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    Correlated transcriptional responses provide insights into the synergy mechanisms of the furazolidone, vancomycin and sodium deoxycholate triple combination in Escherichia coli
    (American Society for Microbiology, 2021-10-27) Olivera C; Cox MP; Rowlands GJ; Rakonjac J; Bradford PA
    Effective therapeutic options are urgently needed to tackle antibiotic resistance. Furazolidone (FZ), vancomycin (VAN), and sodium deoxycholate (DOC) show promise as their combination can synergistically inhibit the growth of, and kill, multidrug-resistant Gram-negative bacteria that are classified as critical priority by the World Health Organization. Here, we investigated the mechanisms of action and synergy of this drug combination using a transcriptomics approach in the model bacterium Escherichia coli. We show that FZ and DOC elicit highly similar gene perturbations indicative of iron starvation, decreased respiration and metabolism, and translational stress. In contrast, VAN induced envelope stress responses, in agreement with its known role in peptidoglycan synthesis inhibition. FZ induces the SOS response consistent with its DNA-damaging effects, but we demonstrate that using FZ in combination with the other two compounds enables lower dosages and largely mitigates its mutagenic effects. Based on the gene expression changes identified, we propose a synergy mechanism where the combined effects of FZ, VAN, and DOC amplify damage to Gram-negative bacteria while simultaneously suppressing antibiotic resistance mechanisms. IMPORTANCE Synergistic antibiotic combinations are a promising alternative strategy for developing effective therapies for multidrug-resistant bacterial infections. The synergistic combination of the existing antibiotics nitrofurans and vancomycin with sodium deoxycholate shows promise in inhibiting and killing multidrug-resistant Gram-negative bacteria. We examined the mechanism of action and synergy of these three antibacterials and proposed a mechanistic basis for their synergy. Our results highlight much-needed mechanistic information necessary to advance this combination as a potential therapy.
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    Requirements of Escherichia coli to survive stress induced by the secretin, pIV : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Manawatū, New Zealand
    (Massey University, 2018) Bagley, Stefanie Jayne
    Pathogenic Gram-negative bacteria utilise complex multiprotein and functionally unrelated trans-envelope machineries to secrete toxins and other virulence factors. Such machineries are referred to as secretion systems. These contain large, membrane-inserted homologous channels called a secretin. These secretion systems include the type II and III secretion systems (T2SS and T3SS), type IV pili assembly system (T4PS), and the filamentous phage assembly-secretion system (FFSS). Secretins are homomultimers with radial symmetry blocked by an inner gate or septum and have a pore size of up to 10 nm. As determined by previous studies on the FFSS secretin, pIV, and the T3SS secretin, InvG, there is a cost associated with the insertion of large membrane channels. Membrane integrity is disrupted, leaving the bacterial cell highly susceptible to antibiotics and environmental stressors. As a result, Gram-negative bacteria have developed stress response pathways which upregulate genes to mitigate this secretin induced stress. These are the Phage Shock Protein response (Psp), Conjugative plasmid expression (Cpx), Regulation of capsular synthesis (Rcs), and SoxRS Superoxide response (Sox). Not all individual genes within these stress response pathways are necessarily required for the survival of Escherichia coli expressing secretin. Stress can be induced in E. coli by expression of leaky pIV mutants as they are open, not gated, under physiological conditions and imitate the actively secreting channel. A synthetic lethality assay was performed to determine the importance of the key regulators from four stress response pathways (PspF, CpxR, RcsA, RcsB, SoxR, and SoxS) on cell viability in the presence of the leaky secretin mutant, pIV-E292K. Here it was determined that the Psp, Rcs and, (to a lesser extent), Cpx regulons, confer a protective effect on E. coli K-12 experiencing stress induced by pIV-E292K. Expression of pIV-E292K mutant also induced an Rcs-dependent capsular polysaccharide phenotype indicating upregulation of Rcs in response to leaky pIV production. These three responses are potential drug targets in the fight against antibiotic-resistant infections. Inhibition of the stress response may prevent mitigation of membrane stress, thereby killing the channel-expressing bacteria.
