Microbio and Immuno Flashcards
(335 cards)
1
Q
Why transport molecules -> Cells
A
Residency: Most porteins made in ER but have functions elsewhere
Secretion: Molecules -> Extracellular environment(antibodies and cytokines)
2
Q
What is the structure of motif
A
12-16 hydrophobic AA sequence at N-terminus by basic charged residues
3
Q
What is the 12-16 hydrophobic AA at N terminus called
A
Signal peptide/Sequence peptide
4
Q
How is the Signal peptide/Sequence peptide removed?
A
Enzyme signal peptidase
5
Q
COPI(Coat protein 1) function
A
Retrograde(retrieval of protein from Golgi -> ER)
Brings stuff back
6
Q
COPII(Coat protein 2) function
A
Anterograde(Exporting protein from ER in vesicle -> Cis face golgi)
Exports stuff away
7
Q
COPI function
A
8
Q
N-terminal motif function
A
Used as entry ticket for proteins to get into ER
9
Q
C-terminal motif function
A
Localizes the protein in the ER
10
Q
v-SNARE
A
Vesicle SNARE on vesicles surface that binds to t-SNARE
11
Q
SNARE protein function
A
Mediate vesicle fusion(neurotransmitter release, hormone secretion, golgi trafficking) ensuring correct fusion
12
Q
t-SNARE
A
Target snare found on the target membrane
13
Q
Was is intracellular trafficking mediated by
A
Microtubules and microtubule motor proteins
14
Q
Where do the microtubules Kinesin and Dynein do
A
Kinesin drives transport ER -> PM(Anterograde)
Dynein drives transport PM -> ER(Retrograde)
15
Q
Explain the unfolded protein/ER stress response
A
- Protein translation shuts down
- Degradation of mRNA
- Protein degradation
- ER expansion
16
Q
What does ubiquitin do to misfolded proteins
A
Attaches to the protein via ubiquitination and protein becomes targeted for destruction by proteasome
17
Q
What does proteasome do to misfolded proteins
A
Recycles and turns it into small peptides + AA
18
Q
Receptor/Clathrin mediated endocytosis steps
A
- Receptors bind to cargo then Clathrin complexes on cargo-receptor to form calthrin tri-skelia.
- Membrane pits invaginates and dynamin used to pinch pit
- Clathrin cage encloses vesicle
- Clatrhin uncoats and vesicle fuses with early endosome.
19
Q
What is clathrin made out
A
3 polypeptide chain forming TRISKELION(cage around vesicle)
20
Q
What does autophagy do
A
Recycle of organelles
21
Q
What are the processes in autophagy
A
Autophagosome fuses with lysosome and internal contents degrade by hydrolyses.
22
Q
Endocytic pathway vs autophagy
A
Endocytic pathway degrades external particles starting in the cell membrane
Autophagy
Degrading internal organelles(except nucleus) to recycle starting in the cytoplasm
23
Q
Immune system definition
A
Network of cells, tissues, organs that function to protect body against pathogens
24
Q
Immune response definition
A
The coordinated response of immune system
25
What are the 2 types of immunity
Innate
Adaptive
26
What is the epitope
Part of antigen that's recognized by immune system
27
Innate immunity properties
- No memory
- From MYELOID progenitor
- Non -specific response
- Rapid responses
- PAMPs(these will be recognized by receptors)
28
Adaptive immunity properties
- Memory cells
- From Lymphoid progenitor
- Specific response
- Slower response
29
What are some examples of innate immunity
- Cells(Macrophage, Granulocytes)
- Physical barrier(Skin, mucus)
- Protein(Cytokines & Chemokines)
30
What are some examples of adaptive immunity
- Specialized cells(B&T lymphocytes)
- Proteins(Antibodies, cytokines, chemokines)
31
What are 2 forms of adaptive immunity
Humoral and Cellular
32
Humoral response
- Antibodies derived from B-LYMPHOCYTES
- For EXTRACELLULAR pathogen
33
Cellular(cell-mediated response) response
- Mediated by T-lymphocytes
- For INTRACELLULAR pathogen
34
What are the 2 types of t -lymphocytes?
CD8+ & CD4+
CD8+(=Killer t-cell or cytotoxic t-cell)
CD4+(helper t-cell)
35
CD8+ T-cell function
-Cytotoxic T-lymphocytes(CTL)
- Recognizes and kills viruses in infected host cells via secretion of anti-viral & inflammatory cytokines(IFNy, TNFa)
36
CD4+ T-cell function
- Helper T cells
- Produces cytokine
- Maturation of response(differentiation)
- Helps B-cells and macrophages
via secertion of anti-viral & inflammatory cytokines(IFNy, TNFa)
37
1° lymphoid organs
Bone marrow(B-cells) and Thymus(T-cells)
38
2° lymphoid organs
Lymph node, spleen, MALT(Mucosa associated lymphoid tissue), Appendix
39
2° lymphoid organs function
Activate lymphocytes against pathogen by exposing them
40
1° lymphoid organs function
Mature and develop lymphocytes
41
How does the lymph node prepare lymphocytes for pathogen
Very compartmentalized thus giving distinct areas where ANTIGEN is delivered
42
How does the spleen prepare lymphocytes for pathogen
Filters blood removing faulty RBCs promoting lymphocytes to fight infection
43
Coordinated response of the 2° lymphoid organs
Naive Lymphocytes recirculate through the lymph nodes & spleen via blood
Antigens/pathogens funnelled from Periphery -> Local lymph nodes
Lymphocytes encounter Antigen in 2° lymphoid organs
44
How does the complement system the first act as the first responder
Marks pathogen activating proteins sequentially leading to:
- Phagocyte migration to infection site
- Opsonization and phagocytosis
- Pathogen lysis
45
List the sequence of proteins activated in the complement system when a pathogen is encountered
C3 broken into C3a that recruits immune cells and C3b that tags for phagocytosis.
Eventually open pores in microbes after complement cascade.
