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

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.

枯草芽孢杆菌的孢子对食品安全构成威胁，因其对常用于使食品微生物安全的热或酸处理具有高度抵抗力。孢子可能在这些处理中存活，并在随后在食品上或摄入后进入肠道时恢复生长，它们能够产生毒素，导致恶心或腹泻。孢子的外层，包括孢子壁和外壳，主要由可能作为检测潜在生物标志物的蛋白质组成。主要的形态发生蛋白CotE对于最外层外壳的正确组装和附着至关重要，进而保留了众多蛋白质。然而，删除CotE影响的蛋白质的特性分析仅限于电泳模式。在本研究中，我们通过液相色谱-傅里叶变换串联质谱（LC-FTMS/MS）分析，报告了CotE缺失对孢子蛋白组不溶性组分的影响。在突变型和野生型孢子壁分离物中总共鉴定出560种蛋白质。另外，有163种蛋白质仅在野生型孢子分离物中被鉴定出来，表明它们依赖于CotE与其结合。其中一些新确认与外壳相关，包括BC_2569（BclF）、BC_3345、BC_2427、BC_2878、BC_0666、BC_2984、BC_3481和BC_2570。在ΔCotE孢子分离物中仅鉴定出153种蛋白质。这包括已知或可能通过与CotE相互作用或被其包裹的蛋白质。关键的孢子蛋白使用QconCAT参考标准进行量化，这是首次在生物化学异质系统中使用。这使我们能够确定21种蛋白质的绝对丰度，这些蛋白质跨越三个数量级，共同占孢子总重量的5.66% ± 0.51。将QconCAT方法应用于ΔCotE突变体，使我们能够量化孢子总重量的4.13% ± 0.14，并揭示了在CotE缺失后大多数已知外壳相关蛋白丰度的减少。相反，一些已知或可能通过与CotE相互作用或被其包裹的蛋白质（例如GerQ）更为丰富。获得的结果为深入理解孢子层状结构提供了更深入的见解，例如哪些蛋白质暴露在孢子外部。这些信息对于开发针对食品安全环境中孢子的检测方法至关重要。此外，蛋白质化学计量和萌发介导酶的丰度测定为萌发和生长模型的发展提供了有用信息。