Microbial Genetics BI 302 Lecture 2 (Centennial College) PDF

Microbial Genetics BI 302 Dr. Nalina Nadarajah Centennial College Lecture 2: Bacteriophage Genetics Image Attribution: Generated with DALL-E in ChatGPT-4o Agenda for This Week Bacteriophage Structure Bacteriophage Life Cycle – Lytic Cycle – Lysoge...

Microbial Genetics BI 302 Dr. Nalina Nadarajah Centennial College Lecture 2: Bacteriophage Genetics Image Attribution: Generated with DALL-E in ChatGPT-4o Agenda for This Week Bacteriophage Structure Bacteriophage Life Cycle – Lytic Cycle – Lysogenic Cycle Phage Assays Virus in the News Image Attribution: Dr. Victor Padilla-Sanchez, PhD https://www.drvictorpadillasanchez.com, CC BY-SA 4.0, via Wikimedia Commons Vaccines A vaccine: a biological preparation that improves immunity to a particular disease Viral vaccines: immune response to surface antigens of a virus A bacterial vaccine (BV) contains bacterial lysates or whole, live attenuated or dead cells of bacterial pathogens Image Attribution: Alachua County. COVID-19 vaccine. Close-up medical syringe with a vaccine. https://www.flickr.com/photos/alachuacounty/50802098581  Do you have any Concerns about Vaccines Thimerosal - an ethyl mercury derivative used as a preservative no vaccine made in Canada since March 2001 for routine use in children contains thimerosal (exception flu vaccine) Flu vaccine also available without thimerosal Does the MMR vaccine cause autism? NO MMR vaccine routinely used in Canada never contained thimerosal Has there been a recent increase in autism rates? Broader diagnostic criteria Inclusion of a wider range of behaviors and learning disorders under the autism spectrum disorder category  Types of Vaccines 1. Live-attenuated vaccines: live pathogens that have been "attenuated" or weakened. e.g. MMR vaccine, varicella (chickenpox) vaccine 2. Inactivated vaccines: live pathogen that is inactivated or killed. e.g. polio vaccine, influenza vaccine 3. Subunit vaccines: based on purified surface antigens of the virus. e.g. pneumococcal vaccine, shingles vaccine, hepatitis B vaccine 4. Toxoid vaccines: inactivated toxins to target the toxic activity, instead of targeting the bacteria itself. e.g. Tetanus vaccine, diphtheria vaccine 5. Viral vector vaccines: use a harmless virus to deliver the genetic code of an antigen you want the immune system to fight. e.g. Ebola vaccine, COVID-19 vaccine (AstraZeneca and Johnson & Johnson) 6. mRNA vaccines: using mRNA to instruct our body on how to produce a specific antigen that is unique to the virus. e.g. Pfizer-BioNTech COVID-19 vaccine Problems in Vaccine Development Understanding Pathogens: some (e.g., HIV and malaria) are highly complex Emerging Pathogens and Pandemics: little is known about the pathogen Rapid Mutation: come viruses mutate rapidly (e.g., influenza) Antigenic drift and shift antigenic drift: gradual, small changes in the genes of influenza viruses that occur over time. antigenic shift: a process where two or more different virus strains combine to create a new subtype. e.g., H1N1 combination of swine, bird, and human flu viruses Image Attribution: Urheber 1: CDC/ Alissa Eckert, MS; Dan Higgins, MAM Urheber 2: Tom-b, CC0, via Wikimedia Commons  Canada’s Latest Vaccine Discovery World's first approved Ebola vaccine Developed in Canada's National Microbiology Laboratory (NML) Developed in early 2004 – 100% efficacy Sat on shelf until 2014 Major Ebola outbreak in 2014 – 11,000 killed Merck partnered to produce commercially Timeline: clinical trial in West Africa in 2014-15 compassionate use protocol in Guinea in 2015 eastern DRC outbreak in 2018-2020 Image Attribution: https://www.cbc.ca/news/health/ebola-vaccine-national- microbiology-laboratory-pharmaceutical-industry-scientists-1.5429060 Edible Vaccines Transgenic plant or animal products that trigger an immune response Foods under study: potato, banana, tomato Currently being developed for cholera & Hep B Advantages Disadvantages No needle; no skilled professional needed Need to eat raw – cooking may destroy the antigen No adjuvants or chemicals May take a long time to mature (banana) Stimulate both mucosal and systemic immunity May get spoilt quickly (tomato, banana) Low cost (no refrigeration, sterile equipment needed; local Consistency from fruit-fruit, plant-plant, generation- plants) generation Feeding & immunizing at the same time Dosage evaluation Improved safety (no attenuated pathogens) Public attitude towards