MCBL 121 final

Why might we use anti-viral treatments combining different targets of action?

Why might we use anti-viral treatments combining different targets of action?

Viruses can mutate rapidly to avoid the action of some drugs

How is the Baltimore classification system organized?

Virus replication mechanisms and genomes

You run an animal infection experiment with a mutant and wild-type pathogenic bacterium, mixed together and phenotypically tagged, in order to calculate a competitive index. The input ratio (mutant/wt) is 10/1 and the output ratio (mutant/wt) is 10/1. What can you conclude?

The two genotypes have similar fitness in the host

What is a primary function of antibodies?

opsonization- using opsonins to tag foreign pathogens for elimination by phagocytes

Which of the following accurately characterizes innate immunity?

active at birth

A phage genome integrated into the bacterial chromosome is known as a(n):

prophage

A culture of E coli does not exhibit visible turbidity after growth with antibiotic A at 100ug/ml, while a culture with 10ug/ml of antibiotic A shows about the same level of growth as a culture with no antibiotic. Which of the following statements best describes the MLC?

Not determinable with the given information

An operon contains 4 genes (in order: A, B, C, D). What best describes the effects of a missense mutation in B?

All genes are expressed

A bacterial surface protein that binds antibody Fc would inhibit which immune functions?

Neutralization

What is a measure for how easily pathogens can move from one human to another?

Infectivity

What does the viral capsid do?

packages viral genome and delivers it into the host cell

Envelope

encloses capsid, formed from cell membrane. contains glycoprotein spikes which are encoded by the virus

• tegument proteins may be found between envelope and capsid

filamentous virus

capsid forms a long tube of protein with the virus coiled inside it; capsid monomers form a tube around genome, which usually winds helically w/in the tube

What are the seven groups of the Baltimore Classification?

I. dsDNA virus

II. ssDNA

III. dsRNA

IV. +ssRNA

V. -ssRNA

VI. RNA reverse transcribing viruses (w/ reverse transcriptase)

VII. DNA transcribing viruses (RNA intermediate)

Are bacteriophages enveloped?

No

What are the different phases of the viral growth curve and what does each represent?

• eclipse period: viruses bound to host cells, and are making proteins, genomes and assembling particles. No free virus can be detected

• rise period: bacterial cell lysis. bacteriophage is making more copies of phages, virions bursting out of cell. A large amount of free packaged virus can be detected

• curve plateaus when cell lysis is complete

Positive sense single-stranded RNA viruses can…

be directly translated into protein

Negative sense single-stranded RNA viruses….

are transcribed to mRNA and then translated into viral protein

What are the two different types of viruses we discussed that infect E.coli & how do they differ?

T4 and lambda coliphages

• T4- intemperate → lytic

• lambda: temperate → lysogenic

Lytic (intemperate) phages

• phage infects bacterium

• Phage DNA replicates & makes phage proteins

• new phage particles are produced

• cell lysis

Lysogenic (temperate) phages

maintain a stable relationship with the host cell in which they neither multiply nor are lost from the cell

• integrate viral genome (prophage) into host DNA- cell is a lysogen

• phage DNA excises upon induction (DNA damage to host cell) & new phage particles are produced

• then cell lyses

What are the different forms of defense against bacteriophage infection?

• genetic resistance

  • altered receptor proteins

• restriction endonucleases

  • cleave viral DNA lacking methylation

• CRISPR integration of phage DNA sequences

  • foreign DNA chewed up and integrated into the genome & can be transcribed, serves as a memory card; will use the sequence to guide trageted degradation of another copy of the viral genome in 2nd infection

What does it mean receptors determine tropism of the virus?

tropism of virus → what kind of cell a virus infects, different receptors of different cells (cellular tropism, tissue tropism, host tropism)

DNA viruses utilize what for replication?

host replication machinery

RNA viruses utilize what for their replication?

use an RNA-dependent RNA Polymerase to transcribe their mRNA

Retroviruses utilize what for their replication?

use reverse transcriptase to copy their genome sequence into DNA for insertion into the host genome

Coronavirus is what kind of virus?

positive sense ssRNA, binds to ACE2 receptor

What kind of virus is HIV?

retrovirus, attaches to CD4 receptor

• uses reverse transcriptase and integrase to get DNA copy of genome & insert it into host DNA

Why is it useful that viruses are error-prone in their replication?

• they can change their tropism by modification of their surface proteins

• they can escape from host immune system

• they can change the interaction with therapeutics by changing molecular targets; enzymes/drugs do not interact

How should viral treatments be made to lessen the likelihood of escape mutations?

make drugs that target multiple viral pathways (fusion, reverse transcription, receptor-binding, integration)

• Highly Active Antiretroviral Therapy for HIV is an example

What is a plaque forming unit?

quantifies the number of viral particles capable of forming plaques in a viral culture, indicating viral infectivity.

• 1 plaque is formed from infection of 1 virion

PFU assay shows 100 plaques at 10-2 dilution, from 100ul plated
from the original phage mixture. What is the concentration of phages in the original sample in PFU/ml?

