Analysis of TcdB Proteins within the Hypervirulent Clade 2 Reveals an Impact of RhoA Glucosylation on Clostridium difficile Proinflammatory Activities

ABSTRACT Clostridium difficile strains within the hypervirulent clade 2 are responsible for nosocomial outbreaks worldwide. The increased pathogenic potential of these strains has been attributed to several factors but is still poorly understood. During a C. difficile outbreak, a strain from this clade was found to induce a variant cytopathic effect (CPE), different from the canonical arborizing CPE. This strain (NAP1V) belongs to the NAP1 genotype but to a ribotype different from the epidemic NAP1/RT027 strain. NAP1V and NAP1 share some properties, including the overproduction of toxins, the binary toxin, and mutations in tcdC. NAP1V is not resistant to fluoroquinolones, however. A comparative analysis of TcdB proteins from NAP1/RT027 and NAP1V strains indicated that both target Rac, Cdc42, Rap, and R-Ras but only the former glucosylates RhoA. Thus, TcdB from hypervirulent clade 2 strains possesses an extended substrate profile, and RhoA is crucial for the type of CPE induced. Sequence comparison and structural modeling revealed that TcdBNAP1 and TcdBNAP1V share the receptor-binding and autoprocessing activities but vary in the glucosyltransferase domain, consistent with the different substrate profile. Whereas the two toxins displayed identical cytotoxic potencies, TcdBNAP1 induced a stronger proinflammatory response than TcdBNAP1V as determined in ex vivo experiments and animal models. Since immune activation at the level of intestinal mucosa is a hallmark of C. difficile-induced infections, we propose that the panel of substrates targeted by TcdB is a determining factor in the pathogenesis of this pathogen and in the differential virulence potential seen among C. difficile strains.

[1]  Kim Rutherford,et al.  Artemis: sequence visualization and annotation , 2000, Bioinform..

[2]  Constantinos Zamboglou,et al.  Clostridial Glucosylating Toxins Enter Cells via Clathrin-Mediated Endocytosis , 2010, PloS one.

[3]  M. Mulvey,et al.  Emergence of Clostridium difficile NAP1 in Latin America , 2009, Journal of Clinical Microbiology.

[4]  L. Lemée,et al.  Multilocus sequence typing for Clostridium difficile. , 2010, Methods in molecular biology.

[5]  M. Rupnik,et al.  New types of toxin A-negative, toxin B-positive strains among clinical isolates of Clostridium difficile in Australia. , 2011, Journal of medical microbiology.

[6]  M. Mann,et al.  Glucosylation of Rho proteins by Clostridium difficile toxin B , 1995, Nature.

[7]  M. Rupnik.  Heterogeneity of large clostridial toxins: importance of Clostridium difficile toxinotypes. , 2008, FEMS microbiology reviews.

[8]  D. Gerding,et al.  Clostridium difficile infection caused by the epidemic BI/NAP1/027 strain. , 2009, Gastroenterology.

[9]  M. Weidmann,et al.  Toxins A and B from Clostridium difficile differ with respect to enzymatic potencies, cellular substrate specificities, and surface binding to cultured cells. , 1997, The Journal of clinical investigation.

[10]  Stuart Johnson,et al.  Distribution of Clostridium difficile variant toxinotypes and strains with binary toxin genes among clinical isolates in an American hospital. , 2004, Journal of medical microbiology.

[11]  Hongjie Yu,et al.  Sequence Variation in tcdA and tcdB of Clostridium difficile: ST37 with Truncated tcdA Is a Potential Epidemic Strain in China , 2014, Journal of Clinical Microbiology.

[12]  Kuo-Bin Li,et al.  ClustalW-MPI: ClustalW analysis using distributed and parallel computing , 2003, Bioinform..

[13]  S. Tzipori,et al.  Essential role of the glucosyltransferase activity in Clostridium difficile toxin-induced secretion of TNF-alpha by macrophages. , 2009, Microbial pathogenesis.

[14]  Brian D. Ondov,et al.  The Harvest suite for rapid core-genome alignment and visualization of thousands of intraspecific microbial genomes , 2014, Genome Biology.

[15]  M. Delmée,et al.  A Novel Toxinotyping Scheme and Correlation of Toxinotypes with Serogroups of Clostridium difficileIsolates , 1998, Journal of Clinical Microbiology.

