FUN_15

Functional Study FUN_15

PI/Lead, Bryan Jensen (Parsons/Myler Labs), Center for Infectious Disease Research

Co-investigator: Christoph Dehio, Biozentrum Basel, Switzerland

Technical support (CX-MS): Institute of Systems Biology, Seattle

SSGCID Lead: Isabelle Phan, Center for Infectious Disease Research

Identification of host-pathogen interactions by chemical crosslinking and mass-spectrometry (CX-MS): targeting Bartonella effector proteins from the Type IV secretion system.

 

Introduction/Background

The SSGCID has determined structures for at least 50 known and potential virulence factors, including experimentally characterized virulence-associated targets from community requests, such as effector proteins from the type IV and VI bacterial secretion systems. In stealth pathogens with type IV secretion systems (T4SSs), for example Bartonella and Brucella, protein effectors are injected into the host cells. Interactions between effector and host proteins subsequently trigger multiple cellular processes within the host in order to establish pathogen persistence [1]. This makes T4SS effectors ideal candidates for studying host-pathogen interactions in the context of understanding mechanisms of virulence. Furthermore, knowledge of the proteins involved in host-pathogen interactions and structural insight into the nature of the protein-protein interfaces are key to develop new antimicrobial therapies that target ‘hotspots’ on contact surfaces [2]. Chemical crosslinking followed by mass-spectrometry (CX-MS) is an emerging technique originally designed to study large molecular assemblies, that are capable of determining both transient and long-lived protein-protein interactions that have been chemically stabilized [3]. The method has been successfully applied to characterize host-protein interactions in human cells infected by multi-drug resistant strains of Acinetobacter baumanii [4].

This project proposes to identify host proteins that interact directly with Bartonella effector proteins (Beps) using CX-MS. Early phylogenetic studies revealed that Beps are a large family of proteins composed of FIC and BID domains, that can be classified into 10 clades (numbered Bep1-10) for Bartonella species in lineage 3 and 9 clades (named BepA-I) for lineage 4 species [5]. Most of the functional work to date has been conducted on Beps from lineage 4 and has shown that Bep domains from different clades performed distinct functions: For example BepA is involved in inhibition of apoptosis via interaction of its BID domain with adenylyl cyclase, whereas BepF triggers invasome formation (reviewed in [6]). The SSGCID has solved 5 FIC and 3 BID domains from multiple clades (see Table 34). While the functions of some of these are known (see details in Specific Aims), their host protein targets are unknown. The proposed work will be conducted in close collaboration with the Dehio group who are established leaders in the field and have been in a successful partnership with the SSGCID for over 5 years. It will complement functional studies currently on-going in the Dehio lab and, where indicated, prioritization of targets will depend on results obtained by the Dehio group by the time work on this project is initiated.

PDB

Target

Bartonella species

Domain

Clade

4XI8

BaclA.17330.o

clarridgeiae

FIC

Bep5

4YK1

BaroA.17330.c

rochalimae

BID

Bep6

4M16

BasaA.17330.c

sp. AR 15-3

FIC

Bep8

4PY3

BaspA.17330.e

sp. 1-1C

FIC

Bep8

4YK2

BaclA.17330.i

clarridgeiae

BID

Bep9

4N67

BaquA.17330.c

quintana

FIC (+ligand)

BepC

4LU4

BaquA.17330.c

quintana

FIC (apo)

BepC

4WGJ

BatrA.17330.a

tribocorum

FIC

BepC

4YK3

BaheA.17330.a

henselae

BID1

BepE

Table 34. Structures of Beps domains solved by SSGCID.

 

Specific Aim 1:  Identification of host proteins interacting with BepC.
Work to be performed by Bryan Jensen at CIDR carried out during Q(s) Q1-Q4.

Since BepC has been shown to interact genetically with BepF, we will seek to identify host proteins that interact with BepC alone or in concert with BepF (we already have clones for both B.quintana and B.tribocorum BepF). HEK293 or other appropriate cells will be transfected with a C-terminally TAP-tagged BepC alone or together with BepF. Following transfection, proteins will be cross-linked, cells lyzed, TAP-tagged protein purified and interacting proteins identified by mass spectrometry.

Specific Aim 2:  Identification of host proteins targeted by Beps containing only BID domains.
Work to be performed by Bryan Jensen at CIDR, carried out during Q(s) Q1-Q4. Target selection to be performed by Dehio lab, carried out during Q1.

