Biothreat Agents Research Articles

Single domain antibody–quantum dot conjugates for ricin detection by both fluoroimmunoassay and surface plasmon resonance

The combination of stable biorecognition elements and robust quantum dots (QDs) has the potential to yield highly effective reporters for bioanalyses. Llama-derived single domain antibodies (sdAb) provide small thermostable recognition elements that can be easily manipulated using standard DNA methods. The sdAb was self-assembled on dihydrolipoic acid (DHLA) ligand-capped CdSe–ZnS core–shell QDs made in our laboratory through the polyhistidine tail of the protein, which coordinated to zinc ions on the QD surface. The sdAb–QD bioconjugates were then applied in both fluorometric and surface plasmon resonance (SPR) immunoassays for the detection of ricin, a potential biothreat agent. The sdAb–QD conjugates functioned in fluoroimmunoassays for the detection of ricin, providing equivalent limits of detection when compared to the same anti-ricin sdAb labeled with a conventional fluorophore. In addition, the DHLA-QD–sdAb conjugates were very effective reporter elements in SPR sandwich assays, providing more sensitive detection with a signal enhancement of ∼10-fold over sdAb reporters and 2–4 fold over full sized antibody reporters. Commercially prepared streptavidin-modified polymer-coated QDs also amplified the SPR signal for the detection of ricin when applied to locations where biotinylated anti-ricin sdAb was bound to target; however, we observed a 4-fold greater amplification when using the DHLA-QD–sdAb conjugates in this format.

Recovery and detection of botulinum toxin type A from drinking water

s Toxins 2011 / Toxicon 68 (2013) 60–123 94 Double anchorage to the membrane and intact interchain disulfide bond are required for the lowpH-induced entry of tetanus and botulinum neurotoxins into neurons M. Pirazzini , O. Rossetto , P. Bolognese , C.C. Shone , C. Montecucco a Dipartimento di Scienze Biomediche and Istituto CNR di Neuroscienze, Universita di Padova, Padova, Italy Health Protection Agency, Porton Down, Salisbury, UK E-mail address: marcopiraz@yahoo.it (M. Pirazzini). Purpose of study: To study themembrane translocation of the clostridial neurotoxins in neurons. Methods used: We have developed a protocol based on an experiment first performed with diphtheria toxin. Conditions were found that by-pass the SV endocytosis step and induce the entry of the L chain directly from the plasma membrane. The neurotoxin, bound to the plasma membrane in the cold, was exposed to a warm, low pH extracellular medium, and the entry of the L chain was monitored by measuring its specific metalloprotease activity with a ratiometric method. Summary of results: We found that the neurotoxin has tobebound to themembraneviaat least twoanchorage sites in order for a productive low-pH-induced structural change to take place. In addition, this process can only occur if the single inter-chain disulfide bond is intact. The pH dependence of the conformational change of tetanus neurotoxin and botulinum neurotoxins B, C and D is similar and take places in the same slightly acidic range, which comprises that present inside synaptic vesicles. Furthermore, using PROPKA3.0 software, we found that tetanus and botulinum neurotoxins share a pool of conserved acid residues that are predicted to be protonated in the pH range 4.5-6. Conclusions: Tetanus and botulinum B, C and D neurotoxins undergo to a conformational change in a very similar pH range (4.5-6), which leads to light chain internalization. These neurotoxins share a pool of acidic residues which could be involved in the initial steps of the pHdependent conformational change. http://dx.doi.org/10.1016/j.toxicon.2012.07.099 Recovery and detection of botulinum toxin type A from drinking water B.H. Raphael , M. Lautenschlager , A. Kahler , S. Pai , B.A. Parks , S. Kalb , S. Maslanka , S. Shah , M. Magnuson , V. Hill a Division of Foodborne, Waterborne, and Environmental Diseases, National Center of Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA Division of Laboratory Science, National Center of Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, USA National Homeland Security Research Center, U.S. Environmental Protection Agency, Cincinnati, OH, USA E-mail address: braphael@cdc.gov (B.H. Raphael). Purpose of study: Botulinum neurotoxin (BoNT) represents a possible biothreat agent for drinking water systems. However, a standard method for monitoring drinking water potentially contaminated with BoNT is not currently available to response laboratories. Methods used: Both the botulinum toxin ELISA and Endopep-MS (a mass spectrometric-based endopeptidase method) were adapted to quantify complex BoNT/A in deionized water and in chlorine-demand-free (CDF) water. Summary of results: The method detection limit (MDL) of the ELISA and theMSmethods in CDF water was 500 and 50 pg/ml of BoNT/A complex toxin, respectively. While adapting these methods to municipal tap water samples, residual chlorine in such samples was neutralized with 50 mg/L sodium thiosulfate prior to toxin spiking because free chlorine rapidly (<5 minutes) inactivates BoNT. Prior to testing by either ELISA or MS, municipal tap water samples from 5 U.S. states having distinct water quality properties were processed using ultrafiltration (UF) to concentrate the toxin by up to 200-fold. In order to evaluate the efficiency of toxin concentration using UF, 100 L tap water samples were spiked with 5 ug BoNT/A, which represents a final concentration equivalent to the MDL of the Endopep-MS method (i.e., 50 pg/ml). Recovery efficiencies of the UF method for BoNT/A complex, quantified by ELISA, ranged from 11%-36%, while efficiencies quantified by MS ranged from 26%-55%. Conclusions: Both the botulinum toxin ELISA and the Endopep-MS methods were effective for the quantification of BoNT in municipal tap water samples of varying properties. Additionally, UF was capable of significantly increasing the BoNT/A concentration in tap water samples for analysis by either ELISA or EndopepMS. http://dx.doi.org/10.1016/j.toxicon.2012.07.100 Simultaneous detection and differentiation of botulinum toxin serotypes A and B by internallyquenched fluorogenic peptides

