MICROBIOLOGY UNIT EXAM 3 Pt.1 Flashcards
Microorganisms: Bacteria is part of what kingdom. This kingdom is broken up into two.
Protista (single celled, or multicellular with little tissue differentiation)
Prokaryotes (no nucleus) - bacteria
Eukaryotes (true nucleus) - fungi, protozoa
Genus and species is used when classifying a microorganism. What is helpful of the serotype and subspecies?
Helpful in diagnosis and treatment. Serotypes highlights the differences in antigens among the same species. Serotyping allows for the classification of subspecies.
Gross morphology of bacteria.
spherical forms Chains Clusters Pairs rod forms intermediate of spherical and rod forms spirals comma shaped, truncated helices
Cocci
- Strepto (cocci)
- Staphylo (cocci)
- Diplo (cocci)
Bacilli
Coccobacilli
Spirochetes
Vibrios
Gram negative stains _____ (color)
Gram positive stains _____ (color)
Acid-fast stains _____
red
dark purple
Certain bacteria (Mycobacterium tuberculosis, mycoplasma) are difficult to stain by the Gram procedure, but they can be stained by a procedure in which the stained bacteriaresist acid alcohol destaining (so-called “acid-fast”bacteria).
Determinant of Gram and acid fast reactions and the major difference between bacteria and human cells is the _____. How does this lead to treatment and interaction with an immune response?
Cell Envelope
Cell envelope can be the target for antibiotics and main interface between bacteria and immune response.
Note: Differences between the cell envelopes of gram negative and positive bacteria present different challenges.
What’s the location of cytoplasmic membrane (AKA ___) relative to the peptidoglycan in Gram +/- bacteria? Which Gram bacteria may have its cytoplasmic membrane be contiguous with outer membrane?
What is the membrane structure of the cytoplasmic membrane and its functions?
In general membrane integrity is maintained by _____ or _____. This marks a difference in mammalian cells/ specific genus of bacteria.
What cytoplasmic maneuver elicits septum and endospore formation?
“Plasma membrane” is interior to the peptidoglycan in both Gram (+) and Gram (-) bacteria.
In gram negative bacteria, may be contiguous with outer membrane
Typical trilaminar (protein layer, lipid bilayer, protein layer) unit membrane structure (lipid, protein and small amounts of bound carbohydrate). Functions in cell wall synthesis, protein secretion, active transport, selective permeability, energy generation.
(membrane integrity is maintained by sterols or cell wall)
Bacterial plasma membrane has No sterols!!
Mycoplasma, which, like mammalian cells, requires sterols to compensate for lack of cell wall; provides rigidity and osmotic protection.
Membrane invagination of cytoplasmic membrane during cell division.
Bacterial Cell Envelopes: Gram-negative bacteria cell envelopes
Cell envelope is a complex outer surface the most medically important are ____(2). What is the most general difference between the outer membrane of the cell envelope and cytoplasmic membrane?
Define cell envelope.
rods and diplococci.
Outer membrane is asymmetrical.
Cell envelope is the membrane(s) and other structures that surround and protect the cytoplasm (i.e. outer membrane (gram-), periplasma (gram-), cell wall, cytoplasmic membrane)
Bacterial Cell Envelopes: Gram-negative bacteria cell envelopes
The outer membrane of the cell envelope contains lipopolysaccharide (LPS) and porins. What makes up the LPS (what parts are antigenic or not- what is the endotoxin).
Define porins and what is the selectivity based on?
What’s the advantage of the outer membrane of the cell envelope to gram negative bacteria?
Lipopolysaccharide (LPS) consisting of [inside out] Lipid A (not antigenic), corepolysaccharide, and O antigens (antigenic; repeat 40 units). LPS is often referred to as Endotoxin, but Lipid A is the Endotoxin.
Porins –Diffusion channels that allow the membrane to act as a sieve. Selectivity is based on size. The outer membrane contributes to resistance of gram negative bacteria to peptidoglycan antibiotics.
Discuss how the peptidoglycan is arranged (thick/think, strongly/poorly linked) for the gram negative bacteria.
In what space is the peptidoglycan hosted in and give an example of an enzyme concentrated in this space for some bacteria.
Thin peptidoglycan sheath with poorly linked pieces forming a gel
The gel (thin peptidoglycan sheath) is found in the periplasmic space and various enzymes -e.g. β-lactamases are concentrated here in some bacteria.
Host response to LPS from gram negative bacteria (or _____/_____ from gram positive bacteria). Also a response to Fungi.
Discuss the Host response interaction of macrophage and endothelial cells with LPS. What molecules specifically are involved?
What’s the danger of the levels of molecules being produced during this type of host response?
- teichoic acid / peptidoglycan
- LPS binds to LPB (LPS Binding Protein), which interacts with macrophage membrane receptors (CD14/TLR4).
- The binding stimulates production of cytokines and inflammatory mediators TNF alpha, IL-1, IL-6, IL-8 and IFN gamma from macrophages and endothelial cells.
- Low level production of these proteins is protective, but overproduction results in development of life threatening bacterial sepsis; systemic inflammatory response that can lead to organ failure.
Note the molecules being produced can activate the coagulation cascade, complement cascade, leukotrienes and/or prostaglandins.
Clinical Definition of Sepsis (5+ categories)
- Suspected or documented bacteremia
- Fever or hypothermia;
- Tachycardia
- Tachypnea
- Hypotension or 2 of the following symptoms
- unexplained metabolic acidosis
- arterial hypoxia
- acute renal failure
- recent unexplained coagulation abnormalities
- sudden decrease in mental acuity
- elevated cardiac index with low systemic vascular resistance
Gram-positive bacterial cell envelopes:
Most gram-positive bacteria are morphologically ____.
What are the characteristics of the cell wall (Discuss how the wall is held together, how many layers, and what it contains)?
- most are cocci (most prevalent are Staphylococciand Streptococci)
- Thick, extensively cross-linked cell wall. Up to 40 layers of peptidoglycan. Cross linking by pentaglycine bridge builds layers of sugar tetrapeptide into a three dimensional structure
- Contain teichoic acids; wall teachoic and lipoteichoic (extends into cytoplasm) acid.
Gram-positive bacterial cell envelopes:
Define teichoic acids. What is the function of teichoic acids? What do teichoic acids contain (molecularly)? Describe two kinds of teichoic acids and how or what they attach to.
What percentage does teichoic acids contribute to the cell wall?
Give the etiology of teichoic acids (2).
Teichoic acids -polymers of ribitol or glycerol with phosphodiester links.
Provide further cross linking and integrity to the cell wall.
Teichoic acids contain amino acid residues or GlcNAc (N-acetylglucosamine) residues or both.
Ribitol teichoic acids usually attached to the MurNAc (N-acetylmuramic acid) residues of peptidoglycan.
Glycerol teichoic acids are anchored in the cytoplasmic membrane via linkage to a membrane glycolipid (extend from the membrane through the cell wall).
May contribute up to 50% of the cell wall
Function in disease:
- Serve as adhesins
- cause host cells to release inflammatory mediators (highly antigenic, although Lipoteichoic acid is 20X less potent than LPS in eliciting release of inflammatory mediators)
Note: GlcNAc is a glucosamine (glucose with an N-group) with with an acetyl attached to the N. MurNAc is GlcNAc with an ether of lactic acid.
Fine structure of Bacteria: Extracellular and surface components: Usually clearly defined chemically and frequently antigenic and/or otherwise biologically active.
Name 4 extracellular and surface components.
