Generation of A Mucor circinelloides Reporter Strain-A Promising New Tool to Study Antifungal Drug Efficacy and Mucormycosis

Abstract

Invasive fungal infections caused by Mucorales (mucormycosis) have increased worldwide. These life-threatening infections affect mainly, but not exclusively, immunocompromised patients, and are characterized by rapid progression, severe tissue damage and an unacceptably high rate of mortality. Still, little is known about this disease and its successful therapy. New tools to understand mucormycosis and a screening method for novel antimycotics are required. Bioluminescent imaging is a powerful tool for in vitro and in vivo approaches. Hence, the objective of this work was to generate and functionally analyze bioluminescent reporter strains of Mucor circinelloides, one mucormycosis-causing pathogen. Reporter strains were constructed by targeted integration of the firefly luciferase gene under control of the M. circinelloides promoter Pzrt1. The luciferase gene was sufficiently expressed, and light emission was detected under several conditions. Phenotypic characteristics, virulence potential and antifungal susceptibility were indifferent to the wild-type strains. Light intensity was dependent on growth conditions and biomass, being suitable to determine antifungal efficacy in vitro. This work describes for the first time the generation of reporter strains in a basal fungus that will allow real-time, non-invasive infection monitoring in insect and murine models, and the testing of antifungal efficacy by means other than survival.

Keywords: Mucor circinelloides; bioluminescence; firefly luciferase; mucormycosis; reporter strain.

Figure 1

Growth phenotypes of recipient and luciferase-expressing strains grown for 24 h on different media at 30 °C (panels (A,B)), 37 °C (panels (C,D)) under normoxic (panels (A,C)) and hypoxic conditions (panels (B,D)). Hypoxia was induced by reducing the oxygen concentration in the incubator to 1%. SUP: supplemented minimal agar; YPG: yeast peptone glucose; YNB: yeast nitrogen base; RPMI: RPMI₁₆₄₀.

Figure 2

(A) Light emission in dependency of inoculum density. YNB medium was inoculated with different concentrations of R7B_luc spores, light emission was induced by addition of luciferin after 24 h and detected by plate reader (upper panel). Relative light units (RLUs) represent the average of three independent measurements. Error bars indicate standard deviation. The lower panel shows fungal growth at the various spore concentrations after 24 h of incubation. (B) Light emission in dependency of substrate concentration. 2 × 10⁵ spores/mL were inoculated in YNB and light emission was induced by addition of different concentrations (undiluted, 1:2, 1:5; 1:10) of luciferin dissolved according to the manufacturer´s protocol (Roche, Basel, Switzerland). Light was detected immediately (dark grey bars) and 10 min after substrate addition (light grey bars). Error bars indicate standard deviation.

Figure 3

Light emission of R7B_luc in different growth media. YNB medium was inoculated with 2 × 10⁵ spores/mL of R7B_luc, incubated for 16 h, and then replaced by fresh YNB, YPG, or RPMI, respectively. Light emission was induced by addition of substrate (luciferin 1:5) 3 h after medium exchange and detected by using a plate reader. RLUs were determined immediately after addition of substrate (black bars), 10 min (light grey bars) and 30 min (dark grey bars) after the addition of substrate. Error bars indicate standard deviation. RLUs emitted in each media were significantly different from the other media tested (Two-way analysis of variance (ANOVA), p < 0.05).

Figure 4

Graphical analysis of drug efficacy by detection of light emission. Luminescence was measured by a plate reader in wells containing 2 × 10⁵ spores/mL of R7B_luc grown in YNB in the presence of amphotericin B (AMB) (A) or posaconazole (POS) (B), and with AMB (C) or POS (D) added to hyphae, respectively. Light emission was detected after 24 h of incubation (A,B) and subsequent luciferin (1:5, Roche) addition. Pre-grown cultures (16 h) were further incubated for 4 h once antifungals were added (C,D). Average values from three independent wells are given, error bars represent standard deviation.

Figure 5

Survival of larvae infected with M. circinelloides strains. Larvae were infected with 10⁶ spores of the respective strains and incubated at 30 °C. (A) represents Kaplan-Meier curves of larvae infected with R7B or R7B_luc strain. (B) represents Kaplan-Meier curves of larvae infected with MU402 or MU402_luc strain. Survival was monitored every 24 h up to 144 h. Untouched larvae and larvae injected with IPS buffer served as controls. Results are expressed as the mean of three independent experiments (60 larvae in total).