[37]

[37]. a gram-negative bacterium found in both freshwater and marine environments. Some strains of cause hemorrhagic septicemia in marine animals, resulting in great economic losses in China every year [16]. expresses several virulence factors, including hemolysins, cytotoxins, lipases, and extracellular proteases as well as biofilm formation, and all of these phenotypes are regulated by AHL-mediated quorum sensing [17]. Previous studies have shown that sp. strain QSI-1 can produce quorum quenching enzyme (AiiAQSI-1), has probiotic properties and also, can decrease the pathogenicity of infection in zebrafish (against experimental infection. 2. Results and Discussion 2.1. Expression and Purification of Recombinant AHL Lactonase Xanthiside AiiAQSI-1 In a search using the BLAST program, we found that gene has 100% similarity in nucleotide sequence with the genes of several species of such as and The gene amplified from sp. strain QSI-1 by PCR, which consists of 747 nucleotides and the gene sequence has been deposited in the GenBank database (“type”:”entrez-nucleotide”,”attrs”:”text”:”MN227141″,”term_id”:”1818942256″MN227141). This gene was then cloned into pET30a(+) vector between Nde I and Xho I restriction sites (Figure 1), which resulted in the expression of the recombinant polypeptide containing 249 amino acids. The transformed strain BL21(DE3) PET30-BL21(DE3)pET30a-aiiAQSI-1 cell lysate; 2: Supernatant of the sonication product of IPTG-induced BL21(DE3)pET30a-aiiAQSI-1; 3: Precipitate of the sonication product of IPTG-induced BL21(DE3)pET30a-aiiAQSI-1; 4: purified AiiAQSI-1 protein (as indicated by arrow). The results of the in vitro bioassays suggested that AiiAQSI-1 has the ability to degrade N-Hexanoyl-L-homoserine lactone (C6-HSL), as indicated by a reduction of the AHL-regulated purple violacein pigment diameter of CV026 reporter CD81 strain (Figure 3). Open in a separate window Figure 3 AHL-degrading activity bioassays in vivo. Ten L C6-HSL (10 M) was mixed with 0 L (a), 20 L (b), 50 L (c), 100 L (d) AiiAQSI-1, respectively and incubated at 30 C. 2.2. Effect of AiiAQSI-1 on the Motility, Virulence Factors Production and Biofilm Formation in Aeromonas hydrophila In previous studies, we found that sp. strain QSI-1 was able to Xanthiside inhibit virulence in vitro and in vivo [20]. In order to investigate whether QQ activity in strain QSI-1 was responsible for reduced virulence, we cloned and expressed gene from strain QSI-1 in BL21(DE3). Our results show that the QQ enzyme AiiAQSI-1 obtained by heterologous expression in could exhibit a similar bioprotective effect as the parent sp. strain QSI-1. Potential anti-QS activity of AiiAQSI-1 was investigated on YJ-1 characteristics including swimming motility, production of virulence factors and biofilm formation. As seen in Figure 4, AiiAQSI-1 inhibits YJ-1 swimming motility (Figure 4A). The extracellular proteolytic activity and hemolytic activity were also inhibited by AiiAQSI-1. Measurements of transparent zones, indicative of proteolytic activity and hemolytic activity, are listed in Table 1. Of note, at the highest AiiAQSI-1 concentration tested, 0.1149 mg/mL, extracellular proteolytic activity of YJ-1 was reduced by 66.5% and hemolytic activity reduced by 68.9% when compared to untreated controls. The reduction in virulence was not due to YJ-1 planktonic culture growth inhibition as growth of this Xanthiside pathogen was slightly enhanced by the presence of AiiAQSI-1 (Figure 4B). After cultured in the microtiter plates for 36 h, the planktonic bacteria growth has no significant difference with or without AiiAQSI-1 treatment. However, YJ-1 biofilm growth in the microtiter assay was reduced at the two highest AiiAQSI-1 concentrations tested (Figure 3C). Open in a separate window Open in a separate window Figure 4 Effect of AiiAQSI-1 on the motility, production of virulence factors and biofilm, and growth of YJ-1. Swimming motility (A), Growth curve of planktonic bacteria (B), and biofilm production (C). Table 1 Diameters of transparent zone for proteolytic activity and hemolytic activity. sp. S1-5 showed the QQ efficacy against quorum sensing-mediated virulence traits of and Bai et al. [22] discovered that the over-expressed recombinant AiiA protein from the gene of a strain BF1, had the ability to inhibit the QS-regulated bioluminescence in Although is not considered to be a pathogen, the association of bioluminenscence with QS in this organism is well-documented [23]. These previous studies did not address biofilm formation by In the present study, we found the AiiAQSI-1 can stimulate the planktonic growth of YJ-1 (Figure 3D) but not biofilm formation Xanthiside (Figure 4E). Planktonic growth stimulation may be due to being able to use degraded AHLs or alternatively use the.