Visualizing Pyrazinamide Action by Live Single-Cell Imaging of Phagosome Acidification and Mycobacterium tuberculosis pH Homeostasis

Santucci, Pierre; Aylan, Beren; Botella, Laure; Bernard, Elliott M.; Bussi, Claudio; Pellegrino, Enrica; Athanasiadi, Natalia; Gutierrez, Maximiliano G.

doi:10.1128/mbio.00117-22

Cited by 20 publications

(8 citation statements)

References 58 publications

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“…Previous work described POA T -induced acidification of the cytoplasm of M. tuberculosis H37Ra and suggested the uncoupling of proton motive force by POA N ( Fontes et al, 2020 ). Similarly, PZA has been shown to acidify the mycobacterial cytoplasm ( Peterson et al, 2015 ; Santucci et al, 2022 ). However, it was not clear that cytoplasmic acidification was a specific effect of POA T or of anti-TB drugs and protonophores in general.…”

Section: Resultsmentioning

confidence: 99%

Pyrazinoic acid, the active form of the anti-tuberculosis drug pyrazinamide, and aromatic carboxylic acid analogs are protonophores

Fontes,

Rooker,

Lynn-Barbe

et al. 2024

Front. Mol. Biosci.

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Pyrazinoic acid is the active form of pyrazinamide, a first-line antibiotic used to treat Mycobacterium tuberculosis infections. However, the mechanism of action of pyrazinoic acid remains a subject of debate, and alternatives to pyrazinamide in cases of resistance are not available. The work presented here demonstrates that pyrazinoic acid and known protonophores including salicylic acid, benzoic acid, and carbonyl cyanide m-chlorophenyl hydrazone all exhibit pH-dependent inhibition of mycobacterial growth activity over a physiologically relevant range of pH values. Other anti-tubercular drugs, including rifampin, isoniazid, bedaquiline, and p-aminosalicylic acid, do not exhibit similar pH-dependent growth-inhibitory activities. The growth inhibition curves of pyrazinoic, salicylic, benzoic, and picolinic acids, as well as carbonyl cyanide m-chlorophenyl hydrazone, all fit a quantitative structure–activity relationship (QSAR) derived from acid–base equilibria with R2 values > 0.95. The QSAR model indicates that growth inhibition relies solely on the concentration of the protonated forms of these weak acids (rather than the deprotonated forms). Moreover, pyrazinoic acid, salicylic acid, and carbonyl cyanide m-chlorophenyl hydrazone all caused acidification of the mycobacterial cytoplasm at concentrations that inhibit bacterial growth. Thus, it is concluded that pyrazinoic acid acts as an uncoupler of oxidative phosphorylation and that disruption of proton motive force is the primary mechanism of action of pyrazinoic acid rather than the inhibition of a classic enzyme activity.

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Section: Resultsmentioning

confidence: 99%

Pyrazinoic acid, the active form of the anti-tuberculosis drug pyrazinamide, and aromatic carboxylic acid analogs are protonophores

Fontes,

Rooker,

Lynn-Barbe

et al. 2024

Front. Mol. Biosci.

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“…Further reduction in pH of medium resulted in growth inhibition of several strains of bacteria (68). Similarly, in human monocyte-derived macrophages, treatment with concentrations as high as 100-400 µg/mL of PZA only resulted in about 70% reduction in bacteria as determined by fluorescence imaging (65,90). Thus, our platform is potentially an excellent substitute for current in vitro models and a starting point for more accurate drug testing and to give deeper insights into host-pathogen interaction before large-scale animal studies are performed.…”

Section: Thp-1 Cells Accumulate Lipid Bodies In Response To Infection...mentioning

confidence: 99%

Lung-mimicking 3-Dimensional hydrogel culture system recapitulates key tuberculosis phenotypes and demonstrates pyrazinamide efficacy

Kolluru

Abhirami

Jyothsna

et al. 2023

Preprint

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Faithful mimics of tuberculosis (TB) infection are warranted needed to providefor mechanistic insights into the complex host-pathogen interactions and for accelerating drug discovery. The current in vitro models provide a short duration to investigate the biology of this slow-growing pathogen, present divergent transcriptional signatures to human infections, and are unreliable drug discovery platforms. We developed and characterized a 3D collagen culture system mimicking the lung microenvironment (collagen fibres, pore size and stiffness), where we incorporated Mycobacterium tuberculosis (Mtb) infected THP-1 or primary monocytes. Dual RNA-sequencing revealed high similarity with human infections. Phenotypes observed in humans infection, such as foamy macrophages and mycobacterial cords (never seen in any other in vitro culture system), were reproduced in our culture system. The system overcomes many challenges associated with the traditional platforms for studying Mtb infections, including showing remarkable efficacy with clinically relevant concentrations of first-line anti-TB drug pyrazinamide, not seen in any other in vitro model, making it reliable, readily adoptable for tuberculosis studies and drug screening.

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“…In addition, Mtb can reside within various compartments inside innate immune cells, which can impact antibiotic efficacy. For example, pyrazinamide preferentially accumulates and is maximally active against Mtb in acidified compartments within the macrophage ( Santucci et al., 2021 ; Santucci et al., 2022 ).…”

Section: Factors That Contribute To Mtb Surviving ...mentioning

confidence: 99%

Understanding the contribution of metabolism to Mycobacterium tuberculosis drug tolerance

Samuels

Wang

Harrison

et al. 2022

Front. Cell. Infect. Microbiol.

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Treatment of Mycobacterium tuberculosis (Mtb) infections is particularly arduous. One challenge to effectively treating tuberculosis is that drug efficacy in vivo often fails to match drug efficacy in vitro. This is due to multiple reasons, including inadequate drug concentrations reaching Mtb at the site of infection and physiological changes of Mtb in response to host derived stresses that render the bacteria more tolerant to antibiotics. To more effectively and efficiently treat tuberculosis, it is necessary to better understand the physiologic state of Mtb that promotes drug tolerance in the host. Towards this end, multiple studies have converged on bacterial central carbon metabolism as a critical contributor to Mtb drug tolerance. In this review, we present the evidence that changes in central carbon metabolism can promote drug tolerance, depending on the environment surrounding Mtb. We posit that these metabolic pathways could be potential drug targets to stymie the development of drug tolerance and enhance the efficacy of current antimicrobial therapy.

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Visualizing Pyrazinamide Action by Live Single-Cell Imaging of Phagosome Acidification and Mycobacterium tuberculosis pH Homeostasis

Cited by 20 publications

References 58 publications

Pyrazinoic acid, the active form of the anti-tuberculosis drug pyrazinamide, and aromatic carboxylic acid analogs are protonophores

Pyrazinoic acid, the active form of the anti-tuberculosis drug pyrazinamide, and aromatic carboxylic acid analogs are protonophores

Lung-mimicking 3-Dimensional hydrogel culture system recapitulates key tuberculosis phenotypes and demonstrates pyrazinamide efficacy

Understanding the contribution of metabolism to Mycobacterium tuberculosis drug tolerance

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