Interactions between the Trimeric Autotransporter Adhesin EmaA and Collagen Revealed by Three-Dimensional Electron Tomography.

Bacterial adhesion to host tissues is considered the first and critical step of microbial infection. The extracellular matrix protein adhesin A (EmaA) is a collagen-binding adhesin of the periodontal pathogen Aggregatibacter actinomycetemcomitans Three 202-kDa EmaA monomers form antenna-like structures on the bacterial surface with the functional domain located at the apical end. The structure of the 30-nm functional domain has been determined by three-dimensional (3D) electron tomography and subvolume averaging. The region exhibits a complex architecture composed of three subdomains (SI to SIII) and a linker between subdomains SII and SIII. However, the molecular interaction between the adhesin receptor complexes has yet to be revealed. This study provides the first detailed 3D structure of reconstituted EmaA/collagen complexes obtained using 3D electron tomography and image processing techniques. The observed interactions of EmaA with collagen were not to whole, intact fibrils, but rather to individual collagen triple helices dissociated from the fibrils. The majority of the contacts with the EmaA functional domain encompassed subdomains SII and SIII and in some cases the tip of the apical domain, involving SI. These data suggest a multipronged mechanism for the interaction of Gram-negative bacteria with collagen.IMPORTANCE Bacterial adhesion is a crucial step for bacterial colonization and infection. In recent years, the number of antibiotic-resistant strains has dramatically increased; therefore, there is a need to search for novel antimicrobial agents. Thus, great efforts are being devoted to develop a clear understanding of the bacterial adhesion mechanism for preventing infections. In host/pathogen interactions, once repulsive forces are overcome, adhesins recognize and tightly bind to specific receptors on the host cell or tissue components. Here, we present the first 3D structure of the interaction between the collagen-binding adhesin EmaA and collagen, which is critical for the development of endocarditis in humans.

On the importance of crystallographic texture in the biocompatibility of titanium based substrate.

The role of grain size and crystallographic orientation on the biocompatibility of commercially pure titanium was investigated. Samples, with significant differences in crystallographic texture and average grain size (from 0.4 to 40 µm) were produced by equal channel angular pressing (ECAP) and post deformation annealing. X-ray diffraction and electron back scattered diffraction (EBSD) were used to evaluate differences in texture and microstructural characteristics. The titanium oxide film present on the surface of the samples was analyzed to determine the oxidation state of titanium and the chemical bonds between oxygen and titanium using X-ray photoelectron spectroscopy (XPS). Biocompatibility experiments were conducted using MC3T3 preosteoblast cells. Cell attachment was found to be texture-sensitive, where the number of attached cells was higher on the samples with higher number of (0002) planes exposed to the surface, regardless of the grain size. A relationship was also found between the titanium oxide species formed on the surface and the crystallographic texture underneath. The surface texture consisting of more densely packed basal planes promote the formation of Ti-OH on the surface, which in turn, enhances the cell-substrate interactions. These surface characteristics are deemed responsible for the observed difference in cell attachment behaviour of surfaces with different textures. Finally, it is inferred that texture, rather than the grain size, plays the major role in controlling the surface biocompatibility of biomedical devices fabricated from pure metallic titanium.

Ultrastructural analysis of the rugose cell envelope of a member of the Pasteurellaceae family.

Bacterial membranes serve as selective environmental barriers and contain determinants required for bacterial colonization and survival. Cell envelopes of Gram-negative bacteria consist of an outer and an inner membrane separated by a periplasmic space. Most Gram-negative bacteria display a smooth outer surface (e.g., Enterobacteriaceae), whereas members of the Pasteurellaceae and Moraxellaceae families show convoluted surfaces. Aggregatibacter actinomycetemcomitans, an oral pathogen representative of the Pasteurellaceae family, displays a convoluted membrane morphology. This phenotype is associated with the presence of morphogenesis protein C (MorC). Inactivation of the morC gene results in a smooth membrane appearance when visualized by two-dimensional (2D) electron microscopy. In this study, 3D electron microscopy and atomic force microscopy of whole-mount bacterial preparations as well as 3D electron microscopy of ultrathin sections of high-pressure frozen and freeze-substituted specimens were used to characterize the membranes of both wild-type and morC mutant strains of A. actinomycetemcomitans. Our results show that the mutant strain contains fewer convolutions than the wild-type bacterium, which exhibits a higher curvature of the outer membrane and a periplasmic space with 2-fold larger volume/area ratio than the mutant bacterium. The inner membrane of both strains has a smooth appearance and shows connections with the outer membrane, as revealed by visualization and segmentation of 3D tomograms. The present studies and the availability of genetically modified organisms with altered outer membrane morphology make A. actinomycetemcomitans a model organism for examining membrane remodeling and its implications in antibiotic resistance and virulence in the Pasteurellaceae and Moraxellaceae bacterial families.

