Genome-scale analyses of health-promoting bacteria: probiogenomics

The human body is colonized by an enormous population of bacteria (microbiota) that provides the host with coding capacity and metabolic activities. Among the human gut microbiota are health-promoting indigenous species (probiotic bacteria) that are commonly consumed as live dietary supplements. Recent genomics-based studies (probiogenomics) are starting to provide insights into how probiotic bacteria sense and adapt to the gastrointestinal tract environment. In this Review, we discuss the application of probiogenomics in the elucidation of the molecular basis of probiosis using the well-recognized model probiotic bacteria genera Bifidobacterium and Lactobacillus as examples.

Development of tumour imaging and therapy strategies utilising unengineered bacteria

The ability of systemically administered bacteria to target and replicate to high numbers within solid tumours is well established. Tumour localising bacteria can be exploited as biological vehicles for the delivery of nucleic acid, protein or therapeutic payloads to tumour sites and present researchers with a highly targeted and safe vehicle for tumour imaging and cancer therapy. This work aimed to utilise bacteria to activate imaging probes or prodrugs specifically within target tissue in order to facilitate the development of novel imaging and therapeutic strategies. The vast majority of existing bacterial-mediated cancer therapy strategies rely on the use of bacteria that have been genetically modified (GM) to express genes of interest. While these approaches have been shown to be effective in a preclinical setting, GM presents extra regulatory hurdles in a clinical context. Also, many strains of bacteria are not genetically tractably and hence cannot currently be engineered to express genes of interest. For this reason, the development of imaging and therapeutic systems that utilise unengineered bacteria for the activation of probes or drugs represents a significant improvement on the current gold standard. Endogenously expressed bacterial enzymes that are not found in mammalian cells can be used for the targeted activation of imaging probes or prodrugs whose activation is only achieved in the presence of these enzymes. Exploitation of the intrinsic enzymatic activity of bacteria allows the use of a wider range of bacteria and presents a more clinically relevant system than those that are currently in use. The nitroreductase (NTR) enzymes, found only in bacteria, represent one such option. Chapter 2 introduces the novel concept of utilising native bacterial NTRs for the targeted activation of the fluorophore CytoCy5S. Bacterial-mediated probe activation allowed for non-invasive fluorescence imaging of in vivo bacteria in models of infection and cancer. Chapter 3 extends the concept of using native bacterial enzymes to activate a novel luminescent, NTR activated probe. The use of luminescence based imaging improved the sensitivity of the system and provides researchers with a more accessible modality for preclinical imaging. It also represents an improvement over existing caged luciferin probe systems described to date. Chapter 4 focuses on the employment of endogenous bacterial enzymes for use in a therapeutic setting. Native bacterial enzymatic activity (including NTR enzymes) was shown to be capable of activating multiple prodrugs, in isolation and in combination, and eliciting therapeutic responses in murine models of cancer. Overall, the data presented in this thesis advance the fields of bacterial therapy and imaging and introduce novel strategies for disease diagnosis and treatment. These preclinical studies demonstrate potential for clinical translation in multiple fields of research and medicine.

The exploration of potato-associated bacteria in the Central Andean Highlands and their application in integrated crop management systems

Potato is the most important food crop after wheat and rice. A changing climate, coupled with a heightened consumer awareness of how food is produced and legislative changes governing the usage of agrochemicals, means that alternative more integrated and sustainable approaches are needed for crop management practices. Bioprospecting in the Central Andean Highlands resulted in the isolation and in vitro screening of 600 bacterial isolates. The best performing isolates, under in vitro conditions, were field trialled in their home countries. Six of the isolates, Pseudomonas sp. R41805 (Bolivia), Pseudomonas palleroniana R43631 (Peru), Bacillus sp. R47065, R47131, Paenibacillus sp. B3a R49541, and Bacillus simplex M3-4 R49538 (Ecuador), showed significant increase in the yield of potato. Using – omic technologies (i.e. volatilomic, transcriptomic, proteomic and metabolomic), the influence of microbial isolates on plant defence responses was determined. Volatile organic compounds of bacterial isolates were identified using GC/MS. RT-qPCR analysis revealed the significant expression of Ethylene Response Factor 3 (ERF3) and the results of this study suggest that the dual inoculation of potato with Pseudomonas sp. R41805 and Rhizophagus irregularis MUCL 41833 may play a part in the activation of plant defence system via ERF3. The proteomic analysis by 2-DE study has shown that priming by Pseudomonas sp. R41805 can induce the expression of proteins related to photosynthesis and protein folding in in vitro potato plantlets. The metabolomics study has shown that the total glycoalkaloid (TGA) content of greenhouse-grown potato tubers following inoculation with Pseudomonas sp. R41805 did not exceed the acceptable safety limit (200 mg kg-1 FW). As a result of this study, a number of bacteria have been identified with commercial potential that may offer sustainable alternatives in both Andean and European agricultural settings.

