Identification of Ser/Thr kinase and forkhead associated domains in Mycobacterium ulcerans: characterization of novel association between protein kinase Q and MupFHA.

Gunjan Arora,Andaleeb Sajid,Richa Virmani,Yogendra Singh,Virginie Molle,Meetu Gupta,Anshika Singhal,Amit K Pandey,Grégory Baronian,Abhijit Maji,Jayadev Joshi,Richa Misra,Nupur Verma

doi:10.1371/journal.pntd.0003315

Abstract

Background Mycobacterium ulcerans, the causative agent of Buruli ulcer in humans, is unique among the members of Mycobacterium genus due to the presence of the virulence determinant megaplasmid pMUM001. This plasmid encodes multiple virulence-associated genes, including mup011, which is an uncharacterized Ser/Thr protein kinase (STPK) PknQ.Methodology/Principal FindingsIn this study, we have characterized PknQ and explored its interaction with MupFHA (Mup018c), a FHA domain containing protein also encoded by pMUM001. MupFHA was found to interact with PknQ and suppress its autophosphorylation. Subsequent protein-protein docking and molecular dynamic simulation analyses showed that this interaction involves the FHA domain of MupFHA and PknQ activation loop residues Ser170 and Thr174. FHA domains are known to recognize phosphothreonine residues, and therefore, MupFHA may be acting as one of the few unusual FHA-domain having overlapping specificity. Additionally, we elucidated the PknQ-dependent regulation of MupDivIVA (Mup012c), which is a DivIVA domain containing protein encoded by pMUM001. MupDivIVA interacts with MupFHA and this interaction may also involve phospho-threonine/serine residues of MupDivIVA.Conclusions/SignificanceTogether, these results describe novel signaling mechanisms in M. ulcerans and show a three-way regulation of PknQ, MupFHA, and MupDivIVA. FHA domains have been considered to be only pThr specific and our results indicate a novel mechanism of pSer as well as pThr interaction exhibited by MupFHA. These results signify the need of further re-evaluating the FHA domain –pThr/pSer interaction model. MupFHA may serve as the ideal candidate for structural studies on this unique class of modular enzymes.

Highlights

Buruli ulcer is a disease of skin and soft tissues caused by the bacteria Mycobacterium ulcerans [1]
PknQ, MupFHA and MupDivIVA are regulatory proteins encoded by the virulence determining plasmid pMUM001 of M. ulcerans
In silico analysis of M. ulcerans Ser/Thr protein kinase (STPK) To identify the total number of STPKs present in M. ulcerans, we performed a BLASTp search using the sequence of the catalytic domain of M. tuberculosis PknB (1–331 aa), corresponding to the most conserved mycobacterial STPK [37]

Summary

Introduction

Buruli ulcer is a disease of skin and soft tissues caused by the bacteria Mycobacterium ulcerans [1]. M. ulcerans evolved from an Mycobacterium marinum ancestor through reductive evolution and acquired a large virulence determinant plasmid (pMUM001) [4]. This plasmid encodes genes for mycolactone synthesis that are required to circumvent the host immune response, as a strain lacking this plasmid is avirulent. Mycobacterium ulcerans, the causative agent of Buruli ulcer in humans, is unique among the members of Mycobacterium genus due to the presence of the virulence determinant megaplasmid pMUM001. This plasmid encodes multiple virulence-associated genes, including mup, which is an uncharacterized Ser/Thr protein kinase (STPK) PknQ

Methods

Results

Discussion

Conclusion