A label-free approach for relative spatial quantitation of c-di-GMP in microbial biofilms

Abstract

Microbial biofilms represent an important lifestyle for bacteria and are dynamic three dimensional structures. Cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous signaling molecule that is known to be tightly regulated with biofilm processes. While measurements of global levels of c-di-GMP have proven valuable towards understanding the genetic control of c-di-GMP production, there is a need for tools to observe the local changes of c-di-GMP production in biofilm processes. We have developed a label-free method for the direct detection of c-di-GMP in microbial colony biofilms using matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). We applied this method to the enteric pathogen Vibrio cholerae, the marine symbiont V. fischeri, and the opportunistic pathogen Pseudomonas aeruginosa PA14 and detected spatial and temporal changes in c-di-GMP signal that accompanied genetic alterations in factors that synthesize and degrade the compound. We further demonstrated how this method can be simultaneously applied to detect additional metabolites of interest in a single experiment.

Matrix-assisted laser desorption/ionization mass spectrometry imaging for quorum sensing

“…MALDI has already been applied for multiple microbiological applications, including bacterial imaging, providing novel insights into QS signalling, distribution, and metabolite quantification. (Feucherolles and Frache 2022;McCaughey et al 2023) Indeed, MALDI has been shown as a valuable technique to gain insight into QS pathways within biofilms, as it, for example, revealed the existence of an N-Acyl-homoserine lactone (AHL) QS system in Pseudomonas putida biofilms, coordinating bacterial cell density during biofilm maturation, and a relationship between biofilm development and spatial production of QS metabolites has been shown. (Pitchapa et al 2022) The AHL signalling involved in QS of gram-negative bacteria has been extensively studied due to the involvement in physiological activities of the biofilm, such as nutrient acquisition, conjugation and biofilm formation.…”