Bidirectional pilus processing in the Tad pilus system motor CpaF.

The bacterial tight adherence pilus system (TadPS) assembles surface pili essential for adhesion and colonisation in many human pathogens. Pilus dynamics are powered by the ATPase CpaF (TadA), which drives extension and retraction cycles in Caulobacter crescentus through an unknown mechanism. Here we use cryogenic electron microscopy and cell-based light microscopy to characterise CpaF mechanism. We show that CpaF assembles into a hexamer with C2 symmetry in different nucleotide states. Nucleotide cycling occurs through an intra-subunit clamp-like mechanism that promotes sequential conformational changes between subunits. Moreover, a comparison of the active sites with different nucleotides bound suggests a mechanism for bidirectional motion. Conserved CpaF residues, predicted to interact with platform proteins CpaG (TadB) and CpaH (TadC), are mutated in vivo to establish their role in pilus processing. Our findings provide a model for how CpaF drives TadPS pilus dynamics and have broad implications for how other ancient type 4 filament family members power pilus assembly.

Fluid flow drives phenotypic heterogeneity in bacterial growth and adhesion on surfaces.

Bacteria often thrive in surface-attached communities, where they can form biofilms affording them multiple advantages. In this sessile form, fluid flow is a key component of their environments, renewing nutrients and transporting metabolic products and signaling molecules. It also controls colonization patterns and growth rates on surfaces, through bacteria transport, attachment and detachment. However, the current understanding of bacterial growth on surfaces neglects the possibility that bacteria may modulate their division behavior as a response to flow. Here, we employed single-cell imaging in microfluidic experiments to demonstrate that attached Escherichia coli cells can enter a growth arrest state while simultaneously enhancing their adhesion underflow. Despite utilizing clonal populations, we observed a non-uniform response characterized by bistable dynamics, with co-existing subpopulations of non-dividing and actively dividing bacteria. As the proportion of non-dividing bacteria increased with the applied flow rate, it resulted in a reduction in the average growth rate of bacterial populations on flow-exposed surfaces. Dividing bacteria exhibited asymmetric attachment, whereas non-dividing counterparts adhered to the surface via both cell poles. Hence, this phenotypic diversity allows bacterial colonies to combine enhanced attachment with sustained growth, although at a reduced rate, which may be a significant advantage in fluctuating flow conditions.

Microbiology: Murder as a solution to promiscuity?

Strain-specific pili enable Vibrio cholerae bacteria to adhere to each other and form aggregates in liquid culture. A new study focuses on strains with less specific, promiscuous pili and suggests a role for contact-dependent bacterial killing in shaping the composition of these aggregates.

Difference in formation of a dental multi-species biofilm according to substratum direction.

OBJECTIVES: The aim of this study was to investigate the difference in dental biofilm formation according to substratum direction, using an artificial biofilm model. METHODS: A three-species biofilm, consisting of Streptococcus mutans, Streptococcus oralis, and Actinomyces naeslundii, was formed on saliva-coated hydroxyapatite (sHA) discs oriented in three directions: downward (the discs placed in the direction of gravity), vertical (the discs placed parallel to the direction of gravity), and upward (the discs placed in opposite direction of gravity). The biofilms at 22 h and 46 h of age were analyzed using microbiological and biochemical methods, fluorescence-based assays, and scanning electron microscopy to investigate difference in bacterial adhesion, early and mature biofilm formation. RESULTS: The biofilms formed in the upward direction displayed the most complex structure, with the highest number and biovolume of bacteria, as well as the lowest pH conditions at both time points. The vertical and downward directions, however, had only scattered and small bacterial colonies. In the 22-h-old biofilms, the proportion of S. oralis was similar to, or slightly higher than, that of S. mutans in all directions of substratum surfaces. However, in the 46-h-old biofilms, S. mutans became the dominant bacteria in all directions, especially in the vertical and upward directions. CONCLUSIONS: The direction of the substratum surface could impact the proportion of bacteria and cariogenic properties of the multi-species biofilm. Biofilms in an upward direction may exhibit a higher cariogenic potential, followed by those in the vertical and downward directions, which could be related to gravity.

Identification of Anaplasma marginale adhesins for entry into Dermacentor andersoni tick cells using phage display.

