Sporulation and Germination in Clostridial Pathogens

Abstract

As obligate anaerobes, clostridial pathogens depend on their metabolically dormant, oxygen-tolerant spore form to transmit disease. However, the molecular mechanisms by which those spores germinate to initiate infection and then form new spores to transmit infection remain poorly understood. While sporulation and germination have been well characterized in Bacillus subtilis and Bacillus anthracis, striking differences in the regulation of these processes have been observed between the bacilli and the clostridia, with even some conserved proteins exhibiting differences in their requirements and functions. Here, we review our current understanding of how clostridial pathogens, specifically Clostridium perfringens, Clostridium botulinum, and Clostridioides difficile, induce sporulation in response to environmental cues, assemble resistant spores, and germinate metabolically dormant spores in response to environmental cues. We also discuss the direct relationship between toxin production and spore formation in these pathogens.

FIGURE 1

Lifecycle of endospore formers. (A) Sporulation. Upon sensing certain environmental conditions, endospore formers activate Spo0A and initiate sporulation. The first morphological event is the formation of a polar septum, which creates a larger mother cell and smaller forespore. The mother cell engulfs the forespore, and the two cells work together to assemble the dormant spore. Calcium dipicolinic acid (Ca-DPA) is synthesized in the mother cell and transported into the forespore in exchange for water. The cortex is formed between the two membranes, and coat proteins polymerize on the surface of the mother cell-derived membrane. Once the spore is mature, the mother cell lyses and releases the dormant spore into the environment. (B) Germination. Upon sensing the appropriate small molecule germinants, the spore initiates a signaling cascade that leads to activation of cortex hydrolases and core hydration, which is necessary for metabolism to resume in the germinating spore.

FIGURE 2

Sporulation initiation via Spo0A phosphorylation. Sigma factors are shown as circles, histidine kinases and phosphatases as hexagons (adapted from Al-Hinai et al. [85]). Positive regulators are shown in green (with the exception of σ^K, which is shown in purple), and negative regulators are shown in red. In B. subtilis, the KinA-E orphan histidine kinases phosphorylate the phosphotransfer protein Spo0F, the first component in the phosphorelay (25). The Rap phosphatases remove phosphates from phosphorylated Spo0F. The phosphate is transferred from Spo0F to Spo0B to Spo0A. In C. difficile, the orphan histidine kinases CD1579 and CD2492 appear to phosphorylate Spo0A (32), while CD1492 likely dephosphorylates Spo0A (47). A more detailed description of C. difficile Spo0A regulation is shown in Fig. 3. Although all the putative orphan histidine kinases with the potential to phosphorylate Spo0A in C. perfringens and C. botulinum are shown, whether they act as positive or negative regulators remains unstudied. The stationary factor σ^H activates expression of spo0A in B. subtilis and C. difficile (101), while σ^K activates spo0A transcription in C. botulinum (74) and possibly C. perfringens, the latter of which induces sporulation during log-phase growth (65).

FIGURE 3

Regulatory pathway controlling Spo0A activation in C. difficile. Early sporulation factors experimentally determined to function as positive regulators of Spo0A are highlighted in green, and those that inhibit Spo0A are highlighted in red (32, 47, 51, 55, 56, 59, 61, 62, 101). Hexagons indicate histidine kinase/phosphatases, rounded rectangles demarcate transcription factors, and circles highlight sigma factors. Red lines indicate negative regulation, and black lines indicate positive regulation. SinR and SinR′, C. difficile orthologs for regulatory proteins characterized in B. subtilis (gray), were recently shown to promote sporulation (60). Solid lines indicate defined regulatory interactions, and dashed lines suggest proposed, and potentially indirect, regulatory effects. Branched-chain amino acids are a CodY cofactor (59), and their precursors are likely imported primarily through the Opp and App oligopeptide transporters (55, 61). CcpA-independent carbon-specific regulation is not shown (56). The reciprocal transcriptional regulation of early sporulation factors by Spo0A has also been omitted for simplicity (100).

