Characterisation of the Vibrio cholerae antibiotic resistance var operon

The discovery and use of antibiotics in the chemotherapy of bacterial infections has revolutionised medicine as it is today. Unfortunately, the progressive use of antibiotics has promoted the evolution of bacterial defences against these mediators and thus the emergence of antibiotic resistance. Multidrug resistance (MDR) in bacterial pathogens has grown with such rapid progression that it now threatens to compromise the effective chemotherapy of a plethora of diseases. This thesis aspires to elucidate the molecular resistance mechanisms adopted by these bacteria, in order to expand our knowledge and to assist in the development of new therapeutic approaches to circumvent these mechanisms. On this basis, this thesis presents insights into a novel Vibrio cholerae antibiotic resistance, var, operon that encodes a metallo- β -lactamase (Msl), VarG, and a tripartite ATP-binding cassette-type (ABC-type) transport system, VarACDEF that has substrate specificities for antimicrobial peptides and macrolide antibiotics. Msls are fast emerging as a primary resistance mechanism, possibly as a consequence of the introduction of newer s-lactam antibiotics such as the carbapenems in response to increasing Gram-negative bacterial resistance. Fascinatingly, the ABC transporter, through secondary structure predictions, has been envisaged to adopt a tripartite structure similar to the MDR transporter, AcrAB-TolC, from the resistance nodulation and cell division (RND) family. The structural characterisation of this system would be the first such tripartite system to be elucidated and may bring new insights into how Gram-negative bacteria may have evolved to tackle the issue that threatens its existence. The resistance mechanisms in the var Operon are believed to be under the control of a LysR-type transcriptional regulatory protein (LTTR), VarR. LTTR proteins form one of the largest transcriptional regulatory families with extremely diverse functions ranging from amino acid biosynthesis to CO(_2) fixation. VarR binds to three distinct promoter regions, varRG, varGA and varBC located upstream and adjacent to VarG, VarA an AcrA-like membrane fusion protein and VarC a TolC-like outer membrane protein, respectively. VarR has also been shown to act as a repressor at the varRG promoter region in the absence of its substrate. Interestingly, the mechanism of regulation by VarR is strikingly similar to the well documented LTTR, AmpR and serine s-lactamase AmpC system that are found in many pathogenic bacteria. It could be that V. cholerae has evolved from this regular system and developed a s-lactamase that would prove more beneficial in light of current selective pressures. Contrary to LTTRs being notoriously recalcitrant to purification due to their low solubility, this thesis reports the successful purification and crystallisation of full-length VarR in the presence and absence of its cognate promoter DNA. Elucidating the structural characteristics of VarR would be the first such regulator associated with MDR in the LTTR family. This would advance the knowledge on the only currently existing full-length crystal structure of a LTTR, CbnR, and will provide further insights into how structural conformations may lead to dissociation from the promoter and induction of gene expression. Understanding the mechanism by which VarR induces expression of these resistance mechanisms is paramount for future strategies to prevent the emergence of MDR microorganisms. Although these mechanisms of MDR maybe elucidated in V. cholerae, the evolutionary relatedness and conservation of structure and function in all families will enable this information to be related to similar systems in alternative bacterial species.