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Lipopolysaccharides of Actinobacillus pleuropneumoniae bind pig hemoglobin
M Bélanger1, C Bégin, M Jacques
1Département de pathologie et microbiologie, Faculté de Médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, Canada.
Abstract:
A previous study indicated that lipopolysaccharides (LPS) extracted from Actinobacillus pleuropneumoniae bind two low-molecular-mass proteins, of approximately 10 and 11 kDa, present in porcine respiratory tract secretions (M. Bélanger, D. Dubreuil, and M. Jacques, Infect. Immun. 62:868-873, 1994). In the present study, we determined the N-terminal amino acid sequences of these two proteins, which revealed high homology with the alpha and beta chains of pig hemoglobin. Some isolates of A. pleuropneumoniae were able to use hemoglobin from various animal species as well as other heme compounds as sole sources of iron for growth, while other isolates were unable to use them. Immunoelectron microscopy showed binding of pig hemoglobin at the surface of all A. pleuropneumoniae isolates as well as labeling of outer membrane blebs. We observed, using Western blotting (immunoblotting), that the lipid A-core region of LPS of all isolates was binding pig hemoglobin. Furthermore, lipid A obtained after acid hydrolysis of LPS extracted from A. pleuropneumoniae was able to bind pig hemoglobin and this binding was completely abolished by preincubation of lipid A with polymyxin B but was not inhibited by preincubation with glucosamines. Fatty acids constituting the lipid A of A. pleuropneumoniae, namely, dodecanoic acid, tetradecanoic acid, 3-hydroxytetradecanoic acid, hexadecanoic acid, and octadecanoic acid, were also binding pig hemoglobin. Our results indicate that LPS of all A. pleuropneumoniae isolates tested bind pig hemoglobin and that lipid A is involved in this binding. Our results also indicate that some A. pleuropneumoniae isolates are, in addition, able to use hemoglobin for growth. Binding of hemoglobin to LPS might represent an important means by which A. pleuropneumoniae acquires iron in vivo from hemoglobin released from erythrocytes lysed by the action of its hemolysins.
Insights
Actinobacillus pleuropneumoniae lipopolysaccharides bind pig hemoglobin, with lipid A being crucial for this interaction. Some bacterial strains can also utilize hemoglobin as an iron source for growth.
Area of Science:
- Microbiology
- Bacterial Pathogenesis
- Molecular Biology
Background:
- Lipopolysaccharides (LPS) from Actinobacillus pleuropneumoniae bind specific proteins in porcine respiratory secretions.
- Previous research identified 10 and 11 kDa proteins interacting with LPS.
Purpose of the Study:
- To identify the proteins bound by Actinobacillus pleuropneumoniae LPS.
- To investigate the role of LPS and its components in hemoglobin binding and iron acquisition by A. pleuropneumoniae.
Main Methods:
- N-terminal amino acid sequencing of LPS-binding proteins.
- Growth studies using hemoglobin and heme compounds as sole iron sources.
- Immunoelectron microscopy and Western blotting (immunoblotting) to detect hemoglobin binding.
- Analysis of lipid A and its fatty acid components for hemoglobin binding.
Main Results:
- The identified proteins showed homology to pig hemoglobin alpha and beta chains.
- All tested A. pleuropneumoniae isolates bound pig hemoglobin to their LPS, particularly the lipid A region.
- Lipid A and its constituent fatty acids directly bind hemoglobin, a process inhibited by polymyxin B.
- Some A. pleuropneumoniae isolates demonstrated the ability to use hemoglobin as an iron source for growth.
Conclusions:
- Lipopolysaccharides of A. pleuropneumoniae bind pig hemoglobin, with lipid A playing a key role in this interaction.
- Hemoglobin binding to LPS may facilitate iron acquisition for A. pleuropneumoniae in vivo, especially following erythrocyte lysis by hemolysins.
- Differential utilization of hemoglobin as an iron source exists among A. pleuropneumoniae isolates.