Targeting the Metabolic Basis of C. difficile’s Life Cycle

Clostridioides (formerly Clostridium) difficile’s success as a pathogen is rooted in the complexity of its life cycle. The bacterial pathogen’s time as a toxin-producing vegetative cell is flanked by periods as a tough, metabolically dormant spore. C. difficile’s transition from one life stage to the next depends on specific nutrients and metabolic compounds derived from its host and other gut microbiota. By understanding what C. difficile needs (or eats, for that matter) to survive and thrive in the gut, we can develop strategies to prevent or treat C. difficile infection (CDI) at various stages of the pathogen’s life cycle.

Current Treatments Encourage C. difficile Recurrence

 C. difficile causes approximately 500,000 infections in the United States alone every year. This diarrhea-inducing bacterium can do some serious damage, both in and outside the gut. Indeed, the economic costs associated with CDI fall somewhere around $1 billion a year. It is no surprise that C. difficile has landed a spot on the Centers for Disease Control and Prevention’s (CDC) “urgent threat” list.

Most of the time, our gut microbiota protect us from C. difficile by hogging nutrients and space in the intestine, releasing metabolic by-products that inhibit C. difficile growth and secreting compounds that directly target and kill this nasty bug. However, perturbations to the microbial community, often as a result of antibiotic treatment, weaken these barriers to C. difficile colonization. Indeed, antibiotic treatment is the primary risk factor for developing CDI, thanks to its microbiota-obliterating powers. Other risk factors include old age and having a weakened immune system or pre-existing condition, like inflammatory bowel disease. 
 
Clinical syndromes resulting from CDI range from mild diarrhea to life-threatening colitis (inflammation of the lining of the colon). Unfortunately, the primary treatment for CDI also happens to be the key risk factor: antibiotics. As a result, as many as 35% of patients fall into cycles of infection recurrence. These patients take antibiotics to clear the initial infection and then experience CDI symptoms again a few weeks later, largely because their microbiota is depleted and fails to return to its pre-infectious, C. difficile-resistant state. To this end, fecal microbiota transplants (FMTs), in which the microbiota from a healthy donor is transmitted to a CDI patient, are currently the gold standard for treating recurrent CDI. Though effective, FMTs are a black box in terms of their microbial composition. They can sometimes contain pathogens, including antibiotic-resistant bacteria, that lead to disease and complications in FMT recipients. 

The problem of recurrent CDI, coupled with concern for the emergence of antibiotic resistant strains of C. difficile and potential for adverse FMT-associated outcomes, has prompted interest in developing novel preventive and therapeutic strategies for managing CDI. Luckily, C. difficile’s life cycle offers a number of potential targets.

The Beginning of C. difficile Colonization: Germination

 As an anaerobic bacterium, C. difficile survives the oxygen-rich world outside the gut by forming spores. Spores are shed through feces. Without good hygienic practices (e.g., proper hand-washing), they can contaminate food or surfaces, where they are inadvertently passed from one person to the next. While C. difficile spores are ubiquitous within the environment, one is most likely to pick them up in CDI hot-spots, like hospitals or nursing homes. 

Similar to the seeds of a plant, spores contain the building blocks of mature C. difficile cells, like DNA and enzymes, encased in multiple membranes and protein-rich shells. Upon ingestion and safe passage into the host’s intestine, the spores germinate into vegetative cells. During this process, they rehydrate and metabolism kicks into gear. Whether or not germination occurs, however, depends on interactions between the spores and small molecules, called germinants, within the gut. In C. difficile’s case, these germinants come in the form of host-derived primary bile acids. 
 
Primary bile acids are synthesized from cholesterol in the liver and secreted into the digestive tract. Like soap cutting through grease, bile acids are detergent-like molecules that help break down fats in the gut. There are several types of bile acids that differ in their molecular structure. Because of this, not all bile acids support C. difficile germination. For example, taurocholate is a potent C. difficile germinant; it binds to a receptor on the spore outer membrane to initiate the germination process. Chenodeoxycholate, on the other hand, can interact with spores with greater affinity than taurocholate, but does not induce the downstream signalling required for germination to occur. 

While certain spore-bile acid interactions are critical for initiating germination, they are not enough by themselves. Co-germinants, like the amino acids glycine or alanine, and calcium are also required. The exact roles of these compounds, which are presumably derived from host diet, are not well defined. However, they likely signal to C. difficile spores that there are enough nutrients in the gut to support the generation and proliferation of vegetative cells. Indeed, glycine and alanine are components of C. difficile’s peptidoglycan layer, while glycine is also an important nutrient for efficient vegetative growth. Thus, bile acids and co-germinants collectively send a message to C. difficile spores that a) they are in the gut and b) there will be enough food to support their transformation into metabolically active, free-living cells. 

In addition to host-associated compounds, germination is tightly linked to the metabolic output of the gut microbiota. Some gut bacteria can convert primary bile acids into secondary bile acids, such as lithocholate and deoxycholate, which inhibit C. difficile germination and outgrowth into vegetative cells, respectively. Under normal circumstances, secondary bile acids constitute a barrier to C. difficile intestinal colonization. However, antibiotic treatment kills members of the microbiota that produce these inhibitory molecules. As a result, primary bile acids, some of which act as germinants, increase in concentration and increase the likelihood for C. difficile spore germination and expansion in the gut.

