Mar 3, 1992 - J. DEVENISH,1* J. EDWARD BROWN,1 AND S. ROSENDAL2 ..... Lalonde, G., T. V. McDonald, P. Gardner, and P. D. O'Hanley. 1989.
INFEcrION AND IMMUNITY, May 1992, p. 2139-2142 0019-9567/92/052139-04$02.00/0 Copyright © 1992, American Society for Microbiology
Vol. 60, No. 5
Association of the RTX Proteins of Actinobacillus pleuropneumoniae with Hemolytic, CAMP, and Neutrophil-Cytotoxic Activities J. DEVENISH,1* J. EDWARD BROWN,1 AND S. ROSENDAL2 Toaxinology Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland 21702-5011,1 and Department of Veterinary Microbiology and Immunology, University of Guelph, Guelph, Ontario, Canada NIG 2W1I Received 6 January 1992/Accepted 3 March 1992 The immunoglobulin G from a monospecific rabbit antiserum to the 11O-kDa RTX hemolysin of Actinobacilus pleuropneumoniae serotype 1 was used to determine that the related RTX proteins in isolates from serotypes 2 to 12 were also responsible for the hemolytic, CAMP, and neutrophil-cytotoxic activities produced by this bacterium. These proteins share common neutralizing epitopes.
Actinobacilluspleuropneumoniae causes fibrinous pleuropneumonia of swine, a disease that has had severe economic impact on pork producers worldwide (27, 37). The characteristic vascular permeability which leads to hemorrhage and fibrin exudation in diseased animals was thought to be caused by a toxin(s) other than endotoxin released by A. pleuropneumoniae during colonization (1, 25). Several different toxins have been reported elsewhere, including a heat-stable hemolysin (23, 28), a protein causing hemolysis of sensitized sheep erythrocytes (CAMP activity) (16), a neutrophil cytotoxin (19, 20, 35), a pleurotoxin responsible for macrophage cytotoxicity but not hemolysis (36), and a heat-labile protein hemolysin of approximately 110 kDa, produced by serotype 1 strains of A. pleuropneumoniae (4, 9, 14, 24), that requires calcium for biological activity (5). Recent genetic and immunological research has placed this 110-kDa protein among the RTX (repeats in the toxin domain) group of bacterial cytolysins (2, 8, 15, 17). However, there are 12 serotypes of A. pleuropneumoniae, and all are capable of producing the same characteristic pathological condition in infected animals (30, 33), suggesting that all produce the same or very similar virulence factors, such as a common cytolysin. We and others have established by electrophoresis and immunoblot analysis that A. pleuropneumoniae serotypes 2 to 12 produce, in vitro and in vivo, proteins of approximately 110 kDa which cross-react immunologically with the 110-kDa RTX hemolysin of serotype 1 (8, 15). These immunologically related proteins may be the common cytolytic virulence factor of A. pleuropneumoniae and may be responsible for the observed hemolytic, CAMP, and neutrophil-cytotoxic activities of this bacterium. The purpose of this research was to determine whether monospecific antibody against the purified 110-kDa hemolysin of serotype 1 (4, 8) could inactivate the hemolytic, CAMP, and neutrophil-cytotoxic activities observed for all 12 serotypes of A. pleuropneumoniae. The strains of A. pleuropneumoniae used in this study have been described previously (8, 35). Starting with an A615 of 0.025, all strains were cultured in brain heart infusion broth (0.02% NAD and 2.5 mM CaCl2) at 37°C with shaking until theA61. reached 0.7. The cultures were centrifuged, the *
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supernatants were filtered, and the pH was adjusted to 7.3. The supernatants were tested for hemolytic and neutrophilcytotoxic activities as described previously (4, 8, 35). The CAMP assay was modified from the procedure of Phillips et al. (31). Briefly, a 1% sheep erythrocyte suspension was exposed to 1 hemolytic unit of the 3-toxin of Staphylococcus aureus Peoria per ml in the presence of 2 mM MgCl2 for 30 min at 37°C. These sensitized erythrocytes were washed once and used as in the hemolysin assay to measure CAMP activity. Streptococcus agalactiae was cultured in brain heart infusion broth (1% maltose), and the filtered supernatant was used as the CAMP factor control. The immunoglobulin G (IgG) fraction was purified from a monospecific rabbit antiserum prepared to the purified 110kDa hemolysin of A. pleuropneumoniae serotype 1 (strain CM-5) (4, 8) by using a protein G affinity column (Pharmacia LKB Biotechnology Inc., Piscataway, N.J.). For neutralization testing, IgG (400 to 750 ,ug) was added to diluted culture supernatants for 40 min at 37°C. When indicated, 400 ,ul of a 20% heat-killed S. aureus Cowan 1 (S. aureus C) suspension (18) was added for 30 min at 220C. After centrifugation, 1 ml of each supernatant solution was tested for hemolytic, CAMP, and neutrophil-cytotoxic activities. Negative controls used the same concentrations of nonimmune rabbit IgG (Cedarlane Laboratories Ltd., Hornby, Ontario, Canada). A. pleuropneumoniae serotype 1, 5, 7, 9, 10, and 11 strains were hemolytic. Serotype 1 isolates produced the highest levels of hemolytic activity, and the serotype 7 strain produced the lowest levels (Table 1). A >90% reduction in the hemolytic activity of all six isolates was observed when the hemolysin-specific IgG was used (Table 2). In contrast, supematants from all serotypes except serotype 6 lysed sensitized erythrocytes in the CAMP assay (Table 1). Serotype 1 strains produced the highest level of CAMP activity, and those of serotypes 3, 8, and 12 produced the lowest. More than 85% of the CAMP activity in serotypes 1, 2, 5, 7, 9, 10, 11, and 12 was reduced when the hemolysin-specific IgG was used. However, only 29, 53, and 61% inactivation of CAMP activity was observed, respectively, in serotypes 3, 4, and 8 by using this IgG fraction (Table 2). S. agalactiae produced hemolysis only with sensitized erythrocytes (Table 1), and this CAMP response was not inactivated with the hemolysin-specific IgG (Table 2). All 12 serotypes of A. pleuropneumoniae caused lactate dehydrogenase (LDH) re2139
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INFECT. IMMUN.
TABLE 1. Hemolytic and CAMP activities in culture supernatants of A. pleuropneumoniae serotypes 1 to 12 and S. agalactiae Bacterial culture supernatant
Serotype
A. pleuropneumoniae CM-5 Shope 4074 VLS 322 S1421 M62 MG131 FEM0 WF83 405 CVJ13261 B2209 56153 1096
1 1 2 3 4 5 6 7 8 9 10 11 12
S. agalactiae
TABLE 3. LDH release from neutrophils exposed to culture
supernatants of A. pleuropneumoniae serotypes 1 to 12 pretreated with nonimmune IgG, hemolysin-specific IgG, or hemolysinspecific IgG and 20% S. aureus C
Activity (HU/ml)a Hemolytic CAMP
940 409
