CC9 - attack and defence in bacteria

Why is it important to study bacteria?

• they dominate the tree of life

• serve as good model organisms

• humans have only ever existed with bacteria we have evolved with them and a re dependent on them - eh. for diegestive processes, immune system, pathogen invasion protection

example of how some bacterial species can be beneficial and pathogenic?

• eg. E.coli

  • many live in the gut - beneficial

  • but can also give you illness

  → same species but different strains

• Change caused by alterations in body location or acquisition of pathogenicity genes

describe gram positive cell envelopes

• upon treatment of gram stain, stain purple

• Thick, many-layered cell wall.

  • often 40-80 layers

• Single membrane containing proteins based on helical architecture

  • proteins are mostly helical architecture

  • cell wall sits on the outside of the cell membrane - exposed and facing the external environment

• example: staph aureaus

describe gram negative cell envelopes

• Upon treatment of gram stain, stain red

• Thin, cell wall (typically 1-3 layers)

• Two membranes

  • OM contains proteins based on beta barrel architecture

  • IM contains proteins based on helical architecture

  • sandwiched between the two is the cell wall - can be between 1-3 layers

• Example: E.coli

why do gram positive and negative bacteria stain differently?

→ reason for the colour differences - gram positive retains the stain (because the cell wall is very thick - many layers), whereas gram positives do not (because only a single layer)

components of the gram negative cell envelope?

• outer membrane

• periplasm

  • aqueous layer containing the cell wall

• inner membrane

describe the components of the outer memrbane?

• lps in the outerleaflet

• phospholipids in the inner leadlet

describe the LPS in the outerleaflet of the gram negative OM

• this is a complex molecule containing between 4-6 lipid tails which are connected by 2 sugars to form lipidA

  • these 2 sugars are phosphorylated

  • lipidA can cause TSS

  • when LPS is shed by bacteria, the lipid A portion is exposed. this activates the toll-like receptor signalling pathway, leading to sepsis and TSS

• there is a further O-antigen region

  • lab strains lack this, but wild strains have it

  • in the wild, there are layers of sugars

    • these vary between species

    • use these to recognise eachother

    • some of these sugars can also be phosphorylated

• because many of the sugars are phosphorylated, they are negatively charged

  • the packing is mediated by the presence of Mg2+ and Ca2+

  • this makes tight electrostatic interactions, minimising the movement of LPS

    • antibiotics can find it difficult to penetrate this

• LPS moves very slowly

  • due to the electrostatics

  • also lots of tails that pack togetjer - this packing increasing VDWs

  • LPS itself is very big and bulky

describe the phospholipids in the innerleaflet of the gram negative OM

• different combinations of types - eg. lengths of tails, head groups, double bonds

  • always phospholipids

    • phosphatidyl glycerol

    • phosphatidul ethanolamine

    • cardiolipin

how is the peptidoglycan cel wall anchored to the OM?

• contains a direct covalent link via the brauns lipoprotein

  • covalently linked to the cell wall on one side, and on the other side facing the membrane, it is acylated, allowing anchoring into the phospholipid region of the inner region

  • tethers the cell wall to the inner leaflet of the outer membrane

inner membrane components of the gram negative inner membrane?

• At the inner membrane, we get phospholipids

  • phosphatidyl glycerol

  • phosphatidul ethanolamine

  • cardiolipin

  → same as the inner leaflet of the OM

functions of the PG cell wall in gram negative bacteria?

Preserves integrity, withstands changes in turgor pressure in the cytoplasm, eg. osmotic pressure - helps the cell adapt and keep its shape

components of the peptidoglycan cell wall?

• Made of long strands of sugars cross linked by peptides

  • Glycan strands run perpendicular to the long axis of an E. coli cell

  • Crosslinking peptides are between 5-7 residues

  • made of disaccharide units of GlcNAc and MurNAc

describe the variation in cell walls between species

• Variation between species is from the peptides

  • peptides in different species have different sequence

    → Much of the variation in peptidoglycan across species comes from the peptide moiety

structure of the peptidoglycan cell wall?

