Genetic Engineering of Avian Pathogenic E. Coli to Study the Functions of Fimh Adhesin

Indian Journal of Experimental Biology Vol. 47, November 2009, pp. 916-920

Genetic engineering of avian pathogenic E. coli to study the functions of FimH adhesin

H H Musa1,2§, S F He1§, S L Wu1, C H Zhu1, Z H Liu1, Z N Zhang1, V S Raj3, R X Gu 4 & G Q Zhu1* 1College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China 2Faculty of Veterinary Science, University of Nyala, Nyala 155, Sudan 3Ranbaxy Laboratories Limited Research & Development Center, Gurgaon 122015, India 4Institute of Dairy Science and Technology, Yangzhou University, Yangzhou 225009, China Received 13 February 2009; revised 22 July 2009

Adhesion of pathogen to host cells is an important prerequisite for successful colonization and establishment of the pathogenesis. The aim of this study is to examine the function of FimH adhesin in the adherence of avian pathogenic E. coli to porcine intestinal epithelial cell lines (IPEC-J2) and human lung epithelial cell line (A549) in an in vitro infection model. Three strains of avian pathogenic Escherichia coli (APEC) and one strain of non-pathogenic E coli were used. The isogenic FimH mutants were constructed by λ Red-mediated recombination system. The wild types and mutants strains were adhered to the host cells with different adherence patterns in certain incubation time. The results demonstrated that the adherence of the isogenic FimH mutants to the porcine intestinal epithelial cells (IPEC-J2) were similar to those of wild types. However, the adherences of isogenic FimH mutants to human lung epithelial cells (A549) were significantly different from the wild types. A549 cell can be used as a type of cell model for colonization of the chicken extraintestinal. FimH offers a unique opportunity to investigate the role of the strength of adhesion independently from the many other factors that may affect surface colonization.

Keywords: Avian pathogenic E. coli, FimH, Host cell

Avian pathogenic Escherichia coli (APEC) are biological phenomena in nature. This phenomenon responsible for extraintestinal infections in chickens, underlies eukaryotic cell-cell or cell-surface turkeys and other avian species. The presence of attachment, initiates recognition and signaling events, several putative virulence genes has been positively binds bacteria to target cells and mediates biofilm linked to the pathogenicity of APEC strains1,2. They formation on medical implants8. Usually the adhesion include the aerobactin iron transport system, K1 is mediated by protein-protein, protein-lipid or capsule, P and type1 fimbriae3,4. The highly protein-saccharide interactions9. Adhesion has been pathogenic E. coli causing outbreaks at flock level widely demonstrated to often enable bacteria to carry possess virulence factors that are not found in out initial colonization of host mucosa and to commensal strains5. Type 1 fimbriae are characterized overcome nonspecific defense barriers10. A number of by their ability to bind D-mannose and thus bind to experimental models have been established to many types of eukaryotic cells including intestinal, evaluate the pathogenicity of E. coli for poultry. The lung, bladder, kidney epithelial and various model used was focused on the ability of isolates to inflammatory cells6. Binding is conferred by the colonize the host epithelial cell6. adhesin FimH, which is a minor component of the DNA engineering is conducted routinely in 2,7 fimbriae . Escherichia coli, not only for genetic studies in FimH consists of a fimbria-associated pilin domain bacteria, but also for constructing DNA molecules to and a mannose-binding lactin domain, receptor-ligand be used in studies of other organisms11. The DNA specific adhesion is among the most fundamental of double-strand break and repair recombination ______pathway is very efficient in yeast. Functions in this §These authors contributed equally to this work. pathway recombine transformed linear DNA with *Correspondent author homologous DNA in the yeast12. In contrast to yeast, Telephone: 0086-514-87972590 Fax: 0086-514-87311374 E. coli is not readily transformed by linear DNA, E-mails: [email protected]; [email protected] because of the presence of intracellular RecBCD MUSA et al.: AVIAN PATHOGENIC E. COLI & FUNCTIONS OF FimH ADHESIN 917

