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 (Mßl), VarG, and a tripartite ATP-binding cassette-type (ABC-type) transport system, VarACDEF that has substrate specificities for antimicrobial peptides and macrolide antibiotics. Mßls are fast emerging as a primary resistance mechanism, possibly as a consequence of the introduction of newer ß-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 ß-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 ß-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

The Vibrio cholerae var regulon encodes a metallo-β-lactamase and an antibiotic efflux pump, which are regulated by VarR, a LysR-type transcription factor

The genome sequence of V. cholerae O1 Biovar Eltor strain N16961 has revealed a putative antibiotic resistance (var) regulon that is predicted to encode a transcriptional activator (VarR), which is divergently transcribed relative to the putative resistance genes for both a metallo-β-lactamase (VarG) and an antibiotic efflux-pump (VarABCDEF). We sought to test whether these genes could confer antibiotic resistance and are organised as a regulon under the control of VarR. VarG was overexpressed and purified and shown to have β-lactamase activity against penicillins, cephalosporins and carbapenems, having the highest activity against meropenem. The expression of VarABCDEF in the Escherichia coli (ΔacrAB) strain KAM3 conferred resistance to a range of drugs, but most significant resistance was to the macrolide spiramycin. A gel-shift analysis was used to determine if VarR bound to the promoter regions of the resistance genes. Consistent with the regulation of these resistance genes, VarR binds to three distinct intergenic regions, varRG, varGA and varBC located upstream and adjacent to varG, varA and varC, respectively. VarR can act as a repressor at the varRG promoter region; whilst this repression was relieved upon addition of β-lactams, these did not dissociate the VarR/varRG-DNA complex, indicating that the de-repression of varR by β-lactams is indirect. Considering that the genomic arrangement of VarR-VarG is strikingly similar to that of AmpR-AmpC system, it is possible that V. cholerae has evolved a system for resistance to the newer β-lactams that would prove more beneficial to the bacterium in light of current selective pressures

VarR binds to the varBC intergenic region

<b>EMSA of 50ng VarR/ 0.08ng 25bp varBC IR DNA (labelled complexed with titrations of unlabelled 30bp varBC DNA. </b><div>Lanes 1 and 2, 0ng and 50ng VarR with 0.08ng 30bp <i>varRG </i>IR DNA (control). Lanes 3 to 12, competitive assay of 50ng VarR/0.08ng 25bp <i>varBC</i> complexed with titrations of unlabelled 0.08ng 25bp <i>varBC</i> IR DNA (0, 0.125, 0.25, 0.5, 1, 2, 5, 10, 20, 40ng, respectively).</div><div><br></div><div><b><br></b></div

Erythromycin does not dissociate the VarR/varBC DNA complex

<b>EMSA of VarR/25bp varBC IR DNA complex with increasing concentrations of erythromycin. </b>Lane 1 0.08ng 25bp varBC IR DNA only. Lanes 2 to 16 50ng VarR/0.08ng 25 bp varBC IR DNA complex with increasing titrations erythromycin (0, 0.5, 1, 2, 4, 8, 16, 32, 64, 128, 256,512, 1024ng, 10, 100ug, respectively).<div><br></div

Penicillin G does not dissociate the VarR/varRG DNA complex

<div><b>EMSA of VarR/30bp <i>varRG</i> IR DNA complex with increasing titration of Penicillin G. </b></div><div>Lane 1 0.08ng 30bp <i>varRG </i>IR DNA only. Lanes 2 to 16 50ng VarR/0.08ng 30bp varRG DNA complex with increasing titrations of Penicillin G (0, 0.5, 1, 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024ng, 10, 100 ug, respectively).</div

ADAMTS-10 and -6 differentially regulate cell-cell junctions and focal adhesions

ADAMTS10 and ADAMTS6 are homologous metalloproteinases with ill-defined roles. ADAMTS10 mutations cause Weill-Marchesani syndrome (WMS), implicating it in fibrillin microfibril biology since some fibrillin-1 mutations also cause WMS. However little is known about ADAMTS6 function. ADAMTS10 is resistant to furin cleavage, however we show that ADAMTS6 is effectively processed and active. Using siRNA, over-expression and mutagenesis, it was found ADAMTS6 inhibits and ADAMTS10 is required for focal adhesions, epithelial cell-cell junction formation, and microfibril deposition. Either knockdown of ADAMTS6, or disruption of its furin processing or catalytic sites restores focal adhesions, implicating its enzyme activity acts on targets in the focal adhesion complex. In ADAMTS10-depleted cultures, expression of syndecan-4 rescues focal adhesions and cell-cell junctions. Recombinant C-termini of ADAMTS10 and ADAMTS6, both of which induce focal adhesions, bind heparin and syndecan-4. However, cells overexpressing full-length ADAMTS6 lack heparan sulphate and focal adhesions, whilst depletion of ADAMTS6 induces a prominent glycocalyx. Thus ADAMTS10 and ADAMTS6 oppositely affect heparan sulphate-rich interfaces including focal adhesions. We previously showed that microfibril deposition requires fibronectin-induced focal adhesions, and cell-cell junctions in epithelial cultures. Here we reveal that ADAMTS6 causes a reduction in heparan sulphate-rich interfaces, and its expression is regulated by ADAMTS10

mNG-tagged fusion proteins and nanobodies to visualize tropomyosins in yeast and mammalian cells

Tropomyosins are structurally conserved α-helical coiled-coil proteins that bind along the length of filamentous actin (F-actin) in fungi and animals. Tropomyosins play essential roles in the stability of actin filaments and in regulating myosin II contractility. Despite the crucial role of tropomyosin in actin cytoskeletal regulation, in vivo investigations of tropomyosin are limited, mainly due to the suboptimal live-cell imaging tools currently available. Here, we report on an mNeonGreen (mNG)-tagged tropomyosin, with native promoter and linker length configuration, that clearly reports tropomyosin dynamics in Schizosaccharomyces pombe (Cdc8), Schizosaccharomyces japonicus (Cdc8) and Saccharomyces cerevisiae (Tpm1 and Tpm2). We also describe a fluorescent probe to visualize mammalian tropomyosin (TPM2 isoform). Finally, we generated a camelid nanobody against S. pombe Cdc8, which mimics the localization of mNG–Cdc8 in vivo. Using these tools, we report the presence of tropomyosin in previously unappreciated patch-like structures in fission and budding yeasts, show flow of tropomyosin (F-actin) cables to the cytokinetic actomyosin ring and identify rearrangements of the actin cytoskeleton during mating. These powerful tools and strategies will aid better analyses of tropomyosin and F-actin cables in vivo

A diagrammatic representation of the <i>var</i> operon.

<p>The locality of the β-lactamase, <i>varG</i>, the MDR <i>varABCDEF</i> transporter complex and the divergently transcribed regulatory <i>varR</i> genes are shown. Arrows indicate orientation of transcription. Three intergenic regions <i>varRG</i>, <i>varGA</i> and <i>varBC</i> to which VarR is hypothesised to regulate transcription are also illustrated.</p

VarR regulation of <i>Cm</i>^<i>R</i> expression.

<p>VarR regulation of <i>Cm</i>^<i>R</i> expression.</p