Towards Development of a Non-Toxigenic Clostridioides difficile Oral Spore Vaccine against Toxigenic C. difficile.

Clostridioides difficile is an opportunistic gut pathogen which causes severe colitis, leading to significant morbidity and mortality due to its toxins, TcdA and TcdB. Two intra-muscular toxoid vaccines entered Phase III trials and strongly induced toxin-neutralising antibodies systemically but failed to provide local protection in the colon from primary C. difficile infection (CDI). Alternatively, by immunising orally, the ileum (main immune inductive site) can be directly targeted to confer protection in the large intestine. The gut commensal, non-toxigenic C. difficile (NTCD) was previously tested in animal models as an oral vaccine for natural delivery of an engineered toxin chimera to the small intestine and successfully induced toxin-neutralising antibodies. We investigated whether NTCD could be further exploited to induce antibodies that block the adherence of C. difficile to epithelial cells to target the first stage of pathogenesis. In NTCD strain T7, the colonisation factor, CD0873, and a domain of TcdB were overexpressed. Following oral immunisation of hamsters with spores of recombinant strain, T7-0873 or T7-TcdB, intestinal and systemic responses were investigated. Vaccination with T7-0873 successfully induced intestinal antibodies that significantly reduced adhesion of toxigenic C. difficile to Caco-2 cells, and these responses were mirrored in sera. Additional engineering of NTCD is now warranted to further develop this vaccine.

Colonisation Factor CD0873, an Attractive Oral Vaccine Candidate against Clostridioides difficile.

Clostridioides difficile is the main cause of health-care-associated infectious diarrhoea. Toxins, TcdA and TcdB, secreted by this bacterium damage colonic epithelial cells and in severe cases this culminates in pseudomembranous colitis, toxic megacolon and death. Vaccines in human trials have focused exclusively on the parenteral administration of toxin-based formulations. These vaccines promote toxin-neutralising serum antibodies but fail to confer protection from infection in the gut. An effective route to immunise against gut pathogens and stimulate a protective mucosal antibody response (secretory immunoglobulin A, IgA) at the infection site is the oral route. Additionally, oral immunisation generates systemic antibodies (IgG). Using this route, two different antigens were tested in the hamster model: The colonisation factor CD0873 and a TcdB fragment. Animals immunised with CD0873 generated a significantly higher titre of sIgA in intestinal fluid and IgG in serum compared to naive animals, which significantly inhibited the adherence of C. difficile to Caco-2 cells. Following challenge with a hypervirulent isolate, the CD0873-immunised group showed a mean increase of 80% in time to experimental endpoint compared to naïve animals. Survival and body condition correlated with bacterial clearance and reduced pathology in the cecum. Our findings advocate CD0873 as a promising oral vaccine candidate against C. difficile.

The glucosyltransferase activity of C. difficile Toxin B is required for disease pathogenesis.

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.

Characterization of the impact of rpoB mutations on the in vitro and in vivo competitive fitness of Clostridium difficile and susceptibility to fidaxomicin.

Objectives: To establish the role of specific, non-synonymous SNPs in the RNA polymerase ß subunit (rpoB) gene in reducing the susceptibility of Clostridium difficile to fidaxomicin and to explore the potential in vivo significance of rpoB mutant strains. Methods: Allelic exchange was used to introduce three different SNPs into the rpoB gene of an erythromycin-resistant derivative (CRG20291) of C. difficile R20291. The genome sequences of the created mutants were determined and each mutant analysed with respect to growth and sporulation rates, toxin A/B production and cytotoxicity against Vero cells, and in competition assays. Their comparative virulence and colonization ability was also assessed in a hamster infection model. Results: The MIC of fidaxomicin displayed by three mutants CRG20291-TA, CRG20291-TG and CRG20291-GT was substantially increased (>32, 8 and 2 mg/L, respectively) relative to that of the parent strain (0.25 mg/L). Genome sequencing established that the intended mutagenic substitutions in rpoB were the only changes present. Relative to CRG20291, all mutants had attenuated growth, were outcompeted by the parental strain, had lower sporulation and toxin A/B production capacities, and displayed diminished cytotoxicity. In a hamster model, virulence of all three mutants was significantly reduced compared with the progenitor strain, whereas the degree of caecum colonization was unaltered. Conclusions: Our study demonstrates that particular SNPs in rpoB lead to reduced fidaxomicin susceptibility. These mutations were associated with a fitness cost in vitro and reduced virulence in vivo.

