Shuaiqi Guo

Cholera is a diarrheal disease resulting from colonization of the small intestine by the Gram-negative bacterium Vibrio cholerae . The peptide-binding domain (PBD) of the adhesion protein FrhA is critical for V. cholerae intestinal colonization and biofilm formation. Antagonists of the FrhA-PBD were designed based on the tripeptide motif H-Trp-Thr-Asp-OH. Constraining the N-terminal tryptophan as a 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (Tcc) residue and X-ray analysis of a co-crystal with the FrhA-PBD define the minimal inhibitory motif. Moreover, H-(R)-Tcc-Thr-Asp-OH exhibited nanomolar binding affinity (Kd = 101 ± 30.2 nM) to the FrhA-PBD, blocking adhesion protein-mediated bacterial hemagglutination and reducing biofilm formation in vitro . Intestinal epithelial cell adhesion to the FrhA-PBD was also blocked by the Tcc tripeptide (IC□□ = 24 µM; 95% CI, 11–51 µM), which resisted chymotrypsin proteolysis. The activity of this peptidomimetic inhibitor and the atomic-level understanding of FrhA-PBD binding provide insights for developing anti-adhesion therapies to treat cholera.

The tight adherence (Tad) pilus is a broadly distributed and evolutionarily distinct subclass of type IV pili that mediate cell adhesion, biofilm formation, predation, and surface sensing in many bacteria, including Caulobacter crescentus , Myxococcus xanthus , Vibrio vulnificus , and Bifidobacterium breve . Tad pili undergo cycles of extension and retraction powered by a cell-envelope-embedded nanomachine. Despite their biological importance, the architecture and assembly mechanism of the Tad pilus system remain poorly understood. Although cryo-electron tomography (cryo-ET) has elucidated the in situ architectures of other type IV pilus systems, no intact Tad machine structure has previously been reported. Here, we use cryo-ET and subtomogram averaging to resolve the architecture of the C. crescentus Tad pilus within the bacterial cell envelope. A three-dimensional classification further reveals multiple assembly intermediates, and integrative modeling incorporating AlphaFold3 predictions helps define the spatial arrangement of all core components. The resulting structural framework gives insight into the stepwise assembly process of the C. crescentus Tad pilus machine. Altogether, our results provide an in situ architectural model of the Tad pilus machine, establishing a foundation for understanding homologous systems across a broad range of bacteria. IMPORTANCE: Investigating the Tad pilus nanomachine in a genetically tractable, non-pathogenic organism like Caulobacter crescentus provides a powerful model for elucidating the architecture and functional dynamics of this widespread system. Insights gained from studying the Tad machinery can improve our understanding of related Tad pilus systems in pathogenic bacteria such as Aggregatibacter actinomycetemcomitans , where Tad pili are a key determinant of biofilm formation and chronic infection. Additionally, the remarkable functional diversity of Tad systems, ranging from surface sensing in C. crescentus to bacterial predation in Myxococcus xanthus , highlights their broad biological relevance. By revealing the in situ architecture of the Tad pilus biosynthetic machinery, this study advances our understanding of a major class of bacterial nanomachines and may thus provide structural insights that could inform the development of new therapeutic strategies targeting pilus-mediated virulence.

Vibrio cholerae , the causative agent of cholera, uses surface proteins such as the repeats‐in‐toxin (RTX) adhesin FrhA to colonize hosts and initiate infection. Blocking bacterial adhesion represents a promising therapeutic strategy to treat infections without promoting drug resistance. FrhA contains a peptide‐binding domain (PBD) that is key for hemagglutination, human epithelial cell binding, and V. cholerae biofilm formation. Previous studies identified a lead pentapeptide ligand with the sequence Ala‐Gly‐Tyr‐Thr‐Asp (AGYTD) that blocks V. cholerae colonization of the mouse small intestine at high micromolar concentrations. In this study, a structure‐guided approach identified a minimal D‐amino acid‐containing tripeptide motif with higher affinity for the FrhA‐PBD and predicted metabolic stability. Our results contribute to the development of anti‐adhesion strategies to combat infections. Impact statement: Our study elucidates the molecular basis of peptide recognition by the Vibrio cholerae adhesin FrhA and develops minimal D‐amino‐acid peptides that block adhesion with nanomolar affinity. These findings advance understanding of RTX adhesins and provide a structural blueprint for next‐generation anti‐adhesion therapeutics against cholera and related infections.

The Tight adherence (Tad) pilus is a broadly distributed and evolutionarily distinct subclass of type IV pili that mediates cell adhesion, biofilm formation, predation, and surface sensing in many bacteria, including Caulobacter crescentus , Myxococcus xanthus , Vibrio vulnificus , and Bifidobacterium breve . Tad pili undergo cycles of extension and retraction powered by a cell-envelope-embedded nanomachine. Despite their biological importance, the architecture and assembly mechanism of the Tad pilus system have remained poorly understood. Although cryo-electron tomography (cryo-ET) has elucidated the in situ structures of other type IV pilus systems, no intact Tad machine structure has previously been reported. Here, we use cryo-ET and subtomogram averaging to resolve the near-native architecture of the C. crescentus Tad pilus within the bacterial cell envelope. 3D classification further reveals multiple assembly intermediates, and integrative modelling incorporating AlphaFold3 predictions help define the spatial arrangement of all core components. The resulting structural framework gives insight into the stepwise assembly process of the C. crescentus Tad pilus machine. Altogether, our results provide an in situ architectural model of the Tad pilus machine, establishing a foundation for understanding homologous systems across a broad range of bacteria. Importance: Investigating the Tad pilus nanomachine in a genetically tractable, non-pathogenic organism like Caulobacter crescentus provides a powerful model for elucidating the architecture and functional dynamics of this widespread system. Insights gained from studying the Tad machinery can improve our understanding of related Tad pilus systems in pathogenic bacteria such as Aggregatibacter actinomycetemcomitans , where Tad pili are a key determinant of biofilm formation and chronic infection. Additionally, the remarkable functional diversity of Tad systems, ranging from surface sensing in C. crescentus to bacterial predation in M. xanthus , highlights their broad biological relevance. By revealing the in situ structure and assembly mechanism of the Tad pilus biosynthetic machinery, this study advances our understanding of a major class of bacterial nanomachines and may thus provide structural insights that could inform the development of new therapeutic strategies targeting pilus-mediated virulence.

