Clostridioides difficile phosphoproteomics shows an expansion of phosphorylated proteins in stationary growth phase

In this paper, we present a comprehensive analysis of protein phosphorylation in the Gram-positive enteropathogen Clostridioides difficile. To date, only limited evidence on the role of phosphorylation in the regulation of this organism has been published; the current study is expected to form the basis for research on this posttranslational modification in C. difficile.Phosphorylation is a posttranslational modification that can affect both housekeeping functions and virulence characteristics in bacterial pathogens. In the Gram-positive enteropathogen Clostridioides difficile, the extent and nature of phosphorylation events are poorly characterized, though a protein kinase mutant strain demonstrates pleiotropic phenotypes. Here, we used an immobilized metal affinity chromatography strategy to characterize serine, threonine, and tyrosine phosphorylation in C. difficile. We find limited protein phosphorylation in the exponential growth phase but a sharp increase in the number of phosphopeptides after the onset of the stationary growth phase. Our approach identifies expected targets and phosphorylation sites among the more than 1,500 phosphosites, including the protein kinase PrkC, the anti-sigma-F factor antagonist (SpoIIAA), the anti-sigma-B factor antagonist (RsbV), and HPr kinase/phosphorylase (HprK). Analysis of high-confidence phosphosites shows that phosphorylation on serine residues is most common, followed by threonine and tyrosine phosphorylation. This work forms the basis for a further investigation into the contributions of individual kinases to the overall phosphoproteome of C. difficile and the role of phosphorylation in C. difficile physiology and pathogenesis. IMPORTANCE In this paper, we present a comprehensive analysis of protein phosphorylation in the Gram-positive enteropathogen Clostridioides difficile. To date, only limited evidence on the role of phosphorylation in the regulation of this organism has been published; the current study is expected to form the basis for research on this posttranslational modification in C. difficile.Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc

The Signal Sequence of the Abundant Extracellular Metalloprotease PPEP-1 Can Be Used to Secrete Synthetic Reporter Proteins in Clostridium difficile

Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc

Practical observations on the use of fluorescent reporter systems in Clostridioides difficile

Fluorescence microscopy is a valuable tool to study a broad variety of bacterial cell components and dynamics thereof. For Clostridioides difficile, the fluorescent proteins CFPopt, mCherry(Opt) and phiLOV2.1, and the self-labelling tags SNAP(Cd) and HaloTag, hereafter collectively referred as fluorescent systems, have been described to explore different cellular pathways. In this study, we sought to characterize previously used fluorescent systems in C. difficile cells. We performed single cell analyses using fluorescence microscopy of exponentially growing C. difficile cells harbouring different fluorescent systems, either expressing these separately in the cytosol or fused to the C-terminus of HupA, under defined conditions. We show that the intrinsic fluorescence of C. difficile cells increases during growth, independent of sigB or spo0A. However, when C. difficile cells are exposed to environmental oxygen autofluorescence is enhanced. Cytosolic overexpression of the different fluorescent systems alone, using the same expression signals, showed heterogeneous expression of the fluorescent systems. High levels of mCherry(Opt) were toxic for C. difficile cells limiting the applicability of this fluorophore as a transcriptional reporter. When fused to HupA, a C. difficile histone-like protein, the fluorescent systems behaved similarly and did not affect the HupA overproduction phenotype. The present study compares several commonly used fluorescent systems for application as transcriptional or translational reporters in microscopy and summarizes the limitations and key challenges for live-cell imaging of C. difficile. Due to independence of molecular oxygen and fluorescent signal, SNAP(Cd) appears the most suitable candidate for live-cell imaging in C. difficile to date.Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc

New insights into the type A glycan modification of Clostridioides difficile flagellar protein flagellin C by phosphoproteomics analysis

The type A glycan modification found in human pathogen Clostridioides difficile consists of a monosaccharide (GlcNAc) that is linked to an N-methylated threonine through a phosphodiester bond. This structure has previously been described on the flagellar protein flagellin C of several C. difficile strains and is important for bacterial motility. The study of post-translational modifications often relies on some type of enrichment strategy; however, a procedure for enrichment of this modification has not yet been demonstrated. In this study, we show that an approach that is commonly used in phosphoproteomics, Fe3+-immobilized metal affinity chromatography, also enriches for peptides with this unique post-translational modification. Using LC-MS/MS analyses of immobilized metal affinity chromatography-captured tryptic peptides, we observed not only type A-modified C. difficile flagellin peptides but also a variety of truncated/modified type A structures on these peptides. Using an elaborate set of mass spectrometry analyses, we demonstrate that one of these modifications consists of a type A structure containing a phosphonate (2-aminoethylphosphonate), a modification that is rarely observed and has hitherto not been described in C. difficile. In conclusion, we show that a common enrichment strategy results in reliable identification of peptides carrying a type A glycan modification, and that the results obtained can be used to advance models about its biosynthesis.Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc

Practical observations on the use of fluorescent reporter systems in Clostridioides difficile

Fluorescence microscopy is a valuable tool to study a broad variety of bacterial cell components and dynamics thereof. For Clostridioides difficile, the fluorescent proteins CFPopt, mCherry(Opt) and phiLOV2.1, and the self-labelling tags SNAP(Cd) and HaloTag, hereafter collectively referred as fluorescent systems, have been described to explore different cellular pathways. In this study, we sought to characterize previously used fluorescent systems in C. difficile cells. We performed single cell analyses using fluorescence microscopy of exponentially growing C. difficile cells harbouring different fluorescent systems, either expressing these separately in the cytosol or fused to the C-terminus of HupA, under defined conditions. We show that the intrinsic fluorescence of C. difficile cells increases during growth, independent of sigB or spo0A. However, when C. difficile cells are exposed to environmental oxygen autofluorescence is enhanced. Cytosolic overexpression of the different fluorescent systems alone, using the same expression signals, showed heterogeneous expression of the fluorescent systems. High levels of mCherry(Opt) were toxic for C. difficile cells limiting the applicability of this fluorophore as a transcriptional reporter. When fused to HupA, a C. difficile histone-like protein, the fluorescent systems behaved similarly and did not affect the HupA overproduction phenotype. The present study compares several commonly used fluorescent systems for application as transcriptional or translational reporters in microscopy and summarizes the limitations and key challenges for live-cell imaging of C. difficile. Due to independence of molecular oxygen and fluorescent signal, SNAP(Cd) appears the most suitable candidate for live-cell imaging in C. difficile to date

The Bacterial Chromatin Protein HupA Can Remodel DNA and Associates with the Nucleoid in Clostridium difficile

Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc

Identification of the unwinding region in the clostridioides difficile chromosomal origin of replication

Faithful DNA replication is crucial for viability of cells across all kingdoms. Targeting DNA replication is a viable strategy for inhibition of bacterial pathogens.Clostridioides difficileis an important enteropathogen that causes potentially fatal intestinal inflammation. Knowledge about DNA replication in this organism is limited and no data is available on the very first steps of DNA replication. Here, we use a combination ofin silicopredictions andin vitroexperiments to demonstrate thatC. difficileemploys a bipartite origin of replication that shows DnaA-dependent melting atoriC2, located in thednaA-dnaNintergenic region. Analysis of putative origins of replication in different clostridia suggests that the main features of the origin architecture are conserved. This study is the first to characterize aspects of the origin region ofC. difficileand contributes to our understanding of the initiation of DNA replication in clostridia

The C-terminal domain of clostridioides difficile TcdC is exposed on the bacterial cell surface

Clostridioides difficile is an anaerobic Gram-positive bacterium that can produce the large clostridial toxins toxin A and toxin B, encoded within the pathogenicity locus (PaLoc). The PaLoc also encodes the sigma factor TcdR, which positively regulates toxin gene expression, and TcdC, which is a putative negative regulator of toxin expression. TcdC is proposed to be an anti-sigma factor; however, several studies failed to show an association between the tcdC genotype and toxin production. Consequently, the TcdC function is not yet fully understood. Previous studies have characterized TcdC as a membrane-associated protein with the ability to bind G-quadruplex structures. The binding to the DNA secondary structures is mediated through the oligonucleotide/oligosaccharide binding fold (0B-fold) domain present at the C terminus of the protein. This domain was previously also proposed to be responsible for the inhibitory effect on toxin gene expression, implicating a cytoplasmic localization of the OB-fold. In this study, we aimed to obtain topological information on the C terminus of TcdC and demonstrate that the C terminus of TcdC is located extracellularly. In addition, we show that the membrane association of TcdC is dependent on a membrane-proximal cysteine residue and that mutating this residue results in the release of TcdC from the bacterial cell. The extracellular location of TcdC is not compatible with the direct binding of the OB-fold domain to intracellular nucleic acid or protein targets and suggests a mechanism of action that is different from that of the characterized anti-sigma factors.IMPORTANCE The transcription of C. difficile toxins TcdA and TcdB is directed by the sigma factor TcdR. TcdC has been proposed to be an anti-sigma factor. The activity of TcdC has been mapped to its C terminus, and the N terminus serves as the membrane anchor. Acting as an anti-sigma factor requires a cytoplasmic localization of the C terminus of TcdC. Using cysteine accessibility analysis and a HiBiT-based system, we show that the TcdC C terminus is located extracellularly, which is incompatible with its role as anti-sigma factor. Furthermore, mutating a cysteine residue at position 51 resulted in the release of TcdC from the bacteria. The codon-optimized version of the HiBiT (HiBiT(opt)) extracellular detection system is a valuable tool for topology determination of membrane proteins, increasing the range of systems available to tackle important aspects of C. difficile development