Pathogen lights: Fast-testing for food safety [Slides]
This report explains foodborne illnesses and how to apply Pathogen Light for food safety testing.
36 MATERIALS SCIENCE↗
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Ultrasensitive microanalytical diagnostic methods for rickettsial pathogens
A strategic CRADA was established between Sandia National Laboratories (SNL) and the University of Texas Medical Branch (UTMB) at Galveston to address pressing needs for US protection against biological weapons of mass destruction (WMD) and emerging infectious diseases. The combination of unique expertise and facilities at UTMB and SNL enabled interdisciplinary research efforts in the development of rapid and accurate diagnostic methods for early detection of trace priority pathogen levels. Outstanding postdoctoral students were also trained at both institutions to help enable the next generation of scientists to tackle the challenging interdisciplinary problems in the area of biodefense and emerging infectious diseases. Novel approaches to diagnostics were developed and the both the speed of assays as well as the detection sensitivity were improved by over an order of magnitude compared to traditional methods. This is a significant step toward more timely and specific detection of dangerous infections. We developed in situ polymerized porous polymer monoliths that can be used as (1) size exclusion elements for capture and processing of rickettsial infected cells from a sample, (2) photopatternable framework for grafting high densities of functionalized antibodies/fluorescent particles using novel monolith chemistry. Grafting affinity reagents specific to rickettsial particles enables rapid, ultra-sensitive assays by overcoming transport limitations of traditional planar assay approaches. We have selectively trapped particles and bacteria at the cell trap and have also detected picomolar mouse IL-6 captured with only 20 minutes total incubation times using the densely patterned monolith framework. As predicted, the monolith exhibits >10x improvements in both capture speed and capture density compared to traditional planar approaches. The most significant advancements as part of this CRADA is the optimization of techniques allowing the detection of <10 rickettsial cells in a whole blood sample. This detection limit is over 2 orders of magnitude more sensitive that previously reported methods and overcomes a key hurdle in ability to sense dangerous infections before they are too late to treat and contain. We also showed that in the new format, cross-reactivity with interfering species is reduced thereby increasing the specificity of such tests. Promising options to treat whole blood and avoid clogging and non-specific fouling of sensors were also developed.
45 MILITARY TECHNOLOGY, WEAPONRY, AND NATIONAL DEF↗
RapTOR: Automated Sequencing Library Preparation and Suppression for Rapid Pathogen Characterization (7th Annual SFAF Meeting, 2012)
Todd Lane on "RapTOR: Automated sequencing library preparation and suppression for rapid pathogen characterization" at the 2012 Sequencing, Finishing, Analysis in the Future Meeting held June 5-7, 2012 in Santa Fe, New Mexico.
59 BASIC BIOLOGICAL SCIENCES↗
Experimental Reservoirs of Human Pathogens: The Vibrio Cholerae Paradigm (7th Annual SFAF Meeting, 2012)
Rita Colwell on "Experimental Reservoirs of Human Pathogens: The Vibrio cholerae paradigm" at the 2012 Sequencing, Finishing, Analysis in the Future Meeting held June 5-7, 2012 in Santa Fe, New Mexico.
59 BASIC BIOLOGICAL SCIENCES↗
Plague: The Emergence of a Highly Fit Clonal Pathogen that Altered the History of Mankind (7th Annual SFAF Meeting, 2012)
Paul Keim on "Plague: A Highly Fit Clonal Pathogen Emerges and Shapes Human History" at the 2012 Sequencing, Finishing, Analysis in the Future Meeting held June 5-7, 2012 in Santa Fe, New Mexico.
59 BASIC BIOLOGICAL SCIENCES↗
3-minute diagnosis: Researchers develop new method to recognize pathogens
Imagine knowing precisely why you feel sick ... before the doctor's exam is over. Lawrence Livermore researcher Reg Beer and his engineering colleagues have developed a new method to recognize disease-causing pathogens quicker than ever before.
