Rôle de l'agrégation bactérienne sur la physiologie de Escherichia coli

Bacterial colonization of the environment and host tissues depends mostly on their ability to interact and adhere robustly to target surfaces as well as to each other in order to withstand the fluxes and perturbations they may encounter. Bacterial colonization involves proteinaceous cellular appendages called adhesins, composed of fimbrial or afimbrial structures exposed to the bacterial surface by a variety of secretion systems. While the process of adhesion to abiotic or mucosal surfaces is well described, the adhesion of bacteria to other bacteria of the same species (self-aggregation) or to unrelated species (co-aggregation) is less studied. While being much smaller than mature biofilms these aggregates maintain some of their characteristics including enhanced tolerance to various stresses among which antibiotics, predation and immune system. To better understand the aggregation phenomenon observed in E. coli, we identified among its numerous adhesins those capable of mediating self-aggregation mechanisms, some of which displaying such properties in specific conditions. We studied the consequences of these aggregation mechanisms on the physiology of E. coli. In particular, we tried to explain why the aggregates are more resistant than planktonic bacteria to certain stresses. This question has been studied by comparative transcriptomic studies on aggregates formed by E. coli versus planktonic strains of the same species. And finally, we showed that a fluorescent tool used in eukaryotes, FAST, can be used to study extracellular proteins in both gram-positive and gram-negative bacteria and we used it to study the dynamic at the surface of Ag43, the main aggregation adhesin of E. coli.La colonisation bactérienne de l'environnement et des tissus hôtes dépend principalement de leur capacité à interagir et à adhérer de manière robuste aux surfaces ainsi qu'entre elles, afin de résister aux flux et aux perturbations qu'elles peuvent rencontrer. La colonisation bactérienne implique des appendices cellulaires protéiques appelés adhésines, composés de structures fimbriales ou afimbriales exposées à la surface bactérienne par divers systèmes de sécrétion. Si le processus d'adhésion aux surfaces abiotiques ou muqueuses est bien décrit, l'adhésion des bactéries à d'autres bactéries de la même espèce (auto-agrégation) ou à des espèces non apparentées (co-agrégation) est moins étudiée. Bien qu'ils soient beaucoup plus petits que les biofilms matures, ces agrégats conservent certaines de leurs caractéristiques, notamment une meilleure tolérance à divers stress, dont les antibiotiques, la prédation et le système immunitaire. Pour mieux comprendre le phénomène d'agrégation observé chez E. coli, nous avons identifié, parmi ses nombreuses adhésines, celles capables de déclencher des mécanismes d'auto-agrégation. Nous avons étudié les conséquences de ces mécanismes d'agrégation sur la physiologie d'E. coli. En particulier, nous tentons d'expliquer pourquoi les agrégats sont plus résistants que les bactéries planctoniques à certains stress. Cette question a été étudiée via des analyses transcriptomiques comparatives sur des agrégats formés par E. coli. Enfin, nous avons montré qu'un outil fluorescent utilisé chez les eucaryotes, FAST, peut être utilisé pour étudier les protéines extracellulaires chez les bactéries gram-positives et gram-négatives et nous l'avons utilisé pour étudier la dynamique à la surface de l'Ag43, la principale adhésine d'agrégation d'E. coli

