Biology subjects

Maillard, R.

Publications and source records attributed to Maillard, R..

4 recordsLinked to original sources

bioRxiv2025

Transcriptome-wide mapping reveals an RNA-dependent mechanism of platinum cancer drugs.

Off-target interactions frequently compromise the clinical utility of anticancer agents by driving dose-limiting toxicity and therapeutic resistance. Although RNA has been predicted to be an off-target for numerous FDA-approved drugs, the extent and functional significance of RNA off-targeting among anticancer small molecules remain poorly understood. Using a systematic drug-binding screen, we identified cisplatin, a frontline chemotherapeutic that acts canonically through DNA adduct formation, as a prominent RNA binder. We employed cisplatin as a model compound to characterize the mechanistic basis and functional impact of RNA-small molecule off-targeting. To map transcriptome-wide cisplatin-RNA interactions, we developed PlatRNA-seq, a click-chemistry-enabled RNA-binding profiling platform. Genomic and functional analyses reveal that cisplatin preferentially accumulates at RNA G-quadruplex (rG4) structures near 5' transcript ends, inducing R-loop formation. Critically, we demonstrate that cisplatin cytotoxicity is partially mediated through RNA binding, revealing a noncanonical mechanism of action. Collectively, these findings illustrate the functional consequences of RNA-small-molecule off-targeting and provide a generalizable framework for investigating small-molecule-RNA interactions, opening new avenues for therapeutic innovation.

molecular biology↗

bioRxiv2025

Nucleoside Binding by a Surface Lipoprotein Governs Conjugative ICE Acquisition in Ruminant Mycoplasmas

Integrative and conjugative elements (ICEs) are major mediators of horizontal gene transfer (HGT) in bacteria. However, the role of recipient cells in their acquisition has received little attention. Using the ruminant pathogens Mycoplasma agalactiae and Mycoplasma bovis as minimal models, we combined genome-wide transposon mutagenesis with high-throughput mating assays to identify recipient factors required for ICE acquisition. The surface lipoprotein P48 emerged as the primary determinant of ICE uptake in both species. Structural and functional analyses revealed that P48 is the substrate-binding component of an ABC transporter with nucleoside-binding capacity. A single point mutation that abolished nucleoside binding drastically reduced ICE acquisition, demonstrating that P48-mediated nucleoside recognition is essential for conjugative transfer. However, ICE uptake did not require nucleoside transport, as inactivation of the transporter permease blocked nucleoside analog toxicity but not ICE invasion. Loss of P48 also triggered transcriptional activation of vestigial ICE genes, suggesting that surface recognition affects the intracellular state of the recipient. Remarkably, ICE transmission from recipient-derived donors was unaffected by P48 loss, underscoring its acquisition-specific role. Together, these results reveal a previously unrecognized, surface-exposed recipient factor critical for efficient ICE transfer in mycoplasmas and identify nucleotide binding as a central function in conjugation. By demonstrating that recipient-encoded functions can directly control ICE dissemination, this work challenges the donor-centric paradigm of bacterial conjugation and suggests new strategies to restrict horizontal gene flow in pathogenic and synthetic mycoplasmas. IMPORTANCEIntegrative and conjugative elements (ICEs) are mobile DNA elements that drive bacterial conjugation, a major process by which bacteria exchange genes. Although conjugation has been studied for decades, the focus has been almost exclusively on donor cells and the ICE itself, leaving the role of recipient cells largely overlooked. Using the wall-less ruminant pathogens Mycoplasma agalactiae and Mycoplasma bovis as minimal models, we discovered that a single recipient lipoprotein is required for efficient ICE uptake. Our data show that nucleoside recognition by P48, but not transport, is critical for conjugation, revealing an unexpected mechanistic link between nutrient sensing and gene acquisition. These findings shift the paradigm of conjugation from a donor-driven process to one jointly determined by donor and recipient functions. By identifying a recipient-encoded determinant of ICE transfer, this work opens new avenues to control horizontal gene flow in both pathogenic and engineered bacteria. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/677790v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@f7cc15org.highwire.dtl.DTLVardef@77a34eorg.highwire.dtl.DTLVardef@82699corg.highwire.dtl.DTLVardef@1e3be93_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Bacterial Conjugation in the Ruminant Pathogen Mycoplasma agalactiae is Influenced by Eukaryotic Host Factors

