Antigenic homology of the inducible ferric citrate receptor (FecA) of coliform bacteria isolated from herds with naturally occurring bovine intramammary infections

Abstract

Expression of ferric citrate receptor FecA by Escherichia coli and Klebsiella pneumoniae isolated from bovine mastitis was investigated. Transformant E. coli UT5600/pSV66, which produces large quantities of FecA in the presence of citrate, was constructed. The FecA of E. coli UT5600/pSV66 was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and used to prepare polyclonal antiserum in rabbits. All coliform isolates of E. coli (n = 18) and K. pneumoniae (n = 17) from naturally occurring bovine intramammary infections in five herds induced iron-regulated outer membrane proteins when grown in Trypticase soy broth containing 200 microM alpha-alpha'-dipyridyl and 1 mM citrate. Polyclonal antiserum against FecA was used in conjunction with an immunoblot technique to determine the degree of antigenic homology of FecA among isolates. In the presence of citrate, each isolate expressed FecA that reacted with the anti-FecA polyclonal antiserum. The molecular mass of FecA ( approximately 80.5 kDa) was also highly conserved among isolates. Therefore, the ferric citrate iron transport may be induced in coliform bacteria and utilized to acquire iron in milk for survival and growth. The FecA is an attractive vaccine component for controlling coliform mastitis during the lactation period.

FIG. 1

Outer membrane protein profiles of E. coli UT5600 (A) and UT5600/pSV66 (B) separated by SDS-PAGE and stained with Coomassie blue. The amount of protein used per lane was 20 μg. Bacteria were grown in TSB (lane 1) or TSB plus 200 μM α-α′-dipyridyl and 1 mM citrate (lane 2). The left lane (M) contains molecular mass (10³) standards. The approximate position of ferric citrate receptor FecA is indicated.

FIG. 2

Immunoblots of separated outer membrane proteins of E. coli UT5600/pSV66 (lanes 1 and 3) and purified FepA reacted with preimmunization (PRE) and postimmunization (POST) rabbit anti-FecA sera. Both sera were diluted 1:2,000 (vol/vol). E. coli UT5600/pSV66 cells were grown in TSB (lane 1) or TSB plus 200 μM α-α′-dipyridyl and 1 mM citrate (lane 3). The left lane (M) contains molecular mass (10³) standards. The approximate position of ferric citrate receptor FecA is indicated.

FIG. 3

Typical outer membrane protein profiles of E. coli and K. pneumoniae isolates separated by SDS-PAGE and stained with Coomassie blue. The amount of protein used per lane was 20 μg. Bacteria were grown in TSB (lane 1) or TSB plus 200 μM α-α′-dipyridyl and 1 mM citrate (lane 2). The left lane (M) contains molecular mass (10³) standards. (A) E. coli 17. (B) E. coli 414. (C) E. coli 471. (D) K. pneumoniae 531. (E) K. pneumoniae 564. (F) K. pneumoniae 32. The approximate position of FecA is indicated.

FIG. 4

Immunoblots of the separated IROMPs of typical isolates of E. coli and K. pneumoniae reacted with the rabbit anti-FecA serum. The amount of protein used per lane was 20 μg. Bacteria were grown in TSB (lane 1) or TSB plus 200 μM α-α′-dipyridyl and 1 mM citrate (lane 2). Rabbit anti-FecA serum was diluted 1:2,000 (vol/vol). (A) E. coli 17. (B) E. coli 414. (C) E. coli 471. (D) K. pneumoniae 531. (E) K. pneumoniae 564. (F) K. pneumoniae 32. The approximate position of FecA is indicated.