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    Characterization of the secretins, large outer membrane channels of gram-negative bacteria : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand
    (Massey University, 2015) Khanum, Sofia
    Secretins, a family of large outer membrane channels, mediate secretion and/or assembly of virulence factors and/or complex proteinaceous structures, such as rods, type IV pili and filamentous bacteriophage. Secretins form large radially-symmetric channels composed of 12 to 14 identical subunits, with internal diameters of up to 10 nm, whose lumen is interrupted by a septum/plug structure that very likely represents a gate or a valve. The identity of septum in the primary sequence of secretins has not been determined as yet, however the cryo-EM and SPA analyses point to the C-terminal domain forming these structures, whereas mutagenesis specifically identified two regions in this domain, named GATE1 and GATE2, as having an important role in gating of the filamentous phage secretion system secretin pIV. However, it is not known whether these regions are also involved in gating of the secretins from type II and type III secretion systems. In this work, twelve “leaky-gate” mutants in the secretin PulD from the type II secretion system (T2SS), selected from a random mutant library based on ability to utilise 829 Da oligosaccharide maltopentaose in the absence of maltoporin, were analysed in detail. Most of PulD leaky-gate mutants clustered in the GATE1 and GATE2 regions. All point mutants were positive for secretion of the cognate PulD substrate, enzyme Pullulanase (PulA), whereas a 5-residue in-frame deletion (?477-481) was negative. Two severely leaky GATE1 region mutants, G458S and ?477-481,sensitised E. coli to all tested antibiotics whose molecular weight is too high to pass through porins: rifamycin SV (720 Da), bacitracin (1423 Da) vancomycin (1449 Da) and daptomycin (1621 Da). The GATE1 of this secretin is therefore a potential drug target, for design of molecules that can sensitise secretin-containing pathogenic Gram-negative bacteria to > 600 Da antibiotics and/or block the secretion of substrates, including virulence factors. Engineered chimeras between PulD and pIV were used to probe functional compartmentalisation among the secretin domains and the segments involved in gating. This analysis showed that the N-terminal domains, GATE1 region and channel-forming secretin homology domain are interdependent with respect to function in secretion/assembly of the substrates, and to different extends for folding and multimerisation. This work further analysed the gating properties of the type III secretion system (T3SS) secretins EscC and InvG. When expressed in E. coli K12, these secretins were naturally “leaky” and mistargeted to the inner membrane, resulting in growth retardation. The survival of E. coli expressing these secretins depended on the PspF, positive regulator of the key inner membrane stress response Psp. Therefore, in the T3SS secretin-expressing or toxin-secreting cells, PspF is a potential target for design of molecules that could kill T3SS-containing toxin-secreting Gram-negative bacteria.
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    Extracytoplasmic stress responses induced by a model secretin : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatū, New Zealand
    (Massey University, 2015) Spagnuolo, Julian
    Pathogenic bacteria export large proteins and protein complexes, including virulence factors, using dedicated transenvelope multiprotein machinery, collectively called secretion systems. Four of these protein export machines found in Gram-negative bacteria, type 2/3 secretion systems, filamentous phage assembly-secretion system and the type 4 pilus assembly system contain large homologous gated channels, called secretins, in the outer membrane. Secretins are radially symmetrical homomultimers (luminal diameter 6-8 nm) interrupted by an internal septum or gate. Expression of these channels imposes a fitness cost to bacteria. While stress induced by model secretin pIV has been previously investigated using microarrays, this thesis is the first RNAseq characterisation of secretin stress responses. Furthermore, this is the first comparison of stress imposed by a closed-gate secretin (wildtype pIV), vs. an isogenic leaky-gate variant, the latter serving as a model of an open-gate substrate-secreting channel. The high sensitivity to changes in gene expression and low background noise of the RNA-seq approach have greatly expanded the known secretin stress responses to include the SoxS, CpxR and RcsB/RcsAB regulons, in addition to the known involvement of the Psp response. A synthetic lethality analysis of candidate genes in these pathways suggested that the leaky-gate secretins, besides rendering the Psp response essential for survival, also stimulate the SoxS and RcsB/RcsAB regulons for protection of the cells. Knowledge of the secretin stress expanded by this work helped identify potential targets for development of much-needed antibiotics against toxinsecreting Gram-negative bacteria.
