Identification and characterization of CAMP cohemolysin as a potential virulence factor of Riemerella anatipestifer

Abstract

Riemerella anatipestifer is responsible for exudative septicemia in ducks. The genetic determinant of the CAMP cohemolysin, cam, from a strain of R. anatipestifer was cloned and expressed in Escherichia coli. Chromosomal DNA from serotype 19 strain 30/90 was used to construct a gene library in pBluescript II SK(-) vector in E. coli XL-1-Blue strain. The clones containing recombinant plasmids were screened for the CAMP reaction with Staphylococcus aureus. Those that showed cohemolysis were chosen for further analysis by sequencing. One of these clones, JFRA8, was subcloned to identify the smallest possible DNA fragment containing the CAMP cohemolysin determinant, which was located on a 3,566-bp BamHI-BstXI fragment which specified a 1,026-bp open reading frame. Clones containing recombinant plasmids carrying cam obtained by PCR cloning into E. coli M15 strain secreted an active CAMP cohemolysin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses confirmed that the recombinant strain expressed a protein with a molecular mass of 37 kDa and that strains from serotypes 1, 2, 3, 5, 6, and 19 expressed the cohemolysin. The deduced amino acid sequence showed high homology to those of O-sialoglycoprotein endopeptidases. Hydrolysis of radioiodinated glycophorin A confirmed that Cam is a sialoglycoprotease.

FIG. 1.

Restriction enzyme map of recombinant plasmid pJFRA8 and the subclone derivatives. Only restriction sites relevant for cloning are given. Regions between arrowheads specify ORFs. Expression (+) and absence (−) of CAMP cohemolytic activity are indicated.

FIG. 2.

CAMP cohemolysis of recombinant E. coli XL1-Blue containing pHMT2A. Vertical streak, S. aureus; horizontal streaks: 1 and 2, XL1-Blue strains harboring recombinant plasmid pHMT2; 3 and 4, XL1-Blue strains harboring recombinant plasmid pHMT2A; 5, XL1-Blue strain harboring recombinant plasmid pJFRA8 (positive control); 6, competent XL1-Blue (negative control); 7, R. anatipestifer serotype 19 30/90 strain; 8, XL1-Blue with pBluescript II SK(−) vector (negative control).

FIG. 3.

Sequence alignment of the product of the cam gene of R. anatipestifer (RANAT) and sialoglycoprotein endopeptidases of H. influenzae (HAEIN), Haemophilus ducreyi (HAEDU), P. multocida (PASMU), and P. haemolytica (PASHA). Alignment was performed by the ClustalW program. Potential zinc-binding sites (♦) are indicated. Asterisks, identical or conserved residues; colons, conserved substitutions; single dots, semiconserved substitutions; dashes, gaps introduced to maximize alignment. Potential zinc-binding sites are shown in bold face.

FIG. 4.

Overexpression and purification of His-Cam fusion protein. Cell lysates were processed by SDS-PAGE and stained with Coomassie blue. Purified His-Cam was obtained by affinity chromatography with Ni-NTA resin. Lanes: M, protein marker; 1, noninduced pQE30 vector; 2, induced pQE30 vector; 3, supernatant of noninduced sonicated cell lysates; 4, Supernatant of induced sonicated cell lysates; 5, purified His-Cam protein.

FIG. 5.

Detection of the cam gene from genomic DNA of serotypes of R. anatipestifer. Lanes: M, 1-kb marker; 1, E. coli HMT2A (positive control); 2, R. anatipestifer ATCC 11845; 3, serotype 1 (S1) 35/90; 4, S1 105/91; 5, S1 179/9; 6, S1 205/90; 7, S1 1795; 8, S2 17/91; 9, S2 2527; 10, S3 2554; 11, S4 2565; 12, S5 2550; 13, S6 389/82; 14, S7 27179; 15, S7 203/89; 16, S8 26220; 17, S9 1785; 18, S10 2/91; 19, S10 232/89; 20, S11 25055; 21, S11 76/91; 22, S11 84/91; 23, S13 134/90; 24, S13 25012; 25, S14 664/83; 26, S15 743/85; 27, S15 34/90; 28, S15 135/90; 29, S15 110/89; 30, S15 204/88; 31, S16 4801; 32, S17 977/83; 33, S18 540/86; 34, S19 30/90; 35, S19 53/91; 36, S19 59/91. Arrow, cam gene of 1,026 bp.

FIG. 6.

Immunodetection of cohemolysin in total-cell lysates of R. anatipestifer strains. Bands correspond to the Cam protein at 37 kDa. Lanes: M, marker; C, purified Cam from pHMTC1 as a positive control; 1, R. anatipestifer ATCC 11845; 2, serotype 1 (S1) 35/90; 3, S1 105/91; 4, S1 179/9; 5, S1 205/90; 6, S1 1795; 7, S2 17/91; 8, S2 2527; 9, S3 2554; 10, S4 2565; 11, S5 2550; 12, S6 389/82; 13, S7 27179; 14, S7 203/89; 15, S8 26220; 16, S9 1785; 17, S10 2/91; 18, S10 232/89; 19, S11 25055; 20, S11 76/91; 21, S11 84/91; 22, S13 134/90; 23, S13 25012; 24, S14 664/83; 25, S15 743/85; 26, S15 34/90; 27, S15 135/90; 28, S15 110/89; 29, S15 204/88; 30, S16 4801; 31, S17 977/83; 32, S18 540/86; 33, S19 30/90; 34, S19 53/91; 35, S19 59/91.

FIG. 7.

Autoradiograph of SDS-PAGE gel showing hydrolysis of ¹²⁵I-GPA. Left lane, 1.75 μg of ¹²⁵I-GPA; right lane, GPA incubated with 0.2 mg of Cam.