Targeting host cell furin proprotein convertases as a therapeutic strategy against bacterial toxins and viral pathogens

Pathogens or their toxins, including influenza virus, Pseudomonas, and anthrax toxins, require processing by host proprotein convertases (PCs) to enter host cells and to cause disease. Conversely, inhibiting PCs is likely to protect host cells from multiple furin-dependent, but otherwise unrelated, pathogens. To determine if this concept is correct, we designed specific nanomolar inhibitors of PCs modeled from the extended cleavage motif TPQRERRRKKR downward arrowGL of the avian influenza H5N1 hemagglutinin. We then confirmed the efficacy of the inhibitory peptides in vitro against the fluorescent peptide, anthrax protective antigen (PA83), and influenza hemagglutinin substrates and also in mice in vivo against two unrelated toxins, anthrax and Pseudomonas exotoxin. Peptides with Phe/Tyr at P1' were more selective for furin. Peptides with P1' Thr were potent against multiple PCs. Our strategy of basing the peptide sequence on a furin cleavage motif known for an avian flu virus shows the power of starting inhibitor design with a known substrate. Our results confirm that inhibiting furin-like PCs protects the host from the distinct furin-dependent infections and lay a foundation for novel, host cell-focused therapies against acute diseases.     

New biochip technology for label-free detection of pathogens and their toxins

microSERS is a new biochip technology that uses surface-enhanced Raman scattering (SERS) microscopy for label-free transduction. The biochip itself comprises pixels of capture biomolecules immobilized on a SERS-active metal surface. Once the biochip has been exposed to the sample and the capture biomolecules have selectively bound their ligands, a Raman microscope is used to collect SERS fingerprints from the pixels on the chip. SERS, like other whole-organism fingerprinting techniques, is very specific. Our initial studies have shown that the Gram-positive Listeria and Gram-negative Legionella bacteria, Bacillus spores and Cryptosporidium oocysts can often be identified at the subspecies/strain level on the basis of SERS fingerprints collected from single organisms. Therefore, pathogens can be individually identified by microSERS, even when organisms that cross-react with the capture biomolecules are present in a sample. Moreover, the SERS fingerprint reflects the physiological state of a bacterial cell, e.g., when pathogenic Listeria and Legionella were cultured under conditions known to affect virulence, their SERS fingerprints changed significantly. Similarly, nonviable (e.g., heat- or UV-killed) microorganisms could be differentiated from their viable counterparts by SERS fingerprinting. Finally, microSERS is also capable of the sensitive and highly specific detection of toxins. Toxins that comprised as little as 0.02% by weight of the biomolecule-toxin complex produced strong, unique fingerprints when spectra collected from the complexes were subtracted from the spectra of the uncomplexed biomolecules. For example, aflatoxins B(1) and G(1) could be detected and individually identified when biochips bearing pixels of antibody or enzyme capture biomolecules were incubated in samples containing one or both aflatoxins, and the spectra were then collected for 20 s from an area of the biomolecule pixel approximately 1 microm in diameter. In the future, we plan to investigate the use of hyperspectral imaging Raman microscopy for collecting fingerprints from all the pixels on the biochip, individually yet simultaneously, to enable the rapid detection of diverse pathogens and their toxins in a sample, using a single biochip.     

Detection of bacterial pathogens in environmental samples using DNA microarrays

Polymerase chain reaction (PCR) is an important tool for pathogen detection, but historically, it has not been possible to accurately identify PCR products without sequencing, Southern blots, or dot-blots. Microarrays can be coupled with PCR where they serve as a set of parallel dot-blots to enhance product detection and identification. Microarrays are composed of many discretely located probes on a solid substrate such as glass. Each probe is composed of a sequence that is complimentary to a pathogen-specific gene sequence. PCR is used to amplify one or more genes and the products are then hybridized to the array to identify species-specific polymorphism within one or more genes. We illustrate this type of array using 16S rDNA probes suitable for distinguishing between several salmonid pathogens. We also describe the use of microarrays for direct detection of either RNA or DNA without the aid of PCR, although the sensitivity of these systems currently limits their application for pathogen detection. Finally, microarrays can also be used to "fingerprint" bacterial isolates and they can be used to identify diagnostic markers suitable for developing new PCR-based detection assays. We illustrate this type of array for subtyping an important food-borne pathogen, Listeria monocytogenes.     