Capsules
Slime
Flagella
Pili
Fine structure of Bacteria: Extracellular and surface components: Usually clearly defined chemically andfrequently antigenic and/or otherwise biologically active.
Define slime.
Slime - Antigenic polysaccharide with no definite outer margin.
Fine structure of Bacteria: Extracellular and surface components: Usually clearly defined chemically andfrequently antigenic and/or otherwise biologically active.
Define flagella (6).
Flagella -
Long, helical filamentous appendages.
Responsible for motility.
Composed of flagellin.
Attached to basal body in cytoplasmic membrane.
Antigenic ("H" antigen).
Causes swarming of some bacteria.
Fine structure of Bacteria: Extracellular and surface components: Usually clearly defined chemically andfrequently antigenic and/or otherwise biologically active.
Define pili (3). Two or more functional types.
Pili (Fimbriae) -
Short filamentous appendages.
Composed of Pilin (Antigenic).
Originate in cell membrane.
Two or more functional types may appear independently or together on same cell:
a. Sex pili (bacteria-bacteria adhesion for genetic transfer via conjugation)
b. Common pili (bacteria-human cell adhesion or attachment = virulence)
Fine structure of Bacteria: Extracellular and surface components: Usually clearly defined chemically andfrequently antigenic and/or otherwise biologically active.
Define capsules (6).
Capsules -
Not part of the cell proper.
Mainly polysaccharide, although a few polypeptidecapsules.
Gel-like, well-defined border.
Virulence factor due to antiphagocytic properties. Antigenic; sometimes vaccine (e.g.Streptococcus pneumoniae -more than 80 distinct antigenic types).
Identify their Gram-stain and morphology identity:
Actinomyces, Klebsiella, Escherichia,Peptostreptococcus, Streptococcus, Bacillus, Bacteroides, Gemella , Enterococcus, Listeria, Streptomycin, Haemophilus, Moraxella, Corynebacterium, Clostridium, Staphylococcus, Bordetella, Enterobacter, Neisseria
Gram-positive cocci
Enterococcus, Gemella, Peptostreptococcus, Streptococcus, Staphylococcus
Gram-positive bacilli
Actinomyces, Bacillus, Clostridium, Corynebacterium,
Listeria, Streptomycin
Gram-negative cocci
Moraxella, Neisseria
Gram-negative bacilli
Bacteroides, Bordetella, Enterobacter, Escherichia, Haemophilus, Klebsiella
Membrane rigidity and osmotic pressure: Sterols of mammalian cells provide membrane rigidity, which prevents H2O uptake and membrane stretching in response to osmotic pressure. To compensate for the absence of the sterols, most bacteria are surrounded by a mesh-like cell wall.
Cell wall: Peptidoglycan layer (mucopeptide layer, murein layer). List the characteristics of cell wall including the function, polymer constituents, cross linking, difference between gram +/-, and what’s out to break it up!
Cell wall layer found in all bacteria that have rigid walls and shape.
Provides rigidity and osmotic protection.
β-1,4 linked disaccharide units of:
N-acetylglucosamine (GlcNAc)
N-acetylmuramic acid (MurNAc).
A tetrapeptide attached to each N-acetylmuramic acid.
Polymers encircle cells and are cross-linked through the tetra peptide.
- Gram negative cell wall: thin peptidoglycan sheath (usually one layer) with poorly linked pieces forming a gel.
- Gram-positive cell wall: (most prevalent are Staphlococci and Streptococci)
a. Thick, extensively cross-linked cell wall. Up to 40 layers of peptidoglycan. Cross linking by PENTAGLYCINE BRIDGE builds layers of sugar tetrapeptide into a three dimensional structure. - Peptidoglycan synthesis (antibiotic target)
Protoplasts, Spheroplasts, L-forms of bacteria.
Give two ways these morphological changes can occur to bacteria.
Define protoplast, spheroplast, and L-form. In disease state can you expect to see L-forms?
- Peptidoglycan synthesis blocked by antibiotics - result is lysis of cell if not in isoosmotic medium
- Under isosmotic pressure (10% sucrose, 3M NaCl, etc.) with blocked peptidoglycan sythesis –> protoplasts and spheroplasts can form
- Spheroplasts/protoplasts may replicate and form L forms (chronic infections)
Protoplasts: Have their cell wall entirely removed and are derived from gram + (gram-positive)
Spheroplasts: Have their cell wall only partially removed and are gram - (gram-negative)
Endospore of bacteria: what are they, what genus makes them (2). Discuss where endospore develop and what they are resistant to and when do they germinate.
Highlight the important of dipicolinic acid.
Endospore: Refractile body-thick walled
- Clostridium & Bacillus
- Develops intracellularly, highly resistant to heat, drying, chemicals, oxygen
a. Ca2+ chelated by dipicolinate to dehydrate the peptidoglycan
b. Drying of cytoplasm
c. Germination in presence of nutrients (under favorable conditions)
Note: Dipicolinic acid forms a complex with calcium ions within the endospore core. This complex binds free water molecules, causing dehydration of the spore. As a result, the heat resistance of macromolecules within the core increases. The calcium-dipicolinic acid complex also functions to protect DNA from heat denaturation by inserting itself between the nucleobases, thereby increasing the stability of DNA.
Intracytoplasmic components of bacteria
Define nucleoids. Discuss the state DNA is and the circumferential size.
Define plasmids. Function/involvement and what “harm” can they avoid?
Define composition of proteins (% breakdown), it’s function, shape, weight, svedberg unit, sensitive to ____, and can form poly_______.
Nucleoids: Regions of the cell containing the condensed bacterial chromosome.
a. Double-stranded circular DNA molecule - circumference of about 1 mm
b. Not bounded by membranes
- Plasmids: Plasmids are autonomously replicating, circular molecules of double-stranded DNA that can confer resistance to ANTIBIOTICS and are involved in the production of bactericidal proteins and toxins. Plasmids are also involved in BACTERIAL CONJUGATION and synthesize surface antigens and pili.
- Ribosomes: Large complex composed of protein and RNA. Protein synthesis.
a. Protein Composed of RNA (60%) and protein (40%).
b. Spherical, MW 2.8 x 106 daltons (70S divided into 50S & 30S particles).
c. Smaller than those of eukaryotes and sensitive to ANTIBIOTICS
d. form polyribosomes
BACTERIAL NUTRITION, METABOLISM AND GROWTH
A. Environmental factors, nutrition and metabolism
pH: discuss range of tolerance and an example for the two extremes.
atmosphere: Discuss anaerobes and aerobes. What are their respective requirements, where can they be found, and different categorical terms for their atmospheric requirements.
temperature: Define mesophile and can some bacteria require different range of temperatures?
osmolarity: state the two categorical terms.
- Factors influencing growth and reproduction:
a. pH - range of tolerance (Acidophiles – Lactobacillus, Neutralophiles, Alkaliphiles - Vibrio)
b. Atmosphere
- Anaerobic - anaerobes don’t use free 02; strict (obligate) anaerobes are killed by atmospheric 02 (Most in GI, although some exist in the environment as spores)
- Aerobic -only grow if free 02 is present (esp. respiratory or mucosal pathogens)
- Facultative - grow with, or without, 02
c. Temperature requirements
- Mesophilic - flora of the human body (35oC - 37oC is often optimal, but some bacteria have unusual requirements).
d. Osmolarity (halophilic & non halophilic)
- Osmophilic – prefer high osmotic pressure
- Halophilic – prefer high [salt]
BACTERIAL NUTRITION, METABOLISM AND GROWTH
Nutritional Requirements:
Autotrophic vs heterotrophic. What type energy, carbon, nitrogen is used.