Rôle de l'agrégation bactérienne sur la physiologie de Escherichia coli

La colonisation bactérienne de l'environnement et des tissus hôtes dépend principalement de leur capacité à interagir et à adhérer de manière robuste aux surfaces ainsi qu'entre elles, afin de résister aux flux et aux perturbations qu'elles peuvent rencontrer. La colonisation bactérienne implique des appendices cellulaires protéiques appelés adhésines, composés de structures fimbriales ou afimbriales exposées à la surface bactérienne par divers systèmes de sécrétion. Si le processus d'adhésion aux surfaces abiotiques ou muqueuses est bien décrit, l'adhésion des bactéries à d'autres bactéries de la même espèce (auto-agrégation) ou à des espèces non apparentées (co-agrégation) est moins étudiée. Bien qu'ils soient beaucoup plus petits que les biofilms matures, ces agrégats conservent certaines de leurs caractéristiques, notamment une meilleure tolérance à divers stress, dont les antibiotiques, la prédation et le système immunitaire. Pour mieux comprendre le phénomène d'agrégation observé chez E. coli, nous avons identifié, parmi ses nombreuses adhésines, celles capables de déclencher des mécanismes d'auto-agrégation. Nous avons étudié les conséquences de ces mécanismes d'agrégation sur la physiologie d'E. coli. En particulier, nous tentons d'expliquer pourquoi les agrégats sont plus résistants que les bactéries planctoniques à certains stress. Cette question a été étudiée via des analyses transcriptomiques comparatives sur des agrégats formés par E. coli. Enfin, nous avons montré qu'un outil fluorescent utilisé chez les eucaryotes, FAST, peut être utilisé pour étudier les protéines extracellulaires chez les bactéries gram-positives et gram-négatives et nous l'avons utilisé pour étudier la dynamique à la surface de l'Ag43, la principale adhésine d'agrégation d'E. coli.Bacterial colonization of the environment and host tissues depends mostly on their ability to interact and adhere robustly to target surfaces as well as to each other in order to withstand the fluxes and perturbations they may encounter. Bacterial colonization involves proteinaceous cellular appendages called adhesins, composed of fimbrial or afimbrial structures exposed to the bacterial surface by a variety of secretion systems. While the process of adhesion to abiotic or mucosal surfaces is well described, the adhesion of bacteria to other bacteria of the same species (self-aggregation) or to unrelated species (co-aggregation) is less studied. While being much smaller than mature biofilms these aggregates maintain some of their characteristics including enhanced tolerance to various stresses among which antibiotics, predation and immune system. To better understand the aggregation phenomenon observed in E. coli, we identified among its numerous adhesins those capable of mediating self-aggregation mechanisms, some of which displaying such properties in specific conditions. We studied the consequences of these aggregation mechanisms on the physiology of E. coli. In particular, we tried to explain why the aggregates are more resistant than planktonic bacteria to certain stresses. This question has been studied by comparative transcriptomic studies on aggregates formed by E. coli versus planktonic strains of the same species. And finally, we showed that a fluorescent tool used in eukaryotes, FAST, can be used to study extracellular proteins in both gram-positive and gram-negative bacteria and we used it to study the dynamic at the surface of Ag43, the main aggregation adhesin of E. coli

Rôle de l'agrégation bactérienne sur la physiologie de Escherichia coli

Bacterial colonization of the environment and host tissues depends mostly on their ability to interact and adhere robustly to target surfaces as well as to each other in order to withstand the fluxes and perturbations they may encounter. Bacterial colonization involves proteinaceous cellular appendages called adhesins, composed of fimbrial or afimbrial structures exposed to the bacterial surface by a variety of secretion systems. While the process of adhesion to abiotic or mucosal surfaces is well described, the adhesion of bacteria to other bacteria of the same species (self-aggregation) or to unrelated species (co-aggregation) is less studied. While being much smaller than mature biofilms these aggregates maintain some of their characteristics including enhanced tolerance to various stresses among which antibiotics, predation and immune system. To better understand the aggregation phenomenon observed in E. coli, we identified among its numerous adhesins those capable of mediating self-aggregation mechanisms, some of which displaying such properties in specific conditions. We studied the consequences of these aggregation mechanisms on the physiology of E. coli. In particular, we tried to explain why the aggregates are more resistant than planktonic bacteria to certain stresses. This question has been studied by comparative transcriptomic studies on aggregates formed by E. coli versus planktonic strains of the same species. And finally, we showed that a fluorescent tool used in eukaryotes, FAST, can be used to study extracellular proteins in both gram-positive and gram-negative bacteria and we used it to study the dynamic at the surface of Ag43, the main aggregation adhesin of E. coli.La colonisation bactérienne de l'environnement et des tissus hôtes dépend principalement de leur capacité à interagir et à adhérer de manière robuste aux surfaces ainsi qu'entre elles, afin de résister aux flux et aux perturbations qu'elles peuvent rencontrer. La colonisation bactérienne implique des appendices cellulaires protéiques appelés adhésines, composés de structures fimbriales ou afimbriales exposées à la surface bactérienne par divers systèmes de sécrétion. Si le processus d'adhésion aux surfaces abiotiques ou muqueuses est bien décrit, l'adhésion des bactéries à d'autres bactéries de la même espèce (auto-agrégation) ou à des espèces non apparentées (co-agrégation) est moins étudiée. Bien qu'ils soient beaucoup plus petits que les biofilms matures, ces agrégats conservent certaines de leurs caractéristiques, notamment une meilleure tolérance à divers stress, dont les antibiotiques, la prédation et le système immunitaire. Pour mieux comprendre le phénomène d'agrégation observé chez E. coli, nous avons identifié, parmi ses nombreuses adhésines, celles capables de déclencher des mécanismes d'auto-agrégation. Nous avons étudié les conséquences de ces mécanismes d'agrégation sur la physiologie d'E. coli. En particulier, nous tentons d'expliquer pourquoi les agrégats sont plus résistants que les bactéries planctoniques à certains stress. Cette question a été étudiée via des analyses transcriptomiques comparatives sur des agrégats formés par E. coli. Enfin, nous avons montré qu'un outil fluorescent utilisé chez les eucaryotes, FAST, peut être utilisé pour étudier les protéines extracellulaires chez les bactéries gram-positives et gram-négatives et nous l'avons utilisé pour étudier la dynamique à la surface de l'Ag43, la principale adhésine d'agrégation d'E. coli