Correlation of the amino-acid sequence and the 3D structure of the functional domain of EmaA from Aggregatibacter actinomycetemcomitans.

Adhesion to collagen is an important virulence determinant for the periodontal pathogen Aggregatibacter actinomycetemcomitans. Binding to collagen is mediated by the extracellular-matrix protein adhesin-A (EmaA). EmaA is a homotrimeric autotransporter protein that forms flexible antenna-like appendages on the bacterium surface. An ellipsoidal structure at the distal end of the appendage, composed of three subdomains, contains the functional domain of the molecule. A correlation between amino-acid sequence and subdomain structure (SI and SII) was proposed based on an analysis of the volume/molecular weight ratio. EmaA from three mutant strains (deletions of amino-acids 70-206 and 70-386 and a substitution mutation G162S) has been studied by electron microscopy to test this hypothesis. 3D structures were analyzed using single-axis tilt tomography of negatively stained preparations of bacteria combined with subvolume averaging. Additionally, a large number of 2D images of the apical domain of the adhesins from the mutants were extracted from micrographs of the bacterial surface, aligned and classified. The combined data showed that amino-acids 70-206 localize to subdomain SI and 70-386 comprise subdomains SI and SII. Moreover, we showed that the substitution mutation G162S, which abolishes collagen binding activity, does not affect the overall structural integrity of the functional domain. However, the structure of subdomain SI in this mutant is slightly altered with respect to the wild-type strain. These data also have allowed us to interpret the architectural features of each subdomain of EmaA in more detail and to correlate the 3D structure of the functional domain of EmaA with the amino-acid sequence.

Self-assembled multifunctional nanoplexes for gene inhibitory therapy.

AIM: To enhance the stability of siRNA while improving their therapeutic properties and visualization at the target site, a novel nanoplex system was developed. MATERIALS & METHODS: The designed nanoplex system involved functionalizing siRNA with near-infrared quantum dots and loading them into histidylated glycol chitosan (GC-His). RESULTS: Colocalization studies revealed a twofold increase in siRNA uptake after encapsulation with GC-His and nanoparticles were localized in cytoplasm, suggesting that histidine promoted their dissociation from the endosomal membranes. Furthermore, as opposed to siRNAs treated with commercial transfection reagent, siRNAs loaded within GC-His showed a marked reduction (64%) of MDM2 protein expression 24 h after transfection. CONCLUSION: These findings concur that GC-His/siRNA-quantum dot nanoplexes are promising multifunctional vehicles for gene inhibitory therapy.

Real-time QCM-D monitoring of cellular responses to different cytomorphic agents.

Quartz crystal microbalance with dissipation monitoring (QCM-D) is used for real-time in situ detection of cytoskeletal changes in live primary endothelial cells in response to different cytomorphic agents; namely, the surfactant Triton-X 100 (TX-100) and bacterial lipopolysaccharide (LPS). Reproducible dissipation versus frequency (Df) plots provide unique signatures of the interactions between endothelial cells and cytomorphic agents. While the QCM-D response for TX-100 can be described in two steps (changes in the osmotic pressure of the medium prior to observing the expected cell lysis), LPS results in a different single-phase signal. A complementary analysis is carried out to evaluate the possible competitive effects of TX-100 and LPS through the QCM-D response to BAEC stress by analyzing the Df plots obtained. Experiments with non-toxic components (fibronectin or serum) produce a different QCM-D response than that observed for the toxic chemicals, suggesting the use of Df plot signatures for the possible differentiation between cytotoxic or non-cytotoxic effects. Observations obtained by QCM-D signals are confirmed by conducting fluorescence microscopy at the same time. Our results show that a fast (few minutes) sensing response can be obtained in situ and in real-time. The conclusions from this study suggest that QCM-D can potentially be used in biodetection for applications in drug screening tests and diagnosis.

The response of fibrinogen, platelets, endothelial and smooth muscle cells to an electrochemically modified SS316LS surface: towards the enhanced biocompatibility of coronary stents.

Modification of a biomedical-grade stainless steel 316LS surface by electrochemical cyclic potentiodynamic passivation (CPP) and the response of fibrinogen (Fg), platelets, endothelial cells (ECs) and smooth muscles cells (SMCs) to this surface was investigated. Polarization modulation infrared reflection absorption spectroscopy revealed a significant difference between the secondary structure of Fg adsorbed on the unmodified and CPP surface, the latter being closer to that of native Fg. This was postulated as the origin of the significantly lower surface density of attached platelets on the CPP surface. The competitive interaction of ECs and SMCs with the surface showed that the ECs/SMCs surface density ratio is significantly higher on the CPP surface over the first 2h of attachment, suggesting faster initial attachment kinetics of ECs on the CPP surface. The presented results thus clearly demonstrate an increase in biocompatibility of the CPP 316LS surface.

