Liu Quin, Mittal Rahul, Emami Claudia N, Iversen Carol, Ford Henri R, Prasadarao Nemani V
Department of Gastroenterology, Children's Hospital Los Angeles, Los Angeles, California 90027, USA.
J Surg Res. 2012 Aug;176(2):437-47. doi: 10.1016/j.jss.2011.10.030. Epub 2011 Nov 15.
Cronobacter sakazakii (CS) is an emerging opportunistic pathogen that causes life-threatening infections in infants. This pathogen has been implicated in the outbreaks of necrotizing enterocolitis (NEC) with associated rates of high mortality and morbidity. In this study, we compared the abilities of CS strains isolated from human and environmental sources to bind to intestinal epithelial cells and trigger apoptosis.
CS strains were isolated from human and environmental sources and their abilities to bind to intestinal epithelial cells were determined. Monolayer permeability was determined by transepithelial electrical resistance (TEER) and horseradish peroxidase (HRP) leakage. Apoptosis was examined by ApoTag and AnnexinV-7AAD staining. PKC activation was evaluated by non-radioactive PepTag assay.
Human isolates of CS bind to rat and human enterocytes more efficiently than environmental strains. Additionally, these strains induced increased enterocyte monolayer permeability as indicated by a decrease in TEER and an increase in transcellular leakage of exogenously added HRP. Human isolates also caused tight junction disruption and significant apoptosis of enterocytes compared with environmental strains due to increased production of inducible nitric oxide. We also observed that human CS isolates caused 2-fold increase in the activation of phosphokinase C (PKC) than environmental strains. Blocking the PKC activity in enterocytes by an inhibitor, Gö 6983, suppressed CS-mediated tight junction disruption, monolayer permeability, and apoptosis of the cells.
These results suggest that human isolates of CS more efficiently bind to and cause damage to intestinal epithelial cells compared with environmental strains.
阪崎克罗诺杆菌（CS）是一种新出现的机会致病菌，可在婴儿中引起危及生命的感染。这种病原体与坏死性小肠结肠炎（NEC）的暴发有关，且伴有高死亡率和高发病率。在本研究中，我们比较了从人类和环境来源分离的CS菌株与肠上皮细胞结合并引发细胞凋亡的能力。
从人类和环境来源分离CS菌株，并测定其与肠上皮细胞结合的能力。通过跨上皮电阻（TEER）和辣根过氧化物酶（HRP）渗漏来测定单层通透性。通过ApoTag和膜联蛋白V-7AAD染色检测细胞凋亡。通过非放射性PepTag分析评估蛋白激酶C（PKC）的激活情况。
与环境菌株相比，从人类分离的CS菌株与大鼠和人类肠上皮细胞的结合效率更高。此外，这些菌株导致肠上皮细胞单层通透性增加，表现为TEER降低和外源性添加的HRP跨细胞渗漏增加。与环境菌株相比，由于诱导型一氧化氮产生增加，人类分离株还导致肠上皮细胞紧密连接破坏和显著凋亡。我们还观察到，与环境菌株相比，人类CS分离株导致磷酸激酶C（PKC）的激活增加了2倍。用抑制剂Gö 6983阻断肠上皮细胞中的PKC活性可抑制CS介导的紧密连接破坏、单层通透性和细胞凋亡。
这些结果表明，与环境菌株相比，从人类分离的CS菌株能更有效地与肠上皮细胞结合并对其造成损伤。
