Extraintestinal Pathogenic Escherichia Coli:Screening Immunogenic Proteins And The Mechanism Of Lysozyme Resistance

Escherichia coli(E.coli)is Gram-negative bacteria exhibiting considerable physiological and metabolic versatility.Based on the infection localization and the characteristics of pathogenicity,E.coli has been grouped into commensal Escherichia coli,Intestinal pathogenic Escherichia coli(IPEC)and Extraintestinal pathogenic Escherichia coli(ExPEC).ExPEC are part of normal intestinal microflora and usually asymptomatically colonize the gut.Once they get access to infection niches outside of the gut,ExPEC are,however,able to efficiently colonize these niches and cause diseases in host.There are three well described ExPEC:Avian pathogenic Escherichia coli(APEC),Neonatal meningitis-associated Escherichia coli(NMEC)and Uropathogenic Escherichia coli(UPEC).APEC causes systemic infections of internal organs in poultry,leading to a diversity of symptoms.Human ExPEC cause diseases including urinary tract infection(UTI),septicemia,and neonatal meningitis in newborns.China is No.1 in the world in chicken and egg proudction.Because of intensive poultry farming,outbreaks of APEC infection have frequently been reported and resulted in significant economic losses.APEC is also zoonotic potential,which is evidenced by sharing similar serotypes,phylogenetic backgrounds,virulence factors,and genomic islands with human ExPEC and confirmed by animal studies using rat model of neonatal meningitis and chicken model.However,the prevention and control of both human and animal ExPEC infection are currently still problematic.To develop effective control strategy against ExPEC infection,new candidate immunogens of APEC have been screened by using immunoproteomic analyses and further characterized in this study.In addition,molecular mechanisms of ExPEC's resistance to lysozyme have been investigated in order to better understand the ExPEC's resistance to host innate immunity.1.Pathogenicity asessment of avian pathogenic Escherichia coli clinical isolatesThirty-two APEC strains,selected from 467 APEC strains collection isolated in 2007,were assessed for their phylogenetic background and virulence genotypes by PCR and the pathogenicity and ability to cross blood brain barrier(BBB)by rat model of neonatal meningitis.Results showed that twenty-six APEC strains with different serotypes caused the mortalities ranging from 0%to 100%,were able to be reisolated from CSF,suggesting the zoonotic potential of APEC.Phylogenetic analysis showed that all tested strains belonged to phylogenetic group B2 could cross BBB and 4 of them caused nearly 100%mortalities,indicating phylogenetic group B2 had a closer relationship with the occurrence of meningitis and pathogenicity.Except for hlyA,all the virulence associated genes were present in the tested strains.In addition,GimB and neuC genes were only found in the APEC strains causing meningitis in the rat model,suggesting their role in crossing BBB.2.Screening new candidate immunogens of avian pathogenic Escherichia coli and evaluation of immune response and protective effectThe diversity of serotype and low cross protection between heterologous strains in E.coli have hampered the development of vaccine.To identify immunogenic proteins of APEC as vaccine candidates,immunoproteomics and matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS)were applied.Proteins from total cell lysates of APEC DE205B isolated from the brain of a duck with septicemia and neurological symptom in China were separated by two-dimensional electrophoresis(2-DE)and reacted with hyperimmune duck serum against DE205B.Fourteen immunoreactive spots were identified,representing 11 distinct proteins.These included two predominant immunogenic components,outer membrane protein A(OmpA)and flagellin(FliC).GroEL,which is a member of the molecular chaperone family and structurally identical to eukaryotic heat shock protein 60(Hsp60),and eight other antigens are for the first time reported here as immunoreactive proteins of APEC.Subsequently,nine genes encoding the identified proteins were successfully cloned and expressed in E.coli BL21(DE3).Seven of the recombinant proteins were able to react with hyperimmune duck serum and three of them,GroEL,OmpA and FliC,showed stronger immunoreactivity.Challenge studies revealed that,just like OmpA and FliC,recombinant GroEL stimulated a strong antibody response and supported protective efficacy against APEC infection in ducks.With high phylogenetic conservation,it is considered that GroEL would be an ideal immunogen of APEC for vaccine development.3.Screening the genes associated with lysozyme resistance in neonatal meningitis-associated Escherichia coli and mutants constructionNeonatal meningitis-associated Escherichia coli(NMEC),belonged to Extraintestinal pathogenic Escherichia coli(ExPEC),is the most common cause of Gram-negative neonatal meningitis.To cause diseases,NMEC have to evade the defense systems encountered in the host and survive in the blood.One of the most important compounds of the host innate defense system is lysozyme,which is widely distributed in body fluids and in a variety of cells.Although several lysozyme inhibitors have been discovered in E.coli recently,the mechanism to evade lysozyme-mediated killing in E.coli is still not fully understood.Here we describe the construction of a PUT mini-Tn5 Km mutant library to identify the critical genes related to the lysozyme resistance.A total of 15000 transposon insertion mutants were screened,among which 25 mutants,presenting 20 genes,were identified as candidates for further characterization.14 genes encode the enzymes involved in biosynthesis,including polysaccharides;1 encodes transporters and regulator respectively and 4 encode putative proteins with unknown function.Interestingly,6 genes,including the gene identified in the mutant strain with the highest lysozyme sensitivity,are located in the gene cluster from galF to gnd.In order to determine the roles of these gene,corresponding mutants were constructed by ?,red recombination system.Compared to the wild type,all the mutants significantly reduced lysozyme resistance.Meanwhile,the membrane permeability of mutants has been slightly increased.4.LPS of neonatal meningitis-associated Escherichia coli inhibit lysozyme activityLipopolysaccharide,with various biological activities,is the major components of the outer surface of Gram-negative bacteria.In previous study,among the 20 genes related to lysozyme resistance,6 genes were located in the gene cluster from galF to gnd,which are related to the synthesis of O-specific polysaccharide of LPS in E.coli.T o determine the roles of these genes,the LPS were extracted from wild type strain NMEC38,mutants and complemented strains.SDS-PAGE showed that LPS from wild type had many O-antigen chains giving a characteristic "laddered" appearance on SDS-PAGE gel after electrophoresis,while LPS profiles of mutants showed defects in the O-antigen region,and western blotting further showed that the LPS from mutants is trucated because it lost the O-specific polysaccharide.These results confirmed that 6 genes contribute to the synthesis of O-specific polysaccharide of LPS.The results of lysozyme inhibition and gel filtration chromatograph assays confirmed that the purified LPS from wild type of NMEC38 could bind to the lysozyme,thus inhibited the bacterial killing and enzymatic hydrolytic activity of lysozyme.Using purified O-SP of the LPS further confirmed that only O-SP could inhibit the hydrolytic activity of lysozyme,although both the Lipid A and O-SP could bind to lysozyme.These results suggested that O-SP of LPS confers the lysozyme resistance to NMEC.