Xenorhabdus khoisanae SB10 produces Lys-rich PAX lipopeptides and a Xenocoumacin in its antimicrobial complex.

BACKGROUND: Xenorhabdus spp. live in close symbiosis with nematodes of the Steinernema genus. Steinernema nematodes infect an insect larva and release their symbionts into the haemocoel of the insect. Once released into the haemocoel, the bacteria produce bioactive compounds to create a semi-exclusive environment by inhibiting the growth of bacteria, yeasts and molds. The antimicrobial compounds thus far identified are xenocoumacins, xenortides, xenorhabdins, indole derivatives, xenoamicins, bicornutin and a number of antimicrobial peptides. The latter may be linear peptides such as the bacteriocins xenocin and xenorhabdicin, rhabdopeptides and cabanillasin, or cyclic, such as PAX lipopeptides, taxlllaids, xenobactin and szentiamide. Thus far, production of antimicrobial compounds have been reported for Xenorhabdus nematophila, Xenorhabdus budapestensis, Xenorhabdus cabanillasii, Xenorhabdus kozodoii, Xenorhabdus szentirmaii, Xenorhabdus doucetiae, Xenorhabdus mauleonii, Xenorhabdus indica and Xenorhabdus bovienii. Here we describe, for the first time, PAX lipopeptides and xenocoumacin 2 produced by Xenorhabdus khoisanae. These compounds were identified using ultraperformance liquid chromatography, linked to high resolution electrospray ionisation mass spectrometry and tandem mass spectrometry. RESULTS: Cell-free supernatants of X. khoisanae SB10 were heat stable and active against Bacillus subtilis subsp. subtilis, Escherichia coli and Candida albicans. Five lysine-rich lipopeptides from the PAX group were identified in HPLC fractions, with PAX1' and PAX7 present in the highest concentrations. Three novel PAX7 peptides with putative enoyl modifications and two linear analogues of PAX1' were also detected. A small antibiotic compound, yellow in colour and λmax of 314 nm, was recovered from the HPLC fractions and identified as xenocoumacin 2. The PAX lipopeptides and xenocoumacin 2 correlated with the genes and gene clusters in the genome of X. khoisanae SB10. CONCLUSION: With UPLC-MS and MSe analyses of compounds in the antimicrobial complex of X. khoisanae SB10, a number of PAX peptides and a xenocoumacin were identified. The combination of pure PAX1' peptide with xenocoumacin 2 resulted in high antimicrobial activity. Many of the fractions did, however, contain labile compounds and some fractions were difficult to resolve. It is thus possible that strain SB10 may produce more antimicrobial compounds than reported here, as suggested by the APE Ec biosynthetic complex. Further research is required to develop these broad-spectrum antimicrobial compounds into drugs that may be used in the fight against microbial infections.

Modelling the variable incorporation of aromatic amino acids in the tyrocidines and analogous cyclodecapeptides.

AIMS: A mathematical model of the nonribosomal synthesis of tyrocidines and analogues by Brevibacillus parabrevis was constructed using a competitive binding mechanism (CBM) for the incorporation of the three variable aromatic amino acid (Aaa) residues in their sequence. These antimicrobial peptides have a conserved structure (D-Phe1 -Pro2 -Aaa3 -D-Aaa4 -Asn5 -Gln6 -Aaa7 -Val8 -Orn9 -Leu10 ), apart from the Aaa in positions 3, 4 and 7 containing either Phe, Trp or Tyr. METHODS AND RESULTS: Ultra-performance liquid chromatography linked mass spectrometry was used to profile peptides from extracts of cultures grown in media with various Phe : Trp ratios. The CBM model describes the production of peptides as a function of growth medium Aaa concentration. The model accounts for variable Aaa incorporation by simultaneously considering the influence of maximal incorporation rate and cooperativity, despite similar KM' s of synthetase modules. CONCLUSIONS: Our CBM model can be utilized to predict the Aaa composition of produced peptides from the concentration of Aaas in the growth medium. SIGNIFICANCE AND IMPACT OF THE STUDY: Subtly exploiting the inherent promiscuity of the nontemplate coded peptide synthesis allows for external control of peptide identity, without using genetic manipulation. Such versatility is exploitable in the production of targeted peptide complexes and rare peptides where production processes are reliant on nonribosomal synthesis.

