General Microbiology Unit 4

UIowa MICR:2157 Fall '25 Unit 4 Exam

Sickle Cell Disease

Autosomal Recessive disorder

Makes RBCs sickle-shaped & difficult to travel through capillaries

Mutated gene for hemoglobin via single bp substitution

CRISPR Discovery

1. Found directed repeats with dyad symmetry in E. coli

2. These regions were found in 10 different bacteria, archaea, and mitochondria

3. CRISPR repeats are separated by nonhomologous spacers, and 4 Cas proteins predicted

4. Phage-resistant strains in bacteria used in food production showed spacers homologous to genomes of phage

CRISPR Function

• tracrRNA + crRNA direct Cas 9 to cut target DNA at a region that bp with crRNA

• repaired via NHEJ (deletion) or HR (editing)

CRISPR to correct sickle cell disease

• BCL11A binds and represses gamma-globulin, repressing fetal Hb expression

• Cas9 edits non-coding region of BCL11A, repressing expression

Principles for the governance of human genome editing

• Promoting Well-being

• Responsible science

• Due care

• Transparency

• Respect for persons

• Fairness

• Transnational cooperation

Regulation for Germline Genome Editing

• Absence of reasonable alternatives

• Existing preclinical data supports efforts

• Vigorous ongoing oversight and transparency

• Long-term multigenerational and population studies

Basic CRISPR Research

• Discovery of strange loci in the genomes of bacteria and archaea

• Elucidating the role of CRISPR in defense against phage

• Investigating the mechanism of CRISPR-mediated cuts to DNA

Translational CRISPR Research

• Engineering sgRNA to direct CRISPR to target any sequence

• Evaluating the ability of CRISPR to cut DNA in human cell lines

Clinical CRISPR Research

• Demonstrating CRISPR can modify cells of patients with genetic disorders as a therapy

PCR Ingredients & Reaction

Primers, DNA Pol, template DNA, dNTPs, buffer

1. Denaturation: >94C, breaks H bonds

2. Annealing: 55-72C, primers bind

3. Extension: 68-72C, DNA Pol copies the template

Restriction Enzyme Discovery

Derived from bacteria, function as defense against phage infection

Cleaves unmethylated DNA at specific nucleotide sequences

Multiple Cloning Site

Region where plasmid can be cut to have complementary overhangs to the insert

Chemical Competence

Exposure of E. coli to CaCl2 makes it competent in the presence of a heat shock

Electrical Competence

exposure of E. coli to cold water washes makes it take up DNA when zapped with electric shock

Agrobacterium tumefaciens

Forms a parasitic symbiosis with plants, causing crown gall disease

Crown galls are plant tumors induced by A. tumefaciens tumor induction plasmid

T-DNA can be modified to insert foreign genes into plants (Bt toxin, etc.)

Virus Characteristics

Acellular

Obligate Intracellular Parasites

Shared structure (nucleic acids + capsid)

Host range

Spectrum of hosts that a pathogen can infect

Depends upon susceptibility and permissibility

Virion structure

Virion = mature virus particle, nucleocapsid

• Nucleic Acids = DNA or RNA

• Capsid = protomers that form capsomeres

Capsid shapes

Helical = protomers form rigid, hollow tube

Icosahedral = regular polyhedron, composed of capsomeres made of 5-6 protomers

Complex symmetry = not helical or icosahedral, poxviruses + complex phage

Naked Capsid vs. Enveloped Viruses

Naked capsid = only nucleocapsid, infectious outside the body longer than enveloped

Enveloped = nucleocapsid + envelope, require bodily fluids for transmission

• Lipid bilayer acquired from host + glycoprotein/spikes

Lysozyme

Often encoded by phage

Makes a hole to allow for entry of nucleic acids, also produced to lyse the host cell

