Katharios-Lanwermeyer, S. | Search bioRxiv

bioRxiv2021

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

Many bacteria, including the major human pathogen Pseudomonas aeruginosa, are naturally found in multicellular, antibiotic-tolerant biofilm communities, where 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 pre-existing 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.

microbiology↗

bioRxiv2020

Pseudomonas aeruginosa uses c-di-GMP phosphodiesterases RmcA and MorA to regulate biofilm maintenance

While the early stages of biofilm formation have been well characterized, less is known about the requirements for Pseudomonas aeruginosa to maintain a mature biofilm. We utilized a P. aeruginosa-phage interaction to find that rmcA and morA, two genes which encode for c-di-GMP-degrading phosphodiesterase (PDEs) enzymes, are important for the regulation of biofilm maintenance. Deletion of these genes initially results in an elevated biofilm phenotype characterized by increased production of c-di-GMP, Pel polysaccharide and biofilm biomass. In contrast to the wild-type strain, these mutants were unable to maintain the biofilm when exposed to carbon-limited conditions. The susceptibility to nutrient limitation, and subsequent loss of biofilm viability of these mutants, was phenotypically reproduced with a stringent response mutant ({Delta}relA {Delta}spoT), indicating that the {Delta}rmcA and {Delta}morA mutants may be unable to appropriately respond to nutrient limitation. Genetic and biochemical data indicate that RmcA and MorA physically interact with the Pel biosynthesis machinery, supporting a model whereby unregulated Pel biosynthesis contributes to the death of the {Delta}rmcA and {Delta}morA mutant strains in an established biofilm when nutrient-limited. These findings provide evidence that c-di-GMP-mediated regulation is required for mature biofilms of P. aeruginosa to effectively respond to changing availability of nutrients. Furthermore, the PDEs involved in biofilm maintenance are distinct from those required for establishing a biofilm, thus indicating that a wide variety of c-di-GMP metabolizing enzymes in organisms like P. aeruginosa likely allows for discrete control over the formation, maintenance or dispersion of biofilms. ImportanceRecent advances in our understanding of c-di-GMP signaling have provided key insights into the regulation of biofilms. Despite an improved understanding of how they initially form, the processes that facilitate the long-term maintenance of these multicellular communities remain opaque. We found that P. aeruginosa requires two phosphodiesterases, RmcA and MorA, to maintain a mature biofilm and that P. aeruginosa biofilms lacking these PDEs succumb to nutrient limitation and die. The biofilm maintenance deficiency observed in {Delta}rmcA and {Delta}morA mutants was also found in the stringent response defective {Delta}relA {Delta}spoT strain, suggesting that a regulatory intersection between c-di-GMP signaling, EPS biosynthesis and the nutrient limitation response is important for persistent surface growth. We uncover components of an important regulatory system needed for P. aeruginosa to persist in nutrient-poor conditions, and provide some of the first evidence that maintaining a mature biofilm is an active process.

microbiology↗