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Title: Quorum sensing mediated adaptability, pathogenicity and rhamnolipid production in Pseudomonas aeruginosa
Author: Ahmed, Syed A. K. Shifat
ISNI:       0000 0004 7229 0203
Awarding Body: Ulster University
Current Institution: Ulster University
Date of Award: 2017
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Quorum sensing (QS) acts as an important communication wiring in Pseudomonas aeruginosa. The bacteria use the system to regulate production of autoinducers for several adaptation and survival advantages. The system controls the production of key factors like proteases and rhamnolipid. Due to the substantial genome size and plasticity of P. aeruginosa, other factors are increasingly being linked with QS calling for the need of a better understanding of this complex system. The initial part of the thesis acknowledges the QS adaptation in P. aeruginosa clinical isolates from Cystic Fibrosis (CF) patients. Much of the factors that are deemed important for early infection establishment become redundant, as observed through loss of QS behaviour in most of the late isolates investigated in this study. This was particularly validated through complete loss of QS gene expressions and virulence factors like elastase, pyocyanin and rhamnolipid in a late clinical isolate, PA80. However, this cannot be regarded as an "universal adage" as isolates with and without QS activity were detected from the same patient in late or chronic stage of infection. The observations recorded in the study helps us to sketch a natural system where isolates having both QS activity and inactivity can co-exist in harmony in a chronic CF lung. In a stressful lung environment, it would be beneficial to have only certain isolates producing the expensive QS metabolites, in concentrations sufficient for the entire community, allowing the cell energy to be reserved for other functions. P. aeruginosa is a notorious nosocomial pathogen which is intractable with most of the common antibiotics. The resistance towards most antibiotics emphasises the need for alternative strategies to reduce virulence without eliciting a selection pressure on the bacterial population. Targeting QS with natural compounds is a promising strategy. This work, using a reliable and accurate RT-qPCR method developed in­house, demonstrated that trans-cinnamaldehyde and salicylic acid could attenuate expressions of QS genes at sub-inhibitory concentrations. Trans-cinnamaldehyde was effective in downregulating the Iasi and lasR genes by 13- and 7-fold respectively while salicylic acid caused smaller reductions of 3-fold in Iasi and 2-fold in lasR compared to untreated samples. The ability to interfere with the master QS genes had a consequent inhibitory effect on the production levels of QS controlled virulence factors (elastase, protease, pyocyanin). Rhamnolipid, an important QS metabolite, was extensively studied and showed to be significantly downregulated following treatment with the inhibitors. The use of QS inhibitors alone may not completely abrade the infection but can reduce the pathogenic load by decreasing the levels of QS regulated virulence factors. This could allow for effective use of antibiotics at much lower concentrations than used now. Rhamnolipid is not only regarded as an important clinical factor but also holds promise as "green surfactant" due to its amphipathic structures allowing for characteristics like chemical surfactants. Therefore, an ability to regulate the rhamnolipid production would be useful in both industry and medicine. However, there is a lack of quantitative understanding on the role of QS regulatory pathways and rhamnolipid metabolic pathways in rhamnolipid biosynthesis in P. aeruginosa. The gap has been reduced in the final study of the thesis which showed a highly conserved and regulated gene expression profiles for rhamnolipid production. The rhl QS genes are indispensable to produce rhamnolipid while the expression of rhIC only initiates after rhlAB genes have been maximally expressed. This makes the rhlAB the rate limiting factor in rhamnolipid production. The stringent regulation explains why rhamnolipids are synthesized in low yields. Therefore, there is a need to look for associate factors that could be engineered for increasing rhamnolipid production. One of these factors could be the transport proteins that are involved in extracellular movement of rhamnolipid or accumulation of precursors. HPLC-MS/MS chemical characterisation of rhamnolipid from PAO1 with transposon mutation in transport genes revealed that the inactivation does not affect the composition of rhamnolipid with C10-C10 fatty acid congeners being most dominant. However, the yields reported from the mutants were lower than the wild type suggesting these genes may also have a more indirect effect on rhamnolipid production. The thesis therefore suggests that the contribution of QS in P. aeruginosa adaptability, pathogenicity and rhamnolipid production is indeed significant and has contributed novel data which will aid our understanding of these important processes.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available