46
What type of cells are used when the complement system is not enough to eliminate pathogens
Effector cells
47
What are some functions if effector cells
- Phagocytosis
- Cytotoxicity
- Secretion of proteins (cytokines, chemokines, complement)
48
What are the 5 antibody Isotopes
IgM
IgA
IgG
IgD
IgE
49
What does affinity maturation mean in terms of b cell receptors
The antigen binding site develops HIGHER affinity for antigen(Somatic hypermutation)
50
IgM antibody
- Pentamer linked by J chain
- Can be precursor to class switching
- Efficient at acting
51
IgA antibody
- Expressed as MONOMER secreted as DIMER held by J-chain
- Efficient at activating compliment
52
IgD antibody
Expressed as naive B cells with IgM
53
IgE antibody
Responds to parasites
Can cause allergy(demerit)
54
IgG antibody
Can cross placenta in babies
55
What determines the isotype in immunoglobulin
The variation in heavy chain in the constant regions
Effector function
56
Simply explain what the Fab and Fc regions do
Fab = Binds to antigens. "Finds the enemy."
Fc = "Calls the cavalry and issues orders."
57
What are the 3 genes that encode the variable domain of the Heavy chain gene
V, D, J
58
What are the 2 genes that encode the variable domain of the Light chain gene
V, J
59
What is the structure of immunoglobulin
4 polypeptide chains(2 heavy+2 light) connnected by S-S bonding
60
What happens with PAMPs and MAMPs are recognized by macrophages
Pattern recognition receptors lead to SIGNALING CASCADE(transcription & translation)
Cytokine & Chemokine produced. leading to phagocytosis and eventually antigen presentation for the adaptive immune response
61
Explain how cytokine and chemokine production aids in response after PAMPs/MAMPs are recognized
Cytokine and chemokine productipn induces inflammation->Δ blood vessel permeability recruiting cells.
Cytokines:
- BLOCK viral spread(INTERFERONS block transcription/translation)
- Activate macrophages,killer cells
- Upregulate MHC
62
What are granulocytes
short lived phagocytic cells with secretory granules and multilobbed nucleus
63
What are the 3 types of granulocytes
Neutrophils
Eosinophils
Basophils
64
Neutrophils
Bactericidal mechanisms: IL(interleukin) 8 triggers/recruits from bone marrow
65
Eosinophils
Kills PARASITES: triggers cytotoxic granules found in tissue
66
Basophils
Kills parasites & inflammation(histamine release)
67
Mast cells(not granulocyte) functions
- Recruits cells
- Found in tissues
- Promotes allergy
68
Dendritic cells
- Detects & Phagocytose pathogens
- Share antigen & info with lymphocytes -> adaptive immune response
- Activates T-cell
69
Natural Killer(NK) cells : Danger response
- Inflammatory Cytokine & Interferon gamme(IFN-y) antiviral cytokines secreted
- Activated NK releases PEROFIN(pore forming) & GRANZYMES(enzyme) triggering APOPTOSIS
70
B cell development in bone marrow steps
1. Developing B-cells acquire functional BCR through arrangement of immunoglobulin
2. B cells with Ig receptors binding to SELF are ELIMINATED
3. Remaining B cells leave bone marrow -> 2° lymphoid tissue
Lymphoid tissue
4. Cells circulate in 2° lymphoid tissue searching for specific antigens. On contact, B-cells proliferate &differentiate -> Plasma cells or memory b cells
71
What are the 2 forms of immunoglobulin binding to infectious agents via Epitope
Surface Ig: Antigen receptor and can transduce singals
Secreted Ig(antibodies): plasma cells = antibodies
72
T-cell development
Hematopoietic stem cells in bone marrow undergoing thymic education in thymus. Naive T-cells transform into effector T-cells which are then able to mount immune responses.
73
Thymic t-cell education process
Positive selection and negative selection
74
Explain positive selection(MHC restriction) in Thymic t-cell education
If TCR cant bind to self MHC moecules, T cell will die(apoptosis). Only TCR that binds weakly will sruvive
75
Explain negative selection(self tolerance) Thymic t-cell education
TCR that binds TOO strongly to MHC/peptide complexes, T cell will die
If let go it will attack host cells
76
T-cell receptor structure
heterodimer(2 different monomers) of A chain and B chain
membrane bound(always)
77
T-cell receptor function
Acessory molecules on T-cell facilitate signaling within the cell helping T&B cell receptors
78
What are the 2 types of MHC molecules
MHCI
MHCII
79
Where are MHCII expressed
Macrophages, B cells and dendritic cells(All are Antigen Presenting Cells, APCs)
79
What is the difference of MHCI and MHCII in terms of how they associate with the membrane
MHCI has 1 transmembrane domain (a3)
MHCII has 2 transmembrane domain (a2&B2)
79
Where are MHCI expressed
All nucleated cells(except blood cells)
80
Does CD8+ or CD4+ recognize peptides presented by MHCI
CD8+
81
Does CD8+ or CD4+ recognize peptides presented by MHCII
CD4+
82
Where are the peptides that MHCI largely derived from?
Endogenous(inside) proteins produced in the cell
83
Where are the peptides that MHCII largely derived from?
Exogenous(outside) proteins produced outside of cell
84
Antigen processing and presentation MHCI
Peptides created by proteolysis in the cytoplasm. Loaded into MHCI in lumen of the ER
85
Antigen processing and presentation MHCII
Peptides generated in endosomal/lysosomal network.
Invariant chain cleaved in MHCII after protecting peptides destined for MHCII to bind with MHCI
86
APC role
Network of cells that are specialized to take antigens & display peptides on MHC molecules.
87
Does the activation of naive T cells require activated Dendritic cells
Yes, secretion of CD80 and CD86 from DCs activate t-cell
88
What does dendritic cell activation result in
- Δ morphology = Lose capacity to capture antigen
- ↑surface expression of MHCI & MHCII
- Costimulatory molecules expressed(CD80&CD86)
- Cytokines secreted
- Migration of DC from tissue to 2° lymphoid tissue
89
What are 3 signals of dendritic cell that activate T cell?