• Glycan strands are built from alternating N acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues connected by a β-1,4 glycosidic bond

  • made from alternating GlcNAc and MurNAc residues connected by a B1-4 glycosidic bond

  • get 20-40 of these per strand

• Peptide crosslinks often contain L- and D-amino acids (usually Ala and Glu) & diaminopimelic acid

• Cell wall is porous

  • pores are thought to be 5-10nm in size in E.coli

  • pores are not likley the same size - not sure on their distribution

evidence for structure of teh peptidoglycan cell wall?

• AFM and cryoET images provide some insights into organisation, but at fairly low resolution

  • emerging cryoET data

  • resolution is low - can get an idea of the uniformity - not much

  • AFM is difficult - squidgy, need to go through the OM to get to it

    • in AFM, get height data - to go throguh the layers of sugars, this is tough

    • have now isolated the entire intact sacculus, but this could change the distribution

2 main steps in cell wall synthesis?

• synthesis and transport

• polymerisation

describe the process of syntehsis and transport of peptidoglycan precursors

• UDP-MurNAc is generated by enzymes MurA and B

  • MurNAc - one of the sugars

• We then use different enzymes to sequentially attach amino acids to this UDP-MurNAc

  • Mur ligase enzymes (MurC-F) attach these amino acids

  • each enzyme adds amino acids once we produce UDP-MurNAc-pentapeptide

    • this contains 5 amino acids

→ this all occurs in the cytoplasm

• MurNAc-pentapeptide is then transferred from UDP to Undecaprenyl Phosphate by MraY

  • this contains a phosphate region and a single lipid tail

  • this is in the inner leaflet of the inner membrane

  • MurNAc pentapeptide is transferred from the UDP to the Und-Phosphate

  • this produces lipid-I

    • MurNAc-pentapeptide—Und-phos anchored into the inner leflet of the inner membrane

    • this only contains 1 glycan - need to add one more to form the complete precursor

    • need to add on GlcNAc

• MurG transfers GlcNAc from UDP-GlcNAc to lipid I, generating lipid-II - the final precursor molecule

  • this contains the disaccharide pentapeptide - this is the building block of PG

• But lipid-II is in the inner leaflet of the inner membrane, need it to be on the outer leaflet, exposed to the periplasm

• MurJ, a flippase flips lipid-II to the outerleaflet

  • now it is exposed to the periplasm, and so different enzymes can polymerise

• This is changed by MurJ - flippase

  • flips lipid-II into the outerleaflet, so not exposed to the periplasm, and so different enymes can polymeroise

describe the process of polymerisatio of the peptidoglycan cell wall

• Polymerisation and cross linking of lipid-II by various enzymes to form the PG matrix

  • process relies on PG synthase enzymes

    • eg. class A penicillin binding proteins

      • have a large extracytoplasmic domain

      • have polymerase and transpeptidase activity - can both polymerise and transport the precursors

  • Once lipid-II is flipped into the periplasmic leaflet, the PGTases can attach the non-Und-P portion of lipid-II to the existing PG polymer

    • existing polymer is sequentiallly extended

    • the lipid region will come off as it is attached to the growing polymer

  • This forms the 4-3 cross links

    • these enzymes are the target of penicillin and other B-lactam antibiotics

      • covalently modify the active site, stop-ing cross link formation and stopping cell wall formation

  → the cell wall is continuously synthesised - grows with the cell

what systems can be used to produce different OMP components?

• Bam

• Lol

• Lpt

function of the Bam system?

Bam system inserts newly formed B-barels into the OM

function of the LOL system?

• LOL system inserts lipoproteins

function of the Lpt system?

LPS is synthesised in the cytoplasm, LPT is the transport machinery to move to the outerleaflet of the OM

structure of the Bam system?

• BamA is a B-barrel in the OM

  • associated with it are lipoproteins - B, C, E and D

  • potra domains are soluble proteins, sitting the in periplasm and helping assembly of the B-barell complex

  • relies on the SEC system

describe why chaperones are needed in the Bam system?

