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Hussain, Farah (2016) The effect of secreted products from Pseudomons aeruginosa on immune cells. PhD thesis, University of Nottingham.

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Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that represents a major burden to the healthcare system as it accounts for a vast number of hospital-acquired infections. P. aeruginosa is a particular threat in Cystic Fibrosis (CF) patients. This bacterium expresses an arsenal of secreted virulence factors that play a fundamental role in the invasion of the damaged physical barriers of the host and abrogate immune responses which aid the persistence of P. aeruginosa infections. The aim of this study is to elucidate the role of P. aeruginosa secretions in the modulation of the immune system and determine the key factors and their specific impact on immune cells such as T helper cells proliferation and monocytes viability.

The present study employed peripheral blood mononuclear cells from healthy donors as a source of immune cells and bacterial supernatants as a source of exoproducts. In this context, the P. aeruginosa strain PAO1 was used throughout this project and two sublines of this strain were mainly involved in this work; the PAO1 sublines Lausanne (PAO1-L) and Nottingham (PAO1-N). Different sublines of PAO1 are genetically diverse and this variation was utilised as a tool to identify the key molecules produced by P. aeruginosa that are responsible for the observed effect.

The present study demonstrated that P. aeruginosa supernatants obtained from PAO1-L has an inhibitory effect on T cell proliferation in response to Staphylococcus Enterotoxin B (SEB), but not α-CD3 and α-CD28 stimulation in peripheral blood mononuclear cells cultures. This effect appears to be mediated by the cytotoxic effect of P. aeruginosa products on monocytes, a precursor of antigen presenting cells. Analysis of different P. aeruginosa mutants showed that the cytotoxic activity is controlled by toxR. ToxR is a regulatory protein which enhances the production of one of the most important virulence factors of P. aeruginosa, exotoxin A (ToxA; ETA).

The impact of ETA production by P. aeruginosa on immune cells was investigated using a newly generated ETA-deficient mutant ΔtoxA in PAO1-L (Lausanne background) in comparison with its isogenic wild type (WT). Preliminary observations showed that the generation of apoptotic monocytes correlated with the high expression of ETA in the bacterial supernatants as both PAO1-LΔtoxR and PAO1-LΔtoxA are less toxic strains than PAO1-L. These findings suggest that the presence of monocytes is crucial to mediate ETA down regulation of T cell proliferation. However, the effect of PAO1-LΔtoxA supernatants on T cell proliferation in response to SEB could not be fully assessed because SEB stopped inducing T cell proliferation as before. To overcome this obstacle, phytohaemagglutinin (PHA) was employed as a promoter of T cell division. However, the suppressor effect was only clear on the CD3+/CD4+ T cell subset after stimulation with PAO1-L (ETA+) supernatant in response to PHA and full T cell proliferation was not recovered in the absence of ETA (PAO1-LΔtoxA).

Finally, P. aeruginosa and Candida albicans are typical opportunistic respiratory tract pathogens and the nature of the relationship between these two pathogens is still not entirely clear. Thus, in this work, an in vitro assay to evaluate the impact of P. aeruginosa secretions on C. albicans was developed. Our results suggest that P. aeruginosa secreted products promote C. albicans hyphal growth and the filament formation is not correlated with either the presence of the toxR gene or the production of ETA and rhamnolipids. Interestingly, a supernatant prepared from PAO1-L mutant with a 58 Kb deletion induces the growth of the yeast form suggesting that the gene that encodes the key element, which is responsible of inducing the hyphal growth, is located within this region. This work might deliver new understandings on the interactions between P. aeruginosa and C. albicans in polymicrobial infections particularly in susceptible individuals like those with Cystic Fibrosis and burn victims.

Further work is currently being carried out in our group to identify the active molecules that trigger filamentous growth in C. albicans and to determine the contribution of this agent on the interaction between P. aeruginosa and C. albicans during co-infection.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Martinez-Pomares, L. Camara Garcia, M.M.
Subjects:	Q Science > QR Microbiology > QR180 Immunology
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID:	31661
Depositing User:	Hussain, Farah
Date Deposited:	11 Jul 2016 07:58
Last Modified:	12 Oct 2017 20:12
URI:	https://eprints.nottingham.ac.uk/id/eprint/31661

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