Pathogens & Pathogenicity Lecture Notes PDF

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Introduction to Viruses and Human disease -Lecture 1Overview of VirusesOutline Understanding structural and genetic properties of a virus provides insight into how the virus replicates, spreads and causes disease  General overview of viruses o Structure & morphology o Viral replication cyc...

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Pathogens and Pathogenicity Introduction to Viruses and Human disease -Lecture 1 Overview of Viruses Outline  

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Understanding structural and genetic properties of a virus provides insight into how the virus replicates, spreads and causes disease General overview of viruses o Structure & morphology o Viral replication cycle End of the session you should be able to: Describe the general structures of viral particles Overview of viral replication cycle o Viral entry o Genome types o Viral exit

What is a virus?  

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A miniscule, acellular infectious agent with nucleic acid covered by a proteinaceous capsomere Structure o Naked virus = Capsid protein coat (lack cytoplasmic membrane) o Enveloped virus = membrane-like envelope o Contains one or several pieces of DNA or RNA o Lack cytosol and functional organelles Virus do not have metabolic activity alone. They hijack the host’s metabolism to synthesis additional viral genome and proteins for new viruses

Extracellular or Intracellular?  Outside the cell a virus is called a virion o Contained by the capsid (or envelope)

Pathogens and Pathogenicity

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o Provides protection and recognition sites to bind to host cells Once the virus becomes internalised the capsid is removed – virus genome is released

Virus Morphology

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Viruses range in size from the smallest virus are 17 nm in diameter the largest 500 nm (currently known) The shape of a virus is also used in classification There are three basic shapes: o Helical o Polyhedral o Complex Complex viruses o Generally have capsids of many different shapes o Example: Smallpox virus has several covering layers including lipid but no capsid

Virus Replication Cycle Viruses cannot replicate without a host • lack the genes encoding essential proteins for survival • Hijack host cell machinery Once a virus enters the host, it forces it to: • Replicate the viral genome • Translate viral proteins The Replication Cycle:

Pathogens and Pathogenicity 1. Attachment of the virion to the host cell 2. Entry of the virion into the host cell 3. Synthesis of new nucleic acids and viral proteins by host enzymes and ribosomes 4. Assembly of new viruses within the host 5. Release of the new virions (sometimes host cell death (lysis)) Virus Envelop     

All viruses lack membranes but some surround their capsids in phospholipid bilayer and proteins Matrix proteins may fill the gap between the envelope and the capsid – tegument Enveloped viruses acquire their envelop from the host during replication or release The proteins are virally coded glycoproteins or Spikes that protrude outward from the envelopes surface. These often play a role in host cell recognition by the virus Naked viruses (those without an envelope) are more stable. But because their surfaces are not similar to the host often they cannot hide from the immune system

Viral Entry 

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Receptors o Viral attachment proteins (VAP) are specific molecules pathogens produce enables viral binding to a host receptor either on the body or within tissues o Host receptors are functional for the host – the pathogen takes advantage of this = tissue specificity o Viral entry is extremely diverse due to receptor recognition Viral attachment proteins (VAP) either: o glycoproteins that oligomerize to form Spikes (enveloped virus) o capsid proteins (non-enveloped virus)

Types of viral entry o Direct penetration

Pathogens and Pathogenicity 

Capsid of naked viruses attaches to the host cell and “sinks” into the cytoplasmic membrane  Creating a pore for the genome to enter & uncoating of the viral capsid o Membrane fusion  The entire capsid and contents enter the host cell by the envelope fusing with the cytoplasmic membrane  Releasing the capsid into the hosts cytoplasm o Endocytosis  Most enveloped and some naked viruses trigger endocytosis  Receptor mediated internalisation Viral Nucleic Acids 

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Genetic material o The viral genome can be DNA or RNA (2kb- 1mb) o The nucleic strands can be single or double stranded o dsDNA, ssDNA, dsRNA or ssRNA o Positive or negative sense o These can be linear, circular, individual or segments strands This is how viruses are characterised o But not how viruses are named!

Genome Types  Synthesis of new genetic material depends on the type of viral nucleic acid

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Depending on the type of viral genome, dictates the when viral mRNA and proteins are synthesised for macromolecule production

Viral Assembly and release  Once the viral components are synthesised, virus particles are assembled ready to be released from the host o RNA viruses assemble in the cytosol o DNA viruses assemble in the nucleus and then are released into the cytosol  Enveloped viruses are released by budding, where they pass through one of the cell’s membranes o A portion of the host’s membrane is then incorporated into the capsuling the virus  Budding of enveloped viruses does not instantly kill the infected host, instead viral particles are shed slowly and steadily. This is called persistent infections  Naked viruses can be released by o Exocytosis o Lysis of the host cell