Anaplasma marginale is an obligate, intracellular, tick-borne bacterial pathogen that causes bovine anaplasmosis, an often severe, production-limiting disease of cattle found worldwide. Methods to control this disease are lacking, in large part due to major knowledge gaps in our understanding of the molecular underpinnings of basic host-pathogen interactions. For example, the surface proteins that serve as adhesins and, thus, likely play a role in pathogen entry into tick cells are largely unknown. To address this knowledge gap, we developed a phage display library and screened 66 A. marginale proteins for their ability to adhere to Dermacentor andersoni tick cells. From this screen, 17 candidate adhesins were identified, including OmpA and multiple members of the Msp1 family, including Msp1b, Mlp3, and Mlp4. We then measured the transcript of ompA and all members of the msp1 gene family through time, and determined that msp1b, mlp2, and mlp4 have increased transcript during tick cell infection, suggesting a possible role in host cell binding or entry. Finally, Msp1a, Msp1b, Mlp3, and OmpA were expressed as recombinant protein. When added to cultured tick cells prior to A. marginale infection, all proteins except the C-terminus of Msp1a reduced A. marginale entry by 2.2- to 4.7-fold. Except OmpA, these adhesins lack orthologs in related pathogens of humans and animals, including Anaplasma phagocytophilum and the Ehrlichia spp., thus limiting their utility in a universal tick transmission-blocking vaccine. However, this work greatly advances efforts toward developing methods to control bovine anaplasmosis and, thus, may help improve global food security.

Optogenetic Control of Bacterial Cell-Cell Adhesion Dynamics: Unraveling the Influence on Biofilm Architecture and Functionality.

The transition of bacteria from an individualistic to a biofilm lifestyle profoundly alters their biology. During biofilm development, the bacterial cell-cell adhesions are a major determinant of initial microcolonies, which serve as kernels for the subsequent microscopic and mesoscopic structure of the biofilm, and determine the resulting functionality. In this study, the significance of bacterial cell-cell adhesion dynamics on bacterial aggregation and biofilm maturation is elucidated. Using photoswitchable adhesins between bacteria, modifying the dynamics of bacterial cell-cell adhesions with periodic dark-light cycles is systematic. Dynamic cell-cell adhesions with liquid-like behavior improve bacterial aggregation and produce more compact microcolonies than static adhesions with solid-like behavior in both experiments and individual-based simulations. Consequently, dynamic cell-cell adhesions give rise to earlier quorum sensing activation, better intermixing of different bacterial populations, improved biofilm maturation, changes in the growth of cocultures, and higher yields in fermentation. The here presented approach of tuning bacterial cell-cell adhesion dynamics opens the door for regulating the structure and function of biofilms and cocultures with potential biotechnological applications.

Adhesion Inhibition Assay for Helicobacter pylori to Mucin by Lactobacillus.

The ability of Lactobacillus to adhere to mucin is a parameter for evaluating the effectiveness of probiotics. In particular, a competitive inhibition assay of pathogenic bacteria using mucin-adherent lactobacilli is useful for identifying Lactobacillus strains capable of preventing mucus from being colonized by pathogens. Here, we describe an adhesion inhibition assay method for Helicobacter pylori to porcine gastric mucin by Limosilactobacillus reuteri.

Etiology, pathology, and host-impaired immunity in medical implant-associated infections.

Host impaired immunity and pathogens adhesion factors are the key elements in analyzing medical implant-associated infections (MIAI). The infection chances are further influenced by surface properties of implants. This review addresses the medical implant-associated pathogens and summarizes the etiology, pathology, and host-impaired immunity in MIAI. Several bacterial and fungal pathogens have been isolated from MIAI; together, they form cross-kingdom species biofilms and support each other in different ways. The adhesion factors initiate the pathogen's adherence on the implant's surface; however, implant-induced impaired immunity promotes the pathogen's colonization and biofilm formation. Depending on the implant's surface properties, immune cell functions get slow or get exaggerated and cause immunity-induced secondary complications resulting in resistant depression and immuno-incompetent fibro-inflammatory zone that compromise implant's performance. Such consequences lead to the unavoidable and straightforward conclusion for the downstream transformation of new ideas, such as the development of multifunctional implant coatings.

Geometric constraint-triggered collagen expression mediates bacterial-host adhesion.

