Muricholic acids inhibit Clostridium difficile spore germination and growth

Abstract

Infections caused by Clostridium difficile have increased steadily over the past several years. While studies on C. difficile virulence and physiology have been hindered, in the past, by lack of genetic approaches and suitable animal models, newly developed technologies and animal models allow these processes to be studied in detail. One such advance is the generation of a mouse-model of C. difficile infection. The development of this system is a major step forward in analyzing the genetic requirements for colonization and infection. While important, it is equally as important in understanding what differences exist between mice and humans. One of these differences is the natural bile acid composition. Bile acid-mediated spore germination is an important step in C. difficile colonization. Mice produce several different bile acids that are not found in humans. These muricholic acids have the potential to impact C. difficile spore germination. Here we find that the three muricholic acids (α-muricholic acid, β-muricholic acid and ω-muricholic acid) inhibit C. difficile spore germination and can impact the growth of vegetative cells. These results highlight an important difference between humans and mice and may have an impact on C. difficile virulence in the mouse-model of C. difficile infection.

Figure 1. Structures of common muricholic acids.

The primary bile acids (cholic acid, chenodeoxycholic acid, α-muricholic acid and β-muricholic acid) are listed. Deoxycholic acid and ω-muricholic acid are secondary bile acids and are products of the normal microbiota.

Figure 2. α-muricholic acid inhibits germination by C. difficile UK1 spores.

(A) Germination of Clostridium difficile UK1 spores in complex medium supplemented with taurocholic acid (TA) or (B) medium supplemented with TA and 1 mM CDCA or (C) medium supplemented with TA and 1 mM α-muricholic acid. • 0 mM TA, ▪ 2 mM TA, ▴ 5 mM TA, ▾ 10 mM TA,♦ 20 mM TA or ○ 50 mM TA. (D) The inverse rate (1/v [sec/OD₆₀₀]), versus the inverse taurocholate concentration (1/S [mM⁻¹], was plotted. Apparent K_m values for TA alone (•) and in the presence of α-muricholic acid (▪) were determined from the linear best fit.

Figure 3. α-muricholic acid inhibits germination by C. difficile M68 spores.

(A) Germination of Clostridium difficile M68 spores in complex medium supplemented with taurocholic acid (TA) or (B) medium supplemented with TA and 1 mM α-muricholic acid. • 0 mM TA, ▪ 2 mM TA, ▴ 5 mM TA, ▾ 10 mM TA,♦ 20 mM TA or ○ 50 mM TA. (C) The inverse rate (1/v [sec/OD₆₀₀]), versus the inverse taurocholate concentration (1/S [mM⁻¹], was plotted. Apparent K_m values for TA alone (•) and in the presence of α-muricholic acid (▪) were determined from the linear best fit of the plotted data. (D) Hill Plot was generated to determine the apparent K_m values for each condition.