Biology subjects

Mignolet, J.

Publications and source records attributed to Mignolet, J..

8 recordsLinked to original sources

bioRxiv2026

A streamlined ABC extruder-repressor module drives multi-bacteriocin resistance in streptococci

Bacteria inhabiting competitive microbial environments must rapidly detect and neutralize antimicrobial peptides (AMPs) produced by rivals. The activation of defense pathways relies on dedicated sensors and complex regulatory cascades. Here, we uncover a streamlined, membrane-embedded mechanism in Gram-positive bacteria that directly links detection to transcriptional control. We show that a YtrA-family transcriptional repressor is regulated through an unconventional direct physical interaction with its cognate ABC transporter. In Streptococcus salivarius, the MbrAB efflux pump sequesters the YtrA-like repressor MbrR through a competitive binding mechanism. Upon bacteriocin sensing, MbrR shifts from promoter-proximal DNA repression sites to membrane-associated sequestration via interaction with MbrA, thereby activating the bacteriocin defense system. ATP binding by MbrA facilitates MbrR recruitment, whereas ATP hydrolysis promotes its release, providing a dynamic, flux-responsive feedback loop finely tuned to environmental threat. Structural modeling, synteny, and conservation analyses reveal that this transporter-mediated sequestration mechanism is highly conserved across Gram-positive bacteria, suggesting a widespread and efficient strategy for AMP resistance that bypasses the need for classical sensor kinases. These findings expand the known repertoire of bacterial sensing and resistance systems and provide new insight into how Gram-positive bacteria swiftly adapt to interbacterial antagonism. IMPORTANCEBacteria in crowded microbial communities face constant chemical warfare, yet how they sense and counteract antimicrobial peptides (AMPs) remains incompletely understood. Here we uncover a minimalist sensing-response module in streptococci in which YtrA-family transcriptional repressors are directly regulated by their partner ABC transporters. In Streptococcus salivarius, the MbrAB transporter detects incoming bacteriocins and physically sequesters the MbrR repressor, triggering rapid induction of a multi-gene defense program. ATP binding drives MbrR capture, while hydrolysis resets the system, providing a fast, energy-coupled switch that bypasses canonical two-component signaling. Comparative genomics shows that this transporter-repressor circuit is highly conserved across Bacillota, pointing to a broadly distributed and efficient strategy for AMP resistance. These findings reveal a direct physical link between membrane transport and transcriptional control, redefining how Gram-positive bacteria can sense and respond to microbial threats. ONE SENTENCE SUMMARYWe identify a conserved transporter-repressor module in streptococci that directly couples bacteriocin detection to transcriptional activation, revealing a minimalist and rapid mechanism for antimicrobial peptide resistance.

microbiology↗

bioRxiv2026

Phage Endolysin Enables Targeted Manipulation of the Small Intestinal Microbiota and Uncovers Niche Overlap Between Oral and Butyrate-Producing Taxa

Oral bacterial overgrowth in the small intestine has been associated with dysbiosis, impaired nutrient absorption, and stunted growth in undernourished children, a condition referred to as small intestinal oral bacterial overgrowth (SIOBO). Here, we explore the use of a phage-derived lysin as a precision antimicrobial to selectively target Streptococcus salivarius within complex microbial communities. Through a newly developed, medium-throughput bioinformatic and wet-lab pipeline we identified, cloned and produced a prophage-encoded lysin from S. salivarius and demonstrated its potent and specific lytic activity against a panel of 49 clinical S. salivarius strains from stunted children, while sparing related species such as S. mitis, S. parasanguinis and S. thermophilus. Application of the lysin to human stool-derived in vitro communities and to mice colonized with S. salivarius led to an approximate 2-3 log reduction in S. salivarius abundance while preserving overall bacterial community composition. However, we observed a negative correlation between S. salivarius and Coprococcus comes in vitro, and Eubacterium xylanophilum, Akkermansia, Lactobacillus and Ruminococcus in vivo. Spent medium assays confirmed niche overlap between 10 clinical strains of S. salivarius and 28 different taxa involved in butyrate production. Together, these results suggest that phage-derived lysins can offer multiple benefits by selectively removing ectopically colonized oral taxa and indirectly promoting beneficial anaerobes.

