The opportunistic human pathogen Pseudomonas aeruginosa is a threat for immunocompromised individuals, a major cause of nosocomial infections, and is prevalent in patients with cystic fibrosis. The bacterium can form biofilms that help it evade the immune response. It adheres to host cells using molecular adhesins, such as pili assembled by chaperone-usher pathways (Cup). Understanding the adhesion could, therefore, help develop treatments that prevent the establishment of infections. This thesis considers the CupB system, consisting of an usher (CupB3), two chaperones (CupB2 and CupB4) and two pilin subunits (CupB1 and CupB6). The chaperones target the pilin subunits to the usher assembling a CupB1-containing pilus with a putative CupB6 adhesin at its tip. The cupB operon also encodes the TpsA-like protein (two partner secretion) CupB5, previously suggested to be secreted by CupB3. The aim of this work was to understand the CupB1-containing pilus assembly and CupB5 secretion mechanism. Genetic and biochemical techniques were used, such as deletion or point mutation, qRT-PCR, pull-down assays, shearing assays, and protein structure prediction or resolution. They led to the following results. First, each chaperone likely has a cognate substrate: CupB1 interacts with CupB2 and CupB6 with CupB4. Second, the crystal structure solved for CupB6 showed that it has a pilin and a putative adhesin domain, connected by a poly-proline linker. Third, CupB5 secretion was observed to be CupB3-independent and TpsB4-dependent. tpsB4 is encoded with its substrate tpsA4. The expression of the cupB and tpsB4/tpsA4 operons was shown to be controlled by the same regulatory pathway, Roc1, and deletion of the tpsB4 transporter gene abolished CupB5 secretion. Fourth, a structural analysis indicated that TpsB4 has two POTRA domains, and POTRA-1 interacts with the highly homologue TPS motifs of CupB5 and TpsA4. Based on these results, the thesis presents a model of CupB pilus assembly and CupB5 secretion.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:666477 |
| Date | January 2014 |
| Creators | Muhl, Daniela |
| Contributors | Filloux, Alain |
| Publisher | Imperial College London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10044/1/25986 |
Page generated in 0.0077 seconds