Identification of potential common cyclic-di-GMP regulated biomarkers among three gram negative bacteria via transcriptomic and metabolomic profiling

• Main Author: Cai, Zhao
• Other Authors: Staffan Kjelleberg
• Format: Theses and Dissertations
• Language: English
• Published: 2017
• Subjects: DRNTU::Science::Biological sciences
• Online Access: http://hdl.handle.net/10356/69468
• Institution: Nanyang Technological University

Bacterial cells can switch between two life styles, namely planktonic and biofilm, depending on different environmental cues. Biofilms are involved in almost 80% of microbial infections. Biofilm infections can evade host immune attack and develop into chronic conditions, which cannot be efficiently eradicated by conventional antimicrobial treatments. In most Gram-negative bacteria, biofilm formation can be regulated by a secondary messenger, bis-(3'-5')-cyclic dimeric guanosine monophos-phate, c-di-GMP. Variations in intracellular c-di-GMP levels may change cellular metabolic profile and lead to the expression of specific biomarkers. The identification of such biomarkers modulated by c-di-GMP might provide alternative methods to diagnose biofilm-related infections. In this study, we applied RNA-sequencing (RNA-seq) and High Performance Liquid Chromatography (LC/MS) techniques to analyse the transcript-me and metabolome of Pseudomonas aeruginosa, Burkholderia cenocepacia and Klebsie-lla pneumoniae cells which are genetically modified to have either excessive or reduced intracellular c-di-GMP levels, in hope to discover possible common cross-species biomarkers. At transcriptional level, one common gene, metE that encodes 5-methyl-tetrahydropte-royl-triglutamate-homocysteine S-methyltransferase was found to be significantly upregulated in the presence of excessive amount of c-di-GMP in both P. aeruginosa PAO1 and B. cenocepacia H111 strains. At metabolic level, 2 common metabolites were found to be significantly overproduced in both P. aeruginosa and K. pneumonia under high intracellular c-di-GMP content, whereas 15 of that were found in both P. aeruginosa and B. cenocepacia. Transcriptomics analysis also indicated that global regulators such as c-di-GMP, quorum sensing and alternative sigma factors coordinately have regulatory capability on the production of virulence factors in P. aeruginosa. We therefore took a step further to investigate the regulation of virulence factor secretion by alternative sigma factor RpoN by both genotypic and phenotypic analysis. The results indicated that RpoN modulates virulence secretion through a PQS quorum sensing regulator, pqsR. This study provides evidence for the possible detection and diagnosis of biofilm infections in clinical prospective using c-di-GMP regulated metabolites; it also demonstrates the complex connections and regulations among global regulators at the same time.