Single domain antibody–alkaline phosphatase fusion proteins for antigen detection — Analysis of affinity and thermal stability of single domain antibody

Single domain antibody (sdAb)–alkaline phosphatase (AP) fusion proteins have been demonstrated to be useful immunodiagnostic reagents for bio-threat agent detection. The bivalent nature of sdAb–AP fusion proteins significantly increases effective affinity and thus the sensitivity of detection, but the thermal stability of the fusion protein had not been explored. This property is critical for the development of immunoassays for use in austere environments. In this study four sdAbs with specificity for MS2 phage coat protein (CP) were expressed as fusions with AP in order to evaluate the thermal stability and affinity of the resulting constructs. The melting temperature (Tm) of the sdAb and sdAb–AP fusion proteins was measured by a combination of Circular Dichroism (CD), differential scanning calorimetry (DSC) and Fluorescence-based Thermal Shift assay. Binding kinetics were assessed using surface plasmon resonance (SPR). Our results indicated that the AP fusion protein did not increase the Tm or enhance thermal stability of the sdAb, but did provide the expected increase in binding affinity as compared to the original sdAb.

Comparative Analysis of Chlamydia psittaci Genomes Reveals the Recent Emergence of a Pathogenic Lineage with a Broad Host Range

ABSTRACTChlamydia psittaci is an obligate intracellular bacterium. Interest in Chlamydia stems from its high degree of virulence as an intestinal and pulmonary pathogen across a broad range of animals, including humans. C. psittaci human pulmonary infections, referred to as psittacosis, can be life-threatening, which is why the organism was developed as a bioweapon in the 20th century and is listed as a CDC biothreat agent. One remarkable recent result from comparative genomics is the finding of frequent homologous recombination across the genome of the sexually transmitted and trachoma pathogen Chlamydia trachomatis. We sought to determine if similar evolutionary dynamics occurred in C. psittaci. We analyzed 20 C. psittaci genomes from diverse strains representing the nine known serotypes of the organism as well as infections in a range of birds and mammals, including humans. Genome annotation revealed a core genome in all strains of 911 genes. Our analyses showed that C. psittaci has a history of frequently switching hosts and undergoing recombination more often than C. trachomatis. Evolutionary history reconstructions showed genome-wide homologous recombination and evidence of whole-plasmid exchange. Tracking the origins of recombinant segments revealed that some strains have imported DNA from as-yet-unsampled or -unsequenced C. psittaci lineages or other Chlamydiaceae species. Three ancestral populations of C. psittaci were predicted, explaining the current population structure. Molecular clock analysis found that certain strains are part of a clonal epidemic expansion likely introduced into North America by South American bird traders, suggesting that psittacosis is a recently emerged disease originating in New World parrots.