Cells living in geometrically confined microenvironments are ubiquitous in various physiological processes, e.g., wound closure. However, it remains unclear whether and how spatially geometric constraints on host cells regulate bacteria-host interactions. Here, we reveal that interactions between bacteria and spatially constrained cell monolayers exhibit strong spatial heterogeneity, and that bacteria tend to adhere to these cells near the outer edges of confined monolayers. The bacterial adhesion force near the edges of the micropatterned monolayers is up to 75 nN, which is ~3 times higher than that at the centers, depending on the underlying substrate rigidities. Single-cell RNA sequencing experiments indicate that spatially heterogeneous expression of collagen IV with significant edge effects is responsible for the location-dependent bacterial adhesion. Finally, we show that collagen IV inhibitors can potentially be utilized as adjuvants to reduce bacterial adhesion and thus markedly enhance the efficacy of antibiotics, as demonstrated in animal experiments.

LppA is a novel plasminogen receptor of Mycoplasma bovis that contributes to adhesion by binding the host extracellular matrix and Annexin A2.

Mycoplasma bovis is responsible for various inflammatory diseases in cattle. The prevention and control of M. bovis are complicated by the absence of effective vaccines and the emergence of multidrug-resistant strains, resulting in substantial economic losses worldwide in the cattle industry. Lipoproteins, vital components of the Mycoplasmas cell membrane, are deemed potent antigens for eliciting immune responses in the host upon infection. However, the functions of lipoproteins in M. bovis remain underexplored due to their low sequence similarity with those of other bacteria and the scarcity of genetic manipulation tools for M. bovis. In this study, the lipoprotein LppA was identified in all examined M. bovis strains. Utilizing immunoelectron microscopy and Western blotting, it was observed that LppA localizes to the surface membrane. Recombinant LppA demonstrated dose-dependent adherence to the membrane of embryonic bovine lung (EBL) cells, and this adhesion was inhibited by anti-LppA serum. In vitro binding assays confirmed LppA's ability to associate with fibronectin, collagen IV, laminin, vitronectin, plasminogen, and tPA, thereby facilitating the conversion of plasminogen to plasmin. Moreover, LppA was found to bind and enhance the accumulation of Annexin A2 (ANXA2) on the cell membrane. Disrupting LppA in M. bovis significantly diminished the bacterium's capacity to adhere to EBL cells, underscoring LppA's function as a bacterial adhesin. In conclusion, LppA emerges as a novel adhesion protein that interacts with multiple host extracellular matrix proteins and ANXA2, playing a crucial role in M. bovis's adherence to host cells and dissemination. These insights substantially deepen our comprehension of the molecular pathogenesis of M. bovis.

[Visualization method of type â £ pili and its application in the study of pili function].

As an important protein structure on the surface of bacteria, type Ⅳ pili (TFP) is the sensing and moving organ of bacteria. It plays a variety of roles in bacterial physiology, cell adhesion, host cell invasion, DNA uptake, protein secretion, biofilm formation, cell movement and electron transmission. With the rapid development of research methods, technical equipment and pili visualization tools, increasing number of studies have revealed various functions of pili in cellular activities, which greatly facilitated the microbial single cell research. This review focuses on the pili visualization method and its application in the functional research of TFP, providing ideas for the research and application of TFP in biology, medicine and ecology.

Binding site plasticity regulation of the FimH catch-bond mechanism.

The bacterial fimbrial adhesin FimH is a remarkable and well-studied catch-bond protein found at the tip of E. coli type 1 pili, which allows pathogenic strains involved in urinary tract infections to bind high-mannose glycans exposed on human epithelia. The catch-bond behavior of FimH, where the strength of the interaction increases when a force is applied to separate the two partners, enables the bacteria to resist clearance when they are subjected to shear forces induced by urine flow. Two decades of experimental studies performed at the single-molecule level, as well as x-ray crystallography and modeling studies, have led to a consensus picture whereby force separates the binding domain from an inhibitor domain, effectively triggering an allosteric conformational change in the former. This force-induced allostery is thought to be responsible for an increased binding affinity at the core of the catch-bond mechanism. However, some important questions remain, the most challenging one being that the crystal structures corresponding to these two allosteric states show almost superimposable binding site geometries, which questions the molecular origin for the large difference in affinity. Using molecular dynamics with a combination of enhanced-sampling techniques, we demonstrate that the static picture provided by the crystal structures conceals a variety of binding site conformations that have a key impact on the apparent affinity. Crucially, the respective populations in each of these conformations are very different between the two allosteric states of the binding domain, which can then be related to experimental affinity measurements. We also evidence a previously unappreciated but important effect: in addition to the well-established role of the force as an allosteric regulator via domain separation, application of force tends to directly favor the high-affinity binding site conformations. We hypothesize that this additional "local" catch-bond effect could delay unbinding between the bacteria and the host cell before the "global" allosteric transition occurs, as well as stabilizing the complex even more once in the high-affinity allosteric state.

