Germination and Sporulation of Clostridioides Difficile

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Germination and Sporulation of Clostridioides difficile

Derek Tan March 10, 2020 MMIC 7050

Historical Perspective

Since its discovery, Clostridioides difficile has become one of the leading nosocomial infections in the world. This opportunistic pathogen is responsible for an estimated 453 000 hospitalizations resulting in 29 300 deaths annually in the United States alone (1). Being an opportunistic pathogen, the most substantial risk of contracting a C. difficile infection (CDI) is antibiotic use resulting in dysbiosis of the gut microbiota (2). Dysbiosis often occurs when broad spectrum antibiotics are used which disrupt the gut microbiota. Consequently, this creates a niche for C. difficile to colonize. Old age is an additional risk factor. It is estimated that those aged 65 and older are 8.65 times more likely in contracting a C. difficile infection (CDI) than those younger than 65 years of age (1).

Clostridioides difficile was first isolated and described in 1935 by Ivan Hall and Elizabeth

O’Toole (3). Hall isolated the novel pathogen by coincidence during a study where he was investigating the bacterial flora of infants. Stool samples were obtained from healthy infants and analyzed (3). Technological limitations of the 1930’s prevented sequence-based phylogeny to be performed. This resulted in the novel pathogen being named after its morphology. The pathogen was first described as a “hitherto undescribed obligate anaerobic pathogen” that was “a large

Gram-positive rod with elongate subterminal spores of about the same width as the rods” (3). As

C. difficile is a strict anaerobe and is extremely sensitive to oxygen, the pathogen was difficult to isolate and culture. Hall and O’Toole therefore named their new discovery Bacillus difficilis;

Bacillus due to its morphology and difficilis for being difficult to culture (3).

From the time that C. difficile was first discovered, it has had its name changed twice. In

1978, RH George’s research concluded that C. difficile was the cause of pseudomembranous

2 colitis (4). With this revelation, Bacillus difficilis was then reclassified as Clostridium difficile. The pathogen’s name remained as Clostridium difficile until 2016 when the pathogen was once again reclassified. Clostridium difficile then became how we know it today: Clostridioides difficile. In

2015, the Clostridium genus was restricted to include only Clostridium butyricum and its closely related species (5). Genetic analysis allowed researchers to determine that the pathogen was divergent enough from Clostridium butyricum to warrant a reclassification (5). During the early phases of reclassifying Clostridium difficile, creating a new genus named Peptoclostridium, was considered (6). However, severe backlash from the scientific community prompted re-evaluation of the new genus name. The overlying issue was that Peptoclostridium would cause widely known acronyms to become obsolete. Examples included but are not limited to: CDAD (C. difficile associated disease), CDI (C. difficile infection) and C. diff. Eventually, the new Clostridioides genus was created for C. difficile. Therefore, for what is now often muscle memory, C. difficile no longer stands for Clostridium difficile but stands for Clostridioides difficile. Currently, there are 2 species that belong to the Clostridioides genus: Clostridioides difficile and Clostridioides mangenotii.

Purpose of Spores

Identical to other spore forming bacteria, C. difficile exists in 2 different states: active vegetative cell and latent spore (5). Following an oral fecal lifecycle, in order to disseminate, C. difficile must leave its current host to colonize another host. While in this host transition period, the pathogen must be capable of surviving in aerobic environments (2). It has been shown that strict anaerobic conditions, 0% oxygen, is the best growth condition for C. difficile (Figure1) (2). C. difficile grown in an environment containing 2% oxygen severely impedes bacterial replication

