Carolyn A. Haller

Dissimilatory Fe(III) and Mn(IV) Reduction by Shewanella putrefaciens Requires ferE, a Homolog of the pulE (gspE) Type II Protein Secretion Gene

Shewanella putrefaciens strain 200 respires anaerobically on a wide range of compounds as the sole terminal electron acceptor, including ferric iron [Fe(III)] and manganese oxide [Mn(IV)]. Previous studies demonstrated that a 23.3-kb S. putrefaciens wild-type DNA fragment conferred metal reduction capability to a set of respiratory mutants with impaired Fe(III) and Mn(IV) reduction activities (T. DiChristina and E. DeLong, J. Bacteriol. 176:1468-1474, 1994). In the present study, the smallest complementing fragment was found to contain one open reading frame (ORF) (ferE) whose translated product displayed 87% sequence similarity to Aeromonas hydrophila ExeE, a member of the PulE (GspE) family of proteins found in type II protein secretion systems. Insertional mutants E726 and E912, constructed by targeted replacement of wild-type ferE with an insertionally inactivated ferE construct, were unable to respire anaerobically on Fe(III) or Mn(IV) yet retained the ability to grow on all other terminal electron acceptors. Nucleotide sequence analysis of regions flanking ferE revealed the presence of one partial and two complete ORFs whose translated products displayed 55 to 70% sequence similarity to the PulD, -F, and -G homologs of type II secretion systems. A contiguous cluster of 12 type II secretion genes (pulC to -N homologs) was found in the unannotated genome sequence of Shewanella oneidensis (formerly S. putrefaciens) MR-1. A 91-kDa heme-containing protein involved in Fe(III) reduction was present in the peripheral proteins loosely attached to the outside face of the outer membrane of the wild-type and complemented (Fer+) B31 transconjugates yet was missing from this location in Fer mutants E912 and B31 and in uncomplemented (Fer-) B31 transconjugates. Membrane fractionation studies with the wild-type strain supported this finding: the 91-kDa heme-containing protein was detected with the outer membrane fraction and not with the inner membrane or soluble fraction. These findings provide the first genetic evidence linking dissimilatory metal reduction to type II protein secretion and provide additional biochemical evidence supporting outer membrane localization of S. putrefaciens proteins involved in anaerobic respiration on Fe(III) and Mn(IV).

Site-specific multivalent carbohydrate labeling of quantum dots and magnetic beads.

