Impact of Binary Toxin on Recurrent Clostridium difficile Infection

 描述（由申请人提供）： 在过去十年中，艰难梭菌感染（CDI）已成为退伍军人和非退伍军人患者日益常见和复杂的治疗问题。最困难的管理问题之一是经常性的CDI。约20%的患者成功地用甲硝唑或万古霉素治疗后会复发。那些已经复发的患者有约50%的机会发生第三次复发，有些患者会复发数月或数年。此外，复发性CDI对医疗机构产生了重大影响，因为急诊护理机构的频繁再入院率。几个宿主因素被认为是CDI复发的风险。然而，相对较少关注可能影响复发风险的病原体或细菌因素。在过去的十年中，CDI和并发症（如复发性CDI）发生率的增加与流行性丙型肝炎的出现相吻合。艰难梭菌BI/027/NAP 1菌株。已经提出了由该菌株表达的几种潜在毒力因子，但最近的证据表明，由该菌株产生的二元毒素（CDT）和其他临床重要的（例如，BK/078/NAP 7）株在CDI的发病中可能起着重要的促发作用。C.艰难梭菌二元毒素（CDT）与大的单一单位糖基化毒素毒素A和B无关。这种双组分（二元）毒素通过细胞骨架蛋白肌动蛋白的ADP-核糖基化起作用，导致微管结构变化和上皮细胞膜突起，从而增加C.很难我们的研究目的是通过两个实验研究来验证二元毒素增加复发性CDI风险的假设。首先，我们将使用仓鼠模型的CDI证明二元毒素的效果，通过使用的同基因突变的C。其中二元基因毒素被敲除的艰难梭菌。其次，我们将使用新开发的二元毒素免疫测定法检测患者粪便标本，并将粪便二元毒素的存在与CDI的临床复发相关联。该提案的具体目标包括：1.建立可重复的CDI复发仓鼠模型。 目的1b）使用二元毒素突变体（CDTneg）在仓鼠模型中建立可再现的复发率，并使用各自的亲本（CDTpos）菌株确认复发率增加。目的2a）使用人肠上皮细胞系确定体外机制目的2b）确定仓鼠体内机制3.将二元毒素在患者中的表达与CDI复发相关联目的3a）将粪便二元毒素检测与CDI复发相关联目的3b）将二元毒素阳性C. CDI复发的艰难菌株

 DESCRIPTION (provided by applicant): Clostridium difficile infection (CDI) has been an increasingly common and complicated treatment issue for veteran and non-veteran patients over the past decade. One of the most difficult management issues is that of recurrent CDI. ~20% of patients who are successfully treated with either metronidazole or vancomycin will have a recurrent episode. Those who have already had a recurrent episode have ~50% chance of third diarrheal episode and some patients will have recurrent CDI for months or years. In addition, recurrent CDI has had a major impact on healthcare facilities because of frequent readmission rates to acute care facilities. Several host factors have been implicated as risks for recurrent CDI. However, there has been relatively little attention paid to pathogen or bacterial factors that might influence the risk of recurrence. The increase in rates of CDI and complications such as recurrent CDI over the past decade has coincided with the emergence of the epidemic C. difficile BI/027/NAP1 strain. Several potential virulence factors expressed by this strain have been proposed, but recent evidence suggests that binary toxin (CDT) produced by this strain and other clinically important (e.g., BK/078/NAP7) strains may play a critical adjunctive role in CDI pathogenesis. C. difficile binary toxin (CDT) is unrelated to the large, single unit glycosylating toxins, toxin A and B. This 2-component (binary) toxin acts by ADP-ribosylation of the cytoskeletal protein, actin, resulting in microtubular structural changes and epithelial cell membrane protrusions which increase adherence and colonization of C. difficile. Our research aims are designed to test the hypothesis that binary toxin increases the risk of recurrent CDI by two experimental lines of investigation. First, we will use the hamster model of CDI to demonstrate the effect of binary toxin by using isogenic mutants of C. difficile in which the binary gene toxin is knocked out. Second, we will use a newly developed immunoassay for binary toxin to test patient stool specimens and correlate the presence of fecal binary toxin with clinical recurrence of CDI. The specific aims of this proposal include: 1. Establish a reproducible hamster model of CDI recurrence. Aim 1a) Construct binary toxin mutants in the epidemic-associated BI and BK strains (CDTneg) for the baseline experiments in the hamster model Aim 1b) Establish a reproducible recurrence rate in the hamster model using binary toxin mutants (CDTneg) & confirm increased recurrence rates using respective parent (CDTpos) strains 2. Determine the mechanism of increased CDI recurrence due to binary toxin Aim 2a) Determine the mechanism in vitro using a human intestinal epithelial cell line Aim 2b) Determine the mechanism in vivo in hamsters 3. Correlate the expression of binary toxin in patients with CDI recurrence Aim 3a) Correlate fecal binary toxin detection with CDI recurrence Aim 3b) Correlate the presence of binary toxin-positive C. difficile strains with CDI recurrence