Lipopolysaccharides of Actinobacillus pleuropneumoniae bind pig hemoglobin

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 ␣ and ␤ 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.

Reverse transcriptase polymerase chain reaction (RT‐PCR) detection of HLP gene expression in Candida glabrata and its possible role in in vitro haemolysin production

“…As there is essentially no free iron in the human host, most pathogens acquire this indirectly from commonly available iron-containing compounds such as haemoglobin (17). Many are therefore equipped with a mechanism that destroys the heme moiety and extracts the elemental iron from the mammalian host.…”

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

“…We and others demonstrated that A. pleuropneumoniae can utilize pig Hb as a sole source of iron for growth in vitro [5,6]. We recently reported the binding of pig Hb to A. pleuropneumoniae LPS [5,7] and hypothesized that LPS might perform a`docking' function. We also suspect that surface proteins are then involved in the iron uptake from pig Hb.…”

“…Several bacterial components, including RTX toxins, lipopolysaccharides (LPS), capsular polysaccharides, outer membrane proteins (OMPs), and proteases appear to contribute to the disease process [4]. We and others demonstrated that A. pleuropneumoniae can utilize pig Hb as a sole source of iron for growth in vitro [5,6]. We recently reported the binding of pig Hb to A. pleuropneumoniae LPS [5,7] and hypothesized that LPS might perform a`docking' function.…”

“…Western blot assays using radioiodinated-Hb [13] or radioiodinatedheme:hemopexin [14] and spectrophotometrical method [15] were also used to determine the Hb-binding activity. We have previously visualized the binding of pig Hb to a limited number of cells of A. pleuropneumoniae using immunogold labeling and electron microscopy [5]. To the best of our knowledge, no study has used £ow cytometry to evaluate the expression of Gram-negative bacterial surface molecules with Hb-binding activity.…”

Expression of Actinobacillus pleuropneumonia gene coding for Apx I protein in Escherichia coli

“…Detailed studies of the clinical symptoms of the disease and its characteristic lung lesions, its experimental induction in pigs with viable and sonicated A. pleuropneumoniae, and the endobronchial inoculation of Apx toxins exist [4,5]. The virulence of A. pleuropneumoniae may be considered multifactorial, as is the case with most pathogenic bacteria; the factors involved in pathogenesis include capsular polysaccharides [6], lipopolysaccharides [7], membrane proteins [8,9], adhesion factors [10], exotoxins [5], and urease [11]. Epidemiological data suggest, however, that virulence is strongly correlated with the presence of Apx toxins [4,17].…”