microbiology↗

bioRxiv2025

Chemical dysbiosis byproducts trigger predation via alternative activation of a peptide quorum sensor in salivarius streptococci

Cell-to-cell communication in Gram-positive bacteria is predominantly orchestrated by cytoplasmic sensors of the RRNPPA family. To date, all characterized members of this family are activated by small, unmodified peptide pheromones that mediate bacterial signaling. In the human commensal Streptococcus salivarius, the RRNPPA sensor ComR controls both competence (DNA transformation) and predation (bacteriocin production). Here, we reveal that ComR can be dually activated by its cognate peptide (XIP) and a distinct class of small organic molecules. A targeted screen of [~]200 organic compounds identified hydroxyphenylacetic acid (HPAA), a bacterial dysbiosis byproduct accumulating in human fluids, as a potent inducer of ComR. Using in vivo and in vitro approaches, we demonstrated that HPAA and structurally related carboxylic acids derived from bulky hydrophobic amino acids bind the pheromone-accommodating pocket, leading to ComR activation. Strikingly, while XIP-mediated activation is transient and regulates both competence and predation, HPAA induces a sustained, predation-oriented response. Furthermore, we showed that Porphyromonas gingivalis, an oral pathogen, produces sufficient (H)PAA quantity to trigger bacteriocin production in S. salivarius, revealing a previously unrecognized chemical interplay between oral microbiota members. These findings highlight the remarkable versatility of cytoplasmic sensors to integrate diverse environmental cues, shedding new light on bacterial peptide-based communication and microbial homeostasis in the human microbiome. IMPORTANCEBacterial communication through quorum sensing (QS) is crucial for coordinating key physiological processes. While QS in Gram-positive bacteria has been predominantly associated with peptide pheromones, our study uncovers an undescribed alternative signaling mechanism. We demonstrate that ComR, a cytoplasmic receptor of the RRNPPA family, can be activated by its canonical peptide signal and by small organic molecules derived from the anaerobic breakdown of hydrophobic amino acids. This alternative activation pathway enhances the ability of S. salivarius to respond to microbial dysbiosis, maintaining ecological balance in the digestive tract. Beyond revealing a novel layer of bacterial communication, our findings suggest that many RRNPPA-family receptors previously considered "orphan" may respond to yet-undiscovered chemical signals. This work expands our understanding of bacterial sensing and opens new avenues for modulating microbial interactions in health and disease.

microbiology↗

bioRxiv2025

CRISPRi-seq in Haemophilus influenzae reveals genome-wide and medium-specific growth determinants

Work in the human pathobiont Haemophilus influenzae has pioneered functional genomics in bacteria such as genome-wide transposon mutagenesis combined with deep sequencing. These approaches unveiled a large set of likely essential genes, but functional studies are hampered due to a limited molecular toolbox. To bridge this gap, we engineered a titratable anhydrotetracycline (aTc)-inducible CRISPRi (Clustered Regularly Interspaced Short Palindromic Repeats interference) platform for efficient regulation of gene expression in H. influenzae. Genome-wide fitness analyses in two different in vitro culture media by CRISPRi-seq revealed growth medium-dependent fitness cost for a panel of H. influenzae genes. We demonstrated that CRISPRi-programmed fitness defects can be rescuable, and refined previous Tn-seq based essentialome studies. Finally, we introduce HaemoBrowse, an extensive user-friendly online resource for visual inspection of H. influenzae genome annotations, including sgRNA spacers. The inducible CRISPRi platform described here represents a valuable tool enabling functional genomics and the study of essential genes, thereby contributing to the identification of therapeutic targets for developing drugs and vaccines against H. influenzae. ImportanceCRISPRi-seq is a robust method to study bacterial gene fitness and essentiality via relative quantification and comparison of sgRNA abundance at a genome-wide scale. Here, we present a novel CRISPRi system for individual genes or pooled libraries knockdown in Haemophilus influenzae. A genome-wide CRISPRi library designed to cover 99.27% of all total genetic features in the genome of RdKW20 strain was constructed and screened in two laboratory growth media through CRISPRi-seq, uncovering growth medium-dependent fitness cost, further confirmed with individual knockdown/knockout mutants. We also introduce HaemoBrowse (https://HaemoBrowse.VeeningLab.com), through which genome annotations and sgRNA design on H. influenzae genomes can be readily inspected. This platform provides a valuable tool for gene function and essentiality analyses in a notorious human pathobiont.