What's a SNP between friends: The influence of single nucleotide polymorphisms on virulence and phenotypes of Clostridium difficile strain 630 and derivatives.

Clostridium difficile is a major cause of antibiotic induced diarrhea worldwide, responsible for significant annual mortalities and represents a considerable economic burden on healthcare systems. The two main C. difficile virulence factors are toxins A and B. Isogenic toxin B mutants of 2 independently isolated erythromycin-sensitive derivatives (630E and 630Δerm) of strain 630 were previously shown to exhibit substantively different phenotypes. Compared to 630, strain 630E and its progeny grow slower, achieve lower final cell densities, exhibit a reduced capacity for spore-formation, produce lower levels of toxin and are less virulent in the hamster infection model. By the same measures, strain 630Δerm and its derivatives more closely mirror the behavior of 630. Genome sequencing revealed that 630Δerm had acquired 7 unique Single Nucleotide Polymorphisms (SNPs) compared to 630 and 630E, while 630E had 9 SNPs and a DNA inversion not found in the other 2 strains. The relatively large number of mutations meant that the identification of those responsible for the altered properties of 630E was not possible, despite the restoration of 3 mutations to wildtype by allelic exchange and comparative RNAseq analysis of all 3 strains. The latter analysis revealed large differences in gene expression between the 3 strains, explaining in part why no single SNP could restore the phenotypic differences. Our findings suggest that strain 630Δerm should be favored over 630E as a surrogate for 630 in genetic-based studies. They also underline the importance of effective strain curation and the need to genome re-sequence master seed banks wherever possible.

A roadmap for gene system development in Clostridium.

Clostridium species are both heroes and villains. Some cause serious human and animal diseases, those present in the gut microbiota generally contribute to health and wellbeing, while others represent useful industrial chassis for the production of chemicals and fuels. To understand, counter or exploit, there is a fundamental requirement for effective systems that may be used for directed or random genome modifications. We have formulated a simple roadmap whereby the necessary gene systems maybe developed and deployed. At its heart is the use of 'pseudo-suicide' vectors and the creation of a pyrE mutant (a uracil auxotroph), initially aided by ClosTron technology, but ultimately made using a special form of allelic exchange termed ACE (Allele-Coupled Exchange). All mutants, regardless of the mutagen employed, are made in this host. This is because through the use of ACE vectors, mutants can be rapidly complemented concomitant with correction of the pyrE allele and restoration of uracil prototrophy. This avoids the phenotypic effects frequently observed with high copy number plasmids and dispenses with the need to add antibiotic to ensure plasmid retention. Once available, the pyrE host may be used to stably insert all manner of application specific modules. Examples include, a sigma factor to allow deployment of a mariner transposon, hydrolases involved in biomass deconstruction and therapeutic genes in cancer delivery vehicles. To date, provided DNA transfer is obtained, we have not encountered any clostridial species where this technology cannot be applied. These include, Clostridium difficile, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium botulinum, Clostridium perfringens, Clostridium sporogenes, Clostridium pasteurianum, Clostridium ljungdahlii, Clostridium autoethanogenum and even Geobacillus thermoglucosidasius.

Improving the reproducibility of the NAP1/B1/027 epidemic strain R20291 in the hamster model of infection.

Comparative analysis of the Clostridium difficile BI/NAP1/027 strain R20291 and ClosTron-derived ermB mutants in the hamster infection model are compromised by the clindamycin susceptibility of the parent. Mutants can appear more virulent. We have rectified this anomaly by genome engineering. The variant created (CRG20291) represents an ideal control strain for virulence assays of ClosTron mutants.

Clostridium difficile secreted Pro-Pro endopeptidase PPEP-1 (ZMP1/CD2830) modulates adhesion through cleavage of the collagen binding protein CD2831.

The Clostridium difficile cd2830 gene product is a secreted metalloprotease, named Pro-Pro endopeptidase (PPEP-1). PPEP-1 cleaves C. difficile cell surface proteins (e.g. CD2831). Here, we confirmed that PPEP-1 has a unique preference for prolines surrounding the scissile bond. Moreover, we show that it exhibits a high preference for an asparagine at the P2 position and hydrophobic residues at the P3 position. Using a PPEP-1 knockout C. difficile strain, we demonstrate that the removal of the collagen binding protein CD2831 is fully attributable to PPEP-1 activity. The PPEP-1 knockout strain demonstrated higher affinity for collagen type I with attenuated virulence in hamsters.

