In Vitro Staphylococcal Aggregate Morphology and Protection from Antibiotics Are Dependent on Distinct Mechanisms Arising from Postsurgical Joint Components and Fluid Motion.

Considerable progress has been made toward elucidating the mechanism of Staphylococcus aureus aggregation in synovial fluid. In this study, aggregate morphology was assessed following incubation under several simulated postsurgical joint conditions. Using fluorescently labeled synovial fluid polymers, we show that aggregation occurs through two distinct mechanisms: (i) direct bridging between S. aureus cells and host fibrinogen and (ii) an entropy-driven depletion mechanism facilitated by hyaluronic acid and albumin. By screening surface adhesin-deficient mutants (clfA, clfB, fnbB, and fnbA), we identified the primary genetic determinant of aggregation in synovial fluid to be clumping factor A. To characterize this bridging interaction, we employed an atomic force microscopy-based approach to quantify the binding affinity of either wild-type S. aureus or the adhesin mutant to immobilized fibrinogen. Surprisingly, we found there to be cell-to-cell variability in the binding strength of the bacteria for immobilized fibrinogen. Superhigh-resolution microscopy imaging revealed that fibrinogen binding to the cell wall is heterogeneously distributed at both the single cell and population levels. Finally, we assessed the antibiotic tolerance of various aggregate morphologies arising from newly deciphered mechanisms of polymer-mediated synovial fluid-induced aggregation. The formation of macroscopic aggregates under shear was highly tolerant of gentamicin, while smaller aggregates, formed under static conditions, were susceptible. We hypothesize that aggregate formation in the joint cavity, in combination with shear, is mediated by both polymer-mediated aggregation mechanisms, with depletion forces enhancing the stability of essential bridging interactions. IMPORTANCE The formation of a bacterial biofilm in the postsurgical joint environment significantly complicates the resolution of an infection. To form a resilient biofilm, incoming bacteria must first survive the initial invasion of the joint space. We previously found that synovial fluid induces the formation of Staphylococcus aureus aggregates, which may provide rapid protection during the early stages of infection. The state of the host joint environment, including the presence of fluid flow and fluctuating abundance of synovial fluid polymers, determines the rate and size of aggregate formation. By expanding on our knowledge of the mechanism and pathogenic implications of synovial fluid-induced aggregation, we hope to contribute insights for the development of novel methods of prevention and therapeutic intervention.

Host blood proteins as bridging ligand in bacterial aggregation as well as anchor point for adhesion in the molecular pathogenesis of Staphylococcus aureus infections.

Fibronectin (Fn) and fibrinogen (Fg) are major host proteins present in the extracellular matrix, blood, and coatings on indwelling medical devices. The ability of Staphylococcus aureus to cause infections in humans depends on favorable interactions with these host ligands. Closely related bacterial adhesins, fibronectin-binding proteins A and B (FnBPA, FnBPB) were evaluated for two key steps in pathogenesis: clumping and adhesion. Experiments utilized optical spectrophotometry, flow cytometry, and atomic force microscopy to probe FnBPA/B alone or in combination in seven different strains of S. aureus and Lactococcus lactis, a Gram-positive surrogate that naturally lacks adhesins to mammalian ligands. In the absence of soluble ligands, both FnBPA and FnBPB were capable of interacting with adjacent FnBPs from neighboring bacteria to mediate clumping. In the presence of soluble host ligands, clumping was enhanced particularly under shear stress and with Fn present in the media. FnBPB exhibited greater ability to clump compared to FnBPA. The strength of adhesion was similar for immobilized Fn to FnBPA and FnBPB. These findings suggest that these two distinct but closely related bacterial adhesins, have different functional capabilities to interact with host ligands in different settings (e.g., soluble vs. immobilized). Survival and persistence of S. aureus in a human host may depend on complementary roles of FnBPA and FnBPB as they interact with different conformations of Fn or Fg (compact in solution vs. extended on a surface) present in different physiological spaces.

A meta-analysis framework to assess the role of units in describing nanoparticle toxicity.

