Identification of potential common cyclic-di-GMP regulated biomarkers among three gram negative bacteria via transcriptomic and metabolomic profiling
Date of Issue2017-01-24
Interdisciplinary Graduate School (IGS)
Singapore Centre for Environmental Life Science Engineering (SCELSE)
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.