Week 12 Lecture: Toxin Analysis & Nucleic Acid Analysis PDF

Week 12 Lecture: Toxin Analysis & Nucleic Acid Analysis FRSC/BIOL 4570 Microbial Forensics Reading: Ch13, 14, 15, 16 Introduction to Toxins FRSC/BIOL 4570 Microbial Forensics Toxins “…molecules produced by living organisms that are poisonous to other species, such as humans.” Small molecule,...

Week 12 Lecture: Toxin Analysis & Nucleic Acid Analysis FRSC/BIOL 4570 Microbial Forensics Reading: Ch13, 14, 15, 16 Introduction to Toxins FRSC/BIOL 4570 Microbial Forensics Toxins “…molecules produced by living organisms that are poisonous to other species, such as humans.” Small molecule, peptide or protein toxin May require separation from substrates or preanalytical treatment before analysis Key analytical techniques include liquid chromatography tandem mass spectrometry (LC- MS/MS) and matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF) (Budowle et al., 2011)  Small Molecule Toxins Chemical structure of: A. Saxitoxin (paralytic shellfish toxins, dinoflagellates) B. Domoic acid an excitotoxin from red algae C. Tetrodotoxin (potent neurotoxins produced by bacteria Pseudomonas and Vibrio) Vilariño et al. 2018. Human Poisoning from Marine Toxins: Unknowns for Optimal Consumer Protection. Toxins 10(8), 324; https://doi.org/10.3390/toxins10080324 Peptide Toxins Structure of: A. Alpha-amanitin (death cap fungus; RNA polymerase II inhibitor) B. Phallacidin (a related compound) https://www.pnas.org/doi/10.1073/pnas.0707340104 Peptide Toxins Sequences related to AMA1 and PHA1 https://www.pnas.org/doi/10.1073/pnas.0707340104 Protein Toxins Staphylococcal Enterotoxin B (SEB) 11+ proteins secreted by Staphylococcus aureus Most common cause of food poisoning in the U.S. though it results in low fatality Enterotoxin = a toxin that targets the intestines Aerosol release could provide a greater death rate? SEB can cross-link T cells → binding sites for major MHC II molecules and Vβ T-cell receptor Activation of proinflammatory response Protein Toxins Staphylococcal Enterotoxin B (SEB) Pinchuck et al 2010. Toxins 2010, 2(8), 2177- 2197; https://doi.org/10.3390/toxins2082177 Protein Toxins Clostridium perfringens Epsilon Toxin Very small protoxin (311 aa = 933 bp long) that is cleaved into 14 aa peptide Causes toxemia in herbivores when ingested Symptoms are pulmonary edema, renal failure, cardiovascular collapse Therefore, aerosol/inhalation route would be suitable for bioterrorism attack No reported of human cases have occurred Biological Toxins Biothreat agents: Category A Botulinum neurotoxins Category B Ricin Staphylococcal enterotoxin B Clostridium perfringens epsilon toxin Reminder of Category Attributes Category A can be easily disseminated (note a toxin would not be transmitted from person to person) result in high mortality rates and have the potential for major public health impact; might cause public panic and social disruption; and require special action for public health preparedness Category B are moderately easy to disseminate; result in moderate morbidity rates and low mortality rates; and require specific enhancements of CDC's diagnostic capacity and enhanced disease surveillance Biological Toxins Distinguished from bacteria and viruses Not contagious Not a living organism (not able to culture) Proteins made of amino acids – not PCR or DNA hybridization detectable Difficult to detect in forensic (or other) applications Detection relies on antibody-based methods or mass spectrometry Ricin and Botulinum Neurotoxin (BoNT) FRSC/BIOL 4570 Microbial Forensics Ricin From castor bean on castor plant (Ricinus communis) Why is ricin a threat? No antidote available Castor grows in many places in the world Ricin extraction from beans is not difficult Ricin purification methods found on www LD = 3-15 g/kg via injection or inhalation Castor Plants Forensic ricin typing depends on the identification of the toxin and the assessment of the source plants Castor oil is produced from the castor seeds Used to make lubricants, cosmetics, soaps, paints, and polyesters Laxative or additives in drugs Toxins are not oil soluble; remains in the “cake” Source plant can be identified via contaminating DNA in toxin preparations Taxonomy must distinguish between closely related species Ricin Proteins Heterodimeric Glycoprotein: composed of A and B chains A chain (N-glycosidase): Removes an adenine residue from the 28S rRNA in the 60S subunit of the ribosome leading to the inhibition of protein synthesis B chain (lectin): Binds to galactose-containing glycolipids as well as cell surface glycoproteins facilitating entry of the toxin into cells  Ricin Mechanism of Action Audi, J., Belson, M.G., Patel, M.M., Schier, J.G., & Osterloh, J.D. (2005). Ricin poisoning: a comprehensive review. JAMA, 294 18, 2342-51.  