Despite ample research on nanoparticles, their environmental toxicity is still debatable. The lack of consensus is due in part to the challenge of comparing studies because of variability in parameters like test organism, test medium, and duration of experiment. However, the unit used to compare the toxicology of nanoparticles is one variable that experimentalists can control. Traditionally, mass per volume is the most common unit used to make comparisons, but there is growing evidence that alternative units such as surface area per volume or particles per volume may provide a better and more mechanistic measure of toxicity. Herein, we propose and test a meta-analytic framework to study the effect of units on nanotoxicology using data from the NanoE-Tox database, a freely available database containing 1518 toxicology values from 224 published articles of which 42 records met our basic inclusion criteria. These data were augmented with more recent data published over the past five years as archived by the Web of Science citation index. An additional 27 records from 1676 papers met the inclusion criteria and were also included in the analysis. The meta-analysis framework measures the degree of heterogeneity for each of three units (grams/L, particles/L, surface area/L) grouped by the type of test organism, particle chemistry, and manner in which a nanoparticle's size was measured (e.g., nominal particle size reported by the manufacturer vs. measurement of size for particles suspended in the liquid medium used in a subsequent toxicity experiment). The result of the meta-analysis reveals that surface area per volume reduces the heterogeneity in the Ag crustacean subgroup when nanoparticle size was measured in the test medium, and the ZnO crustacean subgroup when nanoparticle size was measured out the test medium and may therefore be a more appropriate estimate of the toxicity of soluble nanoparticles. No subgroups in our analysis showed a reduction in heterogeneity for particles per volume in either soluble or insoluble nanoparticles. The lack of conclusion on insoluble nanoparticles was not due to a limitation of our meta-analysis but rather highlights a critical deficiency in the primary literature. The majority of published studies fail to report common measures of error that are essential for further analysis (i.e. error of the measured nanoparticle size and/or interoperable error of the measured half-maximal concentration of the toxic endpoint). If future nanotoxicity studies report such error, as they should, then the framework of our meta-analysis could be used more broadly to provide a simple, statistically rigorous way to assess the role of units on the toxicity of nanoparticles.

Thrust and Power Output of the Bacterial Flagellar Motor: A Micromagnetic Tweezers Approach.

One of the most common swimming strategies employed by microorganisms is based on the use of rotating helical filaments, called flagella, that are powered by molecular motors. Determining the physical properties of this propulsive system is crucial to understanding the behavior of these organisms. Furthermore, the ability to dynamically monitor the activity of the flagellar motor is a valuable indicator of the overall energetics of the cell. In this work, inherently magnetic bacteria confined in micromagnetic CoFe traps are used to directly and noninvasively determine the flagellar thrust force and swimming speed of motile cells. The technique permits determination of the ratio of propulsive force/swimming speed (the hydrodynamic resistance) and the power output of the flagellar motor for individual cells over extended time periods. Cells subjected to ultraviolet radiation are observed to experience exponential decays in power output as a function of exposure time. By noninvasively measuring thrust, velocity, and power output over time at a single-cell level, this technique can serve as the foundation for fundamental studies of bacterial hydrodynamics and also provides a novel, to our knowledge, tether-free probe of single-cell energetics over time.

Fibrinogen binding is affected by amino acid substitutions in C-terminal repeat region of fibronectin binding protein A.

Fibronectin-binding protein A (FnBPA), a protein displayed on the outer surface of Staphylococcus aureus, has a structured A-domain that binds fibrinogen (Fg) and a disordered repeat-region that binds fibronectin (Fn). Amino acid substitutions in Fn-binding repeats (FnBRs) have previously been linked to cardiovascular infection in humans. Here we used microtiter and atomic force microscopy (AFM) to investigate adhesion by variants of full-length FnBPA covalently anchored in the outer cell wall of Lactococcus lactis, a Gram-positive surrogate that otherwise lacks adhesins to mammalian ligands. Fn adhesion increased in five of seven FnBPA variants under static conditions. The bond targeting Fn increased its strength with load under mechanical dissociation. Substitutions extended bond lifetime (1/koff) up to 2.1 times for FnBPA-Fn. Weaker adhesion was observed for Fg in all FnBPA variants tested with microtiter. However, mechanical dissociation with AFM showed significantly increased tensile strength for Fg interacting with the E652D/H782Q variant. This is consistent with a force-induced mechanism and suggests that the dock, lock, and latch (DLL) mechanism is favored for Fg-binding under mechanical stress. Collectively, these experiments reveal that FnBPA exhibits bimodal, ligand-dependent adhesive behavior. Amino acid substitutions in the repeat-region of FnBPA impact binding to both ligands. This was unexpected for Fg since all variants have the same A-domain sequence, and the Fg-binding site is distant from the repeat region. This indicates that FnBRs may fold back on the A-domain in a way that impacts the DLL binding mechanism for Fg.