GM food Image Attribution: Mahmood, N.; Nasir, S.B.; Hefferon, K., CC BY 4.0, via Wikimedia Commons Bacteriophage A bacteriophage (phage) is a virus that infects bacteria First described by Frederick Twort (1915) and Felix d’Herelle (Canadian) (1917) Bacteriophages are obligate intracellular parasites When not in contact with a host cell, remains entirely dormant: virion When the virion comes in contact with the appropriate host, it becomes active: virus Image Attribution: Professor Graham Beards, CC BY-SA 3.0, via Wikimedia Commons  Structure of Bacteriophages Typically consist of a protein coat (capsid) enclosing genetic material, Head which can be either DNA or RNA Head: Contains the genetic material (DNA or RNA). Tail: Acts like a syringe to inject the Tail genetic material into the bacterial host. Tail fiber Tail Fibers: Help the phage attach to specific receptor sites on the bacterial surface. Image Attribution: Adenosine, CC BY-SA 3.0, via Wikimedia Commons  The Role of Phages in Science Discoveries Hershey-Chase: conducted their experiments with T2 phage Confirmed DNA as the genetic material: Nobel prize for Hershey in 1969 Image Attribution: The original uploader was Adenosine at English Wikipedia., CC BY-SA 2.5, via Wikimedia Commons  Current Uses of Bacteriophages in Different Sectors Phage Therapy: using phages to treat bacterial infections, particularly those resistant to antibiotics (e.g., MRSA, Pseudomonas aeruginosa) Wound Care and Burns: used in wound dressings or topical treatments to reduce bacterial infections in burn wounds Biocontrol Agents: applied to combat bacterial pathogens (e.g., controlling Xanthomonas and Erwinia) in agriculture & Vibrio and Aeromonas in fish farming Food Safety: used to reduce the contamination of food products by pathogens like Listeria monocytogenes (GRAS) Biosensors: developed to quickly identify bacterial contamination in industries like food processing, hospital sanitation and water treatment  Dangers and Concerns Associated with Phage Use Emergence of Phage-Resistant Bacteria: leading to new superbugs that are resistant to both antibiotics & phages Narrow Host Range: selecting wrong phage could result in treatment failure Mutations in Bacteria: Rapid mutations in bacterial populations can change the phage-host interaction Horizontal gene transfer (transduction): inadvertently enhancing the pathogenicity (toxins, resistance) of bacteria Impact on the Microbiome: could still affect non-target bacteria, including beneficial members of the human/animal/enviro microbiome Biofilms: less effective against bacteria that form biofilms  Bacteriophage Life Cycles Bacteriophage particles are less than 1% the size of the bacteria they attack They are composed of an icosahedral head, hollow protein sheath, and sometimes a set of tail fibers The head contains a single chromosome from 5,000 to 100,000 base pairs; replication and gene expression require enzymes and factors of the host cell 18 Size Comparison of Bacteria & Viruses Small, obligate, intracellular parasites (TEM needed to study viruses) 450 nm 300 nm 125 nm 80 nm 30 nm 19 Source:  The Structure of Phage Consists of a nucleic acid chromosome and a protective coating head (capsid) – either DNA or RNA, not both sheath – phages often contain unusual or modified bases protect phage nucleic acid from nucleases that break down host cell nucleic acids during phage infection – # of genes depend on the type of phage: 3-5 or >100 Two major parts – capsid or protein coat (e.g., head, tail) – phage genome with DNA or RNA 20  Phage Structure Three basic phage structures observed are: – icosahedral tailless phage - has an icosahedral head but no tail – icosahedral phage with tail – filamentous phage icosahedral phage icosahedral phage with filamentous phage e.g. MS2 tail e.g. CTX phage that causes e.g. T4 cholera Photo source: http://bacteriophageucd.wordpress.com/oh-the-phages-you-will-see/  Structure of Phage Genetics Analysis: An Integrated Approach Copyright © 2012 Pearson Education Inc.  