exonuclease that degrades linear DNA13. Inactivation and Wanner11. Briefly, P1 and P2 primers were used of host RecBCD exonuclease activity, either by to amplify FRT flanked antibiotic resistance cassettes mutation or production of the anti-RecBCD Gam of plasmids pKD3; the resulting PCR products were function, is required for efficient Red-promoted purified. Avian pathogenic E. coli strains containing recombination with linear dsDNA substrates14. The λ the plasmid pKD46, which carried the λ Red was used to knock-out gene in Salmonella recombination genes gam, bet, and exo were grown at enterica Serovar typhimurium15,16 and Salmonella 30°C until OD600 of 0.6. L-arabinose was then added enterica Serovar enteritidis17. In the present study to a final concentration of 0.2% and induced for 1h. isogenic FimH mutant was constructed by Red The competent cells were prepared and electroporated system, and the adherence properties of the isogenic with purified PCR products, 1ml of LB were added FimH mutants were compared with avian pathogenic and incubated for 1h at 37°C, cell were plated on LB and non-pathogenic E. coli isolates from different media with Cm at 37°C and incubated overnight19. ecological niches. PCR was carried out by P3 and P4 primers to confirm that FRT flanked Cm antibiotic resistance cassettes Materials and Methods (cat) were integrated in the correct region on the Bacterial strains and culture condition—Avian chromosome. Thereafter, PCR products were cloned pathogenic Escherichia coli (APEC) isolated from the into pGEM-T Easy vector and sequenced. To infected chicken belong to the serotypes O78:K89, eliminate the Cm cat, mutants containing a FRT Cm O1:K89 and O2:K89 and non-APEC strain of were transformed with the helper plasmid pCP20, and serotype O24:K89 from the healthy chicken were ampcillin resistance transformants were selected at selected as a prototype of Escherichia coli type 1 30°C, after which a few were colony-purified once fimbriae. The isogenic FimH mutants for these strains non-selectively at 42°C and then tested for loss of were constructed by λ Red-mediated recombination antibiotic resistances. system. Expressed FimH from all the wild types and Cell line and cell culture—Human lung epithelial non-expressed mutants were confirmed by the ability cell line (A549) and porcine intestinal epithelial cell of agglutination reaction with both guinea pig line (IPEC-J2) were grown in Dulbecco’s modified erythrocytes and yeast cells18. All strains were eagle medium (DMEM)/Ham’s F-12 (1:1) medium subjected to 48h cultures in LB broth at 37°C to allow supplemented with 10% calf sera and 1% non- a high level of expression of type 1 fimbriae. essential amino acids (Gibco). Cells were maintained

Construction of APEC mutant strains—Luria- in an atmosphere of 5% CO2 at 37°C, and passaged by Bertani (LB) media was used for growth of bacteria trypsinization every 3-4 days. and the recovery of transformants. Bacterial strains Bacterial adhesion—The A549 and IPEC-J2 cells were routinely grown at 37°C except for strains were seeded in 24 well tissue culture plates at containing the temperature sensitive plasmids, pCP20 concentration of 4×105 cells/well and grown for 20h. or pKD46, which were grown at 30°C. Primer P1 5'- Cell monolayers were washed three times with sterile ATG AAA CGA GTT ATT ACC CTG TTT GCT phosphate-buffered saline (PBS, pH 7.2). Each well GTA CTG CTG ATG GGC TGG TCG GTT GTG monolayer was infected at a multiplicity of infection TGA GCT GGA GCT GCT TCG-3' and primer P2 5'- (MOI) of 100 bacteria/epithelial cell in 1ml of cell TTA TTG ATA AAC AAA AGT CAC GCC AAT culture medium without antibiotic. After 1 or 4h of

