MIC 102 A01 - Final

Mutations

Change in genetic material

• Change in DNA → change in RNA → change in protein

• May be neutral, beneficial, or disadvantageous

• Beneficial mutations give rise to diversity, adaptation to environment

• Natural selection removes individuals with disadvantageous genotypes

• IMPORTANT: Mutations arise BEFORE selection!

Disadvantageous errors will be most costly to a cell and its progeny if they occur during….

A. DNA replication

B. Transcription

C. Translation

A. DNA replication

Redundancy, wobble allows for silent mutations (encode the same amino acid)

E.g., 6 different codons for Leucine, a highly abundant amino acid:

• CUU

• CUC

• CUA

• CUG

• UUA

• UUG

Missense mutations can have a range of impacts on protein structure and activity

Impact of amino acid substitution depends on:

• Similarity of WT and mutant amino acids

• Role of WT amino acid in catalytic activity, structure, interactions with other proteins

The Frequency of Mutation

Spontaneous point mutation

• DNA polymerase (DNAP) incorporates the wrong nucleotide every ~1 in 10^6 bp

• Bases can spontaneously deaminate, causing them to be misread during DNA replication

Dinal mutation rate is ~1 in 10^10 bp (after proofreading, mismatch repair, etc)

• Mutagens increase the mutation rate 100-1000x

Types of mutagens

• Radiation: Ultraviolet (UV), ionizing (x-rays, gamma)

  • UV causes pyrimidine dimers; e.g., neighboring thymine or cytosine residues on the same strand will be covalently linked

• Chemicals: e.g., base analogs

  • 5-Bromouracil (5-BrU) is an analog of T that can pair with A or G

  • Result: T:A → C:G transition

  • Biological “mutator” strains that lack proofreading or repair

  • Recombination events

  • Insertion of transposons, viral genomes

Recombination

Mixing of genetic material

• Occurs between regions with identical sequence

• The scale is variable; entire genes or operons can be exchanged

• Can result in a new strain that is different from the donor and recipient

Transposons

Cut-and-paste transposition model

• Transposon elements:

  • Inverted repeats (IR)

  • Transposase

  • Other genes (e.g., AbxR)

How are genes passed between bacteria?

Conjugation: Single strand DNA gets passed from one living bacterium to another through a pore/channel

Transformation: “funeral pass”

• Bacterium takes up naked DNA (from lysed cell?)

• Recipient maintains circularized replicon or incorporates the DNA into its genome

Transduction: “viral pass”

• A bacteriophage packages bacterial DNA from infected (lysed) cell and transfers it into new host bacterium during infection

Conjugation

A donor cell passes DNA to a recipient

• F Factor: “fertility” plasmid with origin of transfer & pilus assembly genes

Transformation

“Naked” DNA is taken up, which “transforms” the phenotype

• Frederick Griffith (1879-1941): Bacteriologist studying pneumonia

• Streptococcus pneumoniae: Still responsible for ~22,000 deaths/year, particularly among the

• Relative virulence of S. pneumoniae strains:

  • “Rough” - less virulent

  • “Smooth” - more virulent

Barriers to transformation:

• DNA must cross permeability boundaries (CM, OM, PG)

• Negatively-charged DNA must get close to the negatively-charged bacterial cell membrane

Competence: the ability to actively take up free, foreign DNA; often transient and inducible

Natural competence:

• ~1% of bacteria readily import DNA from their environment (in vitro) under permissive conditions

• Inducible by high [cell], damage

Artificially-induced competence:

• Used in a laboratory to transform bacterial cells

• Makes transient pores in the cell membrane

• Can transform linear or circular DNA

• Methods to induce competence:

  • Electroporation

  • Chemical competence:

    • A high concentration of cations (usually Ca²+)

    • Shock the cells with heat

Conclusions from Griffith’s experiment

The “transforming principle” (DNA) released from dead cells was incorporated by living cells, resulting in their genetic transformation.

Therefore:

• Traits can be acquired

• DNA can be exchanged between organisms

• Bacteria are a powerful research model to study basic principles

What is genetic engineering?

