Molecular cloning and characterization of a hemolysin gene from Actinobacillus (Haemophilus) pleuropneumoniae.

This article describes the molecular cloning and expression of a hemolysin gene from a serotype 1 strain of Actinobacillus pleuropneumoniae. The hemolysin was a thermolabile protein with an apparent molecular weight of 29,500 (29.5K hemolysin). Unlike expression of the recently described 105K hemolysin of A. pleuropneumoniae (J. Frey and J. Nicolet, FEMS Microbiol. Lett. 55:41-46, 1988), expression of this hemolysin was not regulated by Ca2+. Antiserum prepared against the 105K hemolysin did not neutralize the activity of the 29.5K hemolysin; conversely, antiserum prepared against the 29.5K hemolysin did not neutralize the activity of the 105K hemolysin. The hemolytic activity was not neutralized with antisera against hemolytic Escherichia coli, Streptococcus agalactiae, or purified streptolysin O, but antisera prepared against recombinants containing the 29.5K gene and convalescent pig sera abrogated hemolytic activity. Although hemolytic activity could be detected in several strains of E. coli K-12 and in minicells expressing several different constructs encoding the 29.5K hemolysin, we could not rigorously exclude the possibility that the gene which we have isolated encodes a regulator of hemolytic activity rather than a hemolysin per se.     

Analysis of major antigens of Haemophilus (Actinobacillus) pleuropneumoniae and related organisms.

Outer membrane protein (OMP)-enriched extracts and whole-cell protein preparations of Haemophilus (Actinobacillus) pleuropneumoniae and related organisms were examined by polyacrylamide gel electrophoresis and immunoblotting. Both the OMP-enriched and whole-cell protein profiles of Actinobacillus suis, A. pleuropneumoniae (NAD-independent biovar), A. lignieresii, and Pasteurella haemolytica were very similar to those of H. pleuropneumoniae serotypes 1 to 8. Antisera prepared against H. pleuropneumoniae typically recognized three major OMP antigens with approximate molecular weights of 17,000 (17K), 32K, and 42K in immunoblots of H. pleuropneumoniae serotypes 1 to 8, Actinobacillus spp., and P. haemolytica. Antisera prepared against Actinobacillus spp. and Haemophilus sp. "minor group" also recognized these 17K, 32K, and 42K antigens. Using absorbed sera, we demonstrated that the 17K antigen had an epitope (or epitopes) common to all the gram-negative organisms examined, including Escherichia coli. The 32K and 42K antigens had epitopes common to members of the family Pasteurellaceae but, in the case of the 32K antigen, also contained unique epitopes. These results provide a basis for understanding the lack of specificity of serodiagnostic tests for H. pleuropneumoniae infection and provide another line of evidence for the association of H. pleuropneumoniae with the genus Actinobacillus.     

Cross-reactions between Actinobacillus (Haemophilus) pleuropneumoniae strains of serotypes 1 and 9.

Actinobacillus pleuropneumoniae strains of serotypes 1 and 9 were studied for their serological properties by means of agglutination, coagglutination (CoA), indirect hemagglutination (IHA), Co-IHA, ring precipitation (RP), and immunodiffusion (ID) tests. Particulate and soluble antigens of unheated and heat-treated bacterial cells were used in various serological tests. Agglutination, CoA, and RP tests demonstrated common antigens between strains of serotypes 1 and 9. Quantitative estimation of serotype-specific antigenic activity by CoA, RP, and ID tests proved useful in differentiating strains of serotypes 1 and 9. IHA and Co-IHA tests using sheep erythrocytes sensitized with unheated or heat-treated whole-cell saline extract and the ID test using boiled whole-cell saline extract as antigen distinguished the strains of serotypes 1 and 9. In studies of absorption of rabbit antisera with heterologous whole-cell antigens there was no absorption of antibodies in tube agglutination and IHA tests, suggesting that serotype 1 and 9 strains belong to two distinct serogroups. It appears that the cross-reactivity between serotype 1 and 9 strains could be due to common epitopes associated with cell wall antigens.     

Effects of Actinobacillus pleuropneumoniae hemolysin on porcine neutrophil function.

