Yoo Jin Oh

Curli mediate bacterial adhesion to fibronectin via tensile multiple bonds

Many enteric bacteria including pathogenic Escherichia coli and Salmonella strains produce curli fibers that bind to host surfaces, leading to bacterial internalization into host cells. By using a nanomechanical force-sensing approach, we obtained real-time information about the distribution of molecular bonds involved in the adhesion of curliated bacteria to fibronectin. We found that curliated E. coli and fibronectin formed dense quantized and multiple specific bonds with high tensile strength, resulting in tight bacterial binding. Nanomechanical recognition measurements revealed that approximately 10 bonds were disrupted either sequentially or simultaneously under force load. Thus the curli formation of bacterial surfaces leads to multi-bond structural components of fibrous nature, which may explain the strong mechanical binding of curliated bacteria to host cells and unveil the functions of these proteins in bacterial internalization and invasion.

Characterizing the S-layer structure and anti-S-layer antibody recognition on intact Tannerella forsythia cells by scanning probe microscopy and small angle X-ray scattering

Tannerella forsythia is among the most potent triggers of periodontal diseases, and approaches to understand underlying mechanisms are currently intensively pursued. A ~22‐nm‐thick, 2D crystalline surface (S‐) layer that completely covers Tannerella forsythia cells is crucially involved in the bacterium–host cross‐talk. The S‐layer is composed of two intercalating glycoproteins (TfsA‐GP, TfsB‐GP) that are aligned into a periodic lattice. To characterize this unique S‐layer structure at the nanometer scale directly on intact T. forsythia cells, three complementary methods, i.e., small‐angle X‐ray scattering (SAXS), atomic force microscopy (AFM), and single‐molecular force spectroscopy (SMFS), were applied. SAXS served as a difference method using signals from wild‐type and S‐layer‐deficient cells for data evaluation, revealing two possible models for the assembly of the glycoproteins. Direct high‐resolution imaging of the outer surface of T. forsythia wild‐type cells by AFM revealed a p4 structure with a lattice constant of ~9.0 nm. In contrast, on mutant cells, no periodic lattice could be visualized. Additionally, SMFS was used to probe specific interaction forces between an anti‐TfsA antibody coupled to the AFM tip and the S‐layer as present on T. forsythia wild‐type and mutant cells, displaying TfsA‐GP alone. Unbinding forces between the antibody and wild‐type cells were greater than with mutant cells. This indicated that the TfsA‐GP is not so strongly attached to the mutant cell surface when the co‐assembling TfsB‐GP is missing. Altogether, the data gained from SAXS, AFM, and SMFS confirm the current model of the S‐layer architecture with two intercalating S‐layer glycoproteins and TfsA‐GP being mainly outwardly oriented. Copyright

Nanoscale characteristics of antibacterial cationic polymeric brushes and single bacterium interactions probed by force microscopy

Cationic brushes are powerful molecules for antibacterial purposes with permanent activity and mobility. Therefore, clarifying the characteristics of the brush structure and related interactions with microbial cells becomes important. In this study, two different molecular weight (Mn: 1800 and 60 000) polyethyleneimine (PEI) molecules were grafted on a polyurethane (PU) surface. Moreover, an alkylation step was performed to enhance the antibacterial efficiency by increasing the polycationic character of the PEI brushes. The surface potential of the PEI brushes was raised with an additional alkylation step. Nanoscale characterization of the PEI brushes on PU surfaces was examined in terms of topography, roughness, and surface potentials by Kelvin Probe Force Microscopy (KPFM). The nanomechanical behaviour of the PEI brushes was investigated in Milli-Q water and phosphate buffer saline by Atomic Force Microscopy (AFM). The nanomechanical properties were directly affected by the incubation medium and surface properties. Additionally, polymeric brush–single bacterium interactions were probed by Single Cell Force Spectroscopy (SCFS). The antibacterial activity of the PEI brushes was probed at a single bacterium level with the Escherichia coli K-12 bacteria species. As a result, rupture forces between E. coli K-12 and the alkylated PEI grafted surface were calculated to identify the bacterial adhesion process at piconewton force sensitivity. It was found that, alkylated high molecular weight PEI brushes show the lowest rupture force (56.1 pN) with the lowest binding percentage (%5) against single bacterial cells. This study indicated that, increasing the chain length, charge (alkyl groups) and mobility of PEI brushes decreases the binding percentage and force at a single bacterium level, synergistically.

Characterizing the effect of polymyxin B antibiotics to lipopolysaccharide on Escherichia coli surface using atomic force microscopy

Lipopolysaccharide (LPS) on gram‐negative bacterial outer membranes is the first target for antimicrobial agents, due to their spatial proximity to outer environments of microorganisms. To develop antibacterial compounds with high specificity for LPS binding, the understanding of the molecular nature and their mode of recognition is of key importance. In this study, atomic force microscopy (AFM) and single molecular force spectroscopy were used to characterize the effects of antibiotic polymyxin B (PMB) to the bacterial membrane at the nanoscale. Isolated LPS layer and the intact bacterial membrane were examined with respect to morphological changes at different concentrations of PMB. Our results revealed that 3 hours of 10 μg/mL of PMB exposure caused the highest roughness changes on intact bacterial surfaces, arising from the direct binding of PMB to LPS on the bacterial membrane. Single molecular force spectroscopy was used to probe specific interaction forces between the isolated LPS layer and PMB coupled to the AFM tip. A short range interaction regime mediated by electrostatic forces was visible. Unbinding forces between isolated LPS and PMB were about 30 pN at a retraction velocity of 500 nm/s. We further investigated the effects of the polycationic peptide PMB on bacterial outer membranes and monitored its influences on the deterioration of the bacterial membrane structure. Polymyxin B binding led to rougher appearances and wrinkles on the outer membranes surface, which may finally lead to lethal membrane damage of bacteria. Our studies indicate the potential of AFM for applications in pathogen recognition and nano‐resolution approaches in microbiology.

Lipoteichoic acid mediates binding of a Lactobacillus S-layer protein

Abstract The Gram-positive lactic acid bacterium Lactobacillus buchneri CD034 is covered by a two-dimensional crystalline, glycoproteinaceous cell surface (S-) layer lattice. While lactobacilli are extensively exploited as cell surface display systems for applied purposes, questions about how they stick their cell wall together are remaining open. This also includes the identification of the S-layer cell wall ligand. In this study, lipoteichoic acid was isolated from the L. buchneri CD034 cell wall as a significant fraction of the bacterium’s cell wall glycopolymers, structurally characterized and analyzed for its potential to mediate binding of the S-layer to the cell wall. Combined component analyses and 1D- and 2D-nuclear magnetic resonance spectroscopy (NMR) revealed the lipoteichoic acid to be composed of on average 31 glycerol-phosphate repeating units partially substituted with α-d-glucose, and with an α-d-Galp(1→2)-α-d-Glcp(1→3)−1,2-diacyl-sn-Gro glycolipid anchor. The specificity of binding between the L. buchneri CD034 S-layer protein and purified lipoteichoic acid as well as their interaction force of about 45 pN were obtained by single-molecule force spectroscopy; this value is in the range of typical ligand–receptor interactions. This study sheds light on a functional implication of Lactobacillus cell wall architecture by showing direct binding between lipoteichoic acid and the S-layer of L. buchneri CD034.