Mycobacterium avium subsp. paratuberculosis PtpA is an endogenous tyrosine phosphatase secreted during infection

Abstract

Adaptive gene expression in prokaryotes is mediated by protein kinases and phosphatases. These regulatory proteins mediate phosphorylation of histidine or aspartate in two-component systems and serine/threonine or tyrosine in eukaryotic and eukaryote-like protein kinase systems. The genome sequence of Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease, does not possess a defined tyrosine kinase. Nevertheless, it encodes for protein tyrosine phosphatases. Here, we report that Map1985, is a functional low-molecular tyrosine phosphatase that is secreted intracellularly upon macrophage infection. This finding suggests that Map1985 might contribute to the pathogenesis of Mycobacterium avium subsp. paratuberculosis by dephosphorylating essential macrophage signaling and/or adaptor molecules.

FIG. 1.

Map-PtpA alignment to mycobacterial LMWPTPs. Mycobacterial LMWPTPs were aligned to Map-PtpA based on an E score value of <1e⁻⁶⁰. The LMWPTPs from the following strains were aligned (accession numbers): M. avium subsp. paratuberculosis (NP-960919), M. tuberculosis (NP-216750), M. bovis (NP-855907), and M. smegmatis (MSMEG4314). The conserved catalytic site is marked by a box.

FIG. 2.

(A) Purification of the overexpressed Mab-PtpA by metal-chelation affinity chromatography. Soluble recombinant Mab-PtpA was prepared after the induction of M. smegmatis harboring the pHB-1 plasmid. Proteins were purified by using Ni-NTA resin. Aliquots of column fractions were resolved by SDS-12% PAGE and stained with Coomassie blue. Lanes: M, molecular mass marker, 1, uninduced cells (4.3 μg); 2, start material (26.6 μg); 3, wash with 20 mM imidazole (6.7 μg); 4, elution with 250 mM imidazole (8.7 μg). The protein amounts loaded in each fraction are indicated in parentheses. The molecular mass markers are indicated on the left. (B) Time-dependent dephosphorylation of O-phospho substrates. Dephosphorylation of O-phosphorylated amino acids was assayed in a time-dependent manner. The reactions were tracked by the malachite green assay using 500 μM O-phosphotyrosine (⋄), O-phosphothreonine (□), and O-phosphoserine (▵). The data represent the means of three independent experiments, and the error bars indicate the standard deviations.

FIG. 3.

(A) Immunoblotting of Mab-PtpA. Samples obtained after immunoprecipitation were resolved by SDS-15% PAGE, electroblotted onto a nitrocellulose membrane, and exposed to rabbit anti-mt-PtpA antibodies as described in Materials and Methods. Lanes: 1, immunoprecipitate of cell-free filtrate; 2, immunoprecipitate of M. avium subsp. paratuberculosis-infected THP-1 lysate; 3, recombinant Mab-PtpA; 4, THP-1 cell lysate. (B) Fluorescence microscopy of infected macrophages. Macrophages were infected with fluor-labeled bacteria. Samples were obtained at 24, 48, and 72 h postinfection; processed; and permeabilized with primary and secondary antibodies as described in Materials and Methods. Fluorescence microscopy was used for image processing. Black arrows indicate phagosomes within macrophages.