Shannon Ryan

Orally Administered P22 Phage Tailspike Protein Reduces Salmonella Colonization in Chickens: Prospects of a Novel Therapy against Bacterial Infections

One of the major causes of morbidity and mortality in man and economically important animals is bacterial infections of the gastrointestinal (GI) tract. The emergence of difficult-to-treat infections, primarily caused by antibiotic resistant bacteria, demands for alternatives to antibiotic therapy. Currently, one of the emerging therapeutic alternatives is the use of lytic bacteriophages. In an effort to exploit the target specificity and therapeutic potential of bacteriophages, we examined the utility of bacteriophage tailspike proteins (Tsps). Among the best-characterized Tsps is that from the Podoviridae P22 bacteriophage, which recognizes the lipopolysaccharides of Salmonella enterica serovar Typhimurium. In this study, we utilized a truncated, functionally equivalent version of the P22 tailspike protein, P22sTsp, as a prototype to demonstrate the therapeutic potential of Tsps in the GI tract of chickens. Bacterial agglutination assays showed that P22sTsp was capable of agglutinating S. Typhimurium at levels similar to antibodies and incubating the Tsp with chicken GI fluids showed no proteolytic activity against the Tsp. Testing P22sTsp against the three major GI proteases showed that P22sTsp was resistant to trypsin and partially to chymotrypsin, but sensitive to pepsin. However, in formulated form for oral administration, P22sTsp was resistant to all three proteases. When administered orally to chickens, P22sTsp significantly reduced Salmonella colonization in the gut and its further penetration into internal organs. In in vitro assays, P22sTsp effectively retarded Salmonella motility, a factor implicated in bacterial colonization and invasion, suggesting that the in vivo decolonization ability of P22sTsp may, at least in part, be due to its ability to interfere with motility… Our findings show promise in terms of opening novel Tsp-based oral therapeutic approaches against bacterial infections in production animals and potentially in humans.

Single‐Domain Antibody‐Conjugated Nanoaggregate‐Embedded Beads for Targeted Detection of Pathogenic Bacteria

The rapid screening of pathogenic bacteria remains a key issue in the diagnosis of infectious diseases, food safety, and public health assurance. In particular, the emergence of drug-resistant bacteria presents great challenges to the health care sector. Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for one of the better-known hospital-acquired infections. S. aureus is a common pathogen that can colonize various areas of the human anatomy. Healthy individuals may carry MRSA asymptomatically, but patients with a compromised immune system are at a greater risk of symptomatic secondary infection. Due to the drug-resistant nature of S. aureus, preventive measures, such as routine patient screening, remain the most effective way to control the spread of this bacterium in clinical environments. In the clinical setting, routine analysis of pathogenic bacteria typically involves biochemical characterization of cultured microorganisms taken from contaminated sources. Standardized procedures are time consuming, which thus highlights the need for a rapid and targeted detection methodology. Advances in nanotechnology and biotechnology offer new possibilities for the rapid screening of harmful microorganisms. Surface-enhanced Raman scattering (SERS) has been demonstrated to achieve ultra-high sensitivity detection in many bioanalytical assays. Herein, we combine the high sensitivity of a newly developed SERS nanoprobe with the high specificity of single-domain antibody (sdAb) to achieve the targeted detection of a single bacterial pathogen, S. aureus. The ability of metallic nanostructures to localized surface plasmon resonance (LSPR) under appropriate electromagnetic field excitation is largely responsible for the SERS effect. The LSPR is strongly dependent on the size and shape of the nanostructures. It has been demonstrated that extremely high SERS enhancement can be achieved when nanostructures are closely spaced, which allows their LSPR to couple. At the optimal interparticle spacing, LSPR coupling results in the capacitive enhancement of the Raman effect for molecules located between particles in the SERS “hot sites”, which thus enables the detection of very few molecules under optimal excitation conditions. A specially designed SERS nanoprobe called a nanoaggregateembedded bead (NAEB) was fabricated with this optimization in mind. NAEBs are fabricated by controlled formation of small Au nanoparticle (NP) aggregates that are subsequently encapsulated in a protective silica shell (Scheme 1a), and fully utilize the advantage of LSPR coupling of a small NP aggregate; as a result each nanosized bead is an ultrahigh sensitivity SERS nanoprobe. Raman reporter molecules (R6G) were incorporated into the nanoaggregate during the formation process to give each NAEB a unique Raman signature. Compared with other SERS-based bioanalytical applications that utilize Ag or Au NPs, NAEBs have the added advantage of stability. Without the protective silica shell, even passivated Au or Ag NPs immersed in biological buffers are prone to parasitic signals from adsorption of the molecules in the biological fluids or loss of signals due to the desorption of the Raman reporter molecule. This problem was recognized by the groups of Liz-Marzan, Natan, Nie, and Brown, who pioneered the work on encapsulating Au NPs with a protective silica shell to improve their stability. In these earlier works, encapsulation was done without deliberate aggregation, which resulted in a relatively low [a] P.-J. Huang, Prof. L.-K. Chau Department of Chemistry and Biochemistry National Chung Cheng University 168 University Road Min-Hsiung, Chia-Yi (Taiwan) Fax: (+886)5-2721040 E-mail : chelkc@ccu.edu.tw [b] Dr. L.-L. Tay, Dr. J. Tanha, S. Ryan Institute for Microstructural Sciences and Institute for Biological Sciences National Research Council Canada Ottawa, ON K1A 0R6 (Canada) Fax: (+1) 952-6337 E-mail : lilin.tay@nrc-cnrc.gc.ca Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.200901397.

