Exploiting the bioactive potential of marine sponge microbiota

Endospore-forming bacteria are often isolated from different marine sponges, but their abundance varies, and they are frequently missed by culture-independent studies. Within endospore-formers, Bacillus are renowned for the production of antimicrobials and other compounds of medical and industrial importance. Although this group has been well studied in many different environments, very little is known about the actual diversity and properties of sporeformers associated with marine sponges. Identification of the endospore-forming bacteria associated with the marine sponges; Haliclona simulans, Amphilectus fucorum and Cliona celata, has uncovered an abundant and diverse microbial population composed of Bacillus, Paenibacillus, Solibacillus, Halobacillus and Viridibacillus species. This diversity appears to be overlooked by other non-targeted approaches where spore-formers are masked by more dominant species within the ecosystem. In addition to the identification of two antibiotic resistant plasmids, this bank of sporeformers produce a range of bioactive compounds. New antimicrobial compounds are urgently needed to combat the spread of multidrug resistant pathogens, as few new options are entering the drug discovery pipelines for clinical trials. Based on the results of this project, endospore-formers associated with marine sponges may hold the answer. The power of coupling functional based assays with genomic approaches has enabled us to identify a novel class 1 lantibiotic, subtilomycin, which is active against several clinically relevant pathogens. Subtilomycin is encoded in the genomes of all the marine sponge B. subtilis isolates analysed. They cluster together phylogenetically and form a distinct group from other sequenced B. subtilis strains. Regardless of its potential clinical relevance, subtilomycin may be providing these strains with a specific competitive advantage(s) within the stringent confines of the marine sponge environment. This work has outlined the industrial and biotechnological potential of marine sponge endospore-formers which appear to produce a cocktail of bioactive compounds. Genome sequencing of specific marine sponge isolates highlighted the importance of mining extreme environments and habitats for new lead compounds with potential therapeutic applications.

Investigation of lantibiotic regulation, immunity and synergy

Due to the increasing incidence of antibiotic resistant strains, the use of novel antimicrobials, such as bacteriocins, has become an ever more likely prospect. Lacticin 3147 (of which there are two components, Ltnα and Ltnβ) and nisin belong to the subgroup of bacteriocins called the lantibiotics, which has attracted much attention in recent years. The lantibiotics are antimicrobial peptides that contain unusual amino acids resulting from a series of enzyme-mediated post translational modifications. Given that there have been relatively few examples of lantibiotic-specific resistance; these antimicrobials appear to represent valid alternatives to classical antibiotics. However, the fact that lantibiotics are naturally only produced in small amounts often hinders their commercialisation. In order to overcome this bottleneck, several approaches can be employed. For example, we can create a situation that reduces the quantity of a lantibiotic required to inhibit a target by combining it with other antimicrobials. Here, following an initial screen involving lacticin 3147 and several classical antibiotics, it was observed between lacticin 3147 and the commercial antibiotics polymyxin B/E function synergistically. This reduced the amounts of the individual antimicrobials required for kill and broadened the spectrum of inhibition of both agents. Upon combination with polymyxins, lacticin 3147, which has been associated with Gram positive targets only, actively targeted Gram negative species such as Escherichia coli and Cronobacter sp. An alternative means of addressing problems associated with lantibiotic yield is to better understand how production is regulated, and ultimately use this information to enhance peptide levels. With this in mind the regulation of lacticin 3147 production from the promoter Pbac was investigated using a green fluorescent protein (GFP) expression reporter system. This revealed that elements within both of the divergent operons of the lacticin 3147 gene cluster are involved in Pbac regulation. That is, LtnR, already established as a negative regulator of itself and the lacticin 3147 associated immunity genes, also acts as an activator of Pbac transcription. In contrast, an enhanced level of expression is observed in the absence of the lacticin 3147 structural genes, ltnA1 and ltnA2, indicating that these genes/gene products are involved in Pbac repression. In fact, through complementation of the ltnA2 gene, it was revealed that this regulation is more likely to be dependent on the presence of the gene transcript rather that the corresponding prepropeptide or modified Ltnβ. It may be that if lacticin 3147 production is successfully enhanced, the ability of the producing cell to protect itself may become an issue. To prepare for such a possibility a bioengineered derivative of the lacticin 3147 immunity protein LtnI (LtnI I81V) which provides enhanced protection was discovered through an in depth investigation involving the site and saturation mutagenesis of this protein. In addition, the creation of truncated forms of LtnI allowed the identification of important and essential regions of this immunity protein. Finally, as mentioned, self-immunity is essential to prevent self-killing. However the discovery of nisin U immunity and regulatory gene homologues (spiFEGRR’K) within the pathogenic strain S. infantarius subsp. infantarius is a cause for concern as it represents an example of immune mimicry, a form of lantibiotic-specific resistance. The ability of spiFEG to confer protection was apparent when they successfully provided protection to nisin A, F, Z, Q and U when expressed heterologously in the nisin sensitive L. lactis HP host. As a consequence of the studies presented in this thesis, it is likely that strategies will emerge that will facilitate the production of greater levels of lacticin 3147 production and lead to enhanced immunity in lactococcal backgrounds. Alternatively the need for enhanced production could be avoided through the use of antimicrobial combinations. In addition, providing awareness of the threats of the emergence of resistance through immune mimicry can allow researchers to develop strategies to prevent this phenomenon from leading to the dissemination of lantibiotic resistance.

