P-USHER

Structural analysis of the newly described hybrid secretion system of the pathogen Pseudomonas aeruginosa involving the outer membrane transporter CupB3 (‘P-usher’) and the adhesin CupB5

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 Obiettivo del progetto (Objective)

'Bacterial pathogenesis is closely linked to the biogenesis of fimbrial adhesins. Up to date 7 different types of secretion systems involved in assembling such structures are defined. However, recent data on the CupB secretion system described in Pseudomonas aeruginosa, indicate that this is the first time a hybrid secretion system is described, as it contains proteins of both the type Vb Two-Partner-Secretion (TPS) System (adhesin CupB5) and the CU type Chaperon-usher pathway system (CupB3). The proposed research focuses on the CupB pathway components CupB1-6, especially on the hybrid CupB3 and CupB5 proteins, where the so-called P-usher (CupB3), a pore-forming beta-barrel, transports the CupB5 adhesin to cell surface. CupB5 is a TpsA-like adhesin which normally is transported to cell surface through a cognate TpsB-like protein (TPS system). Yet, the CupB system lacks a TpsB-like protein and the P-usher exerts the transporter role. The clue is that the P-usher has a N-terminal POTRA domain, normally found in TpsB-like proteins, which is the site of interaction/recognition of the adhesin CupB5. This is an extraordinary case, where the P-usher is not only able to transport its cognate and fimbriae forming pilin-subunits (CupB1), but also the non-fimbrial CupB5 adhesin. The way the recognition/transport between non-cognate CupB3 and CupB5 proteins is managed will be assessed from a structural point of view using different biochemical (protein purification and characterization) and biophysical techniques, integrating in an ultimate hybrid approach both X-ray crystallography and cryo-electronmicroscopy to determine the dynamics of interaction of the CupB system in a close-to-nature fashion.'

Introduzione (Teaser)

European researchers investigated a structural aspect of bacterial pathogenesis. The ultimate goal was to develop an innovative approach for targeting pathogenic bacteria capable of causing chronic infections like pneumonia and meningitis.

Descrizione progetto (Article)

Bacteria possess hair-like structures on their surface known as pili that act as antennae for host recognition or as anchors for bacterial attachment. As a result, they play an important role in bacterial virulence as they determine the specificity of adhesion to different cell types.

The assembly of pili in bacteria takes place through three different membrane-bound macromolecular systems, including the chaperone-usher (CU) and the Type 4 pilus (TFP) assembly system. The EU-funded (P-USHER) project investigated this complex machinery and the CupB CU pilus assembly system which operates in the pathogenic Pseudomonas aeruginosa. The TFP system in Neisseria meningitides was also analysed.

Scientists successfully cloned the genes encoding the pilus assembly components and performed pull-down assays to characterise the interaction and resolve the binding of the system proteins. They devoted considerable efforts towards optimising crystallisation experiments to obtain a high-resolution structure of the active and inactive forms of this pilus assembly machinery. The long-term goal was to identify specific targets of therapeutic applicability and design small molecule inhibitors which could bind and impair pilus formation.

Dissecting the pathway of pilus formation could provide a novel, targeted therapeutic approach against pathogens such as Pseudomonas aeruginosa and Neisseria meningitides. With the increasing emergence of antibiotic resistance, such new interventions are highly desirable, benefitting the healthcare and pharmaceutical sector as well.