What is the clinical use of media?
Autotrophic: No organic matter used
i. Energy - Light, Chemical, Heat
ii. Carbon - CO2
iii. Nitrogen - N2, N02, N03, NH3
b. Heterotrophic
i. Energy – Chemical
ii. Carbon - (CO2 and at least 1 organic compound)
iii. Nitrogen - N2, N02, N03, NH3, organic
c. Minimal, defined media - useful in clinic for diagnosis
- API strips
-BACTERIAL NUTRITION, METABOLISM AND GROWTH
Metabolism: What compounds are used for metabolism? Provide two examples of how a diagnosis can be made based on metabolism.
Metabolism:
a. Metabolite utilization
i. Simple compounds, amino acids, sugars
ii. Complex molecules, proteins, polysaccharides, lipids
iii. Diagnostic in the clinic:
(a) N. meningitidis ferments maltose, glucose
(b) N. gonorrhea ferments only glucose
BACTERIAL NUTRITION, METABOLISM AND GROWTH
Growth:
Define the goal/result of binary fission, generation times, and growth rate.
Define growth curve. What are parameters, lag phase, log phase, stationary phase, death phase.
How is knowing the phase of growth important?
- Binary fission - simple means of reproduction - EXPONENTIAL increase in cell numbers.
Generation time = time it takes for the bacterial population to double
Growth rate = number of generations (doublings)/hour
- Growth curve - A growth curve is an empirical model of the evolution of a bacterial # over time.
a. Parameters - each bacterium has a characteristic generation time, although this varies considerably with the nutritional status, pH, etc.
b. Terminology
i. Lag phase - no increase in cell number; period of adaptation
ii. Log (EXPONENTIAL) phase - period of active and uniform growth
iii. Stationary phase - rate of multiplication and death balance one another; due to buildup of toxic compounds and nutrient depletion (RESISTANT TO CELL WALL ANTIBIOTICS)
iv. Death phase - cells die rapidly
c. Clinical significance - diagnosis and treatment may be affected by the phase of growth (gram stain, drug susceptibility, etc.).
Mechanism of action:
Bacitracin
Vancomycin
Penicillin
Bacitracin interferes with the dephosphorylation of C55-isoprenyl pyrophosphate, a molecule that carries the building-blocks of the peptidoglycan bacterial cell wall outside of the inner membrane. BACITRACIN - Inhibits regeneration of lipid carrier
The large hydrophilic molecule is able to form hydrogen bond interactions with the terminal D-alanyl-D-alanine moieties of the NAM/NAG-peptides. Under normal circumstances, this is a five-point interaction. This binding of vancomycin to the D-Ala-D-Ala prevents cell wall synthesis of the long polymers of N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) that form the backbone strands of the bacterial cell wall, and it prevents the backbone polymers that do manage to form from cross-linking with each other.
Penicillin:
β-Lactam antibiotics inhibit the formation of peptidoglycan cross-links in the bacterial cell wall; this is achieved through binding of the four-membered β-lactam ring of penicillin to the enzyme DD-transpeptidase. As a consequence, DD-transpeptidase cannot catalyze formation of these cross-links, and an imbalance between cell wall production and degradation develops, causing the cell to rapidly die.
INFECTION AND HOST RESPONSE
Interaction with an infectious agent can lead to a number of manifestations of disease in the infected host:
Name and discuss (5) illnesses.
- Acute illness - clinically apparent disease
- Latent illness - organism not readily detectable but may recur; e.g. HSV-2
- Recurrent illness - reinfection with same organism or recurrence of latent infection
- Subclinical - acute or chronic symptomless infection
- Chronic illness - persisting symptoms
INFECTION AND HOST RESPONSE
Host immune response
Define apparent absence, innate/adaptive immune response, and immunopathology.
- “Apparently” absent- evidence of infection is difficult to document (superficial fungi)
- Innate immunity - first line defense; may serve to limit extent of early infection prior to the development of specific immunity
- Adaptive immune response - lifelong immunity mediated by T cells and antibody
- Immunopathology - inappropriate (overly vigorous) immune response leading to tissue damage
- A combination of the 4.
INFECTION AND HOST RESPONSE
Host response depends on: (5) factors.
- Type or organism
- Dose of organism
- Site of infection
- Natural history of infection
- Host factors
- Age
– Immunocompetence
– Sex
– Genetics
– Nutrition
INFECTION AND HOST RESPONSE
Immunodeficiency syndromes- what this means in terms of opportunistic infections (give some examples).
Immunodeficiency syndromes - increased susceptibility to infections in immunocompromised hosts implicates the intact immune system in protection from these organisms
e.g: in AIDS - opportunistic infections such as pneumocystis carinii, CMV, Candida, some bacterial pathogens
INFECTION AND HOST RESPONSE
Course of immune response to infection: From entry to eradication of infectious agent.
- Entry of microorganism through epithelial surface (skin or mucosal surfaces).
- Innate immune response-helps contain infection and delivers antigen (e.g via DCs) to lymph nodes for elicitation of adaptive immunity.
- Distinct CD4 T cell subsets mediate clearance of diverse pathogens
- “Priming and Proliferation” Adaptive immunity-Activation of T cells and B cells in the local lymph node; production of antibody and effector T cells.
- Eradication of infectious agent.
INFECTION AND HOST RESPONSE
Mechanisms by which infectious agents evade immune response (4)
- Hiding within cells without antigenic expression- MANY organisms
- Lurk on periphery of body - e.g. superficial fungi
- Pool of non-immune individuals - measles, recent outbreaks of whooping cough
- Changing surface structure either at the population level (e.g. influenza virus) or during the normal life cycle - (African trypanosomes)
Balance between Th1 and Th2 cytokines is important in determining type of immune response to infectious organism. Discuss what cytokines Th1/2 release and what actions they tend to favor relative to an immune response.
Discuss the roles of Th17 cells, third signal, and Th cell in immune responses.
Th1 cytokines IFN gamma and IL-2 (esp. INF gamma) favor cell-mediated immunity and class switch to opsonizing antibody (macrophages, CTL, DTH, NK)
Th2 cytokines (IL-4, IL-5, IL-13) favor IgE responses and some Ig subclasses, parasitic infections, which are particularly important in immunity to worms.
Th17 cells, which produce IL17 and activate neutrophils for killing of extracellular bacteria and fungi. Produce IL-17, pro-inflammatory; important for resistance to extracellular G+ organisms, fungi at mucosal surfaces*; activate neutrophils. Are also involved in autoimmunity.
The third signal (cytokines) delivered by APC in part direct the type of T helper cell that is activated.
A fourth type of Th cell, the T regulatory cell, is involved in suppressing T cell responses. Produce regulatory cytokines to control magnitude and duration of immune response; tolerance
*– Trypanosomes – Leishmania – Tuberculosis – Extracellular G+ bacteria – Multiple sclerosis
Note: Dominance of one response may influence outcome
IL-12 and IFN (components of _____ immunity) tend to bias towards ____ and _____.
Which Th2 cytokines (2) are induced by helminthic infections, leading to production of IgE and recruitment of eosinophils.
What response is associated with Resistance/Exacerbation to murine infection with the protozoan parasite Leishmania major.
IL-12 and IFN- are two components of innate immunity that also tend to bias towards Th1 and CMI.
A specific example is the induction of Th2 cytokines IL-4, IL-5 by helminthic infections, leading to production of IgE and recruitment of eosinophils.