The positive influence of electrochemical cyclic potentiodynamic passivation (CPP) of a SS316LS surface on its response to fibronectin and pre-osteoblasts.

The influence of an electrochemical surface passivation technique (cyclic potentiodynamic polarization, CPP) on the physico-chemical surface properties of SS316LS and its subsequent response to fibronectin (Fn) and pre-osteoblasts were investigated. Contact angle and zeta-potential measurements showed that the CPP-modified surface is more hydrophilic and more positively charged than the unmodified surface. Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) was used to investigate the interaction of Fn with both surfaces. The saturated surface concentration of adsorbed Fn was higher on the CPP-modified surface. As well, significant changes were identified in the secondary structure of Fn adsorbed on both surfaces, in comparison to its native state. This data also indicated a higher degree of Fn unfolding on the CPP-modified surface. Cell studies indicated that the attachment, proliferation and morphology of pre-osteoblasts were significantly improved on the CPP-modified surface, which was attributed to the more open conformation of Fn on the CPP-modified surface. Thus, the CPP surface passivation method was demonstrated to yield a SS316LS surface of enhanced biocompatibility.

Titanium crystal orientation as a tool for the improved and regulated cell attachment.

Cell adhesion is a fundamental process that controls cell proliferation, migration, and differentiation and is crucial for biomaterial-tissue integration. Osteoblast attachment on the surfaces of implant materials is, therefore, essential for the proper function of any implant in which osseointegration is required. Although many reports are available on osteoblast attachment using different surface modification, there is no specific report, so far, that investigates the effect of atomic order of specific crystallographic orientation of substrates on cell behavior. A novel coculture system is proposed to show the differential response of preosteoblast and fibroblast cell lines to the titanium single-crystal substrates. Our investigation has shown that surface recognition by the cell is influenced by the atomic structure of the surface leading to cell-type-specific adhesion. The degree of preosteoblast attachment is significantly higher on the Ti-(1120), whereas the fibroblast adhesion is increased on the Ti-(1010). This demonstrates that the three distinct faces of titanium substrates differ greatly in their capacity to serve as cell adhesive substrates. It also provides clear evidence for the role of crystal structure in regulating and improving cell-substrate interactions relevant for the optimal function of bone implant materials.

Modulated release of OP-1 and enhanced preosteoblast differentiation using a core-shell nanoparticulate system.

A release-controlled OP-1 delivery system consisting of a suspension of core-shell nanoparticles was prepared. The nanoparticles were composed of a core of positively-charged large unilamellar liposomes and a shell constructed through the L-b-L assembly of alternating layers of negatively-charged sodium alginate and positively-charged chitosan. Cytotoxicity was assayed with MC3T3-E1.4 mouse preosteoblast cells and cell viability was determined by colorimetry (CellQuanti-MTT kit). The system was loaded with a range of OP-1 concentrations and the release profiles were obtained and fitted into the Higuchi model to determine release kinetics. Alkaline phosphatase (ALP) activity of preosteoblasts was evaluated using a micro-BCA assay. The resulting monodisperse and nontoxic spherical nanoparticles exhibited high physical stability in simulated physiological media as well as an extended shelf-life allowing for immediate protein loading before future administration. ALP activity increased over time with the OP-1 loaded delivery system when compared with control, protein alone, and nanoparticles alone (p < 0.05). The system offers copious compartments for protein entrapment including the aqueous core and within the polyelectrolyte layers in the shell and demonstrates a sustained triphasic linear release of OP-1 over a prolonged period of 45 days, in vitro. This system offers a great advantage for optimum growth factor performance when applied in different anatomical sites of varying defect sizes and vascularity.

Intracellular precipitation of hydroxyapatite mineral and implications for pathologic calcification.

In contrast to physiologic biomineralization occurring in bones, teeth and otoconia in vertebrates, calcification of soft tissues occurs in many pathologic conditions. Although similarities have been noted between the two processes, and despite the important clinical consequences of ectopic calcification, the molecular mechanisms regulating ectopic calcification are poorly understood. Although calcification is mainly extracellular, intracellular calcification has been reported and might indeed contribute to pathologic calcification of soft tissues. To better understand the process of intracellular calcification as a potential origin for pathologic calcification, and to examine the role of proteoglycans in this process, we investigated a pattern of intracellular nucleation and growth of hydroxyapatite in Madin-Darby Canine Kidney (MDCK) epithelial cells using electron microscopy, secondary ion mass spectroscopy (NanoSIMS), cytochemical staining, immunolabeling and biochemical analysis. We report here that under mineralizing cell culture conditions where beta-glycerophosphate (betaGP) was added as an exogenous organic source of phosphate, betaGP-cleaving alkaline phosphatase activity increased and hydroxyapatite crystals subsequently nucleated within intracellular, membrane-bounded compartments. The small, leucine-rich proteoglycan decorin was also upregulated and associated with mineral in these cultures. Such information provides insight into the mechanisms leading to pathologic calcification and describes a process whereby hydroxyapatite deposition in cells might lead to ectopic calcification.

