Polymorphisms of a Collagen-Like Adhesin Contributes to Legionella pneumophila Adhesion, Biofilm Formation Capacity and Clinical Prevalence

Legionellosis is a severe respiratory illness caused by the inhalation of aerosolized water droplets contaminated with the opportunistic pathogen Legionella pneumophila. The ability of L. pneumophila to produce biofilms has been associated with its capacity to colonize and persist in human-made water reservoirs and distribution systems, which are the source of legionellosis outbreaks. Nevertheless, the factors that mediate L. pneumophila biofilm formation are largely unknown. In previous studies we reported that the adhesin Legionella collagen-like protein (Lcl), is required for auto-aggregation, attachment to multiple surfaces and the formation of biofilms. Lcl structure contains three distinguishable regions: An N-terminal region with a predicted signal sequence, a central region containing tandem collagen-like repeats (R-domain) and a C-terminal region (C-domain) with no significant homology to other known proteins. Lcl R-domain encodes tandem repeats of the collagenous tripeptide Gly-Xaa-Yaa (GXY), a motif that is key for the molecular organization of mammalian collagen and mediates the binding of collagenous proteins to different cellular and environmental ligands. Interestingly, Lcl is polymorphic in the number of GXY tandem repeats. In this study, we combined diverse biochemical, genetic, and cellular approaches to determine the role of Lcl domains and GXY repeats polymorphisms on the structural and functional properties of Lcl, as well as on bacterial attachment, aggregation and biofilm formation. Our results indicate that the R-domain is key for assembling Lcl collagenous triple-helices and has a more preponderate role over the C-domain in Lcl adhesin binding properties. We show that Lcl molecules oligomerize to form large supramolecular complexes to which both, R and C-domains are required. Furthermore, we found that the number of GXY tandem repeats encoded in Lcl R-domain correlates positively with the binding capabilities of Lcl and with the attachment and biofilm production capacity of L. pneumophila strains. Accordingly, the number of GXY tandem repeats in Lcl influences the clinical prevalence of L. pneumophila strains. Therefore, the number of Lcl tandem repeats could be considered as a potential predictor for virulence in L. pneumophila isolates

BurrH: a new modular DNA binding protein for genome engineering

The last few years have seen the increasing development of new DNA targeting molecular tools and strategies for precise genome editing. However, opportunities subsist to either improve or expand the current toolbox and further broaden the scope of possible biotechnological applications. Here we report the discovery and the characterization of BurrH, a new modular DNA binding protein from Burkholderia rhizoxinica that is composed of highly polymorphic DNA targeting modules. We also engineered this scaffold to create a new class of designer nucleases that can be used to efficiently induce in vivo targeted mutagenesis and targeted gene insertion at a desired locus

Recent Progress in Genome Editing for Gene Therapy Applications: The French Perspective

International audienceRecent advances in genome editing tools, especially novel developments in the clustered regularly interspaced short palindromic repeats associated to Cas9 nucleases (CRISPR/Cas9)-derived editing machinery, have revolutionized not only basic science but, importantly, also the gene therapy field. Their flexibility and ability to introduce precise modifications in the genome to disrupt or correct genes or insert expression cassettes in safe harbors in the genome underline their potential applications as a medicine of the future to cure many genetic diseases. In this review, we give an overview of the recent progress made by French researchers in the field of therapeutic genome editing, while putting their work in the general context of advances made in the field. We focus on recent hematopoietic stem cell gene editing strategies for blood diseases affecting the red blood cells or blood coagulation as well as lysosomal storage diseases. We report on a genome editing-based therapy for muscular dystrophy and the potency of T cell gene editing to increase anticancer activity of chimeric antigen receptor T cells to combat cancer. We will also discuss technical obstacles and side effects such as unwanted editing activity that need to be surmounted on the way toward a clinical implementation of genome editing. We propose here improvements developed today, including by French researchers to overcome the editing-related genotoxicity and improve editing precision by the use of novel recombinant nuclease-based systems such as nickases, base editors, and prime editors. Finally, a solution is proposed to resolve the cellular toxicity induced by the systems employed for gene editing machinery delivery