In an attempt to gain insight into the events that take place during Actinobacillus pleuropneumoniae infection, the present study was designed to ascertain the effects of bacterial toxicity on porcine neutrophil functions and viability. Incubation of phagocytes (2 x 10(6)) with opsonized A. pleuropneumoniae 4074 (2 x 10(7) CFU) resulted in phagocytic uptake of less than or equal to 4%. At the same bacterium-to-phagocyte ratio, levels of lactate dehydrogenase activity of 74 and 81% were detected in the extracellular medium after 1.5 and 3 h of incubation, respectively. Furthermore, the ingested bacteria were not killed by the phagocytes. These effects were ascribed to hemolysin produced by the bacteria, because the presence of hemolysin-neutralizing antibody prevented overt cellular damage, significantly increased phagocytic uptake (P less than 0.001), and resulted in an approximately 10-fold decrease in the number of CFU of the ingested bacteria. Cytolytic doses of isolated hemolysin caused dose-related loss of cell viability, diminished bactericidal activity of toxin-treated phagocytes for Escherichia coli, and decreased the ability of the phagocytes to undergo a respiratory burst upon stimulation with phorbol myristic acetate. In contrast, sublytic doses of the hemolysin activated the phagocytes and caused them to respond to phorbol myristic acetate with increased generation of superoxide anion. Because heated (100 degrees C, 5 min) hemolysin preparations did not produce similar effects, we contend that the observed effects were not due to contaminating endotoxin. The data presented herein indicate that A. pleuropneumoniae hemolysin is a potent antiphagocytic virulence factor by virtue of its leukocidal activity. Sublytic doses of the toxin may have important effects on the oxidative metabolism of phagocytic cells.     

Nucleotide sequence of the hemolysin I gene from Actinobacillus pleuropneumoniae.

The DNA sequence of the gene encoding the structural protein of hemolysin I (HlyI) of Actinobacillus pleuropneumoniae serotype 1 strain 4074 was analyzed. The nucleotide sequence shows a 3,072-bp reading frame encoding a protein of 1,023 amino acids with a calculated molecular size of 110.1 kDa. This corresponds to the HlyI protein, which has an apparent molecular size on sodium dodecyl sulfate gels of 105 kDa. The structure of the protein derived from the DNA sequence shows three hydrophobic regions in the N-terminal part of the protein, 13 glycine-rich domains in the second half of the protein, and a hydrophilic C-terminal area, all of which are typical of the cytotoxins of the RTX (repeats in the structural toxin) toxin family. The derived amino acid sequence of HlyI shows 42% homology with the hemolysin of A. pleuropneumoniae serotype 5, 41% homology with the leukotoxin of Pasteurella haemolytica, and 56% homology with the Escherichia coli alpha-hemolysin. The 13 glycine-rich repeats and three hydrophobic areas of the HlyI sequence show more similarity to the E. coli alpha-hemolysin than to either the A. pleuropneumoniae serotype 5 hemolysin or the leukotoxin (while the last two are more similar to each other). Two types of RTX hemolysins therefore seem to be present in A. pleuropneumoniae, one (HlyI) resembling the alpha-hemolysin and a second more closely related to the leukotoxin. Ca(2+)-binding experiments using HlyI and recombinant A. pleuropneumoniae prohemolysin (HlyIA) that was produced in E. coli shows that HlyI binds 45Ca2+, probably because of the 13 glycine-rich repeated domains. Activation of the prohemolysin is not required for Ca2+ binding.     

Identification of a maltose-inducible major outer membrane protein in Actinobacillus (Haemophilus) pleuropneumoniae

The addition of maltose to the growth media of Actinobacillus pleuropneumonia (serotype 1) resulted in the induction of an outer membrane protein (OMP) with a molecular mass of 42 kDa. This protein had porin-like properties in that it was peptidoglycan-associated and was resistant to proteolysis by trypsin. A pleuropneumoniae expressing the 42 kDa OMP were unable to bind lambda phage. Similar proteins were also induced in A. pleuropneumoniae isolates representing serotypes 2 to 7 with the exception of serotype 4; however, not all isolates of any given serotype expressed a maltose-inducible OMP. Western immunoblotting using convalescent antiserae against the serotype 1 A. pleuropneumoniae indicated that the 42 kDa OMP was expressed in vivo and was cross-reactive with the maltose-inducible OMPs from other serotypes.     

Effect of iron restriction on the outer membrane proteins of Actinobacillus (Haemophilus) pleuropneumoniae.

The outer membrane protein profile of Actinobacillus (Haemophilus) pleuropneumoniae grown under iron-restricted and iron-replete conditions was studied by polyacrylamide gel electrophoresis and immunoblotting. A virulent serotype 1 isolate synthesized a novel protein with an apparent molecular weight of 105,000 (105K) and increased the synthesis of a 76K protein under iron-restricted conditions. Both proteins were synthesized within 15 min of establishment of iron-restricted conditions. Proteins of equivalent molecular weights could also be induced by iron restriction in serotype 2, 3, 4, 5, and 7 isolates of A. pleuropneumoniae. Convalescent-phase sera from serotype 1-infected pigs contained antibodies which recognized both the 105K and 76K proteins from all six serotypes examined, indicating that these proteins were expressed in vivo and were immunologically conserved. Cells expressing the 105K and 76K proteins also displayed an enhanced ability to bind Congo red and hemin, suggesting that one or both of these proteins functioned to acquire complexed iron during in vivo growth.     

Virulence properties and protective efficacy of the capsular polymer of Haemophilus (Actinobacillus) pleuropneumoniae serotype 5.

The role of the capsule of Haemophilus (Actinobacillus) pleuropneumoniae serotype 5 in bacterial virulence, and the protective efficacy of