Amino acid polymorphisms in the fibronectin-binding repeats of fibronectin-binding protein A affect bond strength and fibronectin conformation.

The Staphylococcus aureus cell surface contains cell wall-anchored proteins such as fibronectin-binding protein A (FnBPA) that bind to host ligands (e.g. fibronectin; Fn) present in the extracellular matrix of tissue or coatings on cardiac implants. Recent clinical studies have found a correlation between cardiovascular infections caused by S. aureus and nonsynonymous SNPs in FnBPA. Atomic force microscopy (AFM), surface plasmon resonance (SPR), and molecular simulations were used to investigate interactions between Fn and each of eight 20-mer peptide variants containing amino acids Ala, Asn, Gln, His, Ile, and Lys at positions equivalent to 782 and/or 786 in Fn-binding repeat-9 of FnBPA. Experimentally measured bond lifetimes (1/koff) and dissociation constants (Kd = koff/kon), determined by mechanically dissociating the Fn·peptide complex at loading rates relevant to the cardiovascular system, varied from the lowest-affinity H782A/K786A peptide (0.011 s, 747 µm) to the highest-affinity H782Q/K786N peptide (0.192 s, 15.7 µm). These atomic force microscopy results tracked remarkably well to metadynamics simulations in which peptide detachment was defined solely by the free-energy landscape. Simulations and SPR experiments suggested that an Fn conformational change may enhance the stability of the binding complex for peptides with K786I or H782Q/K786I (Kdapp = 0.2-0.5 µm, as determined by SPR) compared with the lowest-affinity double-alanine peptide (Kdapp = 3.8 µm). Together, these findings demonstrate that amino acid substitutions in Fn-binding repeat-9 can significantly affect bond strength and influence the conformation of Fn upon binding. They provide a mechanistic explanation for the observation of nonsynonymous SNPs in fnbA among clinical isolates of S. aureus that cause endovascular infections.

Conformational stability of the epidermal growth factor (EGF) receptor as influenced by glycosylation, dimerization and EGF hormone binding.

The epidermal growth factor receptor (EGFR) is an important transmembrane glycoprotein kinase involved the initiation or perpetuation of signal transduction cascades within cells. These processes occur after EGFR binds to a ligand [epidermal growth factor (EGF)], thus inducing its dimerization and tyrosine autophosphorylation. Previous publications have highlighted the importance of glycosylation and dimerization for promoting proper function of the receptor and conformation in membranes; however, the effects of these associations on the protein conformational stability have not yet been described. Molecular dynamics simulations were performed to characterize the conformational preferences of the monomeric and dimeric forms of the EGFR extracellular domain upon binding to EGF in the presence and absence of N-glycan moieties. Structural stability analyses revealed that EGF provides the most conformational stability to EGFR, followed by glycosylation and dimerization, respectively. The findings also support that EGF-EGFR binding takes place through a large-scale induced-fitting mechanism. Proteins 2017; 85:561-570. © 2016 Wiley Periodicals, Inc.

Polymorphisms in Fibronectin Binding Proteins A and B among Staphylococcus aureus Bloodstream Isolates Are Not Associated with Arthroplasty Infection.

