Midterm 2 Flashcards
1
Q
Main difference gram neg vs positive
A
Neg has outer membrane
2
Q
Cytoplasmic membrane has what
A
- lipid bilayer
- semi permeable barrier
3
Q
What determines the lipid composition of cytoplasmic membrane
A
Conditions
Ie. temperature
4
Q
Functions of membrane proteins
A
- transporters
- signal transduction
- energy transduction
5
Q
Energy transduction example
A
Electron transport chain
6
Q
How do bacteria survive hypotonic conditions
A
Peptidoglycan stretches with pressure and prevents lysis
- porous = allows transport
7
Q
How do bacteria survive pep degradation
A
Isotonic condition
- lose shape
- still stable
8
Q
Spheroplasts
A
Gram negative without pep
9
Q
Protoplasts
A
Gram pos without pep
10
Q
How do mycoplasmas survive without cell wall
A
Uses sterols from host to stabilize membrane
11
Q
Why is pep a good antibiotics target
A
Not made by humans
On outside of cell
Made by most bacteria
12
Q
How does bacitracin target pep
A
Binds undecaprenyl, prevents dephosphorylation = no phosphate = no binding
13
Q
How do beta lactams target pep
A
PBP transpeptidase = no cross linking = weakened Pep = lysis
14
Q
Pep structure
A
Glycan backbone
NAM
NAG
NAM has peptide chain
15
Q
Making pep
A
- UDP binds NAG
- Some convert to NAM
- L - ala + alanine racemase = D-ala ( 1 unit)
- D-Ala d-ala ligand makes D-Ala D-ala (2 units)
- MurF adds d-ala and other peptides to NAM
- NAM binds to undercaprenyl phosphate
- Gets phosphate, loses UDP [ LIPID 1]
- NAG binds NAM
- Get second phosphate [LIPID 2]
- Flips to periplasm
- Penicilin binding proteins add lipid 2 to chain
16
Q
What does undercaprenyl phosphate do
A
Provides phosphate groups to detach UDP and attach NAG
17
Q
What does cycloserine do
A
Blocks alanine racemase = no d-ala = incomplete chain
18
Q
Undecaprenol recycling
A
Flips between periplasm and cytoplasm depending on number of phosphates
19
Q
Lysozyme is part of what immune system
A
Innate
20
Q
Where is lysozyme
A
Saliva tears milk mucous
21
Q
What does lysozyme do
A
Cleaved NAG-NAM bond
Therefore weakens cell wall
22
Q
Why is lysozyme more effective on gram pos
A
Pep more exposed
No outer membrane
23
Q
Order of pentapeptides in pep
A
L-alanine
D-isoglutamate
Diamino acid
D-ala
D-ala
24
Q
Cross linking pep gram neg
A
3 and 4
Release terminal d-ala
25
Cross linking gram pos
Interpeptide bridge between diamino and first d-ala
Terminal d-ala released
26
How are cross links formed
PBP transpeptidase
1. Forms complex w peptide
2. Diamino reacts
3. Amide bond forms
27
What are b lactamases
Enzymes degrade b lactams
Resistance mechanism
28
Serine b lactamases
Serine binds to lactam = hydrolysis = inactive lactam
29
B lactamases inhibitors
Co prescribed with b lactams
30
MRSA and b lactam resistance
Has a PBP gene resistant to lactams
[active site blocked until bound to PB]
31
How does vancomycin target pep
Only targets gram pos
Binds to d-ala d-ala = blocks PBP
32
Vancomycin resistance
Replaces d-ala with different d amino acids = vanomycin cant bind
33
Gram neg outer membrane
Outer leaflet and inner leaflet
34
Gram neg outer leaflet
Lipopolysaccharide (LPS) only in negs
35
Inner leaflet gram neg
Phospholipids
36
LPS function
Impermeable
37
LPS traits
- neg charged
- amphipathic
- bulky
38
LPS structure
Lipid a
Core polysaccharides
O antigen
39
Lipid A (LPS) function
Anchors LPS to membrane
40
Lipid A (LPS) structure
Sugars with fatty acids
Phosphorylation = negative charge
41
Lipid A (LPS) toxicity
Releases endotoxin when lysed
Fever inflammation, septic shock
42
Core poly (LPS) function
Links lipid A to O antigen
43
Core poly (LPS) structure
Sugars
Branches
Anionic sugar
Neg charge
44
O antigen (LPS) function
Classification
Antigenic (bacteria changes this to avoid immune response)
45
Divalent cations Outer membrane gram negative
Stabilize
