GCAT-SEEKquence: genome consortium for active teaching of undergraduates through increased faculty access to next-generation sequencing data.

To transform undergraduate biology education, faculty need to provide opportunities for students to engage in the process of science. The rise of research approaches using next-generation (NextGen) sequencing has been impressive, but incorporation of such approaches into the undergraduate curriculum remains a major challenge. In this paper, we report proceedings of a National Science Foundation-funded workshop held July 11-14, 2011, at Juniata College. The purpose of the workshop was to develop a regional research coordination network for undergraduate biology education (RCN/UBE). The network is collaborating with a genome-sequencing core facility located at Pennsylvania State University (University Park) to enable undergraduate students and faculty at small colleges to access state-of-the-art sequencing technology. We aim to create a database of references, protocols, and raw data related to NextGen sequencing, and to find innovative ways to reduce costs related to sequencing and bioinformatics analysis. It was agreed that our regional network for NextGen sequencing could operate more effectively if it were partnered with the Genome Consortium for Active Teaching (GCAT) as a new arm of that consortium, entitled GCAT-SEEK(quence). This step would also permit the approach to be replicated elsewhere.

Bacterial adaptation and infection.

There is evidence that many pathogens co-evolve with their hosts. This is often reflected in species specific virulence factors that can selectively interfere with host defense mechanisms, innate and acquired as well as a range of interactions with host homeostatic pathways that contribute to the course and severity of an infection. In this review, we highlight a number of select examples of these interactions and suggest that understanding of molecular pathogenesis requires a broad systems approach that can evaluate the multiple and dynamics interactions that are occurring during infection.

"Shovel-ready" Sequences as a Stimulus for the Next Generation of Life Scientists.

Genomics and bioinformatics are dynamic fields well-suited for capturing the imagination of undergraduates in both research laboratories and classrooms. Currently, raw nucleotide sequence is being provided, as part of several genomics research initiatives, for undergraduate research and teaching. These initiatives could be easily extended and much more effective if the source of the sequenced material and the subsequent focus of the data analysis were aligned with the research interests of individual faculty at undergraduate institutions. By judicious use of surplus capacity in existing nucleotide sequencing cores, raw sequence data could be generated to support ongoing research efforts involving undergraduates. This would allow these students to participate actively in discovery research, with a goal of making novel contributions to their field through original research while nurturing the next generation of talented research scientists.

Novel Sample Preparation for Mass Spectral Analysis of Complex Biological Samples.

The ability to combine a selective capture strategy with on chip MALDI-TOF analysis allows for rapid, sensitive analysis of a variety of different analytes. In this overview a series of applications of capture enhanced laser desorption ionization time of flight (CELDI-TOF) mass spectrometry are described. The key feature of the assay is an off-chip capture step that utilizes high affinity bacterial binding proteins to capture a selected ligand. This allows large volumes of sample to be used and provides for a concentration step prior to transfer to a gold chip for traditional mass spectral analysis. The approach can also be adapted to utilize specific antibody as the basis of the capture step. The direct and indirect CELDI-TOF assays are rapid, reproducible and can be a valuable proteomic tool for analysis of low abundance molecules present in complex mixtures like blood plasma.

Immunoglobulin cleavage by the streptococcal cysteine protease IdeS can be detected using protein G capture and mass spectrometry.

The immunoglobulin degrading enzyme of Streptococcus pyogenes, IdeS, is an unusual cysteine protease produced by group A streptococci for which the only known substrate is immunoglobulin G (IgG). To date, IdeS has not been found to cleave any of the known synthetic substrates that other cysteine proteases hydrolyse, thus making the development of an IdeS detection assay difficult. Furthermore, at high doses of substrate, product generation is inhibited potentially due to the need for a dimeric enzyme complex with IgG. In this study we have developed a mass spectral assay for IdeS activity based on the detection of an Mr approximately 25,300 Fc fragment that retains the ability to bind streptococcal protein G. Using this assay procedure, evidence for a multimeric enzyme-substrate complex was obtained as well as identifying isolated heavy chains as a non-substrate inhibitor of IdeS activity. Under appropriate experimental conditions the assay could be used to detect IdeS activity in bacterial culture media or in human plasma without a requirement for purified reactants. The availability of a rapid and sensitive assay for IdeS should facilitate the detailed biochemical characterization of this unusual bacterial cysteine protease.

Use of protein chip mass spectrometry to monitor biotinylation reactions.

Surface-enhanced laser desorption/ionization time-of-flight analysis was used to monitor both the kinetics and heterogeneity of product formation during the biotinylation of a number of model proteins and peptide targets. The selected molecules were the IgG-binding protein, protein A, human serum albumin, and a synthetic peptide corresponding to the N terminus of a streptococcal M1 protein. The extent of biotinylation was determined by kinetic analysis of the shift in molecular mass from the native material. Each residue modified by reaction with N-hydroxysuccinimide biotin resulted in an addition of approximately 341 amu to the native protein or polypeptide. The novelty of the method was in the ability to determine the molecular mass shift, without first separating the targeted molecule from the biotinylating reagent. The analysis was rapid, simple, and provided information on the average number of biotin molecules added and the homogeneity of the resulting product.

