The signal based relationship between the green seaweed Ulva and its indigenous bacterial community

This project has focused on the relationship between the green seaweed Ulva, commonly found in the intertidal zone of the UK coastline and its cognate bacterial community. It has previously been reported that motile Ulva zoospores are attracted to N-Acylhomoserine lactones (AHLs), signalling molecules utilised by Gram-negative bacteria in a density dependent form of cellular communication termed quorum sensing (QS) and produced by several biofilm dwelling species of marine bacteria. The species represented in the bacterial community associated with Ulva spp. were identified by generating a 16S rDNA phylogenetic clone library from bacterial DNA isolated from the surface of the seaweed. These data revealed that the majority of the population belonged to the Proteobacteria or Bacteroidetes phyla. In order to investigate whether QS signalling affected the rate of zoospore germination in addition to zoospore attraction, Ulva zoospores were settled and allowed to grow on synthetic AHLs, biofilms derived from AHL-producing model organisms and strains relevant to the Ulva epiphytic population which were shown to produce AHLs. Results from these experiments revealed that AHLs affected zoospore germination and the early growth of the Ulva germling as zoospores germinated and grown in the absence of AHLs were significantly longer than those germinated in the presence of AHLs. We therefore hypothesise that reduced germling growth in the presence of AHLs allows Ulva to obtain a healthy epiphytic bacterial community that is vital for the seaweed’s later development. Further understanding of Ulva growth biology could have potential applications in preventing marine biofouling by this genus of seaweed. This study progressed to characterise AHL production in a number of strains of Shewanella and Bacteroidetes bacteria, which, for differing reasons were deemed relevant to Ulva biology. Although data presented by this thesis showed AHL production in these bacterial groups, AHL synthase and response regulator sequences could not be identified in the published genome sequences from either Shewanella or the Bacteroidetes. This study also identified an AHL inactivating acylase enzyme in an environmental Shewanella isolate. This acylase, AacS, was shown to degrade a variety of synthetic AHLs and the AHLs produced by Yersinia pseudotuberculosis. This study has therefore increased the range of marine bacteria known to be producing AHLs, however the lack of AHL synthase and response regulator genes in the genomes of these bacteria leads to the conclusion that many marine bacteria possess novel, yet to be characterised AHL-mediated QS systems. Finally, this study screened a number of extracts from marine microalgae for compounds that act as agonists or antagonists to AHL-mediated QS. Although no AHL mimics were identified data presented by this thesis showed extracts to affect the luminescence produced in lux-based AHL bio-reporters in the presence of exogenously added signal, affect a number of QS regulated phenotypes in marine pathogens and effect QS regulated genes in the human pathogen Pseudomonas aeruginosa. As such, we hypothesise that these microalgae have the ability to produce quorum-quenching compound(s). Further characterisation of quorum-quenching compound(s) produced by microalgae may be beneficial in the bio-control of pathogenic bacteria in aquaculture and may act as candidates for novel antibiotics

The signal based relationship between the green seaweed Ulva and its indigenous bacterial community

This project has focused on the relationship between the green seaweed Ulva, commonly found in the intertidal zone of the UK coastline and its cognate bacterial community. It has previously been reported that motile Ulva zoospores are attracted to N-Acylhomoserine lactones (AHLs), signalling molecules utilised by Gram-negative bacteria in a density dependent form of cellular communication termed quorum sensing (QS) and produced by several biofilm dwelling species of marine bacteria. The species represented in the bacterial community associated with Ulva spp. were identified by generating a 16S rDNA phylogenetic clone library from bacterial DNA isolated from the surface of the seaweed. These data revealed that the majority of the population belonged to the Proteobacteria or Bacteroidetes phyla. In order to investigate whether QS signalling affected the rate of zoospore germination in addition to zoospore attraction, Ulva zoospores were settled and allowed to grow on synthetic AHLs, biofilms derived from AHL-producing model organisms and strains relevant to the Ulva epiphytic population which were shown to produce AHLs. Results from these experiments revealed that AHLs affected zoospore germination and the early growth of the Ulva germling as zoospores germinated and grown in the absence of AHLs were significantly longer than those germinated in the presence of AHLs. We therefore hypothesise that reduced germling growth in the presence of AHLs allows Ulva to obtain a healthy epiphytic bacterial community that is vital for the seaweed’s later development. Further understanding of Ulva growth biology could have potential applications in preventing marine biofouling by this genus of seaweed. This study progressed to characterise AHL production in a number of strains of Shewanella and Bacteroidetes bacteria, which, for differing reasons were deemed relevant to Ulva biology. Although data presented by this thesis showed AHL production in these bacterial groups, AHL synthase and response regulator sequences could not be identified in the published genome sequences from either Shewanella or the Bacteroidetes. This study also identified an AHL inactivating acylase enzyme in an environmental Shewanella isolate. This acylase, AacS, was shown to degrade a variety of synthetic AHLs and the AHLs produced by Yersinia pseudotuberculosis. This study has therefore increased the range of marine bacteria known to be producing AHLs, however the lack of AHL synthase and response regulator genes in the genomes of these bacteria leads to the conclusion that many marine bacteria possess novel, yet to be characterised AHL-mediated QS systems. Finally, this study screened a number of extracts from marine microalgae for compounds that act as agonists or antagonists to AHL-mediated QS. Although no AHL mimics were identified data presented by this thesis showed extracts to affect the luminescence produced in lux-based AHL bio-reporters in the presence of exogenously added signal, affect a number of QS regulated phenotypes in marine pathogens and effect QS regulated genes in the human pathogen Pseudomonas aeruginosa. As such, we hypothesise that these microalgae have the ability to produce quorum-quenching compound(s). Further characterisation of quorum-quenching compound(s) produced by microalgae may be beneficial in the bio-control of pathogenic bacteria in aquaculture and may act as candidates for novel antibiotics

