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Architecture and Self-Assembly of Clostridium sporogenes and Clostridium botulinum Spore Surfaces Illustrate a General Protective Strategy across Spore Formers.

ABSTRACT: Spores, the infectious agents of many Firmicutes, are remarkably resilient cell forms. Even distant relatives can have similar spore architectures although some display unique features; they all incorporate protective proteinaceous envelopes. We previously found that Bacillus spores can achieve these protective properties through extensive disulfide cross-linking of self-assembled arrays of cysteine-rich proteins. We predicted that this could be a mechanism employed by spore formers in general, even those from other genera. Here, we tested this by revealing in nanometer detail how the outer envelope (exosporium) in Clostridium sporogenes (surrogate for C. botulinum group I), and in other clostridial relatives, forms a hexagonally symmetric semipermeable array. A cysteine-rich protein, CsxA, when expressed in Escherichia coli, self-assembles into a highly thermally stable structure identical to that of the native exosporium. Like the exosporium, CsxA arrays require harsh "reducing" conditions for disassembly. We conclude that in vivo, CsxA self-organizes into a highly resilient, disulfide cross-linked array decorated with additional protein appendages enveloping the forespore. This pattern is remarkably similar to that in Bacillus spores, despite a lack of protein homology. In both cases, intracellular disulfide formation is favored by the high lattice symmetry. We have identified cysteine-rich proteins in many distantly related spore formers and propose that they may adopt a similar strategy for intracellular assembly of robust protective structures.IMPORTANCE Bacteria such as those causing botulism and anthrax survive harsh conditions and spread disease as spores. Distantly related species have similar spore architectures with protective proteinaceous layers aiding adhesion and targeting. The structures that confer these common properties are largely unstudied, and the proteins involved can be very dissimilar in sequence. We identify CsxA as a cysteine-rich protein that self-assembles in a two-dimensional lattice enveloping the spores of several Clostridium species. We show that apparently unrelated cysteine-rich proteins from very different species can self-assemble to form remarkably similar and robust structures. We propose that diverse cysteine-rich proteins identified in the genomes of a broad range of spore formers may adopt a similar strategy for assembly.

SUBMITTER: Janganan TK

PROVIDER: S-EPMC7333573 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Publications

Architecture and Self-Assembly of Clostridium sporogenes and Clostridium botulinum Spore Surfaces Illustrate a General Protective Strategy across Spore Formers.

Janganan Thamarai K TK Mullin Nic N Dafis-Sagarmendi Ainhoa A Brunt Jason J Tzokov Svetomir B SB Stringer Sandra S Moir Anne A Chaudhuri Roy R RR Fagan Robert P RP Hobbs Jamie K JK Bullough Per A PA

mSphere 20200701 4

Spores, the infectious agents of many <i>Firmicutes</i>, are remarkably resilient cell forms. Even distant relatives can have similar spore architectures although some display unique features; they all incorporate protective proteinaceous envelopes. We previously found that <i>Bacillus</i> spores can achieve these protective properties through extensive disulfide cross-linking of self-assembled arrays of cysteine-rich proteins. We predicted that this could be a mechanism employed by spore former ...[more]

PMID: 32611700

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Project description:The spores of Clostridium botulinum Group II strains pose a significant threat to the safety of modern packaged foods due to the risk of their survival in pasteurization and their ability to germinate into neurotoxigenic cultures at refrigeration temperatures. Moreover, spores are the infectious agents in wound botulism, infant botulism, and intestinal toxemia in adults. The identification of factors that contribute to spore formation is, therefore, essential to the development of strategies to control related health risks. Accordingly, development of a straightforward and versatile gene manipulation tool and an efficient sporulation-promoting medium is pivotal. Our strategy was to employ CRISPR-Cas9 and homology-directed repair (HDR) to replace targeted genes with mutant alleles incorporating a unique 24-nt "bookmark" sequence that could act as a single guide RNA (sgRNA) target for Cas9. Following the generation of the sporulation mutant, the presence of the bookmark allowed rapid generation of a complemented strain, in which the mutant allele was replaced with a functional copy of the deleted gene using CRISPR-Cas9 and the requisite sgRNA. Then, we selected the most appropriate medium for sporulation studies in C. botulinum Group II strains by measuring the efficiency of spore formation in seven different media. The most effective medium was exploited to confirm the involvement of a candidate gene in the sporulation process. Using the devised sporulation medium, subsequent comparisons of the sporulation efficiency of the wild type (WT), mutant and "bookmark"-complemented strain allowed the assignment of any defective sporulation phenotype to the mutation made. As a strain generated by complementation with the WT gene in the original locus would be indistinguishable from the parental strain, the gene utilized in complementation studies was altered to contain a unique "watermark" through the introduction of silent nucleotide changes. The mutagenesis system and the devised sporulation medium provide a solid basis for gaining a deeper understanding of spore formation in C. botulinum, a prerequisite for the development of novel strategies for spore control and related food safety and public health risk management.

Dataset Information

Architecture and Self-Assembly of Clostridium sporogenes and Clostridium botulinum Spore Surfaces Illustrate a General Protective Strategy across Spore Formers.

Publications

Architecture and Self-Assembly of Clostridium sporogenes and Clostridium botulinum Spore Surfaces Illustrate a General Protective Strategy across Spore Formers.

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