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Pseudomonas quinolone signal and two-component systems; Unravelling the intricate network of gene regulation in Pseudomonas aeruginosa

Reference	BB/K003348/1
Principal Investigator / Supervisor	Dr Alan Brown
Co-Investigators / Co-Supervisors	Dr Steven Porter
Institution	University of Exeter
Department	Biosciences
Funding type	Research
Value (£)	349,371
Status	Completed
Type	Research Grant
Start date	01/01/2013
End date	31/12/2015
Duration	36 months

Abstract

In P. aeruginosa, the PhoPQ & PmrAB two-component systems (TCSs) tightly control resistance to the polymyxin class of antibiotics through the regulation of lipid A modifications. However, clinical isolates of polymyxin-resistant P. aeruginosa frequently harbour single amino acid polymorphisms within these TCSs that are associated with their constitutive activation, resulting in a stable polymyxin-resistance phenotype. In the course of studies exploring the wider consequences of these polymorphisms, we found that the activity of the Pseudomonas quinolone signal (PQS) quorum sensing system is significantly enhanced in strains possessing constitutive PhoPQ &/or PmrAB activation. The PQS autoinducer molecule is extremely hydrophobic, and is packaged into membrane vesicles to enable trafficking. We believe that the elevated PQS activity is due to the TCS-mediated lipid A modifications altering the efficiency of that PQS packaging. The proposed multidisciplinary research programme will investigate the nature of the TCS-PQS linkage at every step of the pathway. Biochemical investigations will define the mechanism of PhoPQ & PmrAB activation, and how mutations lead to their permanent activation. Mass spectrometry will define the lipid A modifications that occur in response to this TCS activation, enabling the role of specific lipid A modifications in modulating PQS activity to be assessed. RNA-seq analysis in conjunction with relevant phenotypic characterization of appropriate isogenic strains will be used to establish the extent to which the genome-wide response associated with activation of the PhoPQ & PmrAB TCSs is actually mediated through the actions of the PQS QS system. Our observations to date open up an entirely novel research area in the P. aeruginosa field. The proposed studies will make significant contributions to our understanding of P. aeruginosa biology, LPS biology, membrane vesicle formation, and the interplay between gene regulatory networks.

Summary

P. aeruginosa causes around 10% of all healthcare-associated infections in the UK and is a significant cause of mortality. The organism's intrinsic and acquired antibiotic resistance hinders effective treatment, with multi-drug resistance (MDR) frequently observed. This MDR has increased reliance on the antibiotic colistin, which is frequently considered as a drug of last resort for the treatment of infections caused by MDR bacteria. However, as a result of the increasing reliance on colistin, colistin resistance is increasingly reported, with resistance rates as high as 40%. Colistin resistance in P. aeruginosa is typically mediated by alterations to the bacterial surface that reduce binding of the colistin antibiotic. These surface modifications are normally tightly controlled by regulatory systems within the bacterial cell termed two-component systems (TCSs). However, colistin-resistant P. aeruginosa typically possess mutations within these TCSs that result in their permanent activation, causing the surface modifications to be present all the time. Work underway within our laboratories is investigating the wider significance of these TCS mutations. Unexpectedly, we have found that the permanent activation of these TCSs enhances the activity of another distinct gene regulatory network, termed the PQS quorum sensing system. We believe that the same surface modifications that are switched on by the TCSs to confer colistin resistance are also responsible for enhancing PQS activity. The PQS quorum sensing system is an important regulator of virulence (the ability to cause disease) in P. aeruginosa. Consequently, colistin resistance conferred by permanent TCS activation will impact on the infection process through the action of PQS. These observations provide completely new insight into the interplay between gene regulatory networks in P. aeruginosa, and how they control virulence. The proposed research aims to define the precise mechanism by which TCS activation results in enhanced activity of the PQS quorum sensing system. We will employ a variety of methods to assess this interaction at every step of the pathway, including how the TCSs themselves are activated, which surface modifications occur as a consequence, and how those surface modifications impact on the activity of the PQS system. The overall aim of the research is to assess the extent to which the PQS system is responsible for conferring the multitude of effects previously attributed to the TCSs. These studies will give unique insight into several aspects of the P. aeruginosa biology, thus significantly advancing the scientific field. Long-term, there are potential applications and patient benefit that might be enabled by this research as both TCSs and quorum sensing are widely recognised as novel therapeutic targets for new antibiotics. By defining the nature of the linkage of these two regulatory systems, the proposed studies may promote the rational design of new antimicrobials that target these systems in combination. Given the lack of new antibiotics being developed that possess significant activity against bacteria such as P. aeruginosa, new antimicrobial strategies are urgently required in order to lessen the burden of infectious diseases on society. In addition, by highlighting the unintended consequences of colistin resistance, these studies may be used to guide antibiotic prescribing practices in the future, with a view to improving patient management.

Impact Summary

Approximately 8% of hospital in-patients in England acquire a healthcare-associated infection (HAI). Such infections are estimated to be a direct cause of 5,000 deaths and a contributory factor in a further 15,000 deaths each year, and are believed to cost the NHS over £1 billion per year. Pseudomonas aeruginosa, the organism that is the focus of this proposal, causes around 10% of all HAIs in the UK and is associated with significant mortality. The aim of this proposal is to define the nature of the linkage between two seemingly distinct signalling pathways within P. aeruginosa that are believed to play a pivotal role in regulating the ability of the organism to cause infection. The two signalling pathways being explored are (1) two-component systems (specifically PhoP-PhoQ and PmrA-PmrB), and (2) the Pseudomonas quinolone signal (PQS) quorum sensing system. We have evidence that an 'unintended consequence' of activation of the PhoP-PhoQ and/or PmrA-PmrB two-component systems (TCSs) is the enhanced activity of the PQS system, and that this may have far-reaching effects on the bacterial cell and the infection process. In the long-term (beyond the timeline of the current proposal), patients and associated healthcare services are anticipated to be the primary beneficiaries of the proposed research programme, and activities outlined in the attached Pathways to Impact document will ensure that patients and clinicians are kept informed of the research programme and relevant output. Long-term, it is anticipated that the research outcomes from this study will benefit patients in the following ways: NOVEL ANTIMICROBIAL STRATEGIES. TCSs and quorum sensing systems are widely recognised as novel therapeutic targets for new antibiotics. By defining the nature of the linkage of these two regulatory systems, the proposed studies may promote the rational design of new antimicrobials that target these systems in combination. Given the lack of new antibiotics being developed that possess significant activity against Gram-negative bacteria such as P. aeruginosa, new antimicrobial strategies are urgently required in order to lessen the burden of infectious diseases on society. GUIDING ANTIBIOTIC PRESCRIBING PRACTICES. The knowledge gained from this research may also be used to guide antibiotic prescribing practices in the future. In response to increasing multi-drug resistance (MDR) amongst P. aeruginosa, clinicians frequently have to resort to the use of the antibiotic colistin, and colistin resistance is increasing as a result. This colistin resistance is typically associated with mutations in the PhoP-PhoQ and/or PmrA-PmrB TCSs that lead to their permanent activation, and we have shown that this resistance mechanism is associated with heightened PQS activity. A full awareness of the wider consequences of colistin resistance is essential in order to guide best clinical practice. If colistin resistance mediated by PhoPQ and/or PmrAB dramatically alters the virulence of the organism, it may be advisable to avoid colistin usage (when alternative treatment options are available).

Committee	Research Committee B (Plants, microbes, food & sustainability)
Research Topics	Microbiology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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