Pathogens and Pathogenicity

Viral Genome Replication Virus Replication Replication of viral genomes depend on: • Type of nucleic acid • Transcription to mRNA • Host replication machinery • Viral replication machinery Virus break the central dogma rules Many viruses break the rules of the central dogma: • Molecular Genetics is that information flow is unidirectional: DNA → RNA → PROTEIN Viruses can use RNA to store genetic information • How does replication work? DNA virus replication Replication of dsDNA viruses Double stranded DNA (dsDNA) is similar to normal replication and translation of proteins 1. dsDNA viral genome is transported to the host nucleus and replication is initiated using the host DNA-dependent DNA polymerase 2. mRNA is transcribed in the nucleus by DNA-dependent RNA polymerase and then exported to the cytoplasm 3. Host ribosomes in the cytoplasm translate the viral mRNA to produce macromolecules Assembly of new virions occurs Replication of ssDNA viruses Synthesis of ssDNA is unique for host cells 1. Host DNA-dependent DNA polymerase copies the viral single-stranded DNA 2. A second round of replication using the host polymerase using the negative strand as the template 3. Newly ssDNA is synthesised 4. While RNA-polymerase copies the newly formed negative strand for mRNA synthesis RNA Virus Replication Synthesis of RNA viruses differ significantly than DNA replication Types of RNA viruses: • dsRNA • positive-sense ssRNA (+ssRNA) • negative-sense ssRNA (-ssRNA) • retroviruses

Pathogens and Pathogenicity RNA viruses must encode a copy of RNA-dependent RNA polymerase • Transcriptase • Replicase Because the host cell does not have the machinery to replicate RNA Most RNA viruses replicate in the cytoplasm The genome structure • Determines the mechanism of transcription and translation • How viral mRNA is generated and proteins are processed RNA viruses • Prone to more mutations • Must carry a polymerase (except for (+) sense genomes) • All (-) RNA viruses are enveloped What does ‘sense’ mean? • Positive-sense strand has the information that can be directly translated into protein. • Negative-sense is the non-coding strand, therefore complimentary to mRNA. The negative-sense RNA strand must be converted to positive-sense prior to mRNA synthesis and RNA replication to occur. Double-stranded RNA (dsRNA) • dsRNA unwinds leaving a +ssRNA and –ssRNA • Positive-strand acts as template and mRNA for translation of viral proteins • Protein synthesis occurs immediately upon unwinding • One protein encode RNA-dependent RNA polymerase that transcribes the negativestranded RNA Positive-sense ssRNA (+ssRNA) • Viruses that have positive-sense RNA are retained in the cytoplasm • The RNA acts directly as mRNA, where host cell ribosomes translate the mRNA into viral polypeptides • Using a virally encoded RNA-dependent RNA polymerase, the complementary negative-sense copy of the RNA is transcribed (template for genome replication) Negative-sense ssRNA (-ssRNA) • Negative-sense RNA cannot be used directly as mRNA for translation into proteins • It is 3’ to 5’ therefore the RNA is in the wrong orientation for host cell ribosomes recognition • To overcome this the virus carries an RNA-dependent RNA transcriptase in the capsid (released during uncoating) • Copies –ssRNA to +ssRNA serves as a template for both replication and translation of viral proteins Retroviruses – (+) ssRNA Retroviruses are quite unique Post-entry viral RNA is reverse transcribed by reverse transcriptase (RT) which is retained in the capsid • RNA → DNA • RT is also known as RNA-directed DNA polymerase

Pathogens and Pathogenicity Once the DNA has been transcribed it integrates into the host genome Active transcription of viral mRNA is initiated along with protein synthesis

RNA Viruses - Lecture 2 Poliovirus

Pathogens and Pathogenicity Picornaviridae • Family of viruses • Poliomyelitis - Epidemiology - Vaccination Picornaviridae family • Rhinovirus • Enterovirus • Hepatovirus ‘Pico’ means small = 20nm +ssRNA genome, naked capsid (without an envelope) A subset of the picornaviridae genus are enteroviruses, which are cytolytic (kill their host cell via lysis) Three main types of enterovirus: • Poliovirus • Coxsackievirus • Echovirus Poliovirus • Three serotypes: poliovirus 1, 2 and 3 • Each has a different capsid protein • Extremely virulent and produce the same disease • Recovery from one strain, does not confer immunity from the other strains • R0 value is between 4-7 Extremely stable virus Poliomyelitis History Polio (grey) and myelon (marrow, indicating the spinal cord) derived from Greek words = Classic manifestations of paralysis caused by the virus poliomyelitis Stele from 1570-1342 BCE Polio epidemics did not occur until the end of the 19th century only sporadic Michael Underwood 1789 published the first account of polio in infants as "a debility of the lower extremities." Epidemics initially appeared from 1843 onwards, increasing significantly in the early 1900’s Discovered a virus caused the disease In early 1920’s observations were made that initially infected individuals were rendered immune to the virus The age of vaccination began! Poliomyelitis Highly infectious viral disease, transmitted by the oral-faecal route • Asymptomatic shedding – for up to one month • Associated with poor sanitation & crowded living spaces Intestines site of replication and cause transverse into the nervous system and cause total paralysis - 1 in 200 infections lead to irreversible paralysis (5-10% die)