(Figure1) (2). In an environment containing 5% oxygen, pathogen growth was no longer detected after 4 hours (Figure1) (2). 2% and 5% environmental oxygen concentrations are negligible compared to normal atmospheric oxygen concentration of 21% at sea level (2). The pathogen’s evolutionary solution to oxygen exposure is by producing oxygen-tolerant spores which are shed into the environment. Developing in the gastrointestinal tract, C. difficile spores are shed from the host via the host’s fecal matter (7). C. difficile produces spores not only to disseminate into the environment, but, also as a defence mechanism when exposed to various stresses. Besides oxygen, nutrient deficiencies are a stress signal that indicates less than favourable replication conditions (7). Spores are latent bodies of the bacteria which are extremely robust to a variety of stresses. Due to the metabolic latency of spores, the pathogen only causes disease by producing toxins in their vegetative cell form and not as spores (8). Spores can survive in typically inhospitable environments that exhibit characteristics such as: extreme temperatures, humidity variation, oxygen, pH and even in the presence of common disinfectants (8). This proves to be an issue for the public health as spores may persist in areas that have been cleaned with disinfectants ineffective against spores. An example of such a product would be ethanol. While ethanol can kill C. difficile in its vegetative form, it is ineffective at rendering spores unviable (8).

Killing spores requires sporicidal compounds; some of which are listed by the United States

Environmental Protection Agency (EPA) (9). The EPA’s List K lists registered antimicrobial products effective against Clostridium difficile spores (9). Though List K is not an exhaustive list of all effective cleaners, it lists many of the most commonly used disinfectants (9). Chlorine based disinfectants are a widely used disinfectant against C. difficile spores (10). An example of a chlorine-based disinfectant would be sodium hypochlorite, commonly known as beach (10).

C. difficile spores can be found all over the environment. Vectors for infecting an individual include contaminated water, contaminated food and poor hand hygiene (7). While sporicidal cleaners are often too harsh to be used on skin, preventative measures are the best alternative.

The most effective way to prevent C. difficile spores from entering the body is to wash one’s hands regularly and avoid touching the mouth, food or drink with bare hands (11). Additionally, when working with feces of potentially infected individuals, personal protective equipment such as gloves, gowns and glasses should be worn and promptly discarded once finished (1).

Unfortunately, when it comes to C. difficile spores on food, studies have shown that raising the temperature of meat to the recommended normal cooking temperature of 71°C is not high enough to kill C. difficile spores (12).

Spore Structure

The unique structure and physiology of spores are what allow them to be ultra resistant against a multitude of stresses. Bacterial spores are composed of multiple layers of various materials collectively responsible for its extreme robustness (Figure 2) (13). The center of the spore is known as the spore core. Within the spore core, the pathogen’s DNA is stored along with various other proteins (14). The bacterial DNA is not free floating but is supercoiled and bound to small acid-soluble proteins (SASPs) (14). In this stable state, DNA damage is minimized over time.

Additionally, supercoiled DNA bound to SASPs inhibits transcription from initiating as transcription factors are unable to bind to the DNA (14). This is desirable as the spore is without access to nutrients that would be required to support bacterial replication. Germination when not located in the gastrointestinal tract would ultimately lead to cell death. Other replication proteins are also

5 contained within the spore core ready serve their purposes once the spore beings to germinate.

Compared to vegetative cells, spores contain an extremely low water content (8). The water content within the spore core is inversely associated with a resistance to stresses such as wet heat, hydrogen peroxide and formaldehyde (15). During sporulation, calcium dipicolinic acid (Ca-

DPA) is produced by the mother cell and is transferred to the spore in exchange for water (14).

The Ca-DPA remains in the spore core maintaining its dehydration. To protect the core from harmful chemicals, the core is surrounded by an inner membrane (15). The inner membrane is composed of a peptidoglycan layer which possesses low permeability to chemicals (14). Besides potentially harmful chemicals, the inner membrane also keeps the core well dehydrated by preventing water from entering the core (14). The next layer of the spore is the germ cell wall.

The germ cell wall does not only provide another barrier of protection for the core, but, is also incorporated into the cell wall of the produced vegetative cell once germination occurs. The germ cell wall is not produced but derived from the mother vegetative cell’s cell wall during spore production (14). Therefore, the germ cell wall is the only part of the mother cell which is fully incorporated into the newly germinated vegetative cell. The next layer surrounding the germ cell wall is the cortex. The cortex is composed of a thick layer of modified peptidoglycan (14).