• chaperones bind and deliver the potra domains and into the bam system

  • the chaperones are needed!! - without them, the hydrophobic unfolded proteins will aggregate

what do the C terminal BamA potra domains do?

• C-terminal BamA potra domains make contact with the other bam subunits - these are lipoproteins

  • BamD is essntial - binds the unfolded protein

  • A and D are conserved

how is the structure of the Bam complex optimised for function?

• In BamA, there is a lateral gate between strand 1 and 16

  • allows to open up

    • evidence - various forms in crystallisation - by itself or in co-crystallisation

  • water filled lumen

• at the top of the barrel, loops form a closure over the top of the barrel

• Potra domains act as scaffolds for lipoproteins

  • optimised electrostatics for these interactions, this helps the scaffolding

• Lipoproteins B-D form a ring around the BamA in the membrane

  • captures the unfolded proteins and funnel them into BamA

• BamD recognises the B-signal of the OMP and binds the unfolded OMP

  • others do not recognise! - only invovled in folding

• Lipoproteins and POTRA domains all help to regulate access to the central cavity of BamA

• Lateral gate opens and closes to regulate access to membrane, and the other regions also have a role

how is BamA structure used for folding?

• Membrane spanning B-strands are much shorter on the side of the lateral gate (1 and 16)

  • this is becuase there is a gate that is opened or closed

    • opening of the gae is essential

      • artifical closure via cross linking prevents protein folding

  • fewer H bonds between these shorter1 and 16 strands makes it thermodynamically easier to open the gate

  • the gate opening is controlled y the ring of lipoproteins that sit beneath

    • asymmetry may destabilise the membrane close to BamA

BamA assisted model for OMP foldign?

• BamA plays an indirect role

• distorts the membrane bilayer, reducing the kinetic barrier for pre-folded OMP insertion

  • OMPs are folded and then the membrane becomes disorted, helping insertion

• in vitro folding studies support

BamA budding mdoel for OMP folding?

• acts as a template for folding

• BamA opens at the Lateral gate

• B-hairpins associate with the LG - strands B1

• beta hairpin associates with B1

  • this forms a larger barrel formign

  • new hairpins come along and H bond to the existing gate

• evidence: structures of half inserted barrels

• not sure how this is released into the membrane once formed

how does the LOL system deliver lipoproteins?

• LolCDE transporter transports lipoprotein from synthesis site and delivers into the periplasm

• LolA captures it

  • this has a hydrophobic cavity

  • lipid tails get caught in this cavity

  • carries it towards LolB, anchored in the outermembrane

• LolB also has a hydrophobic cavity, takes the protein

  • affinity for LolB is higher than LolA

  → no energy!! have to go downhill - favourability is from the higher affinity from LolB

why does lipoproteins need to be transported?

Lipoproteins have a variety of functions, including maintaining attachment to peptidoglycan, (covalent and non-covalent, assembly of OMPs, biogenesis of LPS, regulation of peptidoglycan synthesis

describe LPP?

• Braun’s lipoprotein (Lpp) is the most abundant protein in E. coli

  • It is covalently bound to peptidoglycan at its C-terminus (the only known covalently bound protein to the cell wall).

  • Anchors the outer membrane to the cell wall

• It is triacylated at its N-terminus

  • has 3 lipid tails allowing anchoring into the inner leaflet of the OM

• We know now that it is directly involved in maintaining the width of the periplasm

  • can artificially lengthen the protein, find that the periplasm length is altered

what system mediates LPS delivery?

Lpt system is used

describe the Lpt system?

• IM and OM regions, as well as a periplasmic bridge linking the two

  • complete protein link from the inner membrane to the outer membrane

evidence for the role of Lpt system in LPS transport

• LPS is bound in the structure, to the inner membrane part so we know the orientation it binds in

• Problem - periplasm is aqueous, 6hydrocarbon tails need to be moved

• jelly roll region - tails are inside the groove and the polar sugars are exposed into the periplasm (aqueous)

  • move through the groove until it reaches the barrel at the top

  • continuous groove aids transport

new idea of the distributioins of proteins in the e.coli OM?

