Biology subjects

Radicella, J. P.

Publications and source records attributed to Radicella, J. P..

8 recordsLinked to original sources

bioRxiv2026

Unveiling a missing component of the atypical type IV secretion system required for natural transformation of Helicobacter pylori

Exchange of genetic information by natural transformation shapes bacterial evolution. In Helicobacter pylori it is thought to drive its unusually high recombination rate, which has a crucial role in the evolution of virulence and the propagation of antibiotics resistance genes. While in most cases uptake of the incoming DNA into the periplasm is mediated by type IV pili, in H. pylori this initial step of natural transformation requires ComB, a unique competence-specific type IV secretion system (T4SS). The mechanisms by which ComB mediates DNA uptake are still poorly understood, since T4SS are usually involved in an opposite process of DNA export. Here, we identify a gene (hp1421) that is absolutely required for uptake of the transforming DNA into the periplasm, although distant from the comB operons. We show that hp1421 codes for a hexameric ATPase from the VirB11 family. HP1421 is present in the cytoplasm and interacts with ComB4, another ATPase of the T4SS inner membrane subcomplex. The structural modelling and functional analysis of HP1421 and its interaction with ComB4 indicate that HP1421 is a missing component of the ComB inner-membrane subcomplex that we propose to name ComB11. Phylogenetic analyses show that comB11 is a H. pylori core gene and suggest that the competence-dedicated ComB T4SS was a recent acquisition within Helicobacteraceae. Hence, co-option of the T4SS for DNA transformation requires nearly all the proteins that were previously essential for DNA conjugation. Author SummaryThe capacity of bacteria to exchange genetic information contributes in the case of pathogens to the spreading of antibiotic resistance and virulence factors. For Helicobacter pylori, a Gram-negative pathogen that colonises about half of the world population and is at the origin of diseases such as ulcers and gastric cancers, natural transformation is the major mechanism of horizontal gene transfer. However, H. pylori uses a very unusual system to capture and internalise the foreign DNA. Indeed, a Type 4 secretion system mediates this process. Here, we identify a so far missing and essential component of the T4SS, coded by a gene distant from the operon coding the other subunits. Through a combination of structural modelling, biochemical and microscopy approaches we show that this ATPase is an indispensable part of the ComB T4SS. Our study provides new insights into the mechanism by which the peculiar ComB T4SS works backwards to allow the passage of the tDNA from the bacterial environment into the periplasm.

microbiology↗

bioRxiv2025

A tripartite protein complex promotes DNA transport during natural transformation in firmicutes.

Natural genetic transformation is a conserved mechanism of bacterial horizontal gene transfer, which is directed entirely by the recipient cell and facilitates the acquisition of new genetic traits such as antibiotic resistance. Transformation proceeds via the capture of exogenous DNA, its internalisation in single strand form (ssDNA) and its integration into the recipient chromosome by homologous recombination. While the proteins involved in these steps have mainly been identified, the specific mechanisms at play remain poorly characterised. This study takes advantage of recent advances in structural modelling to explore the uptake of ssDNA during transformation. Using the monoderm human pathogen Streptococcus pneumoniae, we model a tripartite protein complex composed of the transmembrane channel ComEC, and two cytoplasmic ssDNA-binding proteins ComFA and ComFC. Using targeted mutation and transformation assays, we propose that pneumococcal ComEC features a narrow channel for ssDNA passage, and we show this channel is conserved in the diderm Helicobacter pylori. We identify key residues involved in protein-protein and protein-ssDNA interactions in the pneumococcal tripartite complex model and we show them to be crucial for transformation efficiency. Structural modelling reveals that this tripartite protein complex and its interaction with ssDNA are conserved in firmicutes. Overall, this study validates a tripartite complex required for the internalisation of ssDNA during transformation in firmicutes, providing new insights into the molecular mechanisms involved in this horizontal gene transfer mechanism central to bacterial adaptation. It also demonstrates the power of recent structural modelling techniques such as AlphaFold3 as hypothesis generators and guides for designing experiments. Significance statementNatural genetic transformation is a key mechanism of horizontal gene transfer, conserved in bacteria. The investigation of transmembrane channel ComEC and its interaction partners was previously hindered by the difficulty of manipulating these proteins experimentally. Thanks to state-of-the-art structural modelling with AlphaFold3 and subsequent thorough experimental validation of this model using targeted mutation and transformation assays, we demonstrate the importance of the ComEC/ComFA/ComFC complex for single strand DNA (ssDNA) uptake during natural transformation in the human pathogen Streptococcus pneumoniae. Similar models in several other species suggest a widely conserved organization of this complex in firmicutes. In addition, we demonstrate that the ComEC transmembrane channel is also crucial for ssDNA uptake during natural transformation in Helicobacter pylori.