BACKGROUND: Nonsynonymous single nucleotide polymorphisms (SNPs) in fibronectin binding protein A (fnbA) of Staphylococcus aureus are associated with cardiac device infections. However, the role of fnbA SNPs in S. aureus arthroplasty infection is unknown. METHODS: Bloodstream S. aureus isolates from a derivation cohort of patients at a single U.S. medical center with S. aureus bacteremia (SAB) and prosthetic hip or knee arthroplasties that were infected (PJI, n = 27) or uninfected (PJU, n = 43) underwent sequencing of fnbA and fnbB. A validation cohort of S. aureus bloodstream PJI (n = 12) and PJU (n = 58) isolates from Germany also underwent fnbA and fnbB sequencing. RESULTS: Overall, none of the individual fnbA or fnbB SNPs were significantly associated with the PJI or PJU clinical groups within the derivation cohort. Similarly, none of the individual fnbA or fnbB SNPs were associated with PJI or PJU when the analysis was restricted to patients with either early SAB (i.e., bacteremia occurring <1 year after placement or manipulation of prostheses) or late SAB (i.e., bacteremia >1 year after placement or manipulation of prostheses). CONCLUSIONS: In contrast to cardiac device infections, there is no association between nonsynonymous SNPs in fnbA or fnbB of bloodstream S. aureus isolates and arthroplasty infection. These results suggest that initial steps leading to S. aureus infection of cardiovascular and orthopedic prostheses may arise by distinct processes.

Endovascular infections caused by methicillin-resistant Staphylococcus aureus are linked to clonal complex-specific alterations in binding and invasion domains of fibronectin-binding protein A as well as the occurrence of fnbB.

Endovascular infections caused by Staphylococcus aureus involve interactions with fibronectin present as extracellular matrix or surface ligand on host cells. We examined the expression, structure, and binding activity of the two major S. aureus fibronectin-binding proteins (FnBPA, FnBPB) in 10 distinct, methicillin-resistant clinical isolates from patients with either persistent or resolving bacteremia. The persistent bacteremia isolates (n = 5) formed significantly stronger bonds with immobilized fibronectin as determined by dynamic binding measurements performed with atomic force microscopy. Several notable differences were also observed when the results were grouped by clonal complex 5 (CC5) strains (n = 5) versus CC45 strains (n = 5). Fibronectin-binding receptors on CC5 formed stronger bonds with immobilized fibronectin (P < 0.001). The fnbA gene was expressed at higher levels in CC45, whereas fnbB was found in only CC5 isolates. The fnbB gene was not sequenced because all CC45 isolates lacked this gene. Instead, comparisons were made for fnbA, which was present in all 10 isolates. Sequencing of fnbA revealed discrete differences within high-affinity, fibronectin-binding repeats (FnBRs) of FnBPA that included (i) 5-amino-acid polymorphisms in FnBR-9, FnBR-10, and FnBR-11 involving charged or polar side chains, (ii) an extra, 38-amino-acid repeat inserted between FnBR-9 and FnBR-10 exclusively seen in CC45 isolates, and (iii) CC5 isolates had the SVDFEED epitope in FnBR-11 (a sequence shown to be essential for fibronectin binding), while this sequence was replaced in all CC45 isolates with GIDFVED (a motif known to favor host cell invasion at the cost of reduced fibronectin binding). These complementary sequence and binding data suggest that differences in fnbA and fnbB, particularly polymorphisms and duplications in FnBPA, give S. aureus two distinct advantages in human endovascular infections: (i) FnBPs similar to that of CC5 enhance ligand binding and foster initiation of disease, and (ii) CC45-like FnBPs promote cell invasion, a key attribute in persistent endovascular infections.

Subcellular localization of the magnetosome protein MamC in the marine magnetotactic bacterium Magnetococcus marinus strain MC-1 using immunoelectron microscopy.

Magnetotactic bacteria are a diverse group of prokaryotes that biomineralize intracellular magnetosomes, composed of magnetic (Fe3O4) crystals each enveloped by a lipid bilayer membrane that contains proteins not found in other parts of the cell. Although partial roles of some of these magnetosome proteins have been determined, the roles of most have not been completely elucidated, particularly in how they regulate the biomineralization process. While studies on the localization of these proteins have been focused solely on Magnetospirillum species, the goal of the present study was to determine, for the first time, the localization of the most abundant putative magnetosome membrane protein, MamC, in Magnetococcus marinus strain MC-1. MamC was expressed in Escherichia coli and purified. Monoclonal antibodies were produced against MamC and immunogold labeling TEM was used to localize MamC in thin sections of cells of M. marinus. Results show that MamC is located only in the magnetosome membrane of Mc. marinus. Based on our findings and the abundance of this protein, it seems likely that it is important in magnetosome biomineralization and might be used in controlling the characteristics of synthetic nanomagnetite.

Repetitive dissociation from crocidolite asbestos acts as persistent signal for epidermal growth factor receptor.

