Midterm 2 Flashcards
Main difference gram neg vs positive
Neg has outer membrane
Cytoplasmic membrane has what
- lipid bilayer
- semi permeable barrier
What determines the lipid composition of cytoplasmic membrane
Conditions
Ie. temperature
Functions of membrane proteins
- transporters
- signal transduction
- energy transduction
Energy transduction example
Electron transport chain
How do bacteria survive hypotonic conditions
Peptidoglycan stretches with pressure and prevents lysis
- porous = allows transport
How do bacteria survive pep degradation
Isotonic condition
- lose shape
- still stable
Spheroplasts
Gram negative without pep
Protoplasts
Gram pos without pep
How do mycoplasmas survive without cell wall
Uses sterols from host to stabilize membrane
Why is pep a good antibiotics target
Not made by humans
On outside of cell
Made by most bacteria
How does bacitracin target pep
Binds undecaprenyl, prevents dephosphorylation = no phosphate = no binding
How do beta lactams target pep
PBP transpeptidase = no cross linking = weakened Pep = lysis
Pep structure
Glycan backbone
NAM
NAG
NAM has peptide chain
Making pep
- 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
What does undercaprenyl phosphate do
Provides phosphate groups to detach UDP and attach NAG
What does cycloserine do
Blocks alanine racemase = no d-ala = incomplete chain
Undecaprenol recycling
Flips between periplasm and cytoplasm depending on number of phosphates
Lysozyme is part of what immune system
Innate
Where is lysozyme
Saliva tears milk mucous
What does lysozyme do
Cleaved NAG-NAM bond
Therefore weakens cell wall
Why is lysozyme more effective on gram pos
Pep more exposed
No outer membrane
Order of pentapeptides in pep
L-alanine
D-isoglutamate
Diamino acid
D-ala
D-ala
Cross linking pep gram neg
3 and 4
Release terminal d-ala
Cross linking gram pos
Interpeptide bridge between diamino and first d-ala
Terminal d-ala released
How are cross links formed
PBP transpeptidase
- Forms complex w peptide
- Diamino reacts
- Amide bond forms
What are b lactamases
Enzymes degrade b lactams
Resistance mechanism
Serine b lactamases
Serine binds to lactam = hydrolysis = inactive lactam
B lactamases inhibitors
Co prescribed with b lactams
MRSA and b lactam resistance
Has a PBP gene resistant to lactams
[active site blocked until bound to PB]
How does vancomycin target pep
Only targets gram pos
Binds to d-ala d-ala = blocks PBP
Vancomycin resistance
Replaces d-ala with different d amino acids = vanomycin cant bind
Gram neg outer membrane
Outer leaflet and inner leaflet
Gram neg outer leaflet
Lipopolysaccharide (LPS) only in negs
Inner leaflet gram neg
Phospholipids
LPS function
Impermeable
LPS traits
- neg charged
- amphipathic
- bulky
LPS structure
Lipid a
Core polysaccharides
O antigen
Lipid A (LPS) function
Anchors LPS to membrane
Lipid A (LPS) structure
Sugars with fatty acids
Phosphorylation = negative charge
Lipid A (LPS) toxicity
Releases endotoxin when lysed
Fever inflammation, septic shock
Core poly (LPS) function
Links lipid A to O antigen
Core poly (LPS) structure
Sugars
Branches
Anionic sugar
Neg charge
O antigen (LPS) function
Classification
Antigenic (bacteria changes this to avoid immune response)
Divalent cations Outer membrane gram negative
Stabilize
Bridge LPS molecules
Neutralizes electric repulsion
Gram neg outer membrane function
Barrier
Prevent antibiotics
Prevents degradation enzymes (too large to pass)
Resistant to detergents
Why are gram negs resistant to detergent
Outer membrane - LPS
Steric and charge
What are divalent cations needed for LPS
Cross-bridging adjacent LPS molecules
What happens limited Mg LPS
Lipid A may be changed to 4AA to cross bridge
Colistin
Cationic antibiotic
Binds to lipid A phosphate groups
Lipid tail can permeate membrane
How do Mg levels impact colistin
Mg sensitive to colistin
4AA prevents colistin binding = resistance
MCR-1 gene
Colistin resistance gene - HGT
Binds smth = positive charge = repels cation colistin
Outer membrane proteins gram negative
Lipoproteins
B barrel proteins
Porins
Nutrient intake gram neg
Form channels
B barrel
Water filled center = selectivity
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
Vesiculation
Vesicles can form when outer membrane of gram negative not attacked to pep
Brauns lipoprotein
Fatty acid chain embedded in outer membrane gram neg
COVALENTLY BOUND TO PEP
Teichoic acids are in what
Gram pos cell wall
Teichoic acids
Linear polymers - glycerol or ribitol
- may have substituents (pos charged)
Phosphate groups neg charged
2 Teichoic acids
Wall Teichoic acids (WTA)
Lipteichoic acid (LTA)
Wall Teichoic acid
Attached to NAM or peptide in pep