Automated thermochemolysis reactor for detection of Bacillus anthracis endospores by gas chromatography–mass spectrometry

An automated sample preparation system was developed and tested for the rapid detection of Bacillus anthracis endospores by gas chromatography–mass spectrometry (GC–MS) for eventual use in the field. This reactor is capable of automatically processing suspected bio-threat agents to release and derivatize unique chemical biomarkers by thermochemolysis (TCM). The system automatically controls the movement of sample vials from one position to another, crimping of septum caps onto the vials, precise delivery of reagents, and TCM reaction times and temperatures. The specific operations of introduction of sample vials, solid phase microextraction (SPME) sampling, injection into the GC–MS system, and ejection of used vials from the system were performed manually in this study, although they can be integrated into the automated system. Manual SPME sampling is performed by following visual and audible signal prompts for inserting the fiber into and retracting it from the sampling port. A rotating carousel design allows for simultaneous sample collection, reaction, biomarker extraction and analysis of sequential samples. Dipicolinic acid methyl ester (DPAME), 3-methyl-2-butenoic acid methyl ester (a fragment of anthrose) and two methylated sugars were used to compare the performance of the autoreactor with manual TCM. Statistical algorithms were used to construct reliable bacterial endospore signatures, and 24 out of 25 (96%) endospore-forming Bacillus species were correctly identified in a statistically designed test.

Rapid Focused Sequencing: A Multiplexed Assay for Simultaneous Detection and Strain Typing of Bacillus anthracis, Francisella tularensis, and Yersinia pestis

BackgroundThe intentional release of Bacillus anthracis in the United States in 2001 has heightened concern about the use of pathogenic microorganisms in bioterrorism attacks. Many of the deadliest bacteria, including the Class A Select Agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis, are highly infectious via the pulmonary route when released in aerosolized form. Hence, rapid, sensitive, and reliable methods for detection of these biothreats and characterization of their potential impact on the exposed population are of critical importance to initiate and support rapid military, public health, and clinical responses.Methodology/Principal FindingsWe have developed microfluidic multiplexed PCR and sequencing assays based on the simultaneous interrogation of three pathogens per assay and ten loci per pathogen. Microfluidic separation of amplified fluorescently labeled fragments generated characteristic electrophoretic signatures for identification of each agent. The three sets of primers allowed significant strain typing and discrimination from non-pathogenic closely-related species and environmental background strains based on amplicon sizes alone. Furthermore, sequencing of the 10 amplicons per pathogen, termed “Rapid Focused Sequencing,” allowed an even greater degree of strain discrimination and, in some cases, can be used to determine virulence. Both amplification and sequencing assays were performed in microfluidic biochips developed for fast thermal cycling and requiring 7 µL per reaction. The 30-plex sequencing assay resulted in genotypic resolution of 84 representative strains belonging to each of the three biothreat species.Conclusions/SignificanceThe microfluidic multiplexed assays allowed identification and strain differentiation of the biothreat agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis and clear discrimination from closely-related species and several environmental background strains. The assays may be extended to detect a large number of pathogens, are applicable to the evaluation of both environmental and clinical samples, and have the potential to be applied in military, public health, and clinical diagnostic settings.

Optimizing Protein Coordination to Quantum Dots with Designer Peptidyl Linkers

Semiconductor quantum dots (QDs) demonstrate select optical properties that make them of particular use in biological imaging and biosensing. Controlled attachment of biomolecules such as proteins to the QD surface is thus critically necessary for development of these functional nanobiomaterials. QD surface coatings such as poly(ethylene glycol) impart colloidal stability to the QDs, making them usable in physiological environments, but can impede attachment of proteins due to steric interactions. While this problem is being partially addressed through the development of more compact QD ligands, here we present an alternative and complementary approach to this issue by engineering rigid peptidyl linkers that can be appended onto almost all expressed proteins. The linkers are specifically designed to extend a terminal polyhistidine sequence out from the globular protein structure and penetrate the QD ligand coating to enhance binding by metal-affinity driven coordination. α-Helical linkers of two lengths terminating in either a single or triple hexahistidine motif were fused onto a single-domain antibody; these were then self-assembled onto QDs to create a model immunosensor system targeted against the biothreat agent ricin. We utilized this system to systematically evaluate the peptidyl linker design in functional assays using QDs stabilized with four different types of coating ligands including poly(ethylene glycol). We show that increased linker length, but surprisingly not added histidines, can improve protein to QD attachment and sensor performance despite the surface ligand size with both custom and commercial QD preparations. Implications for these findings on the development of QD-based biosensors are discussed.

