Bioengineering Lantibiotics for Therapeutic Success

peer-reviewedSeveral examples of highly modified antimicrobial peptides have been described. While many such peptides are non-ribosomally synthesized, ribosomally synthesized equivalents are being discovered with increased frequency. Of the latter group, the lantibiotics continue to attract most attention. In the present review, we discuss the implementation of in vivo and in vitro engineering systems to alter, and even enhance, the antimicrobial activity, antibacterial spectrum and physico-chemical properties, including heat stability, solubility, diffusion and protease resistance, of these compounds. Additionally, we discuss the potential applications of these lantibiotics for use as therapeutics.DF,CH,PC,RR are supported by the Irish Government under the National Development Plan, through a Science Foundation Ireland (SFI) Technology and Innovation Development Award (TIDA14/TIDA/2286) to DF, a SFI Investigator awards to CH and RR (10/IN.1/B3027),SFI-PIfunding(11/PI/1137) to PDC and the Alimentary Pharmabiotic Centre under Grant Number SFI/12/RC/2273

In Vitro Activities of Nisin and Nisin Derivatives Alone and In Combination with Antibiotics against Staphylococcus Biofilms

peer-reviewedThe development and spread of pathogenic bacteria that are resistant to the existing catalog of antibiotics is a major public health threat. Biofilms are complex, sessile communities of bacteria embedded in an organic polymer matrix which serve to further enhance antimicrobial resistance. Consequently, novel compounds and innovative methods are urgently required to arrest the proliferation of drug-resistant infections in both nosocomial and community environments. Accordingly, it has been suggested that antimicrobial peptides could be used as novel natural inhibitors that can be used in formulations with synergistically acting antibiotics. Nisin is a member of the lantibiotic family of antimicrobial peptides that exhibit potent antibacterial activity against many Gram-positive bacteria. Recently we have used bioengineering strategies to enhance the activity of nisin against several high profile targets, including multi-drug resistant clinical pathogens such as methicillin-resistant Staphylococcus aureus, vancomycinresistant enterococci, staphylococci, and streptococci associated with bovine mastitis. We have also identified nisin derivatives with an enhanced ability to impair biofilm formation and to reduce the density of established biofilms of methicillin resistant S. pseudintermedius. The present study was aimed at evaluating the potential of nisin and nisin derivatives to increase the efficacy of conventional antibiotics and to assess the possibility of killing and/or eradicating biofilm-associated cells of a variety of staphylococcal targets. Growth curve-based comparisons established that combinations of derivatives nisin V C penicillin or nisin I4V C chloramphenicol had an enhanced inhibitory effect against S. aureus SA113 and S. pseudintermedius DSM21284, respectively, compared to the equivalent nisin A C antibiotic combinations or when each antimicrobial was administered alone. Furthermore, the metabolic activity of established biofilms treated with nisin V C chloramphenicol and nisin I4V C chloramphenicol combinations revealed a significant decrease in S. aureus SA113 and S. pseudintermedius DSM21284 biofilm viability, respectively, compared to the nisin A C antibiotic combinations as determined by the rapid colorimetric XTT assay. The results indicate that the activities of the nisin derivative and antibiotic combinations represent a significant improvement over that of the wild-type nisin and antibiotic combination and merit further investigation with a view to their use as anti-biofilm agents.DF,CH,PC,RR are supported by the Irish Government under the National Development Plan, through a Science Foundation Ireland (SFI)Technology and Innovation Development Award (TIDA14/TIDA/2286)to DF,a SFI Investigator awards to CH and RR(10/IN.1/B3027),SFI-PI funding(11/PI/1137)to PC and the Alimentary Pharmabiotic Centre under Grant Number SFI/12/RC/2273

Bacteriocins: Novel Solutions to Age Old Spore-Related Problems?

peer-reviewedBacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria, which have the ability to kill or inhibit other bacteria. Many bacteriocins are produced by food grade lactic acid bacteria (LAB). Indeed, the prototypic bacteriocin, nisin, is produced by Lactococcus lactis, and is licensed in over 50 countries. With consumers becoming more concerned about the levels of chemical preservatives present in food, bacteriocins offer an alternative, more natural approach, while ensuring both food safety and product shelf life. Bacteriocins also show additive/synergistic effects when used in combination with other treatments, such as heating, high pressure, organic compounds, and as part of food packaging. These features are particularly attractive from the perspective of controlling sporeforming bacteria. Bacterial spores are common contaminants of food products, and their outgrowth may cause food spoilage or food-borne illness. They are of particular concern to the food industry due to their thermal and chemical resistance in their dormant state. However, when spores germinate they lose the majority of their resistance traits, making them susceptible to a variety of food processing treatments. Bacteriocins represent one potential treatment as they may inhibit spores in the post-germination/outgrowth phase of the spore cycle. Spore eradication and control in food is critical, as they are able to spoil and in certain cases compromise the safety of food by producing dangerous toxins. Thus, understanding the mechanisms by which bacteriocins exert their sporostatic/sporicidal activity against bacterial spores will ultimately facilitate their optimal use in food. This review will focus on the use of bacteriocins alone, or in combination with other innovative processing methods to control spores in food, the current knowledge and gaps therein with regard to bacteriocin-spore interactions and discuss future research approaches to enable spores to be more effectively targeted by bacteriocins in food settings.KE, DF, CH, PC, MR, RR are supported by the Irish Government under the National Development Plan, through the Food Institutional Research Measure, administered by the Department of Agriculture, Fisheries and Food, Ireland (DAFM 13/F/462) to PC and MR, a Science Foundation Ireland (SFI) Technology and Innovation Development Award (TIDA 14/TIDA/2286) to DF, SFI-PI funding (11/PI/1137) to PDC and the APC Microbiome Insitute under Grant Number SFI/12/RC/2273

