Assembly of the Pseudomonas aeruginosa type II secretion system

Abstract

The Gram-negative bacterium Pseudomonas aeruginosa is the most common pathogen responsible for acute respiratory infections in immuno-compromised patients and for chronic infections in patients suffering from cystic fibrosis. Besides the high incidence and the severity of infections, increased resistance to conventional antibiotics forms a problem. Type II secretion largely contributes to the virulence of this bacterium. P. aeruginosa contains two secretion machineries of the type II kind, of which the Xcp system is responsible for the secretion of the majority of the exoproteins. The Xcp system is assembled from 12 constituents and five of these share N-terminal sequence similarity with the structural component of type IV pili, PilA, and are therefore designated pseudopilins. Type IV pilins and pseudopilins are found in various prokaryotic envelope protein complexes, including type IV pili and type II secretion machineries of Gram-negative bacteria, competence systems of Gram-positive bacteria, and flagella and sugar-binding structures within the archaeal kingdom. The precursors of these proteins have highly conserved N termini, consisting of a short positively charged leader peptide, which is cleaved off by a dedicated peptidase during maturation, and a hydrophobic stretch of approximately 20 amino acid residues. The presence of proteins with prepilin-like N termini always coincides with the occurrence of accessory proteins, including a prepilin peptidase, an ATPase and a multispanning transmembrane protein. Inner membrane translocation of pseudopilins has been suggested to depend on these accessory proteins. However, we show that the major pseudopilin of the Xcp system, XcpT, is co-translationally transported across the inner membrane via the SRP/Sec pathway. In support of a general translocation route for pilins and pseudopilins, we demonstrate that the hydrophobic N terminus of XcpT could be substituted by that of PilA without a loss of function. Furthermore, our data reveal that the accessory multispanning transmembrane protein, XcpS, participates in the inner membrane complex of the Xcp system. We show that simultaneous interaction with XcpR and XcpY increased the stability of the XcpS protein and that this interaction requires the large cytoplasmic loop of XcpS. Finally, we describe the reconstitution of the Xcp system in the heterologous hosts Pseudomonas putida and E. coli. These experiments show that targeting of the XcpQ protein to the outer membrane can be an important bottleneck in the reconstitution of the system in a heterologous host. These studies were performed to determine whether the known Xcp components are sufficient for their assembly into a functional machine. Together, the work described in this thesis gives more insight into the assembly of the P. aeruginosa Xcp machinery.