Type Vd secretion: a virulence factor of Pseudomonas aeruginosa?

Pseudomonas aeruginosa er en opportunistisk patogen bakterie som forårsaker en rekke sykdommer, særlig i sykehusomgivelser og hos pasienter som lider av den genetiske sykdommen cystisk fibrose. Flere P. aeruginosa-stammer er antibiotikaresistente, og alternative metoder for å bekjempe P. aeruginosa infeksjoner er derfor nødvendig. I dette prosjektet har vi studert et overflateprotein hos P. aeruginosa, kalt PlpD. PlpD utskilles tilsynelatende fra bakterien via en såkalt autotransportmekanisme, dvs. at proteinet utskiller seg selv fra cellen. Denne utskillelsesmekanismen kalles type Vd sekresjon. Prosjektet består av to deler: i den første delen har vi karakterisert den molekylære mekanismen til PlpD sekresjon. Våre resultater viser at PlpD, på lik måte som andre autotransportere, blir utskilt til bakterieoverflaten via en hårnålsintermediat. I den andre delen undersøkte vi hvorvidt PlpD spiller en rolle i virulensen til P. aeruginosa. PlpD er en lipase med potensialet til å destabiliserere cellemembranene til vertsceller. Likevel viser proteinet seg å ha lav toksisitet hos voksmøllarver. Resultatene våre antyder at PlpD kan ha en subtil rolle i å undergrave intracellulære signaler i vertsceller.

This project has provided basic information about the activity, lipid specificity, toxicity and secretion of type Vd-secreted phospholipases. However, the exact role of these proteins in virulence remains unclear. Outcomes of this have included new international collaborations (USA) and interdisciplinary work together with a structural biology group. This work has also directly resulted in two original research publications, five related research articles and four review articles as well as a commentary note. The work produced in the project will have an impact on the autotransporter field, providing new knowledge on a hitherto poorly studied class of autotransporter proteins and suggesting novel areas of research to investigate the in vivo functions of type Vd-secreted lipases.

Type V secretion systems, also known as autotransporters, are the most widespread protein secretion systems in Gram-negative bacteria. Autotransporters consist of two functional units: the extracellular effector domain or passenger, and the transmembrane translocator, which is responsible for transporting the passenger to the outside of the cell. Currently, five subclasses of autotransporter are known. The aim of this project is to investigate the mechanism of the neglected type Vd secretion pathway and compare it to the other, better characterised type V subclasses. To this end, we will centre on the type Vd-secreted lipase PlpD from Pseudomonas aeruginosa, an important pathogen especially in hospital settings. We will investigate how the passenger is translocated using a combination of biochemical, microbiological and structural biology techniques. The planned experiments will allow us to answer the following questions: Is the PlpD passenger exported via a hairpin (C-terminus first) or by threading (N-terminus first)? Can foreign proteins be exported? Are additional factors, such as the Bam complex, required for PlpD biogenesis? What role does the periplasmic domain of PlpD play in export? What is the atomic structure of the PlpD translocator? PlpD is a possible virulence factor of P. aeruginosa. We will test its role in pathogenicity using two different infection models: the plant Arabidopsis thaliana and zebrafish embryos as a model for animal infection. The results obtained in this project will provide deeper understanding of the biogenesis of PlpD and other type Vd secretion systems, and in addition will help settle a long-standing controversy within the type V secretion field. As possible virulence factors of P. aeruginosa and other pathogens, type Vd secretion systems are a potential drug and vaccine target to combat hard-to-eradicate pathogens such as P. aeruginosa and emerging antibiotic resistance in these organisms.