The advent of cryo-electron microscopy (cryo-EM) has enabled us to image biological macromolecules down to the atomic level in their native form
[15]. It can be used to determine the structure of multiprotein complexes and filaments with known or unknown genetic identities. In our recent work, we used cryo-EM to determine the electron potential map of ex-vivo purified S-Ena appendages at a resolution of 3.2 Å. At this resolution, we could clearly identify amino acids with bulky side chains that led us to recognize a hexapeptide sequence ‘FCMTIRY’ located in the
C-terminal of an Ena subunit. Searching for this hexapeptide in the
B. cereus NVH 0075-95 genome revealed the presence of
ena genes
[7]. Ex-vivo purified S-Ena is composed of Ena1A and Ena1B subunits assembled into a helix of 110 Å diameter and 37.4 Å pitch (
Figure 2B). Each helical turn is made up of 11.6 Ena1A/B subunits (
Figure 1B). Due to their high sequence similarity (identity, 38%; similarity, 58%) and structure, cryo-EM does not help much in unambiguously determining the stoichiometry and arrangement of EnaA/B subunits along the length of the appendages. The Ena1A/B subunits consist of a typical jelly roll fold and a 15-residue long
N-terminal connecter (Ntc) (
Figure 1C). A jelly roll fold generally consists of 8 β-strands arranged in two 4 stranded β-sheets juxtaposed to each other, resembling a jelly or Swiss roll cake
[16]. The jelly roll domains of adjacent Ena1A/B subunits are connected to each other through β-sheet augmentation, which helps in the lateral stabilization of the helix (
Figure 1B). Apart from that, two complementary electrostatic patches on the surfaces enhance inter-subunit contacts. The Ntc of each subunit is connected to two other subunits present in the preceding helical turn to confer longitudinal stability to the helix. Cys10 and Cys11 in the Ntc of each subunit i form disulphide bonds with Cys109 and Cys24 of subunits i-9 and i-10, respectively. The combination of β-sheet augmentation and covalent linkage through Ntc leads to the assembly of an appendage that is extremely stable, both chemically and physically
[7]. Since the Ntcs are present in the luminal side of the helix, they are protected from various environmental assaults. The Ntcs are connected to the jelly roll domain through a flexible five-residue long region that creates a longitudinal gap of 4.5 Å (
Figure 1B) between the two helical turns. Due to the flexibility of this spacer region and the lack of direct protein-protein contact between the two subunits present across different helical turns, the appendages turn out to be highly flexible. At the distal termini of the spore, both S-Ena and L-Ena have short extensions that are dubbed ‘ruffles’ (
Figure 1A). The S-Ena tends to have four to five ruffles, while the L-Ena has one ruffle per appendage. When observed by negative stain TEM, the ruffles appear morphologically similar to collagen-like immunogenic hairy naps (BclA) that are found attached to the exosporium
[17]; however, the protein(s) that constitute the ruffles have not yet been identified.
The jelly roll domain of Ena with its juxtaposed β-sheet architecture has previously been found in many sortase-mediated vegetative pili, such as the XNA domain in BcpA, the major subunit of the vegetative pili of
B. cereus (
Figure 1D)
[9]. In addition to that, CnaA and CnaB-type domains found in BcpA (
Figure 1D), SpaA pilin from
Corynebacterium diphtheriae [18], RrgC from
Streptococcus pneumoniae [19], and FimA and FimP from
Actonomyces oris [20][21] are another class of domains that contain a sandwiched β-sheet architecture. Cna-type domains are IG-like domains that consist of two juxtaposed sheets. The two sheets can be composed of either 4-4 or 4-5 β-strands (in CnaA) or 4-3 β-strands (in CnaB). These domains were first reported in
Staphylococcus aureus as collagen-binding proteins
[22]. Furthermore, triple jelly roll domains found in the C381 turret protein of
Sulfolobus turreted icosahedral virus (STIV) are known to be involved in the interaction with the host pilus
[23]. All this evidence indicates that the jelly roll domain in Enas might also play a role in adherence to host tissue.
At the domain level, Enas are similar to sortase-mediated pili of vegetative cells of other Gram-positive species. However, the arrangement of these domains in the filament is what distinguishes the two types of pili. Sortase-mediated pili consist of a linear chain of CnaA and/or CnaB domains with distinct genes for basal pilin, backbone pilin, and tip pilin and employ specific sortases to catalyze polymerization and anchoring to the peptidoglycan layer. The sortase-mediated pili rely on internal isopeptide bonds between the side chains of Lys and Asn for thermodynamic and physical stability
[24][25]. On the contrary, Ena subunits assemble into a helix and undergo covalent disulphide linkages hidden inside the luminal side to survive extreme physical and chemical stress. Furthermore, Ena1A and Ena1B can self-assemble into filaments nearly identical to S-Ena in vitro
[7] without the need for any specific enzyme.