Bioactivity of K12A, K12S and K12T producers against various Gram-positive targets.

<p>Values are the mean of triplicate deferred antagonism assays and represent zone of inhibition (diameter of zone minus diameter of bacterial growth) expressed as a percentage compared to that of the wild-type nisin producer at 100%. S.D.: Standard Deviation; Relative Standard Deviation<10% for each given value. All values in bold reached statistical significance compared to nisin control (K) (Student's t-test: P<0.05). Strains marked with an asterisk are drug resistant isolates.</p

RP-HPLC Separation of nisin A K12S/Dha and K12T/Dhb.

<p>Representative RP-HPLC peaks showing the appearance of A K12S and B K12T after optimization of solvent gradient. Relative production levels were determined using triplicate peak areas from two independent purifications from first principles. Relative Standard Deviation<12% for each given value.</p

Structure of nisin A.

<p>The structure of nisin A is depicted, showing the location of its five (β-methyl)lanthionine rings (A–E) and modified residues dehydroalanine (yellow) and dehydrobutyrine (green). Position K12 is highlighted in red and the hinge region is boxed.</p

Mass Spectrometry Analysis and Bioactivity Determination of Nisin K12X Bank.

<p>Observed molecular mass (+/−0.25 Da) from MALDI-TOF MS analysis of NZ9800 pDF05-K12X producers. ND: not detected. * represents dehydrated forms (hydrophobic modified residues), i.e. Dhb in the case of T and Dha in the case of S.</p><p>Bioactivity of NZ9800 pDF05-K12X producers against <i>L. lactis</i> HP. Values given are the mean of triplicate deferred antagonism assays and represent zone of inhibition (diameter of zone minus diameter of bacterial growth) expressed as a percentage compared to that of the wild-type nisin producer at 100%. S.D.: Standard Deviation; Relative Standard Deviation <10% for each given value. N/A: not applicable. All values in bold reached statistical significance compared to nisin control (K) (Student's t-test: P<0.05).</p

Bioactivity of purified nisin (WT) and nisin K12A against representative Gram-negative targets.

<p>Results from agarose gel diffusion assays of purified nisin and nisin K12A at a concentration of 40 µM against three Gram-negative strains. Results are expressed as both zone diameter and as K12A bioactivity compared to that of nisin A at 100%. Values represent the mean of triplicate agarose assay results. Standard deviation values in brackets; Relative Standard Deviation<10% for each given value. Values in bold reached statistical significance compared to nisin control (Student's t-test: P<3E⁻⁰⁴).</p

Minimum inhibitory concentrations of purified nisin (WT) and nisin K12A against various Gram-positive targets.

<p>Results from minimum inhibitory concentration assays of purified nisin (WT) and nisin K12A against various Gram-positive targets. Values given are identical results from three independent determinations. Fold Difference represents the improvement of K12A compared to nisin against the relevant indicator.</p

Oligonucleotides used in this study.

<p>PHO indicates 5′ phosphate modification. Underlined sequences represent degenerate codon (N = A+C+G+T, K = G+T, M = A+C). Lower-case letters indicate site-directed mutation.</p

Strains and plasmids used in this study.

<p>DPC: Dairy Products Research Centre, Moorepark; UCC: University College Cork; NCDO: National Collection of Dairy Organisms; DSMZ: German Collection of Microorganisms and Cell Cultures; ATCC: American Type Culture Collection; NCIMB: National Collection of Industrial, Food and Marine Bacteria; BSAC: British Society for Antimicrobial Chemotherapy; ILSI: International Life Science Institute (MartinWiedmann), ARS: Agricultural Research Service, U.S. Department of Agriculture (Todd Ward).</p

Borrelia TOMM Alignment

BorB (A), borC (B) and borD (C) from Borrelia afzelii PKo (Bafz), B. spielmanii A14S (Bspi), and B. valaisiana VS116 (Bval) were aligned using ClustalW. Sites where PCR screening primers annealed are underlined. The primers used to detect borB/C/D were based on bvalB/C/D from B. valaisiana VS11

HIV Protease Inhibitors Block Streptolysin S Production

Streptolysin S (SLS) is a post-translationally modified peptide cytolysin that is produced by the human pathogen <i>Streptococcus pyogenes.</i> SLS belongs to a large family of azole-containing natural products that are biosynthesized via an evolutionarily conserved pathway. SLS is an important virulence factor during <i>S. pyogenes</i> infections, but despite an extensive history of study, further investigations are needed to clarify several steps of its biosynthesis. To this end, chemical inhibitors of SLS biosynthesis would be valuable tools to interrogate the various maturation steps of both SLS and biosynthetically related natural products. Such chemical inhibitors could also potentially serve as antivirulence therapeutics, which in theory may alleviate the spread of antibiotic resistance. In this work, we demonstrate that FDA-approved HIV protease inhibitors, especially nelfinavir, block a key proteolytic processing step during SLS production. This inhibition was demonstrated in live <i>S. pyogenes</i> cells and through <i>in vitro</i> protease inhibition assays. A panel of 57 nelfinavir analogs was synthesized, leading to a series of compounds with improved anti-SLS activity while illuminating structure–activity relationships. Nelfinavir was also found to inhibit the maturation of other azole-containing natural products, namely those involved in listeriolysin S, clostridiolysin S, and plantazolicin production. The use of nelfinavir analogs as inhibitors of SLS production has allowed us to begin examining the proteolysis event in SLS maturation and will aid in further investigations of the biosynthesis of SLS and related natural products