Phase-variable expression of pdcB, a phosphodiesterase, influences sporulation in Clostridioides difficile

Abstract

Clostridioides difficile is the causative agent of antibiotic-associated diarrhea and is the leading cause of nosocomial infection in developed countries. An increasing number of C. difficile infections are attributed to epidemic strains that produce more toxins and spores. C. difficile spores are the major factor for the transmission and persistence of the organism. Previous studies have identified global regulators that influence sporulation in C. difficile. This study discovers that PdcB, a phosphodiesterase, enhances sporulation in C. difficile strain UK1. Through genetic and biochemical assays, we show that phase-variable expression of pdcB results in hypo- and hyper-sporulation phenotypes. In the "ON" orientation, the identified promotor is in the right orientation to drive the expression of pdcB. Production of the PdcB phosphodiesterase reduces the intracellular cyclic-di-GMP (c-di-GMP) concentration, resulting in a hyper-sporulation phenotype. Loss of PdcB due to the pdcB promoter being in the OFF orientation or mutation of pdcB results in increased c-di-GMP levels and a hypo-sporulation phenotype. Additionally, we demonstrate that CodY binds to the upstream region of pdcB. DNA inversion reorients the CodY binding site so that in the OFF orientation, CodY binds a site that is upstream of the pdcB promoter and can further repress gene expression.

Keywords: Clostridioides difficile; gene regulation; phase variation.

Figure 1.. Colony and cell morphologies of UK1_T and UK1_O strains.

A. UK1 strain when grown in TY medium gives rise to phenotypically distinguishable Translucent (UK1_T) and Opaque (UK1_O) colony morphotypes. B. Phase-contrast microscopic images of UK1_T and UK1_O colony morphotypes. C. Average cell length showing UK1_T cells are significantly longer than UK1_O cells. Twenty-five individual cells per image were measured using ImageJ software. At least four images from two replicates were used (n=100). Data was analyzed using unpaired t-test with Welch’s correction where *** indicates p<0.005.

Figure 2.. Phenotypic characterization of UK1_T and UK1_O strains

A. Percentage sporulation of UK1_T and UK1_O cells showing hyper sporulation phenotype in UK1_O morphotype. B. Swarming motility exhibited by UK1_T and UK1_O in TY+1.8% agar. C. Biofilm formation of UK1_T and UK1_O morphotypes. OD₅₇₀ nm was measured to quantify biofilm formation. Three biological replicates were used to carry out the experiments, and data were analyzed by two-tailed unpaired t-test with Welch’s correction where **** indicates p<0.0005 and *** is p<0.005

Figure 3.. DNA inversion upregulates the expression of CDR20291_0685 in UK_O cells.

A. The beta-glucuronidase activity of the PCDR20291_0685 upstream-gusA fusions locked in UK1_T and UK1_O orientation in the UK1 parent strain. The data represent the results from three biological replicates. Data were analyzed using 2-way ANOVA (Sidak’s multiple comparisons test) comparing the mean of UK1_T and UK1_O at each time point. *** indicates p<0.005. B. qRT-PCR results showing overexpression of CDR20291_0685 in UK1_O strain at 16h time point. The representative results from three independent experiments are shown. Data were analyzed by a two-tailed unpaired t-test with Welch’s correction where *** is p<0.005.

Figure 4.. The promoter of pdcB is located within the invertible region.

A. Schematic of the upstream region of pdcB from pdcB (ON) cells (the orientation in which pdcB is expressed) depicting the invertible region flanked by the right and left inverted repeats (RIR and LIR). The forward and the reverse primers designed along the length of the invertible region are shown. The transcription initiation site was found near the RIR, and −35 and −10 sites are marked. B. RT PCR detecting the transcripts of pdcB. Genomic DNA (gDNA) and RNA were used as positive and negative controls, respectively. Only the primer pairs ORG921/ORG853, and ORG922/ORG853 gave amplification products of size 843 bp and 789 bp, respectively.

Figure 5.. Intracellular c-di-GMP levels influence sporulation in UK1 strain

A. Phase-contrast microscopic images along with colony morphology of pdcB (OFF), pdcB (ON), UK1 expressing dccA, UK1::pdcB mutant complemented with pdcB-EAL/ pdcA-EAL. Average cell length is marked at the upper right corner of each image. Using ImageJ software, at least three images from two replicates were used for measuring the cell length (n=100). Long cells appeared upon the induction of dccA were marked with white arrows. The intracellular levels of c-di-GMP (B) and sporulation percentage (C) were measured in these strains. To induce the expression of dccA, pdcB-EAL, pdcA-EAL 100 ngs/ml of ATc was included in the TY agar plates. Three biological replicates were used and data were analyzed by one-way analysis of variance (ANOVA) where ** indicates p<0.05, and *** indicates p<0.005.

Figure 6.. CodY represses the expression of pdcB, which is partially relieved by DNA inversion.

A. Schematic showing the potential CodY binding sites that lie within the invertible region upstream of pdcB. LIR and RIR are in bold and marked with arrows B. Beta-glucuronidase activity of PpdcB-gusA fusions locked in either translucent or opaque orientation in the parent UK1 and UK1::codY mutant. The data shown are the standard errors of the mean of three biological replicates. Statistical analysis was performed using two-way ANOVA.** indicates p<0.05.C. Binding of purified CodY to the upstream region of pdcB. Binding was increased with increasing concentration of CodY. The presence of GTP and ILV further enhanced CodY binding. Ten-fold excess of the cold probe was added with 125 ng of purified CodY in a control reaction to rule out non-specific binding.