1. Display pathogen peptides on MHC
2. Express costimulatory molecules(CD80, CD86)
3. Secrete cytokines and chemokines
90
How do the cytotoxic granules of Granzymes and Perforin leading cell apoptosis in virus infected cells
Perforin creates pores in cell membrane
Granzymes a proteases that cut up EVERY protein it finds
91
Efffector function of T-cell: CD8+ + Virus infected cell =
Dead virus infected cell
92
Efffector function of T-cell: CD4+ + macrophage
Activated macrophage -> Cytokines
92
Effector function of T-cell: CD4+ + B cell
Plasma cell-> Antibodies
93
Can all pathogens be cultured
No
94
Polyphasic taxonomy
Amalgamation of phenetic, phylogenetic and genotypic information
95
What does peptidoglycan of the bacterial cell wall do
- Help determine cell shape
- Protects against osmotic lysis and toxic substances
96
Is the cell wall thinner or thicker in gram - bacteria compared to gram + bacteria
thinner in G-, thicker in G+
97
What are peptidoglycan subunits comprised of?
- Alternating N-acetyl-glucosamine(NAG and N-acetyl-muramic acid(NAM) crosslinked
- Stem peptides
98
What are stem peptide composed of in the peptidoglycan subunits?
5 alternating D & L AA
99
Slime layer
Disorganized polysaccharide material that protects against antibiotics and dissociation also:
- adhere to smooth surfaces
- gliding motility
100
S-layers
lattice or protein or glycoprotein
Function:
- Environmental protection
- CEll shape and rigidity
- Adhesion to surface
- PAthogenesis
101
What are the 3 bacterial cytoskeletons
MreB, FtsZ, and ParM that all play a role in cell SHAPE & DIVISION
102
MreB cytoskeleton
Actin homologue: regulates cell morphology/shape
103
FtsZ
Tubulin homologue: Controls cell diviision
104
ParM
Involved in chromosome segregation(only 1 segregation)
105
What are the components of bacteria's 70S ribosome
50S(larger subunit) + 30S subunit(smaller subunit) = 70S
Sum of parts > sum of whole
106
What is the bacterial genome comprised of?
Plasmid + Nucleoid
107
What are endospores used for?
Backup. Condensed genetic material+proteins required to start up again like seeds in plants
108
What are characteristics for endospores
Resistant dormant structures that occur when there's environmental stress
109
Are spores impermeable to most simple stains
Yes
110
Name some PRR(pattern recognition receptors) that detect PAMPs
Mannose receptors
Toli receptors(most important one)
Lipopoly Saccharide receptors
Glucan
111
What does the Toli receptor(TLR4) recognize?
Is membrane bround, TLR4 recognizes Lipopolysaccharide(LPS) that is found on ALL gram(-) bacteria
112
What are some bacteria external structures that help them survive?
- Capsule & Slime layers
- Flagella
- Fimbriae(Pili)
113
Flagella function in bacteria
Motility/move around
114
Fimbriae(pili) function in bacteria
Attachment, DNA transfer
115
Capsule&Slime layers
Protection and biofilm
116
Are the flagella in Gram "-" and Gram "+" the same?
No
117
How are flagella synthesized?
Subunits are transported through the hollow filament(basal body) -> self assemble into helical array with help of filament cap protein
118
Fimbriae(pili) structure
-Attachment
-motility
-transformation
-uptake of DNA
- conjugation(sex pili)
119
Are Fimbriae(pili) found mroe on GN bacteria or GP bacteria
More on GN than GP bacteria
120
What are the 3 types of bacteria motility?
1. Flagellar motility
2. Twitching motility
3. Gliding motility
121
Describe Flagellar motility
- Rotation of flagella like a propeller
- Change directions by altering direction of flagellar rotation
122
Describe twitching motility
- Short jerky movements on SOLID surfaces
- Associated with type IV pili, slime layer or both
123
Describe gliding motility
- Smooth motility on smooth surfaces
- Associated with type IV pili, slime layer or both
124
Why do bacteria need to move
- Toward attractants(nutrients)
- Away from wastes and harmful substances
125
Is bacterial growth the increase in cell number or cell size?
Cell number
126
What are the 3 ways to measure the no. of cells in defined volume of culture
1. Viable count - counting living colonies
2. Spectrophotometry - Measure TURBIDITY of liquid culture
3. Direct counting - Stains to differentiate dead/alive
127
Explain what higher turbidity in a liquid culture when using spectrophotometry means
More cells = Higher turbidity
More turbid = Lower light transmittance = HIGH absorbance
Indirect count of cells
128
What do bacteria need to grow
- Carbon & energy(carbs,peptones)
- N2 -> peptones(hydrolyzed protein)
- Inorganic elements-> P, S, Fe,
- Growth factors-> blood, serum, vitamins
- Environmental conditions(O2 conc, temp, pH)
129
Anaerobe bacteria
Killed by O2
130
Obligate bacteria
must have O2
131
Facultative anaerobes/aerobes
Can grow with or without O2
132
Microaerophiles
Need O2(but lower than atmospheric conc)
133
Why is O2 dangerous? Think of ROS(reactive oxygen species)
Transfer of e- to O2 in respiration generates ROS that are toxic. ROS DENATURE enzymes and DAMAGE lipids and DNA
134
What are countermeasures that bacteria(except anaerobes ) have come with to combat and detoxify ROS
Enzymes:
- Catalase : 2H2O2 -> 2H2O+O2
- Peroxidase:
H2O2 + NADH + H+ -> 2H2O+O2
- Superoxide dismutase(SOD):
2O2- + 2H+ -> H2O2 + O2
Some pigments:
- Carotenoids
135
pH ranges for these bacteria
Acidophiles
Neutrophiles
Alkalophiles
Acidophiles: 0-5.5
Neutrophiles: 5.5-8
Alkalophiles: 8.5-11.5
136
Defined vs Complex media
Defined:
Defined set of carbs, pprteisn, source of mineral
Complex:
More complex, mictur eof proteins, extracts from plants
136
Selective media
Promote growth of some bacteria, inhibits others.