Another example is murine infection with the protozoan parasite Leishmania major. Resistance to infection is associated with a Th1 response (production of TNF- and IFN-) whereas exacerbation is associated with a dominant Th2 response (IL-4).
IMMUNITY TO BACTERIA
Effective immune response depends upon the mechanisms of pathogenicity. What two factors are we looking at?
Is damage caused by invasive or non-invasive (e.g. toxins) mechanisms?
Is the bacterium extracellular or intracellular?
Immunity to Bacteria depends on:
- Cell wall type (G+/G-)
- Invasiveness
- Toxin
- Intracellular
- Extracellular
Immunity to extracellular bacteria:
Components of both the innate and adaptive system are active. Discuss the actions taken on by both innate and adaptive system.
- Innate aspects have been discussed earlier in the course (complement activation, phagocytosis, and the inflammatory response), production of IL-12 (to activate
Th1 responses). Phagocytes (macrophages and
neutrophils) Complement, IFN-gamma production by NK cells, Acute phase proteins: fibrinogen, CRP - The adaptive immune response to extracellular bacteria includes :
a. Production of antibody that neutralizes the bacterial cells or the toxins they produce, opsonizes, activates complement for direct lysis or induction of inflammation. Opsonization facilitates uptake of the microbes.
b. CD4 cell activation: production of cytokines, IFN-gamma (to activate macrophage killing), TNF (contributes to inflammation). activate antibody response. Although macrophages can respond directly to microbes and kill, this is dramatically increased with Th1 activation.
Mechanisms of immune evasion by extracellular (4) bacteria.
Extracellular bacteria
• Antigenic variation
– Neisseria gonorrhoeae: variation in Pilin expression
– E. coli
– S. typhimurium
• Inhibition of complement activation
– Capsule sialic acid residues
– Pseudomonas - elastase inactivates C3a and C5a
• Resistance to phagocytosis
– Pneumococcus (Streptococcus pneumoniae) - remove capsule, resistance decreases
• Scavenging of reactive oxygen intermediates
– Catalase-positive staphylococci
• IgA-ase
– Secretory component helps protect, but some organisms secrete enzyme to break down IgA
Immunity to intracellular bacteria:
Discuss the innate responses that get tied into the stimulation of adaptive responses. Discuss how the major protective effect is carried out.
- Also associated with innate and adaptive responses
- Innate responses include both phagocytes and NK cells. Phagocytes ingest these bacteria, but many are resistant to the degradation. NK cells can kill either directly, or through the production of IFN-gamma, which activates macrophages for killing. IL-12 production by APC (macrophage) can stimulate the adaptive response.
- The major protective effect is through cell-mediated immunity – T cell activation of macrophages (again, through the production of cytokines like IFN-gamma or through CTL (CD8+ T cells).
Note: Macrophage is activated by IFN-gamma. An activated macrophage will secrete TNF, IL-1, IL-12, chemokine. Increase expression of MHC and costimulators (B7 molecules)
Bacterial cell walls - have a number of adjuvant effects.
How does the cell wall play a role in an effective/noneffective immune response?
List 5 effects of bacterial walls.
The effectiveness of the immune response to bacteria depends partly on the host’s ability to damage the bacterial cell wall.
Adjuvant effects of bacterial cell walls include:
- Trigger inflammatory mechanisms
- Activation of complement
- Activation of macrophages- TLR-4
- Upregulation of costimulatory molecules
- Polyclonal B cell activation
- IL-1 dependent polyclonal T cell activation
Bacterial/Virus “Superantigens”: What’s the mode of action and what’s the danger in them?
Bacterial “Superantigens” activate large numbers of T cells.
Staphlyococcal enterotoxins bind to V region of TCR, stimulating high frequency responses. In excess, this can cause toxic shock or septic shock syndrome. (Viruses
can also encode superantigens)
Other anti-bacterial defenses
Discuss ADCC (Antibody-dependent cell-mediated cytotoxicity; kills ____ bacteria), T cells and heat shock proteins (important in ______ infections), Balance of Th1 and Th2 (mycobacterium leprae infection - 2 outcomes)
Define ADCC mode of action.
- ADCC – various effectors can kill gram negative bacteria this way
- T cells and heat shock proteins – important in mycobacterial infections
- Balance of Th1 and Th2 can affect outcome of infection:
In mycobacterium leprae infection: Th1 dominance leads to Tuberculoid leprosy (more contained), whereas Th2 dominance (or defective Th1), leads to lepromatous leprosy, which has higher bacterial counts in lesions
ADCC- antibodies bind to pathogenic antigen. NK CD16 recognize and bind Fc region of antibody. The NK degranulates into the Ag/Ab/Fc lytic synapse and lysis the antigen.
Bacterial immunopathology - When too much of a good thing isn’t so good!
Discuss:
endotoxic shock,
release of mediators
cross reactivity (specific example associate with strep infections)
granulomatous infection (specific persistent microbes).
- Endotoxic shock (or septic shock) is caused by high systemic levels of TNF induced by bacterial lipopolysaccharide (LPS), characterized by vascular collapse, disseminated intravascular coagulation, organ failure.
- Release of mediators at site of infection
- Cross-reactivity of antigens: strep infections can lead to glomerular nephritis or rheumatic fever
- Granulomatous inflammation: clusters of activated macrophages, particulate antigen form nodules of inflammatory tissue – granulomas. Characteristic of response to some persistent microbes such as TB and
also some fungi. Respiratory difficulty in TB associated with replacement of normal lung tissue with fibrotic tissue.
Types of effector T cells, Main functioning adaptive immune response, pathogens targeted.
CD8 CTL, CD4 Th1, CD4 Th2, CD4 Th17, CD4 regulatory (various types)
CD8 CTL - kill virus infected cell. Target viruses e.g influenza, rabies, vaccinia, and some INTRACELLULAR bacteria
CD4 Th1 - Activated infected macrophages and provide help for B cell production of antibodies. Target microbes that are persistent in macrophage vesicles (e.g. mycobacteria, Listeria, Leishmania donovani, Pneumocystis carinii, and EXTRACELLULAR bacteria.
CD4 Th2 - provide help to B cell for antibody production especially IgE class switch. Target are helminth parasites.
CD4 Th17 - Activate fibroblasts, epithelial cells. Enhance neutrophil response.
CD4 regulatory - Suppress T cell response. Inhibit immature DCs thus surpassing CD4 T cell.
Signal 3 delivery cascade: Treg, Th17, Th1, Th2 (signal 3, TF, Effector cell production signals)
Signal 3 delivered by APCs:
Treg - TGF-beta
Th17 - TGF-beta, IL-6
Th1 - IL-12, IFN-gamma
Th2 - IL-4
From signal 3 to TF - the activation of effector cell:
Treg - FoxP3
Th17 - RORgammaT
Th1 - T-bet
Th2 - GATA-3
From Activation of effector cell we now express :
Treg - TGF-beta, IL-10
Th17 - IL-6, IL-17
Th1 - IL-2, IFN-gamma,
Th2 - IL-4, IL-5
Viruses and some bacteria can induce a cascade between DCs and NK cells and how are naive CD4 T cells influenced?
Other pathogens (e.g. worms) cause a cascade with NK cells and how are naive CD4 T cells influenced?
- Viruses and some bacteria induce IL-12 secretion by DCs that can activate NK cells to produce IFN-gamma. In the presence of IL-12 and IFN-gamma naive CD4 T cell become activated and commit to differentiating into Th1 cells.
- Other pathogens (e.g. worms) may cause NK 1.1+T cellist synthesize and secrete IL-4. Naive CD4 T cells activated in the presence of IL-4 commit to differentiation into Th2 cells.
