Absence of toxin gene transfer from Clostridioides difficile strain 630Δerm to nontoxigenic C. difficile strain NTCD-M3r in filter mating experiments.
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ABSTRACT:
SUBMITTER: Sambol SP
PROVIDER: S-EPMC9242483 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The symptoms of Clostridioides difficile infection (CDI) are largely attributed to two C. difficile toxins, TcdA and TcdB. Significant efforts have been devoted to developing vaccines targeting both toxins through parenteral immunization routes. Recently, we generated a novel chimeric protein (designated Tcd169), comprised of the glucosyltransferase domain (GT), the cysteine protease domain (CPD), and the receptor binding domain (RBD) of TcdB, and the RBD of TcdA. Parenteral immunizations with Tcd169 provide mice effective protection against infection with a ribotype (RT) 027 C. difficile strain. In this study, we expressed Tcd169 in a nontoxigenic C. difficile CCUG37785 strain (designated NTCD), resulting in strain NTCD_Tcd169 to develop an oral vaccine that can target both C. difficile toxins and colonization/adhesion factors. Oral immunizations with NTCD_Tcd169 spores induced systematic and mucosal antibody responses against, not only both toxins, but also C. difficile flagellins (FliC/FliD). Intriguingly yet importantly, anti-Tcd169 sera raised against Tcd169 protein were significantly cross-reactive with FliC/FliD and two surface layer proteins (SlpA and Cwp2). Oral immunizations with NTCD_Tcd169 spores provided mice effective protection against infection with a hypervirulent RT027 C. difficile strain R20291and significantly reduced R20291spore numbers in feces compared with NTCD or PBS immunized mice. These results imply that the genetically modified, nontoxigenic C. difficile strain expressing Tcd169 may represent a novel mucosal vaccine candidate against CDI. IMPORTANCE Clostridioides difficile is an enteric pathogen, and symptoms of C. difficile infection (CDI) are mainly by two exotoxins TcdA and TcdB. Active vaccination is cost-effective approach to prevent CDI and high rates of recurrence. Ideally, vaccines should target both C. difficile toxins and cell/spore colonization. In this study, we expressed immunodominant fragments of TcdA and TcdB (i.e., Tcd169) in a nontoxigenic C. difficile CCUG37785 strain, generating a promising oral/mucosal vaccine candidate against CDI, by targeting both toxins and colonization of pathogenic C. difficile strains. Importantly, anti-Tcd169 sera raised against Tcd169 protein were significantly cross-reactive with FliC/FliD and two surface layer proteins (SlpA and Cwp2), and all of which are involved in C. difficile adhesion/colonization in vitro and in vivo.
| S-EPMC9241731 | biostudies-literature
Project description:The anaerobic spore former Clostridioides difficile causes significant diarrheal disease in humans and other mammals. Infection begins with the ingestion of dormant spores, which subsequently germinate within the host gastrointestinal tract. There, the vegetative cells proliferate and secrete two exotoxins, TcdA and TcdB, which cause disease symptoms. Although spore formation and toxin production are critical for C. difficile pathogenesis, the regulatory links between these two physiological processes are not well understood and are strain dependent. Previously, we identified a conserved C. difficile regulator, RstA, that promotes sporulation initiation through an unknown mechanism and directly and indirectly represses toxin and motility gene transcription in the historical isolate 630Δerm To test whether perceived strain-dependent differences in toxin production and sporulation are mediated by RstA, we created an rstA mutant in the epidemic ribotype 027 strain R20291. RstA affected sporulation and toxin gene expression similarly but more robustly in R20291 than in 630Δerm In contrast, no effect on motility gene expression was observed in R20291. Reporter assays measuring transcriptional regulation of tcdR, the sigma factor gene essential for toxin gene expression, identified sequence-dependent effects influencing repression by RstA and CodY, a global nutritional sensor, in four diverse C. difficile strains. Finally, sequence- and strain-dependent differences were evident in RstA negative autoregulation of rstA transcription. Altogether, our data suggest that strain-dependent differences in RstA regulation contribute to the sporulation and toxin phenotypes observed in R20291. Our data establish RstA as an important regulator of C. difficile virulence traits.IMPORTANCE Two critical traits of Clostridioides difficile pathogenesis are toxin production, which causes disease symptoms, and spore formation, which permits survival outside the gastrointestinal tract. The multifunctional regulator RstA promotes sporulation and prevents toxin production in the historical strain 630Δerm Here, we show that RstA exhibits stronger effects on these phenotypes in an epidemic isolate, R20291, and additional strain-specific effects on toxin and rstA expression are evident. Our data demonstrate that sequence-specific differences within the promoter for the toxin regulator TcdR contribute to the regulation of toxin production by RstA and CodY. These sequence differences account for some of the variability in toxin production among isolates and may allow strains to differentially control toxin production in response to a variety of signals.
