CdtR-mediated regulation of toxin production in Clostridium difficile

Clostridium difficile is the leading cause of hospital-associated diarrhoea in the developed world. Its pathogenicity is elicited by the production of up to three toxins: the monoglucosyltransferases TcdA and TcdB, and the ADP-ribosyltransferase, CDT. This thesis describes the generation and characterisation of twenty one chromosomally distinct mutants of C. difficile, to primarily study the genetic regulation of toxin production by the two-component system (TCS) transcriptional regulator, CdtR. R20291ΔPaLoc model strains devoid of TcdA/TcdB activity, were generated to study CDT and the cdtR gene deleted and reintegrated at the pyrE locus. The application of these strains to in vitro cytotoxicity assays developed herein, established that CdtR was required for the production of CDT to cytotoxic levels in a PCR-ribotype (RT) 027 stain. The creation of a cdtR deletion mutation in the RT 012 strain 630Δerm established that CdtR played no role in TcdA/TcdB production in this strain. Thereafter, model strains expressing (de)phosphomimetic CdtR phospho-variants were generated. Their application provided strong evidence to suggest that CdtR was activated by phosphorylation of Asp61. In contrast, the RT 078 CdtR homolog was shown to be non-functional. Nine potential TCS histidine kinase interaction partners (IPs) for CdtR, were chromosomally altered. One potential IP was identified, CdtS1, which was affected in the production of CDT, TcdA and TcdB

What's a SNP between friends: The lineage of Clostridioides difficile R20291 can effect research outcomes

Clostridioides difficile R20291 is the most studied PCR-Ribotype 027 isolate. The two predominant lineages of this hypervirulent strain, however, exhibit substantive phenotypic differences and possess genomes that differ by a small number of nucleotide changes. It is important that the source of R20291 is taken into account in research outcomes

Clostridioides difficile binary toxin binding component (cdtb) increases virulence in a hamster model

Background Clostridioides difficile is the leading cause of hospital-acquired gastrointestinal infection, in part due to the existence of binary toxin (CDT)-expressing hypervirulent strains. Although the effects of the CDT holotoxin on disease pathogenesis have been previously studied, we sought to investigate the role of the individual components of CDT during in vivo infection. Methods To determine the contribution of the separate components of CDT during infection, we developed strains of C difficile expressing either CDTa or CDTb individually. We then infected both mice and hamsters with these novel mutant strains and monitored them for development of severe illness. Results Although expression of CDTb without CDTa did not induce significant disease in a mouse model of C difficile infection, we found that complementation of a CDT-deficient C difficile strain with CDTb alone restored virulence in a hamster model of C difficile infection. Conclusions Overall, this study demonstrates that the binding component of C difficile binary toxin, CDTb, contributes to virulence in a hamster model of infection

The glucosyltransferase activity of C. difficile toxin b is required for disease pathogenesis

© 2020 Bilverstone et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Enzymatic inactivation of Rho-family GTPases by the glucosyltransferase domain of Clostridioides difficile Toxin B (TcdB) gives rise to various pathogenic effects in cells that are classically thought to be responsible for the disease symptoms associated with C. difficile infection (CDI). Recent in vitro studies have shown that TcdB can, under certain circumstances, induce cellular toxicities that are independent of glucosyltransferase (GT) activity, calling into question the precise role of GT activity. Here, to establish the importance of GT activity in CDI disease pathogenesis, we generated the first described mutant strain of C. difficile producing glucosyltransferase-defective (GT-defective) toxin. Using allelic exchange (AE) technology, we first deleted tcdA in C. difficile 630Δerm and subsequently introduced a deactivating D270N substitution in the GT domain of TcdB. To examine the role of GT activity in vivo, we tested each strain in two different animal models of CDI pathogenesis. In the non-lethal murine model of infection, the GT-defective mutant induced minimal pathology in host tissues as compared to the profound caecal inflammation seen in the wild-type and 630ΔermΔtcdA (ΔtcdA) strains. In the more sensitive hamster model of CDI, whereas hamsters in the wild-type or ΔtcdA groups succumbed to fulminant infection within 4 days, all hamsters infected with the GT-defective mutant survived the 10-day infection period without primary symptoms of CDI or evidence of caecal inflammation. These data demonstrate that GT activity is indispensable for disease pathogenesis and reaffirm its central role in disease and its importance as a therapeutic target for small-molecule inhibition

