Exoproteomics of the Pseudomonas aeruginosa biofilm matrix reveals the role of a novel secretion system

Pseudomonas aeruginosa is a WHO priority pathogen responsible for more than half a million deaths per year, whose reservoir of antibiotic resistance genes is expanding at an alarming rate. Even in the case of infecting strains that are genetically sensitive to drugs administered in the clinic, antibiotic failure is widely observed. This can be partly attributed to the proclivity P. aeruginosa has to forming what are known as biofilms - heterogeneous aggregates of cells living in a matrix of their own secretions. Cells that would otherwise be susceptible to antimicrobials are protected by the modifications that they make, in concert, to their surroundings. Aggregates of as few as 64 cells begin to exhibit elevated tolerance to antibiotics and, indeed, this tolerance can increase as much as 1,000-fold. It has been estimated that such biofilms account for somewhere between 65 and 80% of microbial infections in the human body. There is therefore a lot to gain from improving our understanding of the aggregated cells and the matrix in which they are enmeshed. In this work, I present a new, experimentally tractable model of a P. aeruginosa biofilm. The model offers the benefit of cultivating suspended biofilms, which conform more closely to the free-floating aggregates thought to occupy chronic infection sites. Moreover, these aggregates are encased in a large volume of exopolymeric matrix material, which presents an opportunity to study the matrix in more detail. Herein I describe a method I developed to fractionate the biofilm, permitting the isolation of the cells for time-resolved transcriptomic study, and the matrix and culture supernatant for spatially-resolved proteomic investigation. The transcriptomics revealed a wealth of uncharacterised biology that drives the development of free-floating biofilms. The proteomics provided evidence to support my hypothesis that the biofilm matrix is a distinct extracellular compartment which can trap secreted proteins. Among those proteins trapped by the matrix was PA2668, a member of the interkingdom family of cysteine-rich secretory proteins which are found in extracellular matrices elsewhere. The PA2668 locus is adjacent to an operon that encodes a hypothetical and uncharacterised Type II Secretion System, known as the Hpl Secreton. Based on the common genomic occurrence of protein substrates next to their cognate secretion system, I hypothesised that the Hpl Secreton is functional and secretes PA2668 (SagE), confirming this with a western blot comparing the wild type and a ∆hpl mutant. I then used this mutant to search for other possible substrates for this novel secreton, identifying PA2433 as a likely candidate, whose transcription has elsewhere been shown to be upregulated in concert with Hpl in an epithelial infection model. A DNA-bait pull-down of the Hpl promoter pointed to a possible transcription factor responsible for its regulation, PA1864 (HssR), which has been associated with aggregation. In overexpressing PA1864, I observed a hyperaggregative phenotype, which could be reduced by deletion of the Hpl operon. Meanwhile, overexpression of the Hpl cluster produced the same phenotype, implicating the Hpl Secreton in biofilm formation. These findings are graphically summarised below. Taken as a whole, this work began with the development of a novel method for growing and fractionating biofilms, leading to the discovery of an uncharacterised Type II Secretion System which contributes to the formation of biofilm aggregates.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2025

Author dc:creator

• Nazeer, Rahan

Advisor dc:contributor.advisor

• Welch, Martin

• biofilm
• microbiology
• floating aggregates
• secretion systems

Licence

• https://www.repository.cam.ac.uk/bitstreams/60ba3339-f7d8-48a9-b7ad-27b0ef5ddd9c/download
• https://creativecommons.org/licenses/by/4.0/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.123987

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/393817