Browsing by Author "Smith, Laura J."

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    Cmr is a redox-responsive regulator of DosR that contributes to M. tuberculosis virulence.
    (Oxford University Press, 2017-05-08) Smith, Laura J.; Bochkareva, Aleksandra; Rolfe, Matthew D.; Hunt, Debbie M.; Kahramanoglou, Christina; Braun, Yvonne; Rodgers, Angela; Blockley, Alix; Coade, Stephen; Lougheed, Kathryn E. A.; Hafneh, Nor Azian; Glenn, Sarah M.; Crack, Jason C.; Le Brun, Nick E.; Saldanha, José W.; Makarov, Vadim; Nobeli, Irene; Arnvig, Kristine; Mukamolova, Galina V.; Buxton, Roger S.; Green, Jeffrey
    Mycobacterium tuberculosis (MTb) is the causative agent of pulmonary tuberculosis (TB). MTb colonizes the human lung, often entering a non-replicating state before progressing to life-threatening active infections. Transcriptional reprogramming is essential for TB pathogenesis. In vitro, Cmr (a member of the CRP/FNR super-family of transcription regulators) bound at a single DNA site to act as a dual regulator of cmr transcription and an activator of the divergent rv1676 gene. Transcriptional profiling and DNA-binding assays suggested that Cmr directly represses dosR expression. The DosR regulon is thought to be involved in establishing latent tuberculosis infections in response to hypoxia and nitric oxide. Accordingly, DNA-binding by Cmr was severely impaired by nitrosation. A cmr mutant was better able to survive a nitrosative stress challenge but was attenuated in a mouse aerosol infection model. The complemented mutant exhibited a ∼2-fold increase in cmr expression, which led to increased sensitivity to nitrosative stress. This, and the inability to restore wild-type behaviour in the infection model, suggests that precise regulation of the cmr locus, which is associated with Region of Difference 150 in hypervirulent Beijing strains of Mtb, is important for TB pathogenesis.
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    From formulation to in vivo model: A comprehensive study of a synergistic relationship between vancomycin, carvacrol and cuminaldehyde against Enterococcus faecium
    (Wiley, 2020-02-11) Owen, Lucy; Webb, Joseph P.; Green, Jeffrey; Smith, Laura J.; Laird, Katie
    Vancomycin Resistant Enterococcus faecium (VRE) has become endemic in healthcare settings, reducing treatment options for enterococcal infections. New antimicrobials for VRE infections are a high priority, but the development of novel antibiotics is time-consuming and expensive. Essential Oils (EOs) synergistically enhance the activity of some existing antibiotics, suggesting that EO-antibiotic combinations could re-sensitise resistant bacteria and maintain the antibiotic repertoire. The mechanism of re-sensitisation of bacteria to antibiotics by EOs is relatively understudied. Here, the synergistic interactions between carvacrol (1.98 mM) and cuminaldehyde (4.20 mM) were shown to re-establish susceptibility to vancomycin (0.031 mg/L) in VRE, resulting in bactericidal activity (4.73 log10 CFU/mL reduction). Gene expression profiling, coupled with β-galactosidase leakage and salt tolerance assays suggested that cell envelope damage contributes to the synergistic bactericidal effect against VRE. The EO-vancomycin combination was also shown to kill clinical isolates of VRE (2.33-5.25 log10 CFU/mL reduction) and stable resistance did not appear to develop even after multiple passages. The in vivo efficacy of the EO-vancomycin combination was tested in a Galleria mellonella larvae assay; however no antimicrobial action was observed, indicating that further drug development is required for the EO-vancomycin combination to be clinically useful for treatment of VRE infections.
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    Is the antimicrobial mechanism of action of essential oils against bacteria associated with channels similar to TRPV1 channels found in mammalian cells?