Cell-surface carbohydrates act as receptors for a variety of protein ligands and thereby play a significant role in a wide range of biological processes, including immune-recognition events and the interaction of viruses and bacteria with host cells as well as tissue growth and repair. As such, binding interactions of carbohydrates and proteins provide a starting point for the development of novel diagnostic agents and a framework for new therapies. It is notable that the low affinity and specificity that are typical of monomeric carbohydrate–protein interactions are dramatically enhanced when the carbohydrate component is presented as a multivalent ligand; a phenomenon referred to as the “cluster-glycoside effect”. In response to this observation, considerable effort has focused on the design of unique, multivalent carbohydrate ligands in the form of linear polymers, liposomes, dendrimers, beads, or nanoparticles. In this regard, we have recently described a useful route for the synthesis of glycopolymers by a cyanoxyl-mediated free-radical polymerization scheme that can be performed under aqueous condition and is tolerant of a wide range of monomer functionalities, including OH, COOH, NH2, and OSO3H groups. Conveniently, this synthetic approach facilitates selective derivatization of the polymer-chain terminus. Herein, we report site-specific multivalent carbohydrate labeling of nanocrystal (quantumdot) and magnetic-bead surfaces using a biotin chain-endfunctionalized glycopolymer and demonstrate the potential value of these multivalent carbohydrate polymers in both imaging and biocapture applications (Figure 1). Semiconductor nanocrystals are a new class of size-tunable optical probe. Recently, nanocrystal surfaces have been functionalized with DNA, peptides, proteins, and other small ligands with intended applications as biological reagents and probes. Nanocrystal–streptavidin conjugates, for example, have been used to stain tissues, cells, and intracellular organelles. Likewise, nanocrystal–avidin–antibody conjugates have improved the sensitivity of conventional fluoroimmunoassays. To the best of our knowledge, carbohydrateconjugated nanocrystals have yet to be explored in bioimaging applications although a few nanocrystal–carbohydrate conjugates have been reported (see also note added in proof). In the present study, nanocrystal–multivalent carbohydrate conjugates were produced by incubating nanocrystal–streptavidin (50 mL, 120 mgmL 1 streptavidin in phosphate buffered saline (PBS), Qdot 565 streptavidin conjugate, Quantum Dot Corp. , Hayward, CA) with biotin end-terminated glycopolymer 1 (50 mL, 1 mgmL 1 in PBS) bearing ten pendant lactose groups for one hour at room temperature. RCA120 is a lectin that binds to terminal b-d-galactose. As a model system, RCA120-immobilized agarose beads (100 mL, 2 mgmL , Sigma) were incubated with nanocrystal–carbohydrate conjugates in PBS (100 mL) for 1 h at room temperature and subsequently washed three times with PBS. Confocal microscopy confirmed fluorescent staining of the lectin-modified bead surfaces (Figure 2A). Of particular interest was that staining intensity was dramatically enhanced by the initial exposure of RCA120 beads to biotin end-terminated glycopolymer 1 followed by incubation of the mixture with streptavidin–nanocrystal conjugates (Figure 2B). The weak-intensity staining observed when using the first approach might have been due to the presence of free glycopolymer along with the nanocrystal–carbohydrate conjugates. As a two-step procedure, the sensitivity of staining was increased through the formation in situ of nanocrystal– carbohydrate complexes on the bead surface without the need to purify the conjugate. The absence of staining on treatment with streptavidin nanocrystals alone or with the use of nonbiotinylated glycopolymer 2 confirms the necessity and specificity of both carbohydrate–lectin and streptavidin–biotin interactions (Figure 2C). Biotin end-terminated glycopolymers were also used to extend the versatility of magnetic-bead-based biocapture assays that have been employed for the rapid isolation of a variety of lectin-bearing cells and biomolecules. Indeed, Rye and Bovin have demonstrated that glycopolymer-derivatized magnetic beads provide a useful tool for the selection of cells expressing a specific carbohydrate-binding phenotype. Bundy and Fenselau have also reported that glycopolymerbased affinity capture surfaces are more sensitive than lectinbased systems for microbial capture. While in both reports glycopolymers were effectively attached to the bead and [a] Dr. X.-L. Sun, Dr. W. Cui, Dr. C. Haller Departments of Surgery and Biomedical Engineering Emory University School of Medicine Atlanta, GA 30322 (USA) E-mail : xsun@emory.edu [b] Dr. E. L. Chaikof Department of Surgery and Biomedical Engineering Emory University School of Medicine Atlanta, GA 30322 (USA) Fax: (+1)404-727-3660 E-mail : echaiko@emory.edu Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

The use of microfiber composites of elastin-like protein matrix reinforced with synthetic collagen in the design of vascular grafts

Collagen and elastin networks contribute to highly specialized biomechanical responses in numerous tissues and species. Biomechanical properties such as modulus, elasticity, and strength ultimately affect tissue function and durability, as well as local cellular behavior. In the case of vascular bypass grafts, compliance at physiologic pressures is correlated with increased patency due to a reduction in anastomotic intimal hyperplasia. In this report, we combine extracellular matrix (ECM) protein analogues to yield multilamellar vascular grafts comprised of a recombinant elastin-like protein matrix reinforced with synthetic collagen microfibers. Structural analysis revealed that the fabrication scheme permits a range of fiber orientations and volume fractions, leading to tunable mechanical properties. Burst strengths of 239-2760 mm Hg, compliances of 2.8-8.4%/100 mm Hg, and suture retention strengths of 35-192 gf were observed. The design most closely approximating all target criteria displayed a burst strength of 1483 +/- 143 mm Hg, a compliance of 5.1 +/- 0.8%/100 mm Hg, and a suture retention strength of 173 +/- 4 gf. These results indicate that through incorporation of reinforcing collagen microfibers, recombinant elastomeric protein-based biomaterials can play a significant role in load bearing tissue substitutes. We believe that similar composites can be incorporated into tissue engineering schemes that seek to integrate cells within the structure, prior to or after implantation in vivo.