microbiology↗

bioRxiv2024

G-quadruplex ligand RHPS4 compromises cellular radio-resistance by blocking the increase in mitochondrial mass and activity induced by ionising irradiation.

A major challenge in radiotherapy is to enhance tumor cell sensitivity to radiation while minimizing damage to healthy tissues. Ionising radiation (IR) induces mitochondrial DNA (mtDNA) alterations that can impair mitochondrial function and cell survival. Since mitochondria play a key role in tumor cell proliferation, they represent a promising therapeutic target for cancer treatment. In this study, we characterized the impact of different IR sources on mitochondrial function in radioresistant cancer cells. Our findings revealed several adaptive responses that may contribute to radioresistance, including increased mtDNA content, mitochondrial mass, enhanced activity, and hyperfusion of the mitochondrial network. Notably, the use of mitochondrial-targeted G-quadruplex (G4) ligands, which block mtDNA replication and transcription, disrupted these responses, reducing cancer cell survival in an mtDNA-dependent manner. These results demonstrate that mitochondrial adaptations contribute to radioresistance and highlight mitochondria as a novel target for the radiosensitizing effects of G4-ligands, extending their potential beyond telomere destabilization.

cell biology↗

bioRxiv2024

DprA recruits ComM to facilitate recombination during natural transformation in Gram-negative bacteria

Natural transformation (NT) represents one of the major modes of horizontal gene transfer in bacterial species. During NT, cells can take up free DNA from the environment and integrate it into their genome by homologous recombination. While NT has been studied for >90 years, the molecular details underlying this recombination remain poorly understood. Recent work has demonstrated that ComM is an NT-specific hexameric helicase that promotes recombinational branch migration in Gram-negative bacteria. How ComM is loaded onto the post-synaptic recombination intermediate during NT, however, remains unclear. Another NT-specific recombination mediator protein that is ubiquitously conserved in both Gram-positive and Gram-negative bacteria is DprA. Here, we uncover that DprA homologs in Gram-negative species contain a C-terminal winged helix domain that is predicted to interact with ComM by AlphaFold. Using Helicobacter pylori and Vibrio cholerae as model systems, we demonstrate that ComM directly interacts with the DprA winged-helix domain, and that this interaction is critical for DprA to recruit ComM to the recombination site to promote branch migration during NT. These results advance our molecular understanding of recombination during this conserved mode of horizontal gene transfer. Furthermore, they demonstrate how structural modeling can help uncover unexpected interactions between well-studied proteins to provide deep mechanistic insight into the molecular coordination required for their activity. SIGNIFICANCE STATEMENTBacteria can acquire novel traits like antibiotic resistance and virulence through horizontal gene transfer by natural transformation. During this process, cells take up free DNA from the environment and integrate it into their genome by homologous recombination. Many of the molecular details underlying this process, however, remain incompletely understood. In this study, we identify a new protein-protein interaction between ComM and DprA, two factors that promote homologous recombination during natural transformation in Gram-negative species. Through a combination of bioinformatics, structural modeling, cell biological assays, and complementary genetic approaches, we demonstrate that this interaction is required for DprA to recruit ComM to the site of homologous recombination.

microbiology↗

bioRxiv2022

Identification of key residues of the DNA glycosylase OGG1 controlling efficient DNA scanning and recruitment to oxidized bases in living cells

