A group of bacterial proteases, the Pro-Pro endopeptidases (PPEPs), pos-sess the unique ability to hydrolyze proline-proline bonds in proteins. Sincea protease’s function is largely determined by... Show moreA group of bacterial proteases, the Pro-Pro endopeptidases (PPEPs), pos-sess the unique ability to hydrolyze proline-proline bonds in proteins. Sincea protease’s function is largely determined by its substrate specificity,methods that can extensively characterize substrate specificity are valuabletools for protease research. Previously, we achieved an in-depth characteri-zation of PPEP prime-side specificity. However, PPEP specificity is alsodetermined by the non-prime-side residues in the substrate. To gain a morecomplete insight into the determinants of PPEP specificity, we character-ized the non-prime- and prime-side specificity of various PPEPs using acombination of synthetic combinatorial peptide libraries and mass spec-trometry. With this approach, we deepened our understanding of the P3-P30 specificities of PPEP-1 and PPEP-2, while identifying the endogenoussubstrate of PPEP-2 as the most optimal substrate in our library data. Fur-thermore, by employing the library approach, we investigated the alteredspecificity of mutants of PPEP-1 and PPEP-2. Additionally, we character-ized a novel PPEP from Anoxybacillus tepidamans, which we termed PPEP-4. Based on structural comparisons, we hypothesized that PPEP-4 displaysa PPEP-1-like prime-side specificity, which was substantiated by the experi-mental data. Intriguingly, another putative PPEP from Clostridioides diffi-cile, CD1597, did not display Pro-Pro endoproteolytic activity.Collectively, we characterized PPEP specificity in detail using our robustpeptide library method and, together with additional structural informa-tion, provide more insight into the intricate mechanisms that govern prote-ase specificity. Show less
The bacterial flagellum is involved in a variety of processes including motility, adherence, and immunomodulation. In the Clostridioides difficile strain 630 Delta erm, the main filamentous... Show moreThe bacterial flagellum is involved in a variety of processes including motility, adherence, and immunomodulation. In the Clostridioides difficile strain 630 Delta erm, the main filamentous component, FliC, is post-translationally modified with an O-linked Type A glycan structure. This modification is essential for flagellar function, since motility is seriously impaired in gene mutants with improper biosynthesis of the Type A glycan. The cd0240-cd0244 gene cluster encodes the Type A biosynthetic proteins, but the role of each gene, and the corresponding enzymatic activity, have not been fully elucidated. Using quantitative mass spectrometry-based proteomics analyses, we determined the relative abundance of the observed glycan variations of the Type A structure in cd0241, cd0242, cd0243, and cd0244 mutant strains. Our data not only confirm the importance of CD0241, CD0242, and CD0243 but, in contrast to previous data, also show that CD0244 is essential for the biosynthesis of the Type A modification. Combined with additional bioinformatic analyses, we propose a revised model for Type A glycan biosynthesis. Show less
Proteases comprise the class of enzymes that catalyzesthe hydrolysisof peptide bonds, thereby playing a pivotal role in many aspects oflife. The amino acids surrounding the scissile bond determine... Show moreProteases comprise the class of enzymes that catalyzesthe hydrolysisof peptide bonds, thereby playing a pivotal role in many aspects oflife. The amino acids surrounding the scissile bond determine thesusceptibility toward protease-mediated hydrolysis. A detailed understandingof the cleavage specificity of a protease can lead to the identificationof its endogenous substrates, while it is also essential for the designof inhibitors. Although many methods for protease activity and specificityprofiling exist, none of these combine the advantages of combinatorialsynthetic libraries, i.e., high diversity, equimolar concentration,custom design regarding peptide length, and randomization, with thesensitivity and detection power of mass spectrometry. Here, we developedsuch a method and applied it to study a group of bacterial metalloproteasesthat have the unique specificity to cleave between two prolines, i.e.,Pro-Pro endopeptidases (PPEPs). We not only confirmed the prime-sidespecificity of PPEP-1 and PPEP-2, but also revealed some new unexpectedpeptide substrates. Moreover, we have characterized a new PPEP (PPEP-3)that has a prime-side specificity that is very different from thatof the other two PPEPs. Importantly, the approach that we presentin this study is generic and can be extended to investigate the specificityof other proteases. Show less
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... Show moreThe 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. Show less