e.g. MacConkey agar(MAC)
137
Differential media
Indicate metabolic properties
e.g. blood agar(HBA)
138
Enrichment media
Supports growth of specific bacteria
e.g. Chocolate Agar(CHA)
139
What does MacConkey Agar(MAC) contain
-Bile salts
-Lactose
- Neutral red(inhibits many gram "+" bacteria)
140
What is the function of MacConkey Agar(MAC)
Isolate intestinal bacterial pathogens and also differentiate gram "-" bacteria
141
What does Horse Blood Agar(HBA) contain
Enriched medium
5% blood added after sterile nutrient agar cooled to 50C
142
What is the function of the Horse Blood Agar(HBA)
- Used for fastidious(require "heme" and Fe) bacteria
- Indicates haemolysis
143
What does Chocolate agar(CHA) contain
Enriched medium
7% blood added at high temp - blood cell lyse
Often incubated CO2
144
What is the function of the Chocolate agar(CHA)
Used for VERY fastidious bacteria
145
How to isolate pure cultures
2 techniques that require progressive dilution of cells:
Streak dilution technique OR Limiting dilution followed by spread or pour plate(-> viable count)
146
What are bacteria capsules
Made of polysaccharides
protects against:
- Dehydration/desiccation
- Phagocytosis(from virulence factor)
- Bacteriophage attack
- Toxic substances
147
Describe the steps in binary fission
1. Parent cell ENLARGEs & DNA duplicates
2. Septum forms -> Divides cytoplasm
3. New cell wall forms -> 2 daughter cells
148
E.coli cell cycle step 1: Replication of Chromosome
Initiates at single point(at ori) and completes at opposite terminus
149
E.coli cell cycle step 2: DNA replication and partitioning
Replisome(group of proteins required for replication of chromosome) assembles at origin.
Replication proceeds in both directions and cell ELONGATES
150
E.coli cell cycle step 3: Cytokinesis
Division of cytoplasm and organelles equally.
Synthesize a septum and divide cell
-> 2 daughters
151
E.coli cell cycle step 4: Septation
Forms crosswall(septum) between 2 daughter cells
152
What are critical steps in the Septum phase of the E.coli life cycle?
- FtsZ subunits polymerize to form filaments to assemble Z ring.
- Construction of divisome
- Cell constricted and septum formed
153
Divisome function
Synthesizes the peptidoglycan at the division plane
154
What are the 4 growth phases in bacteria(does NOT apply to continuous cultures)
1. Lag phase
2. Log(exponential) phase
3. Stationary phase
4. Death phase
155
What are 3 types of RNA?
tRNA
mRNA
rRNA
156
RNA polymerase read direction and what strand does it read on.
reads 3'-5' on template strand and synthesizes mRNA 5' -> 3'
157
Does RNA polymerase need free 3' OH to intiate transcription
No
158
What does it mean that RNA is polycistronic
mRNA can encode for multiple proteins
159
What are the 3 stages in transcription
1. Initiation
2. Elongation
3. Termination
160
Transcription: Initiation step
Sigma factor recognizes -10 & -35 box and directs RNA Pol to unwind DNA.
Makes a RNA copy of template strand
161
Transcription: Elongation step
Sigma factors dissociates & mRNA synthesis continutes
162
Transcription: Termination step
RNA Pol reaches terminator sequence. RNA pol ceases transcription & RNA RELEASED
163
What are some features that eukaryote transcription has that prokaryotes don't
1. 3 types of RNA pol
2. 5' caps on euk. mRNAs, long 3' polyA tail
3. Requires several transcription factors
4. mRNA contain exons & introns must be modified by RNA splicing
164
What is the objective of translation and what organelle is used
Turning mRNA into protein
Ribosomes
165
How do ribosomes form polypeptides?
Ribosome form peptide bonds between AA donated by sequence specific binding of tRNA.
166
Can multiple codons code for the same AA?
Yes
167
Translation: Initiation step
Transitional complex forms , tRNA brings first AA to START codon of mRNA
168
Translation: Elongation step
tRNAs bring AA 1 by 1 to add to polypeptide chain
169
Translation: Termination step
Release factor recognizes STOP codon, transitional complex dissociates and completed polypeptide is released
170
Why do bacteria regulate genes?
Regulation allows cells to repsond to different environments. It would be a WASTE of resources to have all genes "on" at once
171
Approaches to regulation:
1. Regulation of gene expression(3 ways)
2. Alter activity of enzymes & proteins (1 way)
Regulation of gene expression:
1. transcription initiation
2. transcription elongation
3. translation
Alter activity of enzymes & proteins
1. post translational
172
Are the regulatory mechanisms in 2 domains of life the same?
No
173
What do you call genes that are expressed all the time?
Housekeeping(constitutive) genes
174
what is the difference between inducible and repressible genes?
Inducible: Expression(transcription) turned ON by stimulus
Repressible genes: Expression(transcription) turned OFF by stimulus
175
Explain the mechanism of B-galactosidase the inducible protein
The Lac Operon is normally OFF due to repressor when lactose in unavailable. When lactose is present, it is converted to allolactose which unbinds the repressor turning on transcription for lactose digesting genes.
176
Explain what trp operon does in low and high concentrations of tryptophan
Low tryptophan = need biosynthesis genes
High tryptophan = No need to biosynthesize
177
Explain the regulation at the level of translation by riboswitches(regulatory RNA elements in mRNA that control gene expression)
- mRNA directly binds metabolite(intermediate or product of metabolism)
- Causes mRNA to fold differently impeding access to
ribosome binding site
- Small RNA regulation
- Antisense RNAs bind to mRNA leader sequence
- Inhibit ribosome attachment & translation
178
4 types of global regulation
Regulatory proteins
Alternative sigma factors(mostly)
2 component signal transduction systems(mostly)
Phosphorelay systems
179
what are σ factors in RNA polymerase complex
σ factors in RNA polymerase complex binds to -10 to -35 promotor(-10 & -35 are RNA pol recognition and binding sites)
180
What are 2 proteins of regulatory systems found in all 3 domains of life
1. Sensor kinase
Extracellular receptor for metabolite & Intracellular communication pathway
2. Response-regulator protein
Activated by sensor kinase
DNA binding protein(activator or repressor for transription)
181
What is the Central Dogma
The flow of genetic information in the cell
e.g. DNA -> RNA -> Protein
182
What are 3 sources that bacteria gain extra information?