[(Plaque count) * (Dilution factor)] / volume mL = PFU / mL

100 plaques * 100(dilution factor)/0.1 mL= 100,000 pfu/mL

Infection

a pathogen grows & multiplies within or on another organism

• infection does not always cause disease

infectious disease

due to presence and multiplication of pathogens, part of all of the host is not capable of performing its normal function

direct transmission

direct contact (touch) or aerosolization (sneeze)

indirect transmission

animal vectors of disease

zoonotic infection

disease spreads from animals → humans or vice versa

pneumonic plague

when Yersenia pestis travels to the lungs, spreads much easier than bubonic plague

What are the steps to infection?

1. Attach & invade host tissues

2. suppress host defenses

3. acquire nutrients from the hosts

4. propagate in the hosts

5. transmit to a new host

Pathogenicity

ability of organism to cause disease

infectivity

how easily pathogen spreads or causes infection

virulence

measure of degree/severity of a disease

What is ID50?

infectious dose; number of organisms to colonize 50% of hosts

• lower ID50 → more infectious, higher ID50 → less infectious

What is LD50?

lethal dose; number of organism to kill 50% of hosts

• lower LD50 → more virulent/pathogenic

What are the pitfalls of using ID50 & LD50?

• Need a lot of animals

• insensistive to attenuated (weakened) or low-pathogenicity

• need to define measurements clearly (time points & sites)

virulence factor

individual characteristics of a pathogen that allow it to invade host and cause disease

• all enhance the disease-producing ability of the pathogen

• encoded by virulence genes, are highly regulated

What are pathogenicity islands?

section of genome that contains multiple virulence genes that often encode related functions

• often planked by a phage or plasmid genes

• often have G-C content different from the rest of the genome

Koch’s Postulates for Pathogenesis

• Suspect microbe is present in diseased tissues but absent from healthy tissues

• Suspect microbe must be isolated and maintained in pure culture

• Inoculation of the cultured microbe into a susceptible host should reproduce original symptoms

• The same microbe must be isolated again from the inoculated diseased host

What do molecular Koch’s Postulates do?

• they can assign role of a gene or gene product to pathogenesis

• only relates to genes of a bacterium

• involves mutagenesis and complementation

Complementation

restoration of mutated gene to original function

polar mutation

A mutation that affects the transcription or translation part of the gene or operon downstream of the mutant site.

If wild type phenotype is restored just by restoring the mutated gene, what does this mean?

no other genes are involved in the phenotype

How can we examine the effects of a mutation?

measure loss of pathogenicity, can do a competition assay

competition assay

based on a corrected ratio of strains after infection as a measure of pathogenicity/fitness in the host

Competitive Index

indicates whether the mutant gene is a putative virulence factor

When CI equals 1…

WT and mutant grow equally; gene does not have anything to do with virulence

When CI < 1,….

WT outcompete mutant bacteria; the gene is important for virulence

When CI > 1,…

mutant outcompete WT bacteria; this gene suppresses virulence

What is the drawback of competition assay?

trans complementation of phenotype can occur, where WT bacteria can compensate for defects of the mutant and allow both to colonize

Why is our epithelium important?

it is the 1st barrier of defense against foreign objects and organisms

• lines body cavities, blood vessels, & organs (mucosa)

• secretes glycoproteins, secretes chemical antimicrobials, keep cell interface w/ environment clear of microbes, physical barrier, etc.

autoimmunity

The immune system mistakenly attacks healthy cells and tissues in the body, leading to chronic inflammation and tissue damage.

allergen

substance that induces immune response against otherwise harmless stimuli

antigen

substance that induces production of antibodies

intrinsic immunity

innate defense system of a cell that provides immediate protection against pathogens without prior exposure or activation of specific immune responses.

innate immunity

present at birth, limited diversity of responses, non-specific defense, no memory, fast responses, constant during response, uses phagocytes, uses pattern recognition receptors (PRRs) of microbes

adaptive/acquired immunity

acquired after contact with pathogen/antigen, includes a vast diversity of responses, is specific (antibodies), memory responses, slow responses, improves during response, uses T-cells & B-cells, generates antigen-specific antibody responses, and uses antigen-specific recognition

Pattern recognition receptors

proteins made by cells within the body that recognize pathogen-associated molecular patterns (PAMPs)

• when PAMPs associate w/ PRRs → immune cells are activated and produce anti-microbial compounds to clear infection

what do phagocytes do?

they ingest foreign particles, microbes, or parts of dead or dying cells.

• form acidified phagosomes, fuse w/ lysosome to form phagolysosome and enzymes digest foreign particles, then waste products are discharged

humoral immunity

involves antibodies produced by B cells to neutralize pathogens in body fluids, providing defense against bacteria, viruses, and toxins.

antibodies (immunoglobulins)

glycoproteins produced by B cells; identify and neutralize specific pathogens based on the antigens that are exposed by the host

What are the 2 major functions of antibodies?

1. opsonization- antibody coats bacteria & promotes phagocytosis

2. neutralization- preventing biological activity by binding Fc r

major histocompability complexes

cell surface proteins that present antigens to immune cells in immune synapse, crucial for immune response and self-recognition.

two-signal hypothesis

proposes that T cell activation requires two signals: antigen recognition by T cell receptor (Signal 1) and co-stimulation (Signal 2) for full activation.

