Dataset Information

Identification of Native Cross-links in Bacillus subtilis Spore Coat Proteins

ABSTRACT: The resistance properties of the bacterial spores are partially due to spore surface proteins, ~30% of which are said to form an insoluble protein fraction. Previous research has also identified a group of spore coat proteins affected by spore maturation, which exhibit an increased level of inter protein cross-linking. However, the proteins and the types of cross-links involved, previously proposed based on indirect evidence, have yet to be confirmed experimentally. To obtain more insight into the structural basis the proteinaceous component of the spore coat we attempted to identify coat cross-links and the proteins involved using new peptide fractionation and bioinformatic methods. Young (day 1) and matured (day 5) Bacillus subtilis spores of wild type and transglutaminase mutant strains were digested with formic acid and trypsin, and cross-linked peptides were enriched using strong cation exchange chromatography. The enriched cross-linked peptide fractions were subjected to FT-ICR MS/MS and the high-quality fragmentation data obtained were analysed using two specialized software tools, pLink2 and XiSearch, to identify cross-links. This analysis identified specific disulfide bonds between coat proteins CotE-CotE and CotJA-CotJC, obtained evidence for disulfide bonds in the spore crust proteins CotX, CotY and CotZ, and identified dityrosine and eplison-(gamma)-glutamyl-lysine cross-linked coat proteins. The findings in this report are the first direct biochemical data on protein cross-linking in the spore coat and the first direct evidence for the crosslinked building blocks of the highly ordered and resistant structure called the spore coat.

INSTRUMENT(S): apex Q

ORGANISM(S): Bacillus Subtilis (ncbitaxon:1423)

SUBMITTER: Stanley Brul

PROVIDER: MSV000086681 | MassIVE | Sat Jan 09 15:03:00 GMT 2021

SECONDARY ACCESSION(S): PXD023492

REPOSITORIES: MassIVE

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Identification of Native Cross-Links in <i>Bacillus subtilis</i> Spore Coat Proteins.

Ursem Rick R Swarge Bhagyashree B Abhyankar Wishwas R WR Buncherd Hansuk H de Koning Leo J LJ Setlow Peter P Brul Stanley S Kramer Gertjan G

Journal of proteome research 20210217 3

The resistance properties of the bacterial spores are partially due to spore surface proteins, ∼30% of which are said to form an insoluble protein fraction. Previous research has also identified a group of spore coat proteins affected by spore maturation, which exhibit an increased level of interprotein cross-linking. However, the proteins and the types of cross-links involved, previously proposed based on indirect evidence, have yet to be confirmed experimentally. To obtain more insight into th ...[more]

PMID: 33596081

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Project description:Bacterial spores play an important role in disease initiation, transmission and persistence. The outermost spore layer, the exosporium, is important as it is the first point of contact between the spore and the environment and may be involved in spore adherence, protection and germination. Clostridium sordellii is a highly lethal, spore forming pathogen that causes soft-tissue infections, enteritis and toxic-shock syndrome. Despite the importance of C. sordellii spores in disease, spore proteins from this bacterium have not been defined or interrogated functionally. In this study, we identified the C. sordellii outer spore proteome and two of the identified proteins, CSA and CSB, were characterised using a genetic and phenotypic approach. Both proteins were essential for the correct formation and positioning of the C. sordellii spore coat and exosporium. The absence of CSA reduced sporulation levels and increased spore sensitivity to heat, sodium hydroxide and hydrochloric acid. By comparison, CSB was required for normal levels of spore adherence to cervical, but not vaginal, cells, with csb mutant spores having increased adherence properties. The establishment of a mouse infection model of the gastrointestinal tract for C. sordellii allowed the role of CSA and CSB to be interrogated in an infected host. Following the oral administration of spores to mice, the wild-type strain efficiently colonized the gastrointestinal tract, with the peak of bacterial numbers occurring at one day post-infection. Colonization was reduced by two logs at four days post-infection. By comparison, mice infected with the csb mutant did not show a reduction in bacterial numbers. The absence of CSB therefore allows the csb mutant to persist within the gastrointestinal tract. We conclude that C. sordellii outer spore proteins are important for the structural and functional integrity of spores, and for colonization and persistence during infection. Finally, Clostridium difficile, Bacillus cereus and Bacillus anthracis encode proteins with homology to CSA and CSB but these bacteria produce spores that are structurally dissimilar to those of C. sordellii, and the function of the proteins in these hosts is different to that in C. sordellii. These findings suggest that, despite their homology, spore proteins can have variable functions in different bacterial species which highlights the necessity of studying each spore protein in the cognate species from which it originates.