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Discovery of uncompetitive inhibitors of SapM that compromise intracellular survival of Mycobacterium tuberculosis.

ABSTRACT: SapM is a secreted virulence factor from Mycobacterium tuberculosis critical for pathogen survival and persistence inside the host. Its full potential as a target for tuberculosis treatment has not yet been exploited because of the lack of potent inhibitors available. By screening over 1500 small molecules, we have identified new potent and selective inhibitors of SapM with an uncompetitive mechanism of inhibition. The best inhibitors share a trihydroxy-benzene moiety essential for activity. Importantly, the inhibitors significantly reduce mycobacterial burden in infected human macrophages at 1 µM, and they are selective with respect to other mycobacterial and human phosphatases. The best inhibitor also reduces intracellular burden of Francisella tularensis, which secretes the virulence factor AcpA, a homologue of SapM, with the same mechanism of catalysis and inhibition. Our findings demonstrate that inhibition of SapM with small molecule inhibitors is efficient in reducing intracellular mycobacterial survival in host macrophages and confirm SapM as a potential therapeutic target. These initial compounds have favourable physico-chemical properties and provide a basis for exploration towards the development of new tuberculosis treatments. The efficacy of a SapM inhibitor in reducing Francisella tularensis intracellular burden suggests the potential for developing broad-spectrum antivirulence agents to treat microbial infections.

SUBMITTER: Fernandez-Soto P

PROVIDER: S-EPMC8027839 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

Project description:The stable infection of host macrophages by Mycobacterium tuberculosis (Mtb) involves, and depends on, the attenuation of the diverse microbicidal responses mounted by the host cell. This is primarily achieved through targeted perturbations of the host cellular signaling machinery. Therefore, in view of the dependency of the pathogen on host molecules for its intracellular survival, we wanted to test whether targeting such factors could provide an alternate route for the therapeutic management of tuberculosis. To first identify components of the host signaling machinery that regulate intracellular survival of Mtb, we performed an siRNA screen against all known kinases and phosphatases in murine macrophages infected with the virulent strain, H37Rv. Several validated targets could be identified by this method where silencing led either to a significant decrease, or enhancement in the intracellular mycobacterial load. To further resolve the functional relevance of these targets, we also screened against these identified targets in cells infected with different strains of multiple drug-resistant mycobacteria which differed in terms of their intracellular growth properties. The results obtained subsequently allowed us to filter the core set of host regulatory molecules that functioned independently of the phenotypic variations exhibited by the pathogen. Then, using a combination of both in vitro and in vivo experimentation, we could demonstrate that at least some of these host factors provide attractive targets for anti-TB drug development. These results provide a "proof-of-concept" demonstration that targeting host factors subverted by intracellular Mtb provides an attractive and feasible strategy for the development of anti-tuberculosis drugs. Importantly, our findings also emphasize the advantage of such an approach by establishing its equal applicability to infections with Mtb strains exhibiting a range of phenotypic diversifications, including multiple drug-resistance. Thus the host factors identified here may potentially be exploited for the development of anti-tuberculosis drugs.

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Discovery of uncompetitive inhibitors of SapM that compromise intracellular survival of Mycobacterium tuberculosis.

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