Ricin Mechanism of Action Audi, J., Belson, M.G., Patel, M.M., Schier, J.G., & Osterloh, J.D. (2005). Ricin poisoning: a comprehensive review. JAMA, 294 18, 2342-51. Ricin Detection - ELISA Immunological assays (ELISA), biological assays, mass spec Detection by ELISA Forms antibody-ricin-antibody sandwich Detector antibody is conjugated to an enzyme, fluorescent molecule, or oligonucleotide Ricin Detection – ELISA Types Ricin Detection - Other Mass spectrometry can provide information on partially denatured ricin and other components of a ricin sample such as fatty acids, carbohydrate and protein composition to aid in forensic investigations Castor bean genotyping Amplified Fragment Length Polymorphisms (AFLPs) – genome sequencing made other techniques preferable Simple sequence repeats (SSRs) – low diversity makes these markers less useful Chloroplast DNA – lacks discrimination power Nuclear SNPs – have good potential  Ricin Poisoning Intentional poisoning is not common! https://www.theguardian.com/world/from- the-archive-blog/2020/sep/09/georgi-markov- killed-poisoned-umbrella-london-1978 Botulinum Neurotoxin & Botulism Produced by bacteria of the genus Clostridium, (C. botulinum, C. baratii, C. butyricum) that cause the rare but life-threatening disease – botulism Prolonged paralysis – requires ICU and mechanical ventilation The most poisonous substance known Human LD 50: - intravenous 0.090-0.15 µg - inhalation 0.7-0.9 µg - oral 70 µg Types of Botulism 1. Food borne – most prevalent before 1980s 2. Wound – drug users (injection) 3. Intestinal (infant) – most prevalent currently 4. Inadvertent – lab workers/cosmetic 5. Intentional or bioterror (biothreat) Wound Botulism Intentional Botulism? No successful use of BoNT as a bioterror agent Aum Shinrikyo made an attempt! BoNT production was/is part of the bioweapons programs of the former Soviet Union, Iraq, Iran, North Korea and Syria Iraq produced 19,000 L of BoNT → 10,000 L weaponized in warheads and bombs Impact of Intentional Attack Treatment requires prolonged ICU hospitalization Most hospitals have ICUs that run at 80% to 100% occupancy Others that need these facilities for emergencies would be collateral damage of the attack Damage estimate: aerosol exposure of 100,000 > 50,000 cases of botulism and 30,000 deaths as well as an estimated 4.2 million hospitals days at a cost of $8.6 billion Cost reduction and lives saved if antitoxin and mechanical ventilators were readily available Positive Uses of Botulinum Toxin! Treating medical conditions: overactive muscle condition cervical dystonia (abnormal squeezing and twisting muscle contractions in the head and neck area) cerebral palsy post-traumatic brain injury post-stroke spasticity “BOTOX Cosmetic is a prescription medicine that is injected into muscles and used to improve the look of moderate to severe frown lines between the eyebrows in adults younger than 65 years of age for a short period of time (temporary)” Clostridial Botulinum Neurotoxins Classified by serotype; BoNT A, B, C, D, E, F and G Differ based on AA sequence Serotypes A, B, E, and F are associated with naturally occurring botulism in humans Aerosolized serotypes C, D and G produce botulism in primates Food borne botulism usually associated with neurotoxins A, B, and E and very rarely F (2000 bases in length and with large numbers of target molecules….. So, this may not work with degraded DNA? WGA method: RCA Rolling circle amplification (RCA) is a highly robust method designed for the amplification of a circular template (e.g., plasmids or viral genomes Using this method, you must first circularize the available single- stranded DNA (ssDNA) by either: 1. T4 DNA Ligase, which catalyzes formation of a phosphodiester bond between 5’-phosphate and 3’-hydroxyl termini in duplex DNA or RNA, 2. CircLigase II, an enzyme that circularizes ssDNA through intrastrand ligation; 3. Molecular inversion probes (MIPs), a circle-based enrichment approach that allows for copying specified targets (e.g., canonical single-nucleotide polymorphisms (SNPs) and permits standard PCR amplification of all targets. Of the ligation approaches CircLigase II is better because it does not produce concatemers under standard conditions WGA method: RCA Essentially, RCA is the continued extension of random oligonucleotide primers that have annealed to a circular template, the procedure replicates the template through a