Long-Term Biostability of Self-Assembling Protein Polymers in the Absence of Covalent Crosslinking

Unless chemically crosslinked, matrix proteins, such as collagen or silk, display a limited lifetime in vivo with significant degradation observed over a period of weeks. Likewise, amphiphilic peptides, lipopeptides, or glycolipids that self-assemble through hydrophobic interactions to form thin films, fiber networks, or vesicles do not demonstrate in vivo biostability beyond a few days. We report herein that a self-assembling, recombinant elastin-mimetic triblock copolymer elicited minimal inflammatory response and displayed robust in vivo stability for periods exceeding 1 year, in the absence of either chemical or ionic crosslinking. Specifically, neither a significant inflammatory response nor calcification was observed upon implantation of test materials into the peritoneal cavity or subcutaneous space of a mouse model. Moreover, serial quantitative magnetic resonance imaging, evaluation of pre- and post-explant ultrastructure by cryo-high resolution scanning electron microscopy, and an examination of implant mechanical responses revealed substantial preservation of form, material architecture, and biomechanical properties, providing convincing evidence of a non-chemically or ionically crosslinked protein polymer system that exhibits long-term stability in vivo.

Acellular vascular grafts generated from collagen and elastin analogs.

Tissue-engineered vascular grafts require long fabrication times, in part due to the requirement of cells from a variety of cell sources to produce a robust, load-bearing extracellular matrix. Herein, we propose a design strategy for the fabrication of tubular conduits comprising collagen fiber networks and elastin-like protein polymers to mimic native tissue structure and function. Dense fibrillar collagen networks exhibited an ultimate tensile strength (UTS) of 0.71±0.06 MPa, strain to failure of 37.1±2.2% and Youngs modulus of 2.09±0.42 MPa, comparing favorably to a UTS and a Youngs modulus for native blood vessels of 1.4-11.1 MPa and 1.5±0.3 MPa, respectively. Resilience, a measure of recovered energy during unloading of matrices, demonstrated that 58.9±4.4% of the energy was recovered during loading-unloading cycles. Rapid fabrication of multilayer tubular conduits with maintenance of native collagen ultrastructure was achieved with internal diameters ranging between 1 and 4mm. Compliance and burst pressures exceeded 2.7±0.3%/100 mmHg and 830±131 mmHg, respectively, with a significant reduction in observed platelet adherence as compared to expanded polytetrafluoroethylene (ePTFE; 6.8±0.05×10(5) vs. 62±0.05×10(5) platelets mm(-2), p<0.01). Using a rat aortic interposition model, early in vivo responses were evaluated at 2 weeks via Doppler ultrasound and CT angiography with immunohistochemistry confirming a limited early inflammatory response (n=8). Engineered collagen-elastin composites represent a promising strategy for fabricating synthetic tissues with defined extracellular matrix content, composition and architecture.

Thrombomodulin Improves Early Outcomes After Intraportal Islet Transplantation

Primary islet nonfunction due to an instant blood mediated inflammatory reaction (IBMIR) leads to an increase in donor islet mass required to achieve euglycemia. In the presence of thrombin, thrombomodulin generates activated protein C (APC), which limits procoagulant and proinflammatory responses. In this study, we postulated that liposomal formulations of thrombomodulin (lipo‐TM), due to its propensity for preferential uptake in the liver, would enhance intraportal engraftment of allogeneic islets by inhibiting the IBMIR. Diabetic C57BL/6J mice underwent intraportal transplantation with B10.BR murine islets. In the absence of treatment, conversion to euglycemia was observed among 29% of mice receiving 250 allo‐islets. In contrast, a single infusion of lipo‐TM led to euglycemia in 83% of recipients (p = 0.0019). Fibrin deposition (p < 0.0001), neutrophil infiltration (p < 0.0001), as well as expression TNF‐α and IL‐β (p < 0.03) were significantly reduced. Significantly, thrombotic responses mediated by human islets in contact with human blood were also reduced by this approach. Lipo‐TM improves the engraftment of allogeneic islets through a reduction in local thrombotic and inflammatory processes. As an enzyme‐based pharmacotherapeutic, this strategy offers the potential for local generation of APC at the site of islet infusion, during the initial period of elevated thrombin production.

