Assembly of the outermost spore layer: pieces of the puzzle are coming together

Summary

Certain endospore-forming soil dwelling bacteria are important human, animal, or insect pathogens. These organisms produce spores containing an outer layer, the exosporium. The exosporium is the site of interactions between the spore and the soil environment and between the spore and the infected host during the initial stages of infection. The composition and assembly process of the exosporium are poorly understood. This is partly due to the extreme stability of the exosporium that has proven to be refractive to existing methods to deconstruct the intact structure into its component parts. Although more than 20 proteins have been identified as exosporium-associated, their abundance, relationship to other proteins, and the processes by which they are assembled to create the exosporium are largely unknown. In this issue of Molecular Microbiology, Terry, Jiang, and colleagues in Per Bullough’s laboratory show that the ExsY protein is a major structural protein of the exosporium basal layer of B. cereus family spores and that it can self-assemble into complex structures that possess many of the structural features characteristic of the exosporium basal layer. The authors refined a model for exosporium assembly. Their findings may have implications for exosporium formation in other spore forming bacteria, including Clostridium species.

Soil dwelling microorganisms lead a stressful life. They are subjected to harsh environmental conditions that include exposure to ultraviolet rays from the sun, periodic desiccation in times of drought, and extremes in ambient temperature. One survival mechanism employed by certain soil bacteria is sporulation. Under unfavorable growth conditions, the bacterium can shift its physiological processes to undergo sporulation, a process that results in a cell producing an endospore. The endospore is a survival structure, consisting of a cell with a partially dehydrated cytoplasm (the core) enveloped by layers of peptidoglycan (the cortex), and a protein shell (the spore coat. The sporulation process, and the composition of the spore, have been well studied in the model organism Bacillus subtilis. Studies on sporulation have contributed greatly to our understanding of molecular genetics, including concepts of gene expression regulation by sigma factor switching, complex processing of sigma factor precursors, and compartmentalization of gene expression. However, there is an aspect of spore formation about which B. subtilis provides no answers. This is because it lacks a spore structure, the exosporium, found on many bacterial spores.

The exosporium is the outermost layer found on some spores. It has best been characterized on the Bacillus cereus family (B. cereus, Bacillus anthracis, and Bacillus thuringiensis) spores, but can also be found on a number of medically important spore-formers including Clostridium botulinum, Clostridium perfringens, and some strains of Clostridium difficile (Bozue et al., 2015; Stewart, 2015). The exosporium layer (Fig. 1) consists of a basal layer and an external hairlike nap comprised principally of the collagen-like glycoprotein BclA. The exosporium layer is an outer protein shell, separated from the spore coat by the interspace layer. It serves as a barrier to penetration by larger molecules while permitting small molecules such as amino acids and nucleosides to diffuse across. Thus small molecule germinants can pass through, but the spore is protected from degradative enzymes in the environment. The exosporium is the surface which, by virtue of its location as the outermost spore layer, interacts with the soil environment. For pathogens for which the spore serves as the infectious form, it is also the layer that interacts with the host and its innate immune system during the early stages of infection.

Figure 1.

Transmission electron micrograph of a B. anthracis spore.

The B. cereus family balloon-like exosporium is deformable, irregularly shaped, and possesses numerous folds and creases. The B. anthracis exosporium basal layer can be of variable thickness but contains at least one crystalline sub-layer, about 5 nm thick. (Ball et al., 2008; Couture-Tosi et al., 2010; Kailas et al., 2011; and Rodenburg et al., 2014). The basal layer has a crystalline structure organization with a six-fold symmetry. It is comprised of a lattice of cups, open to the exterior of the spore and closed off on the interior-facing side. Our understanding of the protein composition and assembly pathway of the exosporium is limited. This is in large part owing to difficulties with extracting exosporium proteins. Exosporium proteins are generally extracted from intact spores or purified exosporia using heat, SDS treatment, reducing agents, and often urea treatment. Despite this rigorous extraction methodology, known exosporium proteins usually appear following SDS-PAGE as high molecular weight complexes (Todd et al., 2003; Redmond et al., 2004; Thompson et al., 2011a). Sorting out the protein interactions in these complexes has been challenging from a biochemical standpoint.

