Research

Our lab is currently focused on these areas:

The Biofilm Matrix

A Pseudomonas aeruginosa aggregate that is stained for all cells (red) and the exopolysaccharide Psl (green). Psl forms a shell around the developing aggregate.

Biofilm formation has been linked to many chronic bacterial infections. Thus, significant research has been directed toward understanding the basic biology behind biofilm formation. Biofilms produce an extracellular matrix that functions, in part, to hold the community together. Pseudomonas aeruginosa represents a paradigm species for the study of biofilms in the laboratory. The regulation of matrix production and the carbohydrate component of the matrix have both been examined. However, the protein component of the biofilm matrix has been relatively understudied. Our groups identified a biofilm matrix protein for P. aeruginosa, CdrA. CdrA provides structural integrity through extracellular interactions with the matrix polysaccharide Psl. We have additionally identified a novel matrix integrity protein, a Psl-binding lectin, LecB. Outside of matrix proteins that provide structural functions, we predict that matrix-associated proteins can play both nutritional and protective roles for the community. Our lab is focused on identifying specific biofilm matrix proteins of P. aeruginosa and the function they play for the biofilm community. We are also interested in the impact of host-protein matrix interactions and their ability to impact biofilm survival and pathogenicity.

Biofilms in the Host Environment

Immunohistochemistry detects P. aeruginosa (green) and Pel (brown) in CF sputum. Slides were imaged with 20X objective and counterstained with hematoxylin (purple) to view host airway material. This figure shows an aggregate of P. aeruginosa stained with P. aeruginosa antisera approximately 50 µm in diameter (green color). This slice was also stained with Pel antisera (brown color). The black bar in the lower left of the panel represents 50 µm.

A critical element of Pseudomonas aeruginosa pathogenesis is its ability to form biofilms in the lungs of CF patients. Biofilm bacteria produce one or more extracellular polymeric substances (EPS) that act as a scaffold, holding biofilm cells together and to a surface. For some time, alginate has been considered the major polysaccharide of the biofilm EPS matrix. Our studies have indicated that although alginate is a key biofilm matrix component of mucoid strains, it’s not a significant component of the matrix of nonmucoid P. aeruginosa strains, which are the first to colonize CF patients. We discovered that P. aeruginosa has the capacity to encode at least two alternative exopolysaccharides, designated Psl and Pel, which play a critical role in biofilm formation. Psl has been reported to consist of a neutral polymer of a pentasaccharide subunit. Recent discoveries by our group have determined that Pel is a positively charged amino sugar polymer.

Most of the research to date on the biofilm EPS matrix has been conducted in vitro. We know quite a bit regarding the regulation of matrix components, their localization within biofilms, and how they can impact antimicrobial tolerance and resistance to host defenses. However, we know very little regarding matrix composition and assembly in the host. We are at a juncture where we are poised to evaluate and extend our in vitro findings in the context of disease. Towards this end, we will probe in vivo aggregates of P. aeruginosa for evidence of matrix production and localization. Additionally, we will explore the potential of host proteins found in the airways to specifically bind matrix polysaccharides. Our research will provide the foundation for future funding geared towards bridging in vitro biofilm work and understanding the nature of bacterial aggregates observed in vivo.

Surface Sensing by P. aeruginosa

Surface attached cells displaying elevated levels of GFP (cyan color) and clustering of the Wsp system (yellow, punctate fluorescence). This figure shows that induction of c-di-GMP production in response to a surface corresponds to clustering of the Wsp system.

The second messenger signaling molecule cyclic diguanylate monophosphate (c-di-GMP) drives the transition from planktonic to the biofilm mode of growth in a variety of bacteria. Pseudomonas aeruginosa has at least two surface sensing chemosensory systems that produce c-di-GMP in response to growth on a surface. P. aeruginosa biofilm cells have elevated c-di-GMP levels relative to their planktonic counterparts, and mutations in genes involved c-di-GMP synthesis lead to defects in biofilm formation. While factors contributing to the formation of mature biofilms have been well-characterized, early biofilm formation, when a bacterium first senses a surface and transitions from a planktonic state to a surface-attached state, remains largely understudied. Here, we describe how heterogeneity in cellular levels of c-di-GMP among a genetically homogenous population of P. aeruginosa leads to a diversification of bacterial behaviors during surface sensing. Surface attached P. aeruginosa cells specialize into physiologically distinct subpopulations of high c-di-GMP early microcolony “founder” polysaccharide producers and low c-di-GMP early surface explorers, each of which contribute to downstream biofilm formation. Diversification in c-di-GMP levels correlates with clustering of the diguanylate cyclase WspR, and mutations in the Wsp system can disrupt the ability of P. aeruginosa to specialize into founders and explorers. Together, these results suggest that a genetically homogenous population of P. aeruginosa cells engages in a division of labor during early biofilm formation, prior to cells becoming irreversibly attached and forming mature biofilm structures.