Application of immunoproteomics to rapid cytokine detection.

Cytokines are pivotal to a balanced innate or cell-mediated immune response, and can be indicative of disease progression and/or resolution. Methods to measure key cytokines rapidly with accuracy, precision, and sensitivity are consequently important. The current assay technologies, which are based on RT-PCR, immunoassays, or bioassays, are limited in their use in the clinic, in particular because of the long time (1-3 h) required to carry out the assays. An alternative, semi-quantitative approach described here, uses an immunological capture step and a mass spectral readout. The goal of the assay is speed rather than sensitivity or precision.

Practical applications of high-affinity, albumin-binding proteins from a group G streptococcal isolate.

Binding proteins that have high affinities for mammalian plasma proteins that are expressed on the surface of bacteria have proven valuable for the purification and detection of several biologically important molecules from human and animal plasma or serum. In this study, we have isolated a high affinity albumin-binding molecule from a group G streptococcal isolate of bovine origin and have demonstrated that the isolated protein can be biotinylated without loss of binding activity and can be used as a tracer for quantification of human serum albumin (HSA). The binding protein can be immobilized and used as a selective capture reagent in a competitive ELISA format using a biotinylated HSA tracer. In this assay format, the sensitivity of detection for 50% inhibition of binding of HSA was less than 1 microg/ml. When attached to the bacterial surface, this binding protein can be used to deplete albumin from human plasma, as analyzed by surface-enhanced laser desorption ionization time of flight mass spectrometry.

Fibrinogen fragment D is necessary and sufficient to anchor a surface plasminogen-activating complex in Streptococcus pyogenes.

In this study, the importance of different domains of the fibrinogen molecule in the binding and assembly of a surface plasminogen (plgn) activator has been analyzed. This was achieved using SELDI technology that enabled dissociation of bound fragments from intact bacteria and accurate distinction between fibrinogen fragments based on their molecular mass. These studies indicate that Streptococcus pyogenes binds directly to human fibrinogen fragment D but not fragment E. The predominant surface proteins binding to fragment D were associated with the mrp gene product. Surface-associated fibrinogen fragment D was capable of anchoring a functional surface plgn activator complex. Taken together, these data indicated that fragment D of fibrinogen is necessary and sufficient to anchor a plgn activator complex on the surface of Streptococcus pyogenes.

Immunoproteomics.

A novel immunoproteomic assay, combining specificity of antibody with precision of mass spectral analysis is described, and a number of practical applications are presented. The assay is carried out in three steps. The first step of the assay involves antibody immobilization, using a bacterial Fc binding support. The second step is antigen capture and washing to remove non-specific binding. The third step involves analysis of the captured antigens by SELDI-TOF. The assay has many advantages in sensitivity, speed, and economy of reagents in detection of specific antigens or antibodies. In addition, under appropriate experimental conditions, semi-quantitative data may be obtained. By combining the increasing range of selective specific antibody reagents available, in part due to advances in antibody engineering technology, and the resolving power available, using mass spectrometry, immunoproteomics is a valuable technique in proteomic analysis. A number of examples of the application of this technique to analysis of biological systems are presented.

Nephritis-associated plasmin receptor and acute poststreptococcal glomerulonephritis: characterization of the antigen and associated immune response.

The role of nephritis-associated antigen as a virulence factor for acute poststreptococcal glomerulonephritis (APSGN) remains to be fully clarified. Nephritis-associated plasmin receptor (NAPlr) was previously isolated from group A streptococcus (GAS) and shown to bind plasmin(ogen). The nucleotide sequence of the naplr gene from GAS isolates obtained from patients with APSGN was determined. The sequence of the putative open reading frame (1011 bp) showed 99.8% identity among isolated strains. Homology screen revealed an exact match with streptococcal glyceraldehyde-3-phosphate dehydrogenase (GAPDH). NAPlr exhibited GAPDH activity in zymography, and it activated the complement pathway in vitro. In APSGN kidney biopsy specimens, NAPlr was observed mainly in the early stage of the disease (1 to 14 d after onset) but was not colocalized with either C3 or IgG as assessed by double immunofluorescence staining. Sera of patients with APSGN, patients with GAS infection without renal involvement, nonrenal pediatric patients, and healthy adults as controls were assayed for anti-NAPlr antibody titers. Anti-NAPlr antibodies were present most frequently in APSGN sera, and antibody titers were also significantly higher than in patients with GAS infection alone or in other control patients. Moreover, antibody titers remained elevated during the entire 10-yr follow-up period.