Extends beyond pep surface to environment
Starts within pep
Lipoteichoic acid
Attached to lipids in cytoplasmic membrane
Extends through pep to environment
Teichoic acids function
- anchor wall to cytoplasm membrane
- binds cations = less repulsion
- regulation of pep degredation during division
- d-ala protects antibiotics and immune
Teichoic acids infections
Pathogensis
- biolfilm
- colonization
- inflammation upon release
Mycobacteria staining
Acid fast staining
Heat cells with stain
Mycobacterial cell wall
Gram pos but have outer membrane
- mycolic acids not LPS
Arabinogalactan
- sugar polymers
Pep
Mycobacteria outer membrane
Asymmetrical bilayer
- inner = mycolic acids
- Outer = glycolipids
Hydrophobic + impermeable
Cells need what 3 things
Energy electrons carbon
Heterotrophs
Organic molecules for carbon
Autotrophs
CO2 for carbon source
Reducing power
Electrons
Needed for
- anabolic reactions
- making atp
Organotrophs
Reduce organic molecules
Lithotrophs
Reduced inorganic molecules
Chemoorganoheterotrophs
Chemo = energy not from light
Organo = reduced organic molecules for electrons
Hetero= organic source of carbon
Most bacteria are what metabolic classification
Chemoorganoheterotrophs
How do Chemoorganoheterotrophs make atp
Oxidizing organic molecules
- aerobic
- anaerobic
- fermentation
Aerobic respiration
When there is adequate oxygen
Glycolysis : glucose —> acetyl coA
Krebs cycle: acetyl coA —> ATP, NADH, FADH2
ETC: NADH/FADH2 —> ATP
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
Kerbs cycle
Acetyl coA —> CO2 + GTP, NADH, FADH2
ETC
Membrane bound electron carriers: Ubiquinone (coenzyme Q) and cytochromes
Carriers reduced via oxidation
ETC in E. coli
- NADH electrons via ubiquinone
- pass through cytochromes
- to terminal electron acceptor (O2)
- proteins to periplasm = proton motive force (PMF)
Proton motive force + ATP synthase
Proton gradient
- cytoplasm = neg
- protons flow from periplasm to cytoplasm via atp synthase
Anaerobic respiration
- Glycolysis
- Krebs cycle
- ETC
** terminal electron receptor is not O2
Could be nitrate, surface, CO2 etc
Ex. NO2- —> NO —> 2NO —> N2O —> N2
Fermentation
When lacking or repressing ETC
- No ETC = still have NAHD
Fermentation = NADH —> pyruvate + NAH+. —> new products
Ex. Ethanol, lactic acid, CO2
Why is it hard to target metabolic activity of bacteria
Most bacteria = Chemoorganoheterotrophs
Humans = Chemoheterotrophs
Tetrahydrofolate
Co factor needed to make purines and pyrimidines and methionine
Importance of tetrahydrofolate antibiotics
Bacteria must produce it
Humans must eat it
Bio synthetic enzymes = targets
Sulfa drugs
Inhibits dihydropteroate synthase (early precursor to tetrahydrofolate)
Competitive inhibitor
Trimethoprim
Inhibits dihydrofolate (can’t reduce to become tri)
Passive diffusion
Moves with gradient across membrane
Facilitates diffusion
Transport proteins aid in transportation across barrier in vesicle like manner
Active transport
Against gradient, needs energy
Primary active transporters
Use ATP to transport against gradient
ATP binding cassette transporters (ABC)
Primary active transporters in bacteria
Import and export
Solute binding proteins
Work with most ABCs
- deliver specific substrate to transported
Where are SBPs in gram neg
Periplasm
Where are SBPs in gram pos
Lipoprotein or pep
Secondary active transport
Ion gradient potential energy transports against gradient
- ETC
- V type ATPase
- Antiporter
V type ATP ase
Reverse of ATP synthase - uses ATP to send them against
Antiporter
ETC generated proton motive force (PMF)
H+ down gradient powers Na+ up gradient
Na+ gradient can then power symporter
Group Translocation
Active transport with modification of substrate
Phosphotransferase system (PTS)
Group translocation
- sugar across cytoplasm membrane
- sugar phosphorylated during transport
- P from PEP —> PTS —> sugar
Outer membrane transport porins types
General
Substrate specific
B barrel proteins
General porins
Channel size = which substrates can enter
Substrate specific porins
Binding site attracts substrate, size impacts selectivity
Ton B dependent receptors
- Receptor inhibited
- Binding to receptor
- Exposes ton B box
- Box binds to Ton B
- Ton B removes plug from receptor
Why do bacteria need iron
Cytochromes and enzyme co factors
How do bacteria find iron
Siderophores
Siderophores
- Bacteria secretes Siderophores
- Bind to Fe3+
- TonB receptor transports into periplasm
- Binds to SBP
- To ABC
- To cytoplasm
Positive taxis
Move toward stimulus
Negative stimulus
Move away from stimulus
Chemotaxis
Move away/towards attractants or repellents
Flagellar swimming