[16]  K. Aktories,et al.  Rho-glucosylating Clostridium difficile toxins A and B: new insights into structure and function. , 2007, Glycobiology.

[17]  Nigel P. Minton,et al.  The role of toxin A and toxin B in Clostridium difficile infection , 2010, Nature.

[18]  M. Hannan,et al.  Emergence and Control of Fluoroquinolone-Resistant, Toxin A–Negative, Toxin B–Positive Clostridium difficile , 2007, Infection Control & Hospital Epidemiology.

[19]  O. Gascuel,et al.  New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. , 2010, Systematic biology.

[20]  H. Kuramitsu,et al.  Evidence for a modular structure of the homologous repetitive C-terminal carbohydrate-binding sites of Clostridium difficile toxins and Streptococcus mutans glucosyltransferases , 1992, Journal of bacteriology.

[21]  J. Lemeland,et al.  Multiplex PCR Targeting tpi (Triose Phosphate Isomerase), tcdA (Toxin A), and tcdB (Toxin B) Genes for Toxigenic Culture of Clostridium difficile , 2004, Journal of Clinical Microbiology.

[22]  Torsten Seemann,et al.  Prokka: rapid prokaryotic genome annotation , 2014, Bioinform..

[23]  Ingo Just,et al.  Difference in the Cytotoxic Effects of Toxin B from Clostridium difficile Strain VPI 10463 and Toxin B from Variant Clostridium difficile Strain 1470 , 2006, Infection and Immunity.

[24]  J. Ballard,et al.  Clostridium difficile Toxins: Mechanism of Action and Role in Disease , 2005, Clinical Microbiology Reviews.

[25]  Klaus Aktories,et al.  Auto-catalytic Cleavage of Clostridium difficile Toxins A and B Depends on Cysteine Protease Activity* , 2007, Journal of Biological Chemistry.

[26]  M. Delmée,et al.  Comparison of PCR-Ribotyping, Arbitrarily Primed PCR, and Pulsed-Field Gel Electrophoresis for Typing Clostridium difficile , 2000, Journal of Clinical Microbiology.

[27]  D. Lacy,et al.  Structural organization of the functional domains of Clostridium difficile toxins A and B , 2010, Proceedings of the National Academy of Sciences.

[28]  김삼묘,et al.  “Bioinformatics” 특집을 내면서 , 2000 .

[29]  N. Oezguen,et al.  Critical Roles of Clostridium difficile Toxin B Enzymatic Activities in Pathogenesis , 2014, Infection and Immunity.

[30]  Jon Brazier,et al.  Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe , 2005, The Lancet.

[31]  S. Fanning,et al.  gyrA Mutations in Fluoroquinolone-resistant Clostridium difficile PCR-027 , 2007, Emerging infectious diseases.

[32]  R. Benz,et al.  Low pH-induced Formation of Ion Channels by Clostridium difficile Toxin B in Target Cells* , 2001, The Journal of Biological Chemistry.

[33]  C. Kelly,et al.  Clostridium difficile--more difficult than ever. , 2008, The New England journal of medicine.

[34]  E. Paul,et al.  Emergence of a ribotype 244 strain of Clostridium difficile associated with severe disease and related to the epidemic ribotype 027 strain. , 2014, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[35]  John W. Chen,et al.  Measuring Myeloperoxidase Activity in Biological Samples , 2013, PloS one.

[36]  J. Ballard,et al.  Variations in TcdB Activity and the Hypervirulence of Emerging Strains of Clostridium difficile , 2010, PLoS pathogens.

[37]  Robert C. Edgar,et al.  MUSCLE: multiple sequence alignment with high accuracy and high throughput. , 2004, Nucleic acids research.

[38]  J. James,et al.  Clostridium difficile 027/BI/NAP1 Encodes a Hypertoxic and Antigenically Variable Form of TcdB , 2013, PLoS pathogens.

[39]  S. Clare,et al.  Defining the Roles of TcdA and TcdB in Localized Gastrointestinal Disease, Systemic Organ Damage, and the Host Response during Clostridium difficile Infections , 2015, mBio.

[40]  M. Rupnik,et al.  New Types of Toxin A-Negative, Toxin B-Positive Strains among Clostridium difficile Isolates from Asia , 2003, Journal of Clinical Microbiology.