Clostridioides difficile is the most common cause of antibiotic-associated gastrointestinal infections. Capillary electrophoresis (CE)-PCR ribotyping is currently the gold standard for C. difficile... Show moreClostridioides difficile is the most common cause of antibiotic-associated gastrointestinal infections. Capillary electrophoresis (CE)-PCR ribotyping is currently the gold standard for C. difficile typing but lacks the discriminatory power to study transmission and outbreaks in detail. New molecular methods have the capacity to differentiate better and provide standardized and interlaboratory exchangeable data. Using a well-characterized collection of diverse strains (N = 630; 100 unique ribotypes [RTs]), we compared the discriminatory power of core genome multilocus sequence typing (cgMLST) (SeqSphere and EnteroBase), whole-genome MLST (wgMLST) (EnteroBase), and single-nucleotide polymorphism (SNP) analysis. A unique cgMLST profile (more than six allele differences) was observed in 82 of 100 RTs, indicating that cgMLST could distinguish most, but not all, RTs. Application of cgMLST in two outbreak settings with RT078 and RT181 (known to have low intra-RT allele differences) showed no distinction between outbreak and nonoutbreak strains in contrast to wgMLST and SNP analysis. We conclude that cgMLST has the potential to be an alternative to CE-PCR ribotyping. The method is reproducible, easy to standardize, and offers higher discrimination. However, adjusted cutoff thresholds and epidemiological data are necessary to recognize outbreaks of some specific RTs. We propose to use an allelic threshold of three alleles to identify outbreaks. Show less
Plasmids are ubiquitous in the bacterial world. In many microorganisms, plasmids have been implicated in important aspects of bacterial physiology and contribute to horizontal gene transfer. In... Show morePlasmids are ubiquitous in the bacterial world. In many microorganisms, plasmids have been implicated in important aspects of bacterial physiology and contribute to horizontal gene transfer. In contrast, knowledge on plasmids of the enteropathogen Clostridioides difficile is limited, and there appears to be no phenotypic consequence to carriage of many of the identified plasmids. Emerging evidence suggests, however, that plasmids are common in C. difficile and may encode functions relevant to pathogenesis, such as antimicrobial resistance and toxin production. Here, we review our current knowledge about the abundance, functions and clinical relevance of plasmids in C. difficile. Show less
Current assays for Clostridioides difficile in nonhospital settings are outsourced and time-intensive, resulting in both delayed diagnosis and quarantining of infected individuals. We designed a... Show moreCurrent assays for Clostridioides difficile in nonhospital settings are outsourced and time-intensive, resulting in both delayed diagnosis and quarantining of infected individuals. We designed a more rapid point-of-care assay featuring a "turn-on " bioluminescent readout of a C. difficile-specific protease, PPEP-1. NanoLuc, a bright and stable luciferase, was "caged " with a PPEP-1-responsive peptide tail that inhibited luminescence. Upon proteolytic cleavage, the peptide was released and NanoLuc activity was restored, providing a visible readout. The bioluminescent sensor detected PPEP-1 concentrations as low as 10 nM. Sensor uncaging was achieved within minutes, and signal was captured using a digital camera. Importantly, the sensor was also functional at ambient temperature and compatible with fecal material, suggesting that it can be readily deployed in a variety of settings. Show less
Paiva, A.M.O.; Jong, L. de; Friggen, A.H.; Smits, W.K.; Corver, J. 2020
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... Show moreClostridioides 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. Show less
Objectives: Clostridium difficile is a major global human pathogen divided into five clades, of which Glade 3 is the least characterized and consists predominantly of PCR ribotype (RT) 023 strains.... Show moreObjectives: Clostridium difficile is a major global human pathogen divided into five clades, of which Glade 3 is the least characterized and consists predominantly of PCR ribotype (RT) 023 strains. Our aim was to analyse and characterize this Glade.Methods: In this cohort study the clinical presentation of C. difficile RT023 infections was analysed in comparison with known 'hypervirulent' and non-hypervirulent strains, using data from the Netherlands national C. difficile surveillance programme. European RT023 strains of diverse origin were collected and whole-genome sequenced to determine the genetic similarity between isolates. Distinctive features were investigated and characterized.Results: Clinical presentation of C. difficile RT023 infections show severe infections akin to those seen with 'hypervirulent' strains from clades 2 (RT027) and 5 (RT078) (35%, 29% and 27% severe CDI, respectively), particularly with significantly more bloody diarrhoea than RT078 and non-hypervirulent strains (RT023 8%, other RTs 4%, p 0.036). The full genome sequence of strain CD305 is presented as a robust reference. Phylogenetic comparison of CD305 and a further 79 previously uncharacterized European RT023 strains of diverse origin revealed minor genetic divergence with >99.8% pairwise identity between strains. Analyses revealed distinctive features among Glade 3 strains, including conserved pathogenicity locus, binary toxin and phage insertion toxin genotypes, glycosylation of S-layer proteins, presence of the RT078 four-gene trehalose cluster and an esculinase-negative genotype.Conclusions: Given their recent emergence, virulence and genomic characteristics, the surveillance of Glade 3 strains should be more highly prioritized. (C) 2019 The Authors. Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases. Show less
