Role of DNA Double-Strand Break Formation in Gyrase Inhibitor-Mediated Killing of Nonreplicating Persistent Mycobacterium tuberculosis in Caseum

Abstract

Tuberculosis is the leading cause of mortality by infectious agents worldwide. The necrotic debris, known as caseum, which accumulates in the center of pulmonary lesions and cavities is home to nonreplicating drug-tolerant Mycobacterium tuberculosis that presents a significant hurdle to achieving a fast and durable cure. Fluoroquinolones such as moxifloxacin are highly effective at killing this nonreplicating persistent bacterial population and boosting TB lesion sterilization. Fluoroquinolones target bacterial DNA gyrase, which catalyzes the negative supercoiling of DNA and relaxes supercoils ahead of replication forks. In this study, we investigated the potency of several other classes of gyrase inhibitors against M. tuberculosis in different states of replication. In contrast to fluoroquinolones, many other gyrase inhibitors kill only replicating bacterial cultures but produce negligible cidal activity against M. tuberculosis in ex vivo rabbit caseum. We demonstrate that while these inhibitors are capable of inhibiting M. tuberculosis gyrase DNA supercoiling activity, fluoroquinolones are unique in their ability to cleave double-stranded DNA at low micromolar concentrations. We hypothesize that double-strand break formation is an important driver of gyrase inhibitor-mediated bactericidal potency against nonreplicating persistent M. tuberculosis populations in the host. This study provides general insight into the lesion sterilization potential of different gyrase inhibitor classes and informs the development of more effective chemotherapeutic options against persistent mycobacterial infections.

Keywords: Mycobacterium tuberculosis; caseum; double-strand break; fluoroquinolones; nonreplicating persistence.

Figure 1

Pathways for gyrase inhibitor-mediated lethality. The gyrase heterotetramer A₂B₂ binds to DNA ahead of a replication fork. FQ binds to gyrase, stabilizing the gyrase–DNA cleaved complex. This traps the gyrase, preventing it from relaxing DNA and stalling both (1) DNA replication and (2) transcription. When the enzyme is released, a double-strand break (DSB) is revealed. The presence of DSB triggers the (3) accumulation of intrabacterial ROS and (4) chromosome fragmentation. Created with BioRender.

Figure 2

Dose response curves for 11 DNA replication inhibitors against nonreplicating persistent M. tuberculosis in ex vivo rabbit caseum. Bacterial burdens are expressed as colony-forming units (CFU) per milliliter of caseum homogenate. Both axes are shown on log scales. Dashed lines indicate the cutoffs for 90% bacterial killing. Dots and error bars represent the means and standard deviations of the triplicate samples.

Figure 3

Inhibition of DNA gyrase supercoiling activity. M. tuberculosis gyrase introduces negative supercoils in the relaxed pBR322 plasmid. The relaxed (R) and supercoiled (SC) forms of pBR322 were separated on a 1% agarose gel. The gel image illustrates the inhibition of DNA supercoiling by nine gyrase inhibitors. Nargenicin and cyclohexyl-griselimycin were included as negative controls. Each assay was performed at least twice, and representative images are shown.

Figure 4

DNA cleavage assay. M. tuberculosis DNA gyrase relaxes positively supercoiled plasmid DNA. Compounds that inhibit this process by stabilizing the cleavage complex and forming DSB produce linearized DNA. The different forms of plasmid pBR322 are separated on a 1% agarose gel and stained with ethidium bromide. N, nicked DNA; L, linear DNA; SC, supercoiled DNA.

Figure 5

Inhibition of DNA gyrase and bactericidal activity of two new fluoroquinolones. (A) M. tuberculosis gyrase introduces negative supercoils into the relaxed pBR322 plasmid. The relaxed (R) and supercoiled (SC) forms of pBR322 were separated on a 1% agarose gel. (B) DNA cleavage activity of delafloxacin (DFX) and sitafloxacin (STFX). Nicked (N), linear (L), and supercoiled (SC) forms of the pBR322 plasmid were separated on a 1% agarose gel. (C) Bacterial burdens in rabbit caseum are expressed as CFU per mL of caseum homogenate. Both axes are shown on log scales. Dashed lines indicate the cutoffs for 90% bacterial killing. Dots and error bars represent the means and standard deviations of triplicate samples.