EXOPOLYSACCHARIDES OF THE PSEUDOMONAS AERUGINOSA BIOFILM MATRIX A Thesis Presented to The Honors Tutorial College Ohio University In Partial Fulfillment of the Requirements for Graduation from the Honors Tutorial College with the degree of Bachelor of Science in Biological Sciences by Elizabeth Mathias April 2014 This thesis has been approved by The Honors Tutorial College and the Department of Biological Sciences Dr. Donald Holzschu Associate Professor, Biological Sciences Thesis Adviser Dr. Soichi Tanda Honors Tutorial College, DOS Biological Sciences Jeremy Webster Dean, Honors Tutorial College 1 Table of Contents Abstract 2 Introduction 4 Pseudomonas aeruginosa Significance 4 Biofilms 12 The Matrix Exopolysaccharides 22 Alginate 22 Psl and Pel 35 Experimental Overview and Research Questions 49 Materials and Methods 52 Results 67 Discussion 82 Conclusion 88 Acknowledgements 89 References 90 2 Abstract Pseudomonas aeruginosa is a common bacterium in the environment. In humans, it is an opportunistic pathogen that is a prevalent cause of hospital-acquired infections. P. aeruginosa is also a frequent cause of pulmonary infections in patients with cystic fibrosis. The majority of patients with cystic fibrosis develop chronic infections of P. aeruginosa that cannot be eradicated. P. aeruginosa is the primary cause of pneumonia in patients with cystic fibrosis. Chronic infection of the cystic fibrosis lung with P. aeruginosa is associated with a major decline in lung function and eventually leads to death in most cases. P. aeruginosa infection of the lung is particularly difficult to treat because these bacteria typically grow in biofilms. Biofilms are complex bacterial communities in which the bacteria are encased in a thick matrix composed mostly of secreted exopolysaccharides. Bacteria within biofilms are protected from environmental stresses as well as from the host immune system and antimicrobials. P. aeruginosa produces three exopolysaccharides that contributes to the biofilm matrix: alginate, Psl, and Pel. Alginate confers additional protection to antimicrobials and the immune system while Psl and Pel contribute to aggregation and adherence. The goal of this research was to use a reporter gene under the control of key promoters from the exopolysaccharides’ biosynthetic operons to elucidate the regulation of exopolysaccharide production over the course of biofilm development. EGFP was placed under the control of the AlgD, PelA, and PslA reporters. Because the PelA and PslA reporters are not well defined, three variations of sequence length upstream of the transcriptional start site were used. These constructs were integrated 3 into P. aeruginosa strains of different morphotypes in order to examine whether promoter activity differs between strains and between biosynthetic operons. Biofilms were then microscopically imaged. However, significant changes in the expression of the reporter were not yet detected. 4 Introduction 1. Pseudomonas aeruginosa Significance Pseudomonas aeruginosa is a Gram-negative, aerobic gammaproteobacterium that is commonly found in soil and water (Iglewski 1996). It is one of the most well- known of the nearly 200 species in the genus Pseudomonas. Rod-shaped and measuring 0.5-0.8 µm by 1.5-3.0 µm, these bacteria typically have one polar flagellum for motility as well as pili. P. aeruginosa is a pathogen of both plants and humans. Not usually found in the normal flora of humans, it is opportunistic and typically affects damaged tissue or individuals who are immunocompromised. P. aeruginosa was first identified in the mid-19th century on surgical wound dressings due to blue and green discoloration on the bandages caused by pigments that these bacteria produce. Today, it is notorious as an antibiotic resistant pathogen. The frequency of the isolation of multidrug resistant P. aeruginosa has increased 10% in almost 20 years (Lister et al. 2009). With a fatality rate of 50%, P. aeruginosa causes 15% of Gram-negative bacteremia as well as 10% of hospital-acquired infections (Iglewski 1996; Aloush 2006). It is the fourth leading cause of nosocomial infections and the second most frequent cause of nosocomial pneumonia (Lister et al. 2009). P. aeruginosa is also a leading cause of pneumonia and chronic infection of the lung in individuals with cystic fibrosis (CF). In 80-95% of patients with CF, respiratory failure ultimately resulting in death is caused by chronic bacterial infection (Lyczak et al. 2002). Because of the incidence and clinical significance of P. aeruginosa infection, this pathogen is being widely studied. 