Neuraminidase

Spike protein encoded by influenza

Enzymatic activity supports separation of envelope from host cell during release

The Baltimore Scheme

Groups viruses based on the relationship between genomes and mRNA

Should not be used to describe relationships between viruses

One-step Growth Curve

Eclipse Phase - No virions detected in or outside of cells

Maturation - Packaging of nucleic acids in capsids, first virions detected

• Burst Size = number of virions released

Latent Period = Eclipse + Maturation

Viral Replication Cycle

Attachment = ligand on virion attaches to receptor on host cell

Entry = genome alone or entire nucleocapsid enters host cell

Synthesis = viral nucleic acids synthesized/replicated and viral proteins synthesized

Assembly = nucleic acids packaged in the capsid

Release = host cell lysis or budding

Bacteriophage Structure

• Icosahedral capsid head, housing nucleic acids

• Sheath/tail: helical capsid, contracts to deliver tail tube through bacterial cell wall

• Tail fibers: associate with host cell receptors

• Tail pins: associate with cell wall to hold tail close to penetration site

• Baseplate: mediates attachment, sheath contraction, phage DNA ejection

Virulent vs. Temperate Phage

Virulent = lytic cycle leads to lysis of cell

Temperate = reproduces using the lytic or lysogenic cycles

• Induction can end the lysogenic phase + induce lytic cycle

T4 Phage

Model virulent phage, infects E. coli

• Attaches to LPS

• Lysozyme forms a pore to inject dsDNA into host

• Synthesis of replication enzymes (early) + head, tail, lysis proteins (late)

• Structural proteins + nucleic acids assemble into virions

• Lysozyme lyses host cell

Prophage

Phage genome integrated into bacterial host chromosome

Mediated by recombinase/integrase

Phage Conversion

Phage infection changes the properties of the cell

• Generalized transduction - any gene in bacterial genome is packaged in the virion

• Specialized transduction - genes near insertion site are repeatedly packaged along with phage DNA

Toxin-antitoxin systems

Antitoxin inhibits induction of cell death by phage toxin

Abortive infection - phage DNA inactivated antitoxin, leading to cell death

• Prevents more virions from spreading, protecting the population

CRISPR as a defense against phage

Cas proteins recognize protospacer adjacent motifs (PAMs) in DNA

Cas proteins cleave near PAM, inserting short piece of DNA into the chromosome

Results in “immunization”; Cas proteins destroy DNA with same protospacer

Fecal Indicators

Ex. coliphage/coliforms

Detects fecal contamination in food, water, treated wastewater

Survive as well or better than pathogens in the substance suspected of contamination

Easy to detect + present in high concentrations in feces

Animal Virus Attachment

Ligand on virion attaches to receptor on host cell

• Specificity of ligand to receptor determines tropism (specificity of virus to particular cell/tissue)

Animal Virus Entry

Fusion: envelope is left at the host cell membrane or at the endosome (receptor-mediated endocytosis)

Uncoating: removal of nucleocapsid due to enzyme/pH change in endosome or following escape

Animal Virus Release

Host cell lysis = viral proteins puncture host plasma membrane

Budding = occurs at the same time as envelope formation, viral proteins incorporated into membrane

dsDNA Viruses

Nucleic acid synthesis occurs in nucleus

Replication: dsDNA → dsDNA

Transcription/translation: dsDNA → mRNA → Protein

Tegument

layer of proteins between nucleocapsid + envelope

Productive infection

Cell lyses, releasing 50,000 - 200,000 virions

Latent infection

Virions are undetected, can reactivate after months/years

Viral genomes remain in cells w/o virion production as proviruses or extrachromosomal elements

Persistent/Chronic infection

Slow increase in viral load, can reactivate after months/years

The cell is not lysed but remains a viral factory

Includes cytopathic effects (changes in host cells and tissues that are distinct from lysis)

dsRNA Viruses

Nucleic acid synthesis occurs in cytoplasm

Replication: dsRNA → ssRNA → dsRNA

Transcription/translation: dsRNA → mRNA → Protein

Needs Rdrp

Positive Sense Viruses

Nucleic acid synthesis occurs in cytoplasm

Replication: +RNA → -RNA → +RNA

Transcription/translation: +RNA → Protein

Needs Rdrp

Negative Sense Viruses

Nucleic acid synthesis occurs in cytoplasm (influenza in nucleus)

