Biology subjects

BELOIN, C.

Publications and source records attributed to BELOIN, C..

13 recordsLinked to original sources

bioRxiv2026

High-resolution lineage tracking maps the multi-phase evolution and adaptive potential of biofilms

Although biofilms represent the dominant bacterial lifestyle and are major contributors to recalcitrant, treatment-resistant infections, their evolutionary dynamics at the individual lineage resolution remain poorly understood. We combine DNA barcoding-mediated lineage tracking and spatial sampling of > 100,000 lineages of Escherichia coli to characterize sequential phases of biofilm evolution quantitatively. Severe founder effects during surface colonization initially reduce the effective population size by nearly three orders of magnitude, establishing significant spatial segregation. This structure is subsequently eroded by a previously underappreciated secondary migration phase, during which cells released upon perturbation recolonize biofilms with a rate 103-fold higher than naive planktonic cells. Adaptive evolution subsequently drives specialization through mutations targeting these distinct stages of the biofilm life cycle. Under severe antibiotic bottlenecks, active secondary migration continuously resuscitates local lineage diversity, enabling the parallel emergence of resistance across hundreds of independent lineages. Once locally established, migration drives a rapid spatial spread of resistant mutants. Together, these findings reveal how spatial segregation and secondary migration shape biofilm dynamics, coupling short-term ecological resilience with long-term adaptive potential.

microbiology↗

bioRxiv2026

3D printed titanium anodized effects on human gingival fibroblasts response and bacterial colonization: a dual approach

Mucointegration is as important as osseointegration to ensure the survival of implant-supported prosthesis. Indeed, effective soft tissue integration (STI) prevents the appearance of complication through bacterial dissemination. To optimize STI, electrochemical anodization can be used to nanostructure the trans-gingival part of the prosthetic component. Moreover, Selective Laser Melting (SLM) is a new 3D-manufacturing technique that enables the production of customized implant-supported prosthesis with complex geometry. ObjectiveThe aim of this study is to evaluate the effect of a SLM manufactured and anodized Ti6Al4V surface on the behaviour of both, human gingival fibroblasts and oral bacteria. MethodSLM-Ti6Al4V discs were polished and anodized with defined parameters to obtain nanotubes (NTs) with specific morphology. Surface characterization was assessed through surface topography and wettability. Human gingival Fibroblasts were cultured, and cell morphology was observed by SEM at day 7. Proliferation, viability (day 1,4,7) and adhesion (6 h and 36 h) were analyzed. Then immunofluorescence and RT-qPCR were used to detect the distribution and the gene expression of vinculin at 48 h. An early colonizer (Streptococcus gordonii) was used for a parallel evaluation of bacteriological adhesion. ResultsSLM-ANO-Ti6Al4V showed similar performances in terms of cytotoxicity, compared with a machined and polished titanium surface currently used in clinics. Interestingly, cell adhesion was enhanced on anodized SLM surfaces, with a difference in the distribution of focal adhesion plaques in HGFs, while biofilm formation of S. gordonii was not affected by anodization. SignificanceSLM anodized surface showed promising ability to promote STI while controlling bacterial adhesion.

biophysics↗

bioRxiv2026

Global analysis of trimeric autotransporters reveals phylogenetically restricted secretion mechanism adaptations

