Biology subjects

Iorga, B. I.

Publications and source records attributed to Iorga, B. I..

10 recordsLinked to original sources

bioRxiv2026

Polysaccharide release factors drive lipid-carrier recycling in bacteria

Gram-negative bacteria build their cell envelope by transporting various sugar-based precursors across the inner membrane on the shared lipid carrier undecaprenyl (also known as C55). Because the cellular pool of undecaprenyl is finite and used by multiple competing pathways, it must be efficiently regenerated to sustain envelope assembly. Yet the enzymes that release polysaccharide cargo from the lipid carrier have remained elusive, leaving a major gap in our understanding of bacterial envelope biogenesis. Here we identify three periplasmic enzymes in Escherichia coli - UprA, UprB, and UprC - that hydrolyze the bond linking polysaccharide precursors to undecaprenyl. UprA releases elongating peptidoglycan glycan strands from the inner membrane, catalyzing the long-sought terminal step of peptidoglycan synthesis. UprB cleaves the undecaprenyl-linked initiating unit common to the enterobacterial common antigen and O-antigen pathways, while UprC is the likely release factor for the colanic acid pathway. These enzymes define a widespread Upr-like protein family (formerly DUF218) as polysaccharide release factors, revealing a unifying mechanism by which bacteria coordinate envelope assembly. Because all three Upr-like proteins share a conserved catalytic core, this family represents a potential target for antibiotics that simultaneously disrupt multiple envelope pathways.

microbiology↗

bioRxiv2026

Stress-induced Membrane Insertion at the β-Barrel Assembly Machinery Complex Regulates BepA Metalloprotease Activity

Proteases must be tightly regulated to prevent uncontrolled degradation, yet the mechanisms ensuring such control remain poorly understood. Members of the widespread M48 metalloprotease family are kept inactive by an autoinhibitory plug that blocks catalytic water activation, but how this plug is released was unknown. Here, using genetic, biochemical and cryo-EM approaches, we discover the activation mechanism of BepA, a quality-control protease that preserves outer membrane integrity by surveilling the {beta}-barrel assembly machinery (BAM) in Gram-negative bacteria. Our cryo-EM analysis of BepA engaged with a stalled BAM-substrate assembly complex revealed that a flexible, unstructured 6-lid covering the active site in the latent protein functions as a molecular harpoon, inserting into the outer membrane when a substrate stalls at BAM and thereby docking BepA at the complex. This membrane anchoring promotes displacement of the autoinhibitory plug and unlocks protease activity precisely where and when it is needed. Thus, a dual enzyme activation mechanism is coupled to membrane association under stress, ensuring that BepA remains inactive until properly localized. Our findings reveal how membranes themselves can license protease activation, a principle that may extend beyond M48 metalloproteases.

microbiology↗

bioRxiv2025

Fine-tuning of outer membrane-peptidoglycan tethering by the redox-active lipoprotein LppB from Salmonella enterica

The bacterial envelope is an essential compartment for survival, acting as a protective and permeability barrier. In diderm bacteria, it comprises an inner membrane, a thin peptidoglycan layer, and an outer membrane. A stable outer membrane-peptidoglycan connection is crucial for envelope integrity, preventing outer membrane detachment and enabling the generation of periplasmic turgor that counterbalances cytoplasmic pressure, thereby limiting susceptibility to antibiotics and environmental stressors. In Escherichia coli, the major lipoprotein Lpp (Brauns lipoprotein) serves as the only covalent linker between the outer membrane and peptidoglycan, through its unique C-terminal lysine residue. Homologs of Lpp are widely conserved among Enterobacteriaceae, including Salmonella enterica serovar Typhimurium, which encodes LppA and LppB. While Lpp and LppA function similarly, the role of LppB remains unclear. Using E. coli as a surrogate model, we investigated the structural and functional properties of Salmonella LppB. Here, we demonstrate that LppB forms disulfide-linked dimers via its unique C-terminal cysteine and crosslinks to peptidoglycan through its terminal lysine, albeit with significantly lower efficiency than Lpp. Notably, LppB interacts with Lpp to form heterotrimers, thereby reducing overall Lpp-peptidoglycan crosslinking. These findings suggest that LppB modulates outer membrane-peptidoglycan tethering by fine-tuning the bacterial envelope architecture in response to environmental conditions. Given the established link between outer membrane-peptidoglycan uncoupling and outer membrane vesicles (OMV) formation, a key mechanism in bacterial pathogenicity, our results suggest a model in which LppB regulates outer membrane-peptidoglycan attachment, potentially influencing OMV production. This study highlights a modulating role for LppB in envelope remodeling, providing new insights into how Salmonella adapts its envelope structure in response to environmental cues to enhance fitness and virulence.

