Stoichiometry, Absolute Abundance, and Localization of Proteins in the Bacillus cereus Spore Coat Insoluble Fraction Determined Using a QconCAT Approach

Abstract

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 dependent on CotE for their association with the spore. Several of these are newly confirmed as associated with the exosporium, namely BC_2569 (BclF), BC_3345, BC_2427, BC_2878, BC_0666, BC_2984, BC_3481, and BC_2570. A total of 153 proteins were only identified in ΔCotE spore isolates. This was observed for proteins that are known or likely to be interacting with or are encased by CotE. Crucial spore proteins were quantified using a QconCAT reference standard, the first time this was used in a biochemically heterogeneous system. This allowed us to determine the absolute abundance of 21 proteins, which spanned across three orders of magnitude and together covered 5.66% ± 0.51 of the total spore weight. Applying the QconCAT methodology to the ΔCotE mutant allowed us to quantify 4.13% ± 0.14 of the spore total weight and revealed a reduction in abundance for most known exosporium associated proteins upon CotE deletion. In contrast, several proteins, either known or likely to be interacting with or encased by CotE (i.e., GerQ), were more abundant. The results obtained provide deeper insight into the layered spore structure such as which proteins are exposed on the outside of the spore. This information is important for developing detection methods for targeting spores in a food safety setting. Furthermore, protein stoichiometry and determination of the abundance of germination mediating enzymes provides useful information for germination and outgrowth model development.

Structural, Metabolic and Evolutionary Comparison of Bacterial Endospore and Exospore Formation

“…Intriguingly, deficiencies in these coat platform proteins also inhibit formation of the cortex ( Ebmeier et al, 2012 ; Benito de la Puebla et al, 2020 ). Additional spore coat morphogenesis proteins of Bacilli include SafA and CotE, which are essential for formation of the inner and outer coat layers, respectively ( Zheng et al, 1988 ; Takamatsu et al, 1999 ; Abhyankar et al, 2013 ; Stelder et al, 2018 ). In Clostridia, a recently identified protein, CotL, has been deemed important for assembly of the coat, although its function is poorly understood ( Alves Feliciano et al, 2019 ).…”

“…In Clostridia, a recently identified protein, CotL, has been deemed important for assembly of the coat, although its function is poorly understood ( Alves Feliciano et al, 2019 ). Altogether, the endospore coat usually comprises multiple layers, each characterized by distinct morphogenic and structural proteins ( McKenney et al, 2010 ; Stelder et al, 2018 ). While morphogenic proteins are relatively conserved, structural proteins can vary by organism and are tailored to its respective environmental niche ( Abhyankar et al, 2013 ).…”

The Membrane Proteome of Spores and Vegetative Cells of the Food-Borne Pathogen Bacillus cereus

“…We previously explored the abundance of insoluble proteins in the spore-coat of B. cereus using a targeted quantification concatemer (QconCAT) approach [18], in our current study we use a label-free approach to estimate protein abundance using iBAQ values (see above). The germinant receptors are of particular interest, as these are thought to be expressed specifically in the inner membrane of spores as is the case for B. subtilis [19].…”

Median based absolute quantification of proteins using Fully Unlabelled Generic Internal Standard (FUGIS)

“…As a complementary approach, absolute quantification determines the exact molar quantity of each protein in each condition. In this way, it is possible to relate the abundances of different proteins, estimate their expression level or determine stoichiometry within a variety of molecular constellations from stable complexes to organelles or metabolic pathways and interaction networks [1]- [10] .…”

“…In contrast, absolute quantification determines the exact molar quantity of each protein in each condition. In this way, it is possible to relate the molar abundances of different proteins, estimate their expression level or determine their stoichiometry within a variety of molecular constellations from stable complexes to organelles or metabolic pathways and interaction networks [1][2][3][4][5][6][7][8][9][10] . Absolute quantification holds an important promise to deliver reference values of individual proteins in liquid and solid biopsies, which is a pre-requisite for robust molecular diagnostics.…”