A quality improvement programme in radiotherapy using workflow audits.

BACKGROUND: Evaluation of the quality of healthcare depends on measures of structures, processes and outcomes. Progress in recording data allows for better measures of processes, such as the completeness of clinical data, the performance of professional tasks and the use of checklists. OBJECTIVES: To report the results of a radiotherapy (RT) workflow audit and a subsequent online survey of user experience. METHODS: The RT workflow audit was developed in 2016 and has been undertaken twice a year at 28 facilities or units, with a total of 32 linear accelerators. Electronic patient folders were reviewed to assess the documentation of 90 task items, of which 64 were scored. The auditor came from another facility. The online survey took place in July 2020. It contained questions on the audit's process, professional value and future use. Invitations were sent by email to the 151 radiotherapist staff at the 28 units where the audit had been implemented. Responses were anonymous. RESULTS: For the RT workflow audit, scores improved from 60% in some units in 2016 to >90% in all units for at least 2 years since 2018. The number of responders to the online survey was 58, giving a responder rate of 38%. The margin of error of the results was 10%. The audit's task items were considered appropriate by 77% of responders, and feedback was reported by 78% of them. The audit was considered very or extremely valuable to their unit's service delivery by 58% of responders. Changes in the unit as a result of the audit were reported by 77% of responders. The audit was very useful or extremely useful to 75% of responders in maintaining personal professional standards. The proportion of responders who were very or extremely supportive of continuing with the audit was 77%. The comments in the online survey will be helpful for ongoing review of the RT workflow audit. CONCLUSIONS: The RT workflow audit extends the scope of accreditation audits by including measures of processes. Users of the audit evaluate its processes favourably and report that it has value both in their unit's clinical service and for their personal professional standards. The audit is effective in developing quality improvement programmes.

Sequence specific stabilization of a linear analog of the antifungal lipopeptide iturin A2 by sodium during low energy electrospray ionization mass spectrometry conditions.

The structures and stability of sodiated species of 8-Beta, a linear lipopeptide analog (beta-aminotetradecanoyl-NYNQPNS) of the antifungal peptide iturin A2, were evaluated by electrospray ionization mass spectrometry (ESI-MS). Association of the lipopeptide, 8-Beta, with sodium afforded protection from fragmentation at high cone voltages and increasing collision energy conditions in the ESI-MS. The order of decreasing stability was found as 8-Beta 1Na > 8-Beta 2Na > 8-Beta 3Na > 8-Beta. Substantial differences were found between fragmentation patterns of the free and sodiated molecular species. Breakage of the N-terminal peptide bond of L-Pro generated the major product ions of the free 8-Beta parent ion. Impaired fragmentation of the sodium adducts of 8-Beta, indicated that this bond is protected by sodium complexation. Fragmentation patterns of the sodiated lipopeptide further revealed two specific binding sites for a nonsolvated sodium ion within the two type II beta-turn sequences (beta-aminotetradecanoyl-NYN and QPNS) of the natural iturin A2. It is proposed that specific interaction with sodium takes place with most of the peptide bond oxygens in these turns, and with the Gln sidechain. This interaction leads to stabilized structures in which the peptide backbone, specifically the peptide bonds in which L-Pro participates, is protected against low-energy fragmentation during ESI-MS.

A sensitive standardised micro-gel well diffusion assay for the determination of antimicrobial activity.