59 BASIC BIOLOGICAL SCIENCES↗
Structural and biochemical analysis of the metallo-β-lactamase L1 from emerging pathogen Stenotrophomonas maltophilia revealed the subtle but distinct di-metal scaffold for catalytic activity
Emergence of Enterobacteriaceae harboring metallo-beta-lactamases (MBL) has raised global threats due to their broad antibiotic resistance profiles and the lack of effective inhibitors against them. We have been studied one of the emerging environmental MBL, the L1 from Stenotrophomonas maltophilia K279a . We determined several crystal structures of L1 complexes with three different classes of beta-lactam antibiotics (penicillin G, moxalactam, meropenem, and imipenem), with the inhibitor captopril and different metal ions (Zn +2 , Cd +2 , and Cu +2 ). All hydrolyzed antibiotics and the inhibitor were found binding to two Zn +2 ions mainly through the opened lactam ring and some hydrophobic interactions with the binding pocket atoms. Without a metal ion, the active site is very similarly maintained as that of the native form with two Zn +2 ions, however, the protein does not bind the substrate moxalactam. When two Zn +2 ions were replaced with other metal ions, the same di-metal scaffold was maintained and the added moxalactam was found hydrolyzed in the active site. Differential scanning fluorimetry and isothermal titration calorimetry were used to study thermodynamic properties of L1 MBL compared with New Deli Metallo-beta-lactamase-1 (NDM-1). Both enzymes are significantly stabilized by Zn +2 and other divalent metals but showed different dependency. These studies also suggest that moxalactam and its hydrolyzed form may bind and dissociate with different kinetic modes with or without Zn +2 for each of L1 and NDM-1. Our analysis implicates metal ions, in forming a distinct di-metal scaffold, which is central to the enzyme stability, promiscuous substrate binding and versatile catalytic activity. Statement: The L1 metallo-beta-lactamase from an environmental multidrug-resistant opportunistic pathogen Stenotrophomonas maltophilia K279a has been studied by determining 3D structures of L1 enzyme in the complexes with several beta-lactam antibiotics and different divalent metals and characterizing its biochemical and ligand binding properties. We found that the two-metal center in the active site is critical in the enzymatic process including antibiotics recognition and binding, which explains the enzyme's activity toward diverse antibiotic substrates. This study provides the critical information for understanding the ligand recognition and for advanced drug development.
59 BASIC BIOLOGICAL SCIENCES↗
Metabolic engineering of non-pathogenic microorganisms for 2,3-butanediol production
2,3-Butanediol (2,3-BDO) is a promising commodity chemical with various industrial applications. While petroleum-based chemical processes currently dominate the industrial production of 2,3-BDO, fermentation-based production of 2,3-BDO pro- vides an attractive alternative to chemical-based processes with regards to economic and environmental sustainability. The achievement of high 2,3-BDO titer, yield, and productivity in microbial fermentation is a prerequisite for the production of 2,3-BDO at large scales. Also, enantiopure production of 2,3-BDO production is desirable because 2,3-BDO stereoisomers have unique physicochemical properties. Pursuant to these goals, many metabolic engineering strategies to improve 2,3-BDO pro- duction from inexpensive sugars by Klebsiella oxytoca, Bacillus species, and Saccharomyces cerevisiae have been developed. Furthermore, this review summarizes the recent advances in metabolic engineering of non-pathogenic microorganisms to enable efficient and enantiopure production of 2,3-BDO.
2,3-BDO stereoisomers↗
Potential biological control agents of Geosmithia morbida restrict fungal pathogen growth via mycoparasitism and antibiosis
Abstract Thousand cankers disease of Juglans (walnut) and Pterocarya (wingnut) spp. (Fagales: Juglandaceae) is caused by the fungal pathogen Geosmithia morbida Kolarík, Freeland, Utley, and Tisserat (Hypocreales: Bionectriaceae) and bark beetle pest/vector, Pityophthorus juglandis Blackman (Coleoptera: Curculionidae). To further the development of biological management strategies for thousand cankers disease, we assessed the ability of 14 endophytic Trichoderma (Hypocreales: Hypocreaceae) isolates and the commercially available isolate T. afroharzianum strain KRL-AG2 to inhibit the in vitro growth of three different G. morbida isolates via mycoparasitism and antibiosis. To identify factors that may affect field success of candidate biological control agents, we quantified the growth responses of Trichoderma spp. and the commercially available entomopathogenic fungus, Beauveria bassiana (Bals.-Criv.) Vuill. (Hypocreales: Cordycipitaceae) strain GHA, to the plant secondary metabolite and antimicrobial compound, juglone in vitro. A total of 12 Trichoderma isolates (from six different Trichoderma species) demonstrated antagonistic activity towards G. morbida in dual-plate assays. Juglone consistently reduced the growth of B. bassiana strain GHA and 14 of the 15 screened Trichoderma isolates in vitro. Additionally, one metabolite-producing Trichoderma isolate, TN4-47, completely inhibited the growth of all three G. morbida isolates across all tested metabolite concentrations and had comparatively greater tolerance to juglone compared to other Trichoderma isolates. Future lines of research should focus on characterizing the active antagonistic compound present in the metabolite filtrates, determine the mode of action of the active component(s), and elucidate how abiotic and biotic factors may influence the growth, persistence, and antagonistic activity of candidate biological control agents in planta .