1. Plasmids
2. Bacteriophages
3. Transposons
183
What are plasmids
Extrachromosomal selfreplicating DNA that add
Flexibility to bacterium
It can be manipulated and used in biotech
184
Plasmid structure features
- Double stranded(some linear) circular DNA supercoiled by DNA gyrase.
- Can have ≥ 1 type of plasmid per cell
185
What is the purpose of plasmids
Contain non-essential genes for bacterial growth
Offer bacteria SELECTIVE advantage under specific conditions(Virulence, resistance, metabolism)
186
What do plasmids need to replicate
-Origin of replication(ori)
- Specific replication proteins(some host derived somefrom own replication)
187
Do plasmids replicate independently of the chromosome
Yes
188
What are 3 common plasmid replication schemes
Unidirectional
Bidirectional
Rolling circle
189
Theta replication in plasmids
Most common for plasmids
Bidirectional depending on if RNA
Similiar to chromosome replication
1 or 2 replication forks from depending on whether RNA primers made
190
Rolling circle replication
Happens in circular DNA:
1 strand is nicked out of the way and then DNA pol synthesizes it which means that the intact strand is the template strand.
191
What is the substitute for a primer in rolling circle replication
The 3' end serves as a primer
192
What are the 2 plasmid properties determined by the origin of replication(ori)
1. Host range
2. Copy number
193
What does Host range mean
Species of bacteria in which the plasmid can replicate in
194
What does it mean to have a narrow or board host range
Broad host range:
- replicate in many species
- encode their OWN replication proteins
Narrow host range:
- restricted to 1 species
- rely on host proteins for replication
195
What does copy number mean
The average number of a single plasmid per cell(vary from 1 - >100s)
196
What does it mean that the copy number in plasmids are well regulated
Regulation mechanism varies between either High or Low copy number plasmids based on the Ori-regulated copy number.
197
Incompatibility groups
Sometimes plasmids with similar replication mechanism cannot survive in the SAME cell.
198
What is the process of conjugation
Donor cell donates plasmid(dna) to recipient bacteria that becomes a transconjugant
199
What are promiscuous plasmids
Conjugative plasmids that can move DNA between unrelated species.
200
Describe the mechanism of DNA transfer in bacteria
Pilus contacts potential recipient and retracts into close contact and pore forms in adjoining cell membranes.
201
Bacterial transformation to a transformant
Naked DNA taken up by bacteria randomly and can be any part of genome
202
Contrast between Lysogenic(forms prophage) and Lytic phages
Lysogenic phage:
Phage genome integrated in host genome = PROPHAGE
Note it can excise itself reverting to lytic growth in some conditions
Lytic phage:
Lyse host cells
203
Explain how transduction transfers bacterial DNA from one another
Virus(bacteriophage) mediated bacterial DNA transfer
204
Compare and contrast Generalised and Specialized transduction
Generalized:
- Any part of bacterial genome transferred
- Occurs at lytic cycle of virulent phage as fragments of host DNA mistakenly packed into phage head during viral assembly
Specialized:
-Specific part of bacterial genome
-Occurs when prophage is incorrectly excised(leads to integration into host chromosome)
205
Why is CRISPR considering adaptive immunity for bacteria against bacteriophage infection?
CRISPR degrades foreign nucleic acids that enter cell from bacteriophage, conjugation, transformation
206
What are the key structural features of CRISPR?
Repeats: Nearly identical short nucleotide sequences.
Spacers: Variable sequences between repeats, matching bacteriophage DNA.
Association: CRISPR arrays are linked to Cas genes (CRISPR-associated genes).
207
What are the 3 stages of CRISPR-Cas immune response?
1. Adaptation
New spacer from phage DNA is added to the CRISPR array.
2. Expression
CRISPR array is transcribed into crRNA (guide for Cas proteins).
3. Interference
crRNA guides Cas proteins to cut matching phage DNA = phage death
208
Explain the expression(biogenesis) phase of the bacteria against bacteriophage
CRISPR array(all Repeats & spacers) transcribed to crRNA which is splcied into crRNA(1 repeat 1 spacer)
209
Explain the interference(biogenesis) phase of the bacteria against bacteriophage
-crRNA recruits(nuclease) and guides
Cas to the target bacteriophage DNA
210
What complexdoes crRNA + Cas9 lead to? What does this complex do?
Ribonucleotide complex that recognizes and cleaves foreign DNA
211
What is the general structure of transposons
A piece of DNA(1-5kbp, 38 kpb) that all contain IR(inverted repeats) on either end
212
What happens during transposition
Target site duplicated at either end to produce direct repeats
213
What are the consequences of high rate of transposition
Lethal mutations
214
What does replicative transposition mean
Transposon replicates/copied to somewhere else
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What are transposable element
Segments of DNA that move around the genome
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What is Horizontal gene transfer
HGT(or LGT) is the transfer of genes from 1 organism to another
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What are 3 mechanisms of HGT
Plasmids: Conjugation
Phages: Transduction
Transposons: Conjugation, transformation
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What is responsible for the little phylogenetic(16s rRNA) diversity of bacteria despite their large phenotypic diversity
Horizontal gene transfer
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How to sequence a bacterial genome? (3 steps)
1. Generate DNA sequence reads
2. Assemble
3. Annotate
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What is a "read"
A length/fragment of readable ssDNA sequence that has been output by a sequencing machine
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Why is important to have a lot of "reads" when dealing a bacterial genome of 3mil bp
Bacterial genome = 3 mil bp
1 read = 300bp
100 reads = 30,000 bp of sequence covered
25 mil reads = 7.5 bil bp of sequence covered
7.5bil/3mil = 2500
2500x coverage of the bacterial genome's 3milk bp
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What are some instruments used to sequence reads
* Sanger sequencing
* Illumina (HiSeq, NextSeq, MiSeq)
* Oxford Nanopore MinION
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Explain the steps of Sanger sequencing
1. Unknown DNA fragment isolated
2. DNA denatured to create single stranded template
3. primer added to ssDNA template
4. Use DNA polymerase + dNTPs (normal nucleotides) + ddNTPs ( didoexy = dye-labeled terminators) to extend.
5. All possible possible positions on fragment labelled by nucleotide
6. Gel electrophoresis gives correct DNA sequence
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Contrast between read-mapping/reference assembly and de novo assembly of bacterial genome.