Crustal structure of the spreading plate boundary in Iceland and the north Atlantic from gravity data

Gravity datasets from two geothermal areas in Iceland, an Iceland-wide gravity dataset, and marine gravity data from the Reykjanes Ridge at 58˚N, which can be seen as the submarine extension of the Icelandic accretionary system, have been studied in this thesis. Results of previous local earthquake seismic tomography studies of the shallow crustal structure of two spreading segments in Iceland have been used, in conjunction with gravity observations, to assess the fidelity of the tomographic method and further refine the description of the crustal structure for these areas. This was accomplished by predicting the component of the observed gravity field which may be produced by the tomographically imaged bodies and in the process attempting to explain any residual anomalies. Results showed additional shallow structure inferred from gravity observation and suggested that local earthquake seismic tomography can 'overlook' some geological structure. The Icelandic gravity field was separated into long and short wavelength components which were attributed to mantle plume and crustal effects, respectively. Physical parameters were attributed to the sources of these anomalies where possible. Results showed that a simple cylinder model (radius -100 km) of anomalous mantle density (~ - 35 kg m(^-3)) could explain the large scale gravity field over Iceland. Shallow density variations in the top 1 km of the crust appeared to be mainly responsible for smaller scale gravity anomalies. A simple Bouguer slab model suggests that the crust may be ~7 km thicker beneath Iceland compared to neighbouring oceanic areas, consistent with an underplating mechanism for crustal accretion in Iceland. Gravity data were acquired on a cruise over the Reykjanes Ridge in the North Atlantic. The marine gravity data were reduced systematically to a residual anomaly and showed that there was the possibility of crustal thinning associated with a bathymetric offset which was interpreted as a second order discontinuity. The form of the residual gravity was similar to other discontinuities of this order on the Mid-Atlantic Ridge, although not of the same amplitude

Wavelength stabilisation for high power CO2 slab waveguide lasers using waveguide surface patterning

RF excited CO2 slab waveguide lasers are now developed as compact, highly efficient, cost-effective high power laser sources, suitable for a large number of uses. However a major problem remains in that the output power, wavelength and beam shape are unstable in time, leading to restrictions in their use in high precision and wavelength dependent work. A new method of wavelength control has been explored, using 2-D periodic patterns machined directly onto the waveguide surface of one of these lasers. These grating structures have been produced using a laser micro-machining technique which has been developed to allow for accurate and repeatable feature periodicity, along with fast prototyping. Several geometries of gratings, both one and two dimensional, have been machined from a number of materials compatible with IR hollow waveguide use, with feature spacings ranging from 80-150 um. Sensitive techniques developed to measure the wavelength dependent transmission of these waveguides out with a laser cavity, have shown a 2-3% loss selectivity using 50mm long patterns. The inclusion of the two dimensional grating in the unstable resonator of an industrial slab laser device is shown to stabilise the output wavelength to the 10.59 um transition and maintain a constant spatial mode

Intensive Mutagenesis of the Nisin Hinge Leads to the Rational Design of Enhanced Derivatives

peer-reviewedNisin A is the most extensively studied lantibiotic and has been used as a preservative by the food industry since 1953. This 34 amino acid peptide contains three dehydrated amino acids and five thioether rings. These rings, resulting from one lanthionine and four methyllanthionine bridges, confer the peptide with its unique structure. Nisin A has two mechanisms of action, with the N-terminal domain of the peptide inhibiting cell wall synthesis through lipid II binding and the C-terminal domain responsible for pore-formation. The focus of this study is the three amino acid ‘hinge’ region (N 20, M 21 and K 22) which separates these two domains and allows for conformational flexibility. As all lantibiotics are gene encoded, novel variants can be generated through manipulation of the corresponding gene. A number of derivatives in which the hinge region was altered have previously been shown to possess enhanced antimicrobial activity. Here we take this approach further by employing simultaneous, indiscriminate site-saturation mutagenesis of all three hinge residues to create a novel bank of nisin derivative producers. Screening of this bank revealed that producers of peptides with hinge regions consisting of AAK, NAI and SLS displayed enhanced bioactivity against a variety of targets. These and other results suggested a preference for small, chiral amino acids within the hinge region, leading to the design and creation of producers of peptides with hinges consisting of AAA and SAA. These producers, and the corresponding peptides, exhibited enhanced bioactivity against Lactococcus lactis HP, Streptococcus agalactiae ATCC 13813, Mycobacterium smegmatis MC2155 and Staphylococcus aureus RF122 and thus represent the first example of nisin derivatives that possess enhanced activity as a consequence of rational design.This work was financed by a grant from the Irish Department of Agriculture, Food and the Marine through the Food Institutional Research Measure (08/RD/C/691) and with Science Foundation Investigator award (10/IN.1/B3027)