• Proteins and lipids are not uniformly distributed across the surface of the membrane

• In the past, view of thew OM was uniformly but randomly distributed proteins across the survace

• AFM as showed regions with no proteins, and other regions of high protein localisation

• OM surface may contain islands of OM proteins connected by trimeric porins, such as OMPF and OMPC

why do bacterial competition systems need to be agressive?

• they need to be this agressive because they live in crowded communities, occupying niches in which nutrients is scarce

  • Bacterial competition systems: bacteria try to outdo eachother using competition

  • because of this, they need to have sophisticated defence mechanisms too

how can competition be done in bacterial defence systems?

• exploitation

• interference

how can exploitation be done in bacterial defence systems?

• Consuming limited resources to induce starvation

  • e.g. bacteroides vs citrobacter

how can interference be done in bacterial defence systems?

• Interference through metabolite production that downregluates virulence gene expression

  • this interferes with protein expression in the competitor

• interference through deployment of antibacterial weaponry

  → more sophisticated

  • bacteriocins

  • CDI

  • T6SS

what are contact dependent methods of bacterial attack?

has to make physical contact with the other one to cause target death

what are contact independent methods of bacterial attack?

no physical contact is needed

examples of some contact dependent weapons in bacteria and how they work?

• T4SS translocate DNA and proteins in Gram negative bacteria.

• T6SS in Gram-negative bacteria allow direct delivery of toxins into competitor cells

  • uses a repurposed contractile phage tail

  • genetically modified to become a weapons

• CDI systems involve a filamentous protein containing a toxic domain and a second protein responsible for export and anchoring to the cell surface of the attacker. Upon contact of the filament to a target cell, the toxic domain is translocated into the victim.

  • toxin on a stick - filamenrous proteins are out of the cell, hit the target and induce death

• Nanotubes create a direct bridge between the two cytoplasms

  • not much known about this

• Outer membrane exchange (OME) mechanisms involve poisoning non-immune neighbours by transferring toxin-containing outer membrane fragments upon cell–cell contact.

examples of contact independent weapons in bacteria and how they work?

• Protein toxins are released often by cell lysis, allowing them to diffuse to target cells.

• Membrane vesicles are produced by diverse bacteria and can kill other cells by, for example, delivery of enzymes that digest the cell wall.

  • The vesicles deliver many molecules, however, and the importance of vesicle production for bacterial competition needs further verification.

• Phages include many viruses integrated into bacterial genomes that when released will kill competitors but not clonemates that also carry the virus.

what different cytotoxic effectors can be delivered in bacterial defence systems?

• nucleases

• pore forming domins

  • make holes in the membrane, causing cell contents to leak out

• PG hydrolases

  • targeting the cell wall biosynthesis

  • beraks the cell wall down

what do organiusms producing weapons produce?

• Organism producing the weapon will also produce immunity proteins

  • this means that when their own kind deploys a weapon, cell will be fine as the immunity protein will neutralise the protein

  • other species will not have this immunity protein

describe the type 6 secretion system

An indiscriminate toxin delivered by a syringe that can be fired multiple times.

mechanism of the T6SS?

• Very very long!!

• Goes right into the cell!!

• Has a sharpened spike at the tip made of proteins

• Has a baseplate assembled to sit undernearth the inner membrane, providing stability

• sheath around the tube is made up of proteins

  • this provides a driving force for the movement and rotation of the tube as it contracts

• when contracted and pushed out, it hits the OM of the target

  • this pierces the OM

    • if gram negative, needs to get through the LPS

    • takes a lot of force to puncture

  • has a range of toxins that can be delivered

  • top region comes off and the poison is delivered

• sheath region is disassembled and recycled

what are the properties of T6SS allowing for contraction>

• When contraction occurs, many favourable PPIs form at once

  • this releases lots of energy

  • Sheath contraction involves increase in VipAB subunit interactions- releases 1000s kcal/mol free energy

what occurs in the T6SS mechanism to deliver the tocin?

• The injection syringe contracts to less than half of its original length in under 5 milliseconds.

