Kahne, Daniel, Outer Membrane Synthesis in Gram Negative Bacteria, Harvard, April 4, 2018

Kahne, Daniel, Molecular Machines Building Membranes, Harvard, April 4, 2018

This is a stellar lecture!  complex topic made simple: how the

outer membrane of gram negative bacteria is constructed.

He is a chem prof. at Harvard;  lectures today in Clark Aud. at Stanford

at 4PM to a packed audience.

traditionally, a carbohydrate chemist...

mentored by the great Gilbert Stork.

best known for work  on outer membrane of gram negative bacteria.

and machines that  transport to that membrane.

nickname = Dan;

interested in natural products...

inhibiting bacterial growth...

this is a biochem talk in the Frontiers Series on Wednesday at 4PM in Clark Aud.

building the outer membrane of gram negative bacteria.

he begins....

while studying vancomycin;  we stumbled upon this

fundamental problem in bacterial physiology...

assembly of the outer membrane.

HC Gram  in 1884 !!!  (famous among student of medicine for his GRAM STAIN)

divided  bacteria into Positive or Negative with respect to his Gram stain.

many bacteria have an outer membrane.

Dan shows it on EM...

there are three concentric spheres:

the outer membrane and then the cell wall inside that and then the inner membrane.

cell wall is a peptidoglycan.

but outside that is another lipid bilayer.. the "outer membrane"

So,  how is one membrane assembled outside of another?

the outer membrane is an asymmetric bilayer containing lipopolysaccharide =LPS...

see MJ Osborn in 1972...

(Mary Jane Osborn)  first to  ID this...

and then H. Nikaido in 1976 at Cal.

LPS = lipopolysaccharide.

so,  how is this membrane topology established?  and does it require energy?

we now have answers...

LPS is an impenetrable barrier... shows a picture.

outermost is the O-antigen   hundreds of sugars and

then a core of   sugars and then

Lipid A with phosphates interconnected via acyl chains and

then long chain fatty acis on the inside of it.

(That's the entire  OM = outer membrane)

but,  the site of synthesis of LPS is NOT the outer membrrane!!!

LPS is actually synthesized at the inner membrane and > 100 genes are involved.

work of Chris Raetz.... 20 years ago.

cell (the bacterium) expends a huge amount of energy to make this outer membrane

 to protect the gram neg bacteria.

molecule is synthesized by the inner membrane and then flipped 180 degrees

to make the OM.

but then need to push it thru the cell wall to get

 to the location of the OM = outer membran.

puts 2-3 Million  of these molecules out every 5 minutes in a cell.

and LPS  moves irreversibly against its concentration gradient.

he started as a synthetic organic chemist at Princeton.

eg see Ge, et al and D Kahne in Science 284, 507  in 1999.

was studying vancomycin and resistance to it that penetrate strains of E. coli.

vanco  used to treat  gram positives...

but cannot get thru OM of gram negatives

to get to the cell wall where it works.

Yfgl...

in 2001...  we discovered the Bam complex, which

puts membrane beta-barrel proteins in the OM.

we got interested in the Bam complex... it puts

the membrane beta barrel proteins in the OM.

the Bam machine... it can fold  a hundred substrates.

one is the LPS  OM translocon...

one machine makes 2nd machine that makes the membrane.

seven  LPS  transproteins = Lpt  proteins  move LPS to the cell surface.

the transport machinery contains an ATPase.

see refs from 2006 to 2011... we call it the PEZ MODEL !!!

shows a PEZ dispenser with a spring at the bottom...

load it and then the candies spring up.

push the LPS up to the mouth of the PEZ to the OM.

how to couple ATP hydrolysis to make this work?

our tool used a tool dev. by Pete Schultz.

it works spectacularly well!

to observe LPS transprot states.

many antibodies to LPS that you can use to illuminate LPS crosslinks.

we were able to detect intermediates in the process o LPS transport.

so, the challenge

we needed to reconstitute LPS to see the transport.

seems that LPS transprot to LptA requires the full inner membrane protein complex

it requires ATP.

you need all the components of the inner membrane!

we can  also see this process unfold over time...

the transprot of LPS from LptC to LptA...

does movement from C to A require additional energy?

yes,  you DO need more ATP hydrolysis to push LPS to the cell surface.

(inhibiting ATP hydrolysis blocks transfer of LPS from LptC to LptA.)

confirms the PEZ model.

wanted to distinguish the chaperone model from the bridge model.

work of Sugura Okuda (one of his postdocs)

this took 8 years!

we wanted to reconstitue the entire process of LPS transport from PURE components.

worked out by David Sherman  (he learned this from Sugura)

all done in vitro (cell free)

showed that all seven  Lpt proteins and ATP are necessary and sufficient for

membrane-to-membrane transport.

Yes,   the bridge model predicts the process...

an LptA-dependent colocalized proteoliposomes.

used flow cytometry to verify...

use FACS and confocal microscopy...

can see the sorted particles...

yes,  the PEZ model explains the two longstanding questions of energy usage and topology.

the particles move on a bridge (rather than a ferry)

the bridges are long-lived and functional.

this is in Science in 2018...

he once trained as a chemist...

the Hero's journey a la Matrix (or Joseph Campbell)

(the hero ventures out into the world and slays dragons and then

returns home.)

now, can I get back to small molecules  (where I began)

... unfortunately the outer membrane keeps most drugs out...

he shows Colistin  (or polymyxin B)

but it is the drug of last resort...

causes irreversible kidney damage.

mech of action of colistin...

colistin binds to the OM and then more colistin can pass thru to

dissolve the inner membran causing the death of bacterial cell.

colistin is like a detergent.

he shows Novobiocin... introduced in the 1970s.. an oral antibiotic.

it targets DNA gyrase.he shows that Novobiocin also binds LptB but not

in the ATP binding pocket.

this is May, Owen, et al and Kahne in J Am Chem Soc in 2017.

and Novobiocin stimulates LPS transport rather than inhibiting it...

but does that matter?

yes... stimulation of LPS transprot makes colistin more effective.

and that enhancement is independent of the gyrase inhibition....

so, we can then lower the required does of colostin

(so that that won't damage the kidneys)

funded by NIH;  stunning lecture!

Kahne, Daniel, Outer Membrane Synthesis in Gram Negative Bacteria, Harvard, April 4, 2018

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