Conservation of the "Outside-in" Germination Pathway in Paraclostridium bifermentans

Abstract

Clostridium difficile spore germination is initiated in response to certain bile acids and amino acids (e.g., glycine). Though the amino acid-recognizing germinant receptor is unknown, the bile acid germinant receptor is the germination-specific, subtilisin-like pseudoprotease, CspC. In C. difficile the CspB, CspA, and CspC proteins are involved in spore germination. Of these, only CspB is predicted to have catalytic activity because the residues important for catalysis are mutated in the cspA and cspC sequence. The CspB, CspA, and CspC proteins are likely localized to the outer layers of the spore (e.g., the cortex or the coat layers) and not the inner membrane where the Ger-type germinant receptors are located. In C. difficile, germination proceeds in an "outside-in" direction, instead of the "'inside-out" direction observed during the germination of Bacillus subtilis spores. During C. difficile spore germination, cortex fragments are released prior to the release of 2,4-dipicolinic acid (DPA) from the spore core. This is opposite to what occurs during B. subtilis spore germination. To understand if the mechanism C. difficile spore germination is unique or if spores from other organisms germinate in a similar fashion, we analyzed the germination of Paraclostridium bifermentans spores. We find that P. bifermentans spores release cortex fragments prior to DPA during germination and the DPA release from the P. bifermentans spore core can be blocked by high concentrations of osmolytes. Moreover, we find that P. bifermentans spores do not respond to steroid-like compounds (unlike the related C. difficile and P. sordellii organisms), indicating that the mere presence of the Csp proteins does permit germination in response to steroid compounds. Our findings indicate that the "outside in" mechanism of spore germination observed in C. difficile can be found in other bacteria suggesting that this mechanism is a novel pathway for endospore germination.

Keywords: Clostridium; DPA; cortex; germination; spore.

FIGURE 1

Characterizing the germinants of P. bifermentans spores. (A) Illustration of the P. bifermentans CspBA and CspC proteins and the location of the would-be catalytic residues. Germination of P. bifermentans spores was analyzed by changes in OD₆₀₀ (B,D,F) and Tb³⁺ fluorescence upon complexing with DPA (C,E,G). Germination assays were conducted in 50 mM HEPES, 100 mM NaCl at pH 7.5 buffer supplemented amino acids. (B,C) Spores alone, 50 mM L-alanine 50 mM L-arginine, Δ; 50 mM L-phenylalanine, open inverted triangle; 50 mM L-alanine, 5 mM L-phenylalanine, 5 mM L-arginine, (D,E). Spores alone, 50 mM L-alanine, 5 mM L-arginine, 50 mM L-alanine, 5 mM L-phenylalanine, Δ; 5 mM L-phenylalanine, 5 mM L-arginine, 50 mM L-alanine, 5 mM L-phenylalanine, 5 mM L-arginine, (F,G) Spores alone, 50 mM L-alanine, 5 mM L-leucine, Data points represent the average from three independent experiments and error bars represent the standard deviation from the mean.

FIGURE 2

Analyzing the influence of bile acids on P. bifermentans spore germination. Germination of P. bifermentans spores was analyzed by changes in OD₆₀₀ (A,C,E,G) and Tb³⁺ fluorescence upon complexing with DPA (B,D,F,H). Spores, 50 mM L-alanine, 5 mM L-phenylalanine, 5 mM L-arginine, 50 mM L-alanine, 5 mM L-phenylalanine, 5 mM L-arginine, indicated compound, Δ. (A,B) 2 mM taurocholic acid; (C,D) 2 mM chenodeoxycholic acid; (E,F) 2 mM deoxycholic acid; (G,H) 2 mM triton X-100. Data points represent the average from three independent experiments and error bars represent the standard deviation from the mean.

FIGURE 3

Cortex degradation precedes DPA release during P. bifermentans spore germination. P. bifermentans spores were germinated in the presence of L-alanine, L-arginine, and L-phenylalanine. At the indicated times, samples were taken to analyze cortex fragment release and for DPA release . Data points represent the average from three independent experiments and error bars represent the standard deviation from the mean. Statistical significant was determined using a two-way ANOVA with Sidak’s multiple comparisons test. The asterisk marked points indicate statistical significance (p-value < 0.05).

FIGURE 4

Analyzing DPA release in the presence of high concentrations of sorbitol. DPA release from germinating P. bifermentans spores (A,C,E,G) or from germinating B. subtilis spores (B,D,F,H) was analyzed germinated in presence of and in absence of sorbitol. (A,B) 10% sorbitol; (C,D) 20% sorbitol; (E,F) 30% sorbitol; (G,H) 38% sorbitol. Data points represent the average from three independent experiments and error bars represent the standard deviation from the mean.

FIGURE 5

Quantifying the effects of sorbitol on spore germination. The maximum rates of spore germination in the presence or absence of sorbitol was determined by applying a first order derivative to the germination curves found in Figure 4 for both P. bifermentans (A,C) and B. subtilis (B,D). The raw data from the first order derivative (A,B) was smoothed using a rolling average of 8 surrounding data points for every data point in the plot (C,D). Experiments were performed in triplicate and the plots are a representative of one of the replicates. The average maximum rate of DPA release from the triplicate samples are tabulated in Table 1.