The signal based relationship between the green seaweed Ulva and its indigenous bacterial community

This project has focused on the relationship between the green seaweed Ulva, commonly found in the intertidal zone of the UK coastline and its cognate bacterial community. It has previously been reported that motile Ulva zoospores are attracted to N-Acylhomoserine lactones (AHLs), signalling molecules utilised by Gram-negative bacteria in a density dependent form of cellular communication termed quorum sensing (QS) and produced by several biofilm dwelling species of marine bacteria. The species represented in the bacterial community associated with Ulva spp. were identified by generating a 16S rDNA phylogenetic clone library from bacterial DNA isolated from the surface of the seaweed. These data revealed that the majority of the population belonged to the Proteobacteria or Bacteroidetes phyla. In order to investigate whether QS signalling affected the rate of zoospore germination in addition to zoospore attraction, Ulva zoospores were settled and allowed to grow on synthetic AHLs, biofilms derived from AHL-producing model organisms and strains relevant to the Ulva epiphytic population which were shown to produce AHLs. Results from these experiments revealed that AHLs affected zoospore germination and the early growth of the Ulva germling as zoospores germinated and grown in the absence of AHLs were significantly longer than those germinated in the presence of AHLs. We therefore hypothesise that reduced germling growth in the presence of AHLs allows Ulva to obtain a healthy epiphytic bacterial community that is vital for the seaweed’s later development. Further understanding of Ulva growth biology could have potential applications in preventing marine biofouling by this genus of seaweed. This study progressed to characterise AHL production in a number of strains of Shewanella and Bacteroidetes bacteria, which, for differing reasons were deemed relevant to Ulva biology. Although data presented by this thesis showed AHL production in these bacterial groups, AHL synthase and response regulator sequences could not be identified in the published genome sequences from either Shewanella or the Bacteroidetes. This study also identified an AHL inactivating acylase enzyme in an environmental Shewanella isolate. This acylase, AacS, was shown to degrade a variety of synthetic AHLs and the AHLs produced by Yersinia pseudotuberculosis. This study has therefore increased the range of marine bacteria known to be producing AHLs, however the lack of AHL synthase and response regulator genes in the genomes of these bacteria leads to the conclusion that many marine bacteria possess novel, yet to be characterised AHL-mediated QS systems. Finally, this study screened a number of extracts from marine microalgae for compounds that act as agonists or antagonists to AHL-mediated QS. Although no AHL mimics were identified data presented by this thesis showed extracts to affect the luminescence produced in lux-based AHL bio-reporters in the presence of exogenously added signal, affect a number of QS regulated phenotypes in marine pathogens and effect QS regulated genes in the human pathogen Pseudomonas aeruginosa. As such, we hypothesise that these microalgae have the ability to produce quorum-quenching compound(s). Further characterisation of quorum-quenching compound(s) produced by microalgae may be beneficial in the bio-control of pathogenic bacteria in aquaculture and may act as candidates for novel antibiotics

Purified Acidic Sophorolipid Biosurfactants in Skincare Applications: An Assessment of Cytotoxic Effects in Comparison with Synthetic Surfactants Using a 3D In Vitro Human Skin Model