microbiology↗

bioRxiv2025

A Modular Genetic Toolbox for Precise Gene Regulation and Multi-Color Imaging in Streptococci

Fluorescent labeling is a powerful tool in microbiology allowing live cell imaging and providing insights into dynamic cellular processes, quantification of gene expression and protein subcellular localization. Although multicolor imaging is widely used in Streptococcus pneumoniae and S. mutans, variants other than the green fluorescent protein (GFP) have rarely been applied in other streptococcal species. To address this gap in the streptococcal molecular toolbox, we benchmarked five different fluorescent proteins. The various fluorescent proteins were fused to the C-terminus of S. pneumoniae HlpA, a small non-specific DNA binding histone-like protein. These reporters, combined with four different antibiotic resistance genes, were engineered with various expression systems (inducible or constitutive) to form versatile cassettes. We provide methods to transfer these cassettes to different streptococcal species including S. salivarius and S. thermophilus. As a proof of concept, we generated a triple labeled S. salivarius strain in which HlpA, FtsZ and DivIVA were fused to three spectrally-distinct compatible fluorescent proteins. Multiple fluorescent labeling has broad applications for deciphering a wide range of scientific problems, from cellular processes to infectious disease mechanisms. The availability of these cassettes should allow for a wider use of single-cell labeling strategies in the Streptococcus clade and other closely related bacteria.

microbiology↗

bioRxiv2024

A bacterial cell wall repair and modification system to resist host antibacterial factors

Pathogenic bacteria have acquired the ability to resist antibacterial defense mechanisms of the host. Streptococci are common in animal microbiota and include opportunistic pathogens like Group A Streptococcus (GAS) and Streptococcus pneumoniae (pneumococcus). While the conserved streptococcal S protein has been identified as a key factor in GAS virulence, its exact function is unclear. Here, we show that the pneumococcal S protein is crucial for resisting against host-derived antimicrobials by coordinating cell wall modification and repair. Specifically, we show that S proteins are septally localized through their transmembrane domain and contain an extracellular peptidoglycan (PG) binding LysM domain which is required for its function. Protein-protein and genetic interaction studies demonstrate that the pneumococcal S protein directly interacts with a PG synthase, class A penicillin binding protein PBP1a, and the PG deacetylase PgdA. Single-molecule experiments reveal that the fraction of circumferentially moving PBP1a molecules is reduced in the absence of S protein. Consistent with an impaired PBP1a function, streptococci lacking S protein exhibit increased susceptibility to cell wall targeting antibiotics and altered cell morphologies. PG analysis showed reduced N-deacetylation of glycans in the S. pneumoniae S protein mutant, indicating reduced PgdA activity. We show that pneumococci lacking the S protein cannot persist transient penicillin treatment, are more susceptible to the human antimicrobial peptide LL-37 and to lysozyme, and show decreased virulence in zebrafish and mice. Our data support a model in which S proteins regulate PBP1a activity and play a key role in coordinating PG repair and modification. This cell wall sentinel control system provides defense against host-derived and environmental antimicrobial attack.