High‐Throughput, Cell‐Based Screens to Identify Small‐Molecule Inhibitors of Ricin Toxin and Related Category B Ribosome Inactivating Proteins (RIPs)

Ricin is a member of the ubiquitous ribosome-inactivating protein (RIP) family of toxins. The Centers for Disease Control and Prevention (CDC) classify ricin and related toxins as Category B biothreat agents. There are currently no antidotes or therapeutics to counteract RIPs in humans. The discovery of effective small-molecule inhibitors of RIPs is increasingly possible, however, due to the availability and accessibility of diverse small-molecule chemical libraries coupled with robust robotics and automated screening methodologies. In this article, we describe a cell-based, high-throughput screening strategy and secondary assays that we have successfully used to identify compounds that target ricin toxin's enzymatic activity and intracellular trafficking, as well as stress-activated signaling pathways associated with cell death. The methods described in the protocol are amenable to the other RIPs.

Development of a Panel of Recombinase Polymerase Amplification Assays for Detection of Biothreat Agents

Syndromic panels for infectious disease have been suggested to be of value in point-of-care diagnostics for developing countries and for biodefense. To test the performance of isothermal recombinase polymerase amplification (RPA) assays, we developed a panel of 10 RPAs for biothreat agents. The panel included RPAs for Francisella tularensis, Yersinia pestis, Bacillus anthracis, variola virus, and reverse transcriptase RPA (RT-RPA) assays for Rift Valley fever virus, Ebola virus, Sudan virus, and Marburg virus. Their analytical sensitivities ranged from 16 to 21 molecules detected (probit analysis) for the majority of RPA and RT-RPA assays. A magnetic bead-based total nucleic acid extraction method was combined with the RPAs and tested using inactivated whole organisms spiked into plasma. The RPA showed comparable sensitivities to real-time RCR assays in these extracts. The run times of the assays at 42°C ranged from 6 to 10 min, and they showed no cross-detection of any of the target genomes of the panel nor of the human genome. The RPAs therefore seem suitable for the implementation of syndromic panels onto microfluidic platforms.

Population Analysis of Bacterial Samples for Individual Identification in Forensics Application

Biodefense preparedness begins with the ability to detect and respond to bio-threats, based on accurate interpretation of genetic information with sophisticated, yet easy-to-use bioinformatics tools. Microbial forensics further enables attribution of microbial pathogen samples back to a suspected source. Sample characterization and traceability back to source are dependent on genome identification of specific targets within samples, comprehensive analysis of mixtures of populations’ present, and detection of major/minor variations in the identified genomes and comparison of sample genetic profile against other samples. Commercial Next Generation Sequencing (NGS) platforms offer the promise of dramatically higher detection sensitivity and resolution of forensic DNA samples than is possible with methods in current use. Before applying these technologies for forensic analyses of bacterial samples, however, it is critical to fully elucidate the benefits, caveats and pitfalls of NGS for hypothesis testing in comparative analyses, as ultimately this will be required for NGS use both as an investigative tool and tool for attribution in courts of law. Methods: We developed and evaluated novel probabilistic algorithms to process metagenomic sequence data from direct sample sequencing to identify genomes present in mixtures. Results: We present a pipeline for reference-free sample-to-sample comparisons to improve target characterization beyond one microorganism to characterization of comprehensive sample content. Our tools strengthen statistical confidence to trace the ancestry of samples and attribute samples to source with probabilistic certainties on many targets instead of a single genome. Conclusion: This study developed a novel reference free, bioinformatics strategy to account for and identify genetic diversity in samples. Sequence variants must be non-arbitrarily confirmed in both forward and reverse reads at a rate above the background noise level of sequencer machine error. A similarity distance metric compares genomes within a range of near relationships. Using sequence data from bio-threat agents, we successfully attributed known related strains together, and excluded near relation of known unrelated strains. The major strengths of this forensic method are the non-arbitrary determinations of data validation and relatedness metrics, as well as the ability to compare microbial genomes with or without a reference database of related genomes.