59 BASIC BIOLOGICAL SCIENCES↗
Cryo-EM of the pathogenic VCP variant R155P reveals long-range conformational changes in the D2 ATPase ring
Single amino acid mutations in valosin containing protein (VCP/p97), a highly conserved member of the ATPases associated with diverse cellular activities (AAA) family of ATPases has been linked to a severe degenerative disease affecting brain, muscle and bone tissue. Previous studies have demonstrated the role of VCP mutations in altering the ATPase activity of the D2 ring; however the structural consequences of these mutations remain unclear. In this study, we report the three-dimensional (3D) map of the pathogenic VCP variant, R155P, as revealed by single-particle Cryo-Electron Microscopy (EM) analysis at 14 Å resolution. We show that the N-terminal R155P mutation induces a large structural reorganisation of the D2 ATPase ring. Results from docking studies using crystal structure data of available wild-type VCP in the EM density maps indicate that the major difference is localized at the interface between two protomers within the D2 ring. Consistent with a conformational change, the VCP R155P variant shifted the isoelectric point of the protein and reduced its interaction with its well-characterized cofactor, nuclear protein localization-4 (Npl4). Together, our results demonstrate that a single amino acid substitution in the N-terminal domain can relay long-range conformational changes to the distal D2 ATPase ring. Our results provide the first structural clues of how VCP mutations may influence the activity and function of the D2 ATPase ring. - Highlights: • p97{sub R155P} and p97{sub A232E} decrease the ability of p97 to bind to its co-factor Npl4. • p97{sub R155P} has a different isoelectric point than that of p97{sub R95G}, p97{sub A232E} and p97{sub WT}. • Mutation R155P changes principally the conformation of the D2 ring. • Mutation R155P modifies the interface between two protomers within the D2 ring.
60 APPLIED LIFE SCIENCES↗
A new comprehensive method for detection of livestock-related pathogenic viruses using a target enrichment system
Highlights: • Development of a comprehensive method to detect viruses using SureSelect system. • A method by which almost the full length of the viral genome can be determined without virus isolation. • Widely available methods for comprehensive analysis of virus genomic information. We tested usefulness of a target enrichment system SureSelect, a comprehensive viral nucleic acid detection method, for rapid identification of viral pathogens in feces samples of cattle, pigs and goats. This system enriches nucleic acids of target viruses in clinical/field samples by using a library of biotinylated RNAs with sequences complementary to the target viruses. The enriched nucleic acids are amplified by PCR and subjected to next generation sequencing to identify the target viruses. In many samples, SureSelect target enrichment method increased efficiencies for detection of the viruses listed in the biotinylated RNA library. Furthermore, this method enabled us to determine nearly full-length genome sequence of porcine parainfluenza virus 1 and greatly increased Breadth, a value indicating the ratio of the mapping consensus length in the reference genome, in pig samples. Our data showed usefulness of SureSelect target enrichment system for comprehensive analysis of genomic information of various viruses in field samples.
60 APPLIED LIFE SCIENCES↗
Vaccine potential of ESAT-6 protein fused with consensus CD4+ T-cell epitopes of PE/PPE proteins against highly pathogenic Mycobacterium tuberculosis strain HN878
Pro-Glu/Pro-Pro-Glu (PE/PPE) family proteins in Mycobacterium tuberculosis (Mtb) are contributors to pathogenesis and immune evasion. These proteins have a unique structure in which the sequence is conserved. We investigated the vaccine potential of ESAT-6 fused with consensus CD4{sup +} T-cell epitopes of PE/PPE proteins against highly pathogenic Mtb strain HN878 in a murine model. We selected consensus CD4{sup +} T-cell epitopes of PE/PPE proteins by multiple alignments, investigated their IFN-γ response during Mtb infection, and produced their fused ESAT-6 vaccine antigens. Our results showed an increased immune response in PE/PPE peptide -ESAT-6 fusion protein immunization group compared to ESAT-6 only immunization group. After challenge with Mtb strain HN878, we observed that induced CD4{sup +} T-cells secreted double-positive cytokine IL-2{sup +}/IFN-γ{sup +}, which is considered to be associated with protective T-cell immunity. Additionally, lower numbers of colony-forming units were observed in the spleen of fusion protein immunization groups than in those of single ESAT-6 group. Therefore, conjugation of consensus CD4{sup +} T-cell epitopes in N terminus of PE/PPE to vaccine antigens could potentially increase the protective efficacy of subunit vaccine.
