Hierarchical recognition of amino acid co-germinants during Clostridioides difficile spore germination

Abstract

Bile acids are an important signal for germination of Clostridioides difficile spores; however, the bile acid signal alone is not sufficient. Amino acids, such as glycine, are another signal necessary for germination by C. difficile spores. Prior studies on the amino acid signal required for germination have shown that there is a preference for the amino acid used as a signal for germination. Previously we found that d-alanine can function as a co-germinant for C. difficile spores at 37 °C but not at 25 °C. Here, we tested the ability of other amino acids to act as co-germinants with taurocholate (TA) at 37 °C and found that many amino acids previously categorized as non-co-germinants are co-germinants at 37 °C. Based on the EC₅₀ values calculated for two different strains, we found that C. difficile spores recognize different amino acids with varying efficiencies. Using this data, we ranked the amino acids based on their effect on germination and found that in addition to d-alanine, other D-forms of amino acids are also used by C. difficile spores as co-germinants. Among the different types of amino acids, ones with branched chains such as valine, leucine, and isoleucine are the poorest co-germinants. However, glycine is still the most effective amino acid signal for both strains. Our results suggest that the yet-to-be-identified amino acid germinant receptor is highly promiscuous.

Keywords: Amino acid; C. difficile; Co-germinant; Germination; Spore.

Figure 1. Comparison of C. difficile spore germination at 25 °C and 37 °C

Purified C. difficile UK1 spores were suspended in buffer supplemented with 10 mM taurocholate and 30 mM of the indicated amino acid. The change in OD₆₀₀ during germination (▲) and the release of DPA (●) was measured over time at 25 °C (A, C, E) or 37 °C (B, D, F). Glycine (A & B), L-alanine (C & D) or D-alanine (E & F) were tested as co-germinants. The data represent the average of three independent measurements and error bars represent the standard deviation from the mean.

Figure 2. Analysis of the effects of different co-germinants on C. difficile spore germination

Purified C. difficile UK1 spores were suspended in buffer supplemented with 10 mM taurocholate and 30 mM of the indicated amino acid. The change in OD₆₀₀ during germination (▲) and the release of DPA (●) was measured over time at 25 °C (A & C) or 37 °C (B & D). L-lysine (A & B) or L-isoleucine (C & D) were tested as co-germinants. The data represent the average of three independent measurements and error bars represent the standard deviation from the mean.