Pre-PCR Processing in Bioterrorism Preparedness: Improved Diagnostic Capabilities for Laboratory Response Networks

Diagnostic DNA analysis using polymerase chain reaction (PCR) has become a valuable tool for rapid detection of biothreat agents. However, analysis is often challenging because of the limited size, quality, and purity of the biological target. Pre-PCR processing is an integrated concept in which the issues of analytical limit of detection and simplicity for automation are addressed in all steps leading up to PCR amplification--that is, sampling, sample treatment, and the chemical composition of PCR. The sampling method should maximize target uptake and minimize uptake of extraneous substances that could impair the analysis--so-called PCR inhibitors. In sample treatment, there is a trade-off between yield and purity, as extensive purification leads to DNA loss. A cornerstone of pre-PCR processing is to apply DNA polymerase-buffer systems that are tolerant to specific sample impurities, thereby lowering the need for expensive purification steps and maximizing DNA recovery. Improved awareness among Laboratory Response Networks (LRNs) regarding pre-PCR processing is important, as ineffective sample processing leads to increased cost and possibly false-negative or ambiguous results, hindering the decision-making process in a bioterrorism crisis. This article covers the nature and mechanisms of PCR-inhibitory substances relevant for agroterrorism and bioterrorism preparedness, methods for quality control of PCR reactions, and applications of pre-PCR processing to optimize and simplify the analysis of various biothreat agents. Knowledge about pre-PCR processing will improve diagnostic capabilities of LRNs involved in the response to bioterrorism incidents.

Aptamers—A Promising Approach for Sensing of Biothreats Using Different Bioinformatics Tools

Intentional release of pathogens or biotoxin against humans, plants, or animals is an impending threat all over the world. Continuous monitoring of environment is required for their detection. These signals can help to distinguish whether the bioattack has occurred or not. Biosensors utilise biological response including different biochemical reactions, antigen antibody reactions, electrochemical reactions, aptameric reactions etc. The currently available biosensors have a limit of detection, specificity and less linearity which affect their sensitivity. Aptamers are single stranded oligonucleotides binding species which are capable of tightly binding to their distinguishing targets. They are evolved from random oligonucleotides pools by using different strategies. These are capable of conscientiously distinguishing their target ligands. They have high sensitivity and a wide range of detection limit. The versatility of nucleic acid based methods allowed for the design of specific aptamer sequences, typically on the order of 10 to 30 base pairs in length, identifying the different biothreat agents in the environment. By using different bioinformatics tools we can design RNA aptamers for toxins of lectin family.

Need for Unambiguous, Multiplex Detection Platforms for Biothreat Agents

Need for Unambiguous, Multiplex Detection Platforms for Biothreat Agents

Conformation-Dependent High-Affinity Potent Ricin-Neutralizing Monoclonal Antibodies

Ricin is a potential biothreat agent with no approved antidote available for ricin poisoning. The aim of this study was to develop potent antibody-based antiricin antidotes. Four strong ricin resistant hybridoma clones secreting antiricin monoclonal antibodies (mAbs) were developed. All four mAbs are bound to conformational epitopes of ricin toxin B (RTB) with high affinity (K D values from 2.55 to 36.27 nM). RTB not only triggers cellular uptake of ricin, but also facilitates transport of the ricin toxin A (RTA) from the endoplasmic reticulum to the cytosol, where RTA exerts its toxic activity. The four mAbs were found to have potent ricin-neutralizing capacities and synergistic effects among them as determined by an in vitro neutralization assay. In vivo protection assay demonstrated that all four mAbs had strong efficacy against ricin challenges. D9 was found to be exceptionally effective. Intraperitoneal (i.p.) administration of D9, at a dose of 5 μg, 6 weeks before or 6 hours after an i.p. challenge with 5 × LD50 of ricin was able to protect or rescue 100% of the mice, indicating that mAb D9 is an excellent candidate to be developed as a potent antidote against ricin poisoning for both prophylactic and therapeutic purposes.