Bridge LPS molecules
Neutralizes electric repulsion
46
Gram neg outer membrane function
Barrier
Prevent antibiotics
Prevents degradation enzymes (too large to pass)
Resistant to detergents
47
Why are gram negs resistant to detergent
Outer membrane - LPS
Steric and charge
48
What are divalent cations needed for LPS
Cross-bridging adjacent LPS molecules
49
What happens limited Mg LPS
Lipid A may be changed to 4AA to cross bridge
50
Colistin
Cationic antibiotic
Binds to lipid A phosphate groups
Lipid tail can permeate membrane
51
How do Mg levels impact colistin
Mg sensitive to colistin
4AA prevents colistin binding = resistance
52
MCR-1 gene
Colistin resistance gene - HGT
Binds smth = positive charge = repels cation colistin
53
Outer membrane proteins gram negative
Lipoproteins
B barrel proteins
54
Porins
Nutrient intake gram neg
Form channels
B barrel
Water filled center = selectivity
55
Outer membrane assembly gram negative
LPS assembled in cytoplasm but needs to cross to periplasm
- chargers make it hard
- LPT proteins make LPT pathway
- LPTD guides it through
56
Vesiculation
Vesicles can form when outer membrane of gram negative not attacked to pep
57
Brauns lipoprotein
Fatty acid chain embedded in outer membrane gram neg
COVALENTLY BOUND TO PEP
58
Teichoic acids are in what
Gram pos cell wall
59
Teichoic acids
Linear polymers - glycerol or ribitol
- may have substituents (pos charged)
Phosphate groups neg charged
60
2 Teichoic acids
Wall Teichoic acids (WTA)
Lipteichoic acid (LTA)
61
Wall Teichoic acid
Attached to NAM or peptide in pep
Extends beyond pep surface to environment
Starts within pep
62
Lipoteichoic acid
Attached to lipids in cytoplasmic membrane
Extends through pep to environment
63
Teichoic acids function
- anchor wall to cytoplasm membrane
- binds cations = less repulsion
- regulation of pep degredation during division
- d-ala protects antibiotics and immune
64
Teichoic acids infections
Pathogensis
- biolfilm
- colonization
- inflammation upon release
65
Mycobacteria staining
Acid fast staining
Heat cells with stain
66
Mycobacterial cell wall
Gram pos but have outer membrane
- mycolic acids not LPS
Arabinogalactan
- sugar polymers
Pep
67
Mycobacteria outer membrane
Asymmetrical bilayer
- inner = mycolic acids
- Outer = glycolipids
Hydrophobic + impermeable
68
Cells need what 3 things
Energy electrons carbon
69
Heterotrophs
Organic molecules for carbon
70
Autotrophs
CO2 for carbon source
71
Reducing power
Electrons
Needed for
- anabolic reactions
- making atp
72
Organotrophs
Reduce organic molecules
73
Lithotrophs
Reduced inorganic molecules
74
Chemoorganoheterotrophs
Chemo = energy not from light
Organo = reduced organic molecules for electrons
Hetero= organic source of carbon
75
Most bacteria are what metabolic classification
Chemoorganoheterotrophs
76
How do Chemoorganoheterotrophs make atp
Oxidizing organic molecules
- aerobic
- anaerobic
- fermentation
77
Aerobic respiration
When there is adequate oxygen
Glycolysis : glucose —> acetyl coA
Krebs cycle: acetyl coA —> ATP, NADH, FADH2
ETC: NADH/FADH2 —> ATP
78
Glycolysic pathways
Embden-Meyehof (EM): most common, ATP NADH, G3P —> PYRUVATE
Entner-Douforoff (ED): some bacteria, NADPH, glucose —> pyruvate + G3P (—> EM)
Penrose phosphate pathway (PPP): biosynthesis, precursor aminos , NADPH
79
Kerbs cycle
Acetyl coA —> CO2 + GTP, NADH, FADH2
80
ETC
Membrane bound electron carriers: Ubiquinone (coenzyme Q) and cytochromes
Carriers reduced via oxidation
81
ETC in E. coli
- NADH electrons via ubiquinone
- pass through cytochromes
- to terminal electron acceptor (O2)
- proteins to periplasm = proton motive force (PMF)
82
Proton motive force + ATP synthase
Proton gradient
- cytoplasm = neg
- protons flow from periplasm to cytoplasm via atp synthase
83
Anaerobic respiration
1. Glycolysis
2. Krebs cycle
3. ETC
