Abramovitch Lab

Mycobacterium tuberculosis (Mtb) uses environmental pH as a cue to adapt its physiology for survival in macrophages. The phoPR two-component regulatory system functions to sense environmental pH and promote Mtb pathogenesis. We have engineered a pH-inducible biosensor in Mtbthat exhibits inducible fluorescence in response to acidic pH in vitro and in macrophages. We have used this biosensor to screen >280,000 compounds for inhibition of Mtb pH-driven adaptation. This screen discovered that ethoxzolamide (ETZ), an FDA approved carbonic anhydrase (CA) inhibitor, down-regulated PhoPR signaling and inhibited Mtb survival in macrophages and mice (Johnson et al., 2015). We are now defining the lin10,653,679ks between CA, pH and PhoPR signaling. A US patent has been awarded for this invention (10,653,679). We have also discovered that PhoPR, specific carbon sources and several newly identified genes, such as ppe51, regulate Mtb growth rate, persistence and antibiotic tolerance at acidic pH (Baker et al., 2014; Baker and Abramovitch, 2018; Baker and Abramovitch, 2019; Baker et al., 2019, Dechow et al., 2022, Dechow et al., 2022, Dechow and Abramovitch, 2024). In a chemical biology screen for inhibitors of Mtb survival at acidic pH, we found that Mtb is highly sensitive to thiol oxidative stress at acidic pH (Coulson et al., 2017, Dechow et al., 2021). Our ongoing work is focused on characterizing these newly discovered small molecules and genes.

Mtb uses hypoxia as a cue to adapts its physiology to survive while trapped inside the granuloma. The dosRST two-component regulatory system functions to sense hypoxia and nitric oxide and promote Mtb persistence (reviewed here, Zheng and Abramovitch, 2019 and Zheng and Abramovitch, 2020). We have engineered a hypoxia-inducible biosensor in Mtb that exhibits inducible fluorescence in response to hypoxia and nitric oxide in vitro, in macrophages and in mice. We have used this biosensor to screen >600,000 compounds for inhibition of Mtb hypoxia-driven adaptation. This screen successfully identified artemisinin and five other compounds as inhibitors of DosRST signaling, persistence and antibiotic tolerance (Zheng et al., 2017 and Zheng et al., 2019). Recently, two patents have been awarded for these inventions (US patents 11,617,740 and 11,717,508). Notably, we have collaborated on a study showing endoperoxide antimalarial DosRS inhibitors are effective at treating non-tuberculosis mycobacteria in animal models of infection (Belardinelli et al., 2022), presenting a clinical path forward for DosRST inhibitors as new drugs. Efforts are underway to further characterize these compounds and optimize them for studies in vivo.

Our lab has screened over 1 million compounds for inhibition of Mtb growth and we are actively characterizing several new compounds that: 1) Kill Mtb at acidic pH, 2) Kill Mtb at both acidic and neutral pH and 3) selectively inhibit Mtb growth inside macrophages. These newly discovered chemical probes will enable the identification of druggable pathways that are essential for Mtb pathogenesis. Several of these molecules are examples of anti-virulence therapies as described in our recently published review (Johnson et al., 2017). A newly described inhibitor from our lab, HC2091, kills Mtb by targeting the MmpL3 mycolic acid transporter (Zheng et al., 2018). Targeted and untargeted screening has identified another 10 new MmpL3 inhibitors (Williams et al., 2019). Structure-function interactions identified by comparing these new compounds and MmpL3 amino acid substitutions driving resistance may help design combinations of molecules or new molecules with a reduced frequency of resistance. These efforts have identified a series with in vivo efficacy (Williams et al., 2024). Our review on MmpL3 function and inhibition can be found here: Williams and Abramovitch 2023. We have also recently described the discovery of new nitro-containing compounds active against Mtb and M.abscessus with in vivo efficacy (Eke et al. 2023).

Socially distanced celebration for John's PhD dissertation defense

University of Notre Dame 2017

RNA-seq data of M. tuberculosis gene expression in response to acidic pH and carbon source (see Baker et al., 2014, Molecular Microbiology, GSE52020).

Table 1. Direct comparison of 10 mM pyruvate to 10 mM glycerol, pH 7.0

Table 2. Direct comparison of 10 mM glycerol at pH 5.7 to pH 7.0

Table 3. Direct comparison of 10 mM pyruvate at pH 5.7 to pH 7.0

Table 4. Direct comparison of 10 mM pyruvate to 10 mM glycerol, pH 5.7

Table 5. Genes exhibiting pH and carbon source dependent regulation

Table 6. Complete list of differential gene expression comparisons of 10 mM glycerol to 10 mM pyruvate, at pH 7.0 and pH 5.7.