Autotransporters are important diderm bacterial cell-surface proteins and are virulence factors enabling surface attachment and adhesion to other bacteria. These proteins are composed of a signal peptide, a {beta}-barrel that serves as an anchor in the outer membrane, and an extracellular passenger domain responsible for adhesion. Autotransporters rely on BamA for their insertion in the outer membrane (OM), but specific helper proteins, such as TpgA and SadB have been described to promote the surface exposure of trimeric autotransporters (TAAs), a specialized subclass of autotransporters forming homotrimer adhesins. To identify domains or proteins that could help TAAs secretion, we analyzed a recent dataset of all trimeric autotransporters found across the bacterial tree of life. While we did not find additional potential helper proteins for OM translocation, we found that the extended signal peptide (ESPR), sometimes found in TAAs, is associated with longer adhesins both for TAAs and type Va autotransporter adhesins. ESPRs are found in all bacteria but Fusobacteriia and Alphaproteobacteria. We also identified in Burkholderia, Veillonellales and Pasteurellales a DUF2827 domain proteins as potential glycosyltransferases constantly associated with TAAs. Finally, e describe the existence of extra periplasmic domains in some TAAs, featuring either a coiled-coil domain or a peptidoglycan-binding domain. Our research show that there is a strong phylogenetic separation between Terrabacteria, almost invariably displaying additional periplasmic domains, and Gracilicutes (represented by Proteobacteria) where they are largely absent. This suggests that the presence these domains might be correlated with specific Terrabacteria OM features. Using the diderm Firmicute Veillonella parvula as a model, we demonstrate that the absence of periplasmic domains in TAAs leads to a significant protein degradation, yet they are not essential for adhesin trimerization or secretion. Additionnaly, we show that the SLH domains of V. parvula TAAs excludes them from the septum during division, but that this exclusion is not crucial for adhesin function or stability in the tested conditions. Altogether, these results illuminate the genetic flexibility and modularity of autotransporters, enhancing our understanding of this important class of adhesins in diderm bacteria.

microbiology↗

bioRxiv2024

Identification of V. parvula and S. gordonii adhesins mediating co-aggregation and its impact on physiology and mixed biofilm structure

The dental plaque is a polymicrobial community where biofilm formation and co-aggregation, the ability to bind to other bacteria, play a major role in the construction of an organized consortium. One of its prominent members is the anaerobic diderm Veillonella parvula, considered as a bridging species, which growth depends on lactate produced by oral Streptococci. Understanding how V. parvula co-aggregates and the impact of aggregation has long been hampered due to the lack of appropriate genetic tools. Here we studied co-aggregation of the naturally competent strain V. parvula SKV38 with various oral bacteria and its effect on cell physiology. We show that V. parvula requires different trimeric autotransporters of the type V secretion system to adhere to oral Streptococci and Actinomyces. In addition, we describe a novel adhesin of Streptococcus gordonii, VisA (SGO_2004), as the protein responsible for co-aggregation with V. parvula. Finally, we show that co-aggregation does not impact cell-cell communication, which is mainly driven by environmental sensing, but plays an important role in the architecture and species distribution within the biofilm.

microbiology↗

bioRxiv2023

An ancestral dual function of OmpM as outer membrane tether and nutrient uptake channel in diderm Firmicutes

The outer membrane (OM) in diderm, or Gram-negative, bacteria must be tethered to peptidoglycan (PG) for mechanical stability and to maintain cell morphology. Most diderm phyla from the Terrabacteria group lack well-characterised OM attachment systems such as Brauns lipoprotein, Pal and OmpA, but instead have the OmpM protein. OmpM has a periplasmic region containing an S-layer homology domain and a {beta}-barrel region that is embedded in the OM. Here, we have determined the structure of the most abundant OmpM protein from the diderm firmicute Veillonella parvula by single particle cryogenic electron microscopy (cryo-EM). We reveal an OM portion similar to well-characterised trimeric general porins and a mobile periplasmic PG-binding region. Combining the cryo-EM data with crystal structures, structure predictions and molecular dynamics simulations, we show that the periplasmic region of OmpM likely adopts multiple conformations. Single-channel electrophysiology demonstrates ion conductance properties similar to E. coli OmpF. Finally, we demonstrate via functional assays that the four OmpM paralogues are likely to be the only general porins in V. parvula. Together, our results show that OM tethering and nutrient acquisition are genetically linked in V. parvula (and other diderm Firmicutes), suggesting that concurrent loss of two key OM-related functions, tethering and nutrient uptake, promoted the multiple OM loss events that have been inferred in the Terrabacteria.

biochemistry↗

bioRxiv2023

Bridges instead of boats? The Mla system of diderm Firmicute Veillonella parvula reveals an ancestral transenvelope core of phospholipid trafficking

