Dissecting virulence pathways of Mycobacterium tuberculosis through protein–protein association

Abstract

The sudden increase in information derived from the completed Mycobacterium tuberculosis (Mtb) genome sequences has revealed the need for approaches capable of converting raw genome sequence data into functional information. To date, an experimental system for studying protein-protein association in mycobacteria is not available. We have developed a simple system, termed mycobacterial protein fragment complementation (M-PFC), that is based upon the functional reconstitution of two small murine dihydrofolate reductase domains independently fused to two interacting proteins. Using M-PFC, we have successfully demonstrated dimerization of yeast GCN4, interaction between Mtb KdpD and KdpE, and association between Esat-6 and Cfp-10. We established the association between the sensor kinase, DevS, and response regulator, DevR, thereby demonstrating the potential of M-PFC to study protein associations in the mycobacterial membrane. To validate our system, we screened an Mtb library for proteins that associate with the secreted antigen Cfp-10 and consistently identified Esat-6 in our screens. Additional proteins that specifically associate with Cfp-10 include Rv0686 and Rv2151c (FtsQ), a component and substrate, respectively, of the evolutionary conserved signal recognition pathway; and Rv3596c (ClpC1), an AAA-ATPase chaperone involved in protein translocation and quality control. Our results provide empirical evidence that directly links the Mtb specialized secretion pathway with the evolutionary conserved signal recognition and SecA͞SecYEG pathways, suggesting they share secretory components. We anticipate that M-PFC will be a major contributor to the systematic assembly of mycobacterial protein interaction maps that will lead to the development of better strategies for the control of tuberculosis.Cfp-10 ͉ DevR ͉ interaction ͉ secretion ͉ virulence

Mycobacterium tuberculosis ESAT-6 Exhibits a Unique Membrane-interacting Activity That Is Not Found in Its Ortholog from Non-pathogenic Mycobacterium smegmatis

“…In addition to PTMs, other factors that show specific association with the heterodimer may also affect MtbESAT-6 and MtbCFP-10 interaction in vivo. For instance, MtbCFP-10 has been shown to interact with several other proteins, including ClpC1, an AAA-ATPase chaperone involved in protein translocation and quality control, and ClpC1 associates with the proteolytic component ClpP2 (33). It has recently been shown that ClpP1 and ClpP2 form a single proteolytic complex and function together in protein degradation, and both are required for bacterial viability in vitro and during infection (34,35).…”

EspR-dependent ESAT-6 Protein Secretion of Mycobacterium tuberculosis Requires the Presence of Virulence Regulator PhoP

“…We further studied PhoP-EspR interaction using the mycobacterial protein fragment complementation (M-PFC) assay as described previously (Fig. 5D) (48). Interestingly, in the presence of 15 g/ml TRIM, M. smegmatis co-expressing M. tuberculosis PhoP and EspR grew just as well compared with cells co-expressing PhoP and PhoR (used as positive control).…”

“…M-PFC Assays-This assay relies on the principle that two interacting mycobacterial proteins independently fused to the domains of murine dihydrofolate reductase if co-expressed in mycobacteria reconstitute functionally active murine dihydrofolate reductase enzyme, conferring bacterial resistance to trimethoprim (48). M. tuberculosis phoP and phoR genes were cloned in integrative vector pUAB400 (Kan r ) and episomal vector pUAB300 (Hyg r ), respectively (Table 1), and expressed in M. smegmatis as described previously (55); espR was cloned in episomal plasmid pUAB300 (Hyg r ) between BamHI/HindIII sites using primer pair mEspRFP/mEspRRP.…”

Regulation of Ergothioneine Biosynthesis and Its Effect on Mycobacterium tuberculosis Growth and Infectivity

“…Protein-Protein Interaction Assay-Protein-protein interactions were investigated using the mycobacterial protein fragment complementation assay as described previously (33). M. tuberculosis EgtD and STPKs (PknA, PknB, PknD, and PknK) were amplified by PCR and cloned into pUAB100 (expressing murine dihydrofolate enzyme fragments F1 and F2) and pUAB200 (expressing murine dihydrofolate fragment F3), respectively.…”

“…To test whether EgtD interacts with the four identified kinases in vivo, a cell-based interaction assay using the mycobacterial protein fragment complementation assay was performed in M. smegmatis (33).The mycobacterial protein fragment complementation assay involves the reassembly of complementary fragments F1 and F2 (expressed by pUAB100) and fragment F3 (expressed by pUAB200) of murine dihydrofolate reductase enzyme, conferring resistance to trimethoprim. As illustrated in Fig.…”