We present Protein-Protein Interaction Genomic Finder (ppigFinder), a standalone, cross-platform desktop application that integrates these steps into a project-oriented graphical workflow for genome-based PPI discovery from nucleotide sequence data. ppigFinder combines ORF prediction, functional annotation, genomic-neighbourhood inspection, AlphaFold 3 job generation, remote job submission, and structural-confidence analysis within a single environment. As a proof of concept, we performed a VirD4-centered AlphaFold 3 interactome screen in Xanthomonas citri pv. citri strain 306, modelling VirD4 (ORF2601) against all 4,303 predicted chromosomal ORFs. Ranking by the minimum interchain predicted aligned error (PAE_min) placed all 14 XVIPCD-containing effector candidates within the top 1% of predictions, with the six top-ranked models corresponding to XVIP candidates. The screen also recovered an XVIPCD-containing protein absent from the reference genome annotation and identified high-confidence candidates predicted to bind VirD4 at a surface opposite to the XVIPCD-binding site.

bioinformatics↗

bioRxiv2025

Pharmacokinetic-Pharmacodynamic Trade-offs in SARS-CoV-2 Main Protease Inhibitors Unveiled through Machine Learning and Molecular Dynamics Simulations

The SARS-CoV-2 main protease (Mpro) is a validated therapeutic target for inhibiting viral replication. Despite the screening of over 55,000 compounds, few candidates have advanced clinically, underscoring the difficulty in optimizing both target affinity and drug-like properties. Thus, developing effective Mpro inhibitors requires balancing high-affinity binding with favorable pharmacokinetic (PK) properties, such as solubility and permeability. To address this challenge, we integrated machine learning (ML) and molecular dynamics (MD) simulations to investigate the trade-offs between pharmacodynamic (PD) and PK properties in Mpro inhibitor design. We developed ML models to classify Mpro inhibitors based on experimental IC50 data, combining molecular descriptors with structural insights from MD simulations. Our Support Vector Machine (SVM) model achieved strong performance (training accuracy = 0.84, ROC AUC = 0.91; test accuracy = 0.79, ROC AUC = 0.86), while our logistic regression model (training accuracy = 0.78, ROC AUC = 0.85; test accuracy = 0.76, ROC AUC = 0.83) identified key molecular features influencing activity, including quantitative estimation of drug-likeness (QED), Log P and molecular weight (ExactMolWt). Notably, PK descriptors often exhibited opposing trends to binding affinity: hydrophilic features enhanced binding affinity but compromised PK properties, whereas hydrogen bonding, hydrophobic and{pi} -{pi} interactions in subsites S2 and S3/S4 are fundamental for binding affinity. Our findings highlight the need for a balanced approach in Mpro inhibitor design, strategically targeting these subsites may balance PD and PK properties. This study provides a computational framework for rational Mpro inhibitor discovery, combining ML and MD to investigate the complex interplay between enzyme inhibition and drug-likeness. These insights may guide in hit-to-lead optimization of the novel next-generation Mpro inhibitors of the SARS-CoV-2 with preclinical and clinical potential.

pharmacology and toxicology↗

bioRxiv2024

Exploring the Educational Potential of Instagram in the New Generations: A Study on the Impact of "Adopt a Bacterium"

In 2013, the "Adopt a Bacterium" project was created, which originally proposed the use of the social network Facebook(R) as an educational tool to engage students in learning microbiology through personalized posts. However, the generational alignment dynamic presents contemporary challenges for educators. Given the relevance and success of the project in recent years, the use of an alternative social network is sought for the application of the methodology. In the present study, we describe the use of the social network Instagram as a tool to understand the impacts of the "Adopt a Bacterium" project on microbiology learning among new generations of students. To verify the effectiveness of the methodology, the following steps were taken: i) voluntary and anonymous questionnaires with an open-ended question about knowledge of the adopted bacterium were applied before the start of the project (Q1), at the end of the project (Q2), and five months after the end (Q3), to Biomedical Sciences students in the years 2020, 2021, and 2022; and ii) a project evaluation questionnaire was administered to students. The data were analyzed using thematic categorization, calculation of the richness of students discourse through the Shannon Diversity Index, and the creation of word clouds to visualize student suggestions, as well as the use of Python language tool to demonstrate citations of specific themes, comparing Facebook and Instagram as Virtual Learning Enviroments. The results revealed an improvement in student learning, as demonstrated by the increase in the frequency of content categories related to bacteriology and the richness of discourse, indicated by higher Shannon Diversity Index values in Q2 and Q3 compared to Q1. In conclusion, the results demonstrate that the methodology of the "Adopt a Bacterium" can be applied in different virtual environments, such as the social network Instagram(R) resulting in increased learning and student interest in Microbiology.

microbiology↗

bioRxiv2024

Genome-directed study uncovers a family of phospholipases and highlights the diversity of Salmonella T6SS effectors.

