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Investigating LasB as a virulence target of Pseudomonas aeruginosa biofilm infections in cystic fibrosis patients

Investigating LasB as a virulence target of Pseudomonas aeruginosa biofilm infections in cystic fibrosis patients
Investigating LasB as a virulence target of Pseudomonas aeruginosa biofilm infections in cystic fibrosis patients
Pulmonary damage as a result of Pseudomonas aeruginosa infection in the mucosal airways of cystic fibrosis patients is facilitated by bacterial virulence factors. LasB is a 33 KDa extracellularly secreted zinc-metalloprotease implicated in tissue damage and inflammation with numerous substrates within the lung including the cystic fibrosis transmembrane regulator, interleukin-6, interleukin-8, and elastin. This quorum sensing dependent protease has been further linked to bacterial physiology, with key roles in the modulation of biofilm formation and motility. These contributions to bacterial virulence make LasB a candidate therapeutic target with a potential scope for novel small molecule inhibitor design. This could provide an anti-virulence strategy to reduce the impact of P. aeruginosa infection on patients’ pulmonary health, improve clearance and minimise the need for eradication therapy for antibiotics.

This thesis explored the prevalence and genetic variation of the lasB gene both within isolated clinical strains and in bacterial genome databases, identifying a series of variant LasB sequences from which phenotypic differences were observed. These variant strains were further explored with the aid of molecular cloning and the creation of protein expression constructs transformed into a P. aeruginosa strain absent of LasB expression. This thesis characterised the differences in both a P. aeruginosa laboratory strain and clinical isolate from a cystic fibrosis patient both with and without the production of the LasB protein in vitro. These strains were further investigated with the aid of a host-pathogen interaction model which provided a eukaryotic cell layer on which bacteria were inoculated into an artificial sputum layer on the apical surface.

Collectively the findings of this thesis indicate that LasB is an important modulator of P. aeruginosa biofilm architecture when assessed both in vitro and on an abiotic substrate in an artificially introduced mucus layer on epithelial cells. Parallel evolution of strains was observed with variant LasB protein sequence conservation present both within a selection of cystic fibrosis patient sputum isolates and in genome databases. These variant sequences were demonstrated not to impede secretion; however, they could provide increased resistance to LasB degradation in the lung environment, or potential scope for alternative post-translational modifications to modulate substrate specificity. Furthermore, LasB amino acid sequence variation was proposed as an important consideration for inhibitor design to promote the success of an anti-virulence treatment strategy.
University of Southampton
Barochia, Bhavik
94c26a03-b10d-464e-82bc-add13e1f2b2d
Barochia, Bhavik
94c26a03-b10d-464e-82bc-add13e1f2b2d
Webb, Jeremy
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Faust, Saul
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Connett, Gary
55d5676c-90d8-46bf-a508-62eded276516
Everett, Martin
3e5a062b-c00f-46c1-94d8-299127d28726

Barochia, Bhavik (2023) Investigating LasB as a virulence target of Pseudomonas aeruginosa biofilm infections in cystic fibrosis patients. University of Southampton, Doctoral Thesis, 278pp.

Record type: Thesis (Doctoral)

Abstract

Pulmonary damage as a result of Pseudomonas aeruginosa infection in the mucosal airways of cystic fibrosis patients is facilitated by bacterial virulence factors. LasB is a 33 KDa extracellularly secreted zinc-metalloprotease implicated in tissue damage and inflammation with numerous substrates within the lung including the cystic fibrosis transmembrane regulator, interleukin-6, interleukin-8, and elastin. This quorum sensing dependent protease has been further linked to bacterial physiology, with key roles in the modulation of biofilm formation and motility. These contributions to bacterial virulence make LasB a candidate therapeutic target with a potential scope for novel small molecule inhibitor design. This could provide an anti-virulence strategy to reduce the impact of P. aeruginosa infection on patients’ pulmonary health, improve clearance and minimise the need for eradication therapy for antibiotics.

This thesis explored the prevalence and genetic variation of the lasB gene both within isolated clinical strains and in bacterial genome databases, identifying a series of variant LasB sequences from which phenotypic differences were observed. These variant strains were further explored with the aid of molecular cloning and the creation of protein expression constructs transformed into a P. aeruginosa strain absent of LasB expression. This thesis characterised the differences in both a P. aeruginosa laboratory strain and clinical isolate from a cystic fibrosis patient both with and without the production of the LasB protein in vitro. These strains were further investigated with the aid of a host-pathogen interaction model which provided a eukaryotic cell layer on which bacteria were inoculated into an artificial sputum layer on the apical surface.

Collectively the findings of this thesis indicate that LasB is an important modulator of P. aeruginosa biofilm architecture when assessed both in vitro and on an abiotic substrate in an artificially introduced mucus layer on epithelial cells. Parallel evolution of strains was observed with variant LasB protein sequence conservation present both within a selection of cystic fibrosis patient sputum isolates and in genome databases. These variant sequences were demonstrated not to impede secretion; however, they could provide increased resistance to LasB degradation in the lung environment, or potential scope for alternative post-translational modifications to modulate substrate specificity. Furthermore, LasB amino acid sequence variation was proposed as an important consideration for inhibitor design to promote the success of an anti-virulence treatment strategy.

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More information

In preparation date: 14 August 2022
Published date: 12 February 2023

Identifiers

Local EPrints ID: 474185
URI: http://eprints.soton.ac.uk/id/eprint/474185
PURE UUID: 15084331-e67c-45f7-9875-4debf243322c
ORCID for Bhavik Barochia: ORCID iD orcid.org/0000-0003-0716-1555
ORCID for Jeremy Webb: ORCID iD orcid.org/0000-0003-2068-8589
ORCID for Saul Faust: ORCID iD orcid.org/0000-0003-3410-7642
ORCID for Gary Connett: ORCID iD orcid.org/0000-0003-1310-3239

Catalogue record

Date deposited: 15 Feb 2023 17:31
Last modified: 06 Jun 2024 02:03

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Contributors

Author: Bhavik Barochia ORCID iD
Thesis advisor: Jeremy Webb ORCID iD
Thesis advisor: Saul Faust ORCID iD
Thesis advisor: Gary Connett ORCID iD
Thesis advisor: Martin Everett

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