Architecture of cell–cell junctions in situ reveals a mechanism for bacterial biofilm inhibition

Abstract

Many bacteria, including the major human pathogen Pseudomonas aeruginosa, are naturally found in multicellular, antibiotic-tolerant biofilm communities, in which cells are embedded in an extracellular matrix of polymeric molecules. Cell–cell interactions within P. aeruginosa biofilms are mediated by CdrA, a large, membrane-associated adhesin present in the extracellular matrix of biofilms, regulated by the cytoplasmic concentration of cyclic diguanylate. Here, using electron cryotomography of focused ion beam–milled specimens, we report the architecture of CdrA molecules in the extracellular matrix of P. aeruginosa biofilms at intact cell–cell junctions. Combining our in situ observations at cell–cell junctions with biochemistry, native 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 inhibit bacterial biofilm formation or disrupt preexisting biofilms in conjunction with bactericidal antibiotics. These results reveal a functional mechanism for cell–cell interactions within bacterial biofilms and highlight the promise of using inhibitors targeting biofilm cell–cell junctions to prevent or treat problematic, chronic bacterial infections.

Molecular architecture of the TasA biofilm scaffold in Bacillus subtilis

“…Further studies into such mechanisms will be important to understand how the ECM is formed, which is of fundamental importance in comprehending how biofilms are built. Since the absence of the B. subtilis eps facilitated disassembly of the biofilm (Figure 5G-5H), this study provides another example of a synergy between filamentous proteins and ECM polysaccharides, described in other model bacterial systems including pathogenic species such as Vibrio cholerae and Pseudomonas aeruginosa [40,41]. Taken together with previous studies, our data suggest that ordered, local interactions between filamentous molecules leading to phase-separation in the ECM might be a general organisational principle for biofilm formation.…”

“…A recurring theme in bacterial biofilms is the abundance of filamentous molecules including polysaccharides [40, 41], proteins [24] and DNA [42] in the ECM [3], which are possibly important for the formation of a phase-separated environment protecting cells within biofilms [43, 44]. Further studies into such mechanisms will be important to understand how the ECM is formed, which is of fundamental importance in comprehending how biofilms are built.…”

Biofilm matrix proteome of clinical strain of P. aeruginosa

“…This adhesin is important for biofilm formation and its binding to Psl results in increased biofilm structural stability. Antibody-mediated blocking of CdrA inhibit biofilm formation[44]. Also, cdrAB is regulated together with Psl.…”

Architecture of the biofilm-associated archaic CupE pilus fromPseudomonas aeruginosa

“…Adhesion of bacterial cells to abiotic and biotic surfaces is crucial for the colonization of new environments, including invasion of hosts during infections and biofilm formation (Klemm and Schembri, 2000, Soto and Hultgren, 1999, Hall-Stoodley et al, 2004, Berne et al, 2015, Melia et al, 2021). Bacterial adhesion is often mediated by proteinaceous, hair-like cell-surface structures known as pili or fimbriae (Nuccio and Baumler, 2007, Sauer et al, 2000).…”