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

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.

The Mammalian Membrane Microenvironment Regulates the Sequential Attachment of Bacteria to Host Cells

Zaïnebe Al-Mayyah, Alexandre Louis André Persat, Xavier Jean-Yves Pierrat

Pathogen attachment to host tissue is critical in the progress of many infections. Bacteria use adhesion in vivo to stabilize colonization and subsequently regulate the deployment of contact-dependent virulence traits. To specifically target host cells, they decorate themselves with adhesins, proteins that bind to mammalian cell surface receptors. One common assumption is that adhesin-receptor interactions entirely govern bacterial attachment. However, how adhesins engage with their receptors in an in vivo-like context remains unclear, in particular under the influence of a heterogeneous mechanical microenvironment. We here investigate the biophysical processes governing bacterial adhesion to host cells using a tunable adhesin-receptor system. By dynamically visualizing attachment, we found that bacterial adhesion to host cell surface, unlike adhesion to inert surfaces, involves two consecutive steps. Bacteria initially attach to their host without engaging adhesins. This step lasts about 1 min, during which bacteria can easily detach. We found that at this stage, the glycocalyx, a layer of glycosylated proteins and lipids, shields the host cell by keeping adhesins away from their receptor ligand. In a second step, adhesins engage with their target receptors to strengthen attachment for minutes to hours. The active properties of the membrane, endowed by the actin cytoskeleton, strengthen specific adhesion. Altogether, our results demonstrate that adhesin-ligand binding is not the sole regulator of bacterial adhesion. In fact, the host cell’s surface mechanical microenvironment mediates the physical interactions between host and bacteria, thereby playing an essential role in the onset of infection.

2021

The role of the inflammasome in cellular responses to toxins and bacterial effectors

Françoise Gisou van der Goot Grunberg

Invading pathogens are recognized by mammalian cells through dedicated receptors found either at the cell surface or in the cytoplasm. These receptors, like the trans-membrane Toll-like Receptors (TLR) or the cytosolic Nod-like Receptors (NLR), initiate innate immunity after recognition of molecular patterns found in bacteria or viruses, such as LPS, flagellin, or double-stranded RNA. Recognition of molecules produced only by a specific pathogen, such as a viral envelop protein or a bacterial adhesin does not appear to occur. Bacterial protein toxins, however, might compose an intermediate class. Considering the diversity of toxins in terms of structure, it is unlikely that cells respond to them via specific molecular recognition. It rather appears that different classes of toxins trigger cellular changes that are sensed by the cells as danger signals, such as changes in cellular ion composition after membrane perforation by pore-forming toxins or type III secretion systems. The signaling pathways triggered through toxin-induced cell alterations will likely play a role in modulating host responses to virulent bacteria. We will here describe the few studied cases in which detection of the toxin by the host cell was addressed. The review will include not only toxins but also bacteria effectors secreted by the bacterium in to the host cell cytoplasm.

Springer-Verlag2007

Structure of viral membrane-penetrating machines by electron cryo-microscopy and tomography

Sergey Nazarov

My PhD thesis aims to obtain the structure of several bacteriophages, including so-called “jumbo” phages, using the electron cryo-microscopy (cryo-EM) and to study the structural transformation of these viruses as they attach and infect the host cell. We aim to understand the process of host cell membrane penetration at the molecular level and to achieve the level of knowledge necessary to understand how the genome and proteins, which are originally packaged into the capsid, are delivered into the host cell. This structural information can be of great importance since bacteriophage therapy is a potentially promising alternative to antibiotics, which are urgently needed. Bacteriophages, or phages are the most abundant and diverse form of life on Earth, and have developed various strategies through which to infect a susceptible bacterial host. A vast majority of phages have evolved to use a special organelle, called a “tail”, for host recognition, attachment and genome delivery into the cell. The host cell envelope is penetrated with the help of the tail. Unlike most eukaryotic viruses, infection of a host by tailed bacteriophages usually requires only one virion per bacterium, indicating that tailed bacteriophages have evolved an extremely efficient infection mechanism. Bacteriophages with contractile tails are complex viruses that infect Bacteria and Archaea. They share a common evolutionary origin and some functional aspect with other large macromolecular machines, such as the Serratia entomophila antifeeding prophage, the Photorhabdus virulence cassette, and the type VI secretion system (T6SS), which function to attack other cells by translocating toxic effector proteins into the target cell’s cytoplasm. The T6SS and the so-called “jumbo” phages are the most complex of these contractile injection systems. Jumbo phages have genomes exceeding 250kb and virus particle sizes of 300 nm and greater. Hundreds of different proteins are involved in the morphogenesis and assembly of a jumbo phage particle. A number of bacteriophages infecting important pathogens were isolated and characterized by means of cryo-EM and bioinformatics tools. Bacteriophage ɸ92 is a virus infecting a wide variety of acapsular Escherichia coli laboratory strains, Salmonella strains, and pathogenic E.Coli strains carrying a polysialic acid, which cause severe invasive infections including meningitis, pneumonia, septicaemia, osteomyelitis, septic arthritis and pyelonephritis. A cryo-EM reconstructions of the ɸ92 capsid, baseplate and long contractile tail were calculated, and complete three-dimensional model of the phage virion was built. Two bacteriophages, ɸEco32 infecting mastitis-causing strains of E.Coli and 7-11 infecting Salmonella strains, were isolated. They are a members of the Podoviridae family with the rare C3 morphotype, which occurs in

EPFL2015

Structure of viral membrane-penetrating machines by electron cryo-microscopy and tomography

Sergey Nazarov

EPFL2015