Three hydrophobic surface binding a proteins link surface hydrophobicity to sporulation, biofilm formation, and host interaction in Mucor lusitanicus

Non-ribosomal Peptides as Structural Determinants of Fungal Hydrophobicity

Fungal surfaces must remain hydrophobic to enable growth, dispersal, and survival under fluctuating environmental conditions, yet the molecular basis of this property remains incompletely understood. Here, we identify fungisporins, fusahexins, and related cyclic non-ribosomal peptides (NRPs) as members of a conserved functional class of fungal metabolites, termed WAter Repellent Peptides (WARPs), that are required for fungal surface hydrophobicity. Across filamentous fungi, WARPs vary substantially in sequence and length but share conserved structural features, including cyclization, hydrophobic amino acid composition, and alternating D- and L-configurations, consistent with a flexible amphiphilic scaffold. Loss of WARP-producing non-ribosomal peptide synthetases results in rapid collapse of aerial hyphae upon water exposure, demonstrating that these peptides are required for maintenance of hydrophobic aerial structures. Using phage-display-derived antibodies, we localize WARPs to the hyphal surface, supporting their role as surface-associated structural components. Together, these findings identify a conserved NRPS-encoded peptide system that contributes to fungal hydrophobicity and establish WARPs as a broadly distributed class of surface-associated metabolites with structural function in filamentous fungi.

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

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

Biofilms serve as a protective mechanism for bacteria, including many pathogens. To form such communities, bacteria secrete macromolecules that form an extracellular matrix serving as a barrier against environmental threats, such as predation, antibiotics, and the host immune system. To be effective, a biofilm must anchor to foreign surfaces and retain sufficient stiffness, in the environment or in a host. However, how this matrix self-organizes to support biofilm formation remains a mystery at the molecular level. The human pathogen Vibrio cholerae produces biofilms primarily composed of an exopolysaccharide called VPS (Vibrio polysaccharide), consisting of an unusually-modified repeating tetrasaccharide core unit. VPS engages with two secreted adhesion proteins, Bap1 and RbmC, which adhere the biofilm to abiotic and biotic surfaces, and serve to strengthen the biofilm by interacting with VPS using a conserved β-propeller. To pinpoint the interaction between the adhesins and purified segments of VPS, we determined the ∼1.6 Å X-ray crystal structure of Bap1 bound to fragmented VPS and used the structure to carry out molecular dynamics simulations. The structure revealed a single binding site consisting of one tetrasaccharide unit involving an induced magnesium binding site. Unexpectedly, the tetrasaccharide adopted a bent state caused by a rotation of the glycosidic bond between the central two monosaccharide units. Using a combination of mutagenesis, light scattering, and in situ fluorescent microscopy, we demonstrate that Bap1 not only facilitates biofilm adhesion, but is also required for proper VPS organization, through the identified binding pocket. Our 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. Our findings provide a generalizable approach for studying the biophysical and biochemical properties of carbohydrate-dependent biofilm assembly, which may lead to new ways to treat disease caused by biofilm-forming bacterial pathogens by disrupting the exopolysaccharide-protein interactions.

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

ABSTRACT Streptococcus gordonii is a gram-positive oral bacterium capable of adhering to a variety of biotic and abiotic surfaces and forming biofilms. To characterize physiological changes associated with biofilm formation in S. gordonii, we investigated the roles of two putative GCN5-related N-acetyltransferases (GNATs), SGO_2030 and SGO_2031, in in vitro biofilm formation on saliva-coated surfaces using the laboratory strain DL1. Our results demonstrate that SGO_2031, but not SGO_2030, seems to contribute to biofilm formation by modulating the abundance of extracellular polysaccharides within the biofilm matrix. This defect in biofilm formation observed by the deletion of SGO_2031 resulted in a significant fitness disadvantage during colonization of the murine oral cavity compared to the wild-type parent strain. Consistent with the role of S. gordonii as an early colonizer of tooth surfaces that influences oral biofilm community structure, inoculation with either the wild-type or the SGO_2031 mutant strain led to distinct alterations in the murine oral microbiome composition. Deletion of SGO_2031 also resulted in changes in protein acetylation patterns, as assessed by Western immunoblot analysis, supporting the role of this enzyme as an acetyltransferase. Given that SGO_2031 is conserved and widely distributed among streptococci, we propose naming this enzyme Streptococcal Lysine Acetyltransferase A (SktA). IMPORTANCE Protein acetylation is a common posttranslational modification conserved across all domains of life. In bacteria, protein acetylation is carried out by homologs of the GCN5-related N-acetyltransferase (GNAT) family. GNATs catalyze the transfer of an acetyl group from acetyl-CoA to the ε-amino group of lysine residues on proteins. This process changes the charge and length of lysine residues, resulting in changes to protein function. Streptococcus gordonii is predicted to encode 17 GNAT homologs. Here, we report that one of them, SGO_2031 (SktA), plays an important role in S. gordonii biofilms. Protein acetylation is a common posttranslational modification conserved across all domains of life. In bacteria, protein acetylation is carried out by homologs of the GCN5-related N-acetyltransferase (GNAT) family. GNATs catalyze the transfer of an acetyl group from acetyl-CoA to the ε-amino group of lysine residues on proteins. This process changes the charge and length of lysine residues, resulting in changes to protein function. Streptococcus gordonii is predicted to encode 17 GNAT homologs. Here, we report that one of them, SGO_2031 (SktA), plays an important role in S. gordonii biofilms.

Tracking Surface Protein Assembly and Peptidoglycan Biosynthesis in Fusobacterium nucleatum.

Divergence in surface protein exposure between reference and clinical-derived Candida glabrata (Nakaseomyces glabratus) strains (CBS138 vs. BG2) – a preliminary proteomic perspective

Candida glabrata (currently classified as Nakaseomyces glabratus) is an opportunistic fungal pathogen notable for its intrinsic antifungal tolerance and ability to persist in host environments. Although strain CBS138 has served as the principal model for genetic and functional studies, accumulating evidence indicates substantial intraspecies diversity that may shape virulence, immune interactions and stress adaptation. In particular, the widely used clinical isolate BG2 differs from CBS138 in genome structure, adhesin regulation and macrophage survival, yet the extent to which these differences are reflected at the fungal cell surface remains unknown. Here, we present a comparative characterization of the surface-exposed proteomes (surfaceomes) of CBS138 and BG2 across three biologically relevant growth conditions: YPD-grown yeast-like cells, RPMI-cultured planktonic aggregates and RPMI-formed biofilms. Using trypsin shaving combined with LC–MS/MS, we identified pronounced strain- and condition-dependent differences in surface protein composition, encompassing adhesins, yapsin proteases and selected moonlighting proteins. Whereas CBS138 showed greater representation of adhesion- and interaction-related surface proteins, BG2 preferentially displayed proteins associated with cell-wall architecture and remodelling, consistent with distinct surface-mediated adaptive strategies. Transmission electron microscopy revealed condition-dependent differences in cell-wall thickness in both strains, with BG2 displaying a broader range of values and the highest thickness under biofilm conditions, providing structural context for variation in protease accessibility and surface-protein detectability. Collectively, our findings highlight substantial surfaceome plasticity in C. glabrata and underscore the importance of considering intraspecies diversity when interpreting host–pathogen interactions and fungal virulence pathways.