We have developed a highly sensitive micro-gel well diffusion assay for the determination of antimicrobial activity. In essence, the normal radial diffusion type assay was adapted to perform it in a microtiter plate. We compared our micro-gel well diffusion assay to a radial diffusion assay and a microtiter broth dilution method, using gramicidin S as model antibiotic, and Micrococcus luteus as the indicator organism. The micro-gel well diffusion assay was as sensitive as the microtiter broth dilution method, and approximately twice as sensitive as the radial diffusion method. Data analysis to calculate minimum inhibitory concentration, 50% microbial growth inhibition and maximum inhibitory concentration was refined by generating dose-response curves with the software package Prism 3.0 (Graphpad Software Inc.). The minimum inhibitory concentrations, determined by the three methods, were significantly different (P<0. 001), highlighting the limitations involved in comparing data obtained from different methods.

Atomic force microscopy study of the effect of antimicrobial peptides on the cell envelope of Escherichia coli.

The influences of the antibacterial magainin 2 and PGLa from the African clawed frog (Xenopus laevis) and the hemolytic bee venom melittin on Escherichia coli as the target cell were studied by atomic force microscopy (AFM). Nanometer-scale images of the effects of the peptides on this gram-negative bacterium's cell envelope were obtained in situ without the use of fixing agents. These high-resolution AFM images of the surviving and intact target cells before and after peptide treatment showed distinct changes in cell envelope morphology as a consequence of peptide action. Although all three peptides are lytic to E. coli, it is clear from this AFM study that each peptide causes distinct morphological changes in the outer membrane and in some cases the inner membrane, probably as a consequence of different mechanisms of action.

The interaction of analogues of the antimicrobial lipopeptide, iturin A2, with alkali metal ions.

Electrospray mass spectrometry was employed as a tool in this first study on the molecular interaction between the alkali metal ions and antifungal lipopeptide iturin A, and some analogues. Cationisation by sodium and signal intensity of lipopeptide species depended on sodium concentration, but was independent of sample solvent, carrier solvent polarity and sample pH between 4 and 11. 8-Beta, a linear analogue of iturin A2 (8-Beta; beta-aminotetradecanoyl-NYNQPNS), and its shorter linear lipopeptide analogues, associated either one or two alkali metal cations, while the N-->C cyclic peptides associated with only one cation. The chirality of the beta-NC14 residue had a limited influence on the cationisation. It was observed that 8-Beta contained at least four interaction sites for a cation of which two, the C-terminal carboxylate and the side-chain of tyrosine, can take part in ionic interaction with a cation. It is proposed that the remaining two interaction centres of alkali metal ions are within the two type II beta-turns found in conformation of natural iturin A. This was corroborated by the diminished capacity of the shorter peptides, in which one of the beta-turns was eliminated to bind a second larger cation. All the lipopeptides showed the same order of alkali metal ion selectivity: Na+ > K+ > Rb+. These results indicated a size limitation in the interaction cavity or cavities. The absence of, or observation of only low abundance, di-cationised complexes of cyclic peptides the indicated association of the cation in the interior of the peptide ring. It is thus hypothesised that alkali metal ions can bind in one of the two beta-turns in the natural iturin A molecule.

Effect of physical exercise on plasma melatonin levels in normal volunteers.

Plasma melatonin, cortisol and prolactin levels were determined in clinically normal adult Black male volunteers before, immediately after, and 1 hour after a period of controlled physical exercise. In all subjects the levels of these three hormones showed highly significant increases immediately after exercise and returned to pre-exercise levels 1 hour after physical exertion. In subjects who exercised in a room with reduced environmental lighting (54 lux), the plasma melatonin level immediately after exercise was significantly higher than in the group who exercised in a room with a lighting level of 320 lux. Reduction of environmental lighting had no effect on the plasma cortisol and prolactin values immediately after exercise. Significant positive relationships between post-exercise melatonin levels and body mass, age and pulse rate were noted in all volunteers. Since plasma melatonin reached a plateau level after 30 minutes, it is suggested that controlled exercise (step-climbing), which induces an energy output of 185 W/m2 body surface, conducted in a room with reduced environmental lighting for a period of 30 minutes, may form the basis for a pineal function test.