Abstract
Antibiotic resistance poses a serious threat to global health. To reinforce the anti-infective arsenal, many novel therapeutic strategies to fight bacterial infections are being explored. Among them, anti-virulence therapies, which target pathways important for virulence, have attracted much attention. Nitric oxide (NO) defense systems have been identified as critical for the pathogenesis of various bacteria, making them an appealing therapeutic target. In this study, we performed chemical screens to identify inhibitors of NO detoxification in Escherichia coli. We found that 2-mercaptobenzothiazole (2-MBT) can potently inhibit cellular detoxification of NO, achieving a level of inhibition that resembled the effect of genetically removing Hmp, the dominant detoxification enzyme under oxygenated conditions. Further analysis revealed that in the presence of NO, 2-MBT impaired the catalysis of Hmp and synthesis of Hmp and other proteins, whereas in its absence there were minimal perturbations to growth and protein synthesis. In addition, by studying the structure-activity relationship of 2-MBT, we found that both sulfur atoms in 2-MBT were vital for its inhibition of NO detoxification. Interestingly, when 2-mercaptothiazole (2-MT), which lacked the benzene ring, was used, differing biological activities were observed, although they too were NO dependent. Specifically, 2-MT could still prohibit NO detoxification, though it did not interfere with Hmp catalysis; rather, it was a stronger inhibitor of protein synthesis and it reduced the transcript levels of hmp, which was not observed with 2-MBT. Overall, these results provide a strong foundation for further exploration of 2-MBT and 2-MT for therapeutic applications.
Highlights
Antibiotic resistance remains a significant threat to modern medicine (Frieri et al, 2017; The Center for Disease, Dynamics Economics and Policy, 2018)
This study identified 2-MBT and 2-MT as compounds that selectively enhance the antibacterial activity of Nitric oxide (NO), elucidated that their mechanisms of action were multifaceted, and delineated chemical moieties that were important for their activities
When NO was present, protein synthesis was generally reduced compared to the growing control, GFPSF abundance steadily increased in the DMSO-only sample (Figure 9B and Supplementary Figure S10), whereas both 2-MBT and 2-MT drastically stifled GFPSF synthesis, achieving a terminal fluorescence level ∼18% and ∼10%, respectively, of that of the DMSO-only control. These results suggest that the inhibition of protein synthesis by 2-MBT and 2-MT is specific to a NO-containing environment and it is not limited to Hmp
Summary
Antibiotic resistance remains a significant threat to modern medicine (Frieri et al, 2017; The Center for Disease, Dynamics Economics and Policy, 2018). Infections by resistant pathogens contribute to more than 2.8 million hospitalizations and result in more than 35,000 deaths every year in the US alone (CDC, 2019). Increasing occurrences of bacteria resistant to last resort antibiotics indicate the precarious state of the antibiotic arsenal (Bratu and Eramo, 2005; Kumarasamy et al, 2010; Meletis, 2016). To address these issues, alternative modalities to treat bacterial infections have been proposed and explored (Clatworthy et al, 2007; Allen et al, 2014; Mahlapuu et al, 2016). These strategies include bacteriophages (Wright et al, 2009; Kutateladze and Adamia, 2010), antimicrobial peptides (Hancock and Sahl, 2006; Mahlapuu et al, 2016), predatory bacteria that prey on pathogens (Kadouri et al, 2013), and agents that target virulence factors that are critical for pathogenesis (Cegelski et al, 2008; Brannon and Hadjifrangiskou, 2016; Dickey et al, 2017; Fleitas Martinez et al, 2019)
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