The Carbonic Anhydrase Inhibitor Ethoxzolamide Inhibits the Mycobacterium tuberculosis PhoPR Regulon and Esx-1 Secretion and Attenuates Virulence

Abstract

bMycobacterium tuberculosis must sense and adapt to host environmental cues to establish and maintain an infection. The twocomponent regulatory system PhoPR plays a central role in sensing and responding to acidic pH within the macrophage and is required for M. tuberculosis intracellular replication and growth in vivo. Therefore, the isolation of compounds that inhibit PhoPR-dependent adaptation may identify new antivirulence therapies to treat tuberculosis. Here, we report that the carbonic anhydrase inhibitor ethoxzolamide inhibits the PhoPR regulon and reduces pathogen virulence. We show that treatment of M. tuberculosis with ethoxzolamide recapitulates phoPR mutant phenotypes, including downregulation of the core PhoPR regulon, altered accumulation of virulence-associated lipids, and inhibition of Esx-1 protein secretion. Quantitative single-cell imaging of a PhoPR-dependent fluorescent reporter strain demonstrates that ethoxzolamide inhibits PhoPR-regulated genes in infected macrophages and mouse lungs. Moreover, ethoxzolamide reduces M. tuberculosis growth in both macrophages and infected mice. Ethoxzolamide inhibits M. tuberculosis carbonic anhydrase activity, supporting a previously unrecognized link between carbonic anhydrase activity and PhoPR signaling. We propose that ethoxzolamide may be pursued as a new class of antivirulence therapy that functions by modulating expression of the PhoPR regulon and Esx-1-dependent virulence. Mycobacterium tuberculosis is a successful pathogen because it overcomes a variety of obstacles raised by the host immune response. The ability of M. tuberculosis to sense host immune pressures and orchestrate adaptive responses to these cues is essential for pathogen virulence. It follows that the identification of chemical compounds that disrupt the ability of M. tuberculosis to sense and respond to host cues may function to attenuate pathogen virulence.M. tuberculosis uses environmental pH as a cue to modulate its physiology. These pH-dependent adaptations play a central role in M. tuberculosis pathogenesis (1). Transcriptional profiling of M. tuberculosis-infected macrophages identified an M. tuberculosis regulon induced at acidic pH (2) that significantly overlaps the PhoPR two-component regulatory system regulon (3-5), suggesting a role for PhoPR in pH-driven adaptation. phoPR-inactivated M. tuberculosis mutant strains are highly attenuated during mouse and guinea pig infections, supporting that this pathway is a suitable target for new drug development (6, 7).Conventional antimicrobial discovery campaigns seeking to identify bactericidal or bacteriostatic compounds are often performed in vitro. However, many targets required for M. tuberculosis pathogenesis, including environmental sensing pathways, are essential only in vivo and would be missed using standard approaches. Compounds that target in vivo essential bacterial virulence factors are known as antivirulence therapies (8). Antivirulence therapies are advantageous over traditional antibiotics because they preserve e...

Identification of Bicarbonate as a Trigger and Genes Involved with Extracellular DNA Export in Mycobacterial Biofilms

“…Ethoxzolamide (EZA) is a sulfonamide carbonic anhydrase inhibitor that has previously been demonstrated to have activity against M. tuberculosis carbonic anhydrases (38, 39). Biofilms were formed and subjected to live-tracking analysis with wild-type M. avium subsp.…”

“…hominissuis A5 biofilm, we investigated eDNA export while using the carbonic anhydrase inhibitor ethoxzolamide. This sulfonamide has been described previously to have activity against M. tuberculosis carbonic anhydrases (38, 39). Even though we observed a significant reduction of the level of eDNA under conditions of coincubation with EZA, complete inhibition of eDNA was not achieved, which could have been due to multiple reasons.…”

Virulence Factor Targeting of the Bacterial Pathogen Staphylococcus aureus for Vaccine and Therapeutics

“…In particular, anti-virulence strategies have been heavily investigated not only for bacterial pathogens such as MRSA, but for many pathogens for which there are clear mechanistic links between the virulence mechanism and pathogenesis. Quorum molecule mimics, pilicides, active site inhibitors of specific toxins, secretion system inhibitors, and phage therapies, have all been extensively investigated as antivirulence strategies to combat mechanisms of bacterial pathogenesis by specific microbes [52–56]. In this review, we specifically highlight several major virulence factors produced by S. aureus for which recent advances in antivirulence approaches may hold promise as an alternative means to treating diseases caused by this pathogen (Fig.…”

Inhibiting Mycobacterium tuberculosis within and without

“…Johnson et al [74] used an acidic pH-inducible fluorescent reporter strain of M. tuberculosis that is fully dependent on PhoPR regulation to identify compounds that might inhibit this two-component system. PhoPR acts pleiotropically in response to acid stress, and among the genes it regulates are several encoding components of the ESX-1 system [75,76].…”