Adhésine bactérienne | EPFL Graph Search

Adhesins are cell-surface components or appendages of bacteria that facilitate adhesion or adherence to other cells or to surfaces, usually in the host they are infecting or living in. Adhesins are a type of virulence factor. Adherence is an essential step in bacterial pathogenesis or infection, required for colonizing a new host. Adhesion and bacterial adhesins are also a potential target for prophylaxis or treatment of bacterial infections. Bacteria are typically found attached to and living in close association with surfaces. During the bacterial lifespan, a bacterium is subjected to frequent shear-forces. In the crudest sense, bacterial adhesins serve as anchors allowing bacteria to overcome these environmental shear forces, thus remaining in their desired environment. However, bacterial adhesins do not serve as a sort of universal bacterial Velcro. Rather, they act as specific surface recognition molecules, allowing the targeting of a particular bacterium to a particular surface such as root tissue in plants, lacrimal duct tissues in mammals, or even tooth enamel. Most fimbria of gram-negative bacteria function as adhesins, but in many cases it is a minor subunit protein at the tip of the fimbriae that is the actual adhesin. In gram-positive bacteria, a protein or polysaccharide surface layer serves as the specific adhesin. To effectively achieve adherence to host surfaces, many bacteria produce multiple adherence factors called adhesins. Bacterial adhesins provide species and tissue tropism. Adhesins are expressed by both pathogenic bacteria and saprophytic bacteria. This prevalence marks them as key microbial virulence factors in addition to a bacterium's ability to produce toxins and resist the immune defenses of the host. Through the mechanisms of evolution, different species of bacteria have developed different solutions to the problem of attaching receptor specific proteins to the bacteria surface. Today many different types and subclasses of bacterial adhesins may be observed in the literature.

Engineering synthetic adhesins for biophysical investigation and rewiring of host-microbe interactions

Xavier Jean-Yves Pierrat

Host attachment is often a critical step in the onset of pathogenesis. To attach to host cells, bacteria have evolved a range of adhesins that bind to specific receptors. Some of these adhesins have been thoroughly characterized using biochemical techniques. However, how adhesins engage with their receptors in a realistic context of host colonization remains obscure. For instance, how target cell surface properties regulate attachment has been overlooked. This hinders our understanding of pathogenicity, thereby limiting our ability to develop new therapeutic approaches. Here, we aimed at characterizing the biophysical rules underlying bacterial attachment to live cells. In this context, we displayed synthetic adhesins on both bacterial and mammalian target cell surfaces to study how the mammalian membrane microenvironment regulates attachment. By leveraging microfluidics and high-temporal resolution confocal microscopy, we tracked the early adhesion of bacteria to target cells and compared it to abiotic surfaces. We modeled the distribution of residence times and uncovered that the binding to mammalian cells is a two-step process, as opposed to one-step binding to an abiotic surface. In particular, we highlight the impact of the mammalian cell glycocalyx and of the actin-mediated cell remodeling. Altogether, our results demonstrate that adhesin-ligand binding is not the only regulator of bacterial adhesion, due to the host mechanical microenvironment playing a critical role on the initiation of infection.With a better knowledge of in vivo adhesion, we repurposed the synthetic adhesin system as a tool for bacterial-based therapy. Our plan consists in using adhesion to rewire host-pathogen interactions. By analogy with pathogenic viruses transformed into therapeutic gene delivery vectors, we focused on pathogenic bacteria injecting DNA to eukaryotic cells. Agrobacterium tumefaciens is a pathogen that delivers DNA to plants using its type IV secretion. It is widely used for gene editing in plants, and sometimes in yeast and fungal cells. It is therefore an attractive candidate as a human gene delivery vector. However, some cell types such as plant monocots or animal cells show extremely low transformation efficiency. Studies demonstrated a positive correlation between adhesion to recalcitrant plants and transformation efficiency. Hence, would a synthetic binding of A. tumefaciens to non-natural target cells increase delivery? To measure the impact of adhesion on delivery efficiency, we repurposed an endogenous autotransporter of A. tumefaciens to display the previously characterized synthetic adhesin. This significantly increased the binding to yeast and mammalian cells displaying the target surface receptor. In addition, we developed a split luciferase assay to quantify the transfer of helper proteins to target mammalian cell. This allowed us to optimize A. tumefaciens-mediated delivery to mammalian cells and to refine hypotheses concerning the translocation mechanisms involved in mammalian cells. Altogether, we show that synthetic adhesins are a valuable tool to improve our understanding of host-microbe interactions and for repurposing pathogens into therapeutic tools.