Rapid rotation of flagella to move in liquid environments
Atrichous
No flagella
Monotrichous
Flagella on one end
Lophotrichous
Multiple flagella at one or both ends
Amphitrichous
One flagella at both ends
Peritrchous
Flagella all over
Flagella structure
Basal body
Filament
Hook
Basal body
Attaches flagellum to cell envelope
Has motor
Filament - flagellum
Helical extending from cell
Rotation moves cell
Hook - flagellum
Transmits rotation from basal body to filament
Basal body structure
Protein structure - central rod
Rings - L,P,MS,C
Basal body function
Export subunits during assembly
Rotate hook and filament
basal body motor
Rotation powered by PMF
Switch determines direction ( C ring in cytoplasm)
Flagella + innate immune
on surface = target
Flagellin = toll like receptor 5 (TLR5)
Flagellin binds TLR5
Activates transcription factor
Pro inflammatory cytokines produces
Flagella adaptive immune system
Antigenic structure
Immune evasion flagella
- alternate flagellins = phase variation
- stop producing once in host
Direction of flagellum rotation determines what
Run vs tumble
Monotrichous and peritrichous flagellum direction
Run = counter clock
Tumble = clockwise
Methyl accepting chemotaxis proteins (MCPs)
Chemoreceptors in cyto membrane
Diff ones sense diff attractants or repellents
Change direction of flagella
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
Tumble no ligand
- CheW detects no attraction
- CheW causes CheA to auto phosphorylate
- CheA phosphorylates CheY
- CheY binds to switch, changes direction to CW
- CheZ dephosphorylates CheY and direction —> CCW
No tumble, ligand
- CheW detects
- Does not cause auto phosphorylation
Therefore no switch
Excitation flagellum
Threshold must be met among many MCPs to determine how long direction will stay same or change
adaptation
After ligand binds:
MCP methylated by CheR
Increases CheA phosp
Increases amount of CheY
- removes CCW bias
Temporal gradients
Bacteria measure [] gradients for chemotaxis
Current = number of MCPs with ligands
Past = number of methylated MCPs
Allows bacteria to swim through gradient
What direction is swimming if Ligand [] increases
Swimming up
More ligands = more methylation (BUT DELAYED) = CCW bias
Fewer tumbles
What direction swimming if ligand [] decreasing
Down gradient
Less ligands = methylation SAME OR MORE = more tumbles
What causes MCP demethylation
CheB over time
What would happen if MCP did not demethylate
More binding wouldn’t equal CCW bias
Swarming motility
Flagella moving on surfaces
- coordinated movement
- requires multiple flagella and surfactants to reduce surface tension
Twitching motility
Pilli - moving on
Inches like catapilar
Extends: adds subunits to base (pillins)
Adheres
Retraction
Pillins removed
Colonize new environments
Glycocalyx
Polysaccharide layer around cell
Adherence
Related to EPS in biol films
Slime layer - easily remove from cell
Capsule - attached to cell
Capsule
Negative staining
Long polymers
Covalent bonding
Capsular polysaccharides
Repeating sugar subunits
Capsule functions
Adherence
Protection
- desiccation
- engulfment
- phage
- antibiotics
Major contributor to serum resistance
Capsule
Serum resistance
Ability to survive and spread in blood
How do capsules protect from antigens
Similar to host cell sugars
Mask antigen components
Conjunctive vaccines
Capsule polysach attached to immunogenicity protein carrier
- modified to evoke immune response
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
Capsule assemble gram negative methods
- Wzx-Wzx dependent pathway
- ABC transporter dependent pathway
Wzx Wzy pathway
- subunit assembled in cytoplasm
- flipped by Wzx
- polymerized by Wzy
- Translocated via Wza channel
- incorporated on cell surface
ABC transporter dependent pathway
- full polysaccharide made in cytoplasm
- transferrred to surface via ABC transporters
Non flagellar appendages
Pilli
Types of pilli
Type IV secretion pilli
Type IV pilli
Type III secretion systems
Chaperone-usher pilli
Type 4 secretion pilli
Sex pilli; conjugation
Type 4 pilli
Twitching motility
Chaperone usher pilli
Virulence factors - adhesion
Parts of chaperone usher pilli
Rod: major pillins
Fibrillum: minor pillins attach end adhesion to rod
Pilli pathogensis
Can only bind to specific molecules depending on the bacteria
UPEC (uropathogenic E. coli)
Causes most UTIs
Type 1 pilli attch to proteins in uroepithelial cells
Forms intracellular bacterial communities
UPEC and pyelonephritis
UPEC —> uterus —> kidneys
Must down regulate T1P production
Increase P pillus production
Immune response to pilli
Targeted
Can be used to make vaccines
How can pilli be used to make vaccine
Purify adhesions
Generate antibodies