Despite extensive characterisation of envelope biogenesis systems in diderm bacteria, glycerophospholipid (GPL) trafficking remains poorly understood, and has only been studied in a handful of model species. Within the Proteobacteria, the maintenance of lipid asymmetry (Mla) system facilitates retrograde GPL trafficking via six proteins, MlaA-F. GPLs are extracted from the outer leaflet of the outer membrane by the lipoprotein MlaA which associates with porin trimers, then shipped through the periplasmic space by the chaperone MlaC, which finally delivers GPLs to the inner membrane complex formed by MlaBDEF. Here, we investigate GPL trafficking in Veillonella parvula, a diderm member of the Firmicutes which encodes an Mla system devoid of MlaA and MlaC. V. parvula {Delta}mla mutants display phenotypes characteristic of disrupted lipid asymmetry such as hypervesiculation and detergent hypersensitivity, and lipid content analysis from outer membrane vesicles reveals an enrichment for the major lipid component phosphatidylethanolamine. Interestingly, suppressor analysis identifies mutations in tamB that rescue detergent hypersensitivity and hypervesiculation of {Delta}mla strains, supporting the involvement of these two systems in antagonistic GPL trafficking functions across diverse bacterial lineages. A combination of structural modeling and subcellular localisation assays shows that MlaDVp is longer than in classical diderm models and forms a transenvelope bridge, encoding both an inner membrane-localised MCE domain and an outer membrane {beta}-barrel. These results strongly suggest that V. parvula possesses a minimal Mla system for GPL trafficking, replacing the need for chaperones and outer membrane lipoproteins by directly connecting the two membranes. Finally, phylogenomic analysis indicates that this MlaEFD self-contained architecture is widely distributed in diderm bacteria and most likely represents the ancestral functional core of the Mla system, which subsequently increased in complexity in Proteobacteria and closely related phyla following the emergence of MlaABC. Our work broadens the diversity of current models of GPL trafficking in diderm bacteria, challenging the paradigm set by classical models and shedding light on the evolution of a crucial system in the biogenesis and maintenance of the bacterial outer membrane.

microbiology↗

bioRxiv2023

Escherichia coli aggregates mediated by native or synthetic adhesins exhibit both core and adhesin-specific transcriptional responses

Bacteria can rapidly tune their physiology and metabolism to adapt to environmental fluctuations. In particular, they can adapt their lifestyle to the close proximity of other bacteria or presence of different surfaces. However, whether these interactions trigger transcriptomic responses is poorly understood. We used a specific set up of E. coli strains expressing native or synthetic adhesins mediating bacterial aggregation to study the transcriptomic changes of aggregated compared to non-aggregated bacteria. Our results show that following aggregation, bacteria exhibit a core response independent of the adhesin type, with differential expression of 56.9% of the coding genome, including genes involved in stress response and anaerobic lifestyle. Moreover, when aggregates were formed via a naturally expressed E. coli adhesin (Antigen 43), the transcriptomic response of the bacteria was more exaggerated compared to aggregates formed via a synthetic adhesin. This suggests that the response to aggregation induced by native E. coli adhesins could have been finely tuned during bacterial evolution. Our study therefore provides insights on the effect of self-interaction in bacteria and allows a better understanding of why bacterial aggregates exhibit increased stress tolerance. ImportanceFormation of bacterial aggregates has an important role in both clinical and ecological contexts. Although these structures have been previously shown to be more resistant to stressful conditions, the genetic basis of this stress tolerance associated with the aggregate lifestyle is poorly understood. Surface sensing mediated by different adhesins can result in varying changes on bacterial physiology. However, whether adhesin-adhesin interactions as well as the type of adhesin mediating aggregation affects bacterial cell physiology is unknown. By sequencing the transcriptomes of aggregated and non-aggregated cells expressing native or synthetic adhesins, we characterized the effects of aggregation and adhesin type on E. coli physiology.

microbiology↗

bioRxiv2022

Bacterial capsular polysaccharides with antibiofilm activity share common biophysical and electrokinetic properties

