New potential eukaryotic substrates of the mycobacterial protein tyrosine phosphatase PtpA: hints of a bacterial modulation of macrophage bioenergetics state

Scientific Reports
Mariana MargenatAndrea Villarino

Abstract

The bacterial protein tyrosine phosphatase PtpA is a key virulence factor released by Mycobacterium tuberculosis in the cytosol of infected macrophages. So far only two unrelated macrophage components (VPS33B, GSK3α) have been identified as PtpA substrates. As tyrosine phosphatases are capable of using multiple substrates, we developed an improved methodology to pull down novel PtpA substrates from an enriched P-Y macrophage extract using the mutant PtpA D126A. This methodology reduced non-specific protein interactions allowing the identification of four novel putative PtpA substrates by MALDI-TOF-MS and nano LC-MS: three mitochondrial proteins - the trifunctional enzyme (TFP), the ATP synthase, and the sulfide quinone oxidoreductase - and the cytosolic 6-phosphofructokinase. All these proteins play a relevant role in cell energy metabolism. Using surface plasmon resonance, PtpA was found to bind immunopurified human TFP through its catalytic site since TFP-PtpA association was inhibited by a specific phosphatase inhibitor. Moreover, PtpA wt was capable of dephosphorylating immunopurified human TFP in vitro supporting that TFP may be a bona fide PtpA susbtrate. Overall, these results suggest a novel scenario where PtpA-mediated...Continue Reading

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Citations

Sep 12, 2015·Annual Review of Microbiology·Andaleeb SajidYogendra Singh
Sep 30, 2015·Biochemical and Biophysical Research Communications·Peifu ZhouYossef Av-Gay
Nov 17, 2020·Frontiers in Microbiology·Marina Andrea ForrelladFabiana Bigi
Oct 28, 2020·Antimicrobial Agents and Chemotherapy·K B ArunR Ajay Kumar
Apr 8, 2021·Microbiology·Leah Isobella Rankine-WilsonYossef Av-Gay

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Methods Mentioned

BETA
circular dichroism
chip
pull down
immunoprecipitation
PCR
size exclusion chromatography
FCS
pull-down

Software Mentioned

MASCOT
SynGene
GBOX ChemiSystem
Mascot engine
K2d
BIAevaluation

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