A Mucosal Subunit Vaccine Protects against Lethal Respiratory Infection with Francisella tularensis LVS

Francisella tularensis (FT) is a highly virulent pathogen for humans and other mammals. Severe morbidity and mortality is associated with respiratory FT infection and there are concerns about intentional dissemination of this organism. Therefore, FT has been designated a category A biothreat agent and there is a growing interest in the development of a protective vaccine. In the present study, we determine the protective potential of a subunit vaccine comprised of the FT heat shock protein DnaK and surface lipoprotein Tul4 against respiratory infection with the live vaccine strain (LVS) of FT in mice. First, we establish an optimal intranasal immunization regimen in C57BL/6 mice using recombinant DnaK or Tul4 together with the adjuvant GPI-0100. The individual immunization regimens induced robust salivary IgA, and vaginal and bronchoalveolar IgA and IgG antigen-specific antibodies. Serum IgG1 and IgG2c antibody responses were also induced, indicative of a mixed type 2 and type 1 response, respectively. Next, we show that immunization with DnaK and Tul4 induces mucosal and systemic antibody responses that are comparable to that seen following immunization with each antigen alone. This immunization regimen also induced IFN-γ, IL-10 and IL-17A production by splenic CD4+ T cells in an antigen-specific manner. Importantly, over 80% of the mice immunized with DnaK and Tul4, but not with each antigen alone, were protected against a lethal respiratory challenge with FT LVS. Protection correlated with reduced bacterial burden in the lung, liver and spleen of mice. This study demonstrates the potential of DnaK and Tul4 as protective antigens and lends support to the notion of combining distinct, immunodominant antigens into an effective multivalent tularemia vaccine.

Lipophilic Prodrugs of FR900098 Are Antimicrobial against Francisella novicida In Vivo and In Vitro and Show GlpT Independent Efficacy

Bacteria, plants, and algae produce isoprenoids through the methylerythritol phosphate (MEP) pathway, an attractive pathway for antimicrobial drug development as it is present in prokaryotes and some lower eukaryotes but absent from human cells. The first committed step of the MEP pathway is catalyzed by 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR/MEP synthase). MEP pathway genes have been identified in many biothreat agents, including Francisella, Brucella, Bacillus, Burkholderia, and Yersinia. The importance of the MEP pathway to Francisella is demonstrated by the fact that MEP pathway mutations are lethal. We have previously established that fosmidomycin inhibits purified MEP synthase (DXR) from F. tularensis LVS. FR900098, the acetyl derivative of fosmidomycin, was found to inhibit the activity of purified DXR from F. tularensis LVS (IC50 = 230 nM). Fosmidomycin and FR900098 are effective against purified DXR from Mycobacterium tuberculosis as well, but have no effect on whole cells because the compounds are too polar to penetrate the thick cell wall. Fosmidomycin requires the GlpT transporter to enter cells, and this is absent in some pathogens, including M. tuberculosis. In this study, we have identified the GlpT homologs in F. novicida and tested transposon insertion mutants of glpT. We showed that FR900098 also requires GlpT for full activity against F. novicida. Thus, we synthesized several FR900098 prodrugs that have lipophilic groups to facilitate their passage through the bacterial cell wall and bypass the requirement for the GlpT transporter. One compound, that we termed “compound 1,” was found to have GlpT-independent antimicrobial activity. We tested the ability of this best performing prodrug to inhibit F. novicida intracellular infection of eukaryotic cell lines and the caterpillar Galleria mellonella as an in vivo infection model. As a lipophilic GlpT-independent DXR inhibitor, compound 1 has the potential to be a broad-spectrum antibiotic, and should be effective against most MEP-dependent organisms.