Lifecycle of a Bacteriophage Phage must recognize bacterium (host range) – λ infects only certain E.coli – Spo1 infects only Bacillus subtilis – E.coli can be infected by λ, M13, P1, T4 and Mu phages Burst size: the number of phages that can be released from one bacterium after infection and growth by one phage – every phage has a characteristic burst size Plaques: circular clear areas in a lawn of bacteria on an agar plate  Phage Functions for its Survival Each phage must perform some minimal functions for continued survival: – protect its nucleic acid from external environment (against degradation, chemical & natural mutation) – deliver its nucleic acid inside of bacterium (~ 3000 bp/sec) – convert bacterium into phage–producing system – Induce lysis of bacterium to release progeny phages from an infected bacterium  Types of Bacteriophage Virulent phage – phages which can only multiply on bacteria and kill the host by lysis at the end of the life cycle (lytic state) e.g. T4 Temperate phage e.g. P1 and λ – may or may not undergo lytic cycle – most commonly integrates and maintains phage chromosome in stable, silent state within bacteria (lysogenic state) – in this condition, the bacterial host is known as lysogen – the incorporated phage is known as prophage lysogens are resistance to subsequent infections due to immunity provided by prophage  Steps of the Lytic Cycle The lytic cycle is a six-step process that leads to lysis of the host cell 1. Attachment of the phage to the host cell 2. Injection of the phage chromosome into the host, followed by circularization of the phage chromosome 3. Replication of phage DNA using host proteins and enzymes Genetics Analysis: An Integrated Approach Copyright © 2012 Pearson Education Inc.  Remaining Steps of the Lytic Cycle 4. Transcription and translation of phage genes, and subsequent production of heads, sheaths, and tail fibers for assembly of progeny phage 5. Packaging of phage chromosomes into phage heads 6. Lysis of the host cell, and release of progeny phage particles Genetics Analysis: An Integrated Approach Copyright © 2012 Pearson Education Inc. Lytic Phage Life Cycle (Virulent)  Summary - Lytic Phage Life Cycle (Virulent) DNA or RNA is injected by phage into host cell Phage genetic information is expressed, taking over host cell and redirecting machinery to phage replication Upon completion of replication, bacterial cell lyses, releasing phage (lysis) When this happens on a lawn of agar-cultured bacteria, a plaque is formed Chapter 7: Bacterial recombination © 2002 by W. H. Freeman and Company  The Lysogenic Cycle Some bacteriophages (temperate phages) have an alternate, temporary life cycle involving integration of the phage chromosome into the bacterial chromosome This is called the lysogenic cycle; integration is called lysogeny Lysogeny can persist for many bacterial cell cycles, but eventually comes to an end, and the lytic cycle is triggered Genetics Analysis: An Integrated Approach Copyright © 2012 Pearson Education Inc.  Steps of the Lysogenic Cycle 1. Attachment of the phage particle to the host cell 2. Injection of the phage DNA into the host, followed by phage-chromosome circularization These two first steps are the same as the lytic cycle 3. Integration of the phage chromosome into the host chromosome via recombination at a specific DNA sequence found in both chromosomes Genetics Analysis: An Integrated Approach Copyright © 2012 Pearson Education Inc. Lysogenic Cycle  Additional Steps of the Lysogenic Cycle Once integrated into the host chromosome, the phage chromosome is called the prophage 4. Excision of the prophage in response to an environmental signal, through a reversal of the site-specific recombination leading to integration 5. Resumption of the lytic cycle, beginning with phage-chromosome replication Genetics Analysis: An Integrated Approach Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Copyright © 2012 Pearson Education Inc.  Phage lamba () A temperate phage of E. coli Upon entering cell,  DNA circularizes It can align with specific region of genome, the  attachment site, and recombine with host genome Upon integration, it can become silent prophage, synthesizing inhibitor of its further replication Upon exit, it may pick up adjacent host genes which can be transduced to next host cell Chapter 7: Bacterial recombination © 2002 by W. H. Freeman and Company  Lytic vs Lysogenic Cycle How a phage decides on lysogenic vs. lytic cycle?  