Discuss the IgA transition from plasma cell in Basal lamina side to lumen side of gut.
IgA transitions across epithelial gut cell at the base of the crypts. Polymeric IgA binds in the mucus layer, and then the IgA neutralized the pathogen and their toxins.
Can the same responses be used for different types of bacteria which for example differ in their capsular polysaccharide?
No new response is warranted.
Mechanisms of immune evasion by intracellular (3) bacteria.
Intracellular bacteria
• Inhibition of phagolysosome formation
– Mycobacterium tuberculosis
– Legionella pneumophilia
• Scavenging of reactive oxygen intermediates
– Mycobacterium leprae
• Inhibition of phagolysosome formation,
escape into cytoplasm
– Listeria monocytogenes (hemolysin protein)
INFECTION WITH MYCOBACTERIUM leprae
Mark the differences between Tuberculoid Leprosy and Lepromatous leprosy
Tuberculoid leprosy: organisms present at a low to undetectable levels, low infectivity, granulomas and local inflammation, peripheral nerve damage, normal serum immunoglobulin levels, Normal T cell responsiveness, specific response to M. leprae antigens.
Lepromatous leprosy: organisms show florid growth in macrophages, high infectivity, disseminated infection, bone, cartilage, and diffuse nerve damage, hypergammaglobulinemia, low or absent T cell responsiveness, No response to M leprae antigens.
List of bacterial superantigens: Enterotoxins (4)
SEB, SEC 2, SEE, TSST-1
SE- Staphylococcus enterotoxin
TSST- Toxic shock syndrome toxin
Bacterial immunopathology - When too much of a good thing isn’t so good!
TNF : low, moderate, high [plasma]
Low: local inflammation
Moderate –> systemic effects; fever (brain), acute phase proteins (liver), leukocytes (bone marrow recruitment)
High: >or equal 10^-7 –> septic shock; low cardiac output, blood vessel is of low resistance/ thrombus formation, hypoglycemia (liver)
Bacteria and Autoimmunity:
List two ways bacteria can cause autoimmunity.
- Activation of APCs while expressing self antigen by microbe –> Induction of costimulators on APCs, presentation of self antigen APCs –> self reactive T cells –> autoimmunity
- Molecular mimicry –> Activation of T cells –> Self-reactive T cells and/or antibody. (e.g. Rheumatic fever) –> autoimmunity.
What is the goal of vaccination?
The goal of vaccination is to mimic this effect so that the individual doesn’t have to have the initial disease yet has the protective effects of the secondary immune response.
Most vaccines induce antibody responses or CTL activity? How does vaccine design change accordingly to targeted pathogen?
The majority of vaccines currently in use induce predominantly antibody responses, with
some of the live vaccines inducing CTL activity.
For example, a vaccine that induces primarily antibody
responses may not be effective for a virus that never goes through a cell-free stage. There, a
development of CTL is likely to be more beneficial. Likewise, many of our current vaccines
favor development of Th2 responses, where as Th1 (cell-mediated) responses might be more
protective.
Passive immunity: Passive immunity provides a rapid, temporary protection.
Explain in what form passive immunity is provided and provide four examples.
What are the pros and cons to passive immunity?
What is a condition that would require passive immunity?
Typically, passive immunity is given in the form of immune globulin or monoclonal antibodies. Passive immunity is used when the exposure is acute and the risk is immediate – i.e. there is no time to actively immunize and wait for the
response.
Examples include snake bite, tetanus exposure in an un-immunized individual, some virus exposures, anti-respiratory syncytial virus antibody in premature babies.
It gives immediate effects but is short-lived (dependent upon the half-life of the antibody) and does not give memory.
Passive immunity can also be given – e.g. pooled human immunoglobulin – to individuals who cannot
mount their own antibody response (e.g. Bruton’s agammaglobulinemia)
Define Active immunity:
Activates the host immune response to produce an immune response that mimics the response to natural infection.
Discuss attenuated vaccines. Give examples of them. Give the pro/con to its use.
Attenuated (live) e.g. live, attenuated viruses (measles, mumps, rubella, polio, flumist) and bacteria (BCG, used in Europe, Asia).
Live vaccines often give the best protection and best induction of CTL activity because they mimic natural
infection, but may cause disease in immunocompromised hosts (e.g. Sabin polio vaccine)
Discuss inactivated and subunit vaccines. Give examples.
Inactivated viruses and bacteria: e.g. Salk polio vaccine, most flu vaccines, old pertussis vaccine.
Subunit vaccines – e.g. tetanus toxoid, diptheria toxoid. Usually administered with an adjuvant to enhance the immune response (e.g. alum)
Discuss conjugate and recombinant vaccines. Give examples.
- Conjugate vaccines – conjugates a relatively weak immunogen for young children (polysaccharide) to an effective immunogen (tetanus toxoid). The Haemophilus influenzae vaccine (note that H. flu is a bacterium, not a
virus!) is the prototype for this. - Recombinant vaccines – hepatitis B
List 3 vaccines in clinical trials or development.
- Viral vectors
- ISCOM – liposome-like structures to deliver antigen to the cytoplasm and initiate MHC Class I presentation and CMI. (in development)
- DNA vaccines (in clinical trials)
WHAT’S NEEDED AND WHAT’S IN DEVELOPMENT for vaccines.
List the 4 types of vaccine modalities for improvement. Provide examples.
What’s Needed? • Effective vaccines for HIV, HSV, EBV, malaria etc. • Better compliance - e.g. measles - Can it be eradicated? • Better adjuvants for human use – – • Asthma connection – , costimulation •
Better adjuvants
Parasite vaccines
Cancer vaccines
Dendritic cell vaccines
- Better adjuvants for human use (Adjuvants increase magnitude and duration of response - stimulate expression of co-stimulatory molecules. Current adjuvant: alum activates inflammasomes) are being developed.
In addition, immunomodulators are being tested as vaccine
augmentors. For example, synthetic bacterial-like DNA sequences (Inclusion of cytokine, plasmid DNA (CpG)) that activate through TLR9 are being tested as adjuvants to induce Th1 responses (Need adjuvants that will favor Th1).
Other strategies being tested include cytokine incorporation such as IL-12. Currently, the only adjuvant in use in humans in the US is alum, which enhances immunity through the inflammasome. (There are some other adjuvants approved in Europe and other parts of the world).
*** Most current vaccines induce Th2 responses; Asthma connection.**
- Parasite vaccines (for example, against malaria) are much-needed.
- Cancer vaccines are under development, and a papilloma virus vaccine has been recently approved – prevents cervical cancer. Recent estimates say that with the
current vaccine penetration in girls of about 45%, 50,000 cases of cervical cancer have been prevented! - Dendritic cell vaccines are in development: let the professional APC choose the epitopes to present. Applications to tumor, infectious disease (HIV). First approved DC vaccine: Dendreon prostate cancer vaccine that uses the patient’s own DC to present the tumor antigens (unfortunately, the company has declared bankruptcy). Therapeutic melanoma vaccines are also in trials.
New challenges we face. List 4 examples.
NEW CHALLENGES – bioterrorism, bird flu and other emerging pathogens – rapid response. Most recent challenge: H1N1 “swine flu”.
A brief history of vaccination
Define the root of the word “immunity”
Concept dates to 430 B.C. when Thucydides, the
historian of the Peloponnesian War wrote _______.
Define variolation. Discuss the history.
1796: Jenner used cow pox to protect from
smallpox. What term come out from this?