** terminal electron receptor is not O2
Could be nitrate, surface, CO2 etc
Ex. NO2- —> NO —> 2NO —> N2O —> N2
84
Fermentation
When lacking or repressing ETC
- No ETC = still have NAHD
Fermentation = NADH —> pyruvate + NAH+. —> new products
Ex. Ethanol, lactic acid, CO2
85
Why is it hard to target metabolic activity of bacteria
Most bacteria = Chemoorganoheterotrophs
Humans = Chemoheterotrophs
86
Tetrahydrofolate
Co factor needed to make purines and pyrimidines and methionine
87
Importance of tetrahydrofolate antibiotics
Bacteria must produce it
Humans must eat it
Bio synthetic enzymes = targets
88
Sulfa drugs
Inhibits dihydropteroate synthase (early precursor to tetrahydrofolate)
Competitive inhibitor
89
Trimethoprim
Inhibits dihydrofolate (can’t reduce to become tri)
90
Passive diffusion
Moves with gradient across membrane
91
Facilitates diffusion
Transport proteins aid in transportation across barrier in vesicle like manner
92
Active transport
Against gradient, needs energy
93
Primary active transporters
Use ATP to transport against gradient
94
ATP binding cassette transporters (ABC)
Primary active transporters in bacteria
Import and export
95
Solute binding proteins
Work with most ABCs
- deliver specific substrate to transported
96
Where are SBPs in gram neg
Periplasm
97
Where are SBPs in gram pos
Lipoprotein or pep
98
Secondary active transport
Ion gradient potential energy transports against gradient
- ETC
- V type ATPase
- Antiporter
99
V type ATP ase
Reverse of ATP synthase - uses ATP to send them against
100
Antiporter
ETC generated proton motive force (PMF)
H+ down gradient powers Na+ up gradient
Na+ gradient can then power symporter
101
Group Translocation
Active transport with modification of substrate
102
Phosphotransferase system (PTS)
Group translocation
- sugar across cytoplasm membrane
- sugar phosphorylated during transport
- P from PEP —> PTS —> sugar
103
Outer membrane transport porins types
General
Substrate specific
B barrel proteins
104
General porins
Channel size = which substrates can enter
105
Substrate specific porins
Binding site attracts substrate, size impacts selectivity
106
Ton B dependent receptors
1. Receptor inhibited
2. Binding to receptor
3. Exposes ton B box
4. Box binds to Ton B
5. Ton B removes plug from receptor
107
Why do bacteria need iron
Cytochromes and enzyme co factors
108
How do bacteria find iron
Siderophores
109
Siderophores
1. Bacteria secretes Siderophores
2. Bind to Fe3+
3. TonB receptor transports into periplasm
4. Binds to SBP
5. To ABC
6. To cytoplasm
110
Positive taxis
Move toward stimulus
111
Negative stimulus
Move away from stimulus
112
Chemotaxis
Move away/towards attractants or repellents
113
Flagellar swimming
Rapid rotation of flagella to move in liquid environments
114
Atrichous
No flagella
115
Monotrichous
Flagella on one end
116
Lophotrichous
Multiple flagella at one or both ends
117
Amphitrichous
One flagella at both ends
118
Peritrchous
Flagella all over
119
Flagella structure
Basal body
Filament
Hook
120
Basal body
Attaches flagellum to cell envelope
Has motor
121
Filament - flagellum
Helical extending from cell
Rotation moves cell
122
Hook - flagellum
Transmits rotation from basal body to filament
123
Basal body structure
Protein structure - central rod
Rings - L,P,MS,C
124
Basal body function
Export subunits during assembly
Rotate hook and filament
125
basal body motor
Rotation powered by PMF
Switch determines direction ( C ring in cytoplasm)
126
Flagella + innate immune
on surface = target
Flagellin = toll like receptor 5 (TLR5)
Flagellin binds TLR5
Activates transcription factor
Pro inflammatory cytokines produces
127
Flagella adaptive immune system
Antigenic structure
128
Immune evasion flagella
- alternate flagellins = phase variation
- stop producing once in host
129