  • very fast!! must be a very big driving force

  • Estimations: sheath may undergo 10 rotations during this time

  → sheath is both rotating and contracting - lots of movement

  • getting shorter and going roound

how does contact dependent frowth inhibtion work? how are these genes encoded?

• Waiting for the right target to come along and touch them

• Gene cluster is often encoded in pathogenicity islands

• CdiB.A is presented on the cell surface

  • CdiB is the transporter, a barrel, CdA is the toxin

  • likely arrangment

• CdiL is also produced, this is an immunity protein → prevents clonemate killing

• Recognise a variety of receptors

  • including BamA

  • not sure how the toxin enters the cell exactly

describe the mechanism of CDI in E.coli

• Toxin is initially half released

• Goes throguh CdiB as an unfolded protein and then begins to fold

• If the receptor binding domain contacts the correct receptor, such as BamA, full release occurs

  • until it constacts, it stops folding

• Contacts correct receptor → full folding → force

• Likely uses another barrel to get the toxin through

• We know the folding occurs in stages - need to hit the correct receptor for full folding

what are bacteriocins?

• Bacteriocins: Highly specific killers that will diffuse through the extracellular medium to reach their target cells

what fo bacteriocins typically ise for their ability to kill cells?

• usually use 2 different b-barels on the target memrbane

  • receptor they bind to

  • translator they go through

    → but the receptor can also be the translcoator

what systems do group A and B bacteriocins use

• Group A use the tol system. GroupB uses the ton system

  • both have stator/rotor proteins in the inner membrane

decribe the structure of nuclease colicoins

• Contain 3 domains

  • T domain, R domain, C domain

  • also have an unstructured N terminus and a tightly bound immunity protein

• All have the same overall organisation

  • killing domains are structurally similar

• unstructured region contains an epitope that binds the target syste

  • very big!! and need to get trhgouh!!

• contain a long region and a bulky region

• different domains are structurally different, producing an irregular shape

  • highly optimised to target very specific proteins

example of a bacteriocin?

colecin

mechanism of ColE9 bacteriocin?

• BtuB is a vitamin 12 transporter

• Colicin ColE9 finds and binds

• Also binds OmpF, a trimeric porin, threads through and loops back into OmpF once it has bound to TolB

  • TolB - soluble prtein in the periplasm, involved in the Tol system

  • physical connection between the tol system and the colicin

  • when rotates, this can pull ColE9 into the cell envelop

→ ColE9 is connected to the energised inner membrane via a tol system. Others use a similar mechanism to target the Ton system

• Effectors them het into the cell and do the killing

  • eg. ColE9 chops up bacterial DNA

how can colicins be used as molecular rulers?

• Can use them as molecular rulers

  • on the surface, there are islands of proteins - dont know the specific proteins, only that there is a protein

  • with colicins, can get information as we know how long the R domain is

  • if using OmpF and BtuB, these cannot be further apart than this legnth

    • not always this length, but sometimes will have to be

    • all of the colicins can be used as rulers to determine which proteins must be close to which

    → used as indirect methods

why study the bacterial cell envelope?

• Connects the bacterial cell to the outside world

  → everything needs to pass through this envelope

  • information

  • resources

  • weapons

• Definition of self for a bacterial organism

  • physical barrier, defining it relative to its environment

  • community interactions

  • defines the cells identitiy - in mixed communities and populations, this tells other cells what this cell is

why does studying the bacterial cell envelope need specialised methods? give 3 reasons

• Size and scale

• Diversity and heterogeneity

• Different context

why does the size and heterogeneity of the bacterial cell enevelope make it difficult to study?

• too many different components and heterogeneity to look at components in isolation

why does the behaviour in different contexts of the bacterial cell enevelope make it difficult to study?

context in which you are studying introduces different questions to ask

describe the size and scal of the bacterial cell envelope

• Most bacterial cells are on the order of 1-2µM

• some are orders of magnitude larger

• Span a huge range of cell sizes

• In the cell envelope, there are different components that have different scales

→ we need methods that can probe these different scales and organisaiton

descrbe the diversity and heteroheneity of cell envelopes

• Cell envelopes are chemically complicated

• Different classes of bacteria use different classes of enzymes to build them

• Gram negative - two membranes, periplasm

  • LPS can shield from immune system

• Gram positives - cytoplasmic membrane and extended cell wall provides structure and protection

example of a vastly heterogenous cell envelope

• mycobacteria have a ‘hybrid’ approach to cell envelope

• Have a cytoplasmic membrane

• very extensive cell wall with coomplicated glycans

• also have a very odd outer membrane, larger than the traditional one with long lipids

examples of why do different contexts alter the cell envelope?

