title: A three-hybrid system to probe in vivo protein-protein interactions: application to the essential proteins of the RD1 complex of M. tuberculosis.
abstract: BACKGROUND
  Protein-protein interactions play a crucial role in enabling a pathogen to survive within a host. In many cases the interactions involve a complex of proteins rather than just two given proteins. This is especially true for pathogens like M. tuberculosis that are able to successfully survive the inhospitable environment of the macrophage. Studying such interactions in detail may help in developing small molecules that either disrupt or augment the interactions. Here, we describe the development of an E. coli based bacterial three-hybrid system that can be used effectively to study ternary protein complexes.
  
  
  RESULTS
  The protein-protein interactions involved in M. tuberculosis pathogenesis have been used as a model for the validation of the three-hybrid system. Using the M. tuberculosis RD1 encoded proteins CFP10, ESAT6 and Rv3871 for our proof-of-concept studies, we show that the interaction between the proteins CFP10 and Rv3871 is strengthened and stabilized in the presence of ESAT6, the known heterodimeric partner of CFP10. Isolating peptide candidates that can disrupt crucial protein-protein interactions is another application that the system offers. We demonstrate this by using CFP10 protein as a disruptor of a previously established interaction between ESAT6 and a small peptide HCL1; at the same time we also show that CFP10 is not able to disrupt the strong interaction between ESAT6 and another peptide SL3.
  
  
  CONCLUSIONS
  The validation of the three-hybrid system paves the way for finding new peptides that are stronger binders of ESAT6 compared even to its natural partner CFP10. Additionally, we believe that the system offers an opportunity to study tri-protein complexes and also perform a screening of protein/peptide binders to known interacting proteins so as to elucidate novel tri-protein complexes.
authors.affiliation: Recombinant Gene Products Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India.
authors.forename: Megha
authors.initials: M
authors.lastname: Tharad
authors.forename: Sachin Kumar
authors.initials: SK
authors.lastname: Samuchiwal
authors.forename: Kuhulika
authors.initials: K
authors.lastname: Bhalla
authors.forename: Anamika
authors.initials: A
authors.lastname: Ghosh
authors.forename: Krishan
authors.initials: K
authors.lastname: Kumar
authors.forename: Sushil
authors.initials: S
authors.lastname: Kumar
authors.forename: Anand
authors.initials: A
authors.lastname: Ranganathan
mesh_terms: D000942:Antigens, Bacterial / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D008722:Methods; D046912:Multiprotein Complexes / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C117940:CFP-10 protein, Mycobacterium tuberculosis; C093276:ESAT-6 protein, Mycobacterium tuberculosis; D046912:Multiprotein Complexes
references.citation: Genes Dev. 1998 Mar 1;12(5):745-54
references.pmid: 9499408
references.citation: J Cell Physiol. 2005 Sep;204(3):750-62
references.pmid: 15880642
references.citation: BMC Biochem. 2009 Jan 05;10:1
references.pmid: 19121228
references.citation: PLoS One. 2009 Nov 10;4(11):e7615
references.pmid: 19901982
references.citation: Open Biochem J. 2008;2:29-37
references.pmid: 18949072
references.citation: J Bacteriol. 2005 Apr;187(7):2233-43
references.pmid: 15774864
references.citation: Chem Biol. 2003 Oct;10(10):917-26
references.pmid: 14583258
references.citation: J Microbiol Methods. 2008 May;73(2):79-84
references.pmid: 18387683
references.citation: J Biol Chem. 1997 Sep 12;272(37):22995-9
references.pmid: 9287295
references.citation: Proc Natl Acad Sci U S A. 1996 Sep 17;93(19):10315-20
references.pmid: 8816797
references.citation: Nat Med. 2003 May;9(5):533-9
references.pmid: 12692540
references.citation: Cell. 2009 Jan 9;136(1):37-49
references.pmid: 19135887
references.citation: Cell Host Microbe. 2008 May 15;3(5):316-22
references.pmid: 18474358
references.citation: FEBS J. 2006 Apr;273(7):1445-62
references.pmid: 16689931
references.citation: J Biol Chem. 2005 Jun 24;280(25):23605-14
references.pmid: 15843374
references.citation: BMC Genomics. 2009 Mar 19;10:118
references.pmid: 19298676
references.citation: Anal Biochem. 1996 Nov 1;242(1):68-72
references.pmid: 8923966
references.citation: Proc Natl Acad Sci U S A. 2003 Oct 28;100(22):13001-6
references.pmid: 14557536
references.citation: Tuberculosis (Edinb). 2005 Sep-Nov;85(5-6):347-55
references.pmid: 16263329
references.citation: Proc Natl Acad Sci U S A. 2006 Jul 25;103(30):11346-51
references.pmid: 16844784
references.citation: Science. 2006 Sep 15;313(5793):1632-6
references.pmid: 16973880
references.citation: PLoS Biol. 2004 Nov;2(11):e367
references.pmid: 15510227
references.citation: J Infect Dis. 2007 Aug 15;196 Suppl 1:S86-107
references.pmid: 17624830
references.citation: EMBO J. 2005 Jul 20;24(14):2491-8
references.pmid: 15973432
references.citation: Infect Immun. 2005 Aug;73(8):4471-7
references.pmid: 16040957
references.citation: Science. 2010 Jan 22;327(5964):466-9
references.pmid: 20007864
references.citation: Proc Natl Acad Sci U S A. 2005 Mar 15;102(11):4033-8
references.pmid: 15753315
references.citation: Nat Biotechnol. 2000 May;18(5):544-7
references.pmid: 10802623
references.citation: Angew Chem Int Ed Engl. 2002 Jul 2;41(13):2327-30
references.pmid: 12203581
references.citation: Annu Rev Microbiol. 2003;57:641-76
references.pmid: 14527294
references.citation: Annu Rev Microbiol. 2010;64:293-311
references.pmid: 20825351
references.citation: J Bacteriol. 2010 Jan;192(1):326-35
references.pmid: 19854905
references.citation: APMIS. 2009 May;117(5-6):440-57
references.pmid: 19400867
references.citation: Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8496-501
references.pmid: 8710898
references.citation: Nature. 1997 Apr 10;386(6625):627-30
references.pmid: 9121589
references.citation: Mol Microbiol. 2009 Sep;73(5):950-62
references.pmid: 19682254
references.citation: Proc Natl Acad Sci U S A. 2003 Oct 14;100(21):12420-5
references.pmid: 14557547
references.citation: J Biol Chem. 2002 Jun 14;277(24):21598-603
references.pmid: 11940590
references.citation: Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):12817-21
references.pmid: 8917502
references.citation: Biochim Biophys Acta. 2010 Mar;1804(3):620-7
references.pmid: 19766738
references.citation: PLoS One. 2009;4(5):e5561
references.pmid: 19440304
references.citation: Curr Top Med Chem. 2007;7(10):922-7
references.pmid: 17508923
references.citation: Protein Sci. 2009 Nov;18(11):2316-25
references.pmid: 19760663
references.citation: Mol Microbiol. 2004 Jan;51(2):359-70
references.pmid: 14756778