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Lactobacillus zeae protects Caenorhabditis elegans from enterotoxigenic Escherichia coli-caused death by inhibiting enterotoxin gene expression of the pathogen.

Zhou M, Yu H, Yin X, Sabour PM, Chen W, Gong J - PLoS ONE (2014)

Bottom Line: 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.The clone with either estA or estB expressed in E. coli DH5α was as effective as ETEC JG280 in killing the nematode.

View Article: PubMed Central - PubMed

Affiliation: 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.

ABSTRACT

Background: 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.

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Comparison of the enterotoxin clones with ETEC JG280 with regard to in-vitro expression of enterotoxin genes in the absence or presence of L. zeae LB1.(A) Relative expression of estA, estB, and elt genes in the absence of isolate LB1. The data of relative expression were log10 2 transformed to acquire a normal distribution after normalization with the housekeeping gene, gapA, as a reference. Data are presented as relative expression changes calculated by comparing three enterotxin expression levels with the housekeeping gene. A value of >1 represents up-regulation; a value of <1 represents down-regulation. (B) Inhibition of gene expression (relative expression) of estA, estB, and elt by isolate LB1. The relative expression of each toxin gene in the absence of LB1 represented 0% inhibition. The bars denoted with different letters represent significant differences (P<0.05).
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pone-0089004-g006: Comparison of the enterotoxin clones with ETEC JG280 with regard to in-vitro expression of enterotoxin genes in the absence or presence of L. zeae LB1.(A) Relative expression of estA, estB, and elt genes in the absence of isolate LB1. The data of relative expression were log10 2 transformed to acquire a normal distribution after normalization with the housekeeping gene, gapA, as a reference. Data are presented as relative expression changes calculated by comparing three enterotxin expression levels with the housekeeping gene. A value of >1 represents up-regulation; a value of <1 represents down-regulation. (B) Inhibition of gene expression (relative expression) of estA, estB, and elt by isolate LB1. The relative expression of each toxin gene in the absence of LB1 represented 0% inhibition. The bars denoted with different letters represent significant differences (P<0.05).

Mentions: In-vitro expression of enterotoxin genes (estA, estB, and elt) in ETEC JG280 or in E. coli DH5α was also examined in the absence or presence of isolate LB1. Clones DH5α-STa and DH5α-LT and JG280 expressed a similar level of estA and elt in vitro (Fig. 6A). However, the expression of estB was increased to more than 2-fold in E. coli DH5α than in ETEC JG280 in the absence of LB1. In the presence of LB1, approximate 40% and 60% reduction in the expression of all enterotoxin genes (estA, estB, and elt) were observed in E. coli DH5α and in ETEC JG280, respectively, compared with the absence of LB1 (Fig. 6B).


Lactobacillus zeae protects Caenorhabditis elegans from enterotoxigenic Escherichia coli-caused death by inhibiting enterotoxin gene expression of the pathogen.

Zhou M, Yu H, Yin X, Sabour PM, Chen W, Gong J - PLoS ONE (2014)

Comparison of the enterotoxin clones with ETEC JG280 with regard to in-vitro expression of enterotoxin genes in the absence or presence of L. zeae LB1.(A) Relative expression of estA, estB, and elt genes in the absence of isolate LB1. The data of relative expression were log10 2 transformed to acquire a normal distribution after normalization with the housekeeping gene, gapA, as a reference. Data are presented as relative expression changes calculated by comparing three enterotxin expression levels with the housekeeping gene. A value of >1 represents up-regulation; a value of <1 represents down-regulation. (B) Inhibition of gene expression (relative expression) of estA, estB, and elt by isolate LB1. The relative expression of each toxin gene in the absence of LB1 represented 0% inhibition. The bars denoted with different letters represent significant differences (P<0.05).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3928337&req=5

pone-0089004-g006: Comparison of the enterotoxin clones with ETEC JG280 with regard to in-vitro expression of enterotoxin genes in the absence or presence of L. zeae LB1.(A) Relative expression of estA, estB, and elt genes in the absence of isolate LB1. The data of relative expression were log10 2 transformed to acquire a normal distribution after normalization with the housekeeping gene, gapA, as a reference. Data are presented as relative expression changes calculated by comparing three enterotxin expression levels with the housekeeping gene. A value of >1 represents up-regulation; a value of <1 represents down-regulation. (B) Inhibition of gene expression (relative expression) of estA, estB, and elt by isolate LB1. The relative expression of each toxin gene in the absence of LB1 represented 0% inhibition. The bars denoted with different letters represent significant differences (P<0.05).
Mentions: In-vitro expression of enterotoxin genes (estA, estB, and elt) in ETEC JG280 or in E. coli DH5α was also examined in the absence or presence of isolate LB1. Clones DH5α-STa and DH5α-LT and JG280 expressed a similar level of estA and elt in vitro (Fig. 6A). However, the expression of estB was increased to more than 2-fold in E. coli DH5α than in ETEC JG280 in the absence of LB1. In the presence of LB1, approximate 40% and 60% reduction in the expression of all enterotoxin genes (estA, estB, and elt) were observed in E. coli DH5α and in ETEC JG280, respectively, compared with the absence of LB1 (Fig. 6B).

Bottom Line: 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.The clone with either estA or estB expressed in E. coli DH5α was as effective as ETEC JG280 in killing the nematode.

View Article: PubMed Central - PubMed

Affiliation: 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.

ABSTRACT

Background: 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.

Show MeSH
Related in: MedlinePlus