• biofilms - collections of different bacteria growing together under a protective surface secreted through the cell envelope

  • here, may want to find other bacteria to establish this

• Predatory bacteria - target and eat other bacteria. Eg. dello vibrio - establish their life cycle in the periplasm of their target, sucking the cytoplasm out

  • Need a cell envelope to protect them inside the other bacterial cell

what methods are commonly used to visualise bacterial envelopes?

• Light and fluorescence microscopy

• Atomic force microscopy

• Electron cryotomography

• Native mass spec

• Molecular dynamics

main problem with light microscopy for bacterial envelope visualisation

• Wavelength of visible light is on the same order of magnitude as the size of most bacterial cells

  • this means we are diffraction limited to see the cell size

    • cannot study anything smaller than a single cell with light microscopy

    • cannot see subcellular organisatoion

why can fluorescence microscopy be good for cell envelope stidies?

• Fluorescence microscopy can provide contrast

  • can provide contrat

  • lots of dyes and stains recognise different chemical components of the cell envelope, so we can see them partitioning

what advanced fluorescence mocroscopy techniques can be used to study bacterial cell envelopes?

• PAINT

• SMLM/STED

• Single moelcule tracking

example of the use of STED in studying bacterial cell envelopes?

• Study used STED to follow cell division, and see how the cell wall and Slayer are built at different times in cell division

  • Not really time resolved, have to fix at different time points

example of the use of single molecule tracking in studying bacterial cell envelopes?

• SM tracking of cytochromes that exist on the bacterial cell surface

• Can watch movement

• Can see cytochrome jumping between cells

  • indicates some sort of communication between cells of the same species using cytochromes

process of AFM?

• Scanning the surface of the sample with a cantilever

  • this has a fine tip

  • provides nm resolution

  • as we scan this across, the laser is used to measure the deflection of the cantilever

    • movement up and down

what components can be measired in AFM?

• can measure the topography - the surface and arrangment of the cells and cell surfces

• can also measure reaction forces

  • how sticky something is

how can you measure adhesion and stickiness using AFM?

• can pull on it and measure force

  • look at different parts of the experiment

  • bring the tip down, and as you start to pull you can measure:

    • adhesion

      • this is the resistance to pulling

    • stiffness

      • how far it can be pulled furhter

what does AFM allow that other techniques fail to?

beuase this can be done on live cells, we can track changes in the outermembrane on a time resolved manner

example of the use of AFM in revealing OM OMP structure?

• Can look at porin organisation in the OM of gram-negative bacteria using AFM

• Can use colicins - genetically modified to have an m-Cherry

  • to be active, needs to be able to pass through an OMP and into the cell

  • mCherry cannot pass into cells - plugs the pore

• Dots seen in the images are M-cherries blocking a pore. The circles seen are individual trimeric porins

• gives high resolution information - localisation of individual porins

• also gives a large scale - can see how this localisation maps across the cell

how do we prepare samples for cryoET?

• Need thin samples - bacteria are slightly too thick

• Try and image in a single cell layer and then use a dual beam SEM

  • has a scanning SEM and a gallium beam

    • this gallium beam cuts the top of of all cells, thinning them si that they are thin enough, allowing us to see through

    • keeps cells frozen, just removes a small region

  • leaves a very thin layer of the cell

• Imaging biomolecules in ‘native hydrated state’ with no stain or preservation artifacts

why are thin samples needed for cryoET?

As samples get thicker, electrons scatter more than once, leading to loss of coherence in imaging

how do we do FIB for studies of bacterial cells?

Gallium ion beam blasts thorough the specific regions, leaving a very thin layer

how does specimen damage limit SNR in cryoET?

