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. 2022 Jul 22:3:924957.
doi: 10.3389/fragi.2022.924957. eCollection 2022.

Mifepristone Increases Life Span in Female Drosophila Without Detectable Antibacterial Activity

Gary N Landis  1 , Luke Riggan  1 , Hans S Bell  1 , William Vu  1 , Tianyi Wang  1 , Ina Wang  1 , Felicia I Tejawinata  1 , Sebastian Ko  1 , John Tower  1
Affiliations

Mifepristone Increases Life Span in Female Drosophila Without Detectable Antibacterial Activity

Gary N Landis et al. Front Aging. .

Abstract

Mifepristone dramatically increases the life span of mated female Drosophila while reducing the expression of innate immune response genes. Previous results indicated that mifepristone also reduced the load of aero-tolerant bacteria in mated females. Experiments were conducted to further investigate the possible role of bacteria in mifepristone life span effects. Life span was assayed in flies grown from sterilized eggs on autoclaved media and in normally cultured controls in two independent assays. Sterilization increased mated female life span (+8.3% and +57%, respectively), and the effect of mifepristone was additive (+53% and +93%, respectively). High-throughput sequencing of 16S sequences revealed that sterilization reduced the abundance of multiple species and the classes Bacteroidia, Bacilli, Actinobacteria, and Cytophagia. By contrast, mifepristone caused no decreases and instead increased the abundance of three species. Five aero-tolerant bacterial species were cultured from extracts of mated female flies, including both Gram-positive and Gram-negative species (Acetobacter sicerae, Enterococcus faecalis, Lactobacillus plantarum, Serratia rubidea, and Paenibacillus glucanolyticus). There was no detectable effect of mifepristone on the growth of these bacteria in vitro, indicating that mifepristone does not have a direct antibiotic effect. To test if antibiotics could mimic the effects of mifepristone in vivo, mated female flies were treated throughout adult life span with high concentrations of the individual antibiotics doxycycline, ampicillin, kanamycin, and streptomycin, in replicate experiments. No significant effect on life span was observed for ampicillin, kanamycin, or streptomycin, and an inconsistent benefit was observed for doxycycline. Finally, supplementation of media with Enterococcus faecalis did not alter adult female life span in the presence or absence of mifepristone. Taken together, the results indicate the life span benefits of mifepristone are not due to an antibiotic effect.

Keywords: Drosophila; aging; bacteria; microbiome; mifepristone; steroid.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Additive life span benefit from egg sterilization and mifepristone. Life span was assayed in virgin females, mated females, and mated females plus mifepristone/RU486 (RU), using control culture conditions (A,C) and using egg sterilization and autoclaved media (B,D). (A) Control conditions, experiment 1. (B) Sterile conditions, experiment 1. (C) Control conditions, experiment 2. (D) Sterile conditions, experiment 2. (E) Statistical summary. For each experiment, mated is compared to virgin, and mated plus drug is compared to mated. Statistical test: log-rank; p-value for significance with two comparisons is 0.025.
FIGURE 2
FIGURE 2
Effect of sterilization and mifepristone on adult female bacterial species and identification of outliers. (A) Top 10 species, including outliers. (B) Top 10 species, excluding outliers. CV, control virgins; CM, control mated; CMRU, control mated plus mifepristone/RU486; SV, sterile virgins; SM, sterile mated; and SMRU, sterile mated plus mifepristone/RU486. Species and classes reported to be aero-tolerant are indicated with asterisk.
FIGURE 3
FIGURE 3
Effect of egg sterilization and mifepristone on adult female bacterial species and classes. (A) Total bacterial load of control flies compared to sterile condition flies, including outliers. Mann–Whitney p = 0.0145 (indicated with asterisk). (B) Total bacterial load of control flies compared to sterile condition flies, excluding outliers. Mann–Whitney p = 0.0173 (indicated with asterisk). (C) Total bacterial load of nontreated flies (−RU) compared to mifepristone (RU486)–treated flies (+RU), excluding outliers. Mann–Whitney p = 0.3286. (D) Average top ten species (excluding outliers). (E) Average top ten classes (excluding outliers). CV, control virgins; CM, control mated; CMRU, control mated plus mifepristone/RU486; SV, sterile virgins; SM, sterile mated; and SMRU, sterile mated plus mifepristone/RU486. Species and classes reported to be aero-tolerant are indicated with asterisk.
FIGURE 4
FIGURE 4
Effect of methoprene and mifepristone on virgin female life span, bacterial species, and classes. (A) Survival curves. C, minus-drug control; RU, mifepristone; and Met, methoprene. Data are replotted from Landis et al. (2021a). (B) Life span statistical summary. Percent change in median life span was calculated by comparing RU to C, and by comparing Met to C, and by comparing Met + RU to Met. Statistical test: log-rank; p-value for significance with three comparisons is 0.017. (C) Average top ten species (excluding outliers). (D) Average top ten classes (excluding outliers). C, control; RU, mifepristone/RU486; and Met, methoprene. For both species and classes, no statistically significant differences were detected between groups using Kruskal–Wallis test. Species and classes reported to be aero-tolerant are indicated with asterisk.
FIGURE 5
FIGURE 5
Mifepristone has no detectable antibacterial properties in vitro. Five abundant bacterial species were purified from single colonies. The colonies were produced by plating extracts of middle-age mated females, using the strain with the largest magnitude response to mating and to mifepristone (progeny of w[1118] X y;Elav-GS). Bacterial species identity was determined by sequencing bacterial 16S DNA. Over-night liquid cultures of the indicated bacterial species were serially diluted as indicated, and 2 μL aliquots were spotted onto control plates (-), and plates adjusted to 200 μg/ml final concentration mifepristone (+). (A) Lactobacillus plantarum. (B) Acetobacter sicerae. (C) Enterococcus faecalis. (D) Paenibacillus glucanolyticus. (E) Serratia rubidaea.
FIGURE 6
FIGURE 6
Effect of high-concentration antibiotics on mated female life span and microbial load. Life span was assayed in replicate experiments, for virgin females (V), mated females (M), and mated females (M), and mated females treated with doxycycline (D), ampicillin (A), kanamycin (K), and streptomycin (S). (A) Experiment 1. (B) Experiment 2. (C) Life span statistical summary. Percent change in median life span was calculated by comparing mated to virgin, and by comparing antibiotic-treated mated to mated. Statistical test: log-rank; p-value for significance with five comparisons is 0.01. (D) Average bacterial species (excluding outliers). (E) Average bacterial classes (excluding outliers). Species and classes reported to be aero-tolerant are indicated with asterisk.
FIGURE 7
FIGURE 7
Effect of Enterococcus faecalis supplementation on adult female life span. Flies with constitutive expression of sex peptide (genotype w[1118];dsx-GAL4/+; UAS-SP/+) were grown on media in absence (-D) and presence (+D) of doxycycline. Virgin females were collected and assayed for life span in absence (-Bact) or presence (+Bact) of added Enterococcus faecalis, and in absence (−RU) or presence (+RU) of 200 μg/ml mifepristone/RU486, in three replicate experiments. (A) Survival curves for experiment 1. (B) Statistical summary for experiments 1–3. For each experiment, +RU is compared to −RU. Statistical test: log-rank; p-value for significance with one comparisons is 0.05.
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