| Avian colibacillosis is one of the most common infectious diseases caused entirely or partly by avian pathogenic Escherichia coli (APEC) in birds. In recent years, it is responsible for worldwide economic losses to the poultry industry.The endotoxic lipopolysaccharide (LPS) is outer monolayer of the outer membranes of most gram negative bacteria. Studies demonstrated that the lipid A portion of the LPS molecule is responsible for activity of LPS. When pathogenic Escherichia coli invades into host, lipid A of LPS releases from the membranes of the bacteria and activates the receptors of the lipid A, then physiological reaction in host was occured and synthesis of inflammation and anti-inflammation cytokine was triggered.Polysaccharide of LPS is differentiation in strains, but construction of lipid A is conserved in some gram negative bacteria. In E. coli, lipid A biosynthesis are10steps, the final steps of lipid A synthesis occur in the inner membrane by the action of LpxL (HtrB) and LpxM (MsbB). At37℃, the late acyltransferases consecutively add lauroyl (C12:0) and myristoyl (C14:0) groups to the tetra-acylated DSMP that synthesis by two UDP-UDP-GlcNAc and four myristate chains. At12℃, however, the cold-temperature-specific late acyltransferase LpxP acts instead of LpxL to add palmitoleate (C16:1). late acyltransferase LpxL, LpxM and LpxP were encoded by genes lpxL,lpxM and lpxP, respectively. The numbers and the construction of the acyl chains are tight related to the potently activates the innate immune system and pathogenesis of gram-negative bacterial infections.To our knowledge, the contribution of the late acyltransferases responsible for lipid A biosynthesis to pathogenicity of APEC has not been elucidated.In this study, we identified three late acyltransferase genes, encoding lauroyl transferase, myristoyl transferase, and palmitoleoyl transferase, from APEC O2:K1strain E058and constructed the corresponding isogenic mutants. The biological and immune characteristics and the pathogenicity of these mutants were determined both in vitro and in vivo, and furthermore development the novel bacterial vaccines.1. Construction and biological characteristics of lpxL、lpxM and lpxPmutants of avian pathogenic Escherichia coli E058In this study, genes in APEC serogroup02strain E058chosen for deletion were lpxL, lpxM and lpxP. Each knockout mutant of both genes was constructed and named as E058ΔlpxL, E058ΔlpxM and E058ΔlpxP, and knockout mutants of both genes were also constructed and named E058AlpxLAlpxP, E058AlpxMAlpxP, and E058ΔlpxLΔlpxM, respectively. And then, the each chlormycetin gene was deletion by plamid pCP20. The ORFs of genes lpxL, lpxM and lpxP were linked to the plamid pGEX-6P-1and constructed the plamids pGEX-6P-1-lpxL pGEX-6P-1-lpxM and pGEX-6P-1-lpxP. These plamids introduced into the mutants E058AlpxL, E058ΔlpxM and E058AlpxP, and the ReE058ΔlpxL, ReE058ΔlpxM and ReE058ΔlpxP were constructed, respectively.Analysis of promoters and open reading frames (ORFs) showed that the lpxL,lpxM, and lpxP DNA fragments each contained a single ORF. Southern blotting and RT-PCR showed that mutants only disrupted the lpxL, lpxM, or lpxP gene transcription, respectively, and gene transcription of the upstreams and downstreams were not effected. Observation by electron microscope showed the construction of cell outer membrane was changed in lpxL or/and lpxM deleted mutants. SDS-PAGE showed E058and E058ΔlpxP owned three bands, lpxM deleted mutants missed one band closing to26KDa, lpxL deleted mutants missed two bands larger than17KDa, and color of17KDa band from lpxL and lpxM double mutant was lighter than that from lpxL single mutant. The result of GC showed when compared with the lipid A of parental strain E058, the lpxL deleted mutants lost one secondary laurate chain (C12:0) in their lipid A, and the lpxM deleted mutants lacked one secondary myristate chain (C14:0) in their lipid A. The fatty acid compositions of lipid A from E058ΔlpxP was similar to those of E058cultured at37℃.All mutants grew in the same rate in minimal medium, and all grew slower than the wild type in LB medium at either37℃or42℃. In LB broth E058AlpxP grew at a rate equal to that of E058at37℃or42℃, while E058ΔlpxZ, E058ΔlpxM, E058ΔlpxLΔlpxP, and E05SΔlpxMΔlpxP showed slightly decreased growth rates compared with E058in logarithmic phase. Meanwhile, the growth rate of E058ΔlpxLΔlpxMwas the slowest.In an LAL assay, whole cell suspensions (OD600=0.1) gave similar endotoxin units (EU)/mL in all strains except E058ΔlpxLΔlpxM. All mutants, especially lpxL deleted mutants, exhibited greater susceptibilities to most antibiotics than that of the parental strain. A bactericidal assay with SPF chicken serum indicated reduced resistance to SPF chicken serum for the lpxL deleted mutants. The invasion and survival in HD11showed that the ability of invasion reduced significantly for lpxL deleted mutants, and survival in HD11reduced significantly to E058after4h.The50%lethal doses (LD50) of all mutants except E058ΔlpxP were reduced to E058. At24h post-challenge, colonies of wild type E058were counted, with the highest levels isolated from the lung (4.4x104CFU.g-1), spleen (3.9×104CFU.g-1) and kidney (9.7×103CFU.g-1). A small number of colonies were obtained from the heart (8.7×101CFU.g-1) and