Gram-negative bacteria attach to host surfaces using ligand-binding domains (LBDs) at the distal tips of fibrillar RTX adhesins. Blocking the initial binding interaction(s) can potentially prevent colonization and subsequent biofilm formation and infection. To this end, adhesins must be identified, and it is also essential to determine the dominant adhesin type for those species that have more than one. RTX adhesins are frequently the largest proteins within each species (ranging from 1500 to 15,000 aa) and are often misannotated as incomplete/pseudogene products because their highly repetitive nature confounds genome assemblies from short-read technologies. Our bioinformatic process collates predicted proteins from long-read assemblies, which are then clustered based on the similarity of their C-terminal regions where the LBDs are typically located. RTX adhesins are identified by their length and domain structure and are modelled using AlphaFold3. An exhaustive search of multiple strains from seven species revealed a total of 35 different RTX adhesins that map to 16 different loci, with differing arrangements of LBDs that include putative carbohydrate-binding modules and von Willebrand Factor A-like domains. Notably, similar adhesins are sometimes found in multiple species, either by descent or through DNA uptake, and three species have an RTX adhesin of uncertain function because it lacks an obvious LBD. Importance: Many bacteria initiate infection by reaching out with large, complex proteins called adhesins to attach themselves to a host cell. The DNA sequences of adhesins are difficult to read, due to their length and repetitiveness. This study leverages recent technological advances like “long-read sequencing” and structural modelling to identify and characterize the adhesins in seven species of harmful bacteria: Acinetobacter baumannii , Aeromonas hydrophila , Aeromonas salmonicida , Bordetella parapertussis , Legionella pneumophila , Vibrio parahaemolyticus , and Vibrio vulnificus . We identified thirty-five unique versions of adhesins and demonstrated their mix-and-match architecture. This research provides a foundation for strategies to block bacteria from binding surfaces, offering a vital alternative treatment as antibiotic resistance continues to rise.

Type IV pili (T4P) produced by the pathogen Pseudomonas aeruginosa play a pivotal role in adhesion, surface motility, biofilm formation, and infection in humans. Despite the significance of T4P as a potential therapeutic target, key details of their dynamic assembly and underlying molecular mechanisms of pilus extension and retraction remain elusive, primarily due to challenges in isolating intact T4P machines from the bacterial cell envelope. Here, we combine cryo-electron tomography with subtomogram averaging and integrative modelling to resolve in-situ architectural details of the dynamic T4P machine in P. aeruginosa cells. The T4P machine forms 7-fold symmetric cage-like structures anchored in the cell envelope, providing a molecular framework for the rapid exchange of major pilin subunits during pilus extension and retraction. Our data suggest that the T4P adhesin PilY1 forms a champagne-cork-shaped structure, effectively blocking the secretin channel in the outer membrane whereas the minor-pilin complex in the periplasm appears to contact PilY1 via the central pore of the secretin gate. These findings point to a hypothetical model where the interplay between the secretin protein PilQ and the PilY1-minor-pilin priming complex is important for optimizing conformations of the T4P machine in P. aeruginosa, suggesting a gate-keeping mechanism that regulates pilus dynamics.

Bacterial adhesins attach their hosts to surfaces that the bacteria will colonize. This surface adhesion occurs through specific ligand‐binding domains located towards the distal end of the long adhesin molecules. However, recognizing which of the many adhesin domains are structural and which are ligand binding has been difficult up to now. Here we have used the protein structure modeling program AlphaFold2 to predict structures for these giant 0.2‐ to 1.5‐megadalton proteins. Crystal structures previously solved for several adhesin regions are in good agreement with the models. Whereas most adhesin domains are linked in a linear fashion through their N‐ and C‐terminal ends, ligand‐binding domains can be recognized by budding out from a companion core domain so that their ligand‐binding sites are projected away from the axis of the adhesin for maximal exposure to their targets. These companion domains are “split” in their continuity by projecting the ligand‐binding domain outwards. The “split domains” are mostly β‐sandwich extender modules, but other domains like a β‐solenoid can serve the same function. Bioinformatic analyses of Gram‐negative bacterial sequences revealed wide variety ligand‐binding domains are used in their Repeats‐in‐Toxin adhesins. The ligands for many of these domains have yet to be identified but known ligands include various cell‐surface glycans, proteins, and even ice. Recognizing the ligands to which the adhesins bind could lead to ways of blocking colonization by bacterial pathogens. Engineering different ligand‐binding domains into an adhesin has the potential to change the surfaces to which bacteria bind.

The bacterium, Vibrio cholerae , which causes cholera, uses an adhesion protein to stick to human cells and begin the infection process. One part of this adhesin protein binds to a particular sugar, fucose, on the surface of the target cells. This binding can lead to colonization and killing of the cells by the bacteria. Adding l -fucose to the bacteria before they bind to the human cells can prevent attachment and has promise as a preventative drug to protect against cholera.