• Immunity: comes from the Latin “immunis” meaning
“exempt”
• Concept dates to 430 B.C. when Thucydides, the
historian of the Peloponnesian War, wrote that those
who had recovered from Plague could care for those
with disease
• Variolation - used in ancient Asia; brought to Europe
in 1721 by Lady Mary Wortley and subsequently
used in the Revolutionary War
- Variation- Variolation or inoculation was the method first used to immunize an individual against smallpox (Variola) with material taken from a patient or a recently variolated individual in the hope that a mild, but protective infection would result.
• 1796: Jenner used cow pox to protect from
smallpox. The term “vaccination” (“vacca” is Latin
for “cow”) derives from this.
What did Koch prove in the 1870’s?
1860’s-1880’s what did Louis Pasteur develop?
- 1870’s: Koch proved that infectious diseases are
caused by microorganisms- anthrax - 1860’s-1880’s: Louis Pasteur developed vaccines
against cholera quite by accident - attenuation.
Coined “vaccine” in honor of Jenner. Also made first
anthrax and rabies vaccines.
Vaccines do not prevent infection
But…
They can ______.
prevent disease
Comparison of unimmunized donor vs immunized donor of B cells.
Compare frequency of specific B cells, isotope of antibody produced, affinity of antibody, and somatic hypermutation.
frequency of specific B cells: 1:10^4 - 1:10^5 vs 1:10^3
isotope of antibody produced: IgM > IgG vs IgG, IgA
affinity of antibody: low vs high
somatic hypermutation: low vs high
Polio: another success story. Discuss what occurred for this success story.
- 1952: 58,000 Americans contract polio
- Salk: inactivated polio vaccine - 1952
- Sabin: live vaccine
- 1994: Western world “free” of polio
• Success leads to modification of recommendation: now
use inactivated polio vaccine
List some infections for which vaccines are not yet available (8) and estimated annual mortality.
Malaria 889K Schistosomiasis 41K Intestinal worm infection 6K Tuberculosis 1.5 million Diarrheal disease 2.2 million Respiratory infections 4 million HIV/AIDS 2 million Measles 400K
Give examples of passive immunity (3)
Natural maternal antibody, immune globulin, humanized monoclonal antibody, antitoxin.
Immune globulin - an antibody-containing solution derived from human blood, obtained by cold ethanol fractionation of large pools of plasma; available in IM and IV preparations.
Antitoxin - An antibody derived from the serum of animals that have been stimulated with specific antigens.
Give examples of active immunity
Natural infection
Vaccines
- attenuated organisms
- inactivated organisms
- purified microbial macromolecules
- Cloned microbial antigens : expressed as recombinant protein, as cloned DNA alone or in virus vectors
- Multivalent complexes
- Toxoid
Vaccines - A suspension of attenuated live or killed microorganisms, or antigenic portions of them, presented to a potential host to induce immunity and prevent disease.
Toxoid- A bacterial toxin that has been modified to be nontoxic but retains the capacity to simulate the formation of antitoxin
Percentage of adult level of serum immunoglobulins
Passively transferred maternal IgG peaks just before birth and approaches zero by month 9.
Transient low IgG levels month 3 - year 1.
IgM increases continuously from less than -6 months to 5 years (stabilizes).
IgA increases continuously from -1.5 months to more than 5 years (stabilizes)
Passive Cellular Immunity
Descrive LAK therapy and transfer of immune T cells; CAR-T
• Transfer of NK cells or activated NK
cells - LAK therapy
• Transfer of immune T cells - Tumor
infiltrating lymphcytes
– Newest therapies use modified chimeric T
cell receptors to more specifically target
tumors – “chimeric antigen receptor T
cells” – CAR-T
Discuss the features of effective vaccines (6).
Safe - vaccine must not itself cause illness of death
Protective - vaccine must protect against illness resulting from exposure to live pathogen
Gives sustained protection - protection against illness must last for several years
Induces neutralizing antibody - Some pathogens (such as polio virus) infect cells that cannot be replaced (e.g. neurons). Neutralizing antibody is essential to prevent infection of such cells
Induces protective T cells - Some pathogens, particularly intracellular, are more effectively health with by cell-mediated responses
Practical considerations - low cost per dose, biological stability, ease of administration, few side-effects.
Live, Attenuated vaccine
Why does it work? Discuss.
• Mimic natural infection without disease
• Can be delivered at appropriate site
• Classically done by passaging virus in foreign
host cells or by temperature
• Often work with one administration - develop
good immunological memory and long-term
protection. Major advantage in developing
world
• Now can be done by deletion of virulence
factors from the organism
How to produce an attenuated vaccine.
- Isolate pathogenic virus from patient and grow in human cultured cells.
- Infect monkey cells with cultured virus
- While infecting the monkey cells the virus acquires many mutations allowing in to grow well in monkey cells.
- As a result the virus no longer grows well in human cells (it is attenuated) and can be used as a vaccine.
Inactivated Vaccines: Advantages vs Disadvantages
- Typically chemicaly inactivated - formaldehyde treated
- Advantages: Stable; safer than live vaccines; refrigeration not required.
- Disadvantages: Weaker immune response; boosters required
• Salk vs. Sabin polio vaccines
– Why the switch?
• Reversion 1:2.4 million; may spread through water
system
Toxoid Vaccines : define toxoid and what’s the theory behind its application?
- Disease is caused by a toxin released by the organism
- Give chemically modified toxin - “toxoid”
- E.g. tetanus, diptheria
Conjugate Vaccines: Define and discuss why this is done and the special population it may be considered for H. Flu.
- H. Flu (also pneumococcal conjugate)
• Haemophilus influenzae
– Infection problematic in young children
– Antibody to capsular polysaccharides is protective
– Young children respond poorly to polysaccharide
vaccines (T independent response weak; poor memory)
– Creation of polysaccharide-toxin conjugate
enables child to respond
• H flu polysaccharides conjugated with tetanus toxoid,
known to induce strong immune responses in children
Note: E.g we can take the polysaccharide from a bacterium and link it with a toxoid to make a conjugate vaccine.
Map out the cascade conjugate vaccine; polysaccharide-toxoid. From initiation to end result.
The polysaccharide component of the conjugate binds to BCR. The BCR-conjugate complex is internalized and degraded (peptide is processed).
B cell then presents processed toxoid peptides via MHC II to Th2 cell. The MHC-antigen binding activated B cell which will differentiate into a plasma cell and produce antibodies against the polysaccharide of the bacteria.
Viral vectors: describe how this is done in vitro.
recombinant DNA containing vaccinia promoter and pathogen gene transfects tissue culture cells along with the infection of the vaccinia virus.
The vaccinia virus links with the vaccinia promoter and pathogen gene portion of the recombinant DNA- homologous recombination.
BUdr selection follows. BrdU is commonly used in the detection of proliferating cells in living tissues.
After the selection we are left with recombinant vaccinia vector vaccine.
Dangers of vaccination: attenuated vs killed vaccines and provide examples.
Attenuate vaccines: (Current recommendation Salk)
- reversion to wild type; polio types 2 & 3
- severe disease in immunodeficient patients; vaccinia, BCG, measles
- persistent infection; varicella-zoster
- hypersensitivity to viral antigens; measles
- hypersensitivity to egg antigens; measles, mumps, flu
Killed vaccines:
- vaccine not killed; polio accidents in the past
- yeast contaminant; hepatitis B
- contamination with animal viruses; polio
- contamination with endotoxin; pertusis (acellular)
How do we know if someone is
immune? (4) ways and (1) aid.