Plasmid biology of natural Lactococcus lactis strains and molecular mechanisms of bacteriophage-host interaction

Lacticin 3147, enterocin AS-48, lacticin 481, variacin, and sakacin P are bacteriocins offering promising perspectives in terms of preservation and shelf-life extension of food products and should find commercial application in the near future. The studies detailing their characterization and bio-preservative applications are reviewed. Transcriptomic analyses showed a cell wall-targeted response of Lactococcus lactis IL1403 during the early stages of infection with the lytic bacteriophage c2, which is probably orchestrated by a number of membrane stress proteins and involves D-alanylation of membrane lipoteichoic acids, restoration of the physiological proton motive force disrupted following bacteriophage infection, and energy conservation. Sequencing of the eight plasmids of L. lactis subsp. cremoris DPC3758 from raw milk cheese revealed three anti-phage restriction/modification (R/M) systems, immunity/resistance to nisin, lacticin 481, cadmium and copper, and six conjugative/mobilization regions. A food-grade derivative strain with enhanced bacteriophage resistance was generated via stacking of R/M plasmids. Sequencing and functional analysis of the four plasmids of L. lactis subsp. lactis biovar. diacetylactis DPC3901 from raw milk cheese revealed genes novel to Lactococcus and typical of bacteria associated with plants, in addition to genes associated with plant-derived lactococcal strains. The functionality of a novel high-affinity regulated system for cobalt uptake was demonstrated. The bacteriophage resistant and bacteriocin-producing plasmid pMRC01 places a metabolic burden on lactococcal hosts resulting in lowered growth rates and increased cell permeability and autolysis. The magnitude of these effects is strain dependent but not related to bacteriocin production. Starters’ acidification capacity is not significantly affected. Transcriptomic analyses showed that pMRC01 abortive infection (Abi) system is probably subjected to a complex regulatory control by Rgg-like ORF51 and CopG-like ORF58 proteins. These regulators are suggested to modulate the activity of the putative Abi effectors ORF50 and ORF49 exhibiting topology and functional similarities to the Rex system aborting bacteriophage λ lytic growth.

Differential and numerical models of hysteretic systems with stochastic and deterministic inputs

Many deterministic models with hysteresis have been developed in the areas of economics, finance, terrestrial hydrology and biology. These models lack any stochastic element which can often have a strong effect in these areas. In this work stochastically driven closed loop systems with hysteresis type memory are studied. This type of system is presented as a possible stochastic counterpart to deterministic models in the areas of economics, finance, terrestrial hydrology and biology. Some price dynamics models are presented as a motivation for the development of this type of model. Numerical schemes for solving this class of stochastic differential equation are developed in order to examine the prototype models presented. As a means of further testing the developed numerical schemes, numerical examination is made of the behaviour near equilibrium of coupled ordinary differential equations where the time derivative of the Preisach operator is included in one of the equations. A model of two phenotype bacteria is also presented. This model is examined to explore memory effects and related hysteresis effects in the area of biology. The memory effects found in this model are similar to that found in the non-ideal relay. This non-ideal relay type behaviour is used to model a colony of bacteria with multiple switching thresholds. This model contains a Preisach type memory with a variable Preisach weight function. Shown numerically for this multi-threshold model is a pattern formation for the distribution of the phenotypes among the available thresholds.