Bioengineering of nisin to enhance functionality against dairy pathogens

The bacteriocin class of antimicrobial peptides have emerged as a viable alternative to at least partially fill the void created by the end of the golden age of antibiotic discovery. Along with this potential use in a clinical setting, bacteriocins also play an important role as bio-preservatives in the food industry. This thesis focuses on a specific bacteriocin group, the lantibiotics (Lanthionine-containing antibiotics). Their numerous methods of appliance in a food setting and how their gene-encoded nature can be modified to improve on overall bioactivity and functionality are explored here. The use of a lantibiotic (lacticin 3147) producing starter culture to control the Crohn’s disease-linked pathogen Mycobacterium paratuberculosis was assessed in a raw milk cheese. Although lacticin 3147 production did not effectively control the pathogen, the study provided an impetus to employ a variety of PCR-based mutagenesis techniques with a view to the creation of enhanced lantibiotic derivatives. Through the use of these techniques, a number of enhanced derivatives were generated from the ‘hinge’ region of the nisin peptide. Furthermore, a derivative in which the three hinge amino acids were replaced with three alanines represents the first enhanced derivative of nisin to have been designed through a rational process. This derivative also formed the backbone for the creation of an active, trypsin resistant, variant. Through the employment of further mutagenesis methods a derivative was created with potential use as an oral anti-bacterial in the future. Finally a number of lead nisin derivatives were investigated to assess their anti- Streptococcus agalactiae ability, a mastitis associated pathogen. Also a system was developed to facilitate the large scale production of these candidates, or other nisin derivatives, from dairy substrates.

Bioengineering strategies to improve functional qualities of nisin

The abuse of antibiotics and the emergence of multi-drug resistant bacterial strains have created the need to explore alternative methods of controlling microbial pathogens. The bacteriocin family of antimicrobial peptides has been proposed as one such alternative to classic antibiotics. Nisin A belongs to the subgroup of bacteriocins called the lantibiotics, which contain several unusual amino acids as a consequence of enzyme-mediated post-translational modifications. As nisin is produced by generally regarded as safe (GRAS) microorganisms, it could potentially be applied in a clinical setting. However, as lantibiotics are naturally produced in such small quantities, this can hinder their industrial potential. In order to overcome this, several approaches can be utilised. For example, given the gene encoded nature of lantibiotics, genetic engineering approaches can be implemented in order to yield variants with enhanced properties. Here, the use of mutagenesis-based strategies was employed to obtain a derivative of nisin with enhanced bioactivity in vitro. Investigations with purified peptide highlighted the enhanced specific activity of this variant, nisin M21V, against food-borne Listeria monocytogenes strains. Furthermore, this specific enhanced bioactivity was evident in a mouse model of listeriosis. Reductions in bioluminescence and microbial counts in organs from infected mice were observed following treatment with nisin M21V compared to that of wild-type nisin A. Peptide bioengineering approaches were also implemented to obtain additional novel derivatives of nisin. The generation of “S5X” and “S33X” banks (representing a change of natural serines at positions 5 and 33 to all possible alternative residues) by a combination of site-saturation and site-directed mutagenesis led to the identification of several derivatives exhibiting improved stability. This allowed the rational design of variants with enhanced stability compared to that of wild type nisin. Another means of tackling issues associated with lantibiotic yield is to combine lantibiotics with other antimicrobials. This could circumvent the need for enhanced production while also reducing concentrations of the peptide antimicrobials. We observed that combinations of nisin variants and low levels of plant essential oils (thymol, carvacrol, trans-cinnamaldehyde) significantly controlled Gram negative foodborne pathogens in in vitro assays compared to nisin A-essential oil combinations. This enhanced control was also evident in model food systems. Nisin variants used in conjunction with carvacrol significantly reduced numbers of E. coli O157:H7 in apple juice while a commercial nisin preparation used in combination with citric acid significantly controlled C. sakazakii in infant milk formula. It is noteworthy that while nisin is generally associated with Gram positive targets, upon combination with plant essential oils the spectrum of inhibition was broadened to Gram negative targets.