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    Biochemical characterization of metal-dependent 3-deoxy-D-manno-octulosonate 8-phosphate synthases from Chlorobium tepidum & Acidithiobacillus ferrooxidans : a thesis presented in partial fulfillment of the requirements for the degree of Masterate of Science in Biochemistry at Massey University, Turitea, Palmerston North, New Zealand
    (Massey University, 2007) Yeoman, Jeffrey Aaron
    3-Deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase is the enzyme responsible for catalyzing the first reaction in the biosynthesis of KDO. KDO is an essential component in the cell wall of Gram-negative bacteria and plants. This compound is not present in mammals; therefore the enzymes responsible for its biosynthesis are potential targets for the development of new antibiotic agents. KDO8P synthase catalyzes the condensation reaction between phosphoenol pyruvate (PEP) and D-arabinose 5-phosphate (A5P) to form KDO8P. Two types of KDO8P synthase have been identified; a metal-dependent type and a non metal-dependent type. KDO8P synthase from the organism Chlorobium tepidum (Cte) has been partially purified and partially characterized. In line with predictions based on sequence alone, the activity of this enzyme is dependent on the presence of a divalent metal ion and is sensitive to the presence of the metal chelating agent EDTA. Cte KDO8P synthase was found to have the highest activity in the presence of Mn2+ or Cd2+. KDO8P synthase from the organism Acidithiobacillus ferrooxidans (Afe) has also been cloned, purified and biochemically characterized. Afe KDO8P synthase was also found to be a metallo enzyme and the catalytic activity is highest in the presence of Mn2+ or Co2+. Afe KDO8P synthase was found to exist as a tetramer in solution and is most active within the pH range of 6.8 to 7.5 and within a temperature range of 35 ºC to 40 ºC. Sequence analysis suggests that this enzyme has characteristics conserved throughout the metallo and the non-metallo KDO8P synthases and is closely related to the metal-dependent 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) synthases. The role of several active-site residues of Afe KDO8P synthase has been investigated. A C21N mutant of Afe KDO8P synthase was found to retain 0.5% of wildtype activity and did not require a divalent metal ion for catalytic activity. This suggests that the metallo and non-metallo KDO8P synthases have similar catalytic mechanisms.
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    Correlated Transcriptional Responses Provide Insights into the Synergy Mechanisms of the Furazolidone, Vancomycin, and Sodium Deoxycholate Triple Combination in Escherichia coli.
    (27/10/2021) Olivera C; Cox MP; Rowlands GJ; Rakonjac J
    Effective therapeutic options are urgently needed to tackle antibiotic resistance. Furazolidone (FZ), vancomycin (VAN), and sodium deoxycholate (DOC) show promise as their combination can synergistically inhibit the growth of, and kill, multidrug-resistant Gram-negative bacteria that are classified as critical priority by the World Health Organization. Here, we investigated the mechanisms of action and synergy of this drug combination using a transcriptomics approach in the model bacterium Escherichia coli. We show that FZ and DOC elicit highly similar gene perturbations indicative of iron starvation, decreased respiration and metabolism, and translational stress. In contrast, VAN induced envelope stress responses, in agreement with its known role in peptidoglycan synthesis inhibition. FZ induces the SOS response consistent with its DNA-damaging effects, but we demonstrate that using FZ in combination with the other two compounds enables lower dosages and largely mitigates its mutagenic effects. Based on the gene expression changes identified, we propose a synergy mechanism where the combined effects of FZ, VAN, and DOC amplify damage to Gram-negative bacteria while simultaneously suppressing antibiotic resistance mechanisms. IMPORTANCE Synergistic antibiotic combinations are a promising alternative strategy for developing effective therapies for multidrug-resistant bacterial infections. The synergistic combination of the existing antibiotics nitrofurans and vancomycin with sodium deoxycholate shows promise in inhibiting and killing multidrug-resistant Gram-negative bacteria. We examined the mechanism of action and synergy of these three antibacterials and proposed a mechanistic basis for their synergy. Our results highlight much-needed mechanistic information necessary to advance this combination as a potential therapy.