Guided by our imaging data, we raised and characterized a CdrA-specific nanobody binder capable of disrupting these cellCcell junctions, thereby increasing the efficacy of antibiotic-mediated bacterial killing in biofilms
Guided by our imaging data, we raised and characterized a CdrA-specific nanobody binder capable of disrupting these cellCcell junctions, thereby increasing the efficacy of antibiotic-mediated bacterial killing in biofilms. the extracellular matrix of biofilms at intact cellCcell junctions. Combining our in situ observations at cellCcell junctions with biochemistry, native SNS-032 (BMS-387032) mass spectrometry, and cellular imaging, we demonstrate that CdrA forms an extended structure that projects from the outer membrane to tether cells together via polysaccharide binding partners. We go on to show the functional importance of CdrA using custom single-domain antibody (nanobody) binders. Nanobodies targeting the tip of functional cell-surface CdrA molecules could be used to SNS-032 (BMS-387032) inhibit bacterial biofilm formation or disrupt preexisting biofilms in conjunction with bactericidal antibiotics. These results reveal a functional mechanism for cellCcell interactions within bacterial biofilms and highlight the promise of using inhibitors targeting biofilm cellCcell junctions to prevent or treat problematic, chronic bacterial infections. Prokaryotic cells including bacteria and archaea are frequently found in nature as part of surface-attached, multicellular communities called biofilms (1C3). Biofilms Mouse monoclonal to CD4 constitute the majority of bacterial biomass on Earth (1), representing a fundamental mode of bacterial existence. While bacterial biofilms may prove beneficial to eukaryotes as host-associated microbiomes (4, 5), the formation of pathogenic bacterial biofilms is associated with the establishment of serious chronic antibiotic-tolerant infections (6). Recently, important advances have been made in understanding early events in biofilm formation (7); however, the molecular mechanisms underlying how mature biofilms are formed and stabilized are still poorly understood. One of SNS-032 (BMS-387032) the hallmarks of mature biofilms is the presence of an extracellular polymeric substance (EPS) matrix that binds bacterial cells together into a sessile community, promoting antibiotic tolerance and providing protection from other predatory organisms (8C11). The EPS matrix of biofilms is a complex mixture of molecules, consisting of proteins, polysaccharides, and extracellular DNA (12). Comprehending the spatial arrangement of molecules in the EPS matrix of biofilms has been problematic (13) due to the inherent difficulty associated with high-resolution microscopic imaging inside the tissue-like environment of a biofilm. As a result, mechanisms of cellular tethering and the architecture of cellCcell junctions within biofilms are incompletely understood at the fundamental molecular level. Nevertheless, elegant optical microscopy studies on biofilms have provided clues to the internal organization of the EPS matrix, revealing that the proteins required for mature biofilm formation (RbmA, Bap1, and RbmC) fail to accumulate at the cell surface in the absence of an exopolysaccharide (called VPS) and that loss of RbmA function dramatically alters biofilm architecture (14, 15). In vitro studies of proteins have revealed an exopolysaccharide-dependent (RbmA) adhesin oligomerization pathway (16), and other studies suggest that direct interactions between the RbmA adhesin and glycans on partner cells SNS-032 (BMS-387032) lead to cellCcell adhesion (17). In is a human pathogen SNS-032 (BMS-387032) of critical concern, posing a significant challenge in hospital settings due to its ability to form antibiotic-tolerant biofilms (19C21). CellCcell interactions in the EPS matrix of biofilms are facilitated by the expression of a 220 kDa adhesin (Fig. 1cells through its membrane protein partner CdrB (22, 23). In these conditions, CdrA promotes cellular aggregation and biofilm formation by directly binding to polysaccharides in the EPS matrix of biofilms, such as the Psl or Pel polysaccharides (22, 24). When cytoplasmic c-di-GMP concentrations are lower, CdrA is cleaved and released into the extracellular milieu by the action of a periplasmic protease, promoting biofilm disaggregation (22). Open in a separate window Fig. 1. CdrAB expression results in the appearance of 70-nm-long, matchstick-shaped protrusions on the surface of cells. ((PAO1 cell expressing CdrAB. ((red dashed line). The outer membrane of the cell (purple) and matchstick-shaped cell surface molecules (green) are shown. ((solid yellow line) with matchstick-shaped protrusions indicated (red arrowheads). (= 108] from 12 tomograms). Refer also to.