Abstract
C.difficile infection (CDI) is not a merely “gut-confined” disease as toxemia could drive the development of CDI-related extra-intestinal effects. These effects could explain the high CDI-associated mortality, not just justified by diarrhea and dehydration. Here, the extra-intestinal effects of toxin A (TcdA) and B (TcdB) produced by C. difficile have been studied in vivo using the zebrafish embryo model. Noteworthy, protective properties of human serum albumin (HSA) towards toxins-induced extra-intestinal effects were also addressed. Zebrafish embryos were treated with TcdA, TcdB and/or HSA at 24 h post-fertilization. Embryos were analyzed for 48 h after treatment to check vital signs and morphological changes. Markers related to cardio-vascular damage and inflammation were evaluated by Real-Time quantitative PCR and/or western blotting. Both toxins induced cardiovascular damage in zebrafish embryos by different mechanisms: (i) direct toxicity (i.e., pericardial edema, cardiac chambers enlargement, endothelial alteration); (ii) increased hormonal production and release (i.e., atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP)), (iii) alteration of the vascular system through the increase of the vascular endothelial growth factor (VEGF-A) levels, as well as of its receptors, (iv) pro-inflammatory response through high cytokines production (i.e., CXCL8, IL1B, IL6 and TNFα) and (v) cell-mediated damage due to the increase in neutrophils number. In addition to cardiovascular damage, we observe skin alteration and inflammation. Finally, our data indicate a protective effect of HSA toward the toxins induced extra-intestinal effects. Together, our findings can serve as a starting point for humans’ studies to substantiate and understand the extra-intestinal effects observed in CDI patients.
Highlights
C. difficile is a gram-positive, anaerobic, spore-forming, toxin-producing bacillus, emerged as the main responsible for nosocomial infections in Western countries [1]
In order to establish the sub-lethal concentrations able to induce the toxins systemic effects, embryos were exposed to increasing doses of each toxin, ranging from 1.5 to 10 μ g/mL for TcdA and embryos were exposed to increasing doses of each toxin, ranging from 1.5 to 10 μg/mL for TcdA
8 μg/mL for TcdA and TcdB respectively because, at these concentrations, toxins were able to induce morphological effects with a similar mortality rate compared to vehicle-treated embryos
Summary
C. difficile is a gram-positive, anaerobic, spore-forming, toxin-producing bacillus, emerged as the main responsible for nosocomial infections in Western countries [1]. The pathogenic effects of C. difficile are mainly caused by the production of toxins A (TcdA) and B (TcdB) in the host’s gut [3]. Both toxins require a receptor-mediated endocytosis to enter the cell and exert their cytotoxic effects [4]. TcdA and TcdB monoglucosylate and inactivate the Rho GTPases expressed in the cytoplasm of host cells, causing cytopathic and cytotoxic effects and colonocytes death and loss of the intestinal barrier [5].
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