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Mechanism of catalysis and inhibition of Mycobacterium tuberculosis SapM, implications for the development of novel antivirulence drugs.

ABSTRACT: Mycobacterium tuberculosis (Mtb) SapM is a secreted virulence factor critical for intracellular survival of the pathogen. The role of SapM in phagosome maturation arrest in host macrophages suggests its potential as a drug target to assist in the clearance of tuberculosis infection. However, the mechanism of action of SapM at the molecular level remains unknown. In this study, we provide new insights into the mechanism of catalysis, substrate specificity and inhibition of SapM, and we identify the critical residues for catalysis and substrate binding. Our findings demonstrate that SapM is an atypical monoester alkaline phosphatase, with a serine-based mechanism of catalysis probably metal-dependent. Particularly relevant to SapM function and pathogenesis, is its activity towards PI(4,5)P₂ and PI3P, two phosphoinositides that function at the early stages of microbial phagocytosis and phagosome formation. This suggests that SapM may have a pleiotropic role with a wider importance on Mtb infection than initially thought. Finally, we have identified two inhibitors of SapM, L-ascorbic acid and 2-phospho-L-ascorbic, which define two different mechanisms by which the catalytic activity of this phosphatase could be regulated. Critically, we demonstrate that 2-phospho-L-ascorbic reduces mycobacterial survival in macrophage infections, hence confirming the potential of SapM as a therapeutic drug target.

SUBMITTER: Fernandez-Soto P

PROVIDER: S-EPMC6635428 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Mechanism of catalysis and inhibition of Mycobacterium tuberculosis SapM, implications for the development of novel antivirulence drugs.

Fernandez-Soto Paulina P Bruce Alexander J E AJE Fielding Alistair J AJ Cavet Jennifer S JS Tabernero Lydia L

Scientific reports 20190716 1

Mycobacterium tuberculosis (Mtb) SapM is a secreted virulence factor critical for intracellular survival of the pathogen. The role of SapM in phagosome maturation arrest in host macrophages suggests its potential as a drug target to assist in the clearance of tuberculosis infection. However, the mechanism of action of SapM at the molecular level remains unknown. In this study, we provide new insights into the mechanism of catalysis, substrate specificity and inhibition of SapM, and we identify t ...[more]

PMID: 31312014

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Project description:Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death due to bacterial infections in mankind, and BCG, an attenuated strain of Mycobacterium bovis, is an approved vaccine. BCG sequesters in immature phagosomes of antigen presenting cells (APCs), which do not fuse with lysosomes, leading to decreased antigen processing and reduced Th1 responses. However, an Mtb derived ΔfbpA attenuated mutant underwent limited phagosome maturation, enhanced immunogenicity and was as effective as BCG in protecting mice against TB. To facilitate phagosome maturation of ΔfbpA, we disrupted an additional gene sapM, which encodes for an acid phosphatase. Compared to the wild type Mtb, the ΔfbpAΔsapM (double knock out; DKO) strain was attenuated for growth in mouse macrophages and PMA activated human THP1 macrophages. Attenuation correlated with increased oxidants in macrophages in response to DKO infection and enhanced labeling of lysosomal markers (CD63 and rab7) on DKO phagosomes. An in vitro Antigen 85B peptide presentation assay was used to determine antigen presentation to T cells by APCs infected with DKO or other mycobacterial strains. This revealed that DKO infected APCs showed the strongest ability to present Ag85B to T cells (>2500 pgs/mL in 4 hrs) as compared to APCs infected with wild type Mtb or ΔfbpA or ΔsapM strain (<1000 pgs/mL in 4 hrs), indicating that DKO strain has enhanced immunogenicity than other strains. The ability of DKO to undergo lysosomal fusion and vacuolar acidification correlated with antigen presentation since bafilomycin, that inhibits acidification in APCs, reduced antigen presentation. Finally, the DKO vaccine elicited a better Th1 response in mice after subcutaneous vaccination than either ΔfbpA or ΔsapM. Since ΔfbpA has been used in mice as a candidate vaccine and the DKO (ΔfbpAΔsapM) mutant is more immunogenic than ΔfbpA, we propose the DKO is a potential anti-tuberculosis vaccine.

Dataset Information

Mechanism of catalysis and inhibition of Mycobacterium tuberculosis SapM, implications for the development of novel antivirulence drugs.

Publications

Mechanism of catalysis and inhibition of Mycobacterium tuberculosis SapM, implications for the development of novel antivirulence drugs.

Similar Datasets

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