Influence of Surface Roughness, Nanostructure, and Wetting on Bacterial Adhesion.

Bacterial fouling is a persistent problem causing the deterioration and failure of functional surfaces for industrial equipment/components; numerous human, animal, and plant infections/diseases; and energy waste due to the inefficiencies at internal and external geometries of transport systems. This work gains new insights into the effect of surface roughness on bacterial fouling by systematically studying bacterial adhesion on model hydrophobic (methyl-terminated) surfaces with roughness scales spanning from ∼2 nm to ∼390 nm. Additionally, a surface energy integration framework is developed to elucidate the role of surface roughness on the energetics of bacteria and substrate interactions. For a given bacteria type and surface chemistry; the extent of bacterial fouling was found to demonstrate up to a 75-fold variation with surface roughness. For the cases showing hydrophobic wetting behavior, both increased effective surface area with increasing roughness and decreased activation energy with increased surface roughness was concluded to enhance the extent of bacterial adhesion. For the cases of superhydrophobic surfaces, the combination of factors including (i) the surpassing of Laplace pressure force of interstitial air over bacterial adhesive force, (ii) the reduced effective substrate area for bacteria wall due to air gaps to have direct/solid contact, and (iii) the reduction of attractive van der Waals force that holds adhering bacteria on the substrate were summarized to weaken the bacterial adhesion. Overall, this study is significant in the context of designing antifouling coatings and systems as well as explaining variations in bacterial contamination and biofilm formation processes on functional surfaces.

Contact Area and Deformation of Escherichia coli Cells Adhered on a Cationic Surface.

When bacteria adhere to surfaces, the chemical and mechanical character of the cell-substrate interface guides cell function and the development of microcolonies and biofilms. Alternately on bactericidal surfaces, intimate contact is critical to biofilm prevention. The direct study of the buried cell-substrate interfaces at the heart of these behaviors is hindered by the small bacterial cell size and inaccessibility of the contact region. Here, we present a total internal reflectance fluorescence depletion approach to measure the size of the cell-substrate contact region and quantify the gap separation and curvature near the contact zone, providing an assessment of the shapes of the near-surface undersides of adhered bacterial cells. Resolution of the gap height is about 10%, down to a few nanometers at contact. Using 1 and 2 µm silica spheres as calibration standards we report that, for flagella-free Escherichia coli (E. coli) adhering on a cationic poly-l-lysine layer, the cell-surface contact and apparent cell deformation vary with adsorbed cell configuration. Most cells adhere by their ends, achieving small contact areas of 0.15 µm2, corresponding to about 1-2% of the cell's surface. The altered Gaussian curvatures of end-adhered cells suggest the flattening of the envelope within the small contact region. When cells adhere by their sides, the contact area is larger, in the range 0.3-1.1 µm2 and comprising up to ∼12% of the cell's total surface. A region of sharper curvature, greater than that of the cells' original spherocylindrical shape, borders the flat contact region in cases of side-on or end-on cell adhesion, suggesting envelope stress. From the measured curvatures, precise stress distributions over the cell surface could be calculated in future studies that incorporate knowledge of envelope moduli. Overall the small contact areas of end-adhered cells may be a limiting factor for antimicrobial surfaces that kill on contact rather than releasing bactericide.

Involvement of the Streptococcus mutans PgfE and GalE 4-epimerases in protein glycosylation, carbon metabolism, and cell division.

Streptococcus mutans is a key pathogen associated with dental caries and is often implicated in infective endocarditis. This organism forms robust biofilms on tooth surfaces and can use collagen-binding proteins (CBPs) to efficiently colonize collagenous substrates, including dentin and heart valves. One of the best characterized CBPs of S. mutans is Cnm, which contributes to adhesion and invasion of oral epithelial and heart endothelial cells. These virulence properties were subsequently linked to post-translational modification (PTM) of the Cnm threonine-rich repeat region by the Pgf glycosylation machinery, which consists of 4 enzymes: PgfS, PgfM1, PgfE, and PgfM2. Inactivation of the S. mutans pgf genes leads to decreased collagen binding, reduced invasion of human coronary artery endothelial cells, and attenuated virulence in the Galleria mellonella invertebrate model. The present study aimed to better understand Cnm glycosylation and characterize the predicted 4-epimerase, PgfE. Using a truncated Cnm variant containing only 2 threonine-rich repeats, mass spectrometric analysis revealed extensive glycosylation with HexNAc2. Compositional analysis, complemented with lectin blotting, identified the HexNAc2 moieties as GlcNAc and GalNAc. Comparison of PgfE with the other S. mutans 4-epimerase GalE through structural modeling, nuclear magnetic resonance, and capillary electrophoresis demonstrated that GalE is a UDP-Glc-4-epimerase, while PgfE is a GlcNAc-4-epimerase. While PgfE exclusively participates in protein O-glycosylation, we found that GalE affects galactose metabolism and cell division. This study further emphasizes the importance of O-linked protein glycosylation and carbohydrate metabolism in S. mutans and identifies the PTM modifications of the key CBP, Cnm.