Direct detection of bacterial pathogens in representative dairy products using a combined bacterial concentration-PCR approach

AIMS: To develop a simple, rapid method to concentrate and purify bacteria and their nucleic acids from complex dairy food matrices in preparation for direct pathogen detection using polymerase chain reaction (PCR). METHODS AND RESULTS: Plain non-fat yogurt and cheddar cheese were each seeded with Listeria monocytogenes or Salmonella enterica serovar. Enteritidis in the range of 10(1)-10(6) CFU per 11-g sample. Samples were then processed for bacterial concentration using high-speed centrifugation (9700 g) followed by DNA extraction, PCR amplification, and amplicon confirmation by hybridization. Bacterial recoveries after centrifugation ranged from 53 to >100% and 71 to >100% for serovar. Enteritidis and L. monocytogenes, respectively, in the non-fat yogurt samples; and from 77 to >100% and 69 to >100% for serovar. Enteritidis and L. monocytogenes, respectively, in the cheddar cheese samples. There were no significant differences in recovery efficiency at different inocula levels, and losses to discarded supernatants were always <5%, regardless of dairy product or pathogen. CONCLUSIONS: When followed by pathogen detection using PCR and confirmation by amplicon hybridization, detection limits of 10(3) and 10(1) CFU per 11-g sample were achieved for L. monocytogenes and serovar. Enteritidis, respectively, in both product types and without prior cultural enrichment. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents progress toward the rapid and efficient direct detection of pathogens from complex food matrices at detection limits approaching those that might be anticipated in naturally contaminated products.     

Thiol-dependent cytolytic bacterial toxins: streptolysin O and prominent toxins

Streptolysin O is the prototype of fifteen bacterial cytolytic protein toxins elaborated by gram-positive bacteria of species Streptococcus, Clostridium, Bacillus and Listeria. These toxins share a number of common properties: they are antigenically related as shown by cross-neutralization and immunoprecipitation; their cytolytic and other reducing agents; these toxins are inactivated by cholesterol and certain related sterols. This group of oxygen-labile cytolytic toxins has been named sulfyhdryl-activated toxins or thiol-activated cytolysins. The mechanism of action of these toxins is very likely identical or at least closely similar.     

Fish as proxies of ecological and environmental change

Anthropogenic impacts have shifted aquatic ecosystems far from prehistoric baseline states; yet, understanding these impacts is impeded by a lack of available long-term data that realistically reflects the organisms and their habitats prior to human disturbance. Fish are excellent, and largely underused, proxies for elucidating the degree, direction and scale of shifts in aquatic ecosystems. This paper highlights potential sources of qualitative and quantitative data derived from contemporary, archived and ancient fish samples, and then, using key examples, discusses the types of long-term temporal information that can be obtained. This paper identifies future research needs with a focus on the Southern Hemisphere, as baseline shifts are poorly described relative to the Northern Hemisphere. Temporal data sourced from fish can improve our understanding of how aquatic ecosystems have changed, particularly when multiple sources of data are used, enhancing our ability to interpret the current state of aquatic ecosystems and establish effective measures to safeguard against further adverse shifts. The range of biological, ecological and environmental data obtained from fish can be integrated to better define ecosystem baseline states on which to establish policy goals for future conservation and exploitation practices.     

Mediterranean fruit fly as a potential vector of bacterial pathogens

The Mediterranean fruit fly (Ceratitis capitata) is a cosmopolitan pest of hundreds of species of commercial and wild fruits. It is considered a major economic pest of commercial fruits in the world. Adult Mediterranean fruit flies feed on all sorts of protein sources, including animal excreta, in order to develop eggs. After reaching sexual maturity and copulating, female flies lay eggs in fruit by puncturing the skin with their ovipositors and injecting batches of eggs into the wounds. In view of the increase in food-borne illnesses associated with consumption of fresh produce and unpasteurized fruit juices, we investigated the potential of Mediterranean fruit fly to serve as a vector for transmission of human pathogens to fruits. Addition of green fluorescent protein (GFP)-tagged Escherichia coli to a Mediterranean fruit fly feeding solution resulted in a dose-dependent increase in the fly's bacterial load. Flies exposed to fecal material enriched with GFP-tagged E. coli were similarly contaminated and were capable of transmitting E. coli to intact apples in a cage model system. Washing contaminated apples with tap water did not eliminate the E. coli. Flies inoculated with E. coli harbored the bacteria for up to 7 days following contamination. Fluorescence microscopy demonstrated that the majority of fluorescent bacteria were confined along the pseudotrachea in the labelum edge of the fly proboscis. Wild flies captured at various geographic locations were found to carry coliforms, and in some cases presumptive identification of E. coli was made. These findings support the hypothesis that the common Mediterranean fruit fly is a potential vector of human pathogens to fruits.     