microbiology↗

bioRxiv2025

A simple workflow to identify novel Small Linear Motif (SLiM)-mediated interactions with AlphaFold

Short linear motifs (SLiMs) are highly compact interaction modules embedded within disordered protein regions and are increasingly recognized for their central role in maintaining cellular homeostasis. Due to their small size, degeneracy and transient binding, SLiMs remain difficult to detect both experimentally and computationally. Here, we show that AlphaFold, used via ColabFold, offers a practical and accessible alternative for in-silico SLiM discovery. Unlike previous studies focused on structural accuracy, we evaluated AlphaFolds capacity to reveal SLiMs independently of model quality. To this end, we benchmarked several scoring metrics and showed that AlphaFold2 combined with MiniPAE yields the best performance, outperforming AlphaFold3 in this context. Building on these findings, we also provide a streamlined and cost-effective workflow for SLiM prediction requiring no installation or local computation. To overcome challenges associated with SLiM validation, we also introduce a highly sensitive detection method based on proximity labeling in living cells. This workflow was used to predict the occurrence of SLiMs that mediate binding to ribosomal protein S6 kinase A3 (RPS6KA3 or RSK2). By leveraging Colabfold and MiniPAE available through Colab notebooks, our approach provides a scalable and widely accessible strategy for identifying functional SLiMs in proteins of interest. MiniPAE can be accessed at https://github.com/martinovein/MiniPAE Short descriptionMartin Veinstein is a PhD student in Biomedical Sciences at the de Duve Institute, UCLouvain, Belgium. He specializes in Small Linear Motifs (SLiMs) in the context of host-virus interactions and has developed strong expertise in bioinformatics, structural biology, and predictive modeling. Victor J is a unfergradiate student at the ECAM Brussels Engineering School, Haute Ecole "ICHEC-ECAM-ISFSC", Brussels, Belgium. His activities span form September to November 2023. B.I. Iorga is a CNRS Research Director at the Institut de Chimie des Substances Naturelles in Gif-sur-Yvette, France. His research focuses among others on methodological developments in molecular modeling and the in-silico prediction of antibiotic resistance using machine learning and deep learning approaches. Raphael Helaers is a Senior Investigator and leads bioinformatics infrastructure at the de Duve Institute, UCLouvain, Belgium. He has developed strong expertise in next-generation sequencing and software development, along with a deep interest in biology, genetics, and evolution. Thomas Michiels is a Full Professor and researcher at the de Duve Institute, UCLouvain, Belgium. His research focuses on virus-mediated subversion of the innate immune response. Frederic Sorgeloos is an adjunct Professor at the INRS, Laval, Canada. He currently focuses on the subversion of cellular homeostasis through small linear peptides encoded by viral and bacterial pathogens. Short abstractVarious AlphaFold2/3 scoring metrics were systematically benchmarked for their ability to detect Small Linear Motifs (SLiMs) Based on this evaluation, a user-friendly and cost-effective in-silico workflow is proposed to identify novel SLiMs-targeting proteins The utility of this workflow is demonstrated through the prediction of previously uncharacterized SLiMs interacting with RSK kinases. A sensitive in-vitro assay is proposed to streamline the validation of low-affinity SLiM-target interactions. Together, our workflow and associated validation assay offer an integrated pipeline for the discovery and validation of SLiM-mediated protein-protein interactions.

bioinformatics↗

bioRxiv2025

Imputing missing minimum inhibitory concentration (MIC) values for Pseudomonas aeruginosa strains with a Denoising AutoEncoder

Pseudomonas aeruginosa is a problematic pathogen with complex antibiotic resistance patterns. In clinical practice, minimum inhibitory concentration (MIC) tests typically focus on a limited subset of antibiotics, hindering a comprehensive assessment of a strains resistance profile. Here, we introduce MICFiller, a Denoising AutoEncoder (DAE) model designed to impute missing MIC values for 14 antibiotics in Pseudomonas aeruginosa within a specific dilution range by leveraging known MIC measurements for other antibiotics in the same strain. We evaluated the performance of DAE against two other commonly used methods: Multiple Imputation by Chained Equations (MICE) and simple median imputation. The DAE achieved the highest balanced 1-tier accuracy for most antibiotics, with performance closely matching that of MICE. MICFiller is freely accessible through a user-friendly web interface at http://iorgalab.org:4567/micfiller, offering clinicians a more complete view of a strains antibiotic resistance profile.

microbiology↗

bioRxiv2025

You Only Look Once (YOLO) Based Machine Learning Algorithm for Real-Time Detection of Loop-Mediated Isothermal Amplification (LAMP) Diagnostics

