Structural studies of pilus biogenesis and bacterial adhesion
Lead Research Organisation:
Birkbeck, University of London
Department Name: Biological Sciences
Abstract
P and type 1 pili are surface fibers of uropathogenic Escherichia coli (UPEC) bacteria that play essential roles in the onset of bacterial infection by mediating attachment to the host kidney (P pili) and bladder (type 1 pili). UPEC are the primary causative agents of urinary tract infections (UTIs), which account for an estimated 8 millions physician-office visits and 100,000 hospital admissions every year in the US or in Europe. Indeed, it has been estimated that 1 in 2 women will contract a UTI during their lives, and that 20-40% of these will experience one or more recurrent infections. P and type 1 pili are assembled by a mechanism that also functions in the biogenesis of surface organelles in many other bacterial pathogens, including the potential bioterrorism agent Yersinia pestis.
Our research programme focuses on two essential goals: i- understanding how pili are assembled at the surface of the bacterium, and ii- discovering novel antibiotics, which will specifically target the assembly of pili.
Assembly of pili requires two specialist proteins: a chaperone that takes up each pilus subunit and ferries them to a site of assembly, and a membrane pore protein termed ?the usher? that serves as assembly platform and site of assembly. In the past we have made significant progress in understanding how the chaperone works. We will therefore focus our research on the usher. Indeed very little is know as to how this membrane protein carries out selection of the subunits, their assembly in a defined order and their secretion through the membrane.
Another goal of this research is the discovery of novel antibiotics able to inhibit pilus biogenesis. Such antibiotics would be very useful as they will disarm the bacterial pathogen only, instead of killing all bacteria (the gut flora includes beneficial bacteria) as is the case for the antibiotics presently available. The added advantage of targeting virulence factors is that the selective pressure for development of resistance is thought to be considerably lowered.
These two goals of our research will have considerable impact on public health. We expect that the research will not only shed light on the processes that lead to disease but also will help understand how secretion through cell membranes, a biological process occurring in all realms of life, is carried out.
Our research programme focuses on two essential goals: i- understanding how pili are assembled at the surface of the bacterium, and ii- discovering novel antibiotics, which will specifically target the assembly of pili.
Assembly of pili requires two specialist proteins: a chaperone that takes up each pilus subunit and ferries them to a site of assembly, and a membrane pore protein termed ?the usher? that serves as assembly platform and site of assembly. In the past we have made significant progress in understanding how the chaperone works. We will therefore focus our research on the usher. Indeed very little is know as to how this membrane protein carries out selection of the subunits, their assembly in a defined order and their secretion through the membrane.
Another goal of this research is the discovery of novel antibiotics able to inhibit pilus biogenesis. Such antibiotics would be very useful as they will disarm the bacterial pathogen only, instead of killing all bacteria (the gut flora includes beneficial bacteria) as is the case for the antibiotics presently available. The added advantage of targeting virulence factors is that the selective pressure for development of resistance is thought to be considerably lowered.
These two goals of our research will have considerable impact on public health. We expect that the research will not only shed light on the processes that lead to disease but also will help understand how secretion through cell membranes, a biological process occurring in all realms of life, is carried out.
Technical Summary
The aim of this proposal is to study adhesive hair-like fibers termed ?pili? which are responsible for the onset of infections caused by uropathogenic Escherichia coli. Pili serve as attachment devices, which allow targeting of the bacteria to specific host tissues.
We are particularly interested in P and type 1 pili, which are associated with pyelonephritis (P pili) and cystitis (type 1 pili) and are assembled by the so-called ?chaperone-usher? pathway. The P pilus is encoded by the Pap gene cluster and is a polymer of 6 subunits (or pilins): PapA, PapK, PapE, PapF, PapH and the adhesin PapG. These subunits are assembled at a site of assembly consisting of an outer-membrane (OM) pore called the PapC usher. Before assembly, each subunit requires association with the molecular chaperone PapD. The type 1 pilus system, encoded by the Fim gene cluster of uropathogenic E. coli, consists of the chaperone FimC, the usher FimD, the subunits FimG, FimF, FimA, and the adhesin FimH.
The proposed research is focused on three overall goals: i- the completion of the structural characterization of the P pilus; ii- the discovery of novel compounds able to inhibit pilus biogenesis; and iii- the structural and functional characterization of the usher.
PapF and PapG are the only two Pap subunits, the structures of which remain to be solved. Crystals have been obtained and structures will be determined. These structures will be placed in the context of a completed structural biology of the P pilus.
We have previously obtained an inhibitor of pilus biogenesis disrupting chaperone-usher interaction and its structure bound to a chaperone has been solved. We are now in a position to improve on the original design.
Finally, we will investigate the structure-function of the usher. Very recently, we have proposed an integrated model for usher-mediated pilus assembly based on our new crystal structure of the outer membrane pore domain of PapC and the cryo-EM structure of the FimD:C:F:G:H complex. We propose to test the implications of this model by determining the X-ray crystal structures of predicted sub-complexes between usher domains and chaperone-subunit complexes. We also propose to solve the X-ray crystal structures of the FimD usher bound to the chaperone-adhesin complex FimC:H and also bound to a quaternary FimC:F:G:H complex. We also propose to use fluorescence resonance energy transfer (FRET) systems of probes judiciously positioned to monitor conformational changes hypothesized to occur during assembly.
We are particularly interested in P and type 1 pili, which are associated with pyelonephritis (P pili) and cystitis (type 1 pili) and are assembled by the so-called ?chaperone-usher? pathway. The P pilus is encoded by the Pap gene cluster and is a polymer of 6 subunits (or pilins): PapA, PapK, PapE, PapF, PapH and the adhesin PapG. These subunits are assembled at a site of assembly consisting of an outer-membrane (OM) pore called the PapC usher. Before assembly, each subunit requires association with the molecular chaperone PapD. The type 1 pilus system, encoded by the Fim gene cluster of uropathogenic E. coli, consists of the chaperone FimC, the usher FimD, the subunits FimG, FimF, FimA, and the adhesin FimH.
The proposed research is focused on three overall goals: i- the completion of the structural characterization of the P pilus; ii- the discovery of novel compounds able to inhibit pilus biogenesis; and iii- the structural and functional characterization of the usher.
PapF and PapG are the only two Pap subunits, the structures of which remain to be solved. Crystals have been obtained and structures will be determined. These structures will be placed in the context of a completed structural biology of the P pilus.
We have previously obtained an inhibitor of pilus biogenesis disrupting chaperone-usher interaction and its structure bound to a chaperone has been solved. We are now in a position to improve on the original design.
Finally, we will investigate the structure-function of the usher. Very recently, we have proposed an integrated model for usher-mediated pilus assembly based on our new crystal structure of the outer membrane pore domain of PapC and the cryo-EM structure of the FimD:C:F:G:H complex. We propose to test the implications of this model by determining the X-ray crystal structures of predicted sub-complexes between usher domains and chaperone-subunit complexes. We also propose to solve the X-ray crystal structures of the FimD usher bound to the chaperone-adhesin complex FimC:H and also bound to a quaternary FimC:F:G:H complex. We also propose to use fluorescence resonance energy transfer (FRET) systems of probes judiciously positioned to monitor conformational changes hypothesized to occur during assembly.
People |
ORCID iD |
| Gabriel Waksman (Principal Investigator) | |
| Han Remaut (Co-Investigator) |