This is a component of genome assembly
read-mapping/reference assembly:
- Faster & easier
- Harder & slower
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Steps of Genome annotation
1. Locates genes by identifying open reading frame(ORF) in genome.
2. Translate DNA sequence -> AA sequence to check if its a functional gene
3. Homology search to check if it matches up with real protein
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Why was sanger sequencing replaced by Novaseq and PacBio Sequel 2
less time and cost
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What is a reading frame
>100 codons not interrupted by a stop codon
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When faced with multiple reading frames from 1 ssDNA, which one is the one to annotate
The one with the OPEN READING FRAME
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Metagenomics extracts DNA from:
EVERYTHING in the sample(e.g. gut biome) and sequences it all at the same time instead of isolation of each
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Contrast between pathogenic bacterium vs opportunistic bacterium
Pathogenic bacterium:
Disease producing bacterium
Opportunistic bacterium
Can cause disease under certain conditions(normally doesn't)
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Contrast between intracellular pathogens vs extracellular pathogens
Extracellular pathogens:
Remains in tissues but NEVER enter host cells
Intracellular pathogens:
Grow & multiply in host cells
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Contrast between Facultative intracellular pathogens vs Obligateextracellular pathogens
Facultative:
Reside in host cell or environment
Obligate:
Must grow in host cell
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What are 3 ways new pathogen can rapidly emerge
1. Hypermutable(high mutation rate)
2. Rapidly spread(air travel, hospital)
3. HGT
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What are some virulence factors expressed by bacterial pathogen
Can mediate:
- Adhesion(ligands for host cell)
- Anti-phagocytic(in capsule)
- Damge(toxin)
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Explain Koch's postulates(determining if bacterium is a pathogen)
1. Microbe associated with symptoms of disease & present at site of infection
2. Microbe isolated & grown in pure culture
3. Pure culture when inoculated(introduced) in susceptible host reproduces the same disease
4. Same microbe must be reisolated in pure culture from experimental infected host
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What is a limitation of Koch's postulates
- Cannot use obligate intracellular pathogens(can't grow in cultures)
- Need animal moduels
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Barrier 1: Physical barriers
What are some physical barriers against pathogens
* High salt, fatty acids, dry, (wounds) → skin
* Acid( stomach)
* Peristalsis (Gastrointestinal tract)
* Mucous & cilia
* Soluble mediators(Lysozyme in salvia/tears)
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How does bacteria invade by bypassing Innate barrier 1: Physical barriers
1. Penetrating the intact skin surface
* Cuts
* Carrier(ticks, mosquitos)
2. Penetrating the mucin layer
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Barrier 2: Innate immune response
What are some Innate immune response against pathogens
Complement
Phagocytosis:
- Neutrophils, macrophages/ monocytes (both professional phagocytes)
Inflammation:
- Toll-like receptor signalling, cytokines, via recognition of bacterial products
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Barrier 3: Adaptive immune response
What are some Innate immune response against pathogens
- Antibodies
- Cytotoxic t cells
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What is the host colonization step of pathogen
Establishes themselves in the host by adhering. Overcoming physical barriers and outcompete normal microbiote
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Adherence to host cells are mediated by external structures on the surface of bacteria are called
Adhesins:
- Pili(fimbriae) = loose connection
- Afimbrial adhesin = close/intimate connection
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What is the structure of Pili/Fimbriae
Ordered helical array of protein subunits called pilin
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Which part of the pili mediates adhesion
The tip of pili which can be pilin or other protein
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Describe the structure of Afrimbial adhesins
Membrane embedded proteins
- cell envelope(for gram + )
- outermembrane protein(for gram - )
This binds to protein or carbohydrate receptors on host cell
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How do capsules help in adherence of bacteria
Its sticky, helps stick to surface, tissue and other bacteria(to aggregate)
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How do biofilms help in adherence of bacteria
Contain extracellular polysaccharide slime and DNA to promote bacterial attachment
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What is the invasion of host cells mediated by
Invasins
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Compare and contrast the zipper and trigger mechanisms of invasion
Zipper mechanism:
Bacteria locks into host receptor and pulls it down via forced entry
e.g. Listeria
Trigger mechanism
Type Secretion system(T3SS) injects effector into host cells
e.g. Salmonella
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How do bacteria evade the host's defence system of Soluble factors(Complement, antibodies, cytokines)
* Avoid complement fixation (serum resistance)
* Destroy antibodies, avoid detection by antibodies
* Shutdown cytokine production
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How do bacteria evade the host's defence system of professional phagocytes(macrophages & neutrophils)
Avoid phagocytosis and / or kill phagocyte
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How do bacteria evade the host's defence system of Cytotoxic(CD8+) T-cells
Shutdown antigen presentation to avoid stimulating T-cells and / or kill T-cell
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What are the 3 major activities of the complement system
1) Stimulate inflammatory response by helping recruit white blood cells
2) Lysing microbial cell membranes
3) Promoting phagocytosis (engulfment and killing) of microbial invaders via Opsonisation
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What are the 3 pathways of the complement system
1. Classical
2. Lectin
3. Alternative
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The classical pathway of the complement system requires what to be activated
Requires specific antibody produced by activated B cells from adaptive immune response
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The lectin pathway of the complement system depends on what to be activated
Dependent on interaction of host mannose-binding lectin (MBL)
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The alternate pathway of the complement system depends on what to be activated
Activated by pathogens with LPS or surface antigens
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Explain the way bacteria avoid complement fixation
Prevent fc end of antibodies from binding to complement
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6 ways the bacteria avoid the immune system
1. Remaining inside host cells
2. Host mimicry
3. Colonise privileged site (with poor access for immune system)
4. Antigenic variation
5. Destroy antibodies(via proteases)
6. Bind antibodies ‘incorrectly
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6 ways bacteria avoid (or survive) Phagocytosis
1. Kill Phagocyte with a toxin
2. Avoid opsonisation(complement, antibody)
3. Prevent contact with phagocyte (capsule) = no opsonization
4. Inhibit phagolysosome fusion = fails to acidify
5. Escape into the cytoplasm
6. Inhibit or resist ROS
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What are 2 major impediments to pathogenic bacteria
- Normal microbiota → competition for space and nutrients
- Pathogenic bacteria are chemoheterotroph(require Fe which is sequestered away)
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Explain Bacterial dissemination
They escape normal microbiota/immune system and may travel to immune privileged or other body sites for new sources of nutrients
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Contrast direct and indirect damage to the host
Direct
– Toxins(Exotoxins & Endotoxin → bacterial LPS)
Indirect
- Immunopathology
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What are the 3 types of exotoxins produced by bacteria
1) A-B toxins
* Simple (AB)
* Compound (AB5)
2) Membrane disrupting toxins
* Pore forming
* Enzymatic
3) Superantigens
* Immunopathology
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What does the A subunit in toxin do? What does the B subunit in toxin do?
