Outer membrane protein U (OmpU) mediates adhesion of Vibrio mimicus to host cells via two novel N-terminal motifs.
Vibrio mimicus (V.mimicus) is a causative agent of ascites disease in aquatic animals. Our previous studies have demonstrated that the outer membrane protein U (OmpU) from V.mimicus is an immunoprotective antigen with six immunodominant linear B-cell epitopes. Although the N-terminus of OmpU contains potential binding motifs, it remained unclear whether OmpU possesses adhesion function. Here, the adhesive capacity of recombinant OmpU and V.mimicus to epithelioma papulosum cyprinid (EPC) cells was determined by immunofluorescence and adherence assay. The results showed that after co-incubated with rOmpU, an obvious visible green fluorescence could be observed on the EPC cell surface and the nuclei exhibited blue fluorescence; while the control cell surface did not show any signal, only nuclei exhibited blue fluorescence. The average number of wild-type strain adhered to each cell was 32.3 ± 4.5. The average adhesion number of OmpU gene deletion mutant was significantly reduced to 10.8 ± 0.5 (P < 0.01) and restored to 31.3 ± 2.8 by complement strain (P >0.05). Pretreatment of cells with rOmpU reduced the average adhesion number of wild-type strain to 9.7 ± 2.9 (P < 0.01). Likewise, binding was significantly decreased to 8.8 ± 3.2 (P < 0.01) due to blocking role of OmpU antibodies. To determine binding motifs of OmpU, six immunodominant B-cell epitope peptides labeled with FITC were employed in flow cytometry-based binding assay. Two FITC-labeled epitope peptides (aa90-101 and aa173-192) showed strong binding to EPC cells (the fluorescence positive cell rate was 99 ± 0.6% and 98 ± 0.3%, respectively), which could be specifically competed by excess corresponding unlabeled peptides, whereas the remaining four showed a low level of background binding. This is the first demonstration that OmpU possesses adhesion function and its N terminal 90-101 and 173-192 amino acid regions are critical sites for cell surface binding.
Key Laboratory of Zoonoses, College of Animal Science and Technology, Anhui Agricultural University, Hefei, P. R. China.
Xueqin
X
Liu
Key Laboratory of Zoonoses, College of Animal Science and Technology, Anhui Agricultural University, Hefei, P. R. China.
Huihui
H
Gao
Key Laboratory of Zoonoses, College of Animal Science and Technology, Anhui Agricultural University, Hefei, P. R. China.
Nin
N
Xiao
Key Laboratory of Zoonoses, College of Animal Science and Technology, Anhui Agricultural University, Hefei, P. R. China.
Yan
Y
Liu
Key Laboratory of Zoonoses, College of Animal Science and Technology, Anhui Agricultural University, Hefei, P. R. China.
Jinnian
J
Li
Key Laboratory of Zoonoses, College of Animal Science and Technology, Anhui Agricultural University, Hefei, P. R. China.
Lin
L
Li
D018829:Adhesins, Bacterial / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D000907:Antibodies, Bacterial / Q000378:metabolism; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001422:Bacterial Adhesion; D001665:Binding Sites; D002460:Cell Line; D018985:Epitopes, B-Lymphocyte / Q000378:metabolism; D005399:Fishes / Q000276:immunology / Q000382:microbiology*; D044164:Vibrio mimicus / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*
D018829:Adhesins, Bacterial; D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D018985:Epitopes, B-Lymphocyte