Bacterial biofilms, the structures created by surface attached bacteria, are the basis of infections that are recalcitrant to antibiotics. For Vibrio cholerae, the Gram negative bacterium that causes the diarrheal disease cholera, these structures facilitate attachment to environmental surfaces. An understanding of the process by which these structures form is essential to blocking the process. V. cholerae responds to specific environmental conditions by synthesizing an adhesive extracellular matrix that promotes biofilm formation. This matrix is comprised of the VPS exopolysaccharide, proteins, and DNA. Through a proteomic analysis of this matrix, we recently identified three secreted proteins, Bap1, RbmC, and RbmA, that are required for the structural integrity of the V. cholerae biofilm. Bap1 and RbmC, which are concentrated between the biofilm and the substratum, mediate adherence of the biofilm structure to the surface. In contrast, RbmA is dispersed throughout the biofilm and surrounds biofilm-associated cells. Based on structural data, RbmA is hypothesized to bind both the bacterial O-antigen and VPS polysaccharide, pulling the biofilm matrix onto the bacterial cell surface. During our studies, we noted that RbmA undergoes proteolytic cleavage in mature biofilms. Our preliminary results suggest that premature cleavage of RbmA augments recruitment of cells to the biofilm. In this application, we propose to evaluate a model in which cleavage increases the affinity of RbmA both for the O-antigen and the VPS polysaccharides, thus locking in the biofilm structure once cell growth within the biofilm is complete. We will tet this model by identifying the RbmA protease or proteases, elucidating the mechanisms by which proteolysis of RbmA is regulated, and exploring the impact of RbmA proteolysis on RbmA function and resistance of mature biofilms to mechanical stress. These studies will define a new paradigm for the role of proteolysis in bacterial biofilm maturation and may suggest new technologies to prevent reinforcement of the biofilm matrix during this process.

Public Health Relevance

Bacterial biofilms, the structures created by surface attached bacteria, are the basis of infections that are recalcitrant to antibiotics. For Vibrio cholerae, th Gram negative bacterium that causes the diarrheal disease cholera, these structures facilitate attachment to environmental surfaces. An understanding of how the biofilm structure is made and reinforced will suggest new technologies to block this process.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21AI115023-02
Application #
9089988
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Hall, Robert H
Project Start
2015-06-15
Project End
2017-05-31
Budget Start
2016-06-01
Budget End
2017-05-31
Support Year
2
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Children's Hospital Boston
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
Liao, Julie; Smith, Daniel R; Brynjarsd├│ttir, J├│hanna et al. (2018) A self-assembling whole cell vaccine antigen presentation platform. J Bacteriol :
Liao, Julie; Gibson, Jacob A; Pickering, Bradley S et al. (2018) Sublingual Adjuvant Delivery by a Live Attenuated Vibrio cholerae-Based Antigen Presentation Platform. mSphere 3:
Smith, Daniel R; Maestre-Reyna, Manuel; Lee, Gloria et al. (2015) In situ proteolysis of the Vibrio cholerae matrix protein RbmA promotes biofilm recruitment. Proc Natl Acad Sci U S A 112:10491-6