Structural Framework for Covalent Inhibition of Clostridium botulinum Neurotoxin A by Targeting Cys165

Clostridium botulinum neurotoxin type A (BoNT/A) is one of the most potent toxins for humans and a major biothreat agent. Despite intense chemical efforts over the past 10 years to develop inhibitors of its catalytic domain (catBoNT/A), highly potent and selective inhibitors are still lacking. Recently, small inhibitors were reported to covalently modify catBoNT/A by targeting Cys(165), a residue located in the enzyme active site just above the catalytic zinc ion. However, no direct proof of Cys(165) modification was reported, and the poor accessibility of this residue in the x-ray structure of catBoNT/A raises concerns about this proposal. To clarify this issue, the functional role of Cys(165) was first assessed through a combination of site-directed mutagenesis and structural studies. These data suggested that Cys(165) is more involved in enzyme catalysis rather than in structural property. Then by peptide mass fingerprinting and x-ray crystallography, we demonstrated that a small compound containing a sulfonyl group acts as inhibitor of catBoNT/A through covalent modification of Cys(165). The crystal structure of this covalent complex offers a structural framework for developing more potent covalent inhibitors catBoNT/A. Other zinc metalloproteases can be founded in the protein database with a cysteine at a similar location, some expressed by major human pathogens; thus this work should find broader applications for developing covalent inhibitors.

Epitope mapping of botulinum neurotoxins light chains

Botulinum neurotoxins (BoNTs) are listed among the most potent biothreat agents. Simultaneously, two out of seven known serotypes of these toxins are used in medicine and cosmetics. This situation calls for development of detailed epitope maps of these toxins. Such maps will help to develop new ways for decreasing damage caused by these toxins if they were to be used as weapons while retaining the therapeutic effect of these toxins used as medicine. Here, we used a library of random fragments of DNA encoding the catalytic domain of botulinum neurotoxin serotype A to identify short epitope-forming sequences. We demonstrated that knowledge of such sequences in a BoNT of one serotype can be used for identification of epitope-forming sequences in other serotypes of BoNTs. We also demonstrated a serodiagnostic value of identified sequences and their ability to retain epitope-specific structures and trigger production of corresponding antibodies, even when they are transferred into a background of a completely alien carrier protein.

Comparison of genome diversity of Brucella spp. field isolates using Universal Bio-signature Detection Array and whole genome sequencing reveals limitations of current diagnostic methods

The detection and identification of bio-threat agents and the study of host-pathogen interactions require a high-resolution detection platform capable of discerning closely related species. Diverse analysis methods are used to identify pathogens, specifically Brucella species or biovars. In this study, we compared four diagnostic approaches including serology-based biochemical test, PCR assay, microarray analysis using a Universal Bio-signature Detection Array (UBDA) and whole genome “deep” sequencing for Brucella organisms including a number of field isolates. We found that although there was frequent agreement among the different tests, some tests gave compound/contradictory results that were a consequence of species diversity due to mixed infections or minor contaminants as measured by UBDA and validated from whole genome sequence. By comparing these analysis techniques, we demonstrate that standard diagnostics used in the field are limited in their ability to identify genomic DNA contaminants in field isolates while UBDA and sequencing analysis are highly sensitive in tracing genomic differences among the isolates.

A multiplex real-time PCR assay for the detection and differentiation of Francisella tularensis subspecies

Francisella tularensis is the aetiological agent of tularaemia, a zoonotic disease with worldwide prevalence. F. tularensis is a highly pathogenic organism and has been designated a category A biothreat agent by the Centers for Disease Control and Prevention. Tularaemia is endemic in much of the USA, Europe and parts of Asia. It is transmitted by numerous vectors and vehicles such as deer flies, ticks and rabbits. Currently, there are four recognized subspecies of F. tularensis: tularensis (type A), holarctica (type B), mediasiatica and novicida. Within the type A classification there are two subclassifications, type A.I and A.II, each with a specific geographical distribution across the USA. F. tularensis subsp. holartica (type B) is found in both the USA and Europe. Because of virulence differences among subtypes, it is important that health departments, hospitals and other government agencies are able to quickly identify each subtype. The purpose of this study was to develop a multiplex real-time PCR assay for the identification and discrimination of type A.I, type A.II, type B and novicida subspecies of F. tularensis. The assay was validated using 119 isolates of F. tularensis, three of its nearest neighbours and 14 other bacterial pathogens. This assay proved to be ~98 % successful at identifying the known subspecies of F. tularensis and could prove to be a useful tool in the characterization of this important pathogen.