Related Experiment Video
Updated: Oct 25, 2025
Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging
Published on: July 9, 2021
Laser capture microdissection to study Bacillus cereus iron homeostasis gene expression during Galleria mellonella in
Laurent Consentino1, Agnès Rejasse1, Nicolas Crapart2,3
1Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, Jouy-en-Josas, France.
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.
Insights
This study investigated iron homeostasis in Bacillus cereus during gut infections using a Galleria mellonella model. Findings show bacterial iron genes are expressed throughout infection, suggesting a role in B. cereus gut colonization.
Area of Science:
- Microbiology
- Pathogen-host interactions
- Molecular biology
Background:
- Bacillus cereus is a Gram-positive opportunistic pathogen causing intestinal infections.
- Iron is crucial for pathogen growth and virulence, but its role in gut infections is poorly understood.
- Understanding bacterial iron homeostasis is key to controlling infections.
Purpose of the Study:
- To assess the expression of B. cereus genes involved in iron homeostasis, virulence, and oxidative stress during gut infection.
- To investigate the correlation between gene expression, colonization stage, and gut cell damage.
- To evaluate the utility of Laser-capture microdissection (LCM) for in situ gene expression analysis of extracellular bacteria.
Main Methods:
- A germ-free Galleria mellonella model was utilized.
- Laser-capture microdissection (LCM) was adapted to isolate specific gut lumen areas from cryo-sections.
- Specific pre-amplification of mRNA followed by quantitative PCR (qPCR) was used to assess gene expression in live larvae.
Main Results:
- LCM proved reliable for bacterial gene expression analysis, despite low RNA yield.
- Bacterial iron homeostasis genes were expressed in the gut lumen at 3 and 16 hours post-infection.
- Host ferritin and transferrin mRNA were also detected, indicating host iron response.
- B. cereus iron gene expression showed slight modulation during infection.
Conclusions:
- Iron homeostasis plays a significant role in Bacillus cereus gut colonization.
- LCM is a valuable technique for in situ gene expression analysis of extracellular bacteria within a whole animal model.
- Further research into host-pathogen iron interactions is warranted.