    (British Society for Antimicrobial Chemotherapy, 2018-01-18) Alhareth, Z; Owen, L.; Dixon, C. J.; McKechnie, K.; Smith, Laura J.; Laird, Katie
    Objectives: The aim of this investigation was to find out if the antimicrobial mechanism of action of Essential Oils (EOs) against Enterococcus spp. may be associated with channels similar to TRPV1 channels found in mammalian cells. A known antimicrobial combination of EOs compounds (Carvacrol 0.2µl and Cuminaldehyde 25µl) against E. faecium and capsaicin were used to investigate this hypothesis. The bacterial membrane has many porin proteins which act as hydrophilic transmembrane channels and allow small hydrophilic solutes to pass through them1, such channels may be the target for the antimicrobial mechanism of action of EOs in the Gram-positive bacterial cell wall. Methods: Transient receptor potential (TRP) channels, are a group of unique ion channels in mammalian cells, that are effected by a wide spectrum of physical and chemical stimuli2, including high temperature and low pH. Capsaicin the EO compound of chilli peppers is known to be a TRP channel antagonist in mammalian cells2,3 whilst AMG 517 specifically blocks TRP channels, and Ruthenium Red (RR) is a wide spectrum blocker for TRPV1 channels4,5. The Minimum Inhibitory Concentration (MIC) of capsaicin against Vancomycin Sensitive E. faecium (VSE) was defined by agar-broth dilution method, a checkerboard assay was conducted to determine the interactions of AMG517 and RR against E. faecium. A growth curve was also conducted to assess the effect of the EO blend in the presence of AMG517. Results: Capsaicin showed antibacterial activity against E. faecium with an MIC of 1.6mM. The checkerboard data demonstrated that AMG517 has no inhibitory effect on E. faecium and therefore was a good candidate to reverse the effect of EOs blend by potentially blocking channels that were similar to TRPV1. RR was antimicrobial at concentrations above 10µM, however, AMG517 above 60nM reverses the inhibitory effect of RR, demonstrating a competitive effect of AMG517 and RR towards a definite site in the bacterial cell wall. Growth curves showed no reverse effect of the EO blend in the presence of AMG517. Conclusions: This study does not support the existence of a TRPVI homologue in the bacterial cell. Future studies will investigate the genes related to transport channels in E. faecium, that are differentially regulated when exposed to the EO blend, in order to further understand the effect of the EOs on transport channels in bacteria.
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    The role of transport channels in the mechanism of action of a novel antimicrobial EO-vancomycin combination against vancomycin-resistant Enterococcus faecium
    (Elsevier, 2022-11-05) Alhareth, Zakia; Owen, Lucy; Laird, Katie; Smith, Laura J.
    Background Novel antimicrobials with new mechanisms of action are critical to circumvent emerging antimicrobial resistant microorganisms (AMR), such as vancomycin resistant Enterococcus faecium (VanREF). Previous research demonstrated, with transcriptomic analysis and phenotypic assays, that the essential oil components (EOCs) carvacrol (CARV) (1.98 mM) and cuminaldehyde (CA) (4.20 mM) with the antibiotic vancomycin (Van) (0.022 µM), restored the susceptibility of VanREF. This finding suggested that an envelope damage has occurred. Several transport channel-related genes were also differentially expressed including bcr, ecfA1, ecsA-1, yloB and nhaC_2, indicating they could contribute to the mechanism of action of the triple combination CARV-CA-Van. Purpose The aim of this study was to elucidate the role of transport channels in the antimicrobial mechanism of action of CARV and CA with Van. Methods The expression levels of bcr, ecfA1, ecsA-1, yloB and nhaC_2 were established using qPCR under the effect of the triple combination, and in the presence of 1 mM calcium or 0.1 mM EDTA (channel blocker) over time. Results Significant (p ≤ 0.05) changes in expression of yloB and bcr genes were observed at 10 and 30 min, and of ecfA1, ecsA-1 and nhaC_2 at later time points (120 and 360 min). Adding Ca2+ to the combination induced significant changes in the expression of yloB (from -5.67 to -50-fold, +3.68 to -4.4-fold, -1.8 to +6.6-fold and +1.32 to +3.08-fold at 10, 30, 60 and 120 min, respectively). Instead, the addition of EDTA significantly changed the expression of bcr (from -13.5 to -1.11-fold, +15.3 to -1.06-fold, +15.71 to -10-fold at 10, 120 and 360 min, respectively). Since that Ca2+ participates in the stability of bacterial cell wall and EDTA blocks efflux pumps, the current findings suggest the involvement of the efflux system in the mechanism of action of the three-drug combination. Conclusion Overall, the findings of this study suggest the involvement of transportation channels in the mechanism of action of the CARV-CA-Van combination against VanREF.
