Enterotoxigenic Escherichia coli Heat-Stable Toxin Increases the Rate of Zinc Release from Metallothionein and Is a Zinc- and Iron-Binding Peptide.
- 作者列表："Kiefer MC","Motyka NI","Clements JD","Bitoun JP
Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in children in low- to middle-income countries. Previous studies have identified heat-stable enterotoxin (ST)-producing ETEC as one of the major diarrhea-causing pathogens in children younger than five years. In this study, we examined iron and zinc binding by both human and porcine ST variants and determined how host metallothionein could detoxify ST. We found that ST purified from ETEC culture supernatants eluted as a doublet during C18 reverse-phase chromatography. Leading edge fractions of the ST doublet were found to be devoid of iron, while trailing edge fractions of the ST doublet were found to contain measurable iron. Next, we found that purified ST could be reconstituted with iron under reducing and anaerobic conditions, and iron-bound ST attenuated the induction of cGMP in T84 epithelial cells. Moreover, we demonstrated that supernatants of ETEC 214-4 grown under increasing iron concentrations were only able to induce cGMP at iron concentrations greater than 5 μM. In vitro studies also demonstrated that ST binds zinc, and once bound, zinc removal from ST required denaturing conditions. Zinc-bound ST also failed to induce cGMP. We found that ST contributes disulfide bonds to the perceived oxidized glutathione pool, increases the rate of zinc release from metallothionein, and can be detoxified by metallothionein. Lastly, we showed ST induces transcriptional changes in genes previously shown to be regulated by deferoxamine. These studies demonstrate ST ETEC pathogenesis may be tied intimately to host mucosal metal status.IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in children in low- to middle-income countries, deployed military personnel, and travelers to regions of endemicity. The heat-stable toxin (ST) is a small nonimmunogenic secreted peptide with 3 disulfide bonds. It has been appreciated that dietary disulfides modulate intestinal redox potential and that ST could be detoxified using exogenous reductants. Using biochemical and spectroscopic approaches, we demonstrated that ST can separately bind iron and zinc under reducing conditions, thereby reducing ST toxicity. Moreover, we demonstrated that ST modulates the glutathione (GSH)/oxidized glutathione (GSSG) ratio and that ST should be considered a toxin oxidant. ST can be detoxified by oxidizing zinc-loaded metallothionine, causing free zinc to be released. These studies help lay a foundation to understand how diarrheal pathogens modulate intestinal redox potential and may impact how we design therapeutics and/or vaccines for the pathogens that produce them.
产肠毒素大肠杆菌 (ETEC) 是中低收入国家儿童的主要腹泻病原体。既往研究已确定产热稳定肠毒素 (ST) ETEC 是 5 岁以下儿童主要致腹泻病原体之一。在这项研究中，我们检测了人和猪 ST 变异体的铁和锌结合，并确定了宿主金属硫蛋白如何解毒 ST。我们发现从 ETEC 培养物上清液中纯化的 ST 在 C18 反相色谱过程中作为双合物洗脱。发现 ST 双合态的前缘分数不含铁，而 ST 双合态的后缘分数含有可测量的铁。接下来，我们发现纯化的 ST 可以在还原和厌氧条件下用铁重组，铁结合的 ST 减弱了 T84 上皮细胞 cGMP 的诱导。此外，我们证明了在增加铁浓度下生长的 ETEC 214-4 上清液仅在铁浓度大于 5 μ m 时能够诱导 cGMP。体外研究还证明 ST 结合锌，一旦结合，从 ST 去除锌需要变性条件。锌结合的 ST 也未能诱导 cGMP。我们发现 ST 向感知的氧化型谷胱甘肽池贡献二硫键，增加金属硫蛋白释放锌的速率，并可被金属硫蛋白解毒。最后，我们发现 ST 诱导先前显示由去铁胺调控的基因的转录变化。这些研究证明 ST ETEC 发病机制可能与宿主粘膜金属状态密切相关。重要性产肠毒素大肠杆菌 (ETEC) 是中低收入国家儿童、部署的军事人员和前往流行地区的旅行者的主要腹泻病原体。热稳定毒素 (ST) 是一种具有 3 个二硫键的小的非免疫原性分泌肽。人们已经认识到，膳食二硫化物调节肠道氧化还原电位，ST 可以使用外源性还原剂解毒。使用生物化学和光谱学方法，我们证明了 ST 可以在还原条件下分别结合铁和锌，从而降低 ST 毒性。此外，我们证明 ST 调节谷胱甘肽 (GSH)/氧化型谷胱甘肽 (GSSG) 比值，ST 应被视为毒素氧化剂。ST 可通过氧化载锌金属硫堇而解毒，使游离锌释放。这些研究有助于为了解腹泻病原体如何调节肠道氧化还原电位奠定基础，并可能影响我们如何为产生它们的病原体设计治疗药物和/或疫苗。
METHODS:Shigella species cause diarrheal disease globally. Shigellosis is typically characterized by bloody stools and colitis with mucosal damage and is the leading bacterial cause of diarrheal death worldwide. After the pathogen is orally ingested, it invades and replicates within the colonic epithelium through mechanisms that rely on its type III secretion system (T3SS). Currently, oral infection-based small animal models to study the pathogenesis of shigellosis are lacking. Here, we found that orogastric inoculation of infant rabbits with Shigella flexneri resulted in diarrhea and colonic pathology resembling that found in human shigellosis. Fasting animals prior to S. flexneri inoculation increased the frequency of disease. The pathogen colonized the colon, where both luminal and intraepithelial foci were observed. The intraepithelial foci likely arise through S. flexneri spreading from cell to cell. Robust S. flexneri intestinal colonization, invasion of the colonic epithelium, and epithelial sloughing all required the T3SS as well as IcsA, a