StoichiometryAbsolute Abundance and Localizationof Proteins in the Bacillus cereus Spore Coat InsolubleFraction Determined Using a QconCAT Approach

摘要

Spores of Bacillus cereus pose a threat to food safety due to their high resistance to the heat or acid treatments commonly used to make food microbiologically safe. Spores may survive these treatments and later resume growth either on foodstuffs or, after ingestion, upon entering the gut they are capable of producing toxins, which cause either vomiting or diarrhea. The outer layers of the spore, the spore coat and exosporium, consist primarily of proteins that may serve as potential biomarkers for detection. The major morphogenetic protein CotE is important for correct assembly and attachment of the outermost layer, the exosporium, and by extension retention of many proteins. However, characterization of the proteins affected by deletion of CotE has been limited to electrophoretic patterns. Here we report the effect of CotE deletion on the insoluble fraction of the spore proteome through liquid chromatography–Fourier transform tandem mass spectrometry (LC–FTMS/MS) analysis. A total of 560 proteins have been identified in both mutant and wild-type spore coat isolates. A further 163 proteins were identified exclusively in wild-type spore isolates indicating that they are dependenton CotE for their association with the spore. Several of these arenewly confirmed as associated with the exosporium, namely BC_2569(BclF), BC_3345, BC_2427, BC_2878, BC_0666, BC_2984, BC_3481, andBC_2570. A total of 153 proteins were only identified in ΔCotEspore isolates. This was observed for proteins that are known or likelyto be interacting with or are encased by CotE. Crucial spore proteinswere quantified using a QconCAT reference standard, the first timethis was used in a biochemically heterogeneous system. This allowedus to determine the absolute abundance of 21 proteins, which spannedacross three orders of magnitude and together covered 5.66% ±0.51 of the total spore weight. Applying the QconCAT methodology tothe ΔCotE mutant allowed us to quantify 4.13% ± 0.14 ofthe spore total weight and revealed a reduction in abundance for mostknown exosporium associated proteins upon CotE deletion. In contrast,several proteins, either known or likely to be interacting with orencased by CotE (i.e., GerQ), were more abundant. The results obtainedprovide deeper insight into the layered spore structure such as whichproteins are exposed on the outside of the spore. This informationis important for developing detection methods for targeting sporesin a food safety setting. Furthermore, protein stoichiometry and determinationof the abundance of germination mediating enzymes provides usefulinformation for germination and outgrowth model development.