AAT CGA TTG CAC ATT CCC TGC CAT ATG incubation at 37°C with 5% CO2, the infected AAT ATC CTC CTT AG-3' have internal overlap with monolayers were gently washed thrice with PBS to the chloramphenicol resistance cassette (Cm) and remove loosely adherent bacteria20. Cells were lysed external overlap with the FimH gene to be deleted. with 1ml of 1% triton x- 100 (Sigma) for 15min. Primers P3 5'-ATG AAA CGA GTT ATT ACC CTG Samples were serially diluted and plated on TT -3' and primer P4 5'-CGC CAA TAA TCG ATT MacKonkey agar plates to determine the number of GCA CAT T-3' were only flank the target FimH gene colony-forming units, the assay was performed in for PCR verification. Primers were synthesized by triplet20. Shanghai Gene Core Biotechnology Company. Data analysis—All values are presented as the Chromosomal mutations were created using the λ mean±SE, and the significant difference between wild Red recombination method as described by Datsenko type and FimH isogenic mutants of APEC in the 918 INDIAN J EXP BIOL, NOVEMBER 2009

adhesion to host cells was determined by Student’s t- been shown that the interaction between APEC and test using SAS 9.0 software. host cells (A549) can be used as a model for the natural colonization of the respiratory surface by the Results APEC and provide information about the response Construction of APEC mutant strains—The Red pathways involved. Since, the wild type strains of all system includes three genes of Gam, Bet and Exo: serotypes demonstrated significantly (P<0.05) a Gam inhabits the host RecBCD exonuclease so that Bet higher association with A549 cells than isogenic and Exo can gain access to DNA ends to promote FimH mutants did (Fig. 2). This is because the recombination. In the present study four isogenic FimH adherence of APEC in this in vitro model depends on the expression of type 1 fimbriae. mutants of APEC were constructed using the λ Red recombination system. A1.1kb of chloramphenicol Discussion resistance cassette was amplified from the pKD3 The construction of FimH mutant gene was plasmid as a template. Avian pathogenic E. coli strains performed by Red recombination system11. The containing the plasmid pKD46, which induced the λ technical advance of the system came from replacing recombination genes, were electroported with the the E coli RecBCD gene cluster with the red genes14. purified PCR product. The resulting colonies The genes gam, beta and exo expressed at moderate characterized by PCR were 1114bp with Cm insertion levels under control of the lac promoter from (△ fimH:: Cat) compared to 883bp of FimH. A multicopy plasmid21. Red system promoted temperature sensitive helper plasmid pCP20 was used recombination between the bacterial chromosome and to eliminate the antibiotic resistance cassette, the loss linear dsDNA molecules introduced into the cell via of Cm cat was confirmed by PCR and the mutant strains were re-named △ fimH Association of avian pathogenic E. coli with host cells—The adhesion of pathogenic bacteria to intestinal epithelial cells is an important prerequisite for their colonization and the establishment of the pathogenesis. In the present study colony forming units (CFU) counting assay was used to investigate the difference between the isogenic FimH mutant and wild type in adherence to porcine intestinal epithelial cell (IPEC-J2) and human lung epithelial cell line (A549). The results showed that the adhesion of all isogenic FimH mutants and wild types to host cells were increased with certain incubation time. The wild type of APEC serotype O78:K89 was significantly (P<0.05) associated with IPEC-J2 cell compared with FimH mutant. However, no significant in vitro adhesion differences were observed between the isogenic FimH mutants and wild-types avian pathogenic Escherichia coli on IPEC-J2 cell for serotypes O1:K89 and O2:K89 (Fig. 1). The results showed that FimH was not required for colonization of the intestinal porcine epithelial cell in vitro by E. coli; it may be concluded that FimH is not a major Fig. 1—Association of wild type and isogenic fimH mutant strains characterization of a porcine intestinal epithelial cell with IPEC-J2 cell [Avian pathogenic E. coli serotype O2:k89, line for in vitro studies of microbial pathogenesis in O78:k89 and O1:k89 and their isogenic fimH mutants ΔfimH swine, and that it is not a major determinant of were used at multiplicity of infection (MOI) of 100 bacteria per bacterial pathogenicity. The A549 cell is the human cell, infection was performed for 1 and 4h, and the adhesion levels were expressed as the number of colony-forming units (CFU) per lung epithelial cell line; in the present study it has well] MUSA et al.: AVIAN PATHOGENIC E. COLI & FUNCTIONS OF FimH ADHESIN 919