Visualization method of type Ⅳ pili and its application in the study of pili function / 生物工程学报

As an important protein structure on the surface of bacteria, type Ⅳ pili (TFP) is the sensing and moving organ of bacteria. It plays a variety of roles in bacterial physiology, cell adhesion, host cell invasion, DNA uptake, protein secretion, biofilm formation, cell movement and electron transmission. With the rapid development of research methods, technical equipment and pili visualization tools, increasing number of studies have revealed various functions of pili in cellular activities, which greatly facilitated the microbial single cell research. This review focuses on the pili visualization method and its application in the functional research of TFP, providing ideas for the research and application of TFP in biology, medicine and ecology.

Exopolysaccharide from Lactobacillus casei NA-2 attenuates Escherichia coli O157:H7 surface adhesion via modulation of membrane surface properties and adhesion-related gene expression.

The natural compound, exopolysaccharide from Lactobacillus casei NA-2 (EPS-cn2), has been shown to inhibit biofilm formation by Escherichia coli O157:H7. Although bacterial adhesion to substrate surfaces is a primary, indispensable step in this process, the mechanisms by which EPS-cn2 can block E. coli O157:H7 adhesion to biotic or abiotic surfaces remain unclear. In this study, investigation of E. coli O157:H7 response to EPS-cn2 revealed that 1 mg/mL EPS-cn2 can decrease adherence to polystyrene and confluent Caco-2 cell surfaces to 49.0% (P＜0.0001) and 57.0% (P＜0.01) of that in untreated E. coli O157:H7, respectively. Moreover, EPS-cn2 significantly reduced outer membrane hydrophobicity by 49.0% and decreased the electronegativity of the membrane surface charge by as much as 1.57 mV (P＜0.05) compared to untreated cells. High throughput RNA sequencing indicated that genes responsible for adhesion through extracellular matrix secretion, such as poly-N-acetyl-glucosamine (PNAG) biosynthesis, locus of enterocyte effacement (LEE) proteins and outer membrane protein (OmpT) were all down-regulated in response to EPS-cn2, while chemotaxis and motility-related flagellar assembly genes were differentially up-regulated, suggesting that the EPS-cn2 may serve as an extracellular signal to attenuate adhesion-related gene expression and alter bacterial surface properties in E. coli O157:H7. These findings support the further development of EPS-cn2 for pathogenic biofilm management in clinical and industrial settings, and suggests the further targeting of adhesion-related genes to limit the persistence of this highly pathogenic strain in sensitive environments.

HfaE Is a Component of the Holdfast Anchor Complex That Tethers the Holdfast Adhesin to the Cell Envelope.

Bacteria use adhesins to colonize different surfaces and form biofilms. The species of the Caulobacterales order use a polar adhesin called holdfast, composed of polysaccharides, proteins, and DNA, to irreversibly adhere to surfaces. In Caulobacter crescentus, a freshwater Caulobacterales species, the holdfast is anchored at the cell pole via the holdfast anchor (Hfa) proteins HfaA, HfaB, and HfaD. HfaA and HfaD colocalize with holdfast and are thought to form amyloid-like fibers that anchor holdfast to the cell envelope. HfaB, a lipoprotein, is required for the translocation of HfaA and HfaD to the cell surface. Deletion of the anchor proteins leads to a severe defect in adherence resulting from holdfast not being properly attached to the cell and shed into the medium. This phenotype is greater in a ΔhfaB mutant than in a ΔhfaA ΔhfaD double mutant, suggesting that HfaB has other functions besides the translocation of HfaA and HfaD. Here, we identify an additional HfaB-dependent holdfast anchoring protein, HfaE, which is predicted to be a secreted protein. HfaE is highly conserved among Caulobacterales species, with no predicted function. In planktonic culture, hfaE mutants produce holdfasts and rosettes similar to those produced by the wild type. However, holdfasts from hfaE mutants bind to the surface but are unable to anchor cells, similarly to other anchor mutants. We showed that fluorescently tagged HfaE colocalizes with holdfast and that HfaE forms an SDS-resistant high-molecular-weight species consistent with amyloid fiber formation. We propose that HfaE is a novel holdfast anchor protein and that HfaE functions to link holdfast material to the cell envelope. IMPORTANCE For surface attachment and biofilm formation, bacteria produce adhesins that are composed of polysaccharides, proteins, and DNA. Species of the Caulobacterales produce a specialized polar adhesin, holdfast, which is required for permanent attachment to surfaces. In this study, we evaluate the role of a newly identified holdfast anchor protein, HfaE, in holdfast anchoring to the cell surface in two different members of the Caulobacterales with drastically different environments. We show that HfaE plays an important role in adhesion and biofilm formation in the Caulobacterales. Our results provide insights into bacterial adhesins and how they interact with the cell envelope and surfaces.