Lytic vs. Lysogenic Cycle Lysis or lysogeny is determined by a contest between two genes that encode repressors – cI gene encodes the l repressor that blocks transcription of all other genes cell enters lysogeny – cro gene encodes a repressor that blocks transcription of cI gene and allows other genes to be transcribed ("anti-repressor“) –thus prevents the establishment of lysogeny –cell enters lytic cycle  Lytic vs. Lysogenic Cycle Cells with sufficient nutrients Lytic Cycle Cells with limited nutrients Lysogenic Cycle Phage Infected E. coli – Before & After  Significance of Lysogeny Lysogenic or phage conversion – in a lysogen, genes carried by the phage get expressed in the cell – these genes can change the properties of the bacterial cell – this is significant clinically – e.g. lysogenic phages have been shown to carry genes that can modify the Salmonella O antigen, which triggers an immune response  Difference between harmless E.coli K12 and O157:H7 Complete sequence of E. coli (K-12) in Science (1997) – genome consists of a single molecule of DNA containing 4.64 x 106 bp – these encode 4288 proteins Complete sequence of the pathogenic strain O157:H7 was reported in the Nature (2001). – contains 5416 genes in 5.44 x 106 base pairs of DNA – include 1,387 genes that are not present in its harmless laboratory relative E. coli K-12 (K-12 has 528 genes that are not found in O157:H7) – here are two strains of the same species that differ in some 25% of their genes  Walkerton E. coli Outbreak – May 2000 Community of about 5,000 people City’s water supply drawn from groundwater became contaminated with O157:H7 strain of E. coli Contamination was due to farm runoff into an adjacent water well Seven people died and 2500 became ill Key recommendations: source water protection, training and certification of operators, a quality management system for water suppliers, and more competent enforcement Virulence Factors Carried on Phage Bacterium Phage Gene Product Phenotype Vibrio cholerae CTX phage cholerae toxin cholera hemorrhagic Escherichia coli lambda phage shiga-like toxin diarrhea Clostridium botulism (food clostridial phages botulinum toxin botulinum poisoning) Corynebacterium corynephage beta diphtheria toxin diphtheria diphtheriae Streptococcus T12 erythrogenic toxins scarlet fever pyogenes Source: San Diego State University - http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/phage/phage-virulence.html  Transduction Some phages accidentally pickup bacterial DNA and carry it from one bacterial cell to another Generalized – phage incorporates random fragments of host DNA for transfer to another host – e.g., phages P1 and P22, temperate phages which accidentally stuff host DNA into phage head Specialized – specific genes are transferred – e.g., phage  which transduces only adjacent genes Chapter 7: Bacterial recombination © 2002 by W. H. Freeman and Company  Assay for Lytic Phage – Plaque Assay Infected bacteria lyses to release ~100-200 phage – each released phage adsorb to nearby bacteria – same process continues for multiple cycles  Plaque Assay Cont. Bacteriophage titre: the concentration of infectious viral particles per millilitre of growth medium (pfu/ml) Multiplicity of infection (MOI) = ratio of infectious agents (e.g. phage) to infection targets (e.g. bacteria)  Plaque Assay Cont. A plaque is a clear area that results from the lysis of bacteria Each plaque arises from a single infectious phage Over time, phage destroys all bacteria at a single localized area in the agar – produce a clear, transparent circular region called ‘plaque’ – 1 phage forms 1 plaque – the infectious particle that gives rise to a plaque is called a pfu (plaque forming unit) 46 Lytic Plaques vs. Lysogenic Plaques Lytic Plaque Lysogenic Plaque  Broth Clearing Assay A dilution series of phage in broth is set up, then inoculated with target bacteria An endpoint is determined based on the highest dilution producing lysis of bacteria and clearing the broth culture This method can be used in parallel with the plaque-forming assay for a given phage, then used later alone as a quick check on concentrations of samples  Phage Typing The identification of bacteria by testing their vulnerability to various bacteriophages A bacterial host may be infected by a range of phages A phage identifies and specifically binds to a host by by binding to specific bacterial surface receptors Host cell may become resistant by surface mutations – lysogenic conversion changes surface receptors and thus protects the host Photo Source: http://classes.midlandstech.edu/carterp/Courses/bio225/chap10/ss3.htm One Step Growth or Phage Burst Size Assay In 1939, Ellis and Delbrück developed a method to study a single round of virus multiplication – one step growth curve  Phases of Life Cycle of a Lytic Phage 1. Eclipse phase – no infectious phage particles can be found inside/ outside host – phage nucleic acid takes over the host biosynthetic machinery and phage specified mRNA's and proteins are made – synthesis of early mRNA's which code for early proteins needed for phage DNA synthesis and for shutting off host DNA, RNA and protein biosynthesis early proteins may degrade the host chromosome – after phage DNA is made late mRNA's & proteins are made the late proteins are the structural proteins that comprise the phage as well as the proteins needed for lysis of host  Phases of Life Cycle of a Lytic Phage 2. Latent Phase (Intracellular Accumulation Phase) – nucleic acid and structural proteins that have been made are assembled – infectious phage particles accumulate within the cell 3. Rise Phase (Lysis and Release Phase) – bacteria begin to lyse due to the accumulation of the phage lysis protein – intracellular phages are released into the medium – the number of particles released per infected bacteria may be as high as 1000 One Step Growth or Phage Burst Size Assay total number of complete virions number of free viruses Burst Size = the ratio of the number of progeny phage to the number of input phages Infectivity Rate (%): pfu/ml at time point (e.g final point)/ Initial conc. of bacteria/ml x 100  Additional Comments on Handling Phage Phages are stored with addition of cations, glycerols and proteins at 4°C to protect from damage – reduces loss of viability – enables phage infection and plaque formation to remain high Host bacterial cells should be healthy, undamaged cells, in their log-growth stage  Virus in the News 1. Giant virus resurrected from 30,000-year-old ice scientists –have revived a giant virus that was buried in Siberian ice for 30,000 years size = 1.5 –μm -bigger than some bacteria! was still able – to infect amoebae despite having spent 30 millennia in a frozen state don’t need a TEM; can be viewed with a light microscope! Pithovirus sibericum Source: https://www.nature.com/news/giant-virus-resurrected-from-30-000-year-old-ice-1.14801  In the News 2. Virus's similarity to body's proteins may explain Autoimmune Diseases – autoimmune disease arise from an inappropriate immune response of the body against its own cells – due to “molecular mimicry”? – e.g. viral infection → human immune cells (macrophage, a dendritic cell or a B cell) engulfs and chops into small fragments → T cells recognize viral proteins and initiate destruction of any tissues containing the virus – problem: if viral fragment mimics one of the proteins in the body → T cells kills virus infected cells as well as the healthy human cells with the similar protein Read: http://www.nytimes.com/1996/12/31/science/virus-s-similarity-to-body-s-proteins-may-explain-autoimmune- diseases.html?pagewanted=all&src=pm  Virus in the News 3. Moving towards a Universal Fu Vaccine – problem: correctly predicting the strains of the flu virus – influenza virus constantly changes its proteins in the outer coating, mutating from year to year – flu vaccines typically contain several strains of killed virus – Injecting this mix prompts the development of antibodies → short term – a team of scientists from the U.S. and China have designed a universal flu vaccine – the new version contains live mutant virus --> a strong reaction from T cells ---> longer term immunity Source: https://www.scientificamerican.com/article/scientists-move-closer-to-a-universal-flu-vaccine/ 8  Virus in the News 4. How Does the Flu Actually Kill People? – 291,000 to 646,000 deaths annually worldwide – in most cases the body kills itself by trying to heal itself – after infection, the influenza virus hijacks human cells to replicate itself --> triggers a strong immune response – body sends white blood cells, antibodies and inflammatory molecules to eliminate – T cells attack and destroy tissue harboring the virus → in respiratory tract and lungs where the virus tends to take hold – immune system's reaction is too strong → destroying so much tissue in the lungs → they can no longer deliver enough oxygen to the blood → hypoxia and death  Reference Chapter 7 – Bacteriophage Genetics from Text Chapter 4 – Bacteriophage Genetics from Modern Microbial Genetics, Second Edition. Edited by Uldis N. Streips, Ronald E. Yasbin ©2002 Wiley-Liss, Inc. Chapter 7 – Bacteriophage from Fundamental Bacterial Genetics, by N. Trun and J.E. Trempy, 2003. Wiley-Blackwell University of South Carolina, School of Medicine – http://pathmicro.med.sc.edu/mayer/phage.htm 62