Bacterial biofilms are surface-attached communities that are difficult to eradicate due to a high tolerance to antimicrobial agents. The use of non-biocidal surface-active compounds to prevent the initial adhesion and aggregation of bacterial pathogens is a promising alternative to antibiotic treatments and several antibiofilm compounds have been identified, including some capsular polysaccharides released by various bacteria. However, the lack of chemical and mechanistic understanding of the activity of these high-molecular-weight polymers limits their use for control of biofilm formation. Here, we screened a collection of 32 purified capsular polysaccharides and identified seven new compounds with non-biocidal activity against biofilms formed by Escherichia coli and/or Staphylococcus aureus. We analyzed the polysaccharide mobility under applied electric field conditions and showed that active and inactive polysaccharide polymers display distinct electrokinetic properties and that all active macromolecules shared high intrinsic viscosity features. Based on these characteristics, we identified two additional antibiofilm capsular polysaccharides with high density of electrostatic charges and their permeability to fluid flow. Our study therefore provides insights into key biophysical properties discriminating active from inactive polysaccharides. This characterization of a specific electrokinetic signature for polysaccharides displaying antibiofilm activity opens new perspectives to identify or engineer non-biocidal surface-active macromolecules to control biofilm formation in medical and industrial settings. Significance statementSome bacteria produce non-biocidal capsular polysaccharides that reduce the adhesion of bacterial pathogens to surfaces. Due to a lack of molecular and structural definition, the basis of their antiadhesion activity is unknown, thus hindering their prophylactic use for biofilm control. Here, we identified nine new active compounds and compared their composition, structure and biophysical properties with other inactive capsular polysaccharides. Despite the absence of specific molecular motif, we demonstrate that all active polysaccharides share common electrokinetic properties that distinguish them from inactive polymers. This characterization of the biophysical properties of antibiofilm bacterial polysaccharide provides key insights to engineer non-biocidal and bio-inspired surface-active compounds to control bacterial adhesion in medical and industrial settings.

microbiology↗

bioRxiv2022

Intermittent antibiotic treatment of bacterial biofilms favors the rapid evolution of resistance

The rise of antibiotic resistance in bacterial pathogens is a major health concern and the determinants of this emergence are actively studied. By contrast, although biofilms are an important cause of infections due to their high tolerance to a broad range of antimicrobials, much less is known on the development of antibiotic resistance within the biofilm environment, an issue potentially aggravating the current antibiotic crisis. Here, we compared the occurrence of resistance mutations in pathogenic Escherichia coli planktonic and biofilm populations exposed to clinically relevant cycles of lethal treatments with the aminoglycoside antibiotic amikacin. This experimental evolution approach revealed that mutations in sbmA and fusA are rapidly selected in biofilm but not in planktonic populations. The apparition of these bona fide resistance --and not tolerance-- mutations is favored by the biofilm preexisting tolerance and high mutation rate. Moreover, we showed that while fusA mutations displayed a high fitness cost in planktonic conditions, these mutations were maintained in biofilms, a phenomenon further possibly amplified by the selection of fimH mutations favoring biofilm formation itself. Our study therefore provides new insights into the dynamic evolution of antibiotic resistance in biofilms, which could lead to clinically practical antibiotic regimen limiting biofilm-associated infections, while mitigating the emergence of worrisome antibiotic resistance mutations.

microbiology↗

bioRxiv2021

Selection for non-specific adhesion is a driver of FimH evolution increasing Escherichia coli biofilm capacity

Bacterial interactions with surfaces rely on the coordinated expression and interplay of surface exposed adhesion factors. However, how bacteria dynamically modulate their vast repertoire of adhesins to achieve surface colonization is not yet well-understood. We used experimental evolution and positive selection for improved adhesion to investigate how an initially poorly adherent Escherichia coli strain increased its adhesion capacities to abiotic surfaces. We showed that all identified evolved clones acquired mutations located almost exclusively in the lectin domain of fimH, the gene coding for the -D-mannose-specific tip adhesin of type 1 fimbriae. While most of these fimH mutants showed reduced mannose- binding ability, they all displayed enhanced binding to abiotic surfaces, indicating a trade-off between FimH-mediated specific and non-specific adhesion properties. Several of the identified mutations were already reported in FimH lectin domain of pathogenic and environmental E. coli, suggesting that, beyond patho-adaptation, FimH microevolution favoring non-specific surface adhesion could constitute a selective advantage for natural E. coli isolates. Consistently, although E. coli deleted for the fim operon still evolves an increased adhesion capacity, mutants selected in the {Delta}fim background are outcompeted by fimH mutants revealing clonal interference for adhesion. Our study therefore provides insights into the plasticity of E. coli adhesion potential and shows that evolution of type 1 fimbriae is a major driver of the adaptation of natural E. coli to colonization.

