To confirm that the produced SGC-CBP30 in vitro antibody is specific and able to recognize not only the ON-01910 manufacturer fusion protein AatAF but also the native wild-type protein AatA, total protein extract of the strain BL21(pET32a:aatAF) prior and after induction of the IPTG-inducible promoter as well as the purified fusion protein AatAF and total protein extracts of strains IMT5155, APEC_O1, CFT073 and MG1655 were separated on an SDS gel and transferred to a polyvinylidene fluoride membrane. As shown in Figure 6 incubation with anti-AatA indeed led to the detection of protein bands of the expected size for AatAF in the total extract of BL21(pET32a:aatAF) and wild-type

AatA protein in APEC strains IMT5155 and APEC_O1, respectively. As expected, no signal was observed for CFT073

and MG1655, which have no aatA homolog in their genomes. Taken together our data show that AatA is suitable for the production of specific antibodies. Furthermore, this antibody recognizes wild-type AatA protein, demonstrating that APEC strains IMT5155 and APEC_O1 express a protein of the expected size, thus the gene in their genomes is likely to encode a functional adhesin. Surprisingly, no band of the expected size for AatA was detectable in strain BL21, which might be due to several BIIB057 cost reasons, including the lower transcription of the gene in this strain probably due to the presence of the different promoter Anacetrapib region as compared to the APEC_O1 and IMT5155 aatA promoter regions. Figure 6 Expression of AatA in different E. coli strains. The purified fusion protein (lane 1) and total protein extract of BL21(pET32a:aatAF) (lanes 2 and 3), expressing AatAF under the control of the IPTG-inducible promoter, AAEC189(pUC18:aatA +P) expressing aatA under the control of the native promoter and AAEC189(pUC18) (lanes 4 and 5), APEC_O1 (lane 6), IMT5155 (lane7), CFT073 (lane 8) and MG1655 (lane 9) were separated on an SDS gel and blotted to polyvinylidene fluoride membrane. The membrane was then incubated

with anti-AatA antibody. Expression of AatA in the fim negative E. coli strain AAEC189 leads to enhanced adhesion abilities Based on sequence analyses it was assumed that also the chromosomal aatA variant encodes a protein with adhesive function. To verify this, adhesion assays were performed using the chicken embryo fibroblast cell line DF-1. For this, aatA was expressed under control of its native promoter in E. coli strain AAEC189. AAEC189 is an MG1655 strain in which the fim operon is deleted leading to a reduced adhesion in in vitro assays [20]. AAEC189(pUC18:aatA +P) and the control strain AAEC189(pUC18) were incubated with DF-1 cells for 3 h. As shown in Figure 7A, the aatA containing strain displayed a 1.9 fold increase in adherence as compared to the adhesion of the negative control (P = 0.009). This suggests that AatA mediates adhesion of E. coli cells to chicken cells.