Single-Domain Antibody-Nanoparticles: Promising Architectures for Increased Staphylococcus aureus Detection Specificity and Sensitivity

Because antibodies are highly target-specific and nanoparticles possess diverse, material-dependent properties that can be exploited in order to label and potentially identify biomolecules, the development of antibody-nanoparticle conjugates (nanoconjugates) has huge potential in biodiagnostics. Here, we describe a novel superparamagnetic nanoconjugate, one whose recognition component is a single-domain antibody. It is highly active toward its target Staphylococcus aureus, displays long shelf life, lacks cross-reactivity inherent to traditional homologue whole antibodies, and captures a few dozen S. aureus cells in a mixed cell population with ~100% efficiency and specificity. We ascribe the excellent performance of our nanoconjugate to its single-domain antibody component and recommend it as a general purpose recognition element.

Protease-resistant single-domain antibodies inhibit Campylobacter jejuni motility

Camelid heavy-chain antibody variable domains (VHHs) are emerging as potential antimicrobial reagents. We have engineered a previously isolated VHH (FlagV1M), which binds Campylobacter jejuni flagella, for greater thermal and proteolytic stability. Mutants of FlagV1M were obtained from an error-prone polymerase chain reaction library that was panned in the presence of gastrointestinal (GI) proteases. Additional FlagV1M mutants were obtained through disulfide-bond engineering. Each approach produced VHHs with enhanced thermal stability and protease resistance. When the beneficial mutations from both approaches were combined, a hyperstabilized VHH was created with superior stability. The hyperstabilized VHH bound C. jejuni flagella with wild-type affinity and was capable of potently inhibiting C. jejuni motility in assays performed after sequential digestion with three major GI proteases, demonstrating the remarkable stability imparted to the VHH by combining our engineering approaches.

Multimodal plasmonic nanosensor for the detection of pathogenic bacteria

Multi-modal sensing scheme significantly improves the detection accuracy but can also introduce extra complexity in the overall design of the sensor. We overcome this difficulty by utilizing the plasmonic properties of metallic nanoparticles. In this study, we will present a simple dual optical sensing mechanism which harvests signals of the resonantly excited metallic nanostructure in the form of surface enhanced Raman scattering (SERS) and resonant Rayleigh scattering. Silver and gold nanoparticles labeled with appropriate antibodies act as signal transduction units and upon exposure to the targeted pathogen render the targeted species optically active. We demonstrate that detection of a single pathogen cell is easily attainable with the dual detection scheme. Furthermore, we explore the markedly different SERS intensity observed from the use of two very different antibody recognition units during the pathogen labeling process.

Detection of Staphylococci aureus cells with single domain antibody functionalized Raman nanoparobes

Raman spectroscopy has demonstrated to be an effective tool in the detection and classification of pathogenic microorganisms. The technique is, however, limited by the inherently low cross-section of the Raman scattering process. Among the many enhanced Raman processes, surface enhanced Raman scattering (SERS) technique provides the highest sensitivity and can be easily adapted in the bio-sensing applications such as DNA hybridization and protein binding events. In this study, we report the targeted detection of the pathogenic bacteria, Staphylococcus aureus, with novel single domain antibody (sdAb) conjugated SERS nanoprobes. A sdAb specific to protein A of S. aureus cells was conjugated to silver nanoparticles (Ag-NP). Bacteria recognition was achieved through specific binding of the sdAb (conjugated to SERS nanoprobe) to protein A. Binding rendered the nanoparticle-labeled S. aureus cells SERS active. As a result, S. aureus cells could be detected rapidly and with excellent sensitivity by monitoring the SERS vibrational signatures. This work demonstrates that the SERS imaging technique offers excellent sensitivity with a detection limit of a single bacterium.