FIGURE 4

Diversity in the regulation of the transcriptional programs controlling sporulation in the Firmicutes. The temporal progression of sporulation is shown from top to bottom. Transcription factors and sigma factors are shown in bold, and proteins enclosed in boxes directly participate in signaling between the mother cell and forespore (dashed boxes indicate that trans-septum signaling has not been tested yet). Text color denotes whether the factor has been detected at both the transcript and protein level (black), at either the transcript or protein level (purple), or has not been tested yet at the transcript or protein level (blue). Black arrows delineate transcriptional control of gene expression, red arrows indicate signaling pathways, dashed lines indicate that the regulatory relationship remains unknown, and thick arrows demarcate notable points of divergence from the pathway defined in B. subtilis. AND gates are indicated. The figure is adapted from Fimlaid et al. (137) under Creative Commons BY 4.0.

FIGURE 5

Spore coat and exosporium structure in C. difficile. (A, B) Transmission electron microscopy sections of C. difficile spores highlighting (from outside to inside) the bumpy, outermost exosporium (Ex) layer with its hair-like projections (HPs), outer coat (OC), inner coat (IC), cortex layer (Cx), germ cell wall (GCW), inner forespore membrane (IM), and spore core (cytosol). (C) Scanning electron microscopy of C. difficile spores reveals the bumpy surface created by the exosporium. Images used without modification from Rabi et al. (280) under Creative Commons BY 4.0. (D) Schematic of spore coat layers highlighting morphogenetic factors identified as being important for the assembly of specific layers. Assembly of the outermost exosporium depends on the BclA collagen-like proteins, which likely create hair-like projections on the spore surface (186, 187), CdeC (185), and CdeM (D. Paredes-Saja, unpublished data). The proteins that make up the outer and inner coat layers are unknown, but CotA and the mucinase, CotE, have been shown to be surface accessible (180, 182). SpoIVA (IVA) and SipL are interacting coat morphogenetic proteins that are essential for recruiting coat proteins to the forespore and forming heat-resistant spores (173, 174). The specific proteins recruited by SpoIVA, SipL, CdeC, and CdeM remain unknown.

FIGURE 6

Putative locations of germination regulators in C. difficile and C. perfringens. Germinant signaling proteins, CspC (pseudoprotease and germinant receptor) and its downstream effectors, the CspB protease, and cortex hydrolase, SleC, are all produced in the mother cell under the control of either σ^E or σ^K (–139). CspB is produced as a fusion to the pseudoprotease, CspA, which is critical for CspC incorporation into mature spores (–235); all three Csp proteins are incorporated into mature spores. GerG is required for optimal incorporation of CspC, CspB, and CspA into mature spores (253). The GerS lipoprotein (248) is produced in the mother cell and does not directly participate in spore germination (O. Diaz and A. Shen, unpublished data), even though it is required for spore germination to proceed. The ATP/GTP binding protein CD3298 presumably localizes to the cytosolic face of the outer forespore membrane and regulates calcium release and possibly internalization (205). Germinant sensing induces the proteolytic activation of SleC by CspB in both organisms, but CspA and/or CspC can cleave SleC in C. perfringens (marked in brackets) (218, 242, 245), since they are active proteases unlike their cognate partners in C. difficile (233). C. perfringens also produces inner membrane-bound germinant receptors, similar to most spore-forming organisms, in the forespore, in contrast to the soluble CspC protein used by C. difficile to sense germinant. The locations of all proteins in mature spores is putative, with the exception of SleC, which has been shown to localize to the C. perfringens cortex region by immuno-electron microscopy (251).

FIGURE 7

Schematic of spore germination signaling pathways. Germinants that are sensed by B. subtilis, C. botulinum, C. perfringens, and C. difficile are shown in dark blue; C. botulinum group I and III germinants are shown in the brackets (and include l-Ala). Amino acid and calcium ion cogerminants are not pictured for C. difficile (199, 204, 205). Germinant receptors are shown in green. The signaling pathway between B. subtilis and C. botulinum groups 1 and III (far left) differs from the other clostridial organisms mainly with respect to cortex hydrolase (shown in orange) activation mechanisms, with SleC being activated by proteolytic cleavage by Csp proteases, and the CwlJ and SleB cortex hydrolases being activated directly or indirectly by DPA release. Accordingly, the order of cortex hydrolysis and DPA release via SpoVAC differs between these two types of mechanisms. C. botulinum groups II and IV encode germinant receptors with variable numbers of A and B components. Adapted from reference .