Efficacy of β-lactam\β-lactamase inhibitor combination is linked to WhiB4 mediated changes in redox physiology of Mycobacterium tuberculosis
- Indian Institute of Science, India
- National Institute of Immunology, India
Abstract
Mycobacterium tuberculosis (Mtb) expresses a broad-spectrum β-lactamase (BlaC) that mediates resistance to one of the highly effective antibacterials, β-lactams. Nonetheless, β-lactams showed mycobactericidal activity in combination with β-lactamase inhibitor, clavulanate (Clav). However, the mechanistic aspects of how Mtb responds to β-lactams such as Amoxicillin in combination with Clav (referred as Augmentin [AG]) are not clear. Here, we identified cytoplasmic redox potential and intracellular redox sensor, WhiB4, as key determinants of mycobacterial resistance against AG. Using computer-based, biochemical, redox-biosensor, and genetic strategies, we uncovered a functional linkage between specific determinants of β-lactam resistance (e.g., β-lactamase) and redox potential in Mtb. We also describe the role of WhiB4 in coordinating the activity of β-lactamase in a redox-dependent manner to tolerate AG. Disruption of WhiB4 enhances AG tolerance, whereas overexpression potentiates AG activity against drug-resistant Mtb. Our findings suggest that AG can be exploited to diminish drug-resistance in Mtb through redox-based interventions. through redox-based interventions.
Data availability
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Transcriptomic analysis of Mtb H37Rv and MtbΔwhiB4 on treatment to 10X Augmentin (AG) i.e. combination of 100 µg/ml of Amoxicillin and 8 µg/ml of Clavulanate and lower Augmentin doses at 1X and 5XPublicly available at the NCBI Gene Expression Omnibus (accession no: GSE93091).
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Transcriptomic analysis of Mtb H37Rrv and MtbΔwhiB4 upon treatment with 0.25 mM CHPPublicly available at the NCBI Gene Expression Omnibus (accession no: GSE73877).
Article and author information
Author details
Funding
Department of Biotechnology , Ministry of Science and Technology (BT/PR5020/MED/29/1454/2012)
- Amit Singh
Wellcome (WT-DBT/500034-Z-09-Z)
- Amit Singh
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: This study was carried out in strict accordance with the guidelines provided by the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA), Government of India. The protocol was approved by the Committee on the Ethics of Animal Experiments of the International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India (Approval number: ICGEB/AH/2011/2/IMM-26). All efforts were made to minimize the suffering.
Copyright
© 2017, MISHRA et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Efficacy of β-lactam\β-lactamase inhibitor combination is linked to WhiB4 mediated changes in redox physiology of Mycobacterium tuberculosis
eLife 6:e25624.
https://doi.org/10.7554/eLife.25624
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Chlamydia trachomatis is an obligate intracellular bacterial pathogen with a unique developmental cycle. It differentiates between two functional and morphological forms: the elementary body (EB) and the reticulate body (RB). The signals that trigger differentiation from one form to the other are unknown. EBs and RBs have distinctive characteristics that distinguish them, including their size, infectivity, proteome, and transcriptome. Intriguingly, they also differ in their overall redox status as EBs are oxidized and RBs are reduced. We hypothesize that alterations in redox may serve as a trigger for secondary differentiation. To test this, we examined the function of the primary antioxidant enzyme alkyl hydroperoxide reductase subunit C (AhpC), a well-known member of the peroxiredoxins family, in chlamydial growth and development. Based on our hypothesis, we predicted that altering the expression of ahpC would modulate chlamydial redox status and trigger earlier or delayed secondary differentiation. Therefore, we created ahpC overexpression and knockdown strains. During ahpC knockdown, ROS levels were elevated, and the bacteria were sensitive to a broad set of peroxide stresses. Interestingly, we observed increased expression of EB-associated genes and concurrent higher production of EBs at an earlier time in the developmental cycle, indicating earlier secondary differentiation occurs under elevated oxidation conditions. In contrast, overexpression of AhpC created a resistant phenotype against oxidizing agents and delayed secondary differentiation. Together, these results indicate that redox potential is a critical factor in developmental cycle progression. For the first time, our study provides a mechanism of chlamydial secondary differentiation dependent on redox status.