Genetic analysis of bacteriophages from clinical and environmental samples

Bacteriophages, viruses infecting bacteria, are uniformly present in any location where there are high numbers of bacteria, both in the external environment and the human body. Knowledge of their diversity is limited by the difficulty to culture the host species and by the lack of the universal marker gene present in all viruses. Metagenomics is a powerful tool that can be used to analyse viral communities in their natural environments. The aim of this study was to investigate diverse populations of uncultured viruses from clinical (a sputum of patient with cystic fibrosis, CF) and environmental samples (a sludge from a dairy food wastewater treatment plant) containing rich bacterial populations using genetic and metagenomic analyses. Metagenomic sequencing of viruses obtained from these samples revealed that the majority of the metagenomic reads (97-99%) were novel when compared to the NCBI protein database using BLAST. A large proportion of assembled contigs were assignable as novel phages or uncharacterised prophages, the next largest assignable group being single-stranded eukaryotic virus genomes. Sputum from a cystic fibrosis patient contained DNA typical of phages of bacteria that are traditionally involved in CF lung infections and other bacteria that are part of the normal oral flora. The only eukaryotic virus detected in the CF sputum was Torque Teno virus (TTV). A substantial number of assigned sequences from dairy wastewater could be affiliated with phages of bacteria that are typically found in the soil and aquatic environments, including wastewater. Eukaryotic viral sequences were dominated by plant pathogens from the Geminiviridae and Nanoviridae families, and animal pathogens from the Circoviridae family. Antibiotic resistance genes were detected in both metagenomes suggesting phages could be a source for transmissible antimicrobial resistance. Overall, diversity of viruses in the CF sputum was low, with 89 distinct viral genotypes predicted, and higher (409 genotypes) in the wastewater. Function-based screening of a metagenomic library constructed from DNA extracted from dairy food wastewater viruses revealed candidate promoter sequences that have ability to drive expression of GFP in a promoter-trap vector in Escherichia coli. The majority of the cloned DNA sequences selected by the assay were related to ssDNA circular eukaryotic viruses and phages which formed a minority of the metagenome assembly, and many lacked any significant homology to known database sequences. Natural diversity of bacteriophages in wastewater samples was also examined by PCR amplification of the major capsid protein sequences, conserved within T4-type bacteriophages from Myoviridae family. Phylogenetic analysis of capsid sequences revealed that dairy wastewater contained mainly diverse and uncharacterized phages, while some showed a high level of similarity with phages from geographically distant environments.

Identification of novel innate immune mechanisms regulating inflammation in the gastrointestinal tract

The Gastro-Intestinal (GI) tract is a unique region in the body. Our innate immune system retains a fine homeostatic balance between avoiding inappropriate inflammatory responses against the myriad commensal microbes residing in the gut while also remaining active enough to prevent invasive pathogenic attack. The intestinal epithelium represents the frontline of this interface. It has long been known to act as a physical barrier preventing the lumenal bacteria of the gastro-intestinal tract from activating an inflammatory immune response in the immune cells of the underlying mucosa. However, in recent years, an appreciation has grown surrounding the role played by the intestinal epithelium in regulating innate immune responses, both in the prevention of infection and in maintaining a homeostatic environment through modulation of innate immune signalling systems. The aim of this thesis was to identify novel innate immune mechanisms regulating inflammation in the GI tract. To achieve this aim, we chose several aspects of regulatory mechanisms utilised in this region by the innate immune system. We identified several commensal strains of bacteria expressing proteins containing signalling domains used by Pattern Recognition Receptors (PRRs) of the innate immune system. Three such bacterial proteins were studied for their potentially subversive roles in host innate immune signalling as a means of regulating homeostasis in the GI tract. We also examined differential responses to PRR activation depending on their sub-cellular localisation. This was investigated based on reports that apical Toll-Like Receptor (TLR) 9 activation resulted in abrogation of inflammatory responses mediated by other TLRs in Intestinal Epithelial Cells (IECs) such as basolateral TLR4 activation. Using the well-studied invasive intra-cellular pathogen Listeria monocytogenes as a model for infection, we also used a PRR siRNA library screening technique to identify novel PRRs used by IECs in both inhibition and activation of inflammatory responses. Many of the PRRs identified in this screen were previously believed not to be expressed in IECs. Furthermore, the same study has led to the identification of the previously uncharacterised TLR10 as a functional inflammatory receptor of IECs. Further analysis revealed a similar role in macrophages where it was shown to respond to intracellular and motile pathogens such as Gram-positive L.monocytogenes and Gram negative Salmonella typhimurium. TLR10 expression in IECs was predominantly intracellular. This is likely in order to avoid inappropriate inflammatory activation through the recognition of commensal microbial antigens on the apical cell surface of IECs. Moreover, these results have revealed a more complex network of innate immune signalling mechanisms involved in both activating and inhibiting inflammatory responses in IECs than was previously believed. This contribution to our understanding of innate immune regulation in this region has several direct and indirect benefits. The identification of several novel PRRs involved in activating and inhibiting inflammation in the GI tract may be used as novel therapeutic targets in the treatment of disease; both for inducing tolerance and reducing inflammation, or indeed, as targets for adjuvant activation in the development of oral vaccines against pathogenic attack.