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    ItemOpen Access
    Structure of a Wbl protein and implications for NO sensing by M. tuberculosis
    (Nature Publishing Group, 2017-12-22) Kudhair, B.K.; Hounslow, A.M.; Rolfe, Matthew D.; Crack, Jason C.; Hunt, Debbie M.; Buxton, Roger S.; Smith, Laura J.; Le Brun, Nick E.; Williamson, M.P.; Green , J.
    Mycobacterium tuberculosis causes pulmonary tuberculosis (TB) and claims ~1.8 million human lives per annum. Host nitric oxide (NO) is important in controlling TB infection. M. tuberculosis WhiB1 is a NO-responsive Wbl protein (actinobacterial iron–sulfur proteins first identified in the 1970s). Until now, the structure of a Wbl protein has not been available. Here a NMR structural model of WhiB1 reveals that Wbl proteins are four-helix bundles with a core of three α-helices held together by a [4Fe-4S] cluster. The iron–sulfur cluster is required for formation of a complex with the major sigma factor (σA) and reaction with NO disassembles this complex. The WhiB1 structure suggests that loss of the iron–sulfur cluster (by nitrosylation) permits positively charged residues in the C-terminal helix to engage in DNA binding, triggering a major reprogramming of gene expression that includes components of the virulence-critical ESX-1 secretion system.
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    Transcriptional response of vancomycin resistant Enterococcus faecium to a synergistic antibiotic-essential oil combination: A strategy to preserve the current antibiotic repertoire?
    (2019-04) Owen, Lucy; Webb, Joseph P.; Green, Jeffrey; Smith, Laura J.; Laird, Katie
    Background: New antimicrobials to treat Vancomycin Resistant Enterococcus faecium (VRE) infections are considered a high priority. Essential oil compounds have been shown to interact synergistically with antibiotics, and so could be used as adjuvants to preserve the antibiotic repertoire. A combination of carvacrol, cuminaldehyde and vancomycin was found to synergistically inhibit VRE, potentially extending the utility of vancomycin against VRE. This study aimed to investigate the mechanism of action of the carvacrol, cuminaldehyde and vancomycin combination against VRE using transcriptomic analysis. Materials/methods: The antimicrobial activity of the combination in 1% DMSO was determined by a time-kill assay. Transcriptomic response of VRE to the combination was determined by microarray analysis. VRE was treated with either 0.031 mg/L vancomycin, 1.98 mM carvacrol, 4.20 mM cuminaldehyde or the ternary combination for 60 minutes. A control of 1% DMSO only was included. RNA was extracted, converted to cDNA labelled with Cy5 and hybridised onto a custom microarray and scanned. Significant (p<0.05) differences in gene expression were determined using a one-way Analysis of Variance (ANOVA) with Benjamini Hochberg FDR multiple testing correction and Tukey’s post-hoc test. Expression changes of genes of interest were confirmed by real time quantitative PCR. Results: The combination of carvacrol, cuminaldehyde and vancomycin reduced VRE by 3.96 log10 Colony Forming Units/mL over 24 hours. Expression of 14 genes were significantly altered by the combination (p<0.05, >2-fold change). Genes with the greatest change in expression mainly related to carbohydrate metabolism; the phosphotransferase system (PTS)-associated genes mtlD, mtlF and agaC6 were downregulated 2.86-2.28-fold and ulaA3 was upregulated 4.07-fold. Glutamine-fructose-6-phosphate aminotransferase (glmS), which is associated with amino sugar biosynthesis was downregulated 2.67-3.83-fold. Vancomycin resistance-associated genes were not altered by the combination or individual components. Conclusions: Carvacrol and cuminaldehyde synergistically enhance the antimicrobial activity of vancomycin against VRE, and so could be useful to preserve the antibiotic repertoire. The combination affected carbohydrate metabolism and biosynthetic processes, indicating that the combination did not directly modulate antibiotic resistance genes. Further research will investigate significantly changed genes to enhance understanding of the synergistic mechanism of action of the combination.