Mesothelioma is an incurable form of cancer located most commonly in the pleural lining of the lungs and is associated almost exclusively with the inhalation of asbestos. The binding of asbestos to epidermal growth factor receptor (EGFR), a transmembrane signal protein, has been proposed as a trigger for downstream signaling of kinases and expression of genes involved in cell proliferation and inhibition of apoptosis. Here, we investigate the molecular binding of EGFR to crocidolite (blue asbestos; Na2(Fe(2+),Mg)3Fe2(3+)Si8O22(OH)2) in buffer solution. Atomic force microscopy measurements revealed an attractive force of interaction (i.e., bond) as EGFR was pulled from contact with long fibers of crocidolite. The rupture force of this bond increased with loading rate. According to the Bell model, the off-rate of bond dissociation (k(off)) for EGFR was 22 s(-1). Similar experiments with riebeckite crystals, the nonasbestiform variety of crocidolite, yielded a k(off) of 8 s(-1). These k(off) values on crocidolite and riebeckite are very rapid compared to published values for natural agonists of EGFR like transforming growth factor and epidermal growth factor. This suggests binding of EGFR to the surfaces of these minerals could elicit a response that is more potent than biological hormone or cytokine ligands. Signal transduction may cease for endogenous ligands due to endocytosis and subsequent degradation, and even riebeckite particles can be cleared from the lungs due to their short, equant habit. However, the fibrous habit of crocidolite leads to lifelong persistence in the lungs where aberrant, repetitious binding with EGFR may continually trigger the activation switch leading to chronic expression of genes involved in oncogenesis.

Magnetotactic bacteria from Pavilion Lake, British Columbia.

Pavilion Lake is a slightly alkaline, freshwater lake located in British Columbia, Canada (50°51'N, 121°44'W). It is known for unusual organosedimentary structures, called microbialites that are found along the lake basin. These deposits are complex associations of fossilized microbial communities and detrital- or chemical-sedimentary rocks. During the summer, a sediment sample was collected from near the lake's shore, approximately 25-50 cm below the water surface. Magnetotactic bacteria (MTB) were isolated from this sample using a simple magnetic enrichment protocol. The MTB isolated from Pavilion Lake belonged to the Alphaproteobacteria class as determined by nucleotide sequences of 16S rRNA genes. Transmission electron microscopy (TEM) revealed that the bacteria were spirillum-shaped and contained a single chain of cuboctahedral-shaped magnetite (Fe3O4) crystals that were approximately 40 nm in diameter. This discovery of MTB in Pavilion Lake offers an opportunity to better understand the diversity of MTB habitats, the geobiological function of MTB in unique freshwater ecosystems, and search for magnetofossils contained within the lake's microbialites.

Collection, isolation and enrichment of naturally occurring magnetotactic bacteria from the environment.

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 1975 from sediment samples collected in salt marshes of Massachusetts (USA). Since then MTB have been discovered in stratified water- and sediment-columns from all over the world. One feature common to all MTB is that they contain magnetosomes, which are intracellular, membrane-bound magnetic nanocrystals of magnetite (Fe3O4) and/or greigite (Fe3S4) or both. In the Northern hemisphere, MTB are typically attracted to the south end of a bar magnet, while in the Southern hemisphere they are usually attracted to the north end of a magnet. This property can be exploited when trying to isolate MTB from environmental samples. One of the most common ways to enrich MTB is to use a clear plastic container to collect sediment and water from a natural source, such as a freshwater pond. In the Northern hemisphere, the south end of a bar magnet is placed against the outside of the container just above the sediment at the sediment-water interface. After some time, the bacteria can be removed from the inside of the container near the magnet with a pipette and then enriched further by using a capillary racetrack and a magnet. Once enriched, the bacteria can be placed on a microscope slide using a hanging drop method and observed in a light microscope or deposited onto a copper grid and observed using transmission electron microscopy (TEM). Using this method, isolated MTB may be studied microscopically to determine characteristics such as swimming behavior, type and number of flagella, cell morphology of the cells, shape of the magnetic crystals, number of magnetosomes, number of magnetosome chains in each cell, composition of the nanomineral crystals, and presence of intracellular vacuoles.

Magnetosomes and magnetite crystals produced by magnetotactic bacteria as resolved by atomic force microscopy and transmission electron microscopy.