Importance of toxin A, toxin B, and CDT in virulence of an epidemic Clostridium difficile strain.

Clostridium difficile infection is the main cause of healthcare-acquired diarrhea in the developed world. In addition to the main virulence factors toxin A and B, epidemic, PCR Ribotype 027 strains, such as R20291, produce a third toxin, CDT. To develop effective medical countermeasures, it is important to understand the importance of each toxin. Accordingly, we created all possible combinations of isogenic toxin mutants of R20291 and assessed their virulence. We demonstrated that either toxin A or toxin B alone can cause fulminant disease in the hamster infection model and present tantalizing data that C. difficile toxin may also contribute to virulence.

The role of flagella in Clostridium difficile pathogenesis: comparison between a non-epidemic and an epidemic strain.

Clostridium difficile is a major cause of healthcare-associated infection and inflicts a considerable financial burden on healthcare systems worldwide. Disease symptoms range from self-limiting diarrhoea to fatal pseudomembranous colitis. Whilst C. difficile has two major virulence factors, toxin A and B, it is generally accepted that other virulence components of the bacterium contribute to disease. C. difficile colonises the gut of humans and animals and hence the processes of adherence and colonisation are essential for disease onset. Previously it has been suggested that flagella might be implicated in colonisation. Here we tested this hypothesis by comparing flagellated parental strains to strains in which flagella genes were inactivated using ClosTron technology. Our focus was on a UK-outbreak, PCR-ribotype 027 (B1/NAP1) strain, R20291. We compared the flagellated wild-type to a mutant with a paralyzed flagellum and also to mutants (fliC, fliD and flgE) that no longer produce flagella in vitro and in vivo. Our results with R20291 provide the first strong evidence that by disabling the motor of the flagellum, the structural components of the flagellum rather than active motility, is needed for adherence and colonisation of the intestinal epithelium during infection. Comparison to published data on 630Δerm and our own data on that strain revealed major differences between the strains: the R20291 flagellar mutants adhered less than the parental strain in vitro, whereas we saw the opposite in 630Δerm. We also showed that flagella and motility are not needed for successful colonisation in vivo using strain 630Δerm. Finally we demonstrated that in strain R20291, flagella do play a role in colonisation and adherence and that there are striking differences between C. difficile strains. The latter emphasises the overriding need to characterize more than just one strain before drawing general conclusions concerning specific mechanisms of pathogenesis.

Gene Dosage Effects at the Imprinted Gnas Cluster.

Genomic imprinting results in parent-of-origin-dependent monoallelic gene expression. Early work showed that distal mouse chromosome 2 is imprinted, as maternal and paternal duplications of the region (with corresponding paternal and maternal deficiencies) give rise to different anomalous phenotypes with early postnatal lethalities. Newborns with maternal duplication (MatDp(dist2)) are long, thin and hypoactive whereas those with paternal duplication (PatDp(dist2)) are chunky, oedematous, and hyperactive. Here we focus on PatDp(dist2). Loss of expression of the maternally expressed Gnas transcript at the Gnas cluster has been thought to account for the PatDp(dist2) phenotype. But PatDp(dist2) also have two expressed doses of the paternally expressed Gnasxl transcript. Through the use of targeted mutations, we have generated PatDp(dist2) mice predicted to have 1 or 2 expressed doses of Gnasxl, and 0, 1 or 2 expressed doses of Gnas. We confirm that oedema is due to lack of expression of imprinted Gnas alone. We show that it is the combination of a double dose of Gnasxl, with no dose of imprinted Gnas, that gives rise to the characteristic hyperactive, chunky, oedematous, lethal PatDp(dist2) phenotype, which is also hypoglycaemic. However PatDp(dist2) mice in which the dosage of the Gnasxl and Gnas is balanced (either 2∶2 or 1∶1) are neither dysmorphic nor hyperactive, have normal glucose levels, and are fully viable. But PatDp(dist2) with biallelic expression of both Gnasxl and Gnas show a marked postnatal growth retardation. Our results show that most of the PatDp(dist2) phenotype is due to overexpression of Gnasxl combined with loss of expression of Gnas, and suggest that Gnasxl and Gnas may act antagonistically in a number of tissues and to cause a wide range of phenotypic effects. It can be concluded that monoallelic expression of both Gnasxl and Gnas is a requirement for normal postnatal growth and development.