A = catalytic (active) subunit
B = binding subunit
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Simple A-B toxin (neurotoxin), binds to what causes flaccid paralysis
Simple A-B toxin (neurotoxin), binds to Synaptotagamin II receptor on neuronal cells
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Cholera has 5 B subunits that binds to cell A subunit enters cell to induce H2O loss, what does the toxin lead to?
* Increase cAMP
* Massive loss of water & electrolytes
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Explain the mechanism of
a-haemolysin(membrane disrupting toxin), the pore forming toxin
Disrupts membrane leading to H2O entering host cell -> cell lysis
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Explain the mechanism of
a-toxin/phospholipase C (membrane disrupting toxin), the enzymatic lysis toxin
Hydrolyses phosphatidylcholine in target host cell membrane. Leads to tissue necrosis
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What do superantigens do and what is the result
Link the MHC class II and TCR(T-cell receptors) non-specifically causing over activation of immune system(excessive cytokine)
Results in organ failure and shock
268
Endotoxin LPS effects
* Fever
* Massive inflammatory response
* Release of IL-1 and TNF
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Define each class of antimicrobial and its effectiveness
Narrow spectrum
Broad spectrum
Minimal inhibitory concentration
Minimal lethal concentration
Narrow spectrum:
Effective against only a limited variety of bacterial pathogens
Broad spectrum:
Effective against many types of bacterial pathogens
Minimal inhibitory concentration:
Lowest concentration of a drug that prevents bacterial pathogen growth
Minimal lethal concentration:
Lowest concentration of a drug that kills the bacterial pathogen
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Define the 3 properties of antimicrobial drugs of: Selective toxicity, Therapeutic dose, Toxic dose
Selective toxicity:
Drug's ability to kill/inhibit pathogen while damaging host as little as it can
Therapeutic dose:
Drug level required for clinical treatment
Toxic dose:
Drug level at which drug becomes too toxic for patient
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What are 2 main ways to determining the effectiveness of an antimicrobial
1. Dilution susceptibility tests for MIC(minimum inhibitory conc)
2. Disk diffusion tests
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In minimum inhibitory conc tests, if broth used, tubes will show no growth. Can they be subcultured into drug-free medium?
Yes
273
Explain the mechanism of antimicrobial drugs that inhibit cell wall synthesis(penicilins)
Inhibits peptidoglycan synthesis(particulary against gram +) via ß-lactam ring that irreversibly binds to enzyme(PBP) responsible for peptidoglycan cell wall synthesis replacing substrat.
274
Explain the mechanism of antimicrobial drugs that inhibit protein synthesis like aminoglycosides. Also which ribosome subunint does aminoglycosides bind to
Bind to bacterial ribosome(30s or 50s) to interfere with mRNA reading &/or cause termination of peptide synthesis causing misreading of mRNA.
Aminoglycosides bind to 30s ribosome
275
Explain the mechanism of antimicrobial drugs that inhibit Cytoplasmic Membrane(plasma membrane) Function
Polymyxins(cyclic peptides) are cationic detergents( produced by gram +) that disrupt plasma membrane against gram (-) They cause pores
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Explain the mechanism of antimicrobial drugs that inhibit Nucleic Acid Synthesis.
Note:
Sulfonamides and Fluroquinolones have different mechanisms
Sulfonamides:
Inhibits PABA->folic acid->purines & pyrimidines
Fluoroquinolones
Binds bacterial DNA-gyrase & topoisomerase IV = blocks DNA coiling
276
How does resistance to Aminoglycosides(drugs that inhibit protein synthesis) work
1. Efflux
2. Modified outer membrane to reduce entry
3. Ribosome mutates to reduce binding
4. Enzyme modified to reduce entry
277
What % of human genome is of retroviral origin
10%
278
Describe the 3 ways viruses could've come from:
Regressive evolution
Cellular origins
Independent entitities
279
What are viruses
Genome(DNA or RNA) surrounded by protective protein coat that are infectious and dependent on host cell to provide machinery needed to reproduce
280
What is a virion and how is it different from a virus
Virion is the extracellular form that is dormant & hasn't replicated yet.
Ex.
When HIV is inside a human cell, it’s a virus (replicating).
