Evaluation of rhizobacterial colonisation and the ability to induce Globodera pallida hatch

Three bacterial isolates, SB13 (Acinetobacter sp.), SB14 (Arthrobacter sp.) and SB15 (Bacillus sp.), were previously isolated from the rhizosphere of sugar beet (Beta vulgaris ssp. vulgaris) plants and shown to increase hatch of potato cyst nematodes in vitro. In this study, the three isolates were assayed for rhizosphere competence. Each isolate was applied to seeds at each of four concentrations (105-108 CFU ml−1) and the inoculated seeds were planted in plastic microcosms containing coarse sand. All three isolates were shown to colonise the rhizosphere, although to differing degrees, with the higher inoculation densities providing significantly better colonisation. The isolates increased sugar beet root and shoot dry weight. Isolates SB14 and SB15 were analysed for their ability to induce in vivo hatch of Globodera pallida in non-sterile soil planted with sugar beet. After 4 and 6 weeks, both isolates had induced significantly greater percentage hatch compared to controls.

The role of bacterial interspecies communication in polymicrobial infection of the cystic fibrosis lung

There is an increasing appreciation of the polymicrobial nature of bacterial infections associated with Cystic Fibrosis (CF) and of the important role for interactions in influencing bacterial virulence and response to therapy. Patients with CF are co-infected with Pseudomonas aeruginosa, Burkholderia cenocepacia and Stenotrophomonas maltophilia. These latter bacteria produce signal molecules of the diffusible signal factor (DSF) family, which are cis-2-unsaturated fatty acids. Previous studies showed that DSF from S. maltophilia leads to altered biofilm formation and increased tolerance to antibiotics in P. aeruginosa and that these responses require the P. aeruginosa sensor kinase PA1396. The work in this thesis aims of further elucidate the influence and mechanism of DSF signalling on P. aeruginosa and examine the role that such interspecies signalling play in infection of the CF airway. Next generation sequencing technologies targeting the 16S ribosomal RNA gene were applied to DNA and RNA isolated from sputum taken from cohorts of CF and non-CF subjects to characterise the bacterial community. In parallel, metabolomics analysis of sputum provided insight into the environment of the CF airway. This analysis revealed a number of observations including; that differences in metabolites occur in sputum taken from clinically stable CF patients and those with exacerbation and DNA- and RNA-based methods suggested that a strong relationship existed between the abundance of specific strict anaerobes and fluctuations in the level of metabolites during exacerbation. DSF family signals were also detected in the sputum and a correlation with the presence of DSFproducing organisms was observed. To examine the signal transduction mechanisms used by P. aeruginosa, bioinformatics with site directed mutagenesis were employed to identify signalling partners for PA1396. A pathway suggesting a role for a number of proteins in the regulation of several factors following DSF recognition by PA1396 were observed.

The relevance of bacterial isoprenoid biosynthetic pathways for host-microbe interactions

This thesis was undertaken to investigate the relevance of two bacterial isoprenoid biosynthetic pathways (Mevalonate (MVAL) and 2-C-methyl-D-erythritol 4-phosphate (MEP)) for host-microbe interactions. We determined a significant reduction in microbial diversity in the murine gut microbiota (by next generation sequencing) following oral administration of a common anti-cholesterol drug Rosuvastatin (RSV) that targets mammalian and bacterial HMG-CoA reductase (HMG-R) for inhibition of MVAL formation. In tandem we identified significant hepatic and intestinal off-target alterations to the murine metabolome indicating alterations in inflammation, bile acid profiles and antimicrobial peptide synthesis with implications on community structure of the gastrointestinal microbiota in statin-treated animals. However we found no effect on local Short Chain Fatty Acid biosynthesis (metabolic health marker in our model). We demonstrated direct inhibition of bacterial growth in-vitro by RSV which correlated with reductions in bacterial MVAL formation. However this was only at high doses of RSV. Our observations demonstrate a significant RSV-associated impact on the gut microbiota prompting similar human analysis. Successful deletion of another MVAL pathway enzyme (HMG-CoA synthase (mvaS)) involved in Listeria monocytogenes EGDe isoprenoid biosynthesis determined that the enzyme is non-essential for normal growth and in-vivo pathogenesis of this pathogen. We highlight potential evidence for alternative means of synthesis of the HMG-CoA substrate that could render mvaS activity redundant under our test conditions. Finally, we showed by global gene expression analysis (Massive Analysis of cDNA Ends (MACE RNA-seq) a significant role for the penultimate MEP pathway metabolite (E)-4-hydroxy-3-methyl-2-but-2-enyl pyrophosphate (HMBPP) in significant up regulation of genes of immunity and antigen presentation in THP-1 cells at nanomolar levels. We infected THP-1 cells with wild type or HMBPP under/over-producing L. monoctyogenes EGDe mutants and determined subtle effects of HMBPP upon overall host responses to Listeria infection. Overall our findings provide greater insights regarding bacterial isoprenoid biosynthetic pathways for host-microbe/microbe-host dialogue.