| Porcine enterotoxigenic Escherichia coli (ETEC) is the most common pathogenic E.coli that can cause neonatal diarrhea. The virulence factors include adhesins, fimbriae, enterotoxin, hemolysin, flagella etc. The porcine post-weaning diarrhea (PWD), caused by ETEC lead to high morbidity in piglets, as well as economic loss in the global swine production. The small non-coding RNA (sRNA) is a new kind of gene regulator that regulates genes expression at post-transcriptional. Although the sRNA chaperone Hfq plays a very important role in regulating bacterial growth, metabolism, pathogenicity and the ability of biofilm formation, the regulatory mechanism and putative target genes of chaperone Hfq in porcine ETEC remain to be further investigate. In this study, we focused on virulence related function and pathogenicity of sRNA chaperone Hfq involved in pocrine ETEC strain C83902.1. The regulatory function of sRNA chaperone Hfq in virulence of K88ac+ enterotoxigenic E.coliWe constructed the hfq delection mutant strain C83902△hfq by λ.-Red recombinant system, as well as the complemented strain C83902Ahfq/phfq, which was constructed by transforming recombinant plasmid pACYC184-hfq into C83902△hfq. At transcriptional level, the expression level of major virulence genes including fimA (the subunit of type I fimbriae). fliC (the subunit of flagella),faeG (the subunit of K88 fimbriae), eltB (the subunit of enterotoxin LT), luxS and pfs (genes involved in Quorum Sensing) were analyzed by qRT-PCR. The results indicated that the mRNA level of fimA, faeG, ecpA in C83902△hfq were notably decreased 63.1%(P<0.01), 38.8%(P<0.01) and 39.4%(P<0.01) respectively as compared with C83902. Otherwise flagella subunit gene fliC enterotoxin subunit eltB and Quorum Sensing related genes luxS and pfs were slightly raised when compared to C83902 (P>0.05). At the protein level, the expression of FimA, FaeG, EcpA, FliC in three stains were determined by Western blot, with GapA protein for internal reference. Quantitative analysis of Western blot gray value of results showed that, the expression of FimA, FaeG, EcpA proteins were decreaseed 29%(P<0.05),23.8%(P<0.05),20% (P<0.05) respectively. But the expression of FliC protein in C83902△hfq was similar to that in protype C 83902. The results of Western blot assay were consistent with the qRT-PCR assay. Based on the above results, it can be inferred that Hfq plays an important role in regulating the expression of ecpA,faeG,fimA genes and its corresponding proteins.2. The regulatory function of sRNA chaperone Hfq in pathogenicity of K88ac+ enterotoxigenic Escherichia coliOn the basis of the above studies, we analyzed the chaperone Hfq affects pathogenicity of ETEC by monitoring the bacterial growth, qualitative and quantitative test of biofilms, IPEC-J2 cell adhesion assay, and mouse challenge experiments. It turns out to be that, compared with C83902, the growth rate of C83902△hfq slowed down significantly. Meanwhile, the biofilm-formation ability and adhesion ability of C83902△hfq to IPEC-J2 cells reduced 80% and 78% respectively compared to C83902. Otherwise, we further determined the status of SPF BALB/c mouse (eating, drinking, mental health and mortality) after challenging with 109 cfu bacteria using the way of gavage,72 h later collecting the intestinal fluid and diluting to coat on plates for counting bacteria. Results show that the mice challenged with WT strain C83902 appear reduced feed intake as well as slowed-movement, and slight blow and congestion were found in the small intestine. The mice challenged with C83902△hfq strain showed no above symptoms. Plate countting results shows that compared with the WT strains, deletion of hfq caused 99% (P<0.01),98%(P<0.01) and 99% (P<0.01) decrease respectively in duodenum, jejunum and ileum. In summary, the data above suggest that chaperone Hfq could affect ETEC pathogenicity by regulating the expression of fimbrial genes. |
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