The P-Usher: A mix and match secretion machine for the assembly of bacterial cell surface appendages.

Fimbriae or pili are filaments attached to the surface of bacteria. These filaments are the result of the assembly of small subunits that come together to form fimbriae of various length and thickness. At the tip of these structures, sits another component whose function is an adhesin. This adhesin is able to connect firmly the bacterium with different types of surface/tissue. In the context of bacterial pathogens, it is an important structure, which allows bacteria to attach to human cells and therefore contribute to the colonization and persistence process. A visual example for this function is given with uropathogenic Escherichia coli. These pathogens are attached to bladder cells and are not eliminated by the urine flow. They resist the pressure of the stream thanks to the flexibility of the fimbriae, and the irreversible attachment via the tip, which could not be disconnected from the host cell. At a molecular level, the occurrence and assembly of the fimbriae at the surface is well understood. It involves a component, which forms a hole (pore) in the envelope of the bacterium, to give the filament access to the surface. This pore component, called the usher, is found in all bacteria, which produce fimbriae. If the usher is absent or does not function properly, fimbriae are not made anymore. Means by which bacteria attach to host cells are numerous. It exists other kinds of bacterial adhesins, of which the filamentous hemagglutinin (FHA) from Bordetella pertussis is an example. B. pertussis is the agent of the whoopping cough and FHA is a major virulence factor involved in host colonization. In contrast to fimbriae the FHA adhesin is not a multi-component structure made of several identical components. Nevertheless, FHA is capable of making a large and helical structure at the cell surface, which will act as an attachment device to host cells. As for fimbriae, FHA can access the cell surface thanks to a pore, which is different from the usher and commonly named TpsB. The TpsB component has a region called POTRA, which is used to fish FHA, before pushing it out to the cell surface. My laboratory is working on Pseudomonas aeruginosa. This bacterial pathogen is feared because of its prevalence in nosocomial infections (third cause world-wide), and because it has a high impact on the morbidity and mortality of hospitalized patients. P. aeruginosa is also known as the main bacterial agent involved in infection of cystic fibrosis individuals, resulting in destruction of lung tissues and patient death. The virulence factors involved in the P. aeruginosa infection process are numerous, but recently a lot of attention has been given to a series of structure, named Cup, which are involved in the formation of fimbriae required in chronic infection. We focused our study on the so-called CupB system and more particularly the usher component CupB3. We discovered that this usher is in fact a hybrid between a 'classical' usher and a TpsB component, since we identified a POTRA domain (see above) in CupB3. We called the CupB3 usher a P-usher for POTRA-containing usher. More interestingly, we realized that CupB3 is not only able to assemble fimbriae, but it also assembles a FHA like protein called CupB5 at the cell surface. Such discovery is an intriguing observation, which reflects evolutionary mechanisms by which bacteria mix and match components to create new molecular machines, which give further improved capacity in colonising and persisting within the host. In this proposal, we want to understand the mechanism by which the P-usher coordinates the assembly of fimbriae and the CupB5 adhesin. We also want to understand how this unique bacterial machine contributes to optimize the colonization process of P. aeruginosa. Finally, by understanding the detailed molecular mechanism, we will be in a situation to design new antimicrobials, which will abolish the CupB3 function and help fighting against P. aeruginosa infection.

