Combinatorial actions of bacterial effectors revealed by exploiting genetic tools in yeast

While yeast has been extensively used as a model system for analysing protein–protein and genetic interactions, in the context of bacterial pathogenesis, the use of yeast-based tools has largely been limited to identifying interactions between pathogen effectors and host targets. In their recent work, Ensminger and colleagues (Urbanus et al, 2016) use the combinatorial power of yeast genetics to systematically screen all known Legionella pneumophila effector proteins for effector–effector interactions. They provide new insights into how bacterial effectors balance host cell perturbation and describe mechanisms used by “meta-effectors” to directly modulate target effector activity [...

A conserved protein, BcmA, mediates motility, biofilm formation, and host colonisation in Adherent Invasive Escherichia coli

Adherent Invasive Escherichia coli (AIEC) is a non-diarrhoeagenic intestinal E. coli pathotype associated with Crohn’s Disease. AIEC pathogenesis is characterised by biofilm formation, adhesion to and invasion of intestinal epithelial cells, and intracellular replication within epithelial cells and macrophages. Here, we identify and characterise a protein in the prototypical AIEC strain LF82 which is required for efficient biofilm formation and dispersal – LF82_p314. LF82 ΔLF82_314 have defective swimming and swarming motility, indicating LF82_p314 is important for flagellar-mediated motility, and thus surface colonisation and biofilm dispersal. Flagellar morphology and chemotaxis in liquid appear unaffected by deletion of LF82_314, suggesting LF82_p314 does not elicit an effect on flagella biogenesis or environmental sensing. Flagellar motility has been implicated in AIEC virulence, therefore we assessed the role of LF82_p314 in host colonisation using a Caenorhabditis elegans model. We found that LF82 ΔLF82_314 have an impaired ability to colonise the C. elegans compared to wild-type LF82. Phylogenetic analysis showed that LF82_314 is conserved in several major enterobacterial pathogens, and suggests the gene may have been acquired horizontally in several genera. Our data suggests LF82_p314 may be a novel component in the flagellar motility pathway and is a novel determinant of AIEC colonisation. Our findings have potential implications not only for the pathogenesis of Crohn’s Disease, but also for the course of infection in several major bacterial pathogens. We propose a new designation for LF82_314, biofilm coupled to motility A, or bcmA

A mechanoresponsive nano-sized carrier achieves intracellular release of drug on external ultrasound stimulus

Control over intracellular release of therapeutic compounds incorporated into nano-carriers will open new possibilities for targeted treatments of various diseases including cancer, and viral and bacterial infections. Here we report our study on mechanoresponsive nano-sized liposomes which, following internalization by cells, achieve intracellular delivery of encapsulated cargo on application of external ultrasound stimulus. This is demonstrated in a bespoke cell reporter system designed to assess free drug in cytoplasm. Biophysical analyses show that drug release is attributable to the action of a mechanoresponsive spiropyran-based compound embedded in the liposomal lipid membrane. Exposure to external ultrasound stimulus results in opening of the molecular structure of the embedded spiropyran, a consequent increase in liposomal lipid membrane fluidity, and size-dependent release of encapsulated model drugs, all pointing to lipid bilayer perturbation. The study hence illustrates feasibility of the proposed concept where intracellular drug release from mechanoresponsive liposomes can be triggered on demand by external ultrasound stimulus

Picosecond ultrasonics for elasticity-based imaging and characterization of biological cells

© 2020 Author(s). Characterization of the elasticity of biological cells is growing as a new way to gain insight into cell biology. Cell mechanics are related to most aspects of cellular behavior, and applications in research and medicine are broad. Current methods are often limited since they require physical contact or lack resolution. From the methods available for the characterization of elasticity, those relying on high frequency ultrasound (phonons) are the most promising because they offer label-free, high (even super-optical) resolution and compatibility with conventional optical microscopes. In this Perspective contribution, we review the state of the art of picosecond ultrasonics for cell imaging and characterization, particularly for Brillouin scattering-based methods, offering an opinion for the challenges faced by the technology. The challenges are separated into biocompatibility, acquisition speed, resolution, and data interpretation and are discussed in detail along with new results

Impaired Autophagy of an Intracellular Pathogen Induced by a Crohn's Disease Associated ATG16L1 Variant

The genetic risk factors predisposing individuals to the development of inflammatory bowel disease are beginning to be deciphered by genome-wide association studies. Surprisingly, these new data point towards a critical role of autophagy in the pathogenesis of Crohn's disease. A single common coding variant in the autophagy protein ATG16L1 predisposes individuals to the development of Crohn's disease: while ATG16L1 encoding threonine at amino acid position 300 (ATG16L1*300T) confers protection, ATG16L1 encoding for alanine instead of threonine (ATG16L1*300A, also known as T300A) mediates risk towards the development of Crohn's disease. Here we report that, in human epithelial cells, the Crohn's disease-associated ATG16L1 coding variant shows impairment in the capture of internalized Salmonella within autophagosomes. Thus, we propose that the association of ATG16L1*300A with increased risk of Crohn's disease is due to impaired bacterial handling and lowered rates of bacterial capture by autophagy

LF82 genes cloned

FASTA files containing the genes cloned for the expression librar

Investigating the innate immune response to Citrobacter rodentium infection of mice

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Building complex biological networks based upon model organisms: Mapping the human autophagy interactome via a hybrid yeast-human protein interaction network

Autophagy is a process whose core machinery is highly conserved from yeast to higher eukaryotes and mammals. However it is becoming clear that multicellular organisms exhibit increased complexity of autophagic regulation and specialization of the non-core apparatus to perform a number of different roles. Using the yeast interaction network as a scaffold we identified the 14 novel human proteins as putative autophagy-associatd proteins. We confirmed one of these, the F-BAR protein FNBP1L ,as being an ATG3 interactor. Using a functional siRNA approach we demonstrated that FNBP1L was essential for autophagy of internalized Salmonella Typhimurium, but dispensable for formation of classical autophagosomes. Our approach illustrates the level of conservation of the autophagy apparatus over large evolutionary distances, but also demonstrates that mammalian cells utilize different autophagy accessory molecules in specific contexts