CdtR-mediated regulation of toxin production in Clostridium difficile

Clostridium difficile is the leading cause of hospital-associated diarrhoea in the developed world. Its pathogenicity is elicited by the production of up to three toxins: the monoglucosyltransferases TcdA and TcdB, and the ADP-ribosyltransferase, CDT. This thesis describes the generation and characterisation of twenty one chromosomally distinct mutants of C. difficile, to primarily study the genetic regulation of toxin production by the two-component system (TCS) transcriptional regulator, CdtR. R20291ΔPaLoc model strains devoid of TcdA/TcdB activity, were generated to study CDT and the cdtR gene deleted and reintegrated at the pyrE locus. The application of these strains to in vitro cytotoxicity assays developed herein, established that CdtR was required for the production of CDT to cytotoxic levels in a PCR-ribotype (RT) 027 stain. The creation of a cdtR deletion mutation in the RT 012 strain 630Δerm established that CdtR played no role in TcdA/TcdB production in this strain. Thereafter, model strains expressing (de)phosphomimetic CdtR phospho-variants were generated. Their application provided strong evidence to suggest that CdtR was activated by phosphorylation of Asp61. In contrast, the RT 078 CdtR homolog was shown to be non-functional. Nine potential TCS histidine kinase interaction partners (IPs) for CdtR, were chromosomally altered. One potential IP was identified, CdtS1, which was affected in the production of CDT, TcdA and TcdB

A laboratory-scale fermentation system and its application to developing predictable regimes for the control of volatile ester formation at production scale

Volatile esters, produced as a result of the metabolism of wort by yeast during fermentation, are essential contributors to the flavor and aroma of beer. Since many esters arise at concentrations close to their flavor thresholds comparatively small variations in the concentrations of individual members can have a large impact on beer organoleptic properties. An essential part of the control of the brewing process is to ensure that esters are produced in the concentration ranges characteristic for individual beer qualities. This is especially the case where a single beer is brewed at several different production sites. The metabolic pathways that are implicated in volatile ester accumulation as a result of yeast activity during fermentation and the underlying genes responsible for the synthesis of the individual enzymes and their regulation are reasonably well-characterized, albeit with several caveats. Similarly, the process factors that are known to have an impact on ester formation have been subject to intensive study over the years, although often with contradictory results. We present volatile ester data relating to a pilsner-type lager beer produced at several international breweries that demonstrate significant site-specific variability although all of the breweries ostensibly used the same yeast strain and identical wort composition and fermentation control parameters. It is assumed that the observed variability must be a consequence of differences in plant and process operation associated with each site—in particular, fermentation vessel design and management. A laboratory model fermentation system is described that has been used to study formation during fermentation of the various groups of volatile esters known to contribute to beer quality as well as the synthesis and fate of their precursors. Trials are described that have been performed with a view to elucidating the cause-and-effect relationships between process conditions and ester accumulation for this combination of yeast strain and wort composition. Preliminary results are presented that demonstrate that this model system can be used to identify production-scale regimes that when implemented will allow ester formation to be controlled in a predictable fashion and so result in improved site-to-site product matching

Manipulation of Conditions during Wort Collection in Production-Scale Fermentations to Regulate Volatile Ester Synthesis as an Aid to Product Matching for Multisite Brewing

© 2015 American Society of Brewing Chemists, Inc. Volatile esters are amongst the most important yeast-derived flavoractive compounds produced during brewery fermentation. Dissolved oxygen concentration has long been recognized as an effector for the synthesis of esters by Saccharomyces spp. Here we demonstrate the effects of oxygen exposure time at vessel filling on the synthesis of esters during fermentation using a series of laboratory-scale experiments. In control fermentations, all the yeast was pitched into 10 L of high-gravity wort. In trial fermentations, all the yeast was pitched at the start of collection and wort was added as three 3.33-L batches or five 2-L batches over 12 or 24 hr, respectively. Wort was oxygenated to 15 ppm of O2 for each experiment and identical pitching rates and temperature profiles were adopted. Compared with the controls, increasing the number of batch fills to three and five reduced the final concentration of isoamyl acetate by 15.7 and 34%, respectively, and ethyl acetate by 25 and 39%, respectively, suggesting that a relationship exists between oxygen exposure time and ester synthesis. In parallel, hydrostatic pressure was applied to each condition, resulting in a further decrease in acetate esters. These data suggest that an inverse correlation exists between vessel size relative to brew length and ester production