Evaluation of the hemoglobin-binding activity of Actinobacillus pleuropneumoniae using fluorescein-labeled pig hemoglobin and flow cytometry

• Published in: FEMS microbiology letters 1999-04, Vol.173 (1), p.17-25
• Main Authors: Archambault, M, Rioux, S, Jacques, M
• Format: Article
• Language: English
• Subjects: Actinobacillus Infections - microbiology; Actinobacillus Infections - veterinary; Actinobacillus pleuropneumoniae; Actinobacillus pleuropneumoniae - genetics; Actinobacillus pleuropneumoniae - growth & development; Actinobacillus pleuropneumoniae - metabolism; animal diseases; animal health; Animals; bacterial infections; Bacteriology; Binding; Biological and medical sciences; Blotting, Western; Capsular polysaccharides; Cell surface; Culture Media; Electrophoresis; Electrophoresis, Polyacrylamide Gel; Evaluation; Flow cytometry; Flow Cytometry - methods; Fluorescein; Fluorescein - metabolism; Fundamental and applied biological sciences. Psychology; Gel electrophoresis; Growth conditions; Hemoglobin; Hemoglobins - metabolism; Iron; Iron - metabolism; Lipopolysaccharides; Membrane proteins; Membranes; Microbiology; Outer membrane proteins; Pathogenicity, virulence, toxins, bacteriocins, pyrogens, host-bacteria relations, miscellaneous strains; Pig hemoglobin; Polyacrylamide; Polysaccharides; Proteins; Receptors; Saccharides; Serotypes; Serotyping; Swine
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1111/j.1574-6968.1999.tb13479.x

In the present study, the hemoglobin (Hb)-binding activity of Actinobacillus pleuropneumoniae was examined using fluorescein-labeled pig Hb and flow cytometry. Comparison of the Hb-binding activity of A. pleuropneumoniae serotype 1 strain 4074 grown under iron-restricted conditions with cells grown under iron-sufficient conditions indicated that iron-restriction in A. pleuropneumoniae promotes the expression of Hb receptors, and that Hb-binding activity is, at least in part, iron-repressible. Hb-binding activity was also observed in representative strains of A. pleuropneumoniae belonging to serotypes 1 and 2. In addition, A. pleuropneumoniae serotype 1 LPS or capsule isogenic mutants were tested in flow cytometry in order to understand the influence of surface polysaccharides on Hb-binding activity. Experiments with an acapsulated mutant indicated that surface molecules with Hb-binding activity are more exposed at the cell surface in the absence of capsular polysaccharides. However, the Hb-binding activity of LPS mutants analyzed in this study was unchanged compared to the parent strain. The outer membrane proteins profile of A. pleuropneumoniae serotype 1 grown under iron-restricted or iron-sufficient conditions was also evaluated by polyacrylamide gel electrophoresis. Iron-regulated outer membrane proteins were observed under iron-restricted growth conditions which suggests that one or more of these outer membrane proteins may play a role in the Hb-binding activity detected by flow cytometry.