Sequence motifs recognized by the casposon integrase of Aciduliprofundum boonei

International audienceCasposons are a group of bacterial and archaeal DNA transposons encoding a specific integrase, termed casposase, which is homologous to the Cas1 enzyme responsible for the integration of new spacers into CRISPR loci. Here, we characterized the sequence motifs recognized by the casposase from a thermophilic archaeon Aciduliprofundum boonei. We identified a stretch of residues, located in the leader region upstream of the actual integration site, whose deletion or mutagenesis impaired the concerted integration reaction. However, deletions of two-thirds of the target site were fully functional. Various single-stranded 6-FAM-labelled oligonucleotides derived from casposon terminal inverted repeats were as efficiently incorporated as duplexes into the target site. This result suggests that, as in the case of spacer insertion by the CRISPR Cas1-Cas2 integrase, casposon integration involves splaying of the casposon termini, with single-stranded ends being the actual substrates. The sequence critical for incorporation was limited to the five terminal residues derived from the 3' end of the casposon. Furthermore, we characterize the casposase from Nitrosopumilus koreensis, a marine member of the phylum Thaumarchaeota, and show that it shares similar properties with the A. boonei enzyme, despite belonging to a different family. These findings further reinforce the mechanistic similarities and evolutionary connection between the casposons and the adaptation module of the CRISPR-Cas systems

Escherichia coli Aggregates Mediated by Native or Synthetic Adhesins Exhibit Both Core and Adhesin-Specific Transcriptional Responses

International audienceBacteria 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 the presence of different surfaces. However, whether these interactions trigger transcriptomic responses is poorly understood. We used a specific setup of E. coli strains expressing native or synthetic adhesins mediating bacterial aggregation to study the transcriptomic changes of aggregated compared to nonaggregated 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 than that of 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 into the effect of self-interaction in bacteria and allows a better understanding of why bacterial aggregates exhibit increased stress tolerance.IMPORTANCE The formation 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 various changes in bacterial physiology. However, whether adhesin-adhesin interactions, as well as the type of adhesin mediating aggregation, affect bacterial cell physiology is unknown. By sequencing the transcriptomes of aggregated and nonaggregated cells expressing native or synthetic adhesins, we characterized the effects of aggregation and adhesin type on E. coli physiology

On the strong connection between nanoscale adhesion of Yad fimbriae and macroscale attachment of Yad-decorated bacteria to glycosylated, hydrophobic and hydrophilic surfaces

Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal's standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.International audienceYad fimbriae are currently viewed as versatile bacterial adhesins able to significantly mediate host or plant-pathogen recognition and contribute to the persistence of Escherichia coli in both the environment and within hosts. To date, however, the underlying adhesion process of Yad fimbriae on surfaces defined by controlled coating chemistries has not been evaluated on the relevant molecular scale. In this work, the interaction forces operational between Yad fimbriae expressed by genetically modified E. coli and self-assembled monolayers (SAM) differing in terms of charge, hydrophobicity or the nature of decorating sugar units are quantified by Single Molecule Force Spectroscopy (SMFS) on the nanoscale. It is found that the adhesion of Yad fimbriae onto probes functionalized with xylose is as strong as that measured with probes decorated with anti-Yad antibodies (ca. 80 to 300 pN). In contrast, the interactions of Yad with galactose, lactose, mannose, -OH, -NH2, -COOH and -CH3 terminated SAMs are clearly non-specific. Interpretation of SMFS measurements on the basis of worm-like-chain modeling for polypeptide nanomechanics further leads to the estimates of the maximal extension of Yad fimbriae upon stretching, of their persistence length and of their polydispersity. Finally, we show for the first time a strong correlation between the adhesion properties of Yad-decorated bacteria determined from conventional macroscopic counting methods and the molecular adhesion capacity of Yad fimbriae. This demonstration advocates the effort that should be made to understand on the nanoscale level the interactions between fimbriae and their cognate ligands. The results could further help the design of potential anti-adhesive molecules or surfaces to better fight against the virulence of bacterial pathogens

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

International audienceBacterial proteins exported to the cell surface play key cellular functions. However, despite the interest to study the localisation 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 used to specifically tag the external side of the bacterial envelop in both diderm and monoderm bacteria. Our work opens new avenues to study the organization and dynamics of the bacterial cell surface proteins

Biophysical insights into sugar-dependent medium acidification promoting YfaL protein-mediated Escherichia coli self-aggregation, biofilm formation and acid stress resistance

International audienceThe ability of bacteria to interact with their environement is crucial to form aggregates, biofilms, and develop acollective stress resistance behavior. Despite its environmental and medical importance, bacterial aggregation is poorlyunderstood and mediated by few known adhesion structures. We identified here a novel surface-exposed Escherichia coliprotein, YfaL, that can self-recognize and induce bacterial autoaggregation. This process occurs only under acidicconditions generated during E. coli growth in the presence of fermentable sugars. These findings were supported byelectrokinetic and atomic force spectroscopy measurements, which revealed changes in the electrostatic, hydrophobic,and structural properties of YfaL upon sugar consumption. Furthermore, YfaL-mediated autoaggregation promotes biofilmformation and enhances E. coli's resistance to acid stress. The prevalence and conservation of YfaL in environmental andclinical E. coli suggest strong evolutionary selection for its function inside or outside the host. Overall, our resultsemphasize the importance of environmental parameters such as low pH as physicochemical cues influencing bacterialadhesins and aggregation, affecting E. coli and potentially other bacteria's resistance to environmental stress

Biophysical insights into sugar-dependent medium acidification promoting YfaL protein-mediated Escherichia coli self-aggregation, biofilm formation and acid stress resistance

International audienceThe ability of bacteria to interact with their environement is crucial to form aggregates, biofilms, and develop acollective stress resistance behavior. Despite its environmental and medical importance, bacterial aggregation is poorlyunderstood and mediated by few known adhesion structures. We identified here a novel surface-exposed Escherichia coliprotein, YfaL, that can self-recognize and induce bacterial autoaggregation. This process occurs only under acidicconditions generated during E. coli growth in the presence of fermentable sugars. These findings were supported byelectrokinetic and atomic force spectroscopy measurements, which revealed changes in the electrostatic, hydrophobic,and structural properties of YfaL upon sugar consumption. Furthermore, YfaL-mediated autoaggregation promotes biofilmformation and enhances E. coli's resistance to acid stress. The prevalence and conservation of YfaL in environmental andclinical E. coli suggest strong evolutionary selection for its function inside or outside the host. Overall, our resultsemphasize the importance of environmental parameters such as low pH as physicochemical cues influencing bacterialadhesins and aggregation, affecting E. coli and potentially other bacteria's resistance to environmental stress