RNA-seq data of M. tuberculosis gene expression in response to ethoxzolamide, acidic pH or disruption of PhoP (see Johnson et al., 2015, Antimicrobial Agents and Chemotherapy, GSE63917).

Supplemental Tables S1-S6

RNA-seq data of M. tuberculosis gene expression in response to chemical or genetic disruption of the DosRST pathway (see Zheng et al., 2017, GEO Database GSE76566).

Differential gene expression data of WT Mtb treated with inhibitors and the DMSO treated DosR mutant (Supplemental Dataset 1).

Differential gene expression data of the DosR mutant treated with the inhibitors (Supplemental Dataset 2).

Complete gene expression tables for transcriptional profiling experiments (Supplemental Dataset 3).

RNA-seq data of M. tuberculosis gene expression in response to AC2P36 (see Coulson et al, 2017, GEO Database GSE89106).

Differential gene expression of Mtb treated with AC2P36 vs DMSO control at pH 7.0 (Supplemental Tables S2).

Differential gene expression of Mtb treated with AC2P36 vs DMSO at pH 5.7 (Supplemental Table S3).

Differential Gene expression of Mtb treated with DMSO pH 5.7 vs pH 7.0 (Supplemental Table S4).

RNA-seq data of M. tuberculosis gene expression in response to MmpL3 inhibitors HC2091 and SQ109 (see Zheng et al., 2018, GEO Database GSE107485)

Differential gene expression of Mtb treated with HC2091 vs DMSO or SQ109 vs DMSO (Supplemental Table S4)

Complete gene lists for Mtb treated with HC2091 vs DMSO or SQ109 vs DMSO (Supplemental Table S5).

Tarn Biosciences is a company, founded by Robert Abramovitch, to translate our discoveries at the bench into new therapies for tuberculosis and non-tuberculous mycobacterial infections. Learn more about this work at www.tarnbio.com.

Ben conducted studies on how Mtb adapts to acidic pH. His studies included the completion of a HTS of a >273,000 compound library for inhibitors of PhoPR signaling. He discovered that ethoxzolamide inhibits PhoPR signaling (leading to US Patent 10,653,679) and that AC2P36 kills Mtb at acidic pH by depleting free thiols. Ben also developed a bioinformatic pipeline to conduct RNA-seq analyses. As part of his PhD training Ben published nine papers.

Following graduation, Ben worked as a bioinformatics specialist and then a postdoc at the Van Andel Institute.

Huiqing conducted studies on chemical biology of hypoxia-driven adaptation. As part of this work, Huiqing screened a >540,000 compound library and discovered six distinct scaffolds that inhibit DosRST signaling. These novel chemical probes have established new mechanisms to study bacterial signaling pathways and develop therapeutics that could shorten the course of therapy. Huiqing published five papers and a book chapter during his training.

Directly from his PhD training, Huiqing was hired as a senior biologist at DuPont.

Jake conducted genetic studies of how Mtb adapts to acidic environments. Jake discovered that Mtb has restricted metabolism at acidic pH and that PhoPR is required to slow Mtb growth at acidic pH. This work allowed Jake to develop a novel model of Mtb non-replicating persistence that we have called Acid Growth Arrest. Leveraging the acid growth arrest model, Jake conducted an innovative forward genetic screen that identified mutants in PPE51 that promote Mtb growth under conditions that normally restrict growth. Jake published three papers during his studies and has two additional studies in preparation.

Following graduation, Jake completed his MD and joined an internal medicine residency program at Spectrum Health.

John conducted genetic and chemical biology studies examining targets in the mycobacterial cell wall. As part of this work, John conducted an innovative, targeted screen that led to the discovery of >10 new inhibitors of a cell envelope transporter MmpL3. He also identified mechanisms by which chelation and osmotic stress kill mycobacteria. John has published 4 papers from his studies, with several more papers in the final stages of preparation. He is also an inventor on a patent application.

Following his PhD, John is continuing his research on mycobacteria in the laboratory of Prof. Christopher Sassetti, at UMass medical school.

Shelby conducted research on how M. tuberculosis adapts to acidic pH, using both chemical biology and genetic approaches (Baker, Dechow, and Abramovitch, 2019). As part of this work, Shelby discovered and characterized a new small molecule AC2P20, that selectively kills M. tuberculosis at acidic pH by depleting free thiols (Dechow et al., 2021). Shelby also conducted studies showing the ppe51 gene promotes glycerol uptake at acidic pH and is required for pathogenesis (Dechow et al., 2022) and that PhoPR plays a role in adaptation to carbon dioxide (Dechow et al. 2022). As part of her research, Shelby authored 3 first author manuscripts and has several additional first and co-author papers in preparation.

Following graduation, Shelby is employed as the Assistant Director of Research Integrity at the University of Michigan.