Bacillus thuringiensis Spores and Vegetative Bacteria: Infection Capacity and Role of the Virulence Regulon PlcR Following Intrahaemocoel Injection of Galleria mellonella

Abstract

Bacillus thuringiensis is an invertebrate pathogen that produces insecticidal crystal toxins acting on the intestinal barrier. In the Galleria mellonella larvae infection model, toxins from the PlcR virulence regulon contribute to pathogenicity by the oral route. While B. thuringiensis is principally an oral pathogen, bacteria may also reach the insect haemocoel following injury of the cuticle. Here, we address the question of spore virulence as compared to vegetative cells when the wild-type Bt407cry- strain and its isogenic ∆plcR mutant are inoculated directly into G. mellonella haemocoel. Mortality dose-response curves were constructed at 25 and 37 °C using spores or vegetative cell inocula, and the 50% lethal dose (LD₅₀) in all infection conditions was determined after 48 h of infection. Our findings show that (i) the LD₅₀ is lower for spores than for vegetative cells for both strains, while the temperature has no significant influence, and (ii) the ∆plcR mutant is four to six times less virulent than the wild-type strain in all infection conditions. Our results suggest that the environmental resistant spores are the most infecting form in haemocoel and that the PlcR virulence regulon plays an important role in toxicity when reaching the haemocoel from the cuticle and not only following ingestion.

Keywords: Bacillus thuringiensis; Galleria mellonella; PlcR-regulon; bio-pesticide; haemocoel; spores; virulence.

Figure 1

Dose–response mortality curves at 48 h. Larvae mortalities are plotted against bacterial doses and are shown as green dots (WT), Bt407WT, or as red dots, (plcR) ∆ plcR mutant. Non-linear regressions fitted to the experimental data are shown as plain lines (WT) or dotted lines (plcR). Mortalities at 48 h post-injection of spores at incubation temperature of 25 °C (A) or 37 °C (B). Mortalities post-injection of vegetative bacteria at 25 °C (C) or 37 °C (D).

Figure 2

Temperature has no significant influence on the LD₅₀. Comparisons between 25 and 37 °C of LD₅₀ values (represented as double arrow lines). Y-axis Bacteria (dose in CFU: colony forming units), X-axis: various conditions). LD₅₀ were considered as significantly different, with p < 0.05 for the null hypothesis, when the CI_95% did not overlap. Non-significant (NS) (blue); significant (* red).