• Refers to the direct manipulation of DNA to alter an organism’s characteristics in a particular way

• This can range from changing one single DNA base to deleting or inserting a whole region of DNA

• For example, this can be used to produce more efficient metabolite production or drug biosynthesis

Steps to genetically engineer a bacterium to synthesize a foreign gene product (e.g., human insulin)

1. Purify [plasmid]

2. [Make many copies] of target gene DNA (insert)

3. Use [restriction enzymes] to “cut” insert & plasmid

4. Ligate cut insert into cut plasmid with [DNA Ligase]

5. Transform cells with recombinant plasmid

   • Perform artificial transformation with recombinant plasmid. Then plate treated cells onto media with selection (e.g., antibiotic) to select for transformants carrying the selectable marker (e.g., abx^R)

6. Induce (activate) expression of gene

7. Purify product

8. Make $$$ and save lives

• [Re-purposed from nature]

Example of a cloning vector: A plasmid

Vectors:

• Small pieces of DNA that can be stably maintained by the recipient

• Foreign DNA fragments can be introduced that encode new properties to the recipient

• Called “vectors” because they have direction; transmit genes from one organism to another

Plasmid properties:

• Origin of replication

• Selectable marker (Abx^R)

• Restriction enzyme cut sites

• High copy number

• Small size

Setting up a PCR “reaction”:

Reagents:

• Water

• Buffer - to keep the pH near neutral, provide KCl (K+)

• dNTPs = deoxyribonucleotides (ATP, GTP, CTP, and TTP)

• Heat-stable DNA polymerase; e.g., Taq DNAP, isolated from the thermophilic bacterium Thermus aquaticus, which has an optimal growth temp ~70C and a maximal growth temp ~80C

• MgCl2 - Mg²+ is a required co-factor for DNAP

• Primer (chemically-synthesized deoxyribo-oligonucleotides/”oligos”)

• Template (DNA)

Materials:

• Microfuge (test) tubes

• Thermocycler machine

Polymerase Chain Reaction (“Amplification”)

~30 cycles

• Denaturation of dsDNA 95C

• Annealing of primers (~60C sequence specific)

• Extension of primers

Restriction enzyme (RE) “digest” of insert & vector

REs are endonucleases that “cut” (hydrolyze the phosphodiester bond of) the phosphate-sugar backbone of dsDNA at specific sequences

• Ex: EcoRI, recognition sequence, GAATTC; cut site: G|AATC

Restriction-Modification (R-M) system protect against viruses

• The activity of restriction enzymes (REs) in the bacterial cytoplasm “restricts” the replication of bacteriophage to certain hosts

• To protect against phage infection: a RE cuts double-strand DNA (e.g., viral genomes) that carry the RE’s recognition sequence, which prevents replication of the viral genome

• To protect host DNA with the recognition sequence: A methylase that recognizes the same DNA sequence as its RE partner modifies the DNA by adding a methyl group; methylation at its recognition site inhibits the partner RE from cutting the DNA.

• RE + methylase = restriction-modification (R-M) system

R-M system

Bacterial immune system

• REase = restriction endonuclease (enzyme)

• MTase = methyltransferase (methylase)

CRISPR-Cas: Immune system in bacteria

Clustered regularly interspaced short palindromic (a word, phrase, or sequence that reads the same backwards as forward, e.g., madam or nurses run.) repeats

1. CRISPR array is assembled on bacterial chromosome by the insertion of phage seqs (spacers) and a CRISPR-specific repeat seq

2. CRISPR array is transcribed to produce precursor RNA (pre-crRNA)

3. crRNA that match viral sequence recruits Cas9 (CRISPR-associated nuclease) which cuts the viral genome, preventing phage replication

   • Task: Remember this phage to fight off this phage

Regulation of enzyme amount

• Microbes are frequently substrate limited

• Transcription and translation are coupled

• Microbial mRNA are short lived

• Genes are organized in operons

Regulation of enzyme activity

Non-covalent modifications:

• Competitive inhibition

• Non-competitive = allosteric

Covalent modifications:

• Phosphorylation

• Methylation

• Acetylation

• Others

Inhibition of folate biosynthesis

Dihydropteroate diphosphate + para-aminobenzoate (PABA)

→ Dihydropteroate reductase (No sulfonamides)→ Dihydropteroate

→ Dihydrofolate → Dihydrofolate reductase → Tetrahydrofolate*

• Tetrahydrofolate is a co-enzyme required for nucleic acid, amino acid synthesis and other essential cell functions

Regulation of flux in central metabolism

Cells regulate flux (flow through the pathways) in response to:

• Concentration of reductant, precursors, & building blocks

• Energy status

Enzyme activity regulated by allosteric activation/inhibition

• High concentration of substrate?