Direction of flagellum rotation determines what
Run vs tumble
130
Monotrichous and peritrichous flagellum direction
Run = counter clock
Tumble = clockwise
131
Methyl accepting chemotaxis proteins (MCPs)
Chemoreceptors in cyto membrane
Diff ones sense diff attractants or repellents
Change direction of flagella
132
How do MCPs direct bacteria
MCP ligand binding domain + ligand = domain
Ligand changed shape of domain
CheW bound to domain
Activity changes based on ligand
133
Tumble no ligand
1. CheW detects no attraction
2. CheW causes CheA to auto phosphorylate
3. CheA phosphorylates CheY
4. CheY binds to switch, changes direction to CW
5. CheZ dephosphorylates CheY and direction —> CCW
134
No tumble, ligand
1. CheW detects
2. Does not cause auto phosphorylation
Therefore no switch
135
Excitation flagellum
Threshold must be met among many MCPs to determine how long direction will stay same or change
136
adaptation
After ligand binds:
MCP methylated by CheR
Increases CheA phosp
Increases amount of CheY
- removes CCW bias
137
Temporal gradients
Bacteria measure [] gradients for chemotaxis
Current = number of MCPs with ligands
Past = number of methylated MCPs
Allows bacteria to swim through gradient
138
What direction is swimming if Ligand [] increases
Swimming up
More ligands = more methylation (BUT DELAYED) = CCW bias
Fewer tumbles
139
What direction swimming if ligand [] decreasing
Down gradient
Less ligands = methylation SAME OR MORE = more tumbles
140
What causes MCP demethylation
CheB over time
141
What would happen if MCP did not demethylate
More binding wouldn’t equal CCW bias
142
Swarming motility
Flagella moving on surfaces
- coordinated movement
- requires multiple flagella and surfactants to reduce surface tension
143
Twitching motility
Pilli - moving on
Inches like catapilar
Extends: adds subunits to base (pillins)
Adheres
Retraction
Pillins removed
Colonize new environments
144
Glycocalyx
Polysaccharide layer around cell
Adherence
Related to EPS in biol films
Slime layer - easily remove from cell
Capsule - attached to cell
145
Capsule
Negative staining
Long polymers
Covalent bonding
146
Capsular polysaccharides
Repeating sugar subunits
147
Capsule functions
Adherence
Protection
- desiccation
- engulfment
- phage
- antibiotics
148
Major contributor to serum resistance
Capsule
149
Serum resistance
Ability to survive and spread in blood
150
How do capsules protect from antigens
Similar to host cell sugars
Mask antigen components
151
Conjunctive vaccines
Capsule polysach attached to immunogenicity protein carrier
- modified to evoke immune response
152
Capsule assembly gram positive
- Components transported to surface
- sugars activated by UDP
- transferred to undecaprenyl phosphate
- grows chain
- flipped to extracellular
- polymerized
- attached to surface
153
Capsule assemble gram negative methods
- Wzx-Wzx dependent pathway
- ABC transporter dependent pathway
154
Wzx Wzy pathway
- subunit assembled in cytoplasm
- flipped by Wzx
- polymerized by Wzy
- Translocated via Wza channel
- incorporated on cell surface
155
ABC transporter dependent pathway
- full polysaccharide made in cytoplasm
- transferrred to surface via ABC transporters
156
Non flagellar appendages
Pilli
157
Types of pilli
Type IV secretion pilli
Type IV pilli
Type III secretion systems
Chaperone-usher pilli
158
Type 4 secretion pilli
Sex pilli; conjugation
159
Type 4 pilli
Twitching motility
160
Chaperone usher pilli
Virulence factors - adhesion
161
Parts of chaperone usher pilli
Rod: major pillins
Fibrillum: minor pillins attach end adhesion to rod
162
Pilli pathogensis
Can only bind to specific molecules depending on the bacteria
163
UPEC (uropathogenic E. coli)
Causes most UTIs
Type 1 pilli attch to proteins in uroepithelial cells
Forms intracellular bacterial communities
164
UPEC and pyelonephritis
