MptpB, a virulence factor from Mycobacterium tuberculosis, exhibits triple-specificity phosphatase activity

Abstract

Bacterial pathogens have developed sophisticated mechanisms of evading the immune system to survive in infected host cells. Central to the pathogenesis of Mycobacterium tuberculosis is the arrest of phagosome maturation, partly through interference with PtdIns signalling. The protein phosphatase MptpB is an essential secreted virulence factor in M. tuberculosis. A combination of bioinformatics analysis, enzyme kinetics and substrate-specificity characterization revealed that MptpB exhibits both dual-specificity protein phosphatase activity and, importantly, phosphoinositide phosphatase activity. Mutagenesis of conserved residues in the active site signature indicates a cysteine-based mechanism of dephosphorylation and identifies two new catalytic residues, Asp165, essential in catalysis, and Lys164, apparently involved in substrate specificity. Sequence similarities with mammalian lipid phosphatases and a preference for phosphoinositide substrates suggests a potential novel role of MptpB in PtdIns metabolism in the host and reveals new perspectives for the role of this phosphatase in mycobacteria pathogenicity.

Figure 1. MptpB exhibits DSP and phosphoinositide phosphatase activity and displays sequence and structural similarities with eukaryotic lipid phosphatases

(A) Alignment of a subset of MptpB-related sequences. Alignments prepared with ClustalX. M. tuberculosis (MYTU0, MptpB), M. bovis (MYBO0), M. flavescens (MYCFV), M. vanbaalenii (MYCVN), M. species (MYCSS), Photorhabdus luminescens (PHLU0), Erwinia carotovora (ERWCT), Bdellovibrio bacteriovorus (BDEBA), Clostridium perfringensgi (CLOPE), Lactobacillus plantarum (LAPL0), Lactococcus lactis (LALA0), Bradyrhizobium japonicum (BRJA0), Listeria monocytogenes (LISMO), Bacteroides fragilis (BACFN). (B) Alignment of P-loop motifs in lipid phosphatases. Indicated are the conserved ‘AGK’ motif found in PTEN and the ‘DRT’ from MTMs (bold), where K and D (shown in italics) are catalytic residues. MptpB has a unique combination of both signatures, with a conserved ‘AGKDRT’ motif (underlined). The DSP IphP from cyanobacteria [27] also contains the conserved ‘AGKDRT’ motif. (C) Specific activity (SA) for MptpB wild-type and mutants towards phosphorylated substrates. Each assay was performed in triplicate. Values are means±S.E.M. The activity of the tyrosine-specific phosphatase from T. brucei (TbPTP1) is shown as a control. (D) Enzyme activity of MptpB wild-type and mutants towards phosphoinositide substrates. (E) Stick representation of MptpB (purple) [25] superimposed into the active site of MTMR2 (pink) with bound PtdIns(3,5)P₂ [22]. Catalytic residues and Asp⁸² are labelled and interactions are displayed as dashed lines. Generated with PyMol (DeLano Scientific LLC). PI, PtdIns; P-Ser, phosphoserine; P-Thr, phosphothreonine; P-Tyr, phosphotyrosine; WT, wild-type.