Rho GTPases as targets of bacterial protein toxins

Several bacterial toxins target Rho GTPases, which constitute molecular switches in several signaling processes and master regulators of the actin cytoskeleton. The biological activities of Rho GTPases are blocked by C3-like transferases, which ADP-ribosylate Rho at Asn41, but not Rac or Cdc42. Large clostridial cytotoxins (e. g., Clostridium difficile toxin A and B) glucosylate Rho GTPases at Thr37 (Rho) or Thr35 (Rac/Cdc42), thereby inhibiting Rho functions by preventing effector coupling. The 'injected' toxins ExoS, YopE and SptP from Pseudomonas aeruginosa, Yersinia and Salmonella ssp., respectively, which are transferred into the eukaryotic target cells by the type-III secretion system, inhibit Rho functions by acting as Rho GAP proteins. Rho GTPases are activated by the cytotoxic necrotizing factors CNF1 and CNF2 from Escherichia coli and by the dermonecrotizing toxin DNT from B. bronchiseptica. These toxins deamidate/transglutaminate Gln63 of Rho to block the intrinsic and GAP-stimulated GTP hydrolysis, thereby constitutively activating the GTPases. Rho GTPases are also activated by SopE, a type-III system injected protein from Salmonella ssp., that acts as a GEF protein.     

Flash detection/identification of pathogens, bacterial spores and bioterrorism agent biomarkers from clinical and environmental matrices.

We propose to develop an integrated rapid, semiportable, prototype point microbial detection/identification system for clinical specimens that is also capable of differentiating microbial bioterrorism attacks from threats or hoaxes by defining the pathogen. The system utilizes "flash" extraction/analytical system capable of detection/identification of microbes from environmental and clinical matrices. The system couples demonstrated technologies to provide quantitative analysis of lipid biomarkers of microbes including spores in a system with near-single cell (amol/microl) sensitivity. Tandem mass spectrometry increases specificity by providing the molecular structure of neutral lipids, phospholipids, and derivatized spore-specific bacterial biomarker, 2,6-dipicolinic acid (DPA) as well as the lipopolysaccharide-amide-linked hydroxy-fatty acids (LPS-ALHFA) of Gram-negative bacteria. The extraction should take about an hour for each sample but multiple samples can be processed simultaneously.     

BTXpred: prediction of bacterial toxins

This paper describes a method developed for predicting bacterial toxins from their amino acid sequences. All the modules, developed in this study, were trained and tested on a non-redundant dataset of 150 bacterial toxins that included 77 exotoxins and 73 endotoxins. Firstly, support vector machines (SVM) based modules were developed for predicting the bacterial toxins using amino acids and dipeptides composition and achieved an accuracy of 96.07% and 92.50%, respectively. Secondly, SVM based modules were developed for discriminating entotoxins and exotoxins, using amino acids and dipeptides composition and achieved an accuracy of 95.71% and 92.86%, respectively. In addition, modules have been developed for classifying the exotoxins (e.g. activate adenylate cyclase, activate guanylate cyclase, neurotoxins) using hidden Markov models (HMM), PSI-BLAST and a combination of the two and achieved overall accuracy of 95.75%, 97.87% and 100%, respectively. Based on the above study, a web server called 'BTXpred' has been developed, which is available at http://www.imtech.res.in/raghava/btxpred/. Supplementary information is available at http://www.imtech.res.in/raghava/btxpred/supplementary.html.     

Immunochemical detection of guanine nucleotide binding proteins mono-ADP-ribosylated by bacterial toxins

Rabbits immunized with ADP-ribose chemically conjugated to carrier proteins developed antibodies reactive against guanine nucleotide binding proteins (G proteins) that had been mono-ADP-ribosylated by bacterial toxins. Antibody reactivity on immunoblots was strictly dependent on incubation of substrate proteins with both toxin and NAD and was quantitatively related to the extent of ADP-ribosylation. Gi, Go, and transducin (ADP-ribosylated by pertussis toxin) and elongation factor II (EF-II) (ADP-ribosylated by pseudomonas exotoxin) all reacted with ADP-ribose antibodies. ADP-ribose antibodies detected the ADP-ribosylation of an approximately 40-kilodalton (kDa) membrane protein related to Gi in intact human neutrophils incubated with pertussis toxin and the ADP-ribosylation of an approximately 90-kDa cytosolic protein, presumably EF-II, in intact HUT-102 cells incubated with pseudomonas exotoxin. ADP-ribose antibodies represent a novel tool for the identification and study of G proteins and other substrates for bacterial toxin ADP-ribosylation.     

Preparation and properties of bacterial chromatophores entrapped in polyacrylamide

The immobilization of Rhodospirillum rubrum chromatophores was successfully performed by entrapping them in polyacrylamide. Their photophosphorylating activity was about 40% of native chromatophores. The temperature and pH optima for immobilized chromatophores were similar to the native photosynthetic apparatus and kinetic parameters showed that the rate of photophosphorylation in polyacrylamide particles was diffusion controlled. Light penetration of the gel particles was not a limiting parameter. Immobilization considerably increased the stability of the chromatophores towards denaturation.