C. difficile Growth: The Vegetative Phase 

After germination, C. difficile enters its hungry phase. Host microbiota normally resist C. difficile colonization, in part by inhibiting the pathogen’s access to nutrients or by releasing inhibitory metabolic compounds, like secondary bile acids. 
 
However, if the microbial community is disrupted, a variety of carbon sources become available for C. difficile to eat. C. difficile is not picky when it comes to food, and can alter its metabolism depending on the nutrients present in its environment at a given time. However, there are some nutrients that are critical for efficient growth, including amino acids like proline, leucine and glycine. These substrates fuel Stickland metabolism, a set of amino acid fermentation reactions common in members of the Clostridium genus, in which electrons are transferred from one amino acid to another to produce adenosine triphosphate (ATP) and other molecular drivers of life. 
 
Many of the nutrients C. difficile consumes are associated with a loss in gut bacterial diversity—the bugs that normally hoard the food C. difficile likes to eat are no longer there (likely due to antibiotic treatment). That being said, the pathogen can also exploit nutrients derived from the microbes that are present. For instance, C. difficile can use sialic acid, a sugar liberated from intestinal mucus by members of the microbiota, to expand in the gut. Other compounds associated with microbiota metabolism, such as succinate, can also feed C. difficile. Thus, the gut microbiota can be both friend and foe when it comes to C. difficile nutrient acquisition.

Hunger and Destruction: C. difficile Toxin Production

C. difficile’s phase as a vegetative cell is marked by intestinal destruction. The pathogen releases up to 3 toxins that disrupt intestinal barrier integrity and promote inflammation. Interestingly, toxin production occurs not in the presence of specific nutritional or metabolic signals, but rather in the absence of such signals. 
 
Several in vitro studies suggest that C. difficile secretes toxins in response to stress, including nutrient limitation. In fact, regulatory proteins involved in starvation response are also involved in toxin regulation. In other words, toxin production is C. difficile’s “hangry”response. New research suggests that toxin-mediated inflammation liberates nutrients that C. difficile can eat, like collagen, from host cells, while inhibiting expansion of bacteria that might compete with the pathogen for those same nutrients. This strategy is similar to what has been reported for other gut bacterial pathogens, like Salmonella enterica serovar Typhimurium and Vibrio cholerae, which exploit the host inflammatory response for food. Thus, C. difficile’s toxins may create a nutritional niche for the pathogen to occupy within the gut.

And So It Begins Again: C. difficile Sporulation 

 After its stint as a free-living cell, C. difficile reverts back to its spore form. While the specific triggers of spore formation remain elusive, there is evidence to suggest that, like toxin production, the process is linked to nutrient availability. Indeed, toxin production and sporulation may occur simultaneously and the same gene regulators implicated in nutrient starvation and toxin production are also involved in sporulation. In any case, once the process is complete, the new spores are shed into the environment, where they bide their time until a new host comes along.

Managing CDI by Starving the Pathogen 

The need for new methods for managing CDI fuels research into strategies that prevent or treat infection by targeting metabolically-distinct phases of C. difficile’s life cycle. Some of these strategies focus on germination. After all, inhibiting germination of C. difficile spores saps the ability of the pathogen to do any damage. Synthesized compounds that mimic host-derived primary bile acids prevent C. difficile spore germination in vitro, sometimes even better than their naturally-occurring molecule. One competitive inhibitor of taurocholate-mediated germination, called CamSa, even prevented/delayed CDI in a mouse model of disease. 
 

Other strategies restore a C. difficile-resistant gut microbiota. While FMTs are undefined in their microbial composition, recent work has focused on identifying and generating specific bacteria or defined bacterial “cocktails” that can inhibit C. difficile germination and growth. For example, Clostridium scindens is one of the gut bacteria that can convert primary bile acids into secondary bile acids. This bacterium is associated with CDI resistance in humans and increases resistance to infection in mouse models of disease, in part by altering the bile acid pool within the gut.
 
Promoting microbial competition with C. difficile for nutrients is another viable option. One recent study identified 5 gut bacteria that can compete with C. difficile for mucus-derived sugars and reduce its growth in the gut. Others have shown that intestinal colonization by strains of C. difficile that lack toxin production genes, some of which are naturally occurring, can protect against subsequent infection by a toxigenic strain. While the mechanism underlying this protection is unclear, nutrient competition may play a role. 

Still others have taken an indirect approach by assessing how host diet affects the microbiota and resistance to CDI. For instance, short chain fatty acids (SCFA) produced from bacterial fermentation of dietary fiber can inhibit C. difficile growth and protect against CDI. To this end, mice fed a diet high in plant-derived polysaccharides had more SCFA-producing microbes in their gut, which were associated with reduced gut concentrations of C. difficile. Decreasing other dietary components, like proline  and protein also reduced C. difficile intestinal expansion in mouse models of disease. Thus, tweaking diet may be one way to reduce the availability of C. difficile-preferred nutrients and enrich for microbes that resist the pathogen. 
 
While the results from these studies are encouraging, many of the proposed strategies for managing CDI are still in the early phases of development—they have yet to become more than intriguing possibilities. Nevertheless, they emphasize that the key to managing CDI may rest in understanding the metabolic underpinnings of C. difficile’s life cycle.