Precise manipulation of the Clostridium difficile chromosome reveals a lack of association between the tcdC genotype and toxin production.

Clostridium difficile causes a potentially fatal diarrheal disease through the production of its principal virulence factors, toxin A and toxin B. The tcdC gene is thought to encode a negative regulator of toxin production. Therefore, increased toxin production, and hence increased virulence, is often inferred in strains with an aberrant tcdC genotype. This report describes the first allele exchange system for precise genetic manipulation of C. difficile, using the codA gene of Escherichia coli as a heterologous counterselection marker. It was used to systematically restore the Δ117 frameshift mutation and the 18-nucleotide deletion that occur naturally in the tcdC gene of C. difficile R20291 (PCR ribotype 027). In addition, the naturally intact tcdC gene of C. difficile 630 (PCR ribotype 012) was deleted and then subsequently restored with a silent nucleotide substitution, or "watermark," so the resulting strain was distinguishable from the wild type. Intriguingly, there was no association between the tcdC genotype and toxin production in either C. difficile R20291 or C. difficile 630. Therefore, an aberrant tcdC genotype does not provide a broadly applicable rationale for the perceived notion that PCR ribotype 027 strains are "high-level" toxin producers. This may well explain why several studies have reported that an aberrant tcdC gene does not predict increased toxin production or, indeed, increased virulence.

The role of toxin A and toxin B in Clostridium difficile infection.

Clostridium difficile infection is the leading cause of healthcare-associated diarrhoea in Europe and North America. During infection, C. difficile produces two key virulence determinants, toxin A and toxin B. Experiments with purified toxins have indicated that toxin A alone is able to evoke the symptoms of C. difficile infection, but toxin B is unable to do so unless it is mixed with toxin A or there is prior damage to the gut mucosa. However, a recent study indicated that toxin B is essential for C. difficile virulence and that a strain producing toxin A alone was avirulent. This creates a paradox over the individual importance of toxin A and toxin B. Here we show that isogenic mutants of C. difficile producing either toxin A or toxin B alone can cause fulminant disease in the hamster model of infection. By using a gene knockout system to inactivate the toxin genes permanently, we found that C. difficile producing either one or both toxins showed cytotoxic activity in vitro that translated directly into virulence in vivo. Furthermore, by constructing the first ever double-mutant strain of C. difficile, in which both toxin genes were inactivated, we were able to completely attenuate virulence. Our findings re-establish the importance of both toxin A and toxin B and highlight the need to continue to consider both toxins in the development of diagnostic tests and effective countermeasures against C. difficile.

Comparative anatomical assessment of the piglet as a model for the developing human medullary serotonergic system.

Because the piglet is frequently used as a model for developmental disorders of the medullary serotonergic (5-HT) system in the human infant, this review compares the topography and developmental profile of selected 5-HT markers between humans in the first year of life and piglets in the first 60 days of life. The distribution of tryptophan hydroxylase-immunoreactive 5-HT neurons in the human infant medulla is very similar, but not identical, to that in the piglet. One notable difference is the presence of compact clusters of 5-HT neurons at the ventral surface of the piglet medulla. While it lacks these distinctive clusters, the human infant medulla contains potentially homologous 5-HT neurons scattered along the ventral surface embedded in the arcuate nucleus. Each species shows evidence of age-related changes in the 5-HT system, but the changes are different in nature; in the human infant, statistically significant age-related changes are observed in the proportional distribution of medullary 5-HT cells, while in the piglet, statistically significant age-related changes are observed in the levels of 5-HT receptor binding in certain medullary nuclei. Analyses of 5-HT receptor binding profiles in selected nuclei in the two species suggest that the equivalent postnatal ages for 5-HT development in piglets and human infants are, respectively, 4 days and 1 month, 12 days and 4 months, 30 days and 6 months, and 60 days and 12 months. Collectively, when certain species differences are considered, these data support the use of the piglet as a model for the human infant medullary 5-HT system.