60 APPLIED LIFE SCIENCES↗
Differences in spore size and atmospheric survival shape stark contrasts in the dispersal dynamics of two closely related fungal pathogens
A frequently ignored but critical aspect of microbial dispersal is survival in the atmosphere. Here, we exposed spores of two closely related, morphologically dissimilar, and economically important fungal pathogens to typical atmospheric environments and modeled their movement in the troposphere. Alternaria solani conidia are nearly 10 times larger than A. alternata conidia, but in our experiments, most died within 24 h, while over half of A. alternata conidia remained viable on day 12. Next, we modeled the movement of spores across North America. We predict 99% of the larger A. solani conidia settle within 24 h, with a maximum dispersal distance of 100 km. By contrast, most A. alternata conidia remain airborne for more than 12 days, and dispersal over long distances(2000 km) is likely. Counterintuitively, the larger A. solani conidia survive poorly, as compared to smaller A. alternata conidia, but also land sooner and move over shorter distances.
59 BASIC BIOLOGICAL SCIENCES↗
Identification and characterization of mono- and bifunctional galactan synthases in the pediatric pathogen Kingella kingae
The emerging pediatric pathogen Kingella kingae elaborates a lipopolysaccharide (LPS) that is extended with a galactofuranose homopolymer called galactan, which is a key virulence determinant that contributes to resistance to complement-mediated and neutrophil-mediated killing. Previous work has demonstrated that the pamABCDE locus is required for galactan synthesis. In this study, mutational studies suggested that the pamC gene product is a UDP-galactofuranose (Galf) transferase and is the galactan synthase. Analysis of genome sequence data revealed two distinct pamC alleles designated pamC1 and pamC2, which correlate with the two galactan structures in K. kingae. Examination of isogenic mutants expressing either pamC1 or pamC2 demonstrated that the pamC alleles are the determinants of galactan structure. Experiments with recombinant PamC1 and PamC2 in vitro established that these proteins are galactan synthases capable of extending synthetic Galf disaccharide acceptors in the presence of UDP-Galf. Homology analysis identified critical amino acids that are essential for PamC1 and PamC2 enzymatic activity both in vitro and in K. kingae. Structural analysis of the in vitro-modified synthetic acceptors implicated PamC1 as a monofunctional enzyme capable of generating a β-(1 → 5) Galf linkage and PamC2 as a bifunctional enzyme capable of generating β-(1 → 3) and β-(1 → 6) Galf linkages. This study advances our understanding of the GT2 family of UDP-galactofuranosyltransferases.
60 APPLIED LIFE SCIENCES↗
Molecular characterization, phylogeny analysis and pathogenicity of a Muscovy duck adenovirus strain isolated in China in 2014
This study aimed to characterize a novel adenovirus (AdV) isolated from diseased Muscovy ducks in China. After the AdV was successfully propagated in duck embryo fibroblasts, the morphological and physicochemical properties of the virions were studied by electron microscopy and different tests. The results of the analyses were in conformity with AdV properties. The full genome sequence was determined and analyzed. The new isolate (named CH-GD-12-2014) shared over 91% sequence identity with duck AdV-2 representing the species Duck aviadenovirus B. The most important distinguishing feature between the two DAdV strains was the presence of a second fiber gene in the Chinese isolate. Phylogeny reconstruction confirmed the affiliation of the virus with goose and duck AdVs in the genus Aviadenovirus. Experimental infection resulted in embryo death, and intramuscular inoculation provoked morbidity and mortality among ducks and chickens. - Highlights: • A duck adenovirus type 3 was isolated and the complete genome of DAdV-3 was obtained. • Physicochemical properties and electron microscopy were researched. • Pathogenicity of duck adenovirus type 3 was researched.