Pathogens and Pathogenicity Mainly affects children 100 types • Costs ....pennies • Used in animal husbandry •

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Challenges of drug resistance o 2.8 million people get an antibiotic-resistant infection, and more than 35,000 people die • US Centre for Disease Control CDC https://www.cdc.gov/drugresistance/index.html European Centre for Disease Prevention and Control https://www.ecdc.europa.eu/en/publications-data/antibiotic-resistance-how-doesantibiotic-resistance-spread “A growing number of infections – such as pneumonia, tuberculosis, gonorrhoea, and salmonellosis – are becoming harder to treat as the antibiotics used to treat them become less effective” WHO

Antibiotic Resistance - Antibiotics are overused. • Overprescribed; used in farm animal feed • Exerts selective pressure for drug-resistant strains • Many strains evolve multiple drug-resistance • Over 80% of Streptococcus pneumoniae infections are now penicillin- resistant in some countries. Antibiotic Resistance Mechanisms See moodle video: Forms of resistance use MS explorer 1) Modify the target so that it no longer binds the antibiotic. - Mutations in ribosomal proteins confer resistance to streptomycin. 2) Destroy the antibiotic before it gets into cell. - The beta-lactamase enzyme specifically destroys penicillins (Amp R ) 3) Add modifying groups that inactivate the antibiotic. - Three classes of enzymes are used to modify and inactivate the aminoglycoside antibiotics eg streptomycin 4) Pump the antibiotic out of the cell. - Specific and nonspecific transport proteins - Similar strategy is used in cancer cells. Antimicrobial Chemotherapy

How Does Drug Resistance Develop?

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See moodle video: Origins of resistance use MS explorer De novo antibiotic resistance develops through gene duplication and/or mutations. Can be acquired via horizontal gene transfer o Conjugation o Transduction o Transformation

Fighting Drug Resistance Several strategies are being used: - Dummy target compounds that inactivate resistant enzymes - Alter antibiotic’s structure so that it sterically hinders access of modifying enzymes - Link antibiotics together Principles of Clinical Microbiology • Which organism is causing the pathology • Is it resistance to common antibiotics (MDR) • What concentrations are needed to kill it • European Antimicrobial Susceptibility testing • https://www.eucast.org/videos_from_eucast/ • Is it contagious • Is it reportable • Respond to patients needs in a timely manner Clinical Microbiology Identification of pathogen and drug susceptibility is critical - Use appropriate treatments. - Antibiotics don’t work on all bacteria. - Many bacteria are now drug-resistant. - Antibacterials don’t work on viruses! - What dose? Minimal Inhibitory Concentration MIC Lowest concentration that prevents growth - Varies for different bacterial species - Test by diluting antibiotic Lowest concentration with no growth = MIC - May still have living (but non-growing) organisms - Plate liquid without antibiotic—do colonies form? - No colonies = Minimal Lethal Concentration (MLC) - MLC always higher than MIC Testing Antibiotic Efficacy • E-test determines MIC • Gradient of antibiotic in paper strip • Drug must be above MIC in tissue to be effective.

Kirby-Bauer disk susceptibility test

Pathogens and Pathogenicity - Test strain sensitivity to multiple antibiotics - Multiple disks with different antibiotics - Size of cleared zones reflects relative sensitivity Molecular Detection methods • Allows culturing of otherwise un-culturable organisms • MDR-TB, XDR-TB • Resistant to most drugs • Safe handling ... • inoculate cartridge, insert in machine, Expensive • ALORITHM Antimicrobial Chemotherapy & Clinical Microbiology • The importance of antibiotics in treating disease was recognised beginning in the 1940s. • Basic concepts of antimicrobial therapy include selective toxicity and spectrum of activity. • Mechanisms of antibiotic resistance include: - Modifying/destroying/pumping out the antibiotic • How to measure bacterial susceptibility to antibiotics. Bacterial Chemotherapeutics Targets of Bacterial Chemotherapeutics • Cell wall synthesis: - Penicillins and cephalosporins: Block transpeptidation - Vancomycin: Blocks transglycosylation - Cycloserine: Blocks formation of D-ala-D-ala dipeptide - Bacitracin: Blocks bactoprenol, the lipid carrier • Cell membrane : gramicidin and polymyxin. • DNA synthesis and integrity: sulfa drugs, metronidazole, quinolones. • RNA synthesis: rifampins and actinomycin D. • Protein synthesis: - Aminoglycosides and tetracyclines: Affect 30S subunit - Macrolides, lincosamides, and streptogramins: Affect 50S Spectrum of Activity • Broad-spectrum - Effective against many species • Narrow-spectrum - Effective against few or a single species • Source of antibiotics - Most discovered as natural products • - Often modified by artificial means - Increase efficacy - Decrease toxicity to humans Microbial Pathogenesis the future • Technology and progress have had the unintended consequence of breeding disease. - Jet travel and suburban growth are prime examples. Classification of Antibiotics

