Supplemental N-acyl homoserine lactonase alleviates intestinal disruption and improves gut microbiota in broilers challenged by Salmonella Typhimurium

Background Salmonella Typhimurium challenge causes a huge detriment to chicken production.N-acyl homoserine lactonase(AHLase),a quorum quenching enzyme,potentially inhibits the growth and virulence of Gram-negative bacteria.However,it is unknown whether AHLase can protect chickens against S.Typhimurium challenge.This study aimed to evaluate the effects of AHLase on growth performance and intestinal health in broilers challenged by S.Typhimurium.A total of 240 one-day-old female crossbred broilers(817C)were randomly divided into 5 groups(6 replicates/group):negative control(NC),positive control(PC),and PC group supplemented with 5,10 or 20 U/g AHLase.All birds except those in NC were challenged with S.Typhimurium from 7 to 9 days of age.All parameters related to growth and intestinal health were determined on d 10 and 14.Results The reductions(P<0.05)in body weight(BW)and average daily gain(ADG)in challenged birds were alleviated by AHLase addition especially at 10 U/g.Thus,samples from NC,PC and PC plus 10 U/g AHLase group were selected for further analysis.S.Typhimurium challenge impaired(P<0.05)intestinal morphology,elevated(P<0.05)ileal inflammatory cytokines(IL-1βand IL-8)expression,and increased(P<0.05)serum diamine oxidase(DAO)activity on d 10.However,AHLase addition normalized these changes.Gut microbiota analysis on d 10 showed that AHLase reversed the reductions(P<0.05)in several beneficial bacteria(e.g.Bacilli,Bacillales and Lactobacillales),along with increases(P<0.05)in certain harmful bacteria(e.g.Proteobacteria,Gammaproteobacteria,Enterobacteriaceae and Escherichia/Shigel a)in PC group.Furthermore,AHLase-induced increased beneficial bacteria and decreased harmful bacteria were basically negatively correlated(P<0.05)with the reductions of ileal IL-1βand IL-8 expression and serum DAO activity,but positively correlated(P<0.05)with the increased BW and ADG.Functional prediction revealed that AHLase abolished S.Typhimurium-induced upregulations(P<0.05)of certain pathogenicity-related pathways such as lipopolysaccharide biosynthesis,shigellosis,bacterial invasion of epithelial cells and pathogenic Escherichia coli infection of gut microbiota.Conclusions Supplemental AHLase attenuated S.Typhimurium-induced growth retardation and intestinal disruption in broilers,which could be associated with the observed recovery of gut microbiota dysbiosis.

Degradation of N-Acyl Homoserine Lactone Quorum Sensing Signals by Bacillus thuringiensis AHL Lactonase

Bacterial cells rely on signaling molecules to communicate with others from the same species and induce certain genes in a process known as quorum sensing (QS). A common molecule is N-acyl homoserine lactone (AHL) which is responsible for the expression of virulence and other factors that allow the organisms to compete in a given environment. On the other hand, other bacteria produce certain enzymes such as AHL-lactonase that break down AHL molecules and prevent gene expression of these factors. The aim of this work was to examine the level of degradation of AHL molecules by AHL-lactonase in 62 Bacillus thuringiensis (Bt) strains isolated from Middle Tennessee, Mississippi, and Alabama. N-hexanoyl-homoserine lactone (C6-HSL) and N-3-oxo-hexanoyl homoserine lactone (3-oxo-C6-HSL), which cause Chromobacterium violaceum (CV026) to produce a purple pigment were tested at different concentrations to view the Bt lactonase activity. In addition, PCR was used to test for the presence of the lactonase gene. The results showed that among the 62 Bt strains, there were 58 that possessed the AHL-lactonase (aiiA) gene and 48 strains were able to degrade C6-HSL. At high concentrations of AHL, only 13 strains were able to completely degrade C6-HSL. In addition, degradation of 3-oxo-C6-HSL was weak compared to C6-HSL. The results also revealed that AHL lactonase was thermostable, and it was concluded that the level of degradation varies in Bt strains. Only 13 of the strains studied have potent inhibitory activity against C6-HSL, which may be good to be used in field applications to control agricultural pest.

N-acyl homoserine lactonase attenuates the virulence of Salmonella typhimurium and its induction of intestinal damages in broilers

This study aimed to investigate the potential mitigating effects of N-acyl homoserine lactonase(AHLase)on the virulence of Salmonella typhimurium and its induction of intestinal damages in broilers.In vitro study was firstly conducted to examine if AHLase treatment could attenuate the virulence of S.typhimurium.Then,an in vivo experiment was performed by allocating 240 broiler chicks at 1 d old into 3 groups(8 replicates per group):negative control(NC),positive control(PC),and PC supplemented with 10,000 U/kg AHLase.All chicks except those in NC were orally challenged by S.typhimurium from 8to 10 d of age.Parameters were measured on d 11 and 21.The results showed that treatment with 1 U/mL AHLase suppressed the biofilm-forming ability(including biofilm biomass,extracellular DNA secretion and biofilm formation-related gene expression),together with swarming motility and adhesive capacity of S.typhimurium.Supplemental 10,000 U/kg AHLase counteracted S.typhimurium-induced impairments(P<0.05)in broiler growth performance(including final body weight,average daily gain and average daily feed intake)during either 1-11 d or 12-21 d,and increases(P<0.05)in the indexes of liver,spleen and bursa of Fabricius on d 11,together with reductions(P<0.05)in ileal villus height and its ratio to crypt depth on both d 11 and 21.AHLase addition also normalized the increased(P<0.05)m RNA expression of ileal occludin on both d 11 and 21 in S.typhimurium-challenged broilers.However,neither S.typhimurium challenge nor AHLase addition altered(P>0.05)serum diamine oxidase activity of broilers.Noticeably,S.typhimurium challenge caused little change in the mRNA expression of ileal inflammatory cytokines except for an increase(P<0.05)in interleukin-8 expression on d 11,whereas AHLase addition normalized(P<0.05)this change.In conclusion,AHLase treatment could attenuate the virulence and pathogenicity of S.typhimurium,thus contributing to alleviate S.typhimurium-induced growth retardation and intestinal damages in broilers.

Constitutive and Secretory Expression of the AiiA in Pichia pastoris Inhibits Amorphophallus konjac Soft Rot Disease

Amorphophallus konjac is an important economic crop widely cultivated in Southeast Asia and Africa. However, A. konjac is seriously infected by soft rot pathogen. The endocellular acyl homoserine lactonase (AiiA) which is generated by Bacillus species has inhibitory effect on soft rot pathogen through disrupting the signal molecules (N-acylhomoserine lactones, AHL) of their Quorum Sensing system. The aim of our study is to obtain recombinant yeast which produces AiiA protein. The recombinant yeast Pichia pastoris GS115 was constructed to constitutive expression of the AiiA gene. The results of reverse transcript PCR analysis showed that the AiiA gene was expressed successfully in the yeast. Proteins extracted from YPDS showed the highest inhibition efficacy to E. carotovora compared with the other two mediums (YPD and LB) under tested conditions.