Metronidazole was until recently used as a first-line treatment for potentially life-threatening Clostridioides difficile (CD) infection. Although cases of metronidazole resistance have been... Show moreMetronidazole was until recently used as a first-line treatment for potentially life-threatening Clostridioides difficile (CD) infection. Although cases of metronidazole resistance have been documented, no clear mechanism for metronidazole resistance or a role for plasmids in antimicrobial resistance has been described for CD. Here, we report genome sequences of seven susceptible and sixteen resistant CD isolates from human and animal sources, including isolates from a patient with recurrent CD infection by a PCR ribotype (RT) 020 strain, which developed resistance to metronidazole over the course of treatment (minimal inhibitory concentration [MIC] = 8 mg L−1). Metronidazole resistance correlates with the presence of a 7-kb plasmid, pCD-METRO. pCD-METRO is present in toxigenic and non-toxigenic resistant (n = 23), but not susceptible (n = 563), isolates from multiple countries. Introduction of a pCD-METRO-derived vector into a susceptible strain increases the MIC 25-fold. Our finding of plasmid-mediated resistance can impact diagnostics and treatment of CD infections. Show less
The major global pathogen Clostridium difficile (recently renamed Clostridioides difficile) has large genetic diversity including multiple mobile genetic elements. In this study, whole genome... Show moreThe major global pathogen Clostridium difficile (recently renamed Clostridioides difficile) has large genetic diversity including multiple mobile genetic elements. In this study, whole genome sequencing of 86 strains from the poorly characterised clade 3, predominantly PCR ribotype (RT)023, of C. difficile revealed distinctive surface architecture characteristics and a large mobile genetic island. These strains have a unique sortase substrate phenotype compared with well-characterised strains of C. difficile, and loss of the phage protection protein CwpV. A large genetic insertion (023_CTnT) comprised of three smaller elements (023_CTn1-3) is present in 80/86 strains analysed in this study, with genes common among other bacterial strains in the gut microbiome. Novel cargo regions of 023_CTnT include genes encoding a sortase, putative sortase substrates, lantibiotic ABC transporters and a putative siderophore biosynthetic cluster. We demonstrate the excision of 023_CTnT and sub-elements 023_CTn2 and 023_CTn3 from the genome of RT023 reference strain CD305 and the transfer of 023_CTn3 to a non-toxigenic C. difficile strain, which may have implications for the use of non-toxigenic C. difficile strains as live attenuated vaccines. Finally, we show that the genes within the island are expressed in a regulated manner in C. difficile RT023 strains conferring a distinct "niche adaptation". Show less
Corver, J.; Sen, J.; Hornung, B.V.H.; Mertens, B.J.; Berssenbrugge, E.K.L.; Harmanus, C.; ... ; Nicolardi, S. 2019
Filamentation induced by cAMP (Fic) domain proteins have been shown to catalyze the transfer of the AMP moiety from ATP onto a protein target. This type of post-translational modification was... Show moreFilamentation induced by cAMP (Fic) domain proteins have been shown to catalyze the transfer of the AMP moiety from ATP onto a protein target. This type of post-translational modification was recently shown to play a crucial role in pathogenicity mediated by two bacterial virulence factors. Herein we characterize a novel Fic domain protein that we identified from the human pathogen Clostridium difficile. The crystal structure shows that the protein adopts a classical all-helical Fic fold, which belongs to class II of Fic domain proteins characterized by an intrinsic N-terminal autoinhibitory alpha-helix. A conserved glutamate residue in the inhibitory helix motif was previously shown in other Fic domain proteins to prevent proper binding of the ATP gamma-phosphate. However, here we demonstrate that both ATP binding and autoadenylylation activity of the C. difficile Fic domain protein are independent of the inhibitory motif. In support of this, the crystal structure of a mutant of this Fic protein in complex with ATP reveals that the gamma-phosphate adopts a conformation unique among Fic domains that seems to override the effect of the inhibitory helix. These results provide important structural insight into the adenylylation reaction mechanism catalyzed by Fic domains. Our findings reveal the presence of a class II Fic domain protein in the human pathogen C. difficile that is not regulated by autoinhibition and challenge the current dogma that all class I-III Fic domain proteins are inhibited by the inhibitory alpha-helix. Show less