UPEC —> uterus —> kidneys
Must down regulate T1P production
Increase P pillus production
165
Immune response to pilli
Targeted
Can be used to make vaccines
166
How can pilli be used to make vaccine
Purify adhesions
Generate antibodies
Binds to adhesions
Prevents attachment
167
Antigenic variation
Change of pilli structure to evade immune response
168
P pilli assembly
- subunits —> periplasm via general sec system
- PapD binds subunits
- Helps them fold (chaperone)
- delivers to PapC
- PapC forms base
- Translocated subunits to surface (Usher)
- PapA (major pillins) Translocated via PapC
169
What happens if a cell doesn’t;t have PapA gene
PapA=major pillins
No major pillins = no rod on pilli
No rod = no pillus
Sill has base and adhesion
Therefore still adherent
170
Regulation of Pilus length
- PapH attaches pillus to cell
- also regulates addition of PapA
171
What happens if cell doesn’t have papH gene
PapH = attached pillus to cell and regulates addition of PapA
no PapH = long pilli not attached to cell
172
What happens if too much papH
Short pilli ( PapA can’t be added enough)
173
Gram positive pilli
Adhesion
Biofilm formations
174
Gram positive structure of pilli
Base Pillin attached to pep
Subunits covalently bonded
Adhesions at end
Long and flexible and thin
175
Gram positive Pilli assembly
- subunits contain cell wall sorting signal (pos charge) LPXTG
- subunits transported via general Sec system
- adhesion folds in periplasm and attaches to subunit
- sortase recognizes signal
- removes signal LPXTG
- sortase binds to subunits and forms complex
- pillus specific sortases form polymers of subunits via covalent bonds
- house keeping sortase trasfers pilli to lipid II
- incorporated into Pep by PBPs
176
Exotoxins
Proteins secreted by pathogens
Lead to injection
Disruption host cells
177
Groups of exotoxins
AB toxins
Pore forming toxins
Superantigens
178
AB toxins
Interfere with internal processes of host cells
179
Structure of AB toxins
A: active component
B: binding component
Form complex
B binds to receptor on host
Triggers endocytosis of host cell
Allows A to enter host
A = enzyme for making toxic compounds
180
Diphtheria
AB toxin
Cardiac and nervous tissue
A blocks elongation factor = no translation
=no proteins
181
Shiga toxin (Stx)
From type of E. coli
Severe inflammation and GI bleeding
Has prophage
AB toxin
A: removed nucleobase from rRNA = disabled ribosome
Vascular
182
Pore forming toxins
forms channels in host membrane
=no gradients = cell swells = cell lyses
183
Hemolysins
Pore forming toxin
Lyses red blood cells
Bacteria steal iron released
184
How do pore forming toxins help bacteria escape phagosomes
Phagosome engulfs
Toxin released
Cell lysed
Can replicate in immune cells
Spreads through blood
185
Superantigens
Force binding of immune cells even when no antigen
=Overproduction of proinflammatory cytokines
= fever, organ failure
Ex. Staphylococcus aureus toxic shock syndrome toxin
186
Sec system - post translational translocation
Chaperons stabilize
Sec A binds signal peptide
Escorts to SecYEG (channel)
Uses ATP
187
Sec system: periplasmic proteins
Signal peptidase recognizes and cuts signal peptide
Chaperones help protein fold in periplasm
188
TAT system
Used when proteins can’t be folded in periplasm
Translocated folded proteins
- protein folds in cytoplasm
- TatABC targets via signal peptide
- Translocation via PMF
- Signal peptide cleaved off
189
ABC exporters
Recognize signal protein
Cleave it off during export
190
Sec system - importing proteins to membrane
- signal peptide translated
- recognized by signal recognition particle
- translation stopped
- escorted to SecYEG
- translation continues into membrane
191
Gram negative BAM complex
- B barrel proteins targeted to outer membrane
- Sec system Translocates
- chaperones fold