• Check for antibody - just knowing the
individual is immunized isn’t enough
– IgG vs. IgM - titer
- Measure T cell proliferation to antigen
- Measure CTL responses
- Skin test - e.g. PPD
• Boosters: stimulate memory cells, raise
affinity, raise Ab titer
Does Immune Response =
Immunity? Give an example.
• Not Necessarily!
– E.g. HIV-1 initially induces a good antibody
response, but it is not protective
– HIV-1 vaccines that induce antibody
haven’t been protective
What’s New….. For Vaccines (5)
• Flumist - live influenza vaccine, cold
attenuated - better protection and more CTL
- Papilloma virus vaccine - cervical cancer
- Viral vectors - e.g. canarypox
- DNA vaccines - stable at room temperature
• DC vaccines - let the DC choose the
epitopes
– tumors (melanoma, others), infectious disease.
Approved Spring 2010: Dendreon prostate cancer
vaccine. 2014: declared bankruptcy
What qualities of vaccines are needed? (4) and public trust.
• Safer vaccines • More effective vaccines • Cheaper vaccines - fewer whole organisms? • Stable vaccines - no refrigeration – DNA vaccines
• Public trust - risks (e.g. asthma, autism)
– “…That vaccines given too early are going to cause
asthma, or anything else is like another big DUH! I mean
is ANYONE out there still swallowing whole the story that
vaccines can’t be dangerous and cause harm? And of
course I am NOT against vaccines (another DUH), but like
anything, you do them in moderation and I don’t care
what the 26 year old smiley faced pediatrician who just
popped out of medical school hearing the “no science to
support vaccines cause harm” mantra says, its not his/her
kid, you have to be CAREFUL with what you do to a baby
under six months…”
Can we eradicate more diseases
by vaccination? Smallpox vs. measles
- Herd immunity: the number of people needed to give
immunity to population depends on two things (smallpox vs. measles). Requirements for lifelong immunity and what is the host for these pathogens?
– Smallpox eradicated; goal to eradicate measles by
2010, but it’s still a major killer in developing world
– Relatively low infectivity (small pox) vs. higher infectivity (measles)
• Herd immunity: the number of people needed to give
immunity to population depends on the infectivity of the
virus and the rate of vaccine “takes”; for measles, this
required rate is much higher than for smallpox
– Man is only host for both (no animal reservoir); no
latency
– Immunity lifelong to both but measles requires two
doses
– Compliance: Timing and dosing; Should
vaccination be a choice?
Herd Immunity:
No one, some, most of population immunized. Contrast the three situations with healthy/ not healthy non-immunized and healthy immunized
No one: contagious disease spreads through the population.
Some: contagious disease spreads through some of the population
Most: Spread of contagious disease contained.
Other Vaccine Strategies: discuss therapeutic vaccination; e.g with tumors vaccines.
• Therapeutic vaccination- enhance host immune response against existing cells
– Tumor vaccines: enhance immunity to existing
tumors. E.g. Dendreon prostate cancer vaccine
– Therapeutic vaccination in infectious disease -
turn on or switch to more protective immunity
• HIV?
What are the three biological kingdoms “Tree of Life”?
What makes up prokaryotes?
Discuss two presumed common progenitors
Bacteria, Archaea, Eukarya.
Prokaryotes = Eubacteria + Archea
Common progenitor for all extant organisms
Common progenitor of archaebacteria and eukaryotes
Describe the differences between prokaryotes and eukaryotes in terms of intracellular structures, transcription and translation.
Contrast morphological features (5).
“Primitive Nucleus”; No nuclear membrane, no histones, no nucleosomes; Transcription & translation “coupled”
“True Nucleus”; nucleus, mitochondria, histones, nucleosomes; Transcription & translation “uncoupled”
Prokaryotes vs. Eukaryotes (Metazoans- animal that undergoes three tissue type development from embryonic stage)
size: 0.2 -2.0 micrometers dia. vs 10-100 micrometers dia
“nucleoid”, no nucleus, no nucleoli vs true nucleus w/ membrane and nucleoli
no membrane-enclosed organelles vs many membrane-enclosed organelles (e.g. lysosomes and chloroplasts)
Cell wall usually present i.e chemically complex and includes peptidoglycan vs normally no cell wall and if present is chemically simple
Plasma membrane usually has no carbs and sterols vs sterols and carbs serving as receptors on plasma membrane
Informational knowledge of differences between prokaryotes and eukaryotes.
Prokaryotes: typically haploid, binary fission, no sexual reproduction (meiosis), horizontal gene transfer of DNA fragments, typically single circular dsDNA molecule, very little “non-essential” DNA, carry extrachromosomal DNA (plasmids and episomes), chromosomal condensation by supercoiling and “architectural proteins”, transcription and translation are coupled, all “information transactions” are in cytoplasm, genes organized into operons (transcription unit with multiple genes), genes typically do not have introns (“intervening”), little to no post transcriptional modification to proceed to translation, ribosomes are smaller (70S).
Eukaryotes: diploid, mitosis, meiosis, multiple linear dsDNA molecules, genomes have large proportion of “non essential”/ repetitive DNA, little extrachromosomal DNA, chromosomes condensed by histones and confined by nuclear membrane, translation in cytoplasm, transcription and RNA processing in nucleus, genes typically singletons (one gene per transcription unit), genes have introns which need to be spliced and transcript is processed and exported into cytoplasm for translation, 80S ribosomes in cytoplasm, 70S in mitochondria.
Discuss the typical set up of bacterial DNA, variations to DNA organization, and extrachromosomal genetic elements; provide examples.
Typical: a single circular double-stranded DNA molecule [E. coli].
Variations: linear dsDNA chromosome [Borrelia burgdorferi (Lyme disease)]; two circular dsDNA chromosomes [Vibrio cholerae; (cholera)]
Extrachromosomal genetic elements: Plasmids, prophages, and transposons–important in pathogenesis
There exists Diversity in Bacterial Genomes in terms of bacterial strain, disease/niche, chromosome #, and size.
Just informational
Discuss the normal bp/turn of DNA in relaxed state, types of supercoiling, and why supercoiling occurs.
B-DNA (“normal DNA”) conformation
10.6 (~10) bp/turn in relaxed (minimal energy) state
Linear ds DNA (Relaxed) 1000 bp / 100 turns
Relaxed circular ds DNA 1000 bp / 100 turns
- negatively supercoiled 1000 bp / 95 turns; “underwound” (required state)
- positively supercoiled; 1000 bp / 105 turns; “overwound”
- The stress of under-winding or over-winding forces supercoiling
note: severity of wound –> +/- # turns with same # bp.
What is the action of quinolones and provide two examples. Expression of what type of topoisomerase increase / decrease when?
Quinolones inhibits topoisomerase II (GYRASE). Expression of topoisomerase II increases with underwound DNA.
- Nalidixic Acid
- Ciprofloxacin
Expression of topoisomerase I increases with overwound DNA.
Overview of DNA Replication in Bacteria
Discuss replicon and difference between bacteria and eukaryote, direction of replication, and separation of two daughter cells.
- The typical bacterial replicon: single origin, single termination sequence for entire chromosome
- Typical eukaryotic replicon: multiple origins (~1 per 100,000 bp of DNA) dispersed throughout each chromosome
Replicon is the unit defined by an origin for replication and a termination site
E. coli chromosome has one* origin (oriC) and a termination site (terC); (eukaryotic chromosome has multiple origins (1 per 100-250 KB)
E. coli DNA replication is bidirectional, i.e., two replication forks start at origin, and move in opposite directions towards terC.