  • More flux through pathway, more enzyme activity

• High concentration of product?

  • Less flux through pathway, less enzyme activity

The cell maintains a high [ATP] relative to [ADP] and [Pi]

• ATP ←→ ADP + Pi

• ATP ←→ AMP + PPi

  • If [ATP] is too low, more flux through fueling is needed

CRP-cAMP activator (Glucose -)

• If glucose is NOT being transported into the cell, cyclic AMP (cAMP) is produced

• cAMP binds to the cAMP Receptor Protein (CRP; aka Catabolite Activator Protein, CAP), which increases its affinity for its specific binding site

• cAMP-CRP binds and enhances RNAP binding to the promoter

CRP-cAMP activator (Glucose +)

• Glucose is transported into the cell → no cAMP produced → CRP does not bind promoter or activate transcription

• There is global regulation of many operons by CRP (~200 transcriptional units in E. coli) including lac and other catabolite-utilization operons (called “catabolite repression” or the “glucose effect”)

Expression dilemma

Consider this paradox:

• Lactose must be transported into the cell in order for its transporter to be synthesized

• Lactose must be converted into allolactose by beta-galactosidase in order for beta-galactosidase to be synthesized

“Two-component” regulatory systems transduce signals

CpxAR, the envelope stress regulon

• Upon detection of mis-folded proteins in the periplasm, the sensory kinase CpxA autophosphorylates & transfers its phosphoryl group to CpxR

• CpXr is the response regulator (a transcription factor) that binds the promoter region and activates expression of protein-folding and protein-degrading enzymes and decreases the synthesis of pili and secretion systems.

Movement in response to the environment

• Goal: To optimize conditions

• Taxis: Directional movement in response to stimuli

• Types of stimuli: Chemicals (chemo-), light (photo-), air (aero-), magnetic field (magneto-), water (hydro-), temperature (thermo-)

  • Positive towards attractant

  • Negative away from repellent

Types of motility

• Swarming (flagella or pili, slime)

• Swimming (flagella)

• Twitching (pili)

• Gliding (varied mechanisms; w/o appendages)

• Sliding (spreading by growth)

Some features of E. coli stationary-phase cells

• First increased, then decreased production of motility structures

• Cells are smaller and rounder

• Cell membrane and LPS are more tightly packed

• DNA is more condensed

• Protein synthesis is reduced, protease activity increases

• Production of secondary metabolites (e.g., antibiotics)

Signals from different stressors are integrated to express stationary-phase genes

• Environmental stress

• sRNA integrators

• Regulators (others involved)

• Genes preparing stationary phase

sRNA DsrA activates translation of rpoS (sigma-s)

• Transcription of DsrA small RNA (sRNA)

• Leads to processing of rpoS transcripts

• Allowing ribosome assembly & synthesis of RpoS (sigma-s)

• Which binds to and directs RNAP to stationary-phase genes

• Which leads to the synthesis of factors that improve survival under non-ideal conditions

sRNA DsrA activates translation of rpoS (sigma-s) mRNA

• Synthesis of DsrA sRNA

• Leads to processing of rpoS transcripts

• Which facilitates ribosome assembly

• Which leads to RpoS (sigma-s)

• Which binds and directs to stationary-phase promoters

• Which leads to the synthesis of factors that improve survival under non-ideal conditions

Biofilm stages of development

1. Monolayer

2. 12 um max. thickness

3. 30 um max. thickness

4. 80 um max. thickness

5. 80 um max. thickness

Some genes required for biofilm development

1. Flagella

2. …

3. Cell division

4. Quorum sensing, pili (twitching motility), exopolymeric substance made of polysaccharides, extracellular DNA. lipids & peptides

5. Detergents, DNases, Proteases

Quorum-sensing system

Express, sense, respond to autoinducer concentration

• Auto = self

• Induce = persuade

High cell density = high [autoinducer]

Quorum-dependent genes: Pilin synthesis, light production, virulence factors, secretion systems, aggregation factors

Bacillus subtilis endospore structure

• Core (compacted chromosomal DNA, proteins, ribosomes, dipicolinic acid)

• Inner forespore membrane

• Cell wall and cortex (modified PG)

• Outer forespore membrane

• Spore Coat Layers (protein

• Exosporium (lipid)