- delivered to B barrel assembly machinery
- inserted into membrane
192
Gram neg secretion systems
Transport proteins out of cell
193
Type 5 secretion system (T5SS)
Tat or Sec
Protein Translocated via B barrel
**protein stays attached to cell (channel)
194
Type 1 secretion system (T1SS)
ABC transporter
Membrane fusion protein
B barrel
Steps
195
Type 3 secretion system (T3SS)
Inject proteins into eukaryotic cells
Substrates = effector proteins
Bacterial cytoplasm —> cytoplasm of host cell
Manipulate host structure and function
Colonization
196
T3SS structure
>20 proteins
Basal body
Needle
197
T3SS assembly
Subunits —> hollow central channel —> extracellular
Plug blocks channel after assembly
198
T3SS effectors
Binds target
Channel opens
Chaperones bring unfolded effectors to T3SS
Effector travels through T3SS
Folds in target
199
T3SS effector proteins
Target host cytoskeleton + signal transduction
Rearrange cytoskeleton of epithelial cells
Forms ruffles on surface
Allows bacteria to enter NON-PHAGOCYTIC cells
200
Protein secretion gram positive
Sec or tat
Sortase = attaches proteins to surface
Virulence factor
Nutrient acquisition
Immune evasion
201
Direct interactions with regulators
Stimuli directly affects transcription
Ex. Lac operon
202
2 component signal transduction systems (TCSs) parts
Sensing + response : different proteins
Sensor kinase: membrane protein
Response regulator: cytoplasmic DNA binding protein
203
TCSs steps
Stimulus
Activates sensor kinase
Kinase auto phosphorylated
Phosphate transferred to response regulator
Phosphorylation changes regulated structure
Can bind to DNA and change transcription
204
How is enterococci resistant to vanomycin
Vanomycin: targets d-ala d-ala
Enterococci senses vancomycin
Changed d-amino to avoid vancomycin targeting
Done only when present because fitness cost
205
Agrobacterium tumefaciens
Plant pathogen
Causes tumour-like growths
Carry tumour inducing plasmid
- enters plant through surface wound
- sensed plant via TCSs (sensor kinase detects)
- response regulator phosphorylated
- Transfer part of plasmid to plant cells
- Transcribed in host plant
- encode type 4 secretion system (pilli)
- connects cells
- proteins transfer dna from plasmid
- T4SS secretes dna into plant
- dna enters plant dna and integrates
- plant cell transcribes dna
- makes phytohormones (forms tumours)
- Makes opines (makes nutrient for bacteria)
206
A tumefaciens uses in bioengineering
Prepare Ti plasmid in lab with desired genes, remove tumour genes
Transfer to plant
Changes plant properties
207
Phosphorelays
More complex version of TCSs
Phosphate transfer protein to protein
More than 2 proteins = more regulation
208
Sporulation Phosphorelays
Endospore formation highly regulated
Each step controlled via different factors
Ensures formation only when necessary
Irreversible once started
209
When does sensing not impact transcription
MCPs
Only alters direction
210
Quorum sensing
Sense population density
High enough = change gene expression (ie make biofilm)
211
Quorum sensing functions
Virulence factor production
Biofilm formation
Competence
212
Autoinducers
Signalling molecules
Enough = enough cells to express
213
QS and luminescence high cell density
Proteins from lux operon —> light
Regulated by LuxI and LuxR
High cell denisity = high [] autoinducers
AI bind luxR
LuxR binds promoter
Recruits RNAP
Lux proteins produced
Cells emit light
214
QS luminescence low density
Low [] AIs
Won’t bind LuxR
Won’t bind promoter
No transcription
No light
215
QS and EHEC
Genome has pathogenicity island
Encodes T3SS
Effector secreted into host cells
Bin to protein on EHEC surface
Remodels host cytoskeleton
Releases Shiga toxin
Regulated via TCS
216
Probiotics and EHEC
QS = potential anti virulence factor
Probiotics contain Lactobacillus spp
That can interfere with EHEC AIs
