Laser capture microdissection to study Bacillus cereus iron homeostasis gene expression during Galleria mellonella in vivo gut colonization

Abstract

Bacillus cereus is a Gram-positive opportunistic pathogen closely related to the entomopathogen, Bacillus thuringiensis , both of which are involved in intestinal infections. Iron is an essential micronutrient for full growth and virulence of pathogens during infection. However, little is known about iron homeostasis during gut infection. Therefore, we aimed to assess the expression of B. cereus genes related to bacterial iron homeostasis, virulence and oxidative stress. The hypothesis is that the expression of such genes would vary between early and later stage colonization in correlation to gut cell damage. To perform the study, a germ-free Galleria mellonella model was set up in order to adapt the use of Laser-capture microdissection (LCM), to select precise areas in the gut lumen from frozen whole larval cryo-sections. Analyses were performed from alive larvae and the expression of targeted genes was assessed byspecific pre-amplification of mRNA followed by quantitative PCR. Firstly, the results reinforce the reliability of LCM, despite a low amount of bacterial RNA recovered. Secondly, bacterial genes involved in iron homeostasis are expressed in the lumen at both 3 and 16 hours post force-feeding. Thirdly, iron gene expression is slightly modulated during gut infection, and lastly, the mRNA of G. mellonella encoding for ferritin and transferrin iron storage and transport are recovered too. Therefore, iron homeostasis should play a role in B. cereus gut colonization. Furthermore, we demonstrate for the first time the value of using LCM for specific in situ gene expression analysis of extracellular bacteria in a whole animal.

Bacillus thuringiensis Spores and Cry3A Toxins Act Synergistically to Expedite Colorado Potato Beetle Mortality

“…Enhancin-like, InhA and other metalloproteases, chitinases, phospholipase, cytolysins and some non-proteinaceous factors (zwittermycin A, β-exotoxins) produced by Bt cells are also linked to virulence and infection outcomes, due to cytotoxic effects or as regulators of major toxin activity [9,10]. There is evidence that bacteria could express some of these virulence and homeostatic factors during insect colonisation in order to overcome host defenses [11,12].…”

A sporulation-independent way of life for Bacillus thuringiensis in the late stages of an infection

“…This is consistent with the activation of the iron homeostasis genes dhbA and isdE1 , belonging to this regulon, during all stages of the infection. Iron is essential for the function of various proteins and iron homeostasis was previously reported as being important for B. cereus pathogenesis in the first stages of G. mellonella infection by oral gavage [39]. However, this metal is also a potential hazard in combination with compounds such as H 2 O 2 because the reaction between the two products generates reactive oxygen species (ROS) that can be harmful by damaging cellular components such as DNA or proteins with [Fe-S] clusters [40].…”

The utilization of Blaptica dubia cockroaches as an in vivo model to test antibiotic efficacy

“…Many previous studies have investigated the use of insects to model mammalian infection and to evaluate antibiotic efficacy 8 – 22 , 24 – 30 . Probably the most extensively utilized insect model for studies involving microbial pathogenesis and antibiotic efficacy is the waxworm, G. mellonella 14 .…”