BID domains are hypothesized to function primarily as protein interaction domains. Based on this hypothesis, we predict that Beps containing just BID domains would form stable interactions with their downstream host protein. Individual BID domains have been solved for 3 Bep clades (see Table 34). We will TAP-tag one of these Beps and identify host interacting proteins as described in Aim 1.

Specific Aim 3:  Identification of host proteins that interact via the FIC domain of a Bep
Work to be performed by Bryan Jensen at CIDR, carried out during Q(s) Q1-Q4. Target selection to be performed by Dehio lab, carried out during Q1.

Unlike the BID domain, the FIC domain is thought to function catalytically by modifying host proteins. The transitory nature of a catalysts interaction with its target makes it more challenging to identify the downstream target. We propose to test the ability of TAP purified cross-linked proteins to identify these targets. SSGCID has solved the structure of five FIC domains. Similar to Aim 1, we will TAP-tag one FIC domain containing protein and transfect it into host cells and isolate interacting proteins as described above. Expression of the FIC domain could be toxic to the host cells. If we are unable to isolate stable transfectants, we will transiently transfect constructs expressing tagged proteins.

 

TIMELINE

MILESTONE

Quarter 1

09.2016-11.2016

  • M1 Generation of TAP-tagged clones (all milestones support Aims 1,2,3)
  • M2 Establishment of tissue culture system

Quarter 2

12.2016-02.2017

  • M3 Transfection
  • M4 Verification of protein expression

Quarter 3

03.2016-05.2017

  • M5 Cross-linking and TAP purification
  • M6 Mass-spec

Quarter 4

06.2016-08.2017

  • M8 Identification of interacting proteins
  • M9 Write manuscript

 

Deliverables will consist in a manuscript describing the interactions and at least one set of crosslinks that might be amenable to macromolecular complex determination using integrative molecular modeling [7]. Results will be analyzed in close collaboration with the Dehio group in the larger context of determining the molecular mechanisms underlying persistent bacterium-host interaction. This study will also serve as a pilot project to evaluate CX-MS as a potential new technology to expand the capabilities of structural genomics to investigate protein-protein complexes in situ.

Estimate of labor hours needed for the work, over a period of 12 months: 0.35 FTE of Bryan Jensen’s efforts.

Other costs: 3K mass-spectrometry time, 5K supplies.

 

References

[1]       E. Cascales, P.J. Christie, The versatile bacterial type IV secretion systems., Nat. Rev. Microbiol. 1 (2003) 137–49. doi:10.1038/nrmicro753.

[2]       J.A. Wells, C.L. McClendon, Reaching for high-hanging fruit in drug discovery at protein-protein interfaces., Nature. 450 (2007) 1001–9. doi:10.1038/nature06526.

[3]       A. Leitner, M. Faini, F. Stengel, R. Aebersold, Crosslinking and Mass Spectrometry: An Integrated Technology to Understand the Structure and Function of Molecular Machines., Trends Biochem. Sci. 41 (2015) 20–32. doi:10.1016/j.tibs.2015.10.008.

[4]       D.K. Schweppe, C. Harding, J.D. Chavez, X. Wu, E. Ramage, P.K. Singh, C. Manoil, J.E. Bruce, Host-Microbe Protein Interactions during Bacterial Infection., Chem. Biol. 22 (2015) 1521–30. doi:10.1016/j.chembiol.2015.09.015.

[5]       H.L. Saenz, P. Engel, M.C. Stoeckli, C. Lanz, G. Raddatz, M. Vayssier-Taussat, R. Birtles, S.C. Schuster, C. Dehio, Genomic analysis of Bartonella identifies type IV secretion systems as host adaptability factors., Nat. Genet. 39 (2007) 1469–76. doi:10.1038/ng.2007.38.

[6]       S. Siamer, C. Dehio, New insights into the role of Bartonella effector proteins in pathogenesis., Curr. Opin. Microbiol. 23C (2014) 80–85. doi:10.1016/j.mib.2014.11.007.

[7]       D. Russel, K. Lasker, B. Webb, J. Velázquez-Muriel, E. Tjioe, D. Schneidman-Duhovny, B. Peterson, A. Sali, Putting the pieces together: integrative modeling platform software for structure determination of macromolecular assemblies., PLoS Biol. 10 (2012) e1001244. doi:10.1371/journal.pbio.1001244.

Featured Structures

Overlay of 3 Bartonella BID domains

Bartonella BID


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