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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.    Methodology/Principal Findings  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/Significance  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.

A Three-Hybrid System to Probe In Vivo Protein-Protein Interactions: Application to the Essential Proteins of the RD1 Complex of M. tuberculosis