Functional genomics reveals that Clostridium difficile Spo0A coordinates sporulation, virulence and metabolism

Abstract

Background: Clostridium difficile is an anaerobic, Gram-positive bacterium that can reside as a commensal within the intestinal microbiota of healthy individuals or cause life-threatening antibiotic-associated diarrhea in immunocompromised hosts. C. difficile can also form highly resistant spores that are excreted facilitating host-to-host transmission. The C. difficile spo0A gene encodes a highly conserved transcriptional regulator of sporulation that is required for relapsing disease and transmission in mice.

Results: Here we describe a genome-wide approach using a combined transcriptomic and proteomic analysis to identify Spo0A regulated genes. Our results validate Spo0A as a positive regulator of putative and novel sporulation genes as well as components of the mature spore proteome. We also show that Spo0A regulates a number of virulence-associated factors such as flagella and metabolic pathways including glucose fermentation leading to butyrate production.

Conclusions: The C. difficile spo0A gene is a global transcriptional regulator that controls diverse sporulation, virulence and metabolic phenotypes coordinating pathogen adaptation to a wide range of host interactions. Additionally, the rich breadth of functional data allowed us to significantly update the annotation of the C. difficile 630 reference genome which will facilitate basic and applied research on this emerging pathogen.

Figure 1

Growth and sporulation kinetics of C. difficile 630 derivatives in broth cultures. a) Growth curves of the C. difficile 630∆erm parental strain and its spo0A isogenic mutant. Shown are the OD₆₀₀ measurements on the left y-axis and ethanol-resistant spore-derived CFUs on the right y-axis. The gray vertical arrow indicates that sampling point. b) Western blot analysis demonstrating that Spo0A is expressed under exponential and stationary growth stages by the parental C. difficile 630∆erm strain but not the isogenic spo0A mutant. Spo0A-his6 protein was purified protein as described before [21].

Figure 2

Correlation between transcriptomic and proteomic datasets of genes differentially expressed in the C. difficile spo0A mutant compared to the parental C. difficile 630Δerm strain. The degree of correlation are plotted as log2 fold change for all gene products that had been quantified in both proteome (y-axis) and RNASeq (x-axis) analyses. Significantly (p-adj < 0.01) disregulated proteins or transcripts are shown with red/brown (upregulated) and light green/dark green (downregulated) symbols. Protein groups that were positive in Significance B test of at least two biological replicates are depicted as significant (dark colours). See methods for analysis details. Note that no proteins were upregulated without being upregulated at the transcript level (“proteome up”).

Figure 3

Updated functional class annotation of C. difficile 630 genome compared to original 2006 genome annotation [5]. Genes are classified according to functional classes for the original 2006 annotation (black bars) compared to this study (white bars). Functional classes are described in more detail in Additional file 2.

Figure 4

Functional classification of C. difficile Spo0A regulated genes. Enriched functional gene classes of genes (a) downregulated or (b) upregulated in the C. difficile spo0A mutant. The number of genes present in each functional class based on RNAseq (grey) and proteomics (black) analysis. The combined RNAseq and proteomics (red) represents unique genes found in each functional class. Transcripts and proteins assigned to functional classes are given in parenthesis and are based on the updated C. difficile 630 annotation presented in this study (Additional file 2).

Figure 5

Proposed sporulation cascade for Clostridium difficile 630. Names of genes and proteins are derived from updated genome annotation (Additional file 2). Solid arrows in the putative regulatory cascade of C. difficile indicate confirmed interactions [15,21], whereas dotted arrows indicate uncharacterized or postulated interactions. Operon structure or genomic region of (conserved) sporulation genes is given when relevant, and in vitro confirmed Spo0A binding sites are indicated with vertical blue bars. Genes are roughly aligned with the stage of sporulation at which they act, except for C. difficile sporulation genes that are not conserved in B. subtilis. When gene names start with spo, this has been omitted for clarity. CD3673 encodes a protein with high similarity to Spo0J. CD0125 encodes a protein with homology to B. subtilis spoIIQ. spoIV encodes a homolog of the B. subtilis YqfD protein. sigK is known as spoIIIC (N-terminal part) and spoIVCB (C-terminal part) in B. subtilis and is interrupted by a skin element. CD1231 encodes the recombinase in skin and is annotated as spoIVCA in B. subtilis. oxaA1 is known as spoIIIJ in B. subtilis. pth is known as spoVC in B. subtilis. spoVE is an FtsW-like protein and is sometimes annotated as such in C. difficile. The product of ftsH2 is the closest homolog of SpoVK of B. subtilis. C. difficile 630 encodes several SpoIIIE/FtsK like proteins. If and which one is associated with sporulation is unknown. cotF/cotCB and cotJB2/cotD are homologs of the B. subtilis genes cotJB and cotJC. SleB is also known as PrsW [44]. CspBA is a serine protease. Proteins from this family in B. subtilis are not directly identified as sporulation specific. CspC is a germination receptor [48]. sipL was hypothesized to encode a functional substitute for B. subtilis SpoVID [49]. CD1613 (cotA), CD1511 (cotB), CD1433 (cotE), CD1567 (cotG) have been given a cot alias in a recent study [37]. For the genes in grey the identification as homologs of the B. subtilis sporulation gene is tentative. Green colors indicate upregulated and red colors indicate downregulated in a spo0A mutant compared to the parental C. difficile 630∆erm strain at the transcriptome or proteome level in this study. Recently, the transcription of many genes - including most of the genes from this scheme - was identified as dependent on sporulation specific sigma factors [28,29,50].

Figure 6

Spo0A is a negative regulator of C. difficile flagellar synthesis. Representative transmission electron micrographs of negatively stained C. difficile 630∆erm and derivatives demonstrating a) no observable flagella on the parental 630∆erm strain but b) hyper-flagellation in the spo0A mutant derivative. Genetic complementation of the c)spo0A mutation greatly reduced flagella levels but did not eliminate their production. Scale bar represents 1 micron.

Figure 7

Spo0A positively regulates glucose fermentation pathways and butyrate production in C. difficile. a) Proposed biochemical pathway for glucose uptake and fermentation leading to the production of butyrate. Genes in red are downregulated in the C. difficile spo0A mutant and genes in green are upregulated in the C. difficile spo0A mutant. Genes in black are not impacted by the spo0A mutation. b) Levels of butyrate from supernatants of C. difficile strains during exponential growth. Analysis was performed in triplicate and levels compared using a Student’s T test; **, P = 0.0005; *, P = 0.6.