In Pursuit of Protein Targets: Proteomic Characterization of Bacterial Spore Outer Layers

Abhyankar, Wishwas; Hossain, Abeer; Djajasaputra, André; Permpoonpattana, Patima; Beek, Alexander Ter; Dekker, Henk; Cutting, Simon M.; Brul, Stanley; Koning, Leo J. de; Koster, Chris G. de

doi:10.1021/pr4005629

Cited by 61 publications

(88 citation statements)

References 83 publications

(147 reference statements)

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“…exosporium proteins, except for the presence of collagen-like proteins (187). However, the lack of B. cereus family exosporium homologs does not rule out the existence of C. difficile exosporium structures, since genomic analyses of known exosporium producers outside the B. cereus family suggest that exosporium proteins differ markedly in various species.…”

Section: Clostridium Difficilementioning

confidence: 99%

The Exosporium Layer of Bacterial Spores: a Connection to the Environment and the Infected Host

Stewart

2015

Microbiol Mol Biol Rev

138

155

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SUMMARYMuch of what we know regarding bacterial spore structure and function has been learned from studies of the genetically well-characterized bacteriumBacillus subtilis. Molecular aspects of spore structure, assembly, and function are well defined. However, certain bacteria produce spores with an outer spore layer, the exosporium, which is not present onB. subtilisspores. Our understanding of the composition and biological functions of the exosporium layer is much more limited than that of other aspects of the spore. Because the bacterial spore surface is important for the spore's interactions with the environment, as well as being the site of interaction of the spore with the host's innate immune system in the case of spore-forming bacterial pathogens, the exosporium is worthy of continued investigation. Recent exosporium studies have focused largely on members of theBacillus cereusfamily, principallyBacillus anthracisandBacillus cereus. Our understanding of the composition of the exosporium, the pathway of its assembly, and its role in spore biology is now coming into sharper focus. This review expands on a 2007 review of spore surface layers which provided an excellent conceptual framework of exosporium structure and function (A. O. Henriques and C. P. Moran, Jr., Annu Rev Microbiol61:555–588, 2007,http://dx.doi.org/10.1146/annurev.micro.61.080706.093224). That review began a process of considering outer spore layers as an integrated, multilayered structure rather than simply regarding the outer spore components as independent parts.

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Section: Clostridium Difficilementioning

confidence: 99%

The Exosporium Layer of Bacterial Spores: a Connection to the Environment and the Infected Host

Stewart

2015

Microbiol Mol Biol Rev

138

155

View full text Add to dashboard Cite

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“…difficile spore morphology resembles that of other sporeforming bacteria; however, the protein composition of the outer layers (i.e., the spore coat and exosporium layer) differs greatly from that of other Gram-positive bacteria (13,14). The composition of the spore coat has been described recently (7); fewer than 25% of the spore proteins and several spore coat proteins have been functionally characterized (8,9). Transmission electron microscopy (TEM) reveals the presence of typical laminations (i.e., lamellae) (6,(10)(11)(12) in the spore coat of other spore-forming bacteria (13).…”

mentioning

confidence: 96%

Ultrastructural Variability of the Exosporium Layer of Clostridium difficile Spores

Pizarro-Guajardo

Calderón-Romero

Castro-Córdova

et al. 2016

Appl Environ Microbiol

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The anaerobic sporeformer Clostridium difficile is the leading cause of nosocomial antibiotic-associated diarrhea in developed and developing countries. The metabolically dormant spore form is considered the transmission, infectious, and persistent morphotype, and the outermost exosporium layer is likely to play a major role in spore-host interactions during the first contact of C. difficile spores with the host and for spore persistence during recurrent episodes of infection. Although some studies on the biology of the exosporium have been conducted ( , there is a lack of information on the ultrastructural variability and stability of this layer. In this work, using transmission electron micrographs, we analyzed the variability of the spore's outermost layers in various strains and found distinctive variability in the ultrastructural morphotype of the exosporium within and between strains. Through transmission electron micrographs, we observed that although this layer was stable during spore purification, it was partially lost after 6 months of storage at room temperature. These observations were confirmed by indirect immunofluorescence microscopy, where a significant decrease in the levels of two exosporium markers, the N-terminal domain of BclA1 and CdeC, was observed. It is also noteworthy that the presence of the exosporium marker CdeC on spores obtained from C. difficile biofilms depended on the biofilm culture conditions and the strain used. Collectively, these results provide information on the heterogeneity and stability of the exosporium surface of C. difficile spores. These findings have direct implications and should be considered in the development of novel methods to diagnose and/or remove C. difficile spores by using exosporium proteins as targets.