Compared to normal peptidoglycan which makes up the germ cell wall, the cortex’s peptidoglycan is modified by replacing peptide side chains (14). By removing the peptide side chain, a lactam ring forms resulting in 50% of the N-acetylmuramic acid being converted to muramic-δ-lactam

(MAL) (14). This process is to ensure that cortex lytic enzymes (CLEs) degrade only the spore’s cortex during germination and not the germ cell wall as they are both made of peptidoglycan.

Unintentionally degrading the germ cell wall would result in cell death. Additionally, 25% of the

6 cortex’s N-acetylmuramic acid (NAM) residues are substituted with short peptides providing the cortex with a lower degree of crosslinking than the germ cell wall (14). The cortex is surrounded by another layer of peptidoglycan, the outer membrane. The outer membrane region of the spore is where the cortex lytic enzymes (CLEs) involved in germination are located (14). The second-last layer of the spore is the spore coat. The spore coat, located on the outside of the outer membrane, serves as another layer of protection for the spore. This layer is composed from a dense layer of various proteins (14). The last layer of the C. difficile spore is the exosporium. This layer is loose fitting and made from a highly permeable complex of carbohydrates (14).

Germination

Germination is the transition of a latent spore into an active vegetative cell. While some aspects of germination are known, many are not. While there are other bacteria that germinate from spores which have been extensively studied, C. difficile lacks some highly conserved germination receptors. GerA, a transmembrane germination receptor is relatively highly conserved among spore formers (14). However, C. difficile does not possess GerA let alone any known transmembrane germination receptor (14). Much more research is required to fully understand the germination pathway of C. difficile.

Bile salts play a crucial role in C. difficile germination. One requirement for the germination of spores is the presence of cholate-derived bile salts (16). Such compounds are exclusively produced in the mammalian gut. Of all the cholate-derived bile salts, taurocholate is the most effective at promoting germination (14). For C. difficile, the presence of taurocholate indicates that the spore is in the correct environment germinate: the colon. Taurocholate is a secondary

7 bile acid which are produced by bacterial processes in the colon (17). Taurocholate levels in the colon also increase when there is dysbiosis in the gut resulting from activities such as antibiotic use (14). Antibiotic use further increases the odds of successful colonization of the newly produced vegetative cell as C. difficile is an opportunistic pathogen. While taurocholate is a promoter of germination, chenodeoxycholate is the opposite. Chenodeoxycholate acts as an efficient competitive inhibitor of taurocholate (18). Chenodeoxycholate is a primary bile acid produced by the liver and present in the earlier parts of the gastrointestinal tract (16).

Evolutionarily, the combination of taurocholate and chenodeoxycholate act as geographical markers for the pathogen as it moves through the gastrointestinal tract. The presence of chenodeoxycholate would signify that the spore has yet to reach its ideal environment. It should also be noted that taurocholate alone is not enough at initiating germination alone (14).

Requiring a co-stimulant, amino acids are one type of co-germinant to bile salts that can initiate germination. Unfortunately, the pathway which amino acids act upon during germination are widely unknown. It has been shown that glycine is the most effective amino acid at stimulating germination in combination with bile salts (14). However, other amino acids have been shown to work which include L-alanine, L-histidine and L-serine (14). D-Alanine and D-serine can also act as co-germinants but require a racemase dependant process (14). The other co- germinants to taurocholate to initiate germination are divalent cations; specifically magnesium or calcium (14). The presence of such cations has been shown to be a suitable substitute for the aforementioned amino acids (19). Once again, the pathway in which these divalent cations act upon during germination are widely unknown. There does however exist synergy between the two pathways. When in the presence of amino acids and divalent cations, the required

8 concentration of either substrate is reduced by 10-fold (14). The interaction between the pathways indicates that a variety of signals is required for germination to commence. These signals all ensure that the spore is in the gastrointestinal tract. All germinant requirement, bile salts, calcium and glycine, are all present in the gastrointestinal tract (14).

