| Multidrug resistant bacterial infections are of a global concern due to the rapidly increasing incidence over the past three decades and alarmingly few new antimicrobial pharmaceuticals currently under development. Nonetheless, simply designing new antibiotics may not be sufficient due to the vast genetic diversity of bacteria and their alarming efficient ability to acquire new and diverse resistance mechanisms. Instead, it has become necessary to understand the virulence and resistance mechanisms by which bacteria infect and persist in their host. Biofilm formation is one type of resistance mechanism of particular concern in pulmonary diseases such as cystic fibrosis and pneumonia. Opportunistic pathogens such as Pseudomonas aeruginosa and Stenotrophomonas maltophilia colonize the lungs of cystic fibrosis patients and secrete an extracellular matrix of protein, DNA, and polysaccharides which acts as a diffusion barrier against dehydration, phagocytosis, and antibiotic treatment. The polysaccharides of many biofilms contain uronic acids. Polysaccharide lyases catalyze the depolymerization of uronic acid-containing polysaccharides via a beta-elimination mechanism and play important roles in microbial biofilm formation and tissue invasion. Furthermore, polysaccharide lyases have pharmaceutical applications in the treatment of biofilm-associated infections and preparation of therapeutic oligosaccharides, as well as industrial applications in biofuel production.;In an effort to understand the potential role that two putative alginate lyases (Smlt1473 and Smlt2602) from S. maltophilia may play in bacterial virulence, each enzyme was heterologously expressed in Escherichia coli, purified in a one-step fashion via affinity chromatography, and assayed for catalytic activity as well as substrate specificity for a range of polysaccharides. Interestingly, Smlt1473 catalyzed the endolytic degradation of not only alginate, but poly-beta-D-glucuronic acid and hyaluronic acid as well. Furthermore, the pH optimum for enzymatic activity was substrate-dependent, with optimal hyaluronic acid degradation at pH 5, poly-beta-D-glucuronic acid degradation at pH 7, and alginate degradation at pH 9. Homology modeling allowed for the selection of residues located in the active site, but not directly involved in the beta-elimination mechanism. These residues were predicted to bind and optimally align the substrate in the active site for catalysis. Mutation of the substrate-binding residues resulted in the significant modification of Smlt1473 substrate specificity. The same nonrandom selection of residues responsible for substrate specificity was applied to Smlt2602, which catalyzed the exolytic degradation of alginate-based substrates. The result was the successful engineering of a completely unique mutant lyase that was exolytically active against both alginate and poly-beta-D-glucuronic acid.;Little is known regarding the specific virulence mechanisms employed by S. maltophlia to infect and invade its host. Therefore we identified and characterized a secreted ankyrin-repeat containing protein (Smlt3054) from S. maltophilia that bound F-actin in vitro and disrupted actin cytoskeletal structure in transfected mammalian cells. Altogether, this work furthers our understanding of S. maltophlia, an emergent, multidrug resistant opportunistic pathogen that is increasingly associated with chronic lung infections, and describes two unique polysaccharide lyases that could be utilized as platforms for the design of highly active enzymes who substrate specificity has been fine-tuned for the problem at hand. |