Gram-negative bacterial pathogens employ a system termed chaperone-usher pathway to assemble multi-subunit fibres on their surface. The mechanism underlying the process relies on two accessory proteins, chaperone and usher. The chaperones bind the pilin subunit and deliver it to the usher. The usher forms a pore into the outer membrane through which pilins are transported to the surface. The assembled fibres display at their tip an adhesin involved in bacteria-host interaction. Other adhesins such as the FHA from Bordetella pertussis, are brought to the surface by another kind of transporter. FHA (generically TpsA) is secreted by the outer membrane protein FhaC (generically TpsB). Recognition between TpsA and TpsB occurs via interaction between the secretion signal (TPS) located at the N terminus of TpsA and the N-terminal domain of TpsB. In case of FhaC, 2 POTRA domains are found at the N terminus and required for interaction with the TPS motif of FHA prior to its transport across the TpsB pore. We discovered in Pseudomonas aeruginosa a composite usher containing a POTRA domain at its N terminus, which we named a P-Usher. The gene encoding the P-usher (cupB3) is part of a cluster involved in the assembly of CupB1 pilin. Importantly, CupB3 is not only required for CupB1 pilin transport but also for secretion of an FHA-like protein, CupB5. CupB5 is encoded within the cupB gene cluster, which also encodes an adhesin (CupB6) and two chaperones (CupB2 and CupB4). We thus identified a mixed and match component between a usher and a TpsB. This observation is very original and raises number of questions as how this unique transporter coordinates translocation of two substrates, which otherwise should use different routes. We address these by using the combined expertise of the Filloux's lab (P. aeruginosa, inventor of the P-usher) and the Waksman's lab (structure of chaperone usher pathways). The approaches combine genetics, biochemistry and crystallography.

Our study on the P-usher will shed light on one of the manifold factors that P. aeruginosa uses to colonize its host and increase our understanding on the mechanisms underlying the pathogenesis of this organism. P. aeruginosa is a major cause of hospital-acquired infection, with an estimated 10,000 cases each year in UK. Infection is severe and life-threatening, leading to pneumonia or septicaemia. P. aeruginosa is also dreaded by cystic fibrosis patients (80% carry the bacteria in their lungs and infection is fatal). A similar system is found in Pseudomonas fluorescens, a plant growth promoter (PGP), and a P-usher is found in Burkholderia ambifaria, the dominant Burkholderia cepacia complex species in natural environments where it is associated with plant roots. Assembly of fimbriae in these organisms supports root colonization. By comparing these systems, we may unravel whether knowledge acquired on mechanisms involved in human colonization by bacterial pathogens could apply to mechanisms involved in plant root colonization by PGP. Academic Impact Research in Filloux and Waksman labs is recognized worldwide by academics in the field of Microbiology and protein structure. A main collaborator for Filloux's lab is Pr Stephen Lory (Harvard Medical School), 5 publications in high impact factor journals on P. aeruginosa and chaperone-usher. The main collaborator for Waksman's lab is Pr Scott Hultgren (Washington University, St Louis), more than 15 top class publications. Waksman and Hultgren have not only solved at high resolution the structure of the chaperone-usher components in E. coli, but in light of these structures understood in subtle details the molecular mechanism of this machinery. This is a remarkable example of the impact a combination of discipline, crystallography and molecular microbiology, may have on significant advances in understanding. This sort of achievement is one main goal in the present proposal. Based on our visibility in the field and a broad network of collaborators we will organize a small size (50 participants) meeting on the site of Imperial College (by the end of the project). The topic on bacterial transporter will attract the main UK players in this field together with other groups from France and US. This way we hope to control of dissemination of our findings not only by attending regular conferences and by publishing in high impact factor journals. Companies representative (Sanofi-Aventis, GSK), and editors (Nature) will also be invited to attend the conference. A Filloux has expertise in organizing conference. He organized the Pseudomonas meeting (Marseille 2005) and chair the Gordon conference on Microbial adhesion (Newport 2009). Economic and Societal impact Our research is basic research. However, research on a human pathogen may lead to biomedical applications, new antimicrobials and societal impact. The P. aeruginosa fimbriae assembled by the P-usher are involved in biofilm formation, which is the lifestyle in chronic infections. Eradicating biofilm is not possible due to elevated antibitotic resistance of these microbial communities. Treatment of such infection is in needs of finding new molecules. The interest of pharmaceutical companies for new antimicrobials is back. The research unit of Sanofi is under restructuration. A Filloux was invited for a meeting at Sanofi in Toulouse (France) to update the research and development units about potential novel antimicrobial targets (October 2009), of which biofilm was presented as the number one target. Interaction and discussion with other pharmaceutical companies will be further extended. Waksman and Hultgren interact with companies for the role of fimbriae in uropathogenic E. coli and the search of 'pilicides' molecules. The P-usher is found in a human pathogen but also in a resident of the rhizosphere, B. ambifaria. Understanding mechanisms promoting root colonization by bacteria could foster the interest of this community.