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INRAE

24, chemin de Borde Rouge -Auzeville - CS52627 31326 Castanet Tolosan cedex - France

Last update: May 2021

Menu Logo Principal AgroParisTech Université Paris-Saclay

INRA GABI Unit

GABI : Génétique Animale et Biologie IntégrativeUnité Mixte de Recherche INRA - AgroParisTech

Laser microdissection allows fine in situ analysis of the interaction between extracellular bacteria and the host

Light microscope observation of G. mellonella larvae infected with B. cereus associated with Cry1C toxin 16 hours after force-feeding. Cross–sections (16 µm) (cresyl violet staining and ethanol and xylene dehydration) with focus on the intestine (midgut). The bacteria appear colored as dark or brighter violet. Different magnifications are shown: (a) 4×; (b)10×; (c) 20×, and (d) 40×. (lum: intestinal lumen; bac: bacteria; gEpi: Gut Epithelium; pm: Peritrophic matrix
Laser capture microdissection to study Bacillus cereus iron homeostasis gene expression during Galleria mellonella in vivo gut colonization.

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.

Contacts: 

  • Claudia Bevilacqua 
  • Christina Nielsen-leroux

See also

Reference

Laurent Consentino, Agnès Rejasse, Nicolas Crapart, Claudia Bevilacqua & Christina Nielsen-LeRoux (2021) Laser capture microdissection to study Bacillus cereus iron homeostasis gene expression during Galleria mellonella in vivo gut colonization, Virulence, 12:1, 2104-2121, DOI: 10.1080/21505594.2021.1959790