factor required for bacterial spreading and adhesion in vitro Expression of the proinflammatory chemokine interleukin 8 (IL-8), detected with in situ mRNA labeling, was higher in animals infected with wild-type S. flexneri versus mutant strains deficient in icsA or T3SS, suggesting that epithelial invasion promotes expression of this chemokine. Collectively, our findings suggest that oral infection of infant rabbits offers a useful experimental model for studies of the pathogenesis of shigellosis and for testing of new therapeutics.IMPORTANCEShigella species are the leading bacterial cause of diarrheal death globally. The pathogen causes bacillary dysentery, a bloody diarrheal disease characterized by damage to the colonic mucosa and is usually spread through the fecal-oral route. Small animal models of shigellosis that rely on the oral route of infection are lacking. Here, we found that orogastric inoculation of infant rabbits with S. flexneri led to a diarrheal disease and colonic pathology reminiscent of human shigellosis. Diarrhea, intestinal colonization, and pathology in this model were dependent on the S. flexneri type III secretion system and IcsA, canonical Shigella virulence factors. Thus, oral infection of infant rabbits offers a feasible model to study the pathogenesis of shigellosis and to develop and test new therapeutics.
METHODS:PURPOSE:To quantify the effects of absorbed radiation dose on healthy liver parenchyma following radioembolisation (RE) using [99mTc]TcMebrofenin to analyse both global and regional liver function. METHODS:Patients having RE to treat hepatic disease underwent a [99mTc]TcMebrofenin hepatobilliary scintigraphy (HBS) study at both baseline and 8 weeks following treatment. Changes in global liver uptake rate were compared with healthy liver absorbed dose measures derived from the post-treatment 90Y PET/CT, including average dose, minimum dose to 70% of the volume (D70) and volume receiving at least 50 Gy (V50). Changes in functional burden associated with treatment and spared liver volumes in patients receiving lobar RE were also assessed, as were changes experienced by regional volumes corresponding to various dose ranges. Standard liver function pathology tests (LFTs) (bilirubin, albumin, ALP, AST, ALT and GGT) were examined for changes between baseline and post-treatment. RESULTS:Thirty-five patients were included in the study, of which, 9 had lobar treatment. A significant linear correlation was found between both baseline global liver uptake rate (negative) and D70 with change in global liver uptake rate. Patients undergoing lobar treatments demonstrated a shift in functional burden, and a significant difference was seen between the mean dose corresponding to liver volumes that increased their functional burden (9 Gy) and those that decreased their functional burden (35 Gy). No baseline LFTs predicted a decrease in global liver function; however, D70 demonstrated a linear correlation with changes in bilirubin and GGT. CONCLUSIONS:Given the significant negative relationship between baseline and change in global liver uptake rate, baseline HBS studies should not be used alone to disqualify patients considered for RE. In terms of treatment planning and evaluation, D70 may be the most appropriate metric of dose, with values greater than 15 Gy indicative of a likely drop in global liver function. The evidence of increasing functional burden in spared liver volumes suggests that patients at risk of complications could benefit from a lobar approach to treatment.
METHODS:NLRP3 inflammasome may serve as a potential target for the development of novel therapeutics for inflammatory bowel diseases. In this study, we found that Libertellenone M (Lib M), a secondary metabolite from the endophytic fungus Phomopsis sp. S12, has anti-inflammatory potential both in vitro and in vivo. Lib M selectively inhibited the expression of proinflammatory cytokine IL-1β and IL-18 in LPS-activated macrophages. The cleavage of pro-caspase 1 was remarkably reduced by Lib M in macrophages stimulated with three NLRP3 inflammasome activators. Administering Lib M attenuated dextran sulfate sodium-induced experimental acute colitis in mice and significantly reduced the production of these cytokines and cleaved caspase 1 in colon tissues. Apart from inhibition of NLRP3 inflammasome assembly, Lib M also suppressed NF-κB nuclear translocation in macrophages. Taken together, these findings suggest that Lib M-mediated inhibition of NLRP3 inflammasome activation could protect against colitis-like inflammatory diseases, and that this compound derived from a plant-associated fungus might inspire the exploration of novel immunosuppressive agents.