Structural basis of biofilm formation mediated by the Pseudomonas aeruginosa fibrillar adhesin CdrA

Structure of a key adhesin-exopolysaccharide interaction provides insights into matrix assembly in Vibrio cholerae biofilms

The structure reveals for the first time the interaction between a biofilm exopolysaccharide and matrix protein, as well as insights into conformational changes of exopolysaccharide induced by this binding, and provides a generalizable approach for studying the biophysical and biochemical properties of carbohydrate-dependent biofilm assembly.

Gene deletions differentially affect surface-associated biofilms and staphylococcal abscess communities

Staphylococcus aureus is a major human pathogen that causes persistent infections characterised by the formation of aggregates such as surface-attached biofilm and staphylococcal abscess communities (SACs). Both consist of dense bacterial populations associated with self-produced matrices that impair immune cell and antibiotic access. Surface-attached biofilms have mostly protein- or polysaccharide-rich matrices, whereas SACs are spherical structures within tissue encased in a fibrin pseudocapsule and microcolony-associated meshwork. It remains unclear whether SAC are simply biofilms within tissue or if they display distinct features with unique genetic regulation. Here, we investigated genetic determinants of SAC and biofilm formation using four S. aureus deletion mutants targeting staphylokinase (Δsak), coagulase (Δcoa), the alternative sigma factor SigB (ΔrpoF), and sortase A (ΔsrtA). SACs were grown in collagen gels, whilst biofilms were grown on titanium discs, with bacterial viability, fibrin deposition, and biofilm biomass assessed by microscopy, image analysis, and crystal violet staining. Although deletion of coa or sak did not significantly impact either SAC or biofilm formation, biofilm surface coverage was markedly increased in ΔsrtA and biomass decreased in ΔrpoF; however, these mutations had no effect on SAC. These findings suggest that certain mutations can have different effects in the two experimental systems, and that the tested gene deletions were more important for surface-associated biofilm development than SAC formation.

Cryo-electron microscopy structure of Jabs, a bacteriophage infecting the multidrug-resistant pathogen Mycobacterium abscessus

Exploring bacteriophage structural diversity is essential for understanding phage biology and for advancing phage-based therapies. Here, we determine the cryo-electron microscopy structure of Jabs, providing, to our knowledge, the first high-resolution view of a phage infecting the multidrug-resistant human pathogen Mycobacterium abscessus. Although Jabs displays the canonical organization of a siphophage, its virion combines several unusual architectural features. The T=9 icosahedral capsid is assembled from two distinct major capsid proteins, with one forming the hexons and the other the pentons, revealing an unprecedented capsid assembly strategy among icosahedral phages. An extensive network of ∼1,700 disulfide bonds stabilize individual structural components and covalently links the capsid, connector, tail, and adhesion device into a continuous assembly. At the distal end of the tail, an elaborate and conformationally dynamic adhesion device comprises multiple candidate receptor-binding proteins organized into complex multidomain architectures, including carbohydrate-binding modules and β-sandwich hetero- and homotrimers resembling the receptor-binding proteins of phages infecting lactic acid bacteria. Together, these findings expand our understanding of phage structural diversity and provide a framework for investigating phage-host interactions and guiding the engineering of therapeutic phages.

Stage V sporulation protein G as a global regulator of biofilm formation and virulence in Staphylococcus species: molecular mechanisms and therapeutic potential

Structural and Functional Interrogation of Active Streptococcus pneumoniae Sortase A

Sortase A (SrtA) enzymes covalently anchor surface proteins to Gram-positive bacterial cell walls, promoting colonization and virulence. In Streptococcus pneumoniae, previous studies identified both a domain-swapped dimer and an active refolded monomer, but the active enzyme has not been characterized at the structural and residue-specific level. Here, we performed quantitative proteomic comparisons of wild-type and SrtA knockout strains that confirmed the loss of multiple LPxTG-containing virulence factors, including ZmpB, NanA, and IgA1 protease, consistent with an essential role for SrtA in surface protein anchoring. To enable mechanistic studies, we established a biochemical framework to produce monomeric Streptococcus pneumoniae SrtA by refolding and developed a gel-based assay using recombinant substrates to monitor catalytic activity. The refolded monomer, but not the swapped dimer, catalyzed cleavage and transpeptidation of a canonical LPxTG substrate in a metal-independent manner under the conditions examined. We further report high-resolution NMR backbone assignments for the active monomer and identify substrate-induced chemical shift perturbations that localize to the active site. Together, these findings provide an integrated proteomic, biochemical, and NMR characterization of monomeric, catalytically active Streptococcus pneumoniae SrtA and reveal residue-specific interactions with a canonical LPNTG recognition peptide.

Identification of a conserved GNAT-family lysine acetyltransferase in Streptococcus gordonii involved in biofilm formation and oral colonization

It is demonstrated that SGO_2031, but not SGO_2030, seems to contribute to biofilm formation by modulating the abundance of extracellular polysaccharides within the biofilm matrix, and proposed naming this enzyme Streptococcal Lysine Acetyltransferase A (SktA) after Streptococcus gordonii.