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“…The mutant contains a chromosomal mutation that replaces the entire cotB1 and cotB2 genes with an Sp r cassette. We also constructed another mutant strain, a ⌬cotG mutant, as a negative control, in which the Sp r cassette replaces the gene encoding another spore coat protein Cot␥ (31) (formerly known as an exosporium protein ExsB [32]; however, recent studies showed that most of this protein is located in the spore coat [31,33]). These mutant strains grew as fast and formed spores at the same rate as the wild type in mR2A medium with 100 g ml Ϫ1 silicate (data not shown), suggesting that the cotB1B2 and cotG disruption did not affect vegetative growth or spore formation.…”

Section: Resultsmentioning

confidence: 99%

The C-Terminal Zwitterionic Sequence of CotB1 Is Essential for Biosilicification of the Bacillus cereus Spore Coat

et al. 2016

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Silica is deposited in and around the spore coat layer of Bacillus cereus, and enhances the spore's acid resistance. Several peptides and proteins, including diatom silaffin and silacidin peptides, are involved in eukaryotic silica biomineralization (biosilicification). Homologous sequence search revealed a silacidin-like sequence in the C-terminal region of CotB1, a spore coat protein of B. cereus. The negatively charged silacidin-like sequence is followed by a positively charged arginine-rich sequence of 14 amino acids, which is remarkably similar to the silaffins. These sequences impart a zwitterionic character to the C terminus of CotB1. Interestingly, the cotB1 gene appears to form a bicistronic operon with its paralog, cotB2, the product of which, however, lacks the C-terminal zwitterionic sequence. A ⌬cotB1B2 mutant strain grew as fast and formed spores at the same rate as wild-type bacteria but did not show biosilicification. Complementation analysis showed that CotB1, but neither CotB2 nor C-terminally truncated mutants of CotB1, could restore the biosilicification activity in the ⌬cotB1B2 mutant, suggesting that the C-terminal zwitterionic sequence of CotB1 is essential for the process. We found that the kinetics of CotB1 expression, as well as its localization, correlated well with the time course of biosilicification and the location of the deposited silica. To our knowledge, this is the first report of a protein directly involved in prokaryotic biosilicification. IMPORTANCEBiosilicification is the process by which organisms incorporate soluble silicate in the form of insoluble silica. Although the mechanisms underlying eukaryotic biosilicification have been intensively investigated, prokaryotic biosilicification was not studied until recently. We previously demonstrated that biosilicification occurs in Bacillus cereus and its close relatives, and that silica is deposited in and around a spore coat layer as a protective coating against acid. The present study reveals that a B. cereus spore coat protein, CotB1, which carried a C-terminal zwitterionic sequence, is essential for biosilicification. Our results provide the first insight into mechanisms required for biosilicification in prokaryotes. Silicon (Si), the second most abundant element in the Earth's crust, is an important mineral for living organisms. It acts as the main component of structural skeletons of diatoms, radiolarians, and siliceous sponges (1). Si is also utilized by some plants (e.g., rice and cucumber) to protect against biotic and abiotic stresses (2). The presence of Si in bacterial spores was first noted several decades ago (3, 4). Until recently, however, there have been no reports on the precise localization of Si and its biological function in prokaryotes. In a recent study, we demonstrated that the Bacillus cereus group, a very homogenous cluster of six species, and its close relatives accumulate Si in and around their spore coat layer and that the Si-containing layer enhances acid resistance of the spores (5). In Si...

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In Pursuit of Protein Targets: Proteomic Characterization of Bacterial Spore Outer Layers

Cited by 61 publications

References 83 publications

The Exosporium Layer of Bacterial Spores: a Connection to the Environment and the Infected Host

The Exosporium Layer of Bacterial Spores: a Connection to the Environment and the Infected Host

Ultrastructural Variability of the Exosporium Layer of Clostridium difficile Spores

The C-Terminal Zwitterionic Sequence of CotB1 Is Essential for Biosilicification of the Bacillus cereus Spore Coat

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