Cellular and molecular interactions between MC3T3-E1 pre-osteoblasts and nanostructured titanium produced by high-pressure torsion.

Ultra-fine surface features are commonly used to modulate cellular activity on a variety of materials. The continuing challenge for materials in contact with bone is the development of a material with both favorable surface and bulk properties to modulate not only the cell-substrate interactions, but also to ensure the long-term stability of the implant. In a combined approach involving material sciences and cell and molecular biology, the nature and mechanism of cell-substrate interaction, in particular, the molecular machinery controlling cell response to the surface of the nanostructured titanium based material produced by the high pressure torsion (HPT) process is assessed. The degree of pre-osteoblast attachment and rate of growth, which are regulated through the activity and interaction of proteins present in the extracellular matrix and associated with cytoskeleton and focal adhesion, are notably increased on the HPT-processed titanium substrates. The improved cell activity is attributed to the nanostructured feature of these substrates consisting of ultra-fine crystals (<50 nm) and a distinct surface oxide layer which provide higher degree of surface wettability. These findings demonstrate the advantages of HPT-processed titanium over the conventional and coated titanium implants, as both mechanical properties and cellular response are improved.

Pyrophosphate inhibits mineralization of osteoblast cultures by binding to mineral, up-regulating osteopontin, and inhibiting alkaline phosphatase activity.

Inorganic pyrophosphate (PP(i)) produced by cells inhibits mineralization by binding to crystals. Its ubiquitous presence is thought to prevent "soft" tissues from mineralizing, whereas its degradation to P(i) in bones and teeth by tissue-nonspecific alkaline phosphatase (Tnap, Tnsalp, Alpl, Akp2) may facilitate crystal growth. Whereas the crystal binding properties of PP(i) are largely understood, less is known about its effects on osteoblast activity. We have used MC3T3-E1 osteoblast cultures to investigate the effect of PP(i) on osteoblast function and matrix mineralization. Mineralization in the cultures was dose-dependently inhibited by PP(i). This inhibition could be reversed by Tnap, but not if PP(i) was bound to mineral. PP(i) also led to increased levels of osteopontin (Opn) induced via the Erk1/2 and p38 MAPK signaling pathways. Opn regulation by PP(i) was also insensitive to foscarnet (an inhibitor of phosphate uptake) and levamisole (an inhibitor of Tnap enzymatic activity), suggesting that increased Opn levels did not result from changes in phosphate. Exogenous OPN inhibited mineralization, but dephosphorylation by Tnap reversed this effect, suggesting that OPN inhibits mineralization via its negatively charged phosphate residues and that like PP(i), hydrolysis by Tnap reduces its mineral inhibiting potency. Using enzyme kinetic studies, we have shown that PP(i) inhibits Tnap-mediated P(i) release from beta-glycerophosphate (a commonly used source of organic phosphate for culture mineralization studies) through a mixed type of inhibition. In summary, PP(i) prevents mineralization in MC3T3-E1 osteoblast cultures by at least three different mechanisms that include direct binding to growing crystals, induction of Opn expression, and inhibition of Tnap activity.

The significance of crystallographic texture of titanium alloy substrates on pre-osteoblast responses.

The aim of this study is to investigate the effects of grain orientation in polycrystalline materials on cell-substrate interactions. Samples are prepared from rods and sheets of Ti-6Al-4V substrates with predominately two distinct crystallographic orientations. X-ray diffraction analysis indicates that 36% of the surfaces of rod samples consist of (1010) plane, while the predominant orientation in the surface of the sheet samples is (1120) plane (29%). Morphological studies and cell biological experiments including cell attachment, proliferation and differentiation are conducted using MC3T3 pre-osteoblast cells cultured on these two different samples. The number of attached cells on the rod Ti-(1010) samples (70% after 1 h and 50% after 2 h) is higher than on the sheet Ti-(1120) samples. Cell proliferation after 3 days is also significantly higher on the Ti-(1010) samples. Alkaline phosphatase activity, however, shows no significant difference between the two samples. Scanning electron microscopy (SEM) analysis of MC3T3 cells grown on samples with different crystallographic texture demonstrate significant differences in morphology with respect to attachment and growth pattern. This study shows that crystal orientation of the substrate can influence cell responses and, therefore, substrate engineering can be used to improve and control cell-substrate interactions.