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

Abstract

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 ironrestriction 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 ironrestricted growth conditions which suggests that one or more of these outer membrane proteins may play a role in the Hbbinding activity detected by flow cytometry. z

Acquisition of haemoglobin-bound iron by Histophilus somni

“…multocida (Bosch et al, 2002;Cox et al, 2003), A.pleuropneumoniae (Archambault et al, 1999(Archambault et al, , 2003Srikumar et al, 2004) and Actinobacillus actinomycetemcomitans (Hayashida et al, 2002) and the genes encoding the TonB-dependent Hb-binding proteins responsible for this binding and utilization have been identified. Interestingly, some members of the Neisseriaceae and Pasteurellaceae, inc1uding H. influenzae Cope et al, 2000), some strains of N. meningitidis (Lewis and Dyer, 1995;Stoji1jkovic etaI., 1996;Lewis etaI., 1999;Perkins-Balding etaI., 2004) andP.…”

“…In H. influenzae and the neisseriae, phase variation involves CCAA repeats and polyguanine tracts in the structural genes, respectively Richardson and Stojiljkovic, 1999 pleuropneumoniae (Srikumar et al, 2004). In A. pleuropneumoniae (Bélanger et al, 1995;Archambault et al, 1999) andA. actinomycetemcomitans (Grenier et al, 1997), the binding of Rb to its receptor might aIso be facilitated by lipopolysaccharides (LPS).…”

Identification of genes involved in biosynthesis of Actinobacillus pleuropneumoniae serotype 1 O-antigen and biological properties of rough mutants

“…The absence of Ochains in the rough LPS mutants did not allow a significant increase of Hb-binding to surface proteins and to the corelipid A region of LPS that are both thought to play a role in Hb-binding. 6,27 We also evaluated the biological activity of LPS of the mutants compared to the LPS of the wild-type strain of A. pleuropneumoniaein a chromogenic LAL test. No significant differences between the biological activity of any of the mutants and the parent strain were observed.…”

“…Overnight cultures of A. pleuropneumoniae were washed and resuspended in PBS to an OD of 0.2 at 540 nm, equivalent to approximately 10 8 CFU/ml. Bacterial suspensions (1 ml) were incubated with FLUOS-labeled pig hemoglobin (Hb; 1 mg/ml) 27 for 60 min at room temperature. Cells were washed twice in PBS and fixed with 2% paraformaldehyde.…”

“…Cells were maintained in the dark at 4°C until analyzed by flow cytometry as previously described. 10,27 Flow cytometry results were expressed as a percentage of gated fluorescent events, which is the percentage of fluorescent cells out of 10,000 cells. The hemoglobinbinding assay was performed with samples in duplicate.…”