Abstract
Tuberculosis continues to kill millions of people each year. The main difficulty in eradication of the disease is the prolonged duration of treatment, which takes at least 6 months. Persister cells have long been associated with failed treatment and disease relapse because of their phenotypical, though transient, tolerance to drugs. By targeting these persisters, the duration of treatment could be shortened, leading to improved tuberculosis treatment and a reduction in transmission. The unique in vivo environment drives the generation of persisters; however, appropriate in vivo mycobacterial persister models enabling optimized drug screening are lacking. To set up a persister infection model that is suitable for this, we infected zebrafish embryos with in vitro-starved Mycobacterium marinumIn vitro starvation resulted in a persister-like phenotype with the accumulation of stored neutral lipids and concomitant increased tolerance to ethambutol. However, these starved wild-type M. marinum organisms rapidly lost their persister phenotype in vivo To prolong the persister phenotype in vivo, we subsequently generated and analyzed mutants lacking functional resuscitation-promoting factors (Rpfs). Interestingly, the ΔrpfAB mutant, lacking two Rpfs, established an infection in vivo, whereas a nutrient-starved ΔrpfAB mutant did maintain its persister phenotype in vivo This mutant was, after nutrient starvation, also tolerant to ethambutol treatment in vivo, as would be expected for persisters. We propose that this zebrafish embryo model with ΔrpfAB mutant bacteria is a valuable addition for drug screening purposes and specifically screens to target mycobacterial persisters.
Highlights
Tuberculosis continues to kill millions of people each year
Exposing M. tuberculosis to nutrient starvation in phosphate-buffered saline (PBS) supplemented with 0.025% Tween 80 induced drug tolerance to TB antimicrobials [27]. This phenotype strongly resembles characteristics of persister cells; we hypothesized that exposure of M. marinum to this condition could induce M. marinum persisters in vitro
When M. marinum was transferred to PBS with 0.025% Tween 80, growth was halted and there was no increase in CFU counts (Fig. 1); the CFU count of these starved M. marinum cells remained stable over the period of 14 days, indicating that this condition induced a nongrowing persister phenotype
Summary
Tuberculosis continues to kill millions of people each year. The main difficulty in eradication of the disease is the prolonged duration of treatment, which takes at least 6 months. The ΔrpfAB mutant, lacking two Rpfs, established an infection in vivo, whereas a nutrient-starved ΔrpfAB mutant did maintain its persister phenotype in vivo This mutant was, after nutrient starvation, tolerant to ethambutol treatment in vivo, as would be expected for persisters. It is believed that prolonged treatment is required because of the presence of so-called M. tuberculosis persister cells These persisters are phenotypically and transiently tolerant to antimicrobials that are used for TB treatment and, are not efficiently eradicated with a shorter treatment regimen. Compounds that target persisters efficiently will reduce the time of treatment and, aid in reducing TB burden To screen for such compounds, models for the generation of M. tuberculosis persisters are required. Formed persisters can withstand the environmental stresses in the host, which are difficult to mimic in vitro
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