Transformation – a mode of horizontal gene transfer (HGT) widespread in bacteria – is defined as the ability of bacteria to bind free DNA, translocate it across membrane(s), and recombine it in the recipient’s genome. Because HGT can have dramatic consequences on human health, such as transmission of antibiotic resistance genes, studying the molecular mechanisms of transformation is an important scientific endeavour. The earliest step in transformation, which consists of DNA binding and uptake close to the cytoplasmic membrane, is usually mediated by type 4 filaments (T4F) and remains one of the least understood steps. In contrast to the T4F involved in DNA uptake in diderm species, the T4aP pilus, which is well characterised, the Com pilus – a radically different T4F involved in DNA uptake in monoderm species (with one membrane) – is understudied and remains poorly understood. We do not understand how Com pili are assembled, how they bind DNA, and how they retract to promote DNA uptake. We therefore propose to answer these fundamental questions by performing a structure/function analysis of the Com pilus in the opportunistic human pathogen Streptococcus sanguinis, which has recently emerged as a monoderm model for studying T4F. This project will have an impact on our understanding of a key step in an important biological property (transformation) and of a filamentous nanomachine found in hundreds of monoderm species. In addition, our findings will have implications for the T4F superfamily as a whole, which is ubiquitous in Bacteria and Archaea.

African trypanosomes are unicellular parasites with a great capacity of adaptation. Indeed, they evolve during their parasitic cycle between an arthropod (the tsetse fly, insect vector) and a mammal (man or wild or farmed animals depending on the species) and must adapt their metabolisms, their morphologies, their antigenic properties in order to survive in very different environments. Lipid droplets are highly dynamic cellular compartments, produced from the endoplasmic reticulum and surrounded by a single layer of phospholipids. They constitute a metabolic platform of great plasticity in close relationship with other energetic compartments such as peroxisomes and mitochondria. Thus, they are involved in energy metabolism, lipid metabolism, membrane remodeling, cell signaling, autophagy etc... Although known for a very long time, their importance has been largely underestimated justifying a growing interest in these compartments during the last 5 years. In trypanosomes their role has not been studied very deeply. OIL is a basic research project that aims to decipher the biology of Lipid Droplets in African trypanosomes. By using a large set of experimental approaches from functional genomics to latest imagery and -omics technics we will investigate the content, the dynamic and the parasitic functions of these organelles.

Gram-negative bacteria represent a major public health concern due to their high resistance to antibiotics resulting in millions of human deaths world-wide each year. Their multilayered envelope contains an outer membrane (OM) that forms an effective permeability barrier shielding against noxious molecules, including several antibiotics. Being exposed to the cell surface, the OM represents a promising target for the development of new antimicrobials that can act from the exterior of the cell. The design of new antimicrobial stategies urges a better understanding of the molecular pathways of OM biogenesis. Integral OM proteins are crucial for envelope homeostasis. The beta-barrel assembly machinery (BAM) plays an essential role in OM protein assembly. The activity of BAM is regulated in space and time ensuring the constant supply of protein components to active sites of OM biogenesis. Many questions remain unresolved concerning the protein folding reaction mediated by BAM and the regulation of its activity throughout the OM. Motivated by the need to better understand the biogenesis of the bacterial OM, we have discovered that in the enterobacterium Escherichia coli, a member of gamma-proteobacteria, the lipoprotein DolP associates with the BAM complex and plays a critical role in OM homeostasis and integrity. DolP is widely conserved in gamma-, beta- and some alpha-proteobacteria contributing to the virulence of several pathogens, as well as to their ability to survive in the presence of some antibiotics. Our preliminary data reveal that DolP directly interacts with BamA, the catalytic subunit of BAM, promoting BamA folding and function. Inactivation of DolP phenocopies BamA depletion and makes cells sensitive to antibiotics that are normally excluded by Gram-negative bacteria. DolP localizes at active sites of OM biogenesis, ideally positioned to support BAM activity. The molecular mechanisms by which DolP contributes to OM assembly by the BAM complex and ensures OM integrity remain to be established. Our project uses an interdisciplinary approach to determine how DolP interacts with the BAM complex, influences its organization with partner complexes and regulatory factors, and supports its OM protein assembly activity. By employing a multiscale experimental strategy, we are investigating the molecular processes mediated by DolP i) at the cell envelope-wide scale, ii) in a chemically defined in vitro system, and iii) at the structural level. We will conduct these studies in the enterobacterial model organism E. coli and test our results in other pathogens of the gamma- and beta-proteobacterial classes. Our results will be important for the research of new antibacterial compounds that can interfere with OM integrity in Gram-negative bacterial pathogens.

The type VI secretion system (T6SS) is a bacterial contractile nanomachine that uses a spring-like mechanism to deliver effectors into target cells, hence involved in competition and pathogenesis. The T6SS is comprised of a tubular edifice called a tail, composed of an inner tube wrapped by a contractile sheath that is assembled in an extended conformation that stores mechanical energy necessary for its contraction and for propelling the inner tube into the target. T6SS biogenesis starts with the assembly of a membrane complex onto which the baseplate (BP) is recruited and serves as a platform onto which the contractile tube/sheath complex (TSC) polymerizes. It is proposed that the polymerization of the TSC is coordinated by the TssA dodecameric complex, which binds to the BP and recruits and inserts tube and sheath subunits from the distal end of the growing structure. TssA remains at the distal extremity of the TSC during its polymerization until it hits the opposed membrane where it interacts with the TagA protein. TagA acts as a stopper and latch by arresting TSC assembly and by maintaining the sheath under the extended conformation. How TagA binds and control TssA activity remains unknown. Recent work from the consortium has allowed to solve the structures of two domains of TagA, to identify key interacting partners and to optimize novel methods to gain further insight into the dynamic of these processes. In this project we propose to use biochemistry, structural biology, microbiology, fluorescence microscopy and biophysical methods to investigate the structural and molecular determinants and dynamics of the interaction between TagA and TssA in vitro and in vivo. Our project will provide a comprehensive analysis of the molecular interplay between these proteins and a better understanding on how sheath polymerization is arrested and how T6SS assembly termination is controlled.