| S-EPMC6941533 | biostudies-literature
Project description:Symptoms of Clostridioides difficile infection (CDI) are attributed largely to two toxins, TcdA and TcdB. About 17-23% of C. difficile isolates produce binary toxin, which enhances C. difficile pathogenesis. Previously, we engineered the nontoxigenic C. difficile strain CCUG37785 (designated as CCUG37785) to express immunogenic fragments of TcdA and TcdB as an oral mucosal CDI vaccine candidate. In this study, we performed genomic and phenotypic analyses of CCUG37785 and evaluated its potential use for preventing and treating CDI. Whole genome sequencing showed that CCUG37785 is ribotype ST3 and lacks toxin genes. Comparative analyses of PaLoc and CdtLoc loci of CCUG37785 revealed 115-bp and 68-bp conserved fragments in these regions, respectively. Phenotypic comparisons between CCUG37785 and C. difficile R20291 (an epidemic hypervirulent BI/NAPI/027 strain, designated as R20291) found that CCUG37785 exhibited significantly higher adhesion and sporulation, significantly lower spore germination and biofilm formation, and comparable motility to R20291. We also showed that oral inoculation of CCUG37785 spores prior to infection with R20291 spores provided mice almost full protection against developing CDI. However, oral inoculation of CCUG37785 spores after infection with R20291 spores only provided minor protection against CDI. Further analysis showed that mice pretreated with CCUG37785 spores secreted significantly less R20291 spores, while mice treated with CCUG37785 spores after infection with R20291 secreted a comparable amount of R20291 spores to mice infected with R20291 spores only. Our data both highlight the potential use of CCUG37785 for the prevention of primary and recurrent CDI in humans and support its use as an oral mucosal vaccine carrier against CDI. IMPORTANCE Clostridioides difficile infection (CDI) symptoms range from diarrhea to intestinal inflammation/lesion and death and are mainly caused by two exotoxins, TcdA and TcdB. Active vaccination provides the attractive opportunity to prevent CDI and recurrence. No vaccine against CDI is currently licensed. Tremendous efforts have been devoted to developing vaccines targeting both toxins. However, ideally, vaccines should target both toxins and C. difficile cells/spores that transmit the disease and cause recurrence. Furthermore, C. difficile is an enteric pathogen, and mucosal/oral immunization would be particularly useful to protect the host against CDI considering that the gut is the main site of disease onset and progression. Data in our current study not only highlight the potential use of CCUG37785 to prevent primary and recurrent CDI in humans but also further support its use as an oral mucosal vaccine carrier against CDI.
| S-EPMC9045287 | biostudies-literature
Project description:Here we report the draft genome sequence of Clostridium difficile strain CD37, the first nontoxigenic strain sequenced. Every sequenced strain of Clostridium difficile has been shown to contain multiple different mobile genetic elements. The draft genome sequence of strain CD37 reveals the presence of two putative conjugative transposons.
| S-EPMC3318468 | biostudies-literature
Project description:BackgroundClostridioides (Clostridium) difficile colonization is common among infants. Serological sequelae of infant C. difficile colonization are poorly understood.MethodsIn this prospective cohort study of healthy infants, stools serially collected between ages 1-2 and 9-12 months were tested for non-toxigenic and toxigenic C. difficile (TCD). Cultured isolates underwent whole-genome sequencing. Serum collected at 9-12 months underwent measurement of IgA, IgG, and IgM against TCD toxins A and B and neutralizing antibody (NAb) titers against toxin B. For comparison, antitoxin IgG and NAb were measured in cord blood from 50 mothers unrelated to study infants.ResultsAmong 32 infants, 16 (50%) were colonized with TCD; 12 were first colonized >1 month before serology measurements. A variety of sequence types were identified, and there was evidence of putative in-home (enrolled siblings) and outpatient clinic transmission. Infants first colonized with TCD >1 month prior had significantly greater serum antitoxin IgA and IgG against toxins A (P = .02 for both) and B (P = .009 and .008, respectively) compared with non-TCD-colonized infants, and greater IgG compared with unrelated cord blood (P = .005). Five of 12 (42%) colonized infants had detectable NAb titers compared with zero non-TCD-colonized infants (P = .02). Breastfeeding was not associated with differences in serological measurements.ConclusionsTCD colonization is associated with a humoral immune response against toxins A and B, with evidence of toxin B neutralization in vitro. The extent and duration of protection against CDI later in life afforded by natural C. difficile immunization events require further investigation.
