Regulation Of Lsr Operon In Avain Pathogenic Escherichia Coli

Avian pathogenic Escherichia coli（APEC） cause poultry severe respiratory and systemic diseases. The prevention of avian colibacillosis is conditioned by its drug resistence, diverse serotypes and complicated pathogenesis.The progress, bacteria communicate with chemical molecule called autoinducers, is called quorum sensing（QS）, allowing bacteria to monitor the population density, alter genes expression and a variety of biological functions. The LuxS/AI-2 type QS system has been widely detected in many Gram-positive and Gram-negative bacteria, and it could regulate different physiological functions with the signal molecule AI-2.Although the regulation of functions of AI-2 is realized through the lsr operon（including 8 genes, lsrK, lsrR, lsrA, lsrC, lsrD, lsrB, lsrF and lsrG, respectively） in Salmonella typhimurium. However, research about AI-2 recognition, signal transduction and pocessing in APEC is not clear. Aiming to provide a new methods for prevention of avian colibacillosis in QS system, this study includes 4 parts: 1. Construction of MG1655ΔlsrB and detection of AI-2 internalization in MG1655ΔlsrB.In order to verify the role of lsrB in AI-2 internalization, the lsrB mutant strain was constructed, and AI-2 internalization of MG1655 and MG1655ΔlsrB was detected. The results showed that MG1655ΔlsrB could not internalize AI-2 compared with the wild strain. The results indicated that the lsr operon was critical for AI-2 intake in Escherichia coli. 2. The distribution of lsr operon in APECFor investigation of the distribution of lsr operon in APEC, the primers of eight genes in lsr operon of MG1655 were designed to detect lsr operon in 60 APEC clinical strains(serotype O₁, O₂ or O₇₈, resepctively) by PCR. The results showed that the distribution of lsr operon is associated with serotypes, with the highest detection rate in O₇₈（95.5%）, while the lowest detection rate in O₂（40.9%）. 3. The binding activity verification of APEC LsrB with AI-2The sequencing results of APEC94 lsr operon showed 99% similarity with MG1655. For verification the capacity of AI-2 binding to LsrB in APEC, the plasmid of pCold TF-LsrB was constructed, and the LsrB was expresssd in BL21（DE3） and BL21?luxS（DE3）, respectively. The assay of AI-2 binding or release to APEC LsrB was evaluated. The results proved that LsrB could bind AI-2. 4. Deletion of the lsr operon in APEC and analysis of the mutant biological characteristicsThe AI-2 could regulate many bacterial behaviors, including biofilm formation, motility and virulence et al. For evaluation the lsr operon function in APEC, the APEC94Δlsr（Cm）mutant strain was constructed by deletion of lsr operon in APEC.The results of LD50 in 9-day-old cherry valley ducks showed that the virulence of APEC94Δlsr（Cm）descended by 294 times compared with the parental strain. In addition, the bacteria load capacities of APEC94 and APEC94Δlsr（Cm）were respectively 2.5×104 and 6.5×102 CFU/mL（in blood, P﹤0.0001）, 5.9×108 and 3.6×105 CFU/mL（in liver, P﹤0.0001）, 1.9×1010 and 6.0×105 CFU/mL（in spleen, P﹤0.0001）, 6.1×108 and 8.1×105 CFU/mL（in kidney, P﹤0.0001）24 h post injection of strains with dose of 1×109 CFU each duck. However, there was no significant difference between the wild strain and the mutant strain in determination of growth curve and biofilm formation. The mechanism of virulence regulation of lsr operon in APEC remains to be further studied.In this study, the results indicates that APEC possesses the lsr operon.The distribution of lsr operon is serotypes related. Furthermore, the LsrB in APEC can bind AI-2 in vitro. Deletion of lsr operon would attenuate virulence of APEC. Our study will provide a new idea for prevention of APEC.