[41]  Mark A. Miller,et al.  Molecular characterization of moxifloxacin resistance from Canadian Clostridium difficile clinical isolates. , 2010, Diagnostic microbiology and infectious disease.

[42]  M. Pirmohamed,et al.  Emergence and global spread of epidemic healthcare-associated Clostridium difficile , 2012, Nature Genetics.

[43]  C. Guzmán-Verri,et al.  R-Ras Glucosylation and Transient RhoA Activation Determine the Cytopathic Effect Produced by Toxin B Variants from Toxin A-negative Strains of Clostridium difficile * , 2003, The Journal of Biological Chemistry.

[44]  P. O'Gaora,et al.  High-level resistance to moxifloxacin and gatifloxacin associated with a novel mutation in gyrB in toxin-A-negative, toxin-B-positive Clostridium difficile. , 2006, The Journal of antimicrobial chemotherapy.

[45]  Julian I. Rood,et al.  Toxin B is essential for virulence of Clostridium difficile , 2009, Nature.

[46]  K. Aktories,et al.  Cellular Uptake of Clostridium difficile Toxin B , 2003, Journal of Biological Chemistry.

[47]  S. Tenzer,et al.  Autocatalytic cleavage of Clostridium difficile toxin B , 2007, Nature.

[48]  K. Katoh,et al.  MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability , 2013, Molecular biology and evolution.

[49]  Ole Lund,et al.  Multilocus Sequence Typing of Total-Genome-Sequenced Bacteria , 2012, Journal of Clinical Microbiology.

[50]  W. Pirovano,et al.  Toward almost closed genomes with GapFiller , 2012, Genome Biology.

[51]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[52]  C. Woods,et al.  Comparison of Seven Techniques for Typing International Epidemic Strains of Clostridium difficile: Restriction Endonuclease Analysis, Pulsed-Field Gel Electrophoresis, PCR-Ribotyping, Multilocus Sequence Typing, Multilocus Variable-Number Tandem-Repeat Analysis, Amplified Fragment Length Polymorphis , 2007, Journal of Clinical Microbiology.

[53]  M. Mulvey,et al.  Emergence of an Outbreak-Associated Clostridium difficile Variant with Increased Virulence , 2015, Journal of Clinical Microbiology.

[54]  Ken Dewar,et al.  A predominantly clonal multi-institutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality. , 2005, The New England journal of medicine.

[55]  M. Weidmann,et al.  A Novel Cytotoxin from Clostridium difficileSerogroup F Is a Functional Hybrid between Two Other Large Clostridial Cytotoxins* , 1999, The Journal of Biological Chemistry.

[56]  E. Birney,et al.  Velvet: algorithms for de novo short read assembly using de Bruijn graphs. , 2008, Genome research.

[57]  H. Feng,et al.  Glucosyltransferase activity of Clostridium difficile Toxin B is essential for disease pathogenesis , 2015, Gut microbes.

[58]  P. Mastrantonio,et al.  Molecular Analysis of the Pathogenicity Locus and Polymorphism in the Putative Negative Regulator of Toxin Production (TcdC) among Clostridium difficile Clinical Isolates , 2002, Journal of Clinical Microbiology.

[59]  M. A. Farrow,et al.  Structural Determinants of Clostridium difficile Toxin A Glucosyltransferase Activity* , 2012, The Journal of Biological Chemistry.

[60]  D. Gerding,et al.  Human Hypervirulent Clostridium difficile Strains Exhibit Increased Sporulation as Well as Robust Toxin Production , 2010, Journal of bacteriology.

[61]  R. Guerrant,et al.  Murine model of Clostridium difficile infection with aged gnotobiotic C57BL/6 mice and a BI/NAP1 strain. , 2010, The Journal of infectious diseases.

[62]  C. Ou,et al.  Identification of toxigenic Clostridium difficile by the polymerase chain reaction , 1991, Journal of clinical microbiology.

[63]  M. Weidmann,et al.  Transcription analysis of the genes tcdA-E of the pathogenicity locus of Clostridium difficile. , 1997, European journal of biochemistry.

[64]  Mark A. Ragan,et al.  Detecting recombination in evolving nucleotide sequences , 2006, BMC Bioinformatics.

[65]  Walter Pirovano,et al.  BIOINFORMATICS APPLICATIONS , 2022 .

[66]  Aaron E. Darling,et al.  Reordering contigs of draft genomes using the Mauve Aligner , 2009, Bioinform..