cascade of strand displacement events to produce tandem copies of the circular template WGA method: RCA Carried out using exonuclease resistant random primers (thiophosphate linkages added to 3’ terminal nucleotides) to prevent their degradation by the 3’-5’ exonuclease proofreading activity of the 29 DNA polymerase WGA method: RCA Do not need to sequence all amplified product from RCA but can carry out Capture, or genomic partitioning Capture enriches the DNA sample for regions of interest prior to sequencing, this could be used in microbial forensic applications to specifically target sequences in a mixture of highly degraded samples enabling increased sensitivity of detecting these specific DNAs WGA: The Capture Process Molecular Inversion probes (MIPs) Another circle-based enrichment approach involves the use of padlock or MIPs A MIP is a single-stranded oligonucleotide that possesses two complementary terminal regions flanking a SNP of interest Molecular Inversion probes (MIPs) When the target complementary regions hybridize to the regions flanking the selected marker, the probe inverts (hence its name) The gap between them is filled with a polymerase and then ligated resulting in the complementary nucleotide of the SNP state of interest being integrated into the circularized MIP The smaller the gap (ideally one base for SNPs), the shorter the template required for analysis Molecular Inversion probes (MIPs) Examples of these Technologies Total transcript amplification assay for a single bacterium, Burkholderia thailandensis Three capture methods were combined to map the transcriptome of Caenorhabditis elegans Isolated and analyzed Picornaviridae from fecal microbiomes of two healthy piglets Employing circularization and RCA to sequence isolates of the Cassava brown streak virus (CBSV) to epidemiologically demonstrate that isolates from East Africa clustered in two distinct clades. How will these techniques Benefit Microbial forensics? These capabilities broaden the microbial forensic toolbox and should allow a wide range of evidence to be analyzed Host Factors FRSC/BIOL 4570 Microbial Forensics Host factors in Microbial forensics Once the microorganism or its toxin is in the living host, it is no longer possible to analyze it in the same way one could an isolated pathogen, except for the possibility to analyze the microbial nucleic acid The host’s response to the biological agent may be available for analysis for clues this is similar to using physical traces of bite marks, scratches, wound trajectories, and sizes of wounds to link the impact on the body to teeth, fingernails, and bullets Host factors in Microbial forensics Responses to a biothreat agent or its toxin may be used to give some chronological indication of exposure Patterns of antibody response which has the most forensic value, by providing a timeframe, is the appearance of IgM first, followed by a B- cell switch to the longer- lasting IgG Microbial Forensics Ch13-14 FRSC/BIOL 4570 Microbial Forensics Ch13. Select Methods for Microbial Forensic Nucleic Acid Analysis of Trace and Uncultivable Specimens Potential Quiz Questions 1. Trace DNA found at a crime scene, or a clandestine lab may be too damaged for traditional amplification strategies - what does this actually mean? 2. An approach for addressing limited quantities of nucleic acid signatures in a sample is whole genome amplification (WGA). How is WGA defined? 3. What polymerase is used for multiple displacement amplification, and what attribute of this polymerase is key for the procedure? 4. What is DOP-PCR? 5. What is the nucleotide sequence 5'-CCGACTCGAGNNNNNNATGTGG-3' an example of? Ch14. The Use of Host Factors in Microbial Forensics Potential Quiz Questions 1. What are two principal methods relating to host responses that have been sufficiently developed to distinguish a potential victim and innocent person from a perpetrator, and to distinguish between a naturally acquired or intentional infection? 2. Once a microorganism or a toxin is in a host, what are forensic scientists no longer able to analyze? 3. Analyzing the host’s response to the biological agent may provide clues in a manner analogous to what other forensic studies? 4. Who is likely to be among the first to recognize that a patient is a victim of a biocrime? 5. What can host responses provide clues for, in a biocrime? Quiz #08: Microbial Forensics Ch11, 12 (& lecture) Multiple choice (no backtracking; 20 minutes) Available: Thursday 9AM Due: Friday by end-of-day Next Week: DNA Sequencing, Genome Assembly, & DNA Diagnostics Microbial Forensics Ch18, 19, 20