Bacterial warfare is a common and ancient phenomenon in nature, where bacterial species use strategies to inhibit the growth or kill competitors. This involves the production and deployment of antibacterial toxins that disrupt essential cellular processes in target cells. The continuous arms race in which bacteria acquire new toxin and immunity proteins to promote increased adaptation to their environment is responsible for the diversification of this toxin repertoire. Here, we deployed in-silico strategies to analyze 10,000 genomes and identify effectors secreted via the type VI secretion system of Salmonella. We identified 128 candidates, which are widespread in a vast array of Salmonella serovars and other bacterial species. Tox-Act1 is among the most frequent candidates and was selected for in-depth characterization. Tox-Act1 contains a permuted NlpC/P60 papain-like catalytic core characteristic of lipid-targeting members rather than the typical peptidases or amidases. Evolutionary analysis revealed the relationship of Tox-Act1 with acyltransferases. Biochemical assays with purified toxin and lipidomics of intoxicated cells showed that Tox-Act1 exhibits phospholipase activity, cleaving off acyl groups from phosphatidylglycerol and phosphatidylethanolamine. In addition, we demonstrate that Tox-Act1 is secreted in a T6SS-dependent manner and provide a competitive advantage during colonization of the gut of infected mice. This work broadens our understanding of toxin domains and provides the first direct characterization of a lipid-targeting NlpC/P60 domain in biological conflicts.

microbiology↗

bioRxiv2022

Molecular dynamics simulations of the Spike trimeric ectodomain of the SARS-CoV-2 Omicron variant: structural relationships with infectivity, evasion to immune system and transmissibility

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant is replacing Delta, the most prevalent variant worldwide from the beginning of 2021 until early 2022. The Omicron variant is highly transmissible and responsible for a new worldwide COVID-19 wave. Herein, we calculated molecular dynamics simulations of the SARS-CoV-2 trimeric spike protein of Wuhan-Hu-1 strain (wild type, WT) and the Omicron variant of concern. Structural analyses reveal that the SpikeOmicron presents more conformational flexibility than SpikeWT, mainly in the N-terminal domain (NTD) and receptor-binding domain (RBD). Such flexibility results in a broader spectrum of different conformations for SpikeOmicron, whereby the RBD can more easily visit an up-conformational state. We reported how the mutations in this variant may influence the intra- and inter-protomer contacts caused by conformational flexibility of the NTD. Based on our analysis, we suggest that the differences in conformational flexibility between SpikeOmicron and SpikeWT may explain the observed gains in infectivity, immune system evasion and transmissibility in this novel variant. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biophysics↗

bioRxiv2021

Antibacterial T6SS effectors with a VRR-Nuc domain induce target cell death via DNA Double-Strand Breaks

The T6SS (Type VI secretion System) secretes antibacterial effectors into target competitors. Salmonella spp. encode five phylogenetically distinct T6SSs. Here we characterize the function of the SPI-22 T6SS of S. bongori, showing that it has antibacterial activity. We identify a group of antibacterial T6SS effectors (TseV1-4) containing an N-terminal PAAR-like domain and a C-terminal VRR-Nuc domain encoded next to cognate immunity proteins that contain the DUF3396 domain (TsiV1-4). TseV2 and TseV3 are toxic when expressed in Escherichia coli and bacterial competition assays confirm that TseV2 and TseV3 are secreted by the SPI-22 T6SS. Phylogenetic analysis reveals that TseV1-4 are evolutionarily related to enzymes involved in DNA repair. TseV2 and TseV3 maintained the ability to bind DNA, but instead cause specific DNA double-strand breaks and induce the SOS response in target cells. The crystal structure of the TseV3:TsiV3 complex reveals that the immunity protein likely blocks the effector interaction with the DNA substrate. These results expand our knowledge on the function of Salmonella pathogenicity islands, the evolution of toxins used in biological conflicts, and the endogenous mechanism regulating the activity of these toxins.

