visualization-germinosomes-inner-membrane-bacillus-subtilis

Visualization of Germinosomes and the Inner Membrane in <em>Bacillus subtilis</em> Spores

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence ...

University of Amsterdam

Amphipathic cationic antimicrobial peptides (AMPs) TC19 and TC84, derived from the major AMPs of human blood platelets, thrombocidins, and Bactericidal Peptide 2 (BP2), a synthetic designer peptide showed to perturb the membrane of Bacillus subtilis. We aimed to determine the means by which the three AMPs cause membrane perturbation in vivo using B. subtilis and to evaluate whether the membrane alterations are dependent on the phospholipid composition of the membrane.

Bactericidal activity of amphipathic cationic antimicrobial peptides involves altering the membrane fluidity when interacting with the phospholipid bilayer.

An empirical approach was taken to screen a novel synthetic compound library designed to be active against Gram-positive bacteria. We obtained five compounds that were active against spores from the model organism Bacillus subtilis and the food-borne pathogen Bacillus cereus during our population based experiments. Using single cell live imaging we were able to observe effects of the compounds on spore germination and outgrowth. Difference in sensitivity to the compounds could be observed between B. subtilis and B. cereus using live imaging, with minor difference in the minimal inhibitory and bactericidal concentrations of the compounds against the spores. The compounds all delayed the bursting time of germinated spores and affected the generation time of vegetative cells at sub-inhibitory concentrations. At inhibitory concentrations spore outgrowth was prevented. One compound showed an unexpected potential for preventing spore germination at inhibitory concentrations, which merits further investigation. Our study shows the valuable role single cell live imaging can play in the final selection process of antimicrobial compounds.

Evaluating novel synthetic compounds active against Bacillus subtilis and Bacillus cereus spores using Live imaging with SporeTrackerX.

Bacterial spores are ubiquitous in nature and can withstand both chemical and physical stresses. Spores can survive food preservation processes and upon outgrowth cause food spoilage as well as safety risks. The heterogeneous germination and outgrowth behavior of isogenic spore populations exacerbates this risk. A major unknown factor of spores is likely to be the inherently heterogeneous spore protein composition. The proteomics methods discussed here help in broadening the knowledge about spore structure and identification of putative target proteins from spores of different spore formers. Approaches to synchronize Bacillus subtilis spore formation, and to analyze spore proteins as well as the physiology of spore germination and outgrowth are also discussed. Live-imaging and fluorescence microscopy techniques discussed here allow analysis, at single cell level, of the 'germinosome', the process of spore germination itself, spore outgrowth and the spore intracellular pH dynamics. For the latter, a recently published improved pHluorin (IpHluorin) under control of the ptsG promoter is applicable. While the data obtained from such tools offers novel insight in the mechanisms of bacterial spore awakening, it may also be used to probe candidate antimicrobial compounds for inhibitory effects on spore germination and strengthen microbial risk assessment.

Article	Year
Bactericidal activity of amphipathic cationic antimicrobial peptides involves altering the membrane fluidity when interacting with the phospholipid bilayer. Biochimica et biophysica acta. Biomembranes\| PubMed ID: 29902419	2018
Evaluating novel synthetic compounds active against Bacillus subtilis and Bacillus cereus spores using Live imaging with SporeTrackerX. Scientific reports\| PubMed ID: 29904100	2018
Proteomics and microscopy tools for the study of antimicrobial resistance and germination mechanisms of bacterial spores. Food microbiology\| PubMed ID: 30910091	2019

Stanley Brul

Biography

JoVE Journal Publications

Other Publications