Binds to adhesions
Prevents attachment
Antigenic variation
Change of pilli structure to evade immune response
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
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
Regulation of Pilus length
- PapH attaches pillus to cell
- also regulates addition of PapA
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
What happens if too much papH
Short pilli ( PapA can’t be added enough)
Gram positive pilli
Adhesion
Biofilm formations
Gram positive structure of pilli
Base Pillin attached to pep
Subunits covalently bonded
Adhesions at end
Long and flexible and thin
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
Exotoxins
Proteins secreted by pathogens
Lead to injection
Disruption host cells
Groups of exotoxins
AB toxins
Pore forming toxins
Superantigens
AB toxins
Interfere with internal processes of host cells
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
Diphtheria
AB toxin
Cardiac and nervous tissue
A blocks elongation factor = no translation
=no proteins
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
Pore forming toxins
forms channels in host membrane
=no gradients = cell swells = cell lyses
Hemolysins
Pore forming toxin
Lyses red blood cells
Bacteria steal iron released
How do pore forming toxins help bacteria escape phagosomes
Phagosome engulfs
Toxin released
Cell lysed
Can replicate in immune cells
Spreads through blood
Superantigens
Force binding of immune cells even when no antigen
=Overproduction of proinflammatory cytokines
= fever, organ failure
Ex. Staphylococcus aureus toxic shock syndrome toxin
Sec system - post translational translocation
Chaperons stabilize
Sec A binds signal peptide
Escorts to SecYEG (channel)
Uses ATP
Sec system: periplasmic proteins
Signal peptidase recognizes and cuts signal peptide
Chaperones help protein fold in periplasm
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
ABC exporters
Recognize signal protein
Cleave it off during export
Sec system - importing proteins to membrane
- signal peptide translated
- recognized by signal recognition particle
- translation stopped
- escorted to SecYEG
- translation continues into membrane
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
Gram neg secretion systems
Transport proteins out of cell
Type 5 secretion system (T5SS)
Tat or Sec
Protein Translocated via B barrel
**protein stays attached to cell (channel)
Type 1 secretion system (T1SS)
ABC transporter
Membrane fusion protein
B barrel
Steps
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
T3SS structure
> 20 proteins
Basal body
Needle
T3SS assembly
Subunits —> hollow central channel —> extracellular
Plug blocks channel after assembly
T3SS effectors
Binds target
Channel opens
Chaperones bring unfolded effectors to T3SS
Effector travels through T3SS
Folds in target
T3SS effector proteins
Target host cytoskeleton + signal transduction
Rearrange cytoskeleton of epithelial cells
Forms ruffles on surface
Allows bacteria to enter NON-PHAGOCYTIC cells
Protein secretion gram positive
Sec or tat
Sortase = attaches proteins to surface
Virulence factor
Nutrient acquisition
Immune evasion
Direct interactions with regulators
Stimuli directly affects transcription
Ex. Lac operon
2 component signal transduction systems (TCSs) parts
Sensing + response : different proteins
Sensor kinase: membrane protein
Response regulator: cytoplasmic DNA binding protein
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
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
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)
A tumefaciens uses in bioengineering
Prepare Ti plasmid in lab with desired genes, remove tumour genes
Transfer to plant
Changes plant properties
Phosphorelays
More complex version of TCSs
Phosphate transfer protein to protein
More than 2 proteins = more regulation
Sporulation Phosphorelays
Endospore formation highly regulated
Each step controlled via different factors
Ensures formation only when necessary
Irreversible once started
When does sensing not impact transcription
MCPs
Only alters direction
Quorum sensing
Sense population density
High enough = change gene expression (ie make biofilm)
Quorum sensing functions
Virulence factor production
Biofilm formation
Competence
Autoinducers
Signalling molecules
Enough = enough cells to express
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
QS luminescence low density
Low [] AIs
Won’t bind LuxR
Won’t bind promoter
No transcription
No light
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
Probiotics and EHEC
QS = potential anti virulence factor
Probiotics contain Lactobacillus spp
That can interfere with EHEC AIs