Maleimide-thiol coupling of a bioactive peptide to an elastin-like protein polymer.

Recombinant elastin-like protein (ELP) polymers display several favorable characteristics for tissue repair and replacement as well as drug delivery applications. However, these materials are derived from peptide sequences that do not lend themselves to cell adhesion, migration, or proliferation. This report describes the chemoselective ligation of peptide linkers bearing the bioactive RGD sequence to the surface of ELP hydrogels. Initially, cystamine is conjugated to ELP, followed by the temperature-driven formation of elastomeric ELP hydrogels. Cystamine reduction produces reactive thiols that are coupled to the RGD peptide linker via a terminal maleimide group. Investigations into the behavior of endothelial cells and mesenchymal stem cells on the RGD-modified ELP hydrogel surface reveal significantly enhanced attachment, spreading, migration and proliferation. Attached endothelial cells display a quiescent phenotype.

Biomolecular surface engineering of pancreatic islets with thrombomodulin.

Islet transplantation has emerged as a promising treatment for Type 1 diabetes, but its clinical impact remains limited by early islet destruction mediated by prothrombotic and innate inflammatory responses elicited upon transplantation. Thrombomodulin (TM) acts as an important regulator of thrombosis and inflammation through its capacity to channel the catalytic activity of thrombin towards generation of activated protein C (APC), a potent anticoagulant and anti-inflammatory agent. We herein describe a novel biomolecular strategy for re-engineering the surface of pancreatic islets with TM. A biosynthetic approach was employed to generate recombinant human TM (rTM) bearing a C-terminal azide group, which facilitated site-specific biotinylation of rTM through Staudinger ligation. Murine pancreatic islets were covalently biotinylated through targeting of cell surface amines and aldehydes and both islet viability and the surface density of streptavidin were maximized through optimization of biotinylation conditions. rTM was immobilized on islet surfaces through streptavidin-biotin interactions, resulting in a nearly threefold increase in the catalytic capacity of islets to generate APC.

Immobilization of Actively Thromboresistant Assemblies on Sterile Blood‐Contacting Surfaces

Rapid one-step modification of thrombomodulin with alkylamine derivatives such as azide, biotin, and PEG is achieved using an evolved sortase (eSrtA) mutant. The feasibility of a point-of-care scheme is demonstrated herein to site-specifically immobilize azido-thrombomodulin on sterilized commercial ePTFE vascular grafts, which exhibit superior thromboresistance compared with commercial heparin-coated grafts in a primate model of acute graft thrombosis.

Collagen-based substrates with tunable strength for soft tissue engineering

Through the use of mechanical reinforcement of collagen matrices, mechanically strong and compliant 3D tissue mimetic scaffolds can be generated that act as scaffolds for soft tissue engineering. Collagen has been widely used for the development of materials for repair, augmentation or replacement of damaged or diseased tissue. Herein we describe a facile method for the layer-by-layer fabrication of robust planar collagen fiber constructs. Collagen gels cast in a phosphate buffer were dried to form dense collagen mats. Subsequent gels were layered and dried atop mats to create multilayer constructs possessing a range of tunable strengths (0.5 - 11 MPa) and stiffness (1 - 115 MPa). Depending on processing conditions and crosslinking of constructs, strain to failure ranged between 9 to 48%. Collagen mats were constructed into hernia patches that prevented hernia recurrence in Wistar rats.