EPFL2022

Identification of genes required for host-cell adhesion and antibiotic persistence of uropathogenic Escherichia coli via high-content screening

Thomas Marie Simonet

Uropathogenic Escherichia coli (UPEC) are the most common bacterial pathogens causing urinary tract infections (UTIs). Driven by the development of antibiotic resistant UPEC strains, UTIs have become a major public health issue and generate substantial healthcare costs. Even in the absence of antibiotic resistance, UTIs are notoriously difficult to treat and have high rates of recurrence, mostly due to: 1. the ability of UPEC to adhere to and invade bladder epithelial cells, which protects them from the action of antimicrobial agents and the immune system 2. the presence in antibiotic-susceptible UPEC populations of a small fraction of so-called persister cells, which are killed at a slower rate than the bulk of the population and can therefore survive prolonged antibiotic exposureThese two features allow some bacteria to remain viable in a protected niche for the whole duration of the antibiotic treatment, potentially leading to infection relapse when the treatment is terminated.High-content screening (HCS), which uses microscopy as a screening tool, allows the visualization of bacterial phenotypes at the single-cell level and can therefore provide a powerful and unbiased tool to understand the genetic basis of these phenotypes. The present thesis describes two complementary high-content screening approaches aimed at identifying UPEC mutants with altered adhesion to bladder cells and with altered antibiotic persistence respectively.In a first chapter, we report preparatory steps that led to the construction of a transposon insertion library of UPEC mutants that is suitable for high-content imaging, using the CFT073 clinical UTI isolate as background strain.In the second chapter, we performed fluorescence microscopy-based high-content screening to identify mutants with defects in early adhesion to bladder epithelial cells. We recovered 82 mutants with decreased adhesion and 54 mutants with increased adhesion. Unexpectedly, nine low-adhesion ÂÂhitsÂÂ mapped to the two P pili operons encoded by CFT073, which are thought to mediate adhesion to kidney cells rather than bladder cells. Additionally, six high-adhesion hits mapped to the operon coding for F1C pili, and we show that this phenotype is linked to increased P pili synthesis. These results therefore reveal a critical role for P pili in UPEC adhesion to bladder epithelial cells, which may inform the development of novel anti-adhesion therapies to prevent UTI recurrence.In the third chapter, we combined microfluidics with time-lapse microscopy to identify mutants with altered persistence to fosfomycin (FOS) âÂÂ a cell-wall inhibitor used as first-line agent in the treatment of UTIs âÂÂ in synthetic human urine (SHU), which mimics the physiology of UPEC in real urine. We recovered four mutants with decreased persistence, three of which had defects in lipopolysaccharide (LPS) synthesis. These results identify the outer membrane of UPEC as a key component for survival to FOS treatment, thus opening up new avenues in the fight against antibiotic persistence.

EPFL2022

Identification of Allobaculum mucolyticum as a novel human intestinal mucin degrader

Guido Van Mierlo

The human gut microbiota plays a central role in intestinal health and disease. Yet, many of its bacterial constituents are functionally still largely unexplored. A crucial prerequisite for bacterial survival and proliferation is the creation and/or exploitation of an own niche. For many bacterial species that are linked to human disease, the inner mucus layer was found to be an important niche. Allobaculum mucolyticum is a newly identified, IBD-associated species that is thought be closely associated with the host epithelium. To explore how this bacterium is able to effectively colonize this niche, we screened its genome for factors that may contribute to mucosal colonization. Up to 60 genes encoding putative Carbohydrate Active Enzymes (CAZymes) were identified in the genome of A. mucolyticum. Mass spectrometry revealed 49 CAZymes of which 26 were significantly enriched in its secretome. Functional assays demonstrated the presence of CAZyme activity in A. mucolyticum conditioned medium, degradation of human mucin O-glycans, and utilization of liberated non-terminal monosaccharides for bacterial growth. The results support a model in which sialidases and fucosidases remove terminal O-glycan sugars enabling subsequent degradation and utilization of carbohydrates for A. mucolyticum growth. A. mucolyticum CAZyme secretion may thus facilitate bacterial colonization and degradation of the mucus layer and may pose an interesting target for future therapeutic intervention.

TAYLOR & FRANCIS INC2021

Identification of genes required for host-cell adhesion and antibiotic persistence of uropathogenic Escherichia coli via high-content screening

Thomas Marie Simonet

EPFL2022

Engineering synthetic adhesins for biophysical investigation and rewiring of host-microbe interactions

Xavier Jean-Yves Pierrat

EPFL2022