Investigations into selective metabolic aspects of bifidobacteria: carbohydrate metabolism, fatty acid biosynthesis and plasmid biology

The gastrointestinal tract (GIT) is a diverse ecosystem, and is colonised by a diverse array of bacteria, of which bifidobacteria are a significant component. Bifidobacteria are Gram-positive, saccharolytic, non-motile, non-sporulating, anaerobic, Y-shaped bacteria, which possess a high GC genome content. Certain bifidobacteria possess the ability to produce conjugated linoleic acid (CLA) from linoleic acid (LA) by a biochemical pathway that is hypothesised to be achieved via a linoleic isomerase. In Chapter two of this thesis it was found that the MCRA-specifying gene is not involved in CLA production in B. breve NCFB 2258, and that this gene specifies an oleate hydratase involved in the conversion of oleic acid into 10-hydroxystearic acid. Prebiotics are defined as non-digestible food ingredients that beneficially affect the host by selectively stimulating growth and/or activity of one or a limited number of bacteria in the colon. Key to the development of such novel prebiotics is to understand which carbohydrates support growth of bifidobacteria and how such carbohydrates are metabolised. In Chapter 3 of this thesis we describe the identification and characterisation of two neighbouring gene clusters involved in the metabolism of raffinose-containing carbohydrates (plus related carbohydrate melibiose) and melezitose by Bifidobacterium breve UCC2003. The fourth chapter of this thesis describes the analysis of transcriptional regulation of the raf and mel clusters. In the final experimental chapter two putative rep genes, designated repA7017 and repB7017, are identified on the megaplasmid pBb7017 of B. breve JCM 7017, the first bifidobacterial megaplasmid to be reported. One of these, repA7017, was subjected to an in-depth characterisation. The work described in this thesis has resulted in an improved understanding of bifidobacterial fatty acid and carbohydrate metabolism, Furthermore, attempts were made to develop novel genetic tools.

Bacteria and tumours: causative agents or opportunistic inhabitants?

Associations between different bacteria and various tumours have been reported in patients for decades. Studies involving characterisation of bacteria within tumour tissues have traditionally been in the context of tumourigenesis as a result of bacterial presence within healthy tissues, and in general, dogma holds that such bacteria are causative agents of malignancy (directly or indirectly). While evidence suggests that this may be the case for certain tumour types and bacterial species, it is plausible that in many cases, clinical observations of bacteria within tumours arise from spontaneous infection of established tumours. Indeed, growth of bacteria specifically within tumours following deliberate systemic administration has been demonstrated for numerous bacterial species at preclinical and clinical levels. We present the available data on links between bacteria and tumours, and propose that besides the few instances in which pathogens are playing a pathogenic role in cancer, in many instances, the prevalent relationship between solid tumours and bacteria is opportunistic rather than causative, and discuss opportunities for exploiting tumour-specific bacterial growth for cancer treatment.

Synergistic nisin-polymyxin combinations for the control of Pseudomonas biofilm formation

The emergence and dissemination of multi-drug resistant pathogens is a global concern. Moreover, even greater levels of resistance are conferred on bacteria when in the form of biofilms (i.e., complex, sessile communities of bacteria embedded in an organic polymer matrix). For decades, antimicrobial peptides have been hailed as a potential solution to the paucity of novel antibiotics, either as natural inhibitors that can be used alone or in formulations with synergistically acting antibiotics. Here, we evaluate the potential of the antimicrobial peptide nisin to increase the efficacy of the antibiotics polymyxin and colistin, with a particular focus on their application to prevent biofilm formation of Pseudomonas aeruginosa. The results reveal that the concentrations of polymyxins that are required to effectively inhibit biofilm formation can be dramatically reduced when combined with nisin, thereby enhancing efficacy, and ultimately, restoring sensitivity. Such combination therapy may yield added benefits by virtue of reducing polymyxin toxicity through the administration of significantly lower levels of polymyxin antibiotics.

Growing up in a bubble: using germ-free animals to assess the influence of the gut microbiota on brain and behavior

There is a growing recognition of the importance of the commensal intestinal microbiota in the development and later function of the central nervous system. Research using germ-free mice (mice raised without any exposure to microorganisms) has provided some of the most persuasive evidence for a role of these bacteria in gut-brain signalling. Key findings show that the microbiota is necessary for normal stress responsivity, anxiety-like behaviors, sociability, and cognition. Furthermore, the microbiota maintains central nervous system homeostasis by regulating immune function and blood brain barrier integrity. Studies have also found that the gut microbiota influences neurotransmitter, synaptic, and neurotrophic signalling systems and neurogenesis. The principle advantage of the germ-free mouse model is in proof-of-principle studies and that a complete microbiota or defined consortiums of bacteria can be introduced at various developmental time points. However, a germ-free upbringing can induce permanent neurodevelopmental deficits that may deem the model unsuitable for specific scientific queries that do not involve early-life microbial deficiency. As such, alternatives and complementary strategies to the germ-free model are warranted and include antibiotic treatment to create microbiota-deficient animals at distinct time points across the lifespan. Increasing our understanding of the impact of the gut microbiota on brain and behavior has the potential to inform novel management strategies for stress-related gastrointestinal and neuropsychiatric disorders.