| S-EPMC7201415 | biostudies-literature
Project description:Stable resistance to metronidazole in a nontoxigenic Clostridium difficile strain was investigated at both the genomic and proteomic levels. Alterations in the metabolic pathway involving the pyruvate-ferredoxin oxidoreductase were found, suggesting that reduction of metronidazole, required for its activity, may be less efficient in this strain. Proteomic studies also showed a cellular response to oxidative stress.
| S-EPMC4135993 | biostudies-literature
Project description:BACKGROUND:Most Clostridioides difficile toxinogenic strains produce both toxins A and B (A+B+), but toxin A-negative, toxin B-positive (A-B+) variants also cause disease. We report the identification of a series of pathogenic clinical C. difficile isolates that produce high amounts of toxin A with low or nondetectable toxin B. METHODS:An ultrasensitive, quantitative immunoassay was used to measure toxins A and B in stool samples from 187 C. difficile infection (CDI) patients and 44 carriers. Isolates were cultured and assessed for in vitro toxin production and in vivo phenotypes (mouse CDI model). RESULTS:There were 7 CDI patients and 6 carriers who had stools with detectable toxin A (TcdA, range 23-17 422 pg/mL; 5.6% of samples overall) but toxin B (TcdB) below the clinical detection limit (<20 pg/mL; median TcdA:B ratio 17.93). Concentrations of toxin A far exceeded B in in vitro cultures of all 12 recovered isolates (median TcdA:B ratio 26). Of 8 toxin A>>B isolates tested in mice, 4 caused diarrhea, and 3 of those 4 caused lethal disease. Ribotyping demonstrated strain diversity. TcdA-predominant samples were also identified at 2 other centers, with similar frequencies (7.5% and 6.8%). CONCLUSIONS:We report the discovery of clinical pathogenic C. difficile strains that produce high levels of toxin A but minimal or no toxin B. This pattern of toxin production is not rare (>5% of isolates) and is consistently observed in vitro and in vivo in humans and mice. Our study highlights the significance of toxin A in human CDI pathogenesis and has important implications for CDI diagnosis, treatment, and vaccine development.
| S-EPMC7286367 | biostudies-literature
Project description: Not available
| S-EPMC11344133 | biostudies-literature
Project description:Clostridioides difficile infections have emerged as the leading cause of healthcare-associated infectious diarrhea. Disease symptoms are mainly caused by the virulence factors, TcdA and TcdB, which are large homologous multidomain proteins. Here, we report a 2.8 Å resolution cryo-EM structure of native TcdA, unveiling its conformation at neutral pH. The structure uncovers the dynamic movement of the CROPs domain which is induced in response to environmental acidification. Furthermore, the structure reveals detailed information about the interaction area between the CROPs domain and the tip of the delivery and receptor-binding domain, which likely serves to shield the C-terminal part of the hydrophobic pore-forming region from solvent exposure. Similarly, extensive interactions between the globular subdomain and the N-terminal part of the pore-forming region suggest that the globular subdomain shields the upper part of the pore-forming region from exposure to the surrounding solvent. Hence, the TcdA structure provides insights into the mechanism of preventing premature unfolding of the pore-forming region at neutral pH, as well as the pH-induced inter-domain dynamics.
| S-EPMC8728606 | biostudies-literature
Project description:Metabolomic and transcriptomic analysis of changes in the exponential and stationary phase of Clostridioides difficile after cultivation in casamino acids medium (reference) and supplemented with L-lactate and the connection to toxin production.
2021-01-31 | GSE139354 | GEO