Germination

1. Vegetative cell

2. Entry into sporulation

3. Polar septation

4. Engulfment

5. Cortex formation

6. Coat formation

7. Release

Changes in gene expression (mRNA transcript levels)

By switching to a different sigma factor, the expression of large sets of genes can be shut off/activated at once

Fungi

• Non-motile, cell size ~5 um

• Cell wall of polysaccharide (glucans, mannan, chitin) and glycoproteins

• Many contain ergosterol in their cell membranes

• Produce asexual and sexual spores for dispersal, diversity

• Lifestyles: Decomposer, mutualistic symbiont, commensal, parasite

• Heterotrophs (heteros = other, trophe = nourishment)

Molds: multicellular fungi

• Form multicellular hyphae (singular: hypha)

  • A mass of hyphae = mycelium (plural: mycelia)

• Vegetative hyphae acquire nutrients

• Aerial hyphae are reproductive structures that aid dispersal

• Primary aerobic metabolism (respiration)

Yeasts: Single-celled fungi

• Unicellular fungi

• Budding yeasts divide asymmetrically; fission yeast divide by binary fission; may stay connected in chains = pseudohyphae

• Facultative anaerobes (mostly fermentative, can respire aerobically when carbon sources are limiting)

• Part of the human microbiome and can be found in the gut, mouth, and skin

Mycorrhizae: A mutualistic relationship between fungi and plants

• Fungi colonize & extend roots of >90% vascular land plants

• Fungal partner:

  • Hyphae increase surface area 100-1000X for nutrient & water absorption

  • Secrete enzymes that help capture organic N, P, Fe

  • Protect against pathogens

• Plant partner supplies carbohydrates for energy, carbon

Fungi as decomposers, pathogens

• Extracellular digestion

  • Secrete enzymes to degrade keratin, chitin, cellulose, lignin

  • Absorb release nutrients

• Can grow well in slightly acidic, low moisture, hypertonic conditions

• Can have capsules

• Fungi infections are pro-inflammatory, they trigger the host’s immune system, which releases cytokines to fight the infection, leading to inflammation

Valley fever: coccidioidomycosis

• Caused by Coccidioides spp.

• > 15,000 new cases/year, ~200 deaths/year

• 60% asymptomatic

• Symptoms:

  • Most with mild flu-like symptoms or uncomplicated pneumonia

  • 5-10% with severe form that can cause chronic pneumonia and lung damage

  • In ~1% of individuals, disseminates to other organs including the brain and spinal cord, leading to skin abscesses, blindness, and death

• Treatment: rest (mild case); antifungals that target ergosterol in membranes (e.g., fluconazole, amphotericin B)

Dimorphic fungus

• Coccidiodes grows in moist soil as a mold (hyphae, nonpathogenic)

• In dry soil, hyphae fragment and form arthroconidia (pathogenic)

• Not just a pathogen, it also breaks down organic matter in the soil and recycle nutrients (decomposer, during hyphae stage)

Acquisition of airborne Coccidioides

Differentiates into yeast-like form at human body temperature

Groups at risk for cocoidiodomycosis

• Weakened immune system (uncontrolled HIV, some immunosuppressive drugs, genetic deficiency in certain immune factors, late stages of pregnancy, diabetes)**

• Male biological sex**

• African-American ancestry**

• Work in agriculture (root crops) & construction**

• People who live or travel in the southwest U.S. (including CA)*

Preventing Valley Fever in our prisons

• Relocate people with weakened immune systems to prisons where Valley Fever is not endemic

• People who are African-American or Filipino are sent to prisons other than Pleasant Valley (PVSP) or Avenal State Prison (ASP)

• Spherusol skin test that detects pre-existing immunity:

  • If test negative (native to fungus), they are at higher risk of infection and therefore should NOT be sent to PVSP or ASV

• Dust masks available for incarcerated persons to wear outside; can stay indoors if high speed wind

Myotoxins

Only some strains of fungi can produce this. Fungi used in the industry do not synthesize toxins. Even when the genome encodes them, they are only synthesized under some conditions (stress).

