4.7 Antimicrobial activity
4.7.2 Vibroxin MIC
The quantitative determination of antimicrobial activity is necessary to evaluate antibiotic potency across di↵erent pathogenic strains. One method of clinical sig- nificance is the minimum inhibitory concentration (MIC) assay using the Clinical Laboratory and Standards Institute (CLSI) broth micro-dilution method. The lowest concentration that inhibits bacterial growth is visually determined for vi- broxin following an overnight incubation. The MIC for vibroxin was determined against six representative pathogens with growing multi-drug resistant virulence,
i.e. the ‘ESKAPE’ panel (Table 4.5). Vibroxin was shown to demonstrate potent activity against A. baumannii with a MIC of 2 µg/mL (cf. 2 µg/mL for enacy- loxin IIa). The growth of all other tested pathogens was not inhibited by vibroxin below the maximum concentration tested. Whilst the MIC value relates to growth inhibition, the minimal bactericidal concentration (MBC) represents the lowest concentration of a bioactive compound required to kill a particular pathogen. The MBC was determined to be 8 µg/mL for vibroxin against A. baumannii which lies on the boundary of what is considered bactericidal or bacteriostatic.
Table 4.5: MIC and MBC values for vibroxin determined against the ESKAPE
panel of pathogens. Organism MIC (µg/mL) MBC (µg/mL) Enterococcus faecium >32 >32 Staphylococcus aureus >32 >32 Klebsiella pneumoniae >32 >32 Acinetobacter baumannii 2 8 Pseudomonas aeruginosa >32 >32 Enterobacter cloacae >32 >32
4.8
Conclusion and further work
Genome mining of V. rhizosphaerae led to the targeted discovery of the antibi- otic vibroxin, revealing the value of rhizosphere-associated Vibrio as a source of
evolution of the homologous enacyloxin biosynthetic gene cluster. In particular, how genetic inversion events and excisions have left relics in terms of domain frag- ments. A number of di↵erences between the enacyloxin and vibroxin biosynthetic pathways have not yet been fully explained. Whilst VbxJ contains an additional KR domain, compared to Bamb 5920, both biosynthetic pathways display KR function at this stage. The origin of the KR enzymatic function in enacyloxin biosynthesis therefore remains unknown. A dehydration event is predicted to take place in the second module of the vibroxin PKS, yet no DH domain is present within VbxE. The underlying mechanism of this di↵erence between vibroxin and enacyloxin is still unclear.
The development of an HMM to identify sMT domains assists with the an- notation of any PKS gene utilising this type of domain. This HMM can provide valuable insight when predicting core molecular structures of putative polyketides, and has uncovered indications of evolutionary development of loading modules within PKSs. The basis for methylation control by sMT domains has not been elucidated in terms of predicting the number of methylations of a starter unit, but provides an opportunity for engineering starter units within biosynthetic path- ways utilising a GNAT-based loading strategy.
The potent antimicrobial activity of vibroxin against the opportunistic pathogen
A. baumannii highlights the value of targeted drug discovery using a genomics-
driven approach. Vibroxin is potentially more exploitable for semi-synthesis or total chemical synthesis due to a simplified molecular structure compared with enacyloxin, and is chemically more stable. Novel molecular features or fragments could be utilised as a reference on which to design more potent, stable vibroxin analogues via biosynthesis or chemical synthesis. The stereochemistry of the hydroxyls in the polyketide chain, and the proximal methyl (C-12 to C-17), is proposed based upon KR stereospecificity, but experimentally determined stereo- chemistry is required, which may be achieved from NMR spectroscopy of vibroxin derivatives or comparison with chemically synthesized standards.
Chapter 5
Materials and Methods
5.1
Materials
5.1.1
Plasmids
Constructs for the expression of domains from the enaclyoxin biosynthetic path- way were provided by Dr P Sydor (University of Warwick), and those encoding watasemycin NRPS domains were provided by S Zhou (University of Warwick), within the pET151 plasmid; these are listed in Table 5.1. The plasmid containing
sfp was acquired from Manuela Tosin (University of Warwick) in pET28b.
Table 5.1: Plasmids for protein production.
Protein domain names Gene name Length (bp)
PCP-COM2C bamb 5917 405 ACP bamb 5919 660 COM2N-C bamb 5915 1638 KS0 bamb 5919 2156 PCP-COM2C sven 0512 453 Sfp sfp B. subtilis -
5.1.2
Microbial Strains
BL21 (DE3) (Invitrogen) and C43 (DE3) (Invitrogen) were used. Vibrio rhi-
zosphaerae MSSRF3 (DSM 18581) was used for vibroxin production. Bioactivity
assays using V. rhizosphaerae or vibroxin were performed using the following organisms:
• Bacillus subtilis
• Methicillin-resistant Staphylococcus aureus DSM 21979
• Staphylococcus aureus NCTC 12981
• Escherichia coli SY397
• Serratia plymuthica RVH1
• Pseudomonas aeruginosa DSM 29239
• Burkholderia multivorans ATCC 17616
• Klebsiella pneumoniae DSM 26371 • Enterobacter cloacae DSM 16690 • Acinetobacter baumannii DSM 25645 • Candida albicans SC 5314 • Enterococcus faecium DSM 25390
5.1.3
Culture media
Lysogeny broth (LB) (Fisher), tryptic soy broth (TSB) (Fisher) and basal salt medium (BSM),175 were made following the suppliers recommended dilutions.
Glycerol or glucose was used as a carbon source for BSM media to a final con- centration of 4 g/L.
M9 minimal media
5 ⇥ M9 salt solution
• 32 g Na2HPO4.7H2O • 6 g KH2PO4
• 2.5 g NaCl
• Add H2O up to 500 mL and autoclave
M9 minimal media
• 200 mL 5 ⇥ M9 salt solution
• 2 mL of 1 mMgSO4 • 100 µL of 1 mCaCl2
• Add H2O up to 1 L and autoclave
• 2 g of d-13C-glucose (or 4 g of d-glucose) in 20 mL H
2O with 0.2 µm
filtration
• 20 mL of 100 ⇥ BME vitamins (Sigma Aldrich)
• 0.5 g of 15NH
4Cl in 2.5 mL H2O with 0.2 µm filtration
• Ampicillin (final concentration 50 µg/mL)
5.2
Molecular Biology
5.2.1
Site-directed mutagenesis
For single, double and truncation mutations, the Q5 site-directed mutagenesis kit (NEB) was used. Primers were designed using the associated NEBaseChanger
tool, where truncation mutants were achieved through substitution mutants us- ing the TGA stop codon. Annealing temperatures, PCR protocols, Kinase- Ligase-DpnI (KLD) reactions, and transformations were conducted according to suppliers guidelines. Typically, three colonies were selected for plasmid isola- tion using the GeneJET Plasmid Miniprep Kit (Thermo Scientific). DNA con- centration was measured by absorbance at 280 nm. Resulting plasmids were confirmed by DNA sequencing. The primers used for site directed mutagene- sis were: PCP 9 f = GGCGCCTTCGGGCGACCAGGACGATGCATGAGAG- TACGAAGAAGGCGTGATCC, PCP 9 r = CCGCGGAAGCCCGCTGGTC- CTGCTACGTACTCTCATGCTTCTTCCGCACTAGG.
5.2.2
Sequencing
Sanger sequencing (GATC-biotech) was used to determine successful mutations of genes within the pET151 plasmid. Samples were prepared according to the company’s requirements. Sequencing primers were T7 forward primer and pET- RP reverse primer.