Detection Of Quorum Sensing Signal Molecules In Family Vibrionaceae And Edwardsiella Ictaluri

Quorum sensing (QS) has been considered as a kind of universal device in bacteria for cell-cell communication. This communication device depends on the synthesis of small signal molecules that either diffuse or are actively transported out of the bacterial cells, and increase in concentration as the bacterial population density increases. When the level of the signal molecules comes to a threshold, QS pathways are activated and regulate the expression of some QS-controlled genes such as bioluminescence and antibiotic production, etc.Although the first acyl-homoserine lactone (AHL) based QS system was found in Vibrio fischeri, little is known about the QS systems in most species of the family Vibrionaceae. Species of the family Vibrionaceae are ubiquitous in marine environments, and several of them are important pathogens for humans and marine organisms. Some pathogenic species in the family Vibrionaceae are reported to utilize quorum sensing (QS) to regulate the expression of virulence-associated genes. The aim of this study was to detect the distribution and diversity of QS signal molecules in 25 Vibrionaceae strains. We detected the production of three kinds of QS signal molecules, i.e. the N-acyl-homoserine lactone (AHL), the autoinducer-2 (AI-2) and the cholerae autoinducer-1-like (CAI-1-like) molecules with different biosensors. Analyzing traces of CAI-1-like molecules was standardized by using report strain V. harveyi JAF375 with diminishing the standard deviation and increasing the reproducibility and reliability of results.The results indicated that QS signal molecules, especially AHL and AI-2 molecules, were widespread in the family Vibrionaceae. Except Salinivibrio costicola VIB288 and Vibrio natriegens VIB299, all the other 23 Vibrionaceae strains could produce one or more kinds of detectable QS signal molecules. Twenty-one of the 25 strains were found to produce AHL signal molecules by using V. harveyi JMH612 and Agrobacterium tumefaciens KYC55 (pJZ372)(pJZ384)(pJZ410) as biosensors. The AHL fingerprints of eight strains were detected by thin layer chromatography (TLC) with Ag. tumefaciens KYC55, and two of them, i.e. V. mediterranei VIB296 and Aliivibrio logei VIB414 had a high diversity of AHLs. Twenty of the 25 strains were found to have the AI-2 activity, and the luxS gene sequences in 18 strains were proved to be conserved by PCR amplification and sequencing. All the sequences showed two conserved motifs, His-Thr-Leu-Glu-His and [Gln/Lys]-Ile-Pro-[Glu/Asp]-Leu-Asn-Glu-Tyr sequences, which matched the sequence formulas of LuxS motifs, His-Xaa-Xaa-Glu-His and [Lys/Arg]-Xaa_2-3-[Asp/Glu]-Xa_a2-3-Tyr sequences, respectively. Only six of the 25 strains possessed the CAI-1-like activity. A. logei VIB414, V. campbellii VIB285, V. furnissii VIB293, V. pomeroyi LMG20537 and two V. harveyi strains VIB571 and VIB645 were found to produce all the three kinds of QS signal molecules.QS signal molecules in an Edwardsiella ictaluri strain Ei-151 were also be detected in this study. Edw. ictaluri is a gram-negative pathogenic bacterium in the family Enterobacteriaceae that causes enteric septicemia of catfish (ESC). ESC has become a significant problem in the aquaculture of striped catfish (Pangasianodon hypophthalmus) in Vietnam. A bacterium designated as Ei-151 was isolated from the diseased fish. Based on the 16S rDNA sequencing and phenotypic tests, the pathogenic bacterium was identified as Edw. ictaluri. Another pathogenic bacterium called Edw. tarda belonging to the same genus Edwardsiella has been reported to have two kinds of quorum sensing (QS) signal molecules, i.e. acylated homoserine lactone (AHL) and autoinducer-2 (AI-2), one of which has been verified to be used for the regulating of some virulence-associated elements. However, there is no information about the QS systems in Edw. ictaluri species. In this study, the presence of QS signal molecules was analysed in the Edw. ictaluri strain Ei-151 with different bioreporter strains. The results showed that at least three kinds of AHL signal molecules were detected in Ei-151 performed with the reporter strain Ag. tumefaciens KYC55, and the AHL fingerprint was similar to that of Edw. tarda LTB-4. This could be supported by the fact that the sequences of putative AHL synthase [Genbank: YP₀02934276] and AHL-dependent transcriptional regulator [Genbank: YP₀02934275] of Edw. ictaluri from the Genebank database, showed 93% and 96% similarity with EdwI and EdwR from Edw. tarda, respectively. Ei-151 produced AHLs and AI-2 signal molecules during its entire growth with their levels increasing accompanied by the growth of cell population density. The two kinds of signal molecules all reached a high level at the exponential phase with OD₆₀₀ around 1.8, and then had a significant decline, which suggested that they may involve in regulating genes expression at the same time. No CAI-1-like activity in Ei-151 as well as its type strain LMG7860^T was detected.In response to a variety of environmental conditions and selection forces, the family Vibrionaceae and Edw. ictaluri produced QS signal molecules with great diversity and complexity. The knowledge we obtained from this study will be useful for further research on the roles of different QS signal molecules in this family.