Replication: -RNA → +RNA → -RNA

Transcription/translation: -RNA → mRNA → Protein

Needs Rdrp, packaged in virion

Retroviruses

Nucleic acid synthesis occurs in the cytoplasm (RNA → DNA) and nucleus (DNA integration)

Replication: +RNA → ssDNA → dsDNA → +RNA

Transcription/translation: DNA → mRNA → Protein

Reverse transcriptase and Integrase needed

Proteins that viruses encode for

Nonstructural Proteins (NSP): enzymes for viral processes (Rdrp, reverse transcriptase)

Structural proteins (SP): spike/glycoproteins

Accessory proteins (AP): Interfering with cellular processes and the innate immune response

Hemagglutinin Spikes (HA)

Attaches to sialic acid moieties linked to glycoproteins and glycolipids on cells in the respiratory epithelium

Neuraminidase Spikes (NA)

Facilitates induction of receptor mediated endocytosis and separation of the virus from the host cell during budding

Cytocidal infection

Disease in which some of the symptoms result from cell death

Oncogenic viruses

Activate/insert oncogenes, inactivate tumor suppressor genes, cause chronic inflammation

Cytokines

Soluble, low molecular weight protein or glycoprotein that acts as an intracellular signaling molecule

• Used by immune response to communicate

Cytokine storms

Dilation of blood vessels, rapid drop in blood pressure, leading to shock

Can also overstimulate fever-inducing pathways, causing unregulated blood clotting

Immune Response to viral infections

Complement proteins → opsonization → phagocytosis

NK cells → look for absence of MHC I or cells tagged with antibodies

Interferons → cytokines produced by infected cells that promote antiviral pathways in neighboring cells

Production of neutralizing antibodies

Cytotoxic T cells → check MHC I signals → induce apoptosis of infected cells

Treatments for viral infection

Neuraminidase inhibitors: inhibit enzymatic activity of NA spikes

Nucleoside/mononucleotide analogs: inhibit DNA viruses which use their own enzyme to phosphorylate nucleotides (incorporation of analogs halts DNA synthesis)

Nucleoside RT inhibitors: inhibit retroviruses by incorporating into DNA during reverse transcription, halting synthesis

Nonnucleoside RT inhibitors: bind to and inhibit reverse transcriptase

Protease inhibitors: block HIV protease (required for protein synthesis)

Integrase inhibitors: prevent DNA integration

Fusion inhibitors: prevent entry

Types of cell lines

Primary: used for viral growth, die after a few generations

Diploid: developed from human embryos, can be maintained for hundreds of generations

Continuous/immortal: cancerous cells, can be maintained indefinitely

Serology

Detection of presence of antibodies specific to pathogen of interest in patient’s serum (ELISA)

Lateral flow/rapid antigen assays

Detect presence of specific antigen using labeled & unlabeled antibodies

Multiplex PCR

multiple primers used in the same rxn to determine the causal agent

Reverse transcriptase PCR

For RNA viruses

RT enzyme makes cDNA, which is then amplified via PCR

Influenza virus

Antisense RNA genome

Enveloped

NA & HA spikes used to designate types

Antigenic drift

Accumulation of point mutations (error-prone Rdrp)

As mutations accumulate, transmission increases & pathogenicity decreases

Antigenic shift

reassortment of influenza genomes in a “mixing vessel” due to co-infection of two different flu strains in the same cell

Plant viruses

Have a wider host range

Prions

Infectious protein that induces progressive degeneration of the brain & eventual death

PrPc (cellular prion protein): localized in neuron plasma membrane, brain development/function

PrPsc (scrapie-associated PrPc): same AA sequence as PrPc but has been irreversibly misfolded

• Can cause PrPc to misfold following direct contact

• Aggregates and causes degeneration

Scrapie

Prion disease first described in sheep in 1732

Variant Creutzfeldt-Jakob disease

Prion disease first observed in humans in 1920

Transmitted by ingesting infected cows (mad cow disease)

Kuru

Prion disease first described in 1959 in Papau New Guinea

Chronic Wasting Disease

Prions disease that effects deer, elk, reindeer, sika deer, moose