Several receptors and effectors of germination are also known. C. difficile lacks a transmembrane germinant receptor present in many other spores. However, CspC has been identified as the major C. difficile germinant receptor (20). This pseudoprotease recognizes taurocholate to initiate downstream signalling (20). Other members of the pathway include CspB whose main function is to enzymatically activate pro-SleC (21). SleC, a cortex lytic enzyme (CLE) in its active state post CspB enzymatic activation, is then able to commence degradation of the cortex (21). Degradation of the cortex allows water to slowly move into the cell to eventually rehydrate the core (21). While the core rehydrates, calcium dipicolinic acid (Ca-DPA) is expelled from the core via SpoVA (21).

Combining currently known information on germination, two major theories exist about the C. difficile germination pathway (Figure 3). While some parts are known, the link between

CspC and CspB activation remain unclear. The first model relies on a yet to be identified protein that acts an intermediate between CspC and CspB (14). Here, all components are proposed to form a complex. The complex is thought to be composed of CspC, CspA as a chaperone, CspB,

GerS and pro-SleC. Activation of the complex would thus occur when taurocholate and co- germinate factors interact with the complex (14). The second model foregoes the concept of a complex and instead recognizes CspC as a gatekeeper. Through some unknown pathway, it believes that CspC interaction with taurocholate facilitates the passage of co-germinate factors

9 into the spore (14). These co-germinate factors are then believed to move deeper into the cell to uncharacterized receptors located on the inner membrane which then activates CspB (14). This model attempts to explain how the co-germinant amino acids and divalent cations enter the spore in the first place.

Other proteins identified as being involved in the germination and sporulation process include GerG, GerS and CD630_32980. GerG is a lipoprotein that is highly expressed during sporulation (20). Studies have shown that GerG mutant C. difficile strains are unable to initiate cortex hydrolysis (20). It is hypothesized that GerG additionally modifies the cortex during germination (14). GerS is a protein which has been shown to affect germination. However, it is thought to affect the spore during sporulation (14). GerS mutant C. difficile strains are also unable to initiate cortex hydrolysis (14). GerS is thought to transport Csp proteins to the cortex during sporulation (14). CD630_32980 encodes for an ATPase. However, since ATP is not required during germination, it is most likely active during sporulation but affects germination (14). CD630_32980 mutants are deficient in both Ca-DPA and calcium. These mutants can respond and germinate when in the presence of taurocholic acid and amino acids but not when in the presence of taurocholic acid and calcium without amino acids (14).

Sporulation

While germination is the transition of a latent spore into an active vegetative cell, sporulation is the opposite. Sporulation produces a latent spore from an active vegetative cell. As with germination, the pathways which involve sporulation are not all completely understood. Like with other spore forming bacteria, C. difficile retained the conserved sporulation factor Spo0A and

10 its spore formation process (22). Vegetative C. difficile cells replicate via binary fission producing more vegetative cells. It should be noted that sporulation is a defence mechanism against stresses. While binary fission produces more bacterial cells, spore formation kills the mother cell in the process inhibiting replication (23). When Spo0A is activated, the sporulation process begins

(22). The first step of sporulation begins with the formation of a polar septum (22). This asymmetric division produces a larger mother cell and a smaller forespore (22). Eventually the mother cell fully engulfs the forespore in its entirely (22). The mother cell’s sole purpose is to complete the construction of the spore while 0sacrificing itself. Calcium dipicolinic acid (Ca-DPA) is synthesized by the mother cell and transported to the forespore (22). When the Ca-DPA enters the spore, it is exchanged for water dehydrating the core of the spore (22). The cortex and spore coat are then formed. Following the completion and maturation of the spore, the mother cell lyses which releases the spore (22). This spore is intended to exit the host so that it can find a fresh host to colonize and reproduce in.

