A DMINISTRADORAS DE F ONDOS DE P ENSIONES

Infections in the upper airways predispose the lower respiratory tract of CF patients to a particular subset of microbial pathogens. Staphylococcus aureus and Haemophilus influenza are amongst the most commonly isolated pathogens in infants and paediatric patients in CF (Hauser et al., 2011). Although infections by these two pathogens are under control since antibiotic intervention was introduced, the breach of epithelial barrier by the above pathogens might therefore cultivate a colonisation niche for subsequent infections from nosocomial pathogens or even opportunistic pathogens. The prevalence of P. aeruginosa infection increases with age, and together with Burkholderia cepacia infection, is associated with rapid declines in the lung function of CF patients (Hauser et al., 2011). B. cepacia is highly transmissible and elicit robust inflammation in the host resulting in acute deterioration (Jones et al., 2004, Kalish et al., 2006). Around 15-20% of CF patients are colonised with Stenotrophomonas maltophilia, meticillin-resistant S. aureus (MRSA) and the colonisation is associated with the deterioration of pulmonary function

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(Dasenbrook et al., 2008). Fungus Aspergillus fumigatus is also frequently isolated in CF patients. A. fumigatus usually does not cause invasive infection but rather induces intense allergic response which is known as allergic bronchopulmonary aspergillosis. This may affect up to 15% of patients with CF and the symptoms include wheezing, pulmonary infiltrates, and bronchiectasis (Stevens et al., 2003, O'Sullivan and Freedman, 2009).

1.2.3.1. Pseudomonas aeruginosa

P. aeruginosa is a Gram-negative aerobic rod-shaped motile bacterium with a single or two flagella categorised in the family Pseudomonadaceae. It is environmentally ubiquitous and thrives in a wide range of ecological niches from soil, water, plants to animal tissues. P. aeruginosa carries a large genome size of 6.3 million base pairs that is believed to provide it with metabolic versatility and environmental adaptability (Stevens et al., 2003, Stover et al., 2000). The intrinsic low susceptibility to antibiotics of P. aeruginosa is attributable to the action of multidrug efflux pumps and P. aeruginosa is naturally resistant to penicillin and the majority of -lactam antibiotics. P. aeruginosa is also an opportunistic pathogen that preferentially infects susceptible individuals with compromised epithelial barrier that can be caused by tracheal intubation, urinary tract catheterisation, mechanical ventilation or burn wounds (Lavoie et al., 2011, Engel and Eran, 2011). The respiratory tract infection by P. aeruginosa is the leading cause of mortality in patients with CF.

Consistent with the large genome size and versatile life pattern, P. aeruginosa exploits tightly regulatory mechanisms to control the expression of virulence determinants such as elastase, exotoxins, pyocyanin and biofilm formation.

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Many of them are regulated in a cell density-dependent manner via cell-cell communication or so-called quorum-sensing (QS). Amongst the QS systems identified in P. aeruginosa, this thesis has focused on the investigation of the contribution of alkyl-quinolone (AQ) QS to P. aeruginosa pathogenesis and that will be further discussed in chapter 2.

1.2.3.2. P. aeruginosa adaptation to the CF lung

According to the cystic fibrosis foundation annual report, half of CF patients under 18 years of age are infected with P. aeruginosa and the prevalence rises up to 80% of patients age above 18 (Hauser et al., 2011). The mechanisms of how P. aeruginosa compete with other pathogens to adapt in the CF lung is still unclear, despite theories on the initial infections of the lung epithelium by S. aureus and H. influenza cultivating a niche suitable for P. aeruginosa persistence (Hauser et al., 2011). Here the evidence of phenotypic changes of P. aeruginosa that are considered superior for adaptation in the CF lung is listed. As mentioned above, in the CF airways, the function of the mucociliary clearance, cationic antimicrobial peptides and neutrophils and macrophages are impaired and inflammatory signal transduction pathways exaggerated. This results in the chronic airway colonisation with opportunistic pathogens and leads to life-threatening lung disease. Being a ubiquitous environmental strain, it is intriguing how P. aeruginosa shifts its lifestyle from an environmental unobtrusive planktonic life-pattern to a biofilm-forming phenotype during infection in the host. There are several virulence factors involved either in initial attachment and invasion or in the later chronic stage.

42 Early colonization

Colonisation of CF airways is mediated by the adhesion of cell appendages such as flagellum and type IV pili to host epithelial cell surfaces. Studies have demonstrated that a type IV pili-associated protein (PilY1) is required for P. aeruginosa adherence to mucosal epithelium and preferentially binds to the exposed basolateral cell surfaces (Heiniger et al., 2010, Bucior et al., 2012). After attachment, P. aeruginosa is thought to inject type III secretion proteins ExoU, S, T, and Y into the host cells. These secreted proteins were identified to have a role in activating pro-apoptotic pathways, modulating small GTPases, enhancing P. aeruginosa invasive capabilities by interacting with multiple eukaryotic cytoskeletal components, such as tight junctions and causing cell death as a consequence (Soong et al., 2008, Kipnis et al., 2006, Engel and Balachandran, 2009, Angus et al., 2010). In addition, P. aeruginosa elastases, LasA and LasB, and alkaline protease are thought to modulate host immunoregulatory proteins and damage epithelia through mitogen-activated protein kinase (MAPK) pathways, increasing IL-8 expression (Azghani, 1996).

Late stage adaptation

The long-term persistence of P. aeruginosa in CF lung is characterised by the process of adaptive radiation, which causes the selection of a variety of genotype and phenotypes (Hogardt and Heesemann, 2010). The adaptability of P. aeruginosa confers a selective advantage to better thrive in the diverse niches and microenvironments of the inflamed and hostile CF lung. Loss-of- function mutations in genes such as lasR, mucA and mexT lead to a general adaptation pattern of P. aeruginosa. More intriguingly, several cellular

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virulence factors including type II secreted elastases and pyoverdines, exotoxins S/T/Y secreted via T3SS, pyocyanin and siderophores were attenuated. The most striking clinically important feature of P. aeruginosa infection is the tendency of this bacterium to adjust its life style from a single cell to mucoid phenotype. This phenotypic change probably initiates the chronic infection stage and therefore, enables P. aeruginosa to survive in the microaerobic or even anaerobic environment in CF lungs. P. aeruginosa mucoid exopolysaccharide, alginate has been shown to reduce chemotaxis of neutrophils into the CF lung and by itself to inhibit activation of the complement system. Mutated mucoid P. aeruginosa is inherently resistant to macrophage and neutrophil phagocytosis (Matsui et al., 2006). Clinical isolates of P. aeruginosa from a single CF sputum sample appear morphologically diverse and include mucoid variants, non-mucoid pyomelanin producing variants, non-pigmented LPS-rough variants and small-colony variants. This high mutation rate of variants that coexist with non-mutated variants is another evidence of how P. aeruginosa accelerates its ecological fitness in the CF lung (Hogardt and Heesemann, 2010, Hauser et al., 2011).

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