liver (9.2×102CFU.g-1). For mutants E058ΔlpxP and E05SΔlpxMΔlpxP, colonies in the heart, liver, spleen, lung, and kidney were equal to those of the parental strain. Meanwhile, on average, the other four mutant strains were re-isolated at levels10-1000times lower than the wild type strain from the tested internal organs at24h post-challenge. With respect to mutants E058Δ/pxL, E058Δ/pxM, E058ΔlpxLΔlpxP, and E058Δ/pxLΔlpxM, no colonies were recovered from the heart. E05SΔlpxL colonies collected from tested organs (1.3CFU.g-1in liver,5.6CFU.g-1in spleen,2.9Δ102CFU.g-1in lung, and9.5×101CFU.g-1in kidney), were significantly decreased compared to parental strain E058. E05SΔlpxM showed reduced bacterial numbers in the liver, spleen, lung, and kidney, but there were no significant differences compared to the wild type strain, except in the liver (3.4CFU.g-1) and spleen (1.4×102CFU.g-1). E058ΔlpxLΔlpxP colonies from liver, spleen, lung and kidney were also sharply reduced compared to E058, or reduced in kidney. No colonies E058ΔlpxLΔlpxMin liver. Colony numbers of E058ΔlpxLΔlpxM in liver, spleen, lung, and kidney were significantly reduced or non-existent in comparison with the wild type strain. Three-week-old white leghorn SPF chickens were inoculated in the left thoracic air sac with1011CFU of the E058AlpxL and E058AlpxLAlpxM mutants. The results showed that increased bacterial numbers of E05SΔlpxL and E05SΔlpxLΔlpxM were recovered from all tested organs;10-100times higher than those of the same strain inoculated with108CFU. The numbers in tested organs were slightly lower than those of the E058challenge group, except for in the heart. However, the chickens challenged with1011CFU E058ΔlpxLΔlpxM had no visible histopathological lesions in heart, liver, spleen, lung, or kidney. The chickens challenged with1011CFU EO58ΔlpxL only developed slight histopathological lesion of the spleen. The recovered colony numbers of ReE058ΔlpxZ, and ReE058ΔlpxM were not significantly different from those of the wild-type strain. All results suggested that lpxL and lpxM were relationship to the pathogenicity of APEC.2. Effect of LPS derived from E058and the mutants E058ΔlpxL, E058ΔlpxM and E058ΔlpxP to the signal pathway related to LPS and apoptosis induced by LPSThe LPS derived from E058and the mutants E058ΔlpxL, E058ΔlpxM and E058AlpxP was used to stimulate the HD11and RAW264.7to detect the change of signal pathway and cytokine. At the same time, the research about expression of signal molecule and cytokine in different organism in chicken stimulated by LPS. The results showed that because of absence of lipopolysaccharide binding protein (LBP), chicken to LPS is more sensitive to mice. The signal molecule of related to LPS, include TLR4, MyD88, IRAK4, TRAF6, TAB, TAK1, IKKα/β and NF-κB were activated with times after2h which the cell stimulated by LPS. Furthermore,IL-1β, IL-6, IL-8, IL-18, IFN-α, IFN-β, IL-10and lysozyme were induced. LPS from mutant E058AlpxP was similar to LPS from E058in activating cells, and it was significantly reduced ability for LPS of E058ΔlpxL and E058ΔlpxM to activating cells compared to that of E058, and descended lower of production for stimulating cells by LPS of E058ΔlpxL and E058ΔlpxM than that by LPS of E058. production of IFN-β was difference to other cytokine due to the absence MyD88-independence pathway in chicken. Production of IFN-P reduced sharply by HD11stimulating by LPS from E058or E058AlpxP than LPS from E058ΔlpxL and E058ΔlpxM after4h, and production of IFN-β was high by LPS from E058ΔlpxL and E058ΔlpxM than LPS from E058or E058ΔlpxP at6h. At8h, there were significantly differen. Expression of NLRC5was up by oscillation and reach its peak at6h after stimulating by LPS. Expression of NLRC5stimulating by LPS from E058AlpxL and E058AlpxM was lower than that stimulated by LPS from E058or E058AlpxP. Changes of NO was similar to other signal molecule and cytokine.Production of TLR4and NF-κB in spleen was high after stimulating by LPS, and production of NLRC5was no difference in different organisms, including heart, liver, spleen, lung and kidney. After stimulating, Production of TLR4, NF-κB and NLRC5was reduced by LPS from E058ΔlpxL and E058ΔlpxM than from E058or E058AlpxP. Apoptosis tested by caspase-3showed that apoptosis was severe stimulating by LPS from E058ΔlpxL and E058AlpxM than from E058or E058AlpxP.3. Evaluation of the immune efficacy of E058ΔlpxLΔlpxMIn this study, E058AlpxLAlpxM inactivated vaccine, attenuated vaccine were developed to evaluate the immune efficacy by ELISA. The results indicated that serum IgG specific to APEC E058, OMPs and LPS antigens could be stimulated in SPF chicken immunized with E058ΔlpxLΔlpxM inactivated vaccine and attenuated vaccine with high density. OD450of IgG from sera against against APEC E058, OMPs and LPS antigens were measured by indirect ELISA respectively. The protection efficacy of vaccinated chicken against challenge of APEC E058were88.9%and81.5%. The attenuated vaccine with low density did not provided the immune efficacy against E058.Stimulated index of apoptosis were significantly different in chicken periphery lymphocytes between stimulating by E058AlpxLAlpxM inactivated vaccine, attenuated vaccine and by PBS tested by MTT. |
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