Protozoa

• Eukaryote, highly diverse group

• Unicellular, ~10 um in diameter (range of sizes with most being smaller than 50 um)

• Most lack a rigid cell wall

• Most with supportive pellicle, a layer of closely-packed protein vesicles associated with the membrane

• Asexual and sexual reproduction

• Vegetative form is a trophozoite “nourish animal”

• Some produce dormant, tough cysts

Classification of protozoans

• Amoeboids: Pseudopodia

• Ciliates: Cilia

• Flagellates: Flagella

• Apicomplexa: Most glide, one cell type flagellated

Protozoan diet

Most are chemoheterotophs, key predators of other microbes

Acquire nutrition through

• Ingestion

  • Phagocytosis or

  • Sweep food into mouth spores

  • Digest food inside vacuoles

  • Discharge waste from anal pore by exocytosis

• Absorption

Protozoa: Apicomplexa

• Intracellular obligate parasites

• Named for apical complex structure used to invade cells

• Causes diseases that affect 1/3 of the world population

• Case study: Plasmodium spp. that cause malaria “bad air”:

  • Transmitted by the Anopheles mosquito vector

  • ~Half the world population is at risk of transmission

  • 608,000 deaths in 2022, 94% in the World Health Organization (WHO) African Region; most are children and immunocompromised adults

Malaria

• Febrile disease caused by infection of red blood cells

  • Each 48 hours: result of RBC rupture

    • Short cold stage (chills, shaking)

    • Hot stage with fever (>40C kills mature forms)

• Severity of disease depends primarily on host immunity, host genetics, & Plasmodium species

• Spread of disease depends on density & feeding preferences of the mosquito vector

Plasmodium (parasite) - Anopheles (vector) - human (host)

1. Infected mosquito bites human; sporozoites migrate through bloodstream to liver of human

2. Sporozoites undergo schizogony in liver cell to form merozoites

3. Merozoites released into bloodstream from liver may infect red blood cells

4. Merozoite develops into ring stage in red blood cell

5. Ring stage grows and divides producing merozoites

6. Merozoites are released when red blood cells ruptures; some develop into male and female gametocytes

7. Another mosquito bites infected human and ingests gametocytes

8. In mosquito’s digestive tract, gametocytes unite to form zygote that invades gut & grows into sporozoites

9. Resulting sporozoites migrate to salivary glands of mosquito

Viruses

• Non-cellular infectious agents (acellular)

• Consists of genetic material (DNA or DNA) inside a protein coat (capsid); some have an envelope

• Cannot reproduce on their own → must hijack a host cell

• Extremely small

• Causes diseases in humans, animals, plants, and bacteria (bacteriophages)

Tobacco mosaic virus

• TMV was the first virus to be discovered

• The filterable “poison”

• Discovery of viruses: “contagium vivum fluidum” = soluble living germ → “virus”

Virus size advantages

Large (genome)

• Encode more efficient enzymes

• More genes to help suppress immunity or control gene expression

Small

• Fit genome into capsid

• Fewer targets for immune system

• Faster replication

Virus particle

• A single infectious viral particle is a “virion”

• Central core

  • Genome of single-stranded (ss) or double-stranded (ds) DNA or RNA molecule(s) or hybrid genomes (few)

  • Enzymes

• Covering

  • Capsid (all viruses)

  • Envelope (some viruses)

  • “Spike” of protein/carbohydrate (some viruses)

• Matrix (some viruses): Links the core/capsid with envelope; mediates assembly, exit, and entry

Viral capsid

• A protein shell that surrounds and protects a virus’s genetic material from nucleases.

• Made up of many copies of capsid proteins that are bonded together by electrostatic interactions

• Helps the virus attach to receptors on a host cell during infection.

• Transporting and releasing the genome: Encapsulates the genome in one host, transports it, tand then releases it in another host cell.

Viral structure and classification

1. Icosahedral nucleocapsid

2. Enveloped icosahedron

3. Helical, nonenveloped

4. Helical, enveloped

5. Complex

Stages of viral replication

Attachment:

• Viral surface ligand collides with and binds to host receptor

• Highly specific

• May be co-receptors

Penetration:

• Virion or viral genome passes through cell membrane

  • Membrane fusion (only for enveloped viruses)

  • Endocytosis (many enveloped viruses, all naked eukrayote viruses)

  • Uncoating: Removal of capsid; releases genome into appropriate compartment

• Injection of genome into cytoplasm (bacteriophage)

Bacteriophages “bacteria eater”

• Lytic cycle: Phage replicates, lysing host cell

• Lysogenic cycle: Prophage DNA of temperate virus integrates into host chromosome or is maintained as a plasmid

Stages of viral replication

Biosynthesis:

• Sequential viral gene expression to:

  • Disrupt/take over host functions

  • Enable viral genome replication

  • Make virion components

• Genome replication

Replication of DNA host genomes & expression

• (+) Sense DNA strand: specifies amino acids

• (-) Antisense DNA strand: serves as template for transcription & DNA rep.