microbiology↗

bioRxiv2020

Lifestyle-specific S-nitrosylation of protein cysteine thiols regulates Escherichia coli biofilm formation and resistance to oxidative stress

Communities of bacteria called biofilms are characterized by reduced diffusion, steep oxygen and redox gradients and specific properties compared to individualized planktonic bacteria. In this study, we investigated whether signaling via nitrosylation of protein cysteine thiols (S-nitrosylation), regulating a wide range of functions in eukaryotes, could also specifically occur in biofilms and contribute to bacterial adaptation to this widespread lifestyle. We used a redox proteomic approach to compare cysteine S-nitrosylation in aerobic and anaerobic biofilm and planktonic Escherichia coli cultures and we identified proteins with biofilm-specific S-nitrosylation status. Using bacterial genetics and various phenotypic screens, we showed that impairing S-nitrosylation in proteins involved in redox homeostasis and amino acid synthesis such as OxyR, KatG and GltD altered important biofilm properties, including motility, biofilm maturation or resistance to oxidative stress. Our study therefore revealed that S-nitrosylation constitutes a physiological basis underlying functions critical for E. coli adaptation to the biofilm environment.

microbiology↗

bioRxiv2020

Visualizing the dynamics of exported bacterial proteins with the chemogenetic fluorescent reporter FAST

Bacterial proteins exported to the cell surface play key cellular functions. However, despite the interest to study the localization of surface proteins such as adhesins, transporters or hydrolases, monitoring their dynamics in live imaging remains challenging, due to the limited availability of fluorescent probes remaining functional after secretion. In this work, we used the Escherichia coli intimin and the Listeria monocytogenes InlB invasin as surface exposed scaffolds fused with the recently developed chemogenetic fluorescent reporter protein FAST. Using both membrane permeant (HBR-3,5DM) and non-permeant (HBRAA-3E) fluorogens that fluoresce upon binding to FAST, we demonstrated that fully functional FAST can be exposed at the cell surface and specifically tagged on the external side of the bacterial envelop in both diderm and monoderm bacteria. Our work opens new avenues to study of the organization and dynamics of the bacterial cell surface proteins.

microbiology↗

bioRxiv2020

Capsular polysaccharides cross-regulation modulates Bacteroides thetaiotaomicron biofilm formation

Bacteroides thetaiotaomicron is one of the most abundant gut symbiont species, whose contribution to host health through its ability to degrade diet polysaccharides and mature the immune system is under untense scrutiny. By contrast, adhesion and biofilm formation, which are potentially involved in gut colonization, microbiota structure and stability, have hardly been investigated in this intestinal bacterium. To uncover B. thetaiotaomicron biofilm-related functions, we performed a transposon mutagenesis in the poor biofilm-forming reference strain VPI 5482 and showed that capsule 4, one of the eight B. thetaiotaomicron capsules, hinders biofilm formation. We then showed that the production of capsules 1, 2, 3, 5 and 6 also inhibits biofilm formation and that decreased capsulation of the population correlated with increased biofilm formation, suggesting that capsules could be masking adhesive surface structures. We also showed that, by contrast, capsule 8 displayed intrinsic adhesive properties. Finally, we demonstrated that BT2934, the wzx homolog of B. thetaiotaomicron glycosylation locus, competes with capsule production and therefore impacts its adhesion capacity. This study identified regulation of capsular polysaccharides as a major determinant of B. thetaiotaomicron biofilm formation, providing new insights into how modulation of different B. thetaiotaomicron surface structures affect in vitro biofilm formation.

microbiology↗