Mouse skin passage of Streptococcus pyogenes results in increased streptokinase expression and activity.

The plasminogen activator streptokinase has been proposed to be a key component of a complex mechanism that promotes skin invasion by Streptococcus pyogenes. This study was designed to compare ska gene message and protein levels in wild-type M1 serotype isolate 1881 and a more invasive variant recovered from the spleen of a lethally infected mouse. M1 isolates selected for invasiveness demonstrated enhanced levels of active plasminogen activator activity in culture. This effect was due to a combination of increased expression of the ska gene and decreased expression of the speB gene. The speB gene product, SpeB, was found to efficiently degrade streptokinase in vitro.

Streptococcus pyogenes infection in mouse skin leads to a time-dependent up-regulation of protein H expression.

Streptococcus pyogenes protein H (sph) is an immunoglobulin-binding protein present in the Mga regulon of certain M1 serotype isolates. Although sph is present in many strains, it is frequently not expressed. In this paper we show that protein H was highly expressed after bacteria were injected into the skin of mice and were recovered from the blood, kidney, or spleen at various times postinfection. The percentage of protein H-positive colonies increased with time, reaching 100% in the spleen and kidney within 24 to 72 h postinfection. The up-regulation of sph expression was also observed in a mga mutant.

Regulation of protein H expression in M1 serotype isolates of Streptococcus pyogenes.

Protein H is an immunoglobulin-binding protein expressed by certain M1 serotypes of Streptococcus pyogenes. In a recent study of invasive group A isolates, it was found that none of the 16 M1 serotype isolates analyzed expressed protein H on their surface despite the presence of the protein H gene (sph) in approximately one-third of the isolates. Selection of stable protein H-expressing variants could be achieved by infection of prtH(+) non-expressing strains into a mouse skin and recovering bacteria from the spleen. This effect was independent of the transcription regulator Mga, since a similar effect was noted in an mga(-) mutant. Thus, host passage of S. pyogenes can lead to stable high level expression of Protein H.

Application of immunoproteomics to analysis of post-translational processing of the antiphagocytic M protein of Streptococcus.

Post-translational modification of the antiphagocytic M1 protein of Streptococcus pyogenes can influence its binding properties for human immunoglobulin G subclasses and its invasive potential. Current methods of monitoring this modification event involve N-terminal sequencing and are cumbersome, slow and not amenable to routine analysis. In this study we demonstrate that surface enhanced laser desorption/ionization-time of flight mass spectrometry can be used to monitor modification of the M1 protein by the secreted bacterial cysteine protease, SpeB. This method, when combined with a specific antibody capture step provides a specific, rapid and sensitive assay for key virulence factors of the important human pathogen Streptococcus pyogenes.

Application of immuno-mass spectrometry to analysis of a bacterial virulence factor.

Here we describe a novel antibody-based assay that combines specificity of antibody with precision of mass spectral analysis. The assay is carried out in three steps using a single antigen capture and transfer reagent. The first step of the assay involves antibody immobilization. The second step is antigen capture and washing to remove unbound proteins. The third step involves the analysis of the captured antigens by surface enhanced laser desorption ionization time-of-flight mass spectrometry. The assay is facilitated by the ability of a single nonviable bacterial preparation expressing immunoglobulin-binding proteins that enable antibody immobilization, specific capture of fluid-phase antigen, and direct sample transfer to a protein chip for mass spectral analysis. Proof-of-concept studies using a model Streptococcus pyogenes virulence factor, the secreted cysteine protease SpeB, are presented.

Viral binding proteins as antibody surrogates in immunoassays of cytokines.

Cytokines are pivotal to a balanced innate or cell-mediated immune response, can be indicative of disease progression and/or resolution, and are being evaluated as therapeutics. There is a need to purify and/or to measure key cytokines rapidly with accuracy, precision, and sensitivity. The current assay technologies, which are based on RT-PCR, immunoassays, or bioassays, are limited in their use in the clinic, in particular because of the long time (1-3 h) required to carry out the assays. An alternative approach explored here is the use of pathogen-encoded cytokine-binding proteins, which have Kd in the nanomolar range. It is anticipated that pathogens have evolved binding proteins, antagonists, and/or specific neutralizing phenotypes directed against key signaling and effector molecules involved in the multifaceted host defense system. Thus, by screening the genomes of a wide range of microbial agents, we would expect to find coding sequences for binding proteins for the most important cytokines. Consistent with this view is the identification of poxvirus genes encoding binding activities for TNF type I and type II interferons, interleukin (IL)-1beta, IL-18, and beta-chemokines. These high-affinity receptors have the potential to act as surrogate antibodies in a number of applications in cytokine quantification and purification and could be potentially useful reagents to complement the existing panel of anti-cytokine, monoclonal, polyclonal, or engineered antibodies that are currently available.