• Issues with radiation damage in tomography due to the use of high energy electrons

• In principle, good at spanning space scales from most of the cell → individual proteins

  • because we are limited by radiation damage, there are tricks we use to look at protein structure

how are SNR limitations overcome in cryoET

Instead of taking lots of flat images, we box out all of the copies of the protein of interest inside of the cell

• Then average together via subtomograph/subvolume averaging

example of the use of cryoET in combination with structural techniques to aid structure determination of the S-layer

• Determining the structure of the S-layer

• Can take the stalk on the same colobacter cell

• see repeating structures in the tomograms

  • these can be boxed out as individual volumes and averaged to produce a structure

• can then take single particle/XRC/NMR structures to dock in and show how this wraps the entire cell

what does native mass spec study?

• Instead of fragmenting the protein, we keep them whole, in complex with binding proteins

• We use mass measurment to determine proteins, stoichiometry and the presence of specific lipids

how does native mass spec work?

• Typically purify a region of the membrane and then process using electrospray ionisation

• This slowly dehydrates the protein, removing water and producing only the protein/complex/lipids/detergents

  • this is then put into the mass spec

• can activate with collisions or voltage to remove things

what can activation in native mass spec do?

• can use activation to strip detergents

• as you increase the removal , get cleaner mass spec information

• but you loose information about binding partners too

• you typically compare the different spectra when you activate/do not

what has native ms revealed about the OM?

• native MS along with cross linkinghas shows that different OMPs associate with eachother in the OM

how can native MS be used directly from bacterial membranes

• Can also take regions of the cell membrane

• lyse cells

• separate membranes

• in order to switch closed vesicles into a mass spec friendly buffer (if too many ions about, low resolution spectra), we need to transfer into a volatile buffer

  • ions need to come off easy

  • usually ammonium acetate

• Low power sonication temporality breaks open vesicles, allows the buffer to exchange and then reseal

• Then use ESI on these vesicles

• Use collision induced dissociation to break these open in the gas phase

• gas phase is good at preserving protein structure

  • there is no water, so the proteins wont change shape

    • good to preserve complexes

describe the fields of view for MD of bacterial cell envelopes and why this happens?

• Very small field of view

• Many molecules need to be simulated for a specific region

  • Many molecules need to be simulated due to the complexity of the cell envelop

• Very difficult

• Need to be able to scale up and look at large or multiple proteins within a context

how can we scale up systems for MD of outer membrane?

Use coarse graining for large systems

how can LPS be modified in MD to allow for longer simulations?

• LPS is very big and chemically complicated

  • Contains a collection of lipid tails

  • phosphate head group

  • sugars

  • complicated LPS

• we can truncate LPS at different points in the chemistry to simplify into specific groups

  • only coarse grain some regions, but not others as not important for protein interaction

• alternatively, can coarse grain regions of the LPS closest to the membrane

• coarse graining allows simulation of much bigger patches of LPS and porins

how can we alter LPS-LPS interactions in MD to reduce somulation times?

• LPS-LPS interactions are quite strong, so not a very mobile system

• so if you want to look at something changing, need to artificially increase the mobility to do it in a reasonable time

• decrease the effective strength of these interactions, so they are likely to separate in the simulation

how can MD complement wet lab experiments for bacterial envelope stidoes?

used a combination of AFM data and structural single particle data to produce a simulation of protein interactions

is antibitoic resistance widespread? evidence?

• yes

• Whole genome sequencing has been useful to look at mutations and the spread of resistance strains across the world

• It is very widespread!!

• with travel, the problem is getting worse!!

main types of antibiotics?

bacteristatic

bacteriocidal

bacteriostatic antibiotics?

• dont kill the bacteria itself

• stunt growth, preventing reproduction

• this generation is still alive, wont develop fully

bactericidal antibiotics?

kills the bacteria itself

why are some antibiotics not successful for treatment of gram negative bacteria?

• this is because many antibiotics are too large to go through porins

• it is also unlikley they they can traverse LPS - must be through proteins

• if dont have a hole big enough, they will be excluded