Regulation of sporulation is regulated by many different factors. Activation of Spo0A via phosphorylation is what ultimately initiates sporulation (22). Across spore formers, while Spo0A may be conserved, the histidine kinases which act upon Spo0A vary across species (24). The main histidine kinases in C. difficile that phosphorylated Spo0A are CD1579 and CD2492 (22). It has been shown that knocking out CD1579 or CD2492 results in a 3-fold reduction in spore production

(22). On the other hand, CD1492 is a histidine kinase which is believed to dephosphorylate Spo0A; negatively regulating sporulation (22). It has been shown that a CD1492 knockout increased sporulation by 4-fold (22).

There are however many additional factors which do affect sporulation. RstA, regulator of sporulation and toxins, an RRNP family orthologue, is one of those factors that also regulates

Spo0A (22). RstA knockout mutants produce 20-fold fewer spores compared to their wild type counterparts (22). RstA homologues in other spore forming bacteria are regulated by Opp and

App, conserved oligopeptide permeases that often interact with quorum-sensing peptide genes

(22). However, is has not been established if such quorum-sensing peptides affect C. difficile’s

RstA. CcpA is another regulator of C. difficile sporulation but who detects carbon availability (22).

Since sporulation is a defence mechanism, to guarantee survival, it would be in the best interest of the bacteria to sporulate during nutrient deprivation. Low carbon availability is one such stress.

CcpA represses Spo0A directly, Opp and SinR (22). SinR encodes sporulation enhancing products

(22). CcpA knockout mutants have been shown to produce 10-fold more spores (22). CodY detects nutrients like with CcpA, but, detects amino acid levels instead of carbon (22). CodY also supresses SinR and Opp (22). Overall, there is more than a single factor that regulates the sporulation of vegetative C. difficile cells. An intertwined network of various receptors exist which come together to decide if the cell is going to being sporulation or not.

Implications

Further research into the pathways of sporulation and germination may reveal information of clinical importance. Sporulation and germination are an area which therapeutics could be based on (25). Deep understanding of the sporulation or germination pathway may lead to the identification of viable clinical targets. A deep understanding of sporulation and germination pathways would be easier for researchers to find methods to best target such

12 pathways to prevent germination or sporulation from occurring. Without the ability to germinate,

C. difficile will not be able to convert from its latent spore form to active vegetative cells. Spores would not be able to colonize an individual and thus not cause any disease. Targeting germination would be a preventative treatment as it could render the human gastrointestinal tract not colonizable by C. difficile. Risks could be mitigated for susceptible people and in areas where there is a high chance of contracting C. difficile spores such as in hospital bathrooms.

Without the ability to sporulate, the C. difficile would not be able to leave its current host and successfully persist in the environment. When an infected patient appears for treatment, they are shedding infectious spores. Those spores, released into the environment, can then potentially infect others. However, preventing sporulation would render the patient essentially non- infectious to others. The anaerobic nature of C. difficile would prevent vegetative cells to persist in normal environmental oxygen levels. Preventing the production and thus spread of C. difficile spores can help contain the pathogen to an individual, thus, eliminating the risk of outbreaks.

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Figure Legend

Figure 1. C. difficile growth in differing oxygen concentrations P. 18

Figure 2. Layers of a Clostridioides difficile spore P. 18

Figure 3. Clostridioides difficile germination theories P. 19

Figure 1. Clostridioides difficile growth in differing oxygen concentrations

A) Clostridioides difficile growth analyzed every hour for 8 hours at different environmental

oxygen concentrations. Adopted from (2).

Figure 2. Layers of a Clostridioides difficile spore

Clostridioides difficile combines multiple layers of varying materials to from a spore. Adopted from (14).

Figure 3. Clostridioides difficile germination theories

Major theories on the mechanism of germination in Clostridioides difficile. A) Germinosome complex theory. B) CspC mediated import theory. Adopted from (14).