Bacterial fungal interactions in the cystic fibrosis lung

Pseudomonas aeruginosa is the major bacterial pathogen found in Cystic Fibrosis (CF) patients, with up to 80% being colonised by the age of 18. Within the CF lung, however, polymicrobial infections are increasingly common. In fact, the saprophytic fungi Aspergillus fumigatus is increasing observed in association with P. aeruginosa, which is correlated with poorer clinical outcomes. The aim of this project was therefore to investigate whether the interaction between P. aeruginosa and A. fumigatus resulted in changes to its virulence potential using a range of in vitro and in vivo experimental methodologies. The series of experiments described herein investigated the effect that A. fumigatus biofilms had on the induction of a surrogate virulence factor elastase from P. aeruginosa through the use of a biochemical assay. Elastase is produced by many strains of P. aeruginosa and causes host damage by cleaving elastin within the CF lung. A. fumigatus biofilms were grown in a 24-well plate format and live and ethanol-killed biofilms of various stages of development were produced (8 h germlings, 12 h monolayer of proliferating mycelia, 24 h and 48 h mature biofilm). These were then exposed to planktonic wild type and clinical strains of P. aeruginosa. Dead A. fumigatus biofilm was shown to increase elastase significantly in multiple strains, including a mucoid strain. It was also shown that the intact biofilm structure was essential to this increase in elastase, as disrupting the dead A. fumigatus biofilm and treating P. aeruginosa with the lysate did not result in a significant increase in elastase. Interestingly, the effect was restricted to dead biofilms, as presence of live A. fumigatus biofilm resulted in a significant decrease in elastase produced by certain P. aeruginosa strains. Despite a significant change in expressed elastase being found for P. aeruginosa type strain PA01 at the phenotypic level, a real time qPCR approach did not show any significant up-regulation of LasB, the gene encoding elastase, over multiple time points in response to dead 24 h A. fumigatus. The possibilities of transient gene expression or mRNA degradation with an increase in protein expression are potential explanations for this. Cell culture studies utilising the A549 human lung epithelial cell line aimed to determine the effects of both supernatants from combinational cultures and elastase itself on a cell line of relevance to the CF lung. Elastase was shown to detach A549 cells from the plastic surface they were grown on and adherent to using light microscopy, with no loss of viability as measured by alamarBlue. The effect of combinational P. aeruginosa/A. fumigatus supernatants in contrast with their respective controls were difficult to determine due to the highly cytotoxic effect of live A. fumigatus in this project. A Galleria mellonella wax worm insect model was also utilised to test for synergy in P. aeruginosa/A. fumigatus co-infections. This was performed by establishing the effect of an optimised P. aeruginosa inoculum on wax worm survival treated with dead A. fumigatus 8 h germlings. No significant difference were shown in the survival rates of this model when worms were injected with combinations of P. aeruginosa and killed A. fumigatus 8 hour germlings, though melanisation of the worms indicated an additive effect on the organisms. In conclusion, these studies demonstrated a relationship between A. fumigatus biofilms and an increased virulence expression from P. aeruginosa using a surrogate virulence marker, elastase. Due to its known negative effects in regards to human health, this suggests that polymicrobial infections of both P. aeruginosa and A. fumigatus could generate increased morbidity in those susceptible to these infections. Future work should be undertaken to unravel the exact mechanisms behind increased virulence expression in P. aeruginosa in response to A. fumigatus, and determine fully whether this is of important clinical significance and therefore a key bacterial/fungal interaction to focus on within CF studies.