What does triggering T-cell receptor alone lead to?

tolerant state (anergy)

T-cell

type of white blood cell that play a central role in the immune response, recognizing and destroying infected or abnormal cells.

How is complement/neutralization activated?

by binding to antibodies (in Fc region) that are bound to antigens (in Fab region), triggering a cascade of reactions to destroy or neutralize the invader (opsonization or membrane attack complex → cell lysis

What does activating complement do?

Activating complement enhances the immune response by promoting inflammation, opsonization, and cell lysis to eliminate pathogens.

How do vaccines work?

by stimulating the immune system to produce antibodies against pathogen-specific antigens, providing immunity by generating memory without causing the disease.

phase variation

a strategy used by bacteria to switch between different phenotypes (cell wall, capsule, external structures) by mutations, aiding in immune evasion and adaptation to changing environments.

What are the different ways in which bacteria can avoid being detected and killed by immune mechanisms?

• phase variation

• binding of host antibodies via Fc to prevent Fab binding & complement activation

• degradation of antibody

• formation of structures to prevent access to immune cells and components (biofilm, capsule)

• go intracellular to avoid humoral immunity

• kill host immune cells (via toxins)

Microbial toxins

products of pathogens that alter normal metabolism of the host

• directly damage host cells

• endotoxins: intrinsic to bacterial cell, but can enter bloodstream → sepsis

• exotoxins: proteins secreted out of the pathogens

AB toxins

common structure of bacterial exotoxins- consist of two subunits - A is the active component causing harm, while B binds to target host cell receptors for entry.

How do you fulfill Molecular Koch’s Postulates?

• need to manipuate presence of microbes in order to observe symptoms and transmission

• inoculating microbe into susceptible host should produce same symptoms, need a good model of disease (animal)

What are characteristics of a good animal model of infection?

anatomical similarity, similar symptoms and courses of diseases, similar bacterial distribtuion, similar routes of transmission, similar immune system, inexpensive to obtain & maintain, no endangered species, & genetic modification available (?)

How can fermentation be used to preserve foods? How do fermentation products contribute to food production?

• fermentation can produce products that inhibit microbial growth, by

  • pH changes (acid/base acts as an effective preservative), &

  • alcohols that act as detergents for growth inhibition

• products of fermentation in food include: cheese, tempeh, miso, soy sauce, pickles, kimchi, natto, century eggs, chocolate, bread, beer, wine, etc.

What is the difference between food spoilage and food contamination?

food spoilage refers to microbial changes that renders a product unfit for consumption; food contamination is food poisoning, which contains human pathogens

What is the difference between rancidity and putrefaction?

• rancidity: due to oxidation of fats

• putrefaction: due to decomposition of proteins

What are some methods for preserving food from microbial breakdown?

• eat it before it spoils

• heat treatment

  • pasteurization

  • sterilization via heat

    • cooking

    • autoclave/UHF

• remove liquid water (changing osmolarity)

  • freezing, drying, salting, jamming

• radiation

• organic acids

• useful microbes in controlled fermentation

Biological Safety levels

BSL-1: safest (Saccharomyces cerevisiae) → BSL-4: most dangerous (extreme health risk to humans; Ebola)

In which BSL are most microbes found in?

BSL-2

Garbage in garbage out (GIGO)

A concept emphasizing that flawed input results in flawed output.

• samples must be collected aseptically

What are the different ways in which microbes can be identified?

by structure, by metabolism, using Triple Iron Sugar Agar, PCR, quantitative real time PCR, antibody binding (fluorescent antibody staining or ELISA), & point-of care tests

In Gram-staining procedure, gram positive & gram negative bacteria show up as what colors?

• gram negative- pink

• gram positive- purple/blue

Why is differential media helpful for identification (MacConkey Agar)?

• strains grow, but differ in indicator color, helps to identify

Triple Iron Sugar (TSI) Agar

a differential medium used to differentiate bacteria based on their ability to ferment sugars and produce hydrogen sulfide gas

• yellow @ acidic pH

• red → purple @ alkaline pH or >

fermentation of lactose/sucrose → yellow

bacteria that cannot ferment break down proteins instead, raising pH → deep purple/red

How are TSI results evaluated?

Score is A (acid/yellow) or K (alkaline/red)

• slant/butt tells us about aerobic v. anaerobic conditions

• hydrogen sulfide gas produced or not (black precipitate)

• gas produced can give cracks in the agar

restriction analysis

technique that uses restriction enzymes to cut DNA at specific sequences, helping to create a genetic fingerprint for identification purposes.

quantitative real time PCR

Quantitative real-time PCR measures DNA amplification during each cycle, providing accurate quantification of initial DNA template.

• utilizes reverse transcriptase to generate complementary DNA from RNA templates

• involves measurement of fluorescence that increases with target copy number

  • either using TaqMan (custom, specific probe) or SYBR (more amplicon → more signal)

Which region of antibody binds antigen?

Fab region, Fc is constant region