Acidic sophorolipids (Acidic SL), congeners of sophorolipid biosurfactants, offer a potential alternative to synthetic sodium lauryl ether sulphate (SLES) in skincare applications. However, major challenges associated with the laboratory-based investigations of the cytotoxic effects of Acidic SL have been the utilisation of impure and/or poorly characterised congeners as well as the use of monolayers of skin cells in in vitro assays. While the former limitation makes glycolipids less attractive for use in academic research and skincare applications, the latter does not provide an accurate representation of the in vivo human skin. The present study, therefore, for the first time, assessed the cytotoxic effects of 96% pure Acidic SL on a 3D in vitro skin model in comparison with SLES, with the aim of investigating a natural alternative to synthetic surfactants for potential use in skincare applications. The 3D in vitro skin model was colonised with Staphylococcus epidermidis for 12 h, and afterwards treated with either Acidic SL or SLES at 100 μg mL−1 for a further 12 h. Subsequently, the cytotoxic effects of Acidic SL in comparison with SLES were assessed using a combination of microbiology, molecular biology techniques, immunoassays, and histological analyses. It was demonstrated that Acidic SL had no deleterious effects on the viability of S. epidermidis, tissue morphology, filaggrin expression, and the production of inflammatory cytokines in comparison to SLES. These findings, in conjunction with the possibility to produce Acidic SL from cheaper renewable natural resources, demonstrate that Acidic SL could offer a potential sustainable alternative to synthetic surfactants

Characterisation of cytotoxicity and immunomodulatory effects of glycolipid biosurfactants on human keratinocytes

ABSTRACT: Skin irritation and allergic reactions associated with the use of skincare products formulated with synthetically derived surfactants such as sodium lauryl ether sulphate (SLES) have encouraged the search for naturally derived and biocompatible alternatives. Glycolipid biosurfactants such as sophorolipids (SL) and rhamnolipids (RL) offer a potential alternative to SLES. However, most studies on the bioactive properties of microbial glycolipids were determined using their mixed congeners, resulting in significant inter-study variations. This study aims to compare the effects of highly purified SL (acidic and lactonic) and RL (mono-RL and di-RL) congeners and SLES on a spontaneously transformed human keratinocyte cell line (HaCaT cells) to assess glycolipids’ safety for potential skincare applications. Preparations of acidic SL congeners were 100% pure, lactonic SL were 100% pure, mono-RL were 96% pure, and di-RL were 97% pure. Cell viability using XTT assays, cell morphological analyses, and immunoassays revealed that microbial glycolipids have differing effects on HaCaT cells dependent on chemical structure. Compared with SLES, acidic SL and mono-RL have negligible effects on cell viability, cell morphology, and production of pro-inflammatory cytokines. Furthermore, at non-inhibitory concentrations, di-RL significantly attenuated IL-8 production and CXCL8 expression while increasing IL-1RA production and IL1RN expression in lipopolysaccharide-stimulated HaCaT cells. Although further studies would be required, these results demonstrate that as potential innocuous and bioactive compounds, microbial glycolipids could provide a substitute to synthetic surfactants in skincare formulations and perform immunopharmacological roles in topical skin infections such as psoriasis. KEY POINTS: • Purified glycolipid congeners have differing effects on human keratinocytes. • Compared with SLES, acidic sophorolipids and mono-rhamnolipids have innocuous effects on keratinocytes. • Di-rhamnolipids and mono-rhamnolipids modulate cytokine production in lipopolysaccharide stimulated human keratinocytes. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00253-022-12302-5

Mono-Rhamnolipid Biosurfactants Synthesized by Pseudomonas aeruginosa Detrimentally Affect Colorectal Cancer Cells

Over the past 15 years, glycolipid-type biosurfactant compounds have been postulated as novel, naturally synthesized anticancer agents. This study utilized a recombinant strain of Pseudomonas aeruginosa to biosynthesize a preparation of mono-rhamnolipids that were purified via both liquid and solid-phase extraction, characterized by HPLC-MS, and utilized to treat two colorectal cancer cell lines (HCT-116 and Caco2) and a healthy colonic epithelial cell line CCD-841-CoN. Additionally, the anticancer activity of these mono-rhamnolipids was compared to an alternative naturally derived anticancer agent, Piceatannol. XTT cell viability assays showed that treatment with mono-rhamnolipid significantly reduced the viability of both colorectal cancer cell lines whilst having little effect on the healthy colonic epithelial cell line. At the concentrations tested mono-rhamnolipids were also shown to be more cytotoxic to the colorectal cancer cells than Piceatannol. Staining of mono-rhamnolipid-treated cells with propidium iodine and acridine orange appeared to show that these compounds induced necrosis in both colorectal cancer cell lines. These data provide an early in vitro proof-of-principle for utilizing these compounds either as active pharmaceutical ingredient for the treatment of colorectal cancer or incorporations into nutraceutical formulations to potentially prevent gastrointestinal tract cancer