- Title
- Investigation of the gram-negative cell envelope and its contribution to antibiotic resistance
- Creator
- Maher, Claire
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2024
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- A lack of knowledge about the Gram-negative cell envelope has been identified as a major barrier in the rational development of new antibiotics, which are urgently needed to combat the growing antibiotic resistance crisis. In order to contribute to this knowledge, this study investigated the contribution of the different components of the Gram-negative cell envelope to antibiotic accumulation using functional genomics, biochemistry and bioinformatic analyses. Ampicillin, a broad-spectrum antibiotic, was developed through the addition of a primary amine group to the Gram-positive antibiotic benzylpenicillin. This modification has since been determined to improve compound uptake through porins in the Gram-negative outer membrane (OM), but its influence on interactions with other cell envelope components has been understudied. This study applied transposon-directed insertion site sequencing (TraDIS) to investigate the role of this chemical group in improving ampicillin accumulation in the model Gram-negative organism Escherichia coli. The importance of the amine group in improving passage through the major porin OmpF was reaffirmed. It was also found that disruption of genes encoding the major multidrug efflux pump AcrAB-TolC were more detrimental to survival under ampicillin than benzylpenicillin, suggesting that maintaining ampicillin efflux may be more significant to E. coli survival than full inhibition of OmpF-mediated uptake. The use of TraDIS was also expanded to investigate the genes involved in the accumulation of a physiochemically diverse range of antibiotic compounds. A transposon mutant library was constructed in a strain of E. coli containing an inducible outer membrane hyperporination system, which essentially removes the OM as a permeability barrier, allowing its contribution to small molecule accumulation to be more clearly defined. A set of genes involved in maintaining OM integrity were found to play a significant role in tolerance across the antibiotics tested, emphasising the role of the OM as a general permeability barrier to most compounds. Disruption of lipopolysaccharide genes were typically more detrimental to fitness for antibiotics that are unable to cross the OM through porins, aligning with expectations. The distribution and spread of genes encoding the major multidrug efflux pump in Acinetobacter, AdeAB(C), was also investigated. While surveys have been conducted on chromosomally-encoded AdeAB(C), variants found in the accessory gene pool (e.g. on plasmids) have only recently been identified and had not yet been investigated in detail. Several variants of AdeAB(C) were identified on a wide variety of plasmids across Acinetobacter species, and in some cases in chromosomal genomic islands. These genes appeared to be highly mobile and possibly the subject of multiple individual mobilisation events through different mobile elements. More distantly related homologs were also identified which may represent distinct, uncharacterised efflux systems. These findings indicate that genes encoding AdeAB(C) are widespread in the Acinetobacter pan-genome and are likely to represent an understudied multidrug resistance threat. Overall, the study demonstrated the utility of TraDIS in investigating factors influencing compound accumulation in Gram-negative bacteria. It also highlighted the understudied threat of plasmid-encoded multidrug efflux pumps. Together, the findings emphasised the need for a holistic approach in investigating the complex problem of Gram-negative permeability.
- Subject
- antibiotic resistance; gram-negative; cell envelope; new antibiotics
- Identifier
- http://hdl.handle.net/1959.13/1510238
- Identifier
- uon:56359
- Rights
- Copyright 2024 Claire Maher
- Language
- eng
- Full Text
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| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details Download | ATTACHMENT01 | Thesis | 12 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Abstract | 602 KB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT03 | Supplementary Dataset 1 | 908 KB | Excel Microsoft Office Open XML Format document | View Details Download | ||
| View Details Download | ATTACHMENT04 | Supplementary Dataset 2 | 1 MB | Excel Microsoft Office Open XML Format document | View Details Download | ||
| View Details Download | ATTACHMENT05 | Supplementary Dataset 3 | 2 MB | Excel Microsoft Office Open XML Format document | View Details Download |