Pathogens and Pathogenicity • Bactericidal antibiotics kill target organisms. • - Many drugs only affect growing cells. - Inhibitors of cell wall synthesis - Only effective if organism is building new cell wall - e.g., Penicillin • Bacteriostatic antibiotics prevent growth of organisms. - Cannot kill organism - Immune system removes infection. Mechanisms of Action The following aspects of a microbe’s physiology are classic targets: - Cell wall - Cell membrane - DNA synthesis - RNA synthesis - Protein synthesis – Metabolism Cell Wall Antibiotics Peptidoglycan synthesis is rather complex, however, it may be summarised as such: 1) Precursors are synthesised in the cytoplasm. - UDP-NAG and UDP-NAM-peptide 2) They are carried across the cell membrane by a lipid carrier – bactoprenol. - The carrier is then recycled. 3) The precursors are polymerised to the existing cell wall structure by transglycosylases. 4) The peptide side chains are cross-linked by transpeptidases. • Beta-lactam antibiotics - Penicillins, cephalosporins - Competitive inhibitors of transpeptidases - R groups can be modified to generate a number of semisynthetic drugs • Other cell wall synthesis inhibitors o Vancomycin: - Binds ends of peptides - Prevents action of transglycosylases and transpeptidases - Same step as penicillin, but different activity o Cycloserine - Inhibits formation of the D-ala-D-ala dipeptide precursor o Bacitracin: - Blocks the lipid carrier - Disaccharide subunits don’t reach Drugs That Disrupt Cell Membranes • Gramicidin - Cyclic peptide produced by Bacillus brevis - Forms a cation channel,through which ions leak • Polymyxin - Produced by Bacillus polymyxa

Pathogens and Pathogenicity - Destroys cell membrane, just like a detergent DNA Synthesis Inhibitors • Quinolones: Nalidixic acid, ciprofloxacin - Blocks bacterial DNA gyrase, and so prevents DNA replication • Metronizadole - Nontoxic, unless metabolized by anaerobe • Sulfa drugs - Analogues of PABA, a precursor of folic acid - Needed for DNA synthesis - Supplied in our diet; thus no folic acid synthesis to inhibit RNA Synthesis Inhibitors • Rifampin - Binds to the beta subunit of RNA polymerase - Prevents the elongation step of transcription • Actinomycin D - Binds to DNA - Prevents the initiation step of transcription - Not selectively toxic Protein Synthesis Inhibitors 1. Drugs that interact with the 30S subunit - Aminoglycosides: Cause the translational misreading of mRNA - Are bactericidal - Include streptomycin - Tetracyclines: - Block the binding of charged tRNAs to the A site of the ribosome - Are bacteriostatic - Include doxycycline 2. Drugs that interact with the 50S subunit - Macrolides: Inhibit translocation - Lincosamides: Inhibit translocation - Chloramphenicol: Inhibits peptidyl transferase activity - Oxazolidinones: Prevent formation of the 70S ribosome initiation complex - Streptogramins - Streptogramin A: Blocks tRNA binding - Streptogramin B: Blocks translocation

Microbial Antibiotic Biosynthesis

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Antibiotics are secondary metabolites. o Not essential for survival o Enhance ability to survive competition Relatively simple pathway for some - Penicillin o Complex synthesis for others Bacteria secrete some antibiotics. o Also make enzymes to disable antibiotics o Keep drug from killing cells that make them = Resistance

The Future of Drug Discovery • Evolutionary pressure is constant. o Requires constant search for new antibiotics • The modern drug discovery process: o Use genomics—identify new targets. o Design compounds to inhibit targets. o Alter compound structure to optimise MIC. o Determine spectrum of compound. - Narrow or broad? o Determine pharmaceutical properties. - Not toxic to animals; persistence in body Biological Weapons • Bioweapons are highly virulent infectious agents or toxins. • Biowarfare o Inflicts massive casualties o Rapid onset; death or temporary incapacitation  ...