Atomic force microscopy (AFM) was used in concert with transmission electron microscopy (TEM) to image magnetotactic bacteria (Magnetospirillum gryphiswaldense MSR-1 and Magnetospirillum magneticum AMB-1), magnetosomes, and purified Mms6 proteins. Mms6 is a protein that is associated with magnetosomes in M. magneticum AMB-1 and is believed to control the synthesis of magnetite (Fe(3)O(4)) within the magnetosome. We demonstrated how AFM can be used to capture high-resolution images of live bacteria and achieved nanometer resolution when imaging Mms6 protein molecules on magnetite. We used AFM to acquire simultaneous topography and amplitude images of cells that were combined to provide a three-dimensional reconstructed image of M. gryphiswaldense MSR-1. TEM was used in combination with AFM to image M. gryphiswaldense MSR-1 and magnetite-containing magnetosomes that were isolated from the bacteria. AFM provided information, such as size, location and morphology, which was complementary to the TEM images.

Dissociation rate constants of human fibronectin binding to fibronectin-binding proteins on living Staphylococcus aureus isolated from clinical patients.

Staphylococcus aureus is part of the indigenous microbiota of humans. Sometimes, S. aureus bacteria enter the bloodstream, where they form infections on implanted cardiovascular devices. A critical, first step in such infections is a bond that forms between fibronectin-binding protein (FnBP) on S. aureus and host proteins, such as fibronectin (Fn), that coat the surface of implants in vivo. In this study, native FnBPs on living S. aureus were shown to form a mechanically strong conformational structure with Fn by atomic force microscopy. The tensile acuity of this bond was probed for 46 bloodstream isolates, each from a patient with a cardiovascular implant. By analyzing the force spectra with the worm-like chain model, we determined that the binding events were consistent with a multivalent, cluster bond consisting of ~10 or ~80 proteins in parallel. The dissociation rate constant (k(off), s(-1)) of each multibond complex was determined by measuring strength as a function of the loading rate, normalized by the number of bonds. The bond lifetime (1/k(off)) was two times longer for bloodstream isolates from patients with an infected device (1.79 or 69.47 s for the 10- or 80-bond clusters, respectively; n = 26 isolates) relative to those from patients with an uninfected device (0.96 or 34.02 s; n = 20 isolates). This distinction could not be explained by different amounts of FnBP, as confirmed by Western blots. Rather, amino acid polymorphisms within the Fn-binding repeats of FnBPA explain, at least partially, the statistically (p < 0.05) longer bond lifetime for isolates associated with an infected cardiovascular device.

Polymorphisms in fibronectin binding protein A of Staphylococcus aureus are associated with infection of cardiovascular devices.

Medical implants, like cardiovascular devices, improve the quality of life for countless individuals but may become infected with bacteria like Staphylococcus aureus. Such infections take the form of a biofilm, a structured community of bacterial cells adherent to the surface of a solid substrate. Every biofilm begins with an attractive force or bond between bacterium and substratum. We used atomic force microscopy to probe experimentally forces between a fibronectin-coated surface (i.e., proxy for an implanted cardiac device) and fibronectin-binding receptors on the surface of individual living bacteria from each of 80 clinical isolates of S. aureus. These isolates originated from humans with infected cardiac devices (CDI; n = 26), uninfected cardiac devices (n = 20), and the anterior nares of asymptomatic subjects (n = 34). CDI isolates exhibited a distinct binding-force signature and had specific single amino acid polymorphisms in fibronectin-binding protein A corresponding to E652D, H782Q, and K786N. In silico molecular dynamics simulations demonstrate that residues D652, Q782, and N786 in fibronectin-binding protein A form extra hydrogen bonds with fibronectin, complementing the higher binding force and energy measured by atomic force microscopy for the CDI isolates. This study is significant, because it links pathogenic bacteria biofilms from the length scale of bonds acting across a nanometer-scale space to the clinical presentation of disease at the human dimension.

Atomic force microscopy to study intermolecular forces and bonds associated with bacteria.