Mechanical Stabilization of a Bacterial Adhesion Complex.

The adhesions between Gram-positive bacteria and their hosts are exposed to varying magnitudes of tensile forces. Here, using an ultrastable magnetic tweezer-based single-molecule approach, we show the catch-bond kinetics of the prototypical adhesion complex of SD-repeat protein G (SdrG) to a peptide from fibrinogen ß (Fgß) over a physiologically important force range from piconewton (pN) to tens of pN, which was not technologically accessible to previous studies. At 37 °C, the lifetime of the complex exponentially increases from seconds at several pN to ∼1000 s as the force reaches 30 pN, leading to mechanical stabilization of the adhesion. The dissociation transition pathway is determined as the unbinding of a critical ß-strand peptide ("latch" strand of SdrG that secures the entire adhesion complex) away from its binding cleft, leading to the dissociation of the Fgß ligand. Similar mechanical stabilization behavior is also observed in several homologous adhesions, suggesting the generality of catch-bond kinetics in such bacterial adhesions. We reason that such mechanical stabilization confers multiple advantages in the pathogenesis and adaptation of bacteria.

Allantodapsone is a Pan-Inhibitor of Staphylococcus aureus Adhesion to Fibrinogen, Loricrin, and Cytokeratin 10.

Staphylococcus aureus infections have become a major challenge in health care due to increasing antibiotic resistance. We aimed to design small molecule inhibitors of S. aureus surface proteins to be developed as colonization inhibitors. We identified allantodapsone in an initial screen searching for inhibitors of clumping factors A and B (ClfA and ClfB). We used microbial adhesion assays to investigate the effect of allantodapsone on extracellular matrix protein interactions. Allantodapsone inhibited S. aureus Newman adhesion to fibrinogen with an IC50 of 21.3 µM (95% CI 4.5-102 µM), minimum adhesion inhibitory concentration (MAIC) of 100 µM (40.2 µg/mL). Additionally, allantodapsone inhibited adhesion of Lactococcus lactis strains exogenously expressing the clumping factors to fibrinogen (L. lactis ClfA, IC50 of 3.8 µM [95% CI 1.0-14.3 µM], MAIC 10 µM, 4.0 µg/mL; and L. lactis ClfB, IC50 of 11.0 µM [95% CI 0.9-13.6 µM], MAIC 33 µM, 13.3 µg/mL), indicating specific inhibition. Furthermore, the dapsone and alloxan fragments of allantodapsone did not have any inhibitory effect. Adhesion of S. aureus Newman to L2v loricrin is dependent on the expression of ClfB. Allantodapsone caused a dose dependent inhibition of S. aureus adhesion to the L2v loricrin fragment, with full inhibition at 40 µM (OD600 0.11 ± 0.01). Furthermore, recombinant ClfB protein binding to L2v loricrin was inhibited by allantodapsone (P < 0.0001). Allantodapsone also demonstrated dose dependent inhibition of S. aureus Newman adhesion to cytokeratin 10 (CK10). Allantodapsone is the first small molecule inhibitor of the S. aureus clumping factors with potential for development as a colonization inhibitor. IMPORTANCE S. aureus colonization of the nares and the skin provide a reservoir of bacteria that can be transferred to wounds that can ultimately result in systemic infections. Antibiotic resistance can make these infections difficult to treat with significant associated morbidity and mortality. We have identified and characterized a first-in-class small molecule inhibitor of the S. aureus clumping factors A and B, which has the potential to be developed further as a colonization inhibitor.