Bacterial conjugation plays a pivotal role in the evolution and adaptation of genome-reduced mycoplasmas. Despite their fast evolution rate, the conjugative properties of these organisms remain largely understudied, particularly in vivo. In the present study, the ruminant pathogen Mycoplasma agalactiae was used as a model organism to document the conjugative properties of mycoplasmas in environments of increasing complexity, from axenic to cell and organotypic culture conditions. Compared to axenic mating conditions, mycoplasma co-cultivation with goat epithelial cells or bovine precision-cut lung slices (PCLS) resulted in enhanced mating frequencies with high rates of M. agalactiae Integrative and Conjugative Element (ICEA) self-dissemination. These results were conditioned by the presence of eukaryotic cells in the culture and influenced by competition between mating partners but were not limited to M. agalactiae, as similar results were observed with Mycoplasma bovis. Mycoplasma conjugation ex vivo was further characterized by analyzing mycoplasma chromosomal transfer (MCT), a newly discovered mechanism of horizontal exchange of chromosomal DNA that generates mosaic genomes. Although closely associated with ICEA transfer, MCT was detected at low rates under cell and organotypic culture conditions suggesting a complex interplay between these two conjugative processes or a poor viability of the MCT progeny. Finally, mating experiments under nutrient-deprived conditions identified nucleotide stress as a potential factor influencing the modulation of mycoplasma conjugation by eukaryotic host cells. In conclusion, these results suggest that horizontal gene transfer in vivo is likely underestimated and provide valuable models to further studying mycoplasma conjugation ex vivo. IMPORTANCEConjugation is an evolutionary shortcut that bacteria use to exchange genetic information with their neighbors. Despite the fast evolution rate of the genome-reduced mycoplasmas, their conjugative properties remain largely understudied, particularly in vivo. Here we used the ruminant pathogen Mycoplasma agalactiae to study how mycoplasmas conjugate in co-culture with hosts-derived cells and tissues. Interestingly, conjugation was stimulated when mycoplasmas were co-cultured with eukaryotic cells. This was documented by monitoring the self-propagation of a mobile genetic element known as Integrative and Conjugative Element (ICE) and the exchange of chromosomal DNA leading to the formation of mosaic genomes. While ICE transfer was observed at high frequency, only a few mosaic genomes were detected in the presence of eukaryotic cells. Further data point towards nucleotide stress as a possible factor modulating mycoplasma conjugation in cellular environments. These results suggest that mycoplasma-host interactions may stimulate conjugation in vivo.

microbiology↗

bioRxiv2024

Allosteric modulation of protein kinase A in individuals affected by NLPD-PKA , a neurodegenerative disease in which the RIβ-L50R variant is expressed

Protein kinase A (PKA) is a crucial signaling enzyme in neurons, with its dysregulation being implicated in neurodegenerative diseases. Assembly of the PKA holoenzyme, comprising a dimer of heterodimers of regulatory (R) and catalytic (C) subunits, ensures allosteric regulation and functional specificity. Recently, we defined the RI{beta}-L50R variant as a causative mutation that triggers protein aggregation in a rare neurodegenerative disease. However, the mechanism underlying uncontrolled PKA allosteric regulation and its connection to the functional outcomes leading to clinical symptoms remain elusive. In this study, we established an in vitro model using patient-derived cells for a personalized approach and employed direct measurements of purified proteins to investigate disease mechanisms in a controlled environment. Structural analysis and circular dichroism spectroscopy revealed that cellular proteins aggregation resulted from misfolded RI{beta}-subunits, preventing holoenzyme assembly and anchoring through A Kinase Anchoring Proteins (AKAPs). While maintaining high affinity to the C subunit, the resulting RI{beta}-L50R:C heterodimer exhibits reduced cooperativity, requiring lower cAMP concentrations for dissociation. Consequently, there was an increased translocation of C-subunit into the nucleus, impacting gene expression. We successfully controlled C subunit translocation by introducing a mutation that decreased RI{beta}:C dissociation in response to elevated cAMP levels. This research thus sets the stage for developing therapeutic strategies that modulate PKA assembly and allostery, thus exerting control over the unique molecular signatures identified in the disease-associated transcriptome profile.

cell biology↗