Clostridioides difficile minimal nutrient requirements for flagellar motility

Abstract

As many gastro-intestinal pathogens, the majority of Clostridioides difficile strains express flagella together with a complete chemotaxis system. The resulting swimming motility is likely contributing to the colonization success of this important pathogen. In contrast to the well investigated general energy metabolism of C. difficile, little is known about the metabolic requirements for maintaining the ion motive force across the membrane, which in turn powers the flagellar motor. We studied here systematically the effect of various amino acids and carbohydrates on the swimming velocity of C. difficile using video microscopy in conjunction with a software based quantification of the swimming speed. Removal of individual amino acids from the medium identified proline and cysteine as the most important amino acids that power swimming motility. Glycine, which is as proline one of the few amino acids that are reduced in Stickland reactions, was not critical for swimming motility. This suggests that the ion motive force that powers the flagellar motor, is critically depending on proline reduction. A maximal and stable swimming motility was achieved with only four compounds, including the amino acids proline, cysteine and isoleucine together with a single, but interchangeable carbohydrate source such as glucose, succinate, mannose, ribose, pyruvate, trehalose, or ethanolamine. We expect that the identified "minimal motility medium" will be useful in future investigations on the flagellar motility and chemotactic behavior in C. difficile, particularly for the unambiguous identification of chemoattractants.

Keywords: Clostridioides difficile; flagella; minimal medium optimization; motility; nutrients.

FIGURE 1

Swimming speed in defined medium. The median swimming speed (μm/s) of Clostridioides difficile cells was determined in (i) BHIS based medium (10% BHIS in PBS); (ii) PBS supplemented with the seven amino acids methionine (200 mg/L), cysteine (200 mg/L), isoleucine (300 mg/L), tryptophan (100 mg/L), proline (2,000 mg/L), leucine (400 mg/L), valine (300 mg/L), and 0.2 g/L glucose; (iii) PBS supplemented with the seven amino acids as before, but without glucose; (iv) PBS supplemented with 0.2 g/L glucose. (A) For each experiment, the median swimming speed of at least 100 bacteria was determined using the YSMR software. Indicated values are the mean from at least three independent experiments (biological replicates) ± standard deviation. Samples with significantly reduced values using the Tukey HSD method (p < 0.05) in comparison to the BHIS control were labeled with asterisks (*). Swimming speed was strongly reduced without glucose (bar 3) and without amino acids (bar 4). (B) One representative experiment. Each dot represents the median speed of one tracked bacterium, with the number of tracked bacteria per experiment given at the bottom (n). Additionally for each condition a box-plot is shown, indicating the interquartile range (IQR) (lower and upper end of the gray box), the median (middle line), and the lower/upper whisker limit as given by 1.5 times the IQR.

FIGURE 2

The median swimming speed (μm/s) of C. difficile cells was determined for different combinations of six amino acids. The control (7AA + Gluc) consists of PBS supplemented with the seven amino acids as described in Figure 1. The other samples include six out of these seven amino acids, without the indicated amino acid. All samples include 0.2 g/L glucose. For each experiment, the median swimming speed of at least 100 bacteria was determined using the YSMR software. Indicated values are the mean from at least three independent experiments (biological replicates) ± standard deviation. Samples with significantly reduced values using the Tukey HSD method (p < 0.05) in comparison to the control sample (7AA + Gluc) were labeled with asterisks (*). Swimming speed was significantly reduced without cysteine and without proline.

FIGURE 3

Identification of the “minimal motility medium.” The median swimming speed (μm/s) of C. difficile cells was determined for various combinations of proline (2,000 mg/L), cysteine (200 mg/L), isoleucine (300 mg/L), and glucose (0.2 g/L). For each experiment, the median swimming speed of at least 100 bacteria was determined using the YSMR software. Indicated values are the mean from at least three independent experiments (biological replicates) ± standard deviation. Samples with significantly reduced values using the Tukey HSD method (p < 0.05) in comparison to the sample containing proline, cysteine, isoleucine + glucose (PCI + Gluc) were labeled with asterisks (*). Swimming speed was strongly reduced in all samples, which lack either cysteine, or proline, or both.

FIGURE 4

Influence of glycine on the swimming motility. The median swimming speed (μm/s) of C. difficile cells was determined (i) with additional glycine (bars 3–6), and (ii) by replacement of proline for glycine (bar 7). Concentration of glycine was as indicated either 0.3 g/l or 3 g/L. For each experiment, the median swimming speed of at least 100 bacteria was determined using the YSMR software. Indicated values are the mean from at least three independent experiments (biological replicates) ± standard deviation. Samples with significantly reduced values using the Tukey HSD method (p < 0.05) in comparison to the sample containing proline, cysteine, isoleucine + glucose (PCI + Gluc) were labeled with asterisks (*). Swimming speed was strongly reduced after replacement of proline for glycine.

FIGURE 5

Influence of different carbohydrates on the swimming motility. The median swimming speed (μm/s) of C. difficile cells was determined in samples containing the three amino acids proline (2,000 mg/L), cysteine (200 mg/L), isoleucine (300 mg/L) (abbreviated as “PCI”) plus one of the indicated carbohydrates at (0.2 g/L). For each experiment, the median swimming speed of at least 100 bacteria was determined using the YSMR software. Indicated values are the mean from at least three independent experiments (biological replicates) ± standard deviation. Samples with significantly reduced values using the Tukey HSD method (p < 0.05) in comparison to the sample containing proline, cysteine, isoleucine + glucose (PCI + Gluc) were labeled with asterisks (*). Swimming speed was significantly reduced in samples with ribose (p < 0,006) and with pyruvate (p < 0.002).