Enigmatic Pilus-Like Endospore Appendages of Bacillus cereus Group Species

Abstract

The endospores (spores) of many Bacillus cereus sensu lato species are decorated with multiple hair/pilus-like appendages. Although they have been observed for more than 50 years, all efforts to characterize these fibers in detail have failed until now, largely due to their extraordinary resilience to proteolytic digestion and chemical solubilization. A recent structural analysis of B. cereus endospore appendages (Enas) using cryo-electron microscopy has revealed the structure of two distinct fiber morphologies: the longer and more abundant "Staggered-type" (S-Ena) and the shorter "Ladder-like" type (L-Ena), which further enabled the identification of the genes encoding the S-Ena. Ena homologs are widely and uniquely distributed among B. cereus sensu lato species, suggesting that appendages play important functional roles in these species. The discovery of ena genes is expected to facilitate functional studies involving Ena-depleted mutant spores to explore the role of Enas in the interaction between spores and their environment. Given the importance of B. cereus spores for the food industry and in medicine, there is a need for a better understanding of their biological functions and physicochemical properties. In this review, we discuss the current understanding of the Ena structure and the potential roles these remarkable fibers may play in the adhesion of spores to biotic and abiotic surfaces, aggregation, and biofilm formation.

Keywords: Bacillus cereus; Ena; appendage; endospore; pili; spore.

Figure 1

B. cereus endospore decorated with multiple hair-like appendages. (A) Illustration showing a cross-section of a B. cereus spore revealing multiple concentric layers, the exosporium and endospore appendages (S-Ena and L-Ena). The sizes of the spore layers and appendages are not drawn to scale. (B) Negative stain transmission electron microscopy (TEM) image of B. cereus NVH0075-95 spore showing the spore body (red arrow) and hair (pilus)-like appendages (S-Ena) (green arrow).

Figure 2

Architecture of the Ena fibers. (A) Illustration of S-Ena and L-Ena fibers with staggered and ladder-like arrangements of Ena subunits, respectively. In their termini distal to the endospore, the S-Ena fiber has ~4–5 ruffles (thin filamentous extensions of ~4 nm diameter), while the L-Ena has a single ruffle. (B) Surface representation of the atomic model of four helical turns of the S-Ena. Side and top view of one S-Ena helical turn featuring a ribbon representation of β–strands. (C) Three connected Ena subunits (as viewed from the central axis of the fiber or exposing the interior) highlighting the jelly roll domain and the N-terminal connector (Ntc) (PDB ID 7A02). Arrows point to Cys10, Cys11 of the Ntc from ith subunit that is involved in the disulphide linkage with Cys109, Cys24 of the i-9th and the i-10th subunits, respectively. Reprinted from [29].(D) Example of Gram-positive pilin with its Cna domains. Shown here is BcpA, the major pilin of B. cereus vegetative pili (PDB ID 3KPT), highlighting the CnaA₂, CnaA₃, and Xna domains in light orange, light cyan, and rainbow, respectively [35]. The CnaA domains are composed of two juxtaposed β-sheets of 3–4 strands each, while the Xna domain with a jelly roll topology consists of β-sheets of four to five strands each.

Figure 3

The ena1 and ena2 gene loci and distribution of these among 735 Bacillus genomes. (A) Distribution of ena1/2A-C among B. cereus s.l. spp. and the presence of genes encoding Ena subunits are indicated on surrounding rings in the following order from inner to outer: presence of enaA, enaB, and enaC, respectively (for all three, ena1: teal, ena2: orange, different locus: cyan). When no homolog or ortholog was found, the ring is gray. Whole genome clustering of the B. cereus s.l. group and B. subtilis created by Mashtree [52,53] and visualized in Microreact [54]. Rooted on B. subtilis. (B) (Inset) Ena1 and Ena2 loci with average amino acid sequence identity indicated between the population of EnaA-C orthologs and homologs found within the B. cereus s.l. group. Reprinted from [29].

Figure 4

An illustration depicting potential roles that Enas may play in Bacillus s.l. spp. Adhesion of endospores to intestinal epithelial cells (A), insect tissues (larvae) (B), and plant roots (C). (D) Autoaggregation of endospores. (E) Biofilm formation. (F) Adhesion of endospores to abiotic surfaces, such as food processing surfaces. The pictures are not drawn to scale.