• (+) Sense RNA strand is equivalent to mRNA

• DNA viruses need the same enzyme to replicate and express genes on their genome!

Replication of RNA virus genomes

• Replicates through a dsRNA stage

• RNA viruses encode RdRp on their genome

• Examples: Rotavirus, Ebola virus, Influenza virus, Measles, Rabies, SARS, Yellow fever, West Nile, Hepatitis A, Polio

Replication of retrovirus genomes

(+) RNA → dsDNA → integration → provirus

Stages of viral replication

Assembly:

• Components concentrated at a particular location in the host cell

• Nucleocapsid forms, virion assembled

Release:

• Lysis of cell (many non-enveloped or “naked” viruses)

• Exocytosis or “budding” (many enveloped viruses)

• Budding can take place from CM, nuclear M, Golgi M, or ER M

Budding takes advantage of natural cell processes to traffic proteins from membranous structures to the cell surface

Maturation: (for some reason, some proteins)

• Viral protein processing; e.g., polyprotein cleavage by host or viral proteases

• Required for immature virus to become infectious

Culturing & counting viruses

• Viruses are dependent upon host cells for replication

• Bacteriophage from plaques on a lawn of bacteria

  • # infectious units = plaque-forming units (PFU)

Human Immunodeficiency Virus (HIV): Enveloped, diploid, positive sense, single-stranded RNA retrovirus

Envelope:

• Two layers of lipids

• Envelope contains proteins from the host cell, and complex HIV protein known as Env (green)

• Env = gp120 + gp41

Matrix:

• Lies the core and the envelope (protein called p17)

Core:

• Capsid

• Two ssRNA strands

• Integrase

• Reverse transcriptase

HIV target

• Targets the immune system

• HIV destroys CD4 T lymphocytes (helper T cells)

HIV facts

• Retroviridae family, Lentivirus

• Causes Acquired Immunodeficiency Syndrome (AIDS)

• Pandemic; ~650,000 people die of HIV-related illness globally per year

• Currently leading cause of disease in Sub-Saharan Africa:

  • 67% of new infections globally

  • 1 in ~10 adults infected

  • ~14 million orphans due to AIDS

• Some good news!

  • 86% of people living with HIV knew their status

  • 75% of people with AIDS were accessing anti-retrovirus therapy (ART)

HIV Transmission

• Direct (injection, transfusion, placenta)

• Via bodily fluids (semen, vaginal secretions, blood, breast milk)

Host defenses

Innate (non-specific)

• Rapid

• Effective against many pathogens

• Includes:

  • Barriers (physical, chemicals, microbiological)

  • White blood cells

  • Inflammation

  • Fever Antimicrobial substances

Adaptive (specific)

• Takes time to develop

• Educated resistance to a specific pathogen

• Provides lasting protection (memory)

• Includes:

  • B cell antibody production

  • T cell activation of immune cells and clearance of infected host cells

Inflammation

1. Recruit and activate immune components at the site of injury

   • Release signaling molecules (cytokines, chemotaxins)

   • Dilate blood vessels

   • Movement of immune cells from blood vessels into infected tissues

2. Destroy, isolate pathogen

3. Initiate tissue repair, clear harmful substances

Diapedesis

WBCs leave circulation, enter tissue

Cellular response

• Patrol and migrate through tissues

• Recognize

  • Self v. non-self

  • Beneficial from harmful (location)

• Eliminate non-self

Cell markers:

• Host: Glycoproteins, others

• Microbes: Microbe-associated molecular patterns (MAMPs)

White blood cells: Neutrophil

• First responder

• Multi-lobed nucleus

• Phagocyte

• Anti-bacterial/fungal

• Short-lived (hours)

• We make ~100 billion/day

• The number of phagocytic events/cell is low (1-few)

• Major component of pus

• Granules carry antimicrobial lysozyme, enzymes, oxidizers, peptides, iron scavengers

White blood cells: Monocyte → Macrophage

Monocytes:

• Circulate in blood 1-3 days, non-phagocytic

• Migrate toward chemoattractants released by infectious agents and host cells at the infection site

• Crawl into tissues, develop into highly phagocytic macrophages

Macrophages (M0):

• Live months-years

• Engulf viruses, bacteria, fungi, debris, worn-out cells

• Release inflammatory signals

• Educate adaptive immune cells to target foreign antigens

Phagocytosis

• Ingestion of microbes or particles by a cell

• Performed by:

  • Neutrophils and macrophages to reduce infectious units

  • Antigen (a substance that can trigger an immune response) -presenting cells (APCs; e.g., macrophages, dendritic cells) to acquire antigen for presentation to B cells & T cells

• Detection of phagocytosed pathogen → alert immune system

  • MAMPs bind to PRRs at the plasma membrane, endosomal membrane, and in the cytosol → triggers expression of immunity-related genes

  • Secrete cytokines to signal to other cells

Steps in phagocytosis

1. Chemotaxis

2. Adherence

3. Engulfment

4. Formation of phagosome

5. Fusion with lysosome → phagolysosome

6. Digestion with enzymes, killing with antimicrobials

7. Release debris

Meanwhile…

• Recognition of non-self

• Release cytokines to recruit help

Mediators of inflammation: Cytokine

• Small proteins

• Secreted by a subset of cells

• Bind specific receptors found on subset of host cells

• May affect the cells tha tproduce them or other cells

  • Some have long-distance effects (via circulation)

• Can be pro-inflammatory or anti-inflammatory

Types:

• Interleukins (IL): leukocyte communication

• Chemokine: Chemoattractant to recruit WBCs

Macrophage (M0) detects intruder, signals for help

• Phagocytosis by M0

• LPS recognized by pattern recognition receptors

• Chemokines secreted by M0. Neutrophils respond

• M0 also produce cytokines that trigger their own differentiation into an activated form

Adaptive Immune Response

“Specific” or “acquired” immunity:

• Specific for a particular pathogen

• Developed over a lifetime

• Long lasting memory (~decades)

Humoral immunity:

• Antibody (Ab) secreted by B cells

• Good against extracellular pathogens

Cell-mediated immunity:

• T cells

• Good against intracellular pathogens

Some key players in adaptive immunity

• Antigen: Material that stimulates an adaptive immune response

  • Protein & carbohydrate groups recognized particularly well

• ANtigen-presenting cells (APCs)

• Lymphocytes: B cells - make antibody

• Lymphocytes: T cells

  • Activate B cells and boost inflammation through cytokine release

  • Kill infected host cells

  • Regulate/suppress immune response

Lymphocytes: B cell

• Form & mature in Bone marrow

• Assemble a B cell receptor by random assortment of DNA receptor elements via recombination

  • Many copies of a unique receptor for each cell

  • Initially embedded in the cell membrane

• Naive B cells migrate to lymph organs

• Receptor-antigen binding leads to differentiation with the help of Helpter T cells that secrete cytokines

• Naive B cells differentiate into:

  • Plasma cells: Secrete a soluble version of the receptor (antibody, immunoglobulin, Ig)

  • Memory B cells: Long lived, quickly differentiate into plasma cells upon re-exposure to antigen

Lymphocyte: T cell

• Form in bone marrow, mature in Thymus

• Assemble a T cell receptor by random assortment of DNA receptor elements via recombination

  • Many copies of a unique receptor on each cell

  • Embedded in the cell membrane (never secreted)

• Naive T cells circulate in blood, lymph organs

• Once bind antigen, native T cells differentiate into:

  • Effector T cells

    • Cytotoxic T cells: Kill infected host cells

    • Helper T cells: Activate B cells, T cells, macrophages

    • Regulatory T cells: Modulate the immune response

  • Memory T cells: Long-lived antigen-specific T cells that rapidly differeniate into T effector cells in response to antigen

Clonal deletion of lymphocytes that recognize self

• Body should not respond to/attack itself!

• B cells/T cells with receptors that recognize self-antigens are destroyed before leaving the bone marrow/thymus

Clonal selection of lymphocytes that recognize non-self antigens

• Lymphocyte binds an antigen through its membrane-bound receptor

• Receptor binding triggers cell division & differentiation of memory cells and either plasma cells (B cell) or effector cells (T cells)