The DNA-glycosylase OGG1 oversees the detection and clearance of the 7,8-dihydro-8-oxoguanine (8-oxoG), which is the most frequent form of oxidized base in the genome. This lesion is deeply buried within the double-helix and its detection requires careful inspection of the bases by OGG1 via a mechanism that remains only partially understood. By analyzing OGG1 dynamics in the nucleus of living human cells, we demonstrate that the glycosylase constantly scans the DNA by rapidly alternating between diffusion within the nucleoplasm and short transits on the DNA. This scanning process, that we find to be tightly regulated by the conserved residue G245, is crucial for the rapid recruitment of OGG1 at oxidative lesions induced by laser micro-irradiation. Furthermore, we show that residues Y203, N149 and N150, while being all involved in early stages of 8-oxoG probing by OGG1 based on previous structural data, differentially regulate the scanning of the DNA and recruitment to oxidative lesions.

cell biology↗

bioRxiv2021

ComF is a key mediator in single-stranded DNA transport and handling during natural transformation

Natural transformation plays a major role in the spreading of antibiotic resistances and virulence factors. Whilst bacterial species display specificities in the molecular machineries allowing transforming DNA capture and integration into their genome, the ComF(C) protein is essential for natural transformation in all Gram-positive and - negative species studied. Despite this, its role remains largely unknown. Here, we show that Helicobacter pylori ComF is not only involved in DNA transport through the cell membrane, but it also required for the handling of the ssDNA once it is delivered into the cytoplasm. ComF crystal structure revealed the presence of a zinc-finger motif and a putative phosphoribosyl transferase domain, both necessary for its in vivo activity. ComF is a membrane-associated protein with affinity for single-stranded DNA. Collectively, our results suggest that ComF provides the link between the transport of the transforming DNA into the cytoplasm and its handling by the recombination machinery.

microbiology↗

bioRxiv2020

DNA oxidation induced by fetal exposure to BPA agonists impairs female meiosis

Many endocrine disruptors have been proven to impair the meiotic process that is mandatory to produce healthy gametes. Bisphenol A is emblematic as it impairs meiotic prophase I and causes oocyte aneuploidy following in utero exposure. However, the mechanisms underlying these deleterious effects remain poorly understood. Furthermore, the increasing uses of BPA analogs raise concerns for public health. Here, we investigated the effect on oogenesis in mouse of fetal exposure to two BPA analogs, Bisphenol A Diglycidyl Ether (BADGE) or Bisphenol AF (BPAF). These analogs delay meiosis initiation, increase MLH1 foci per cell and induce oocyte aneuploidy. We further demonstrate that these defects are accompanied by a deregulation of gene expression and aberrant mRNA splicing in fetal premeiotic germ cells. Interestingly, we observed an increase in DNA oxidation after exposure to BPA analogs. Specific induction of oxidative DNA damages during fetal germ cell differentiation causes similar defects during oogenesis, as observed in 8-Oxoguanine DNA Glycosylase (OGG1) deficient mice or after in utero exposure to potassium bromate (KBrO3), an inducer of oxidative DNA damages. Moreover, the supplementation of N-acetylcysteine (NAC) with BPA analogs counteracts the bisphenol-induced meiotic effect. Together our results position oxidative stress as a central event that negatively impacts the female meiosis with major consequences on oocyte quality. This could be a common mechanism of action for so called endocrine disruptors pollutants and it could lead to novel strategies for reprotoxic compounds.

molecular biology↗

bioRxiv2020

An adaptive pre-DNA-damage-response protects genome integrity

The DNA damage response (DDR) interrupts cell cycle progression to restore genome integrity. However, unchallenged proliferating cells are continually exposed to endogenous stress, raising the question of a stress-threshold for DDR activation. Here, we identified a stress threshold below which primary human fibroblasts, activate a cell-autonomous response that not activates full DDR and not arrests cell cycle progression. We characterized this "pre-DDR" response showing that it triggers the production of reactive oxygen species (ROS) by the NADPH oxidases DUOX1 and DUOX2, under the control of NF-{kappa}B and PARP1. Then, replication stress-induced ROS (RIR) activates the FOXO1 detoxifying pathway, preventing the nuclear accumulation of the pre-mutagenic 8-oxoGuanine lesion, upon endogenous as well as exogenous pro-oxidant stress. Increasing the replication stress severity above the threshold triggers the canonical DDR, leading to cell cycle progression arrest, but also to RIR suppression. These data reveal that cells adapt their response to stress severity, unveiling a tightly regulated "pre-DDR" adaptive response that protects genome integrity without arresting cell cycle progression.

cell biology↗