When HIV is in the bloodstream as a free particle, it’s a virion (waiting to infect)
281
What are the 4 main categories of Viruses based on capsid structure
1. Icosahedral protein coat
2. Helical protein coat
3. Viral envelopes
4. Complex symmetry
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Virus Icosahedral protein coat properties
- Repeating units of protein (capsomers)
- Whole coat is the capsid
Surrounds nucleic acid and “core” protein (nucleocapsid)
283
Virus Helical protein coat properties
- Rod shaped coat consisting of repeating units
- 1 protomer associates with nucleic acid in a spiral or helical arrangement
284
285
Virus Viral envelopes properties
- Icosahedral or helical nucleocapsid surrounded by membrane
-Envelope contains virus-encoded proteins or glycoproteins(SPIKES)
- Envelope is pleiomorphic(flexible)
286
What is the structure of capsomers in virus Isocahdral protein coat
Penons and hexons = symmetrical
287
Virus Complex symmetry properties
Largest and most complex
288
Is there any type of viral nucleic acid it can't be? Can it be:
– s.s. (single stranded) DNA
– d.s. (double stranded) DNA
– s.s. RNA
– d.s. RNA
It can be:
– s.s. (single stranded) DNA
– d.s. (double stranded) DNA
– s.s. RNA(sense or antisense as well)
– d.s. RNA
289
What are the 5 virus classfications/groupings
1. Enteric viruses(intestine tract)
2. Respiratory viruses
3. Arboviruses(insect bites)
4. STI
5. Hepatitis viruses(liver)
290
Explain the simplified stages for a virus cycle
1. Entry
2. RNA replication
3. DNA replication
4. Assembly
5. Exit and maturation
291
What are 3 Fusion & endocytosis strategies used by viruses
1. Uncoating at plasma membrane
2. Uncoating within endosome
3. Uncoating at nuclear membrane
292
What is does the 1st step of virus cycle Attachment/Adsorption do
Viral attachment protein binds to plasma membrane receptor
293
What does the 2nd step of virus cycle: Penetration and uncoating do
Viral genome released from protective capsid either through FUSING or ENDOCYTOSIS = nucleic acid to be transported = Membrane fusion
294
What does the 3rd step of virus cycle: Amplification of the viral genome and viral proteins
mRNA transcribed from viral DNA( formed directly from some RNA viruses) and codes for viral proteins that are translated by host cell
295
Special enzymes – RNA Polymerases job in the virus life cycle
Reverse transcriptase enables viruses to integrate their RNA genome into host DNA chromosome
296
What does the 4th step of virus cycle: Virus assembly and release for non-enveloped viruses
Sponatenous assembly of capsid proteins around nucleic acid genome and virions accumulate and released when cell lyses
297
What does the 4th step of virus cycle: Virus assembly and release for enveloped viruses
Budding from the cell surface/membrane utilizing the secretory pathway(exocytosis)
298
The most common entry for viruses into the human body is
The respiratory tract
299
What is the difference between disseminated infection and systemic infection
disseminated infection - spread beyond primary site
systemic infection -=many organs infected
300
4 mechanisms of spread in the body
- Local spread on epithelial surfaces
- Sub-epithelial invasion and lymphatic spread
- Sub-epithelial invasion & neuronal spread
- viraemia(spread via bloodstream )
301
What are the 2 types of Viraemia
1. Free in plasma = easy to find & short term infection
2. Cell associated viruses = hides in WBCs = hard to find & long term infection
302
Explain viral damage to tissues & organs in terms of mechanisms of disease progression
Cell death caused by viral replicatoin
303
Explain the consequences of the immune response when the disease progresses
Immunopathology:
– inflammation
– fever
– enlarged lymph nodes
– killing of infected cells
Immunosuppression:
– virus grows in cells of the immune system
304
What determines host resistance and susceptibility
genetic factors
age
malnutrition
hormones, pregnancy
dual infections
305
Viral genomes are constantly changing as a result of
* mutation(RdRps purposefully make errors)
* recombination (exchange of nucleic acid sequence)
* reassortment
306
What does antigenic drift result from
RNA copying errors and genetic reassortment
307
Why do some diseases reappear, often in a different form in our lives
Latent infection
308
How do Zanamivir (Relenza) and Oseltamivir (Tamiflu) inhibits influenza
Inhibitors designed to mimic natural ligand, sialic acid
309
Explain how Antiviral drug: HIV protease inhibitors act
Protease activated upon
final assembly and exit of the virion, if cleaved = stops the released virus from becoming infectious
310
Explain how Antiviral drugs: Nucleoside analogues(Acyclovir)
Antiviral drugs work
Acyclovir is a guanosine analogue and when incorporated into DNA, it will cause TERMINATION of infected cell
311
Is Acyclovir a prodrug?
Yes, people unaffected by the active form of herpes are not affected
312
Why is the esophagus rarely infected
Lots of movement
313
What are some properties of virus that infect the intestinal tract
Bile & acid resistannt and DON'T have envelopes
314
Viraemia virus that are free in plasma are neutralized by
The ab response and removed by macrophages
315
Why do we get the same diseases over and over again
- Ineffective immunity(Warts)
- Multiple SEROTYPES despite effective immunity
- constantly evolving virus
316
Cytocidal virus causes disease by
Cell death caused by viral infection
317
What are the 2 viruses that are cytocidal viruses
Rotavirus diarrhoea(enterocytes) & influenza virus infections
318
What viral component allows the Acyclover to become Acyclover-TP that will be incoporated into DNA causing chain termination?
Viral Thymidine Kinase(TK)
319
Explain the mechanism of viral membrane fusion in HIV viruses
Contents released directly -> cytoplasm. CD4 and co(chemokine)receptor bind to HIV's g41 glycoprotein, it twists, bends and genome released after fusion.
320
How does M.Tuberculosis survive phagocytosis
Kill macrophages
Resistant to oxidative killing
Inhibit diffusion of lysosamal enzymes
321
Contrast the mode of transmission between Venereal syphilis and congenital syphilis
Venereal = sexually transmitted
Congenital = acquired in utero from infected mother
322
Which is the toxic component of LPS
Lipid A
323
AB toxins are activated by what
The low pH environment of the endosome
324
What are mecanisms of iron intake in bacteria
- Siderophores: High affinity iron chelators
- Iron binding proteins
- Receptors from Fe-binding proteins of the host
- Toxins
325
What are virulence factors to enchance spreading/dissemination
1. Enzymes(elastase, protease, phospholipase) that degrade connective tissue matrix released
2. travel in blood stream(requires serum resistance )
326
What does transposase recognize to cut transposon
Transposase recognizes IR cuts the transponson out of its original spot
Transposase also cuts target DNA and inserts transposon and gaps are filled by DNA pol.
327
What type of replication does conjugation use
Rolling circle replication