60 APPLIED LIFE SCIENCES↗
Targeting Bacillosamine Biosynthesis in Bacterial Pathogens: Development of Inhibitors to a Bacterial Amino-Sugar Acetyltransferase from Campylobacter jejuni
The glycoproteins of selected microbial pathogens often include highly modified carbohydrates such as 2,4-diacetamidobacillosamine (diNAcBac). These glycoconjugates are involved in host–cell interactions and may be associated with the virulence of medically significant Gram-negative bacteria. In light of genetic studies demonstrating the attenuated virulence of bacterial strains in which modified carbohydrate biosynthesis enzymes have been knocked out, we are developing small molecule inhibitors of selected enzymes as tools to evaluate whether such compounds modulate virulence. We performed fragment-based and high-throughput screens against an amino-sugar acetyltransferase enzyme, PglD, involved in biosynthesis of UDP-diNAcBac in Campylobacter jejuni. Herein we report optimization of the hits into potent small molecule inhibitors (IC 50 < 300 nM). Biophysical characterization shows that the best inhibitors are competitive with acetyl coenzyme A and an X-ray cocrystal structure reveals that binding is biased toward occupation of the adenine subpocket of the AcCoA binding site by an aromatic ring.
59 BASIC BIOLOGICAL SCIENCES↗
De Novo Synthesis of Phosphorylated Triblock Copolymers with Pathogen Virulence-Suppressing Properties That Prevent Infection-Related Mortality
Phosphate is a key and universal "cue" in response to which bacteria either enhance their virulence when local phosphate is scarce or downregulate it when phosphate is adundant. Phosphate becomes depleted in the mammalian gut following physiologic stress and serves as a major trigger for colonizing bacteria to express virulence. This process cannot be reversed with oral supplementation of inorganic phosphate because it is nearly completely absorbed in the proximal small intestine. In the present study, we describe the de novo synthesis of phosphorylated polyethylene glycol compounds with three defined ABA (hydrophilic/-phobic/-philic) structures, ABA-PEG10k-Pi10, ABA-PEG16k-Pi14, and ABA-PEG20k-Pi20, and linear polymer PEG20k-Pi20 absent of the hydrophobic block. The 10k, 16k, and 20k demonstrate the molecular weights of the poly(ethylene glycol) block, and Pi10, Pi14, and Pi20 represent the repeating units of phosphate. Polymers were tested for their efficacy against Pseudomonas aeruginosa virulence in vitro and in vivo by assessing the expression of the phosphate sensing protein PstS, the production of key virulence factor pyocyanin, and Caenorhabditis elegans killing assays. Results indicate that all phosphorylated polymers suppressed phosphate sensing, virulence expression, and lethality in P. aeruginosa. Among all of the phosphorylated polymers, ABA-PEG20kPi20 displayed the greatest degree of protection against P. aeruginosa. To define the role of the hydrophobic core in ABA-PEG20k-Pi20 in the above response, we synthesized PEG20k-Pi20 in which the hydrophobic core is absent. Results indicate that the hypdrophobic core of ABA-PEG20k-Pi20 is a key structure in its protective effect against P. aeruginosa, in part due to its ability to coat the surface of bacteria. Taken together, the synthesis of novel polymers with defined structures and levels of phosphorylation may elucidate their antivirulence action against clinically important and lethal pathogens such as P. aeruginosa
36 MATERIALS SCIENCE↗
Structural Characterization of the Reaction and Substrate Specificity Mechanisms of Pathogenic Fungal Acetyl-CoA Synthetases
Acetyl CoA synthetases (ACSs) are Acyl-CoA/NRPS/Luciferase (ANL) superfamily enzymes that couple acetate with CoA to generate acetyl CoA, a key component of central carbon metabolism in eukaryotes and prokaryotes. Normal mammalian cells are not dependent on ACSs, while tumor cells, fungi, and parasites rely on acetate as a precursor for acetyl CoA. Consequently, ACSs have emerged as a potential drug target. As part of a program to develop antifungal ACS inhibitors, we characterized fungal ACSs from five diverse human fungal pathogens using biochemical and structural studies. ACSs catalyze a two-step reaction involving adenylation of acetate followed by thioesterification with CoA. Our structural studies captured each step of these two half-reactions including the acetyl-adenylate intermediate of the first half-reaction in both the adenylation conformation and the thioesterification conformation and thus provide a detailed picture of the reaction mechanism. We also used a systematic series of increasingly larger alkyl adenosine esters as chemical probes to characterize the structural basis of the exquisite ACS specificity for acetate over larger carboxylic acid substrates. Consistent with previous biochemical and genetic data for other enzymes, structures of fungal ACSs with these probes bound show that a key tryptophan residue limits the size of the alkyl binding site and forces larger alkyl chains to adopt high energy conformers, disfavoring their efficient binding. Together, our analysis provides highly detailed structural models for both the reaction mechanism and substrate specificity that should be useful in designing selective inhibitors of eukaryotic ACSs as potential anticancer, antifungal, and antiparasitic drugs.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