Study On Mechanism Of Porcine Derived Klebsiella Pneumoniae AI-2 Signal Molecule And MrkD Protein Inhibitory Effect By Eugenol

Klebsiella pneumoniae?K.pneumoniae?is the second most common opportunistic bacterium after Escherichia coli.As a zoonotic pathogen,K.pneumoniae is widely distributed in soil,skin,respiratory digestive tract and genitourinary tract.It can cause diseases such as pneumonia,peritonitis,traumatic infection and sepsis in humans and animals.Recently,the emergence of clinical multi-drug resistant and hypervirulent K.pneumonia has posed a serious threat to the health of humans and animals.Therefore,it is urgent to find new anti-infective agents and anti-infective strategies.The pathogenicity of K.pneumoniae depends on virulence factors,including capsular polysaccharide,lipopolysaccharide,adhesive protein and siderophore.Among them,capsular polysaccharide and lipopolysaccharide are the main components of bacterial biofilm.The fimbria type?mainly mediates the adhesion of bacteria to endothelial cells and epithelial cells,and the adhesion to host cells is the primary condition for K.pneumoniae to invade body and cause infection.Bacterial quorum sensing?QS?system can regulate various physiological functions such as bioluminescence,biofilm formation,virulence gene expression,cell differentiation,etc..LuxS/AI-2 type QS,widely presented in G⁺and G^-,acts as both intraspecies signal and interspecies signal,and has a wider range of effects.Eugenol is the main component of essential oils such as clove,camphor and cinnamon.It has various pharmacological effects such as antibacterial,antiseptic,anti-inflammatory,anti-oxidation and analgesic anesthesia.With the deepening research on natural compounds,researchers found that eugenol at sub-inhibitory concentration also exhibits inhibitory activity on QS system,which provides new ideas for prevention and treatment of bacteria infection.In this study,two strains of K.pneumoniae were isolated from clinically infected swine nasal swabs,and identified by morphological observation,culture characteristics,biochemical tests,molecular biological identification,drug sensitivity test and mouse pathogenicity.Both the two isolates were multi-drug resistant K.pneumoniae and were named strain KP1 and strain KP2,respectively.Among them,KP1 is slightly more pathogenic to mice than KP2,and the LD₅₀ of the two strains were 5×10⁸cfu/ml and 1×10⁹cfu/ml respectively;Congo red plate,crystal violet staining,string test and main virulence gene PCR method were used to identifiy the virulent phenotype of KP1 and KP2.The results showed that both KP1 and KP2 could grow black colonies on congo red plates.Crystal violet staining indicated that both isolates could form biofilm,and the biofilm formation ability of KP2 was stronger than KP1,which was consistent with the result of drug sensitivity test.Both KP1 and KP2 did not have a hypermucoviscosity phenotype with rmpA gene negative.Combined with LD50 the two strain was excluded from the possibility of hypervirulent K.pneumoniae.The capsular serotype identification showed that the strain KP1 belongs to the K2 capsule serotype.To further study the effect of eugenol at sub-inhibitory concentration on AI-2 and MrkD of K.pneumoniae,the minimum inhibitory concentration?MIC?and minimum bactericidal concentration?MBC?of eugenol against K.pneumoniae were determined by two-fold dilution method,the results showed that both MIC and MBC of eugenol for KP1 and KP2were 2?g/mL.The minimum biofilm inhibitory concentration?MBIC?of eugenol was1?g/mL,indicating good antibacterial activity.Mice were infected with KP1 strain co-cultured with 1/4MIC eugenol,and it was found that eugenol at subinhibitory concentration could extend the median survival time of K.pneumoniae infected mice.However,the results of cardiac bacteria colony counting and pathological section observation showed that eugenol treated group did not significantly alleviate the infection of mice compared with control group,and there was no difference between the two groups.In addition,prokaryotic transcriptome sequencing technology was used to study the effect of eugenol at subinhibitory concentration on transcription level of luxS,pfs?mtnn?,lsrR,lsrK,the major regulatory genes for AI-2 synthesis,and fimbrial mrkD genes,,and the results were verified by real-time quantitative PCR.Transcriptome sequencing results showed that the FPKM values of luxS,lsrR and mrkD genes in eugenol treated group were lower than that in control group,and the FPKM values of pfs and lsrK genes were higher than that in control group.The results of qPCR verification showed that eugenol at a sub-inhibitory concentration could down-regulate the expressions of luxS and mrkD,and up-regulate the expressions of pfs,lsrR and lsrK.The expressions of lsrR and lsrK were both significantly higher than that in the control group.While,the results of lsrR gene verification differred from transcriptome sequencing.Considering the higher FPKM and log₂?FC?values of lsrR,we speculate that lsrR and lsrK genes expression were both up-regulated after eugenol treated,but overall is transcriptional inhibition,which would inhibit transport of AI-2.To prove the effect of LuxS/AI-2 QS system on growth characteristics,biofilm and virulence of K.pneumoniae strain KP1,the experiment successfully constructed the recombinant suicide plasmid pRE112-luxS homologous arm of upstream and downstream.Homologous recombination method was used to construct luxS gene deletion strain,KP1?luxS.The results showed that the growth rate of KP1?luxS,has no obvious change compared with wild strain,but the ability of biofilm formation and virulence were attenuated,indicating that luxs gene could regulate the biofilm and virulence of bacteria.To study the inhibitory activity of eugenol at subinhibitory concentration on LuxS,Pfs and MrkD proteins in vitro,the coding genes of these three proteins were amplified in this study.After being correctly identified by sequencing and enzyme digestion,the three genes were connected with prokaryotic expression vector pET-28a?+?to construct recombinant prokaryotic expression plasmid.The plasmid was induced to expression using IPTG and then purified by nucleophilic chromatography Ni⁺column.The expression products were further analyzed by SDS-PAGE electrophoresis and Western-blot.The results showed that LuxS,Pfs and MrkD with molecular weights of about 24kDa,30kDa and 35kDa were obtained,respectively.Western-blot analysis showed that eugenol promoted LuxS expression,inhibited Pfs expression and inhibited MrkD expression at1/8MIC and 1/4MIC.In addition,the results of AI-2 in vitro synthesis test showed that eugenol could inhibit the synthesis of AI-2 molecule in a dose dependent manner.Molecular docking prediction showed that eugenol could dock well with LuxS and Pfs proteins and change their conformation by intermolecular forces,but poorly with MrkD.The results of the epithelial cell adhesion and adhesion inhibitory test showed that MrkD protein could promote the adhesion of K.pneumoniae to MDBK cells in a concentration dependent manner,however,the adhesion effect could be inhibited by eugenol,which was also enhanced with the increase of eugenol concentration.