Structural characterisation of bacterial proteins implicated in resistance and adaptation

The emergence and rapid global spread of antibiotic resistance phenotypes amongst different bacterial lineages is threatening a return to a pre-antibiotic era. Two prime mechanisms used by bacteria for defence against antibiotics are lateral gene transfer and efflux systems. This thesis considers protein properties in solution for both mechanisms and assesses their capacities to respond to small molecule binding partners. Part I focuses on novel fold proteins recovered from integron/gene cassette systems of various bacterial isolates. Part II investigates efflux regulator proteins in Acinetobacter baumannii. Part I: Lateral gene transfer allows a dynamic gene pool to give rise to the wide phenotypic diversity and rapid evolution rates characteristic of bacteria. The integron/gene cassette system is an active player in lateral gene transfer, particularly credited with the rapid spread of multi-drug resistance phenotypes. The genes within these cassettes are remarkably diverse: 80% carry open reading frames (ORFs) with either no known homology or homology to ORFs of unknown function. Crystal structures of completely novel folds have been defined for several ORFs, derived from mobile gene cassettes in microbial isolates from Halifax Harbour and Vibrio spp. In this thesis the first biophysical characterisation for three of these proteins has been provided. They are named for the bacterial hosts or locations from which they were recovered and are small oligomeric proteins of α/β and α+β fold class. Hfx1 is an extremely robust trimer which is stabilised bymetals and protamine sulfate. A series of mutant forms were generated to probe its tertiary stability and investigate hypothesised binding pockets. Each mutant, whether it targeted backbone hydrogen bonds, hydrophobic amino acids, cysteines or histidines, still assembled as a trimer, although melting temperatures varied. In addition, histidine mutant forms of Hfx1 did not reduce metal stabilisation, removing them as possible chelating residues in the hypothesised metal-binding pocket. These methods were also used for the hydrophobic dimeric protein Vch14. However, as with Hfx1, mutant forms of Vch14 still formed dimers with varying stabilities. Hfx5, a domain-swapped dimer under crystal conditions, was found to form mixed oligomeric species of monomer and dimer in solution, so the arrangements of these quaternary structures were probed. Conditions such as low protein concentration and more destabilising buffers (using MgCl2 and CaCl2 as salts) resulted in a shift towards the monomeric form of Hfx5. Overall, these small bacterial proteins are highly robust and retain a propensity to oligomerise even when structurally stressed. Many of these novel-fold proteins hold the potential to form the building blocks for larger heteromeric protein structures, either in nature or in vitro. Part II:A. baumannii is a tenacious Gram-negative opportunistic human pathogen, commonly associated with hospital-acquired infections. Membrane drug efflux systems are prominent resistance factors in A. baumannii, likely acting as regulators for virulence, stress and biofilm formation. There are six families of efflux-pump proteins known in Gram-negative bacteria, with a large range of substrates. The structure and function for four efflux-pump regulators (AdeN, AmvR, AdeL, AceR), known to regulate the expression of major facilitator, resistance nodulation division, and proteobacterial antimicrobial compound efflux transporter family pumps have been investigated. These regulator proteins belong to the TetR and LysR-type family of transcriptional regulators. Both AmvR and AdeL display higher melting temperatures in the presence of small molecules of varying chemistries, including polyamines. This responds to previous work demonstrating the ability of AceR to bind polyamines. In particular, both AmvR and AdeL are highly stabilised by cystamine, suggesting that their efflux systems may respond to oxidative stress. Thus, different regulators from both TetR and LysR families appear to have affinity for similar molecules. More work is required to understand the full regulatory role of these proteins in A. baumannii, but this study has demonstrated the viability of studying recombinant forms to address gene regulation mechanisms.

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