Zhou Mengzhou, Yu Hai, Yin Xianhua, Sabour Parviz M, Chen Wei, Gong Joshua
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China ; Guelph Food Research Centre, Agriculture and Agri-Food Canada, Guelph, Ontario, Canada.
Guelph Food Research Centre, Agriculture and Agri-Food Canada, Guelph, Ontario, Canada.
PLoS One. 2014 Feb 18;9(2):e89004. doi: 10.1371/journal.pone.0089004. eCollection 2014.
The nematode Caenorhabditis elegans has become increasingly used for screening antimicrobials and probiotics for pathogen control. It also provides a useful tool for studying microbe-host interactions. This study has established a C. elegans life-span assay to preselect probiotic bacteria for controlling K88(+) enterotoxigenic Escherichia coli (ETEC), a pathogen causing pig diarrhea, and has determined a potential mechanism underlying the protection provided by Lactobacillus.
METHODOLOGY/PRINCIPAL FINDINGS: Life-span of C. elegans was used to measure the response of worms to ETEC infection and protection provided by lactic acid-producing bacteria (LAB). Among 13 LAB isolates that varied in their ability to protect C. elegans from death induced by ETEC strain JG280, Lactobacillus zeae LB1 offered the highest level of protection (86%). The treatment with Lactobacillus did not reduce ETEC JG280 colonization in the nematode intestine. Feeding E. coli strain JFF4 (K88(+) but lacking enterotoxin genes of estA, estB, and elt) did not cause death of worms. There was a significant increase in gene expression of estA, estB, and elt during ETEC JG280 infection, which was remarkably inhibited by isolate LB1. The clone with either estA or estB expressed in E. coli DH5α was as effective as ETEC JG280 in killing the nematode. However, the elt clone killed only approximately 40% of worms. The killing by the clones could also be prevented by isolate LB1. The same isolate only partially inhibited the gene expression of enterotoxins in both ETEC JG280 and E. coli DH5α in-vitro.
CONCLUSIONS/SIGNIFICANCE: The established life-span assay can be used for studies of probiotics to control ETEC (for effective selection and mechanistic studies). Heat-stable enterotoxins appeared to be the main factors responsible for the death of C. elegans. Inhibition of ETEC enterotoxin production, rather than interference of its intestinal colonization, appears to be the mechanism of protection offered by Lactobacillus.
线虫秀丽隐杆线虫越来越多地用于筛选用于病原体控制的抗菌剂和益生菌。它还为研究微生物与宿主的相互作用提供了有用的工具。本研究建立了一种秀丽隐杆线虫寿命测定法，以预选用于控制K88(+)产肠毒素大肠杆菌（ETEC）的益生菌，K88(+)产肠毒素大肠杆菌是一种导致猪腹泻的病原体，并确定了乳酸杆菌提供保护作用的潜在机制。
方法/主要发现：利用秀丽隐杆线虫的寿命来测量线虫对ETEC感染的反应以及产乳酸菌（LAB）提供的保护作用。在13株对秀丽隐杆线虫免受ETEC菌株JG280诱导死亡具有不同保护能力的LAB分离株中，玉米乳酸杆菌LB1提供了最高水平的保护（86%）。用乳酸杆菌处理并未减少ETEC JG280在秀丽隐杆线虫肠道中的定殖。喂食大肠杆菌菌株JFF4（K88(+)但缺乏estA、estB和elt的肠毒素基因）不会导致线虫死亡。在ETEC JG280感染期间，estA、estB和elt的基因表达显著增加，而分离株LB1可显著抑制这种增加。在大肠杆菌DH5α中表达estA或estB的克隆在杀死线虫方面与ETEC JG280一样有效。然而，elt克隆仅杀死了约40%的线虫。分离株LB1也可以阻止克隆的致死作用。同一分离株在体外仅部分抑制了ETEC JG280和大肠杆菌DH5α中肠毒素的基因表达。
结论/意义：所建立的寿命测定法可用于益生菌控制ETEC的研究（用于有效筛选和机制研究）。热稳定肠毒素似乎是导致秀丽隐杆线虫死亡的主要因素。抑制ETEC肠毒素的产生，而不是干扰其在肠道中的定殖，似乎是乳酸杆菌提供保护作用的机制。
