Clostridioides (also Clostridium) difficile is a Gram-positive, spore-forming, obligate anaerobe that causes a spectrum of intestinal diseases. C. difficile diseases are among the most commonly acquired nosocomial infections in the developed world. Treatment of C. difficile infections costs more than $3 billion per year in the U.S. alone. C. difficile produces toxins that damage the intestinal epithelium, resulting in inflammation and diarrheal symptoms. Yet little is known about how this organism senses and adapts to the host intestinal environment. The Tamayo Lab is interested in identifying and characterizing the regulatory mechanisms employed by C. difficile to colonize, survive, and cause disease in a host.

PHASE VARIATION AND PHENOTYPIC HETEROGENEITY

Phase variation is a means by which many bacterial species introduce phenotypic heterogeneity into the population as a strategy to ensure the population’s survival in the face of changing selective pressures. Phase variation occurs in a heritable, reversible manner and typically modulates the ON/OFF production of factors affecting environmental adaptation. These factors are typically surface components that directly interact with the environment, such as fimbriae, flagella, and extracellular carbohydrates. Numerous mucosal pathogens employ phase variation to control the biosynthesis of virulence factors that may be immunostimulatory, thereby promoting immune evasion and persistence in a host. In C. difficile, up to seven loci are subject to phase variation via site-specific DNA recombination. These loci are each preceded by an invertible DNA element, or “switch”, that depending on its orientation determines whether the downstream genes are expressed. Of particular interest are four phase variable loci connected to c-di-GMP signaling: the c-di-GMP regulated flagellar operon, a c-di-GMP regulated signal transduction system operon, and two c-di-GMP hydrolase genes. We aim to determine the mechanisms of switch inversion, how the orientation of the switches controls gene expression, the conditions that influence phase variation, and the impact of phase variation on disease outcome. These studies are tackling fundamental questions about the phenotypic heterogeneity that arises in a C. difficile population, including the molecular basis and phenotypic outcomes of c-di-GMP signaling and phase variation in vitro and during infection.

SECOND MESSENGER SIGNALING IN C. DIFFICILE

The intracellular signaling molecule cyclic diguanylate (c-di-GMP) transmits signals received at the cell surface to control a wide variety of adaptive responses. Most notably, c-di-GMP regulates the switch between motile and non-motile lifestyles in many species. We aim to determine if and how c-di-GMP influences C. difficile colonization of the host intestine and development of disease. We have generated tools for manipulating c-di-GMP levels in C. difficile and have identified numerous regulated pathways. These processes include flagellum-based swimming motility, toxin production, pilus-mediated surface motility, and host colonization, suggesting a fundamental role for c-di-GMP in the ability of C. difficile to colonize and cause disease in the intestine. These processes are regulated directly or indirectly via c-di-GMP riboswitches. We are currently investigating the molecular basis of c-di-GMP regulation of these various pathways and the impact of c-di-GMP signaling on C. difficile virulence. In addition, we are dissecting the c-di-GMP signaling pathway by evaluating the contribution of individual components (i.e., c-di-GMP synthases and hydrolases) to controlling c-di-GMP—regulated processes and by identifying the extracellular stimuli that influence intracellular c-di-GMP levels.