The last step, separation of two daughter chromosomes, requires topoisomerase function (Topo-IV)
Bacterial Replication is Carried Out by a Giant Enzymatic Machine
Discuss them (4) categories. DNA Pols and other protein factors.
Replication is Carried Out by a Giant Enzymatic Machine
DNA polymerases
DNA polymerase III holoenzyme (pol III) –a multiprotein complex–the major replication enzyme
DNA pol I: single polypeptide; fills-in gaps in “lagging strand“, and in DNA damage repair
DNA pol II, IV and V: specialized functions under stress conditions
Other protein factors:
Initiation factors, primase, helicases, topoisomerases, ssDNA-binding proteins, ligase…
Key Players and Drug Targets at Replication Fork
Currently exploited targets for antibiotics/antivirals: (2)
Potential targets for Antibiotics : (3)
Provide drugs.
Currently exploited targets for antibiotics/antivirals:
- Gyrase (Ciprofloxacin and quinolones)
- dNTP synthesis (trimethoprim, sulfonamide and other folate inhibitors)
Potential targets for Antibiotics :
- DNA polymerase, helicase, primase
Variability is required for survival of all organisms, but also leads to disease
Discuss the two major mechanisms generating variability in all organisms.
Evolution absolutely depends on variants
Cancer and inherited diseases
Bacterial pathogenesis, infection control
Two major mechanisms generate variability in all organisms:
- Mutagenesis: “heritable change in nucleic acid sequence or content of an organism as compared to that of a reference organism called wild type”; basis for variability
- Recombination: “physical exchange of DNA between two DNA molecules; mechanism for rapid reassortment of variability”
Recombination (overview)
Define.
Discus two types.
Definition: Physical exchange of DNA between two DNA molecules. Always involves cutting and joining of DNA.
Two Types of recombination:
Homologous: Requires sequence homology; homology means substantial sequence similarity, not necessarily identity. Also called “classical” or “legitimate” recombination
Non-homologous: Requires no homology. Mediated by transposable genetic elements, or related sequence elements. Also called “illegitimate”, or “site-specific” recombination.
Homologous Recombination
Discuss the 2 routes the can be taken.
5’ —> 3’ resection, strand invasion by homologous parental DNA & DNA synthesis –> formation of D loop:
- Second end capture D-loop extension (Double Holiday Junction) —-> resolution —> Double Stranded Break Repair (crossover)
Homologous recombination: requirement for sequence homology.
i. Cutting and covalent linking of two DNA molecules to create “Holliday Junction”
ii. Branch Migration to expand amount of DNA exchanged
iii. Breaking apart (resolution) of the two recombinant molecules
- Dissolution –> annealing –> Synthesis-dependent strand annealing (non-crossover)
This mechanism absolutely requires homology. Minimum length of homology for recombination in E. coli is >300 bp
Transposable Genetic Elements
Discuss the anatomy of TGE, where TGE can move to, what organisms have TGEs, discuss their mediation of “illegitimate recombination”, TGE being replicative vs non-replicative.
Anatomy: one or a few genes flanked by inverted repeat (IR) sequences. A gene specifying a “transposase”; Inverted repeat sequences at ends that define boundaries of the TGE from host DNA
Whole unit can transpose (move) to a different location in the same or different host DNA molecules
Found in all examined organisms, from bacteria to the human
Mediate “illegitimate recombination”: promote cutting and joining of DNA without need for homology
TGEs can be replicative (the original copy stays in place, and a new copy is exported to a new location) or non-replicative (the original copy is cut out from one site and inserted into a second site)
Non-replicative vs. Replicative transposition
Cut and paste (non-replicative)
Replicative
With an RNA intermediate
Map out the process
Cut and paste (non-replicative): excision, insertion, repair
Replicative: cleavage, insertion, replication & repair
Sometimes for replicative an RNA intermediate is used: transcription, reverse transcription, cleavage, insertion, repair
Genetic Islands (GEI) / Pathogenicity Islands (PAI) - Discuss. What advantage may this offer and comment on the specificity of site integration.
GEI are large segments of DNA acquired from plasmids, phages or other unrelated bacterial genomes. When GEI confer pathogenicity to previously non-pathogenic bacteria, they are also called PAI.
Acquisition by site-specific recombination (integrase action), followed by mutations leading to loss of mobility
Integration sites are tRNA genes because sequences are homologous in all bacteria
Experimentally recognizable as “foreign” by differences in GC content and codon usage compared to adjacent chromosome segments
Acquisition of GEI often confers selective advantage to a new environment
Horizontal Gene Transfer:
Discuss the conclusion derived from the Griffith’s Classic Experiment (1928): Transfer of “Pneumococcal” Smooth Phenotype to Rough cells. (4) Treatments
R strain : - protective capsule ; S strain : + protective capsule
Treatment 1: R strain - live
Treatment 2: S strain - die
Treatment 3: heat killed S strain- die
Treatment 4: heat killed S strain + R strain - die (transformation)
Capsule allowed for the evasion from immune response.
Horizontal Gene Transfer Continuously Re-shapes Bacterial Genomes Example: Creation of Pathogenic Bacterial Lineages From Commensals
just informational
Horizontal Gene Transfer in Bacteria
Compare the results of intra-species transfer vs inter-species (and inter-genus) transfer.
Discuss the 3 major mechanisms of gene transfer.
Major Evolutionary Force in Bacteria:
- intra-species transfer allows for reassortment of traits
- inter-species (and inter-genus) transfer plays an extremely important role in bacterial resistance and virulence
Three major mechanisms
- Conjugation (“sexual reproduction”); mediated by a plasmid (prototype of a conjugative plasmid is the “fertility factor” F in E. coli); conjugation requires cell-cell contact; conjugation is the most efficient means for horizontal gene transfer
- Transduction: bacteriophage-mediated gene transfer
- Transformation: uptake of naked DNA from medium
Bacterial Plasmids
Describe the possible structures and three relevant criteria for classification
Structure: typically circular dsDNA; Variants: ssDNA circles; linear dsDNA
Classification: various criteria, but three relevant criteria are:
Mobility: conjugative Vs. non-conjugative plasmids
Pathogenic Significance: “virulence plasmids” encode toxins (e.g., tetanus, anthrax, enerotoxin), adhesins (pili) etc.
Antibiotic Resistance: R Factors (= R Plasmids): bear multiple antibiotic resistance genes contained within transposons embedded within plasmids
Note: virulence and R plasmids can be conjugative or non-conjugative
Significance of Conjugative Plasmids In Bacterial Pathogenesis
Discuss 3 categories: efficiency for transferring genes, carrying virulence determinants, and mobility vs mobilization.
MOST EFFICIENT route for transferring genes among bacteria (e.g., F+/F- mating).
CARRY VIRULENCE DETERMINANTS: encode toxins, adhesins and other virulence factors; genes specifying synthesis of antibiotics; genes specifying resistance to antibiotics. In fact, the most common mechanism for resistance is acquisition of “R” plasmids.
MOBILITY Vs. MOBILIZATION: can transfer themselves; can also “mobilize” other resident non-conjugative plasmids, or even the host chromosome (as in Hfr/F-mating).
“R” Factors (= “R” Plasmids)
History: Discuss
Features (3)
“R” Factors (= “R” Plasmids)
History: unusual antibiotic-resistant bacteria in hospitals in post-war Japan
Features: Traced to the acquisition of R plasmids (= R factors)
1. Simultaneous resistance to several antibiotics
2. High tolerance to antibiotics
3. Ability to spread across species and genera