5 P. aeruginosa is a notable pathogen not only because of the number and severity of P. aeruginosa infections, but also because of the wide range of tissues in which it causes infection. It is a significant cause of wound and urinary tract infections as well as pneumonia, otitis externa, keratitis, and folliculitis (Gellatly and Hancock 2013). Additionally, P. aeruginosa is a significant cause of hospital-acquired infection because its resistance to antimicrobials and its ability to survive in low- nutrient environments make it difficult to eradicate. The ability to infect many tissues and survive in both natural and hospital environments is due to the great adaptability of P. aeruginosa. P. aeruginosa is extremely metabolically versatile; it is able to catabolize a vast number of hydrocarbons, giving it the ability to survive in even the most nutrient poor settings, including surfaces in medical facilities and water lines. The P. aeruginosa genome contributes to its adaptability and metabolic versatility. P. aeruginosa has one of the largest known bacterial genomes, containing ‘core’ regions of conserved genes across different strains as well as regions of genomic plasticity. Within the genome, 10% of genes encode regulatory proteins (Gellatly and Hancock 2013). Many of these proteins are part of two-component regulatory systems, which are signal transduction pathways that facilitate rapid adaptation to environmental changes. Several regulatory systems control quorum sensing, biofilm formation, and virulence factors. This adaptability allows P. aeruginosa to survive in many environments, to be a pathogen of both plants and humans, and to colonize and cause infection in a wide range of tissues in humans. P. aeruginosa is a significant cause of chronic lung infection in patients with 6 CF. CF is a homozygous recessive disease that is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), which is a cAMP-dependent chloride channel in the apical membrane of epithelial cells (Goldberg and Pier 2000). Over 900 different homozygotic mutations in the CFTR gene can cause CF, and the effects can be seen in a wide range of tissues including pancreatic, gastrointestinal, and, most noticeably, pulmonary. Patients with CF are often diagnosed due to elevated salt levels in their sweat, and CF is associated with low body mass due to an inability to properly digest and absorb nutrients in the gut. In the lungs, CF is characterized by thickened airway surface liquid and a dehydrated mucus layer (Fig. 1). These secretions can harbor microbes and inhibit the ability of the immune response to eradicate them (Gellatly and Hancock 2013). Mucociliary clearance, an important mechanism for clearing microbes and other foreign substances in the lung, is inhibited because the mucus is more strongly adhered to the epithelial surface, thereby retarding the movement of beating cilia. Additionally, the high ionic strength of the mucus and lung secretions may inhibit the activity of antimicrobial peptides (Lyczak et al. 2002). Damage to the lung tissue associated with P. aeruginosa infection is largely caused by chronic inflammation (Lyczak et al. 2002). While P. aeruginosa from chronic lung infections typically downregulate virulence factors and lose inflammatory features, P. aeruginosa infection of the lung is marked by recruitment of neutrophils; these release reactive oxygen species and other molecules that, during the course of a chronic infection, lead to long-term inflammation and significant tissue damage. Proteases, exotoxin A, and the pigment pyocyanin secreted by P. aeruginosa 7 also contribute to this damage. P. aeruginosa infections of the CF lung that are untreated often lead to the early death of the patient; with extensive treatment, life expectancy is increased to 35 years (Gaspar et al. 2013). Fig. 1: Differences in the secretions of normal and CF-affected lungs (Lyczak et al. 2002). The dehydrated mucus layer of the CF lung results in increased bacterial adherence and decreased mucociliary clearance, causing chronic infection. Because of its prevalence, the development of P. aeruginosa infection in the CF lung has been well studied. It is thought that the lung is typically first exposed to P. aeruginosa from the environment within three years of birth; the lung is also exposed to other common pathogens including Staphylococcus aureus and Haemophilus influenza (Fig. 2; Lyczak et al. 2002). Pathogens are typically inhaled and then settle into the secretions in the obstructed airways of the lung (Lyczak et al. 2002; Gellatly and Hancock 2013). Serological studies have indicated that P. aeruginosa is present in CF patients prior to their fourth year of age in 97.5% of cases 8 (Burns et al. 2001). However, these bacteria are not often recovered from lung secretions, indicating that P. aeruginosa is being eradicated by the immune system. Through these early years of infection,