microbiology↗

bioRxiv2024

Uncovering the class II-bacteriocin predatiome in salivarius streptococci

Facing the surge of antibiotic resistance, the medical field has a critical need for alternatives to treat bacterial infections. Among these, the use of bacteriocins, ribosomally-synthesized antimicrobial peptides produced by bacteria, is considered to be a promising route. In the human commensal Streptococcus salivarius, the production of unmodified class II bacteriocins is directly controlled at the transcriptional level by the quorum-sensing ComRS system. Here, we used an integrated approach combining bioinformatics and synthetic biology to identify novel bacteriocins from salivarius streptococci active against human pathogens. We developed a bioinformatic pipeline that combines conservation of DNA motifs for genetic regulation and features of bacteriocin primary sequences to uncover cryptic class II bacteriocins. Notably, we discovered more than 50 novel bacteriocin candidates clustered into 21 groups from 100 genomes of S. salivarius. Strain-based analysis of bacteriocin cocktails revealed an important diversity restricted to seven distinct loci, probably resulting from bacteriocin intra- and inter- species exchanges. Using in vitro or in vivo production and synthetic biology tools, we showed that most of them are active against a panel of Gram-positive bacteria, including clinically- relevant pathogens. Overall, this work provides a new search-to-test generic pipeline for the discovery of novel bacteriocins in Gram-positive bacteria that could be used in cocktails for broad applications in the food and biomedical fields. IMPORTANCETo survive in highly challenging environments, streptococci have evolved a competence- predation coupling mechanism for genome plasticity. This developmental process is highly regulated at the transcriptional level, masking the predation killing effects in usual laboratory conditions. Here, we present a general strategy that combines bioinformatics and synthetic biology to unveil class II bacteriocins in streptococci. Its implementation to the beneficial species Streptococcus salivarius revealed around 40 class II salivaricin cocktails explained by the plasticity of seven independent loci. Notably, the salivaricin predatiome includes a subtle blend of fratricins, sobrinicins, and broad-spectrum bacteriocins with overlapping activities against a wide spectrum of low-GC Gram-positive bacteria, including notorious pathogens. Furthermore, most of those bacteriocins are predicted to be variants of a common -hairpin structure, indicating that their mode of action evolved convergently. Finally, the discovery of ca. 50 novel bacteriocins offers perspectives for the rational assembly of potent cocktails active against pathogenic staphylococci, streptococci or enterococci.

microbiology↗

bioRxiv2022

A genome-wide CRISPRi screen reveals a StkP-mediated connection between cell-wall integrity and competence in Streptococcus salivarius

Competence is one of the most efficient bacterial evolutionary and adaptative strategies by synchronizing production of antibacterial compounds and integration of DNA released by dead cells. In most streptococci, this tactic is orchestrated by the ComRS system, a pheromone communication device providing a sharp time window of activation in which only part of the population is responsive. Understanding how this developmental process integrates multiple inputs to fine-tune the adequate response is a long-standing question. However, essential genes involved in the regulation of ComRS have been challenging to study. In this work, we built a conditional mutant library using CRISPR-interference and performed three complementary screens to investigate competence genetic regulation in the human commensal Streptococus salivarius. We show that initiation of competence increases upon cell-wall impairment, suggesting a connection between cell envelope stress and competence activation. Notably, we report a key role for StkP, a serine-threonine kinase known to regulate cell-wall homeostasis. We show that StkP controls competence by a mechanism that reacts to peptidoglycan fragments. Together, our data suggest a key cell-wall sensing mechanism coupling competence to cell envelope integrity. IMPORTANCESurvival of human commensal streptococci in the digestive tract requires efficient strategies which must be tightly and collectively controlled for responding to competitive pressure and drastic environmental changes. In this context, the autocrine signaling system ComRS controlling competence for natural transformation and predation in salivarius streptococci could be seen as a multi-input device integrating a variety of environmental stimuli. In this work, we revealed novel positive and negative competence modulators by using a genome-wide CRISPR- interference strategy. Notably, we highlighted an unexpected connection between bacterial envelope integrity and competence activation that involves several cell-wall sensors. Together, these results showcase how commensal streptococci can fine-tune the pheromone-based competence system by responding to multiple inputs affecting their physiological status in order to calibrate an appropriate collective behavior.

microbiology↗