Loop-mediated isothermal amplification (LAMP) is a widely used rapid and affordable molecular DNA amplification method with minimal resource requirements. However, visual interpretation of results is subjective and prone to errors, leading to potential false-positive and negative results. To address this limitation, a machine-learning approach is proposed for automated LAMP classification based on digital images. The approach utilizes You Only Look Once (YOLOv8), a fast and robust object detection algorithm to locate and classify tubes within LAMP images, enabling automated categorization as positive or negative. The trained model achieved a high overall accuracy of 95.5% in classifying LAMP images into positive or negative. Additionally, the approach had a 98.0% precision and 92.7% recall for positive cases and 93.4% precision and 98.2% recall for negative cases, demonstrating its potential for real-time LAMP diagnosis and enhanced assay performance. This project demonstrated the platforms suitability for real-time testing, offering an easy operation and rapid results.

microbiology↗

bioRxiv2025

Genetic Determinants of pOXA-48 Plasmid Maintenance and Propagation in Escherichia coli

Conjugative plasmids are the main drivers of antibiotic resistance dissemination contributing to the emergence and extensive spread of multidrug resistance clinical bacterial pathogens. pOXA-48 plasmids, belonging to the IncL group, have emerged as the primary vehicle for carbapenem resistance in Enterobacteriaceae. Despite the problematic prevalence of pOXA-48, most research has focused on epidemiology and genomics, leaving gaps in our understanding of the mechanisms behind its propagation. In this study, we used a transposon sequencing approach to identify genetic elements critical for plasmid stability, replication, and conjugative transfer. Our results identified a novel type I toxin-antitoxin system, new essential maintenance factors, and components of the type IV secretion system and regulatory elements crucial for conjugation. This study advances our understanding of pOXA-48 biology, providing key insights into the genetic factors underlying its successful maintenance and spread in bacterial populations.

microbiology↗

bioRxiv2022

A molecular plugin rescues GroEL/ES substrates from pre-folding oxidation

Hsp60 chaperonins and their Hsp10 cofactors assist protein folding in all living cells, constituting the paradigmatic example of molecular chaperones. Despite extensive investigations of their structure and mechanism, crucial questions regarding how these chaperonins promote folding remain unsolved. Here, we report that the bacterial Hsp60 chaperonin GroEL forms a stable, functionally relevant complex with the chaperedoxin CnoX, a protein combining a chaperone and a redox function. Binding of GroES (Hsp10) to GroEL induces CnoX release. Cryo-electron microscopy provided crucial structural information on the GroEL-CnoX complex, showing that CnoX binds GroEL outside the substrate-binding site via a highly conserved C-terminal -helix. Furthermore, the identification of complexes in which CnoX, bound to GroEL, forms mixed-disulfides with GroEL substrates indicates that CnoX likely functions as a redox quality-control plugin for GroEL. Proteins sharing structural features with CnoX exist in eukaryotes, which suggests that Hsp60 molecular plugins have been conserved through evolution.

biochemistry↗

bioRxiv2021

Disorder is a critical component of lipoprotein sorting in Gram-negative bacteria

Gram-negative bacteria express structurally diverse lipoproteins in their envelope. Here we found that approximately half of lipoproteins destined to the Escherichia coli outer membrane display an intrinsically disordered linker at their N-terminus. Intrinsically disordered regions are common in proteins, but establishing their importance in vivo has remained challenging. Here, as we sought to unravel how lipoproteins mature, we discovered that unstructured linkers are required for optimal trafficking by the Lol lipoprotein sorting system: linker deletion re-routes three unrelated lipoproteins to the inner membrane. Focusing on the stress sensor RcsF, we found that replacing the linker with an artificial peptide restored normal outer membrane targeting only when the peptide was of similar length and disordered. Overall, this study reveals the role played by intrinsic disorder in lipoprotein sorting, providing mechanistic insight into the biogenesis of these proteins and suggesting that evolution can select for intrinsic disorder that supports protein function.

biochemistry↗

bioRxiv2020

Defining the function of OmpA in the Rcs stress response

OmpA, a protein commonly found in the outer membrane of Gram-negative bacteria, has served as a paradigm for the study of {beta}-barrel proteins for several decades. In Escherichia coli, OmpA was previously reported to form complexes with RcsF, a surface-exposed lipoprotein that triggers the Rcs stress response when damage occurs in the outer membrane and the peptidoglycan. How OmpA interacts with RcsF and whether this interaction allows RcsF to reach the surface has remained unclear. Here, we integrated in vivo and in vitro approaches to establish that RcsF interacts with the C-terminal, periplasmic domain of OmpA, not with the N-terminal {beta}-barrel, thus implying that RcsF does not reach the bacterial surface via OmpA. Our results reveal a novel function for OmpA in the cell envelope: OmpA competes with the inner membrane protein IgaA, the downstream Rcs component, for RcsF binding across the periplasm, thereby regulating the Rcs response.

microbiology↗