Atomic force microscopy (AFM) operates on a very different principle than other forms of microscopy, such as optical microscopy or electron microscopy. The key component of an AFM is a cantilever that bends in response to forces that it experiences as it touches another surface. Forces as small as a few picoNewtons can be detected and probed with AFM. AFM has become very useful in biological sciences because it can be used on living cells that are immersed in water. AFM is particularly useful when the cantilever is modified with chemical groups (e.g. amine or carboxylic groups), small beads (e.g. glass or latex), or even a bacterium. This chapter describes how AFM can be used to measure forces and bonds between a bacterium and another surface. This paper also provides an example of the use of AFM on Staphylococcus aureus, a Gram-positive bacterium that is often associated with biofilms in humans.

A tactile response in Staphylococcus aureus.

It is well established that bacteria are able to respond to temporal gradients (e.g., by chemotaxis). However, it is widely held that prokaryotes are too small to sense spatial gradients. This contradicts the common observation that the vast majority of bacteria live on the surface of a solid substrate (e.g., as a biofilm). Herein we report direct experimental evidence that the nonmotile bacterium Staphylococcus aureus possesses a tactile response, or primitive sense of touch, that allows it to respond to spatial gradients. Attached cells recognize their substrate interface and localize adhesins toward that region. Braille-like avidity maps reflect a cell's biochemical sensory response and reveal ultrastructural regions defined by the actual binding activity of specific proteins.

Bonds between fibronectin and fibronectin-binding proteins on Staphylococcus aureus and Lactococcus lactis.

Bacterial cell-wall-associated fibronectin binding proteins A and B (FnBPA and FnBPB) form bonds with host fibronectin. This binding reaction is often the initial step in prosthetic device infections. Atomic force microscopy was used to evaluate binding interactions between a fibronectin-coated probe and laboratory-derived Staphylococcus aureus that are (i) defective in both FnBPA and FnBPB (fnbA fnbB double mutant, DU5883), (ii) capable of expressing only FnBPA (fnbA fnbB double mutant complemented with pFNBA4), or (iii) capable of expressing only FnBPB (fnbA fnbB double mutant complemented with pFNBB4). These experiments were repeated using Lactococcus lactis constructs expressing fnbA and fnbB genes from S. aureus. A distinct force signature was observed for those bacteria that expressed FnBPA or FnBPB. Analysis of this force signature with the biomechanical wormlike chain model suggests that parallel bonds form between fibronectin and FnBPs on a bacterium. The strength and covalence of bonds were evaluated via nonlinear regression of force profiles. Binding events were more frequent (p < 0.01) for S. aureus expressing FnBPA or FnBPB than for the S. aureus double mutant. The binding force, frequency, and profile were similar between the FnBPA and FnBPB expressing strains of S. aureus. The absence of both FnBPs from the surface of S. aureus removed its ability to form a detectable bond with fibronectin. By contrast, ectopic expression of FnBPA or FnBPB on the surface of L. lactis conferred fibronectin binding characteristics similar to those of S. aureus. These measurements demonstrate that fibronectin-binding adhesins FnBPA and FnBPB are necessary and sufficient for the binding of S. aureus to prosthetic devices that are coated with host fibronectin.

Oxide-dependent adhesion of the Jurkat line of T lymphocytes.

The adhesion force of Jurkat cells was measured using atomic force microscopy (AFM) in aqueous solution at pH 7.2 on six metal oxide surfaces, namely, two quartz (alpha-SiO2) crystal faces, amorphous SiO2 glass, rutile (alpha-TiO2), muscovite mica (KAl2(AlSi3O10)(OH)2), and polycrystalline corundum (alpha-Al2O3). We show quantitatively for the first time that the T lymphocyte adhesion force and adhesion work correlates with substrate point of zero charge, indicating greater adsorption on surfaces with smaller negative charge. Adhesion events also exhibited sawtooth-shaped force-distance profiles indicative of protein bonds. No significant correlations were found with oxide Hamaker constants, indicating negligible contributions from van der Waals forces, nor with surface roughness. These results suggest that, when cell-surface receptors are not activated, Jurkat cell adhesion is dominated by specific interactions related to the unfolding of modular glycoproteins or other proteins that are not unique to T-cell surfaces and by electrostatic forces between negatively charged glycoproteins and variably charged oxide surfaces. Our results have implications for the interactions of immune system cells with metal oxides present in the human body either by design as in biomedical applications or inadvertently such as inhaled mineral dust particles in the lung.