Research Article: Identification of Ser/Thr kinase and Forkhead Associated Domains in Mycobacterium ulcerans: Characterization of Novel Association between Protein Kinase Q and MupFHA

Date Published: November 20, 2014

Publisher: Public Library of Science

Author(s): Gunjan Arora, Andaleeb Sajid, Anshika Singhal, Jayadev Joshi, Richa Virmani, Meetu Gupta, Nupur Verma, Abhijit Maji, Richa Misra, Grégory Baronian, Amit K. Pandey, Virginie Molle, Yogendra Singh, Pamela L. C. Small.

Abstract: BackgroundMycobacterium 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.

Partial Text: Buruli ulcer is a disease of skin and soft tissues caused by the bacteria Mycobacterium ulcerans[1]. It is the third most important mycobacterial disease after tuberculosis and leprosy [2], and the prevalence continues to increase in tropical and sub-tropical countries [3]. 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. Therefore, the pMUM001 plasmid is considered to be a key determinant of M. ulcerans pathogenesis [5], [6].

In this study we analyzed the STPK-mediated signaling system of M. ulcerans, which is the causative agent of Buruli ulcer. M. ulcerans is a slow growing bacterium (slower than M. marinum and M. tuberculosis) [62], [63] and this slow growth together with restrictive temperature requirements are the major reasons for our limited understanding about this important human pathogen and its signaling systems. Using in silico analysis, we identified 13 STPKs in the M. ulcerans genome that are distinct from its close relative M. marinum that has 24 STPKs [39]. STPKs of M. tuberculosis have been classified in five clades [39], and phylogenetic analysis reveals that M. ulcerans also has individual STPKs related to all five clades with an over-representation of the PknF/PknI/PknJ clade. Analyses of STPKs and FHA domain encoding genes confirmed that M. ulcerans underwent reductive evolution compared to M. marinum.



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