epithelial cells24. In the present study the wild type strains demonstrated significantly a higher association with A549 cells than isogenic FimH mutants for all strains studied. Similarly, Mellata et. al22 indicated that the wild type APEC strain demonstrated a high association with phagocytes (macrophage and heterophils) than FimH mutant strain. Because FimH mutant strains were less able to survive in the lungs of chickens than the parent strains25, and unable to adhere in vitro to chicken epithelial pharyngeal or tracheal cells18. FimH through specific ligand-receptor interactions can trigger various responses in cells involved in host defenses such as macrophage, mastocytes, lymphocytes B and neutrophils22. FimH mediates weak adhesion at low flow but strong adhesion at high flow. There is evidence that this occurs because FimH forms catch Fig. 2—Association of wild type and isogenic fimH mutant strains bonds, defined as bonds that are strengthened by tensile with A549 cell [Avian pathogenic E. coli serotype O2:k89, mechanical force26. The physiological advantage to the O78:k89 and O1:k89 and non-pathogenic E. coli serotype weak adhesion demonstrated by commensal variants of O24:k89 and their isogenic fimH mutants ΔfimH were used at FimH may be to allow rapid surface colonization27. multiplicity of infection (MOI) of 100 bacteria per cell, infection 28 was performed for 1 and 4h, and the adhesion levels were Duncan et. al indicated that the distinct binding expressed as the number of colony-forming units (CFU) per well] specificities of different enterobacterial type 1 fimbriae cannot be ascribed solely to the primary structure of electroporation, the system had been used to replace their respective FimH subunits, but are also modulated gene in Salmonella, enteropathogenic E. coli and 21 by the fimbrial shaft on which each FimH subunit is entrohemorrahagic E. coli . Thus, we have succeeded presented, possibly through conformational constraints to knockout FimH adhesin gene from three avian imposed on FimH by the fimbrial shaft 28. pathogenic E. coli and one non-pathogenic E. coli, It is clear in the present study that FimH plays and this will help to study the function of FimH similar biological roles for commensal bacteria because adhesin and his interaction with host cell. they also have to colonize specific niches and APEC strains adhere to chicken epithelial cells of overcome the host's natural clearing mechanisms22,29. the pharynx and trachea by means of the type 1 Schierack et. al23 indicated that the commensal E. coli fimbriae, comprising a major structural subunit, FimA isolates had a broad spectrum of adhesion to IPEC-J2, and a minor subunit, FimH that mediates the 22 ranging from 0 to more than 100 bacteria per cell due attachment to D-mannose residues . The IPEC-J2 to difference in the FimH genes. It is thought that cell line is a non-transformed intestinal cell line commensal and some pathogenic Escherichia coli originally derived from jejunal epithelia isolated from strains use type 1 pili or curli to colonize human and a neonatal, is suitable as host for a number of 29 23 animal tissues . Commensal bacteria block adhesion pathogenic and non-pathogenic bacteria . In the of pathogens by competition for common receptors and present study no significant in vitro adhesion aid in maintaining the physiological balance of the differences were observed between the isogenic FimH intestinal tract30. It can be concluded that FimH offers a mutants and wild-types avian pathogenic Escherichia unique opportunity to investigate the role of the coli on IPEC-J2 cell. The non- significant difference strength of adhesion independently from the many of adherence between wild type and mutants may be other factors that may affect surface colonization. due to that type 1 fimbriae have mainly involved in the upper respiratory colonization7 instead of Acknowledgement intestinal in the present study. The adhesion patterns This study was supported by grants from the Chinese and lysis of human and porcine epithelial cells by National Science Foundation Grant (No. 30571374 and pathogenic E. coli varied broadly depending on the No. 30771603), China Postdoctoral Science Foundation origin of the bacteria and species origin of the Grant (No. 20070420201), the Chinese National Science 920 INDIAN J EXP BIOL, NOVEMBER 2009

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