Blood group polymorphisms in Southern Africa and innate resistance to plasmodium falciparum

A research report submitted to the faculty of Medicine, University of the Witwatersrand, Johannesburg, in part fulfillment of the requirements for the degree of Master of Medicine (in the branch of Haematology) Johannesburg 1992.The observation by Haldane in 1949 that the distribution of malaria and certain thalassaemias were similar and that the former disease must be a selective force tor the continued existence of the latter by preservation of the heterozygotes. This theory which later became known as lithe malaria hypothesis" has been applied to other inherited conditions such as G6PD deficiency, membranopathies, certain blood group polymorphisms, other heamoglobinopathies such as sickle cell disease, blood group polymorphisms and more recently HLA phenotypes. It has been shown that the Duffy blood group antigens are the receptors for. Plasmodium vivax and since these antigens are lacking in most black Africans this species of malaria is virtually absent in Africa. It has also been shown that the glycophorins are at least in part the receptors for Pfalciparum. Several variants of the glycophorins exist and the biochemistry and, where known, the molecular mechanisms by which these arise is reviewed. Experimental work is carried out to establish the growth characteristics of Pfalciparum in an in vitro culture system using cells with glycophorin variants on their membranes. Three such variants were compared to normal cells and two (S~s-U-and Dantu) were found to be partially resistant to invasion by Pfalciparum merozoites whereas the third (Henshaw) was found to be no different to controls.MT201

Saturation Mutagenesis of Lysine 12 Leads to the Identification of Derivatives of Nisin A with Enhanced Antimicrobial Activity

peer-reviewedIt is becoming increasingly apparent that innovations from the “golden age” of antibiotics are becoming ineffective, resulting in a pressing need for novel therapeutics. The bacteriocin family of antimicrobial peptides has attracted much attention in recent years as a source of potential alternatives. The most intensively studied bacteriocin is nisin, a broad spectrum lantibiotic that inhibits Gram-positive bacteria including important food pathogens and clinically relevant antibiotic resistant bacteria. Nisin is gene-encoded and, as such, is amenable to peptide bioengineering, facilitating the generation of novel derivatives that can be screened for desirable properties. It was to this end that we used a site-saturation mutagenesis approach to create a bank of producers of nisin A derivatives that differ with respect to the identity of residue 12 (normally lysine; K12). A number of these producers exhibited enhanced bioactivity and the nisin A K12A producer was deemed of greatest interest. Subsequent investigations with the purified antimicrobial highlighted the enhanced specific activity of this modified nisin against representative target strains from the genera Streptococcus, Bacillus, Lactococcus, Enterococcus and Staphylococcus.This work was supported by the Irish Government under the National Development Plan; by the Irish Research Council for Science Engineering and Technology (IRCSET); by Enterprise Ireland; and by Science Foundation Ireland (SFI), through the Alimentary Pharmabiotic Centre (APC) at University College Cork, Ireland, which is supported by the SFI-funded Centre for Science, Engineering and Technology (SFI-CSET) and provided P.D.C., C.H. and R.P.R. with SFI Principal Investigator funding

Efficacy of nisin A and nisin V semi-purified preparations alone and in combination with plant essential oils to control Listeria monocytogenes

peer-reviewedThe foodborne pathogenic bacterium Listeria is known for relatively low morbidity and high mortality rates reaching up to 25-30%. Listeria is a hardy organism and its control in foods represents a significant challenge. Many naturally occurring compounds, including the bacteriocin nisin and a number of plant essential oils, have been widely studied and are reported to be effective as antimicrobial agents against spoilage and pathogenic microorganisms. The aim of this study was to investigate the ability of semi-purified preparations (spp) containing either nisin A or an enhanced bioengineered derivative nisin V, alone and in combination with low concentrations of the essential oils thymol, carvacrol and trans-cinnamaldehyde, to control L. monocytogenes in both laboratory media and model food systems. Combinations of nisin V-containing spp (25 μg/ml) with thymol (0.02%), carvacrol (0.02%) or cinnamaldehyde (0.02%) produced a significantly longer lag phase than any of the essential oil/nisin A combinations. In addition, the log reduction in cell counts achieved by the nisin V + carvacrol or nisin V + cinnamaldehyde combinations was twice that of the equivalent nisin A + essential oil treatment. Significantly, this enhanced activity was validated in model food systems against L. monocytogenes strains of food origin. We conclude that the fermentate form of nisin V in combination with carvacrol and cinnamaldehyde offers significant advantages as a novel, natural and effective means to enhance food safety by inhibiting foodborne pathogens such as L. monocytogenes.This work was supported by the Irish Government under the National Development Plan, through Science Foundation Ireland Investigator awards to C.H. and R.P.R. (10/IN.1/B3027), and C.H., R.P.R. and P.D.C. (06/IN.1/B98)