tir faculty seminar

8/15/2019 tir faculty seminar

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Structural mimicry in virulent factors:

Bacteria TIR-like proteins

Jaime Pascual


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Innate immunity:firstline of defense against microbes


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Three arms forinnate immune detection

ofpathogens


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Suzuki et al, Trends in Immunology, 2002

Toll-like receptorsignaling pathway


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TIR 3D fold belongs

to the flavodoxin-like

/ familythat includes

bacterial proteins

Crystal structure of human Toll-like

receptor2 TIR domain


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Model forTIR-TIR signaling interaction:homotypic via E-helix and heterotypic via BB loop


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Sequence alignment between prokaryotic & eukaryotic TIRs


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Genome location ofTIR-like genes:pathogenicity islands of infectious bacteria

(1) TIR-like gene; (20) integrase gene; (12) type IV secretion pilin protein precursor gene


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B.abortus RBV02711 114-217/250

Phyla:

Euryarchaeota

Proteobacteria

Firmicutes

Cyanobacteria

ChlorobiChloroflexi

Actinobacteria

Planctomycetes

Fusobacteria

BacteroidetesMetazoa

Phylogenetic

distribution

ofTIRs


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aims

3D structure of a prokaryote TIR domain Comparison with a eukaryote TIR receptor

TIR signaling: prokar eukar complex Homo- vs. hetero-typic interactions

Function of TIR-like domain in bacteria

Prevent host cell inflammation signals (virulent factor)

Prokaryote programmed cell death


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Prokaryotic TIRs Status

Bacillus subtilis Cloned

Brucella abortus Cloned, no expression

Burkholderia fungorum Cloned, no expression

Escherichia coli CFT073 Cloned, toxic, refolded, unstableParacoccus denitrificans Cloned, expressed, purified, >20mg/ml, xtal trials

Salmonella enteriditis Cloned, toxic

Staphylococus aureus Cloned, no expression

Prokaryote TIR-like proteins

expression trials in E. coli

P. denitrificans TIR-like protein (PdTLP)

could only be expressed in high quantity

using the Rosetta(DE3)-pLysSE. colistrain.


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Limitedproteolysis analysis ofPdTLP


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Mass spectrometry identification of

PdTLP digestion fragments

Full length

C-terminal

fragment

(S146-A-M-K-P)


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158kDa 44kDa 17kDa

Gelfiltration chromatograms forPdTLP

Full length

(33 kDa)

C-terminal

TIR-like

(17 kDa)

N-terminal

Coiled-coil

(16 kDa)

Dimer

Monomer

Oligomer

TIR-likeCoiled-coilpI 5.7pI 10.2 Full length pI 9.6

Leu145 aa299aa1


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Differential scanningcalorimetry profiles forPdTLP full length, coiled-coil & TIR-like domains


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Circular dichroism spectrum of

the coiled-coil region ofPdTLP


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1H-15N 2D HSQC NMR spectrum of PdTLP

TIR-like domain

(pp

m)

(ppm)


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Salmonella Paracoccus Bruc ella

Prokaryote TIR-like proteins suppress

NFkB activation induced by MyD88


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In vitrointeraction between MyD88

and PdTLP TIR domains

GSTGST-mMyD88-

TIR

PdTLP-TIR

GSTGST-mMyD88-TIR

hTLR2-TIR

GSTGST-hTLR2-

TIR

mMyD88-

TIR

GSTGST-hTLR2-

TIR

PdTLP-TIR


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Model forvirulent action by TIR-like mimicryhealthy

cell

infected

cell


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Striking a balance through host mimicry:

Salmonella SopE (GEF) & SptP (GAP) virulent factors

Stebbins, C. Nature 412:701-5 (2001)


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Drugability of the TLR pathway

Beutler, B. Nature 430:257-263 (2004)


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BB loop mimic as inhibitorof prot-prot inter:a low molecular weight pyrrolidine interacts with MyD88 TIR

Davis, CN, Rebek, J & Bartfai, B. PNAS 103:2953-2958 (2006)


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Acknowledgements

Ray Low

Genaro Pimienta

Takashi Mukasa

Robert C. Liddington

John C. Reed

Adam Godzik

Group Collaborators


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Second life avatar:

jamie pascal

See you in the metaverse


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Documents

tir faculty seminar