Assembly of the exosporium in the B. cereus family sporulating cells initiates at the mother cell central pole of the spore (Fig. 2). Genetic studies have provided clues as to the assembly pathway. Mutants lacking the exosporium basal layer protein ExsY were found to produce only the mother cell central pole region of the exosporium (approximately 25% of the exosporium) (Boydston et al., 2006). Turnbough and coworkers have termed this region of the exosporium the “bottle cap”. They discovered that the exosporium bottle cap portion (along with some spore coat material) “pops open” during spore germination and outgrowth freeing the emerging bacterial cell. The cap and non-cap regions of the exosporium basal layer have different protein compositions (Steichen et al., 2007; Thompson et al., 2012). Protein differences reported in the literature are indicated in Fig. 2. The cap only structure produced by the exsY mutant possesses a BclA nap layer, suggesting bottle cap maturation is not dependent on the assembly of the non-cap portion of the exosporium. Anne Moir’s laboratory demonstrated that loss of the ExsY and CotY proteins resulted in B. cereus spores lacking an exosporium but the cotY mutant produced spores with an intact exosporium (Johnson et al., 2006). Two other exosporium proteins thought to be components of the basal layer, ExsFA (also known as BxpB) and ExsFB contribute to exosporium stability (Steichen et al., 2005; Sylvestre et al., 2005). Loss of both of these proteins results in a failure to assemble the BclA nap layer and the exosporium produced by this double mutant is structurally unstable and becomes lost from spores over time (Sylvestre et al., 2005). The observation that the exsFA/exsFB double mutant produces spores with an exosporium layer and the cotY/exsY mutant fails to assemble an exosporium, suggests that the BxpB/ExsFB assembly steps occurs later than the CotY/ExsY step. More detailed studies are needed to determine if this indicates that the former protein pair are located in the outer basal sublayer (adjacent to the BclA nap layer) whereas the latter pair are components of the inner sublayer of the basal layer. In support of an outer exosporium location for ExsFA, fluorescent protein fusion studies found that the N-terminus of the BclA protein is positioned close enough to ExsFA for FRET to occur (Thompson et al., 2011b). FRET was not observed with BclA and ExsY fusions, consistent with ExsY being an inner sublayer basal layer protein and thus further away from the BclA N-terminus (Thompson et al., 2011b). An interesting finding is that the two basal layer protein pairs indicated above each consist of proteins that share extensive amino acid identity (86% for the CotY/ExsY pair and 78% for the BxpB/ExsFB pair). Despite their extensive sequence similarity, the cap versus non-cap distribution of the proteins are markedly different. CotY and ExsFB are primarily cap proteins whereas ExsY and BxpB are found in greater amounts in the non-cap portion of the exosporium (Fig. 2). Genetic studies have provided clues, but details on how exosporium assembly initiates and the nature of the specific steps involved have remained elusive.

Figure 2.

Illustrations of exosporium assembly in a sporulating cell of B. anthracis. The bottlecap portion of the exosporium, at the mother cell-central pole of the spore, is depicted in blue and the non-cap exosporium is in red. In the top figure, the exosporium assembly initiates at the mother cell central pole of the spore and proceeds around the spore and transitions into the non-cap region of the exosporium. BclA incorporation follows basal layer assembly in the same pattern. In the middle figure, exosporium basal layer assembly is complete and the BclA nap layer assembly is being completed at the non-cap spore pole. The mature intracellular spore is shown in the bottom figure. Proteins known to be present in the bottle cap, the non-cap, or showing no cap versus non-cap distribution are listed at the bottom of the Figure.

In this issue of Molecular Microbiology, Terry, Jiang et al. have gained insight as to the composition of the exosporium basal layer of the B. cereus family spores and, more importantly, mechanisms driving the assembly of this exosporium layer. They systematically refined the exosporium extraction procedure to more reliably disrupt the large protein complexes usually seen with exosporium extracts to yield a more reliable population of component proteins, the majority of which were less than 20,000 molecular weight based on Coomassie brilliant blue staining of SDS-PAGE resolved proteins. They identified ExsY as a major protein in these extracts and hence a major component of the exosporium basal layer. The authors expressed His-tagged recombinant ExsY and CotY in E. coli host cells. ExsY was found to self-assemble in the cytoplasm of this heterologous host into sheets of crystalline arrays of remarkable size (micrometer sizes). Electron crystallography studies of these arrays indicated that they exhibit the open lattice of hexameric ring-like structures that was extraordinarily similar to that observed in the native exosporia of the B. cereus family spores. Terry, Jiang et al. propose that ExsY forms the repeating unit comprising most of the crystalline lattice of the exosporium basal layer. The recombinant CotY protein was also found to be capable of self-assembly, but less efficiently compared to ExsY. They incorporated this observation into a model of exosporium assembly. Self-assembly had earlier been shown by this group to be a property of certain spore proteins of B. subtilis (Jiang et al., 2015). In addition, they also provide evidence in support of an outer exosporium location for ExsFA, since they ascribe some of the outward facing density in the wild-type crystal structure to ExsFA and possibly part of BclA.

The Terry et al. report describes results that critically change the landscape in biochemical research related to exosporium-containing spores. Now we will not simply seek to identify proteins in the exosporium layer, but can more specifically investigate the specific steps involved the assembly of this important spore layer. The authors have proposed a model around which hypotheses can be formulated and specifically tested. They have begun this process with suggestions as to why ExsY comprises the major structural protein of the non-cap portion of the exosporium and also why arrays of this protein exhibit the remarkable structural stability characteristic of the exosporium layer. Important questions immediately come to mind for future work. How does ExsY get positioned at the site of exosporium basal layer assembly to begin self-assembly? Does ExsY substitute for CotY in the cap of cotY null spores to permit formation of an intact exosporium? What is the reason that CotY cannot substitute for ExsY in an exsY null mutant to make the non-cap exosporium? Although the ExsY array is quite stable to denaturation, in the absence of ExsFA (BxpB) and ExsFB, the exosporium is fragile and easily lost from the spores. How do these proteins stabilize the exosporium basal layer? The good news is that now the puzzle pieces are falling into place and we can seriously begin addressing these questions.