microbiology↗

bioRxiv2021

Molecular dynamics analysis of fast-spreading severe acute respiratory syndrome coronavirus 2 variants and their effects in the interaction with human angiotensin-converting enzyme 2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is evolving with mutations in the Spike protein, especially in the receptor-binding domain (RBD). The failure of public health measures to contain the spread of the disease in many countries has given rise to novel viral variants with increased transmissibility. However, key questions about how quickly the variants can spread and whether they can cause a more severe disease remain unclear. Herein, we performed a structural investigation using molecular dynamics simulations and determined dissociation constant (KD) values using surface plasmon resonance (SPR) assays of three fastspreading SARS-CoV-2 variants, Alpha, Beta and Gamma ones, as well as genetic factors in the host cells that may be related to the viral infection. Our results suggest that the SARS-CoV-2 variants facilitate their entry into the host cell by moderately increased binding affinities to the human ACE2 receptor, different torsions in hACE2 mediated by RBD variants, and an increased Spike exposure time to proteolytic enzymes. We also found that other host cell aspects, such as gene and isoform expression of key genes for the infection (ACE2, FURIN and TMPRSS2), may have few contributions to the SARS-CoV-2 variants infectivity. In summary, we concluded that a combination of viral and host cell factors allows SARS-CoV-2 variants to increase their abilities to spread faster than wild-type. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

molecular biology↗

bioRxiv2021

Comparative phylogenomic analysis reveals evolutionary genomic changes and novel toxin families in endophytic Liberibacter pathogens

Liberibacter pathogens are the causative agents of several severe crop diseases worldwide, including citrus Huanglongbing and potato Zebra Chip. These bacteria are endophytic and non-culturable, which makes experimental approaches challenging and highlights the need for bioinformatic analysis in advancing our understanding about Liberibacter pathogenesis. Here, we performed an in-depth comparative phylogenomic analysis of the Liberibacter pathogens and their free-living, nonpathogenic, ancestral species, aiming to identify the major genomic changes and determinants associated with their evolutionary transitions in living habitats and pathogenicity. We found that prophage loci represent the most variable regions among Liberibacter genomes. Using gene neighborhood analysis and phylogenetic classification, we systematically recovered, annotated, and classified all prophage loci into four types, including one previously unrecognized group. We showed that these prophages originated through independent gene transfers at different evolutionary stages of Liberibacter and only the SC-type prophage was associated with the emergence of the pathogens. Using ortholog clustering, we vigorously identified two additional sets of genomic genes, which were either lost or gained in the ancestor of the pathogens. Consistent with the habitat change, the lost genes were enriched for biosynthesis of cellular building blocks. Importantly, among the gained genes, we uncovered several previously unrecognized toxins, including a novel class of polymorphic toxins, a YdjM phospholipase toxin, and a secreted EEP protein. Our results substantially extend the knowledge on the evolutionary events and potential determinants leading to the emergence of endophytic, pathogenic Liberibacter species and will facilitate the design of functional experiments and the development of new detection and blockage methods of these pathogens. ImportanceLiberibacter pathogens are associated with several severe crop diseases, including citrus Huanglongbing, the most destructive disease to the citrus industry. Currently, no effective cure or treatments are available, and no resistant citrus variety has been found. The fact that these obligate endophytic pathogens are not culturable has made it extremely challenging to experimentally uncover from the whole genome the genes/proteins important to Liberibacter pathogenesis. Further, earlier bioinformatics studies failed to identify the key genomic determinants, such as toxins and effector proteins, that underlie the pathogenicity of the bacteria. In this study, an in-depth comparative genomic analysis of Liberibacter pathogens together with their ancestral non-pathogenic species identified the prophage loci and several novel toxins that are evolutionarily associated with the emergence of the pathogens. These results shed new lights on the disease mechanism of Liberibacter pathogens and will facilitate the development of new detection and blockage methods targeting the toxins.

microbiology↗

bioRxiv2021

Resource sharing by outer membrane vesicles from a citrus pathogen

The causative agent of citrus canker disease, Xanthomonas citri pv. citri, was found to produce copious amounts of outer membrane vesicles (OMVs), frequently forming long membranous tubes under different culture conditions. Lipidomic analysis revealed significant differences in lipid composition between purified vesicles in relation to whole cells. The results suggest an enrichment in saturated cardiolipins and a decrease in unsaturated lipids in the OMV samples, possibly granting them a more rigid structure while allowing their high degree of curvature caused by their small diameters. The vesicles proteome was found to be significantly enriched in TonB-dependent receptors related to the acquisition of different nutrients. These proteins are known to transport siderophores, which were evidenced to be present in purified X. citri OMVs, along with essential metals including iron, zinc, and manganese quantified by elemental analysis. The availability of vesicle-associated nutrients to be incorporated by cells was demonstrated by the use of OMVs as the sole carbon source for bacterial growth. At last, the vesicles also presented esterase and protease activities, which have been associated with virulence in phytopathogens. These evidences point that X. citri cells can use OMVs to share resources within microbial communities, which has potential implications for microbial interactions and plant colonization, affecting their survival and persistence on the host and in the environment. ImportanceThe shedding of outer membrane vesicles appears to be universal in Gram-negative bacteria and effectively constitutes a unique secretion pathway for diverse molecules and proteins. To study their possible functions in the citrus pathogen Xanthomonas citri, purified vesicles from this bacterium were studied by omics and functional approaches. Nutrient transporters were found associated to these structures, which were evidenced to contain siderophores and essential metals. The availability of these nutrients to be incorporated by cells was then demonstrated by showing that purified vesicles can be used as sole carbon sources for microbial growth. Additionally, the samples also presented esterase and protease activities which can contribute to the release of substrates from plant host tissues. These observations help to establish the developing idea of vesicles as shared bacterial resources which can participate in shaping host-associated microbial communities in contrast to other interactions such as bacterial competition.

microbiology↗

bioRxiv2020

A Superfamily of T6SS Antibacterial Effectors Displaying L,D-carboxypeptidase Activity Towards Peptidoglycan

Type VI secretion systems (T6SSs) are contractile nanomachines used by bacteria to inject toxic effectors into competitors. The identity and mechanism of many effectors remain unknown. We characterized a Salmonella SPI-6 T6SS antibacterial effector called Tae5STM (type VI amidase effector 5). Tae5STM is toxic in target-cell periplasm and is neutralized by its cognate immunity protein (Tai5STM). Microscopy analysis revealed that cells expressing the effector stop dividing and lose cell envelope integrity. Bioinformatic analysis uncovered similarities between Tae5STM and the catalytic domain of L,D-transpeptidase. Point mutations on conserved catalytic histidine and cysteine residues abrogated toxicity. Biochemical assays revealed that Tae5STM displays L,D-carboxypeptidase activity, cleaving peptidoglycan tetrapeptides between meso-diaminopimelic acid3 and D-alanine4. Phylogenetic analysis showed that Tae5STM homologs constitutes a new superfamily of T6SS-associated amidase effectors distributed among -, {beta}- and {gamma}-proteobacteria. This work expands our current knowledge about bacterial effectors used in interbacterial competition.

microbiology↗

bioRxiv2019

Global distribution and evolution of Mycobacterium bovis lineages

Mycobacterium bovis is the main causative agent of zoonotic tuberculosis in humans and frequently devastates livestock and wildlife worldwide. Previous studies suggested the existence of genetic groups of M. bovis strains based on limited DNA markers (a.k.a. clonal complexes), and the evolution and ecology of this pathogen has been only marginally explored at the global level. We have screened over 2,600 publicly available M. bovis genomes and newly sequenced two wildlife M. bovis strains, gathering 823 genomes from 21 countries and 21 host-species, including humans, to complete a phylogenomic analyses. We propose the existence of four distinct global lineages of M. bovis (Lb1, Lb2, Lb3 and Lb4) underlying the current disease distribution. These lineages are not fully represented by clonal complexes and are dispersed based on geographic location rather than host species. Our data divergence analysis agreed with previous studies reporting independent archeological data of ancient M. bovis [(South Siberian infected skeletons at [~]2,000 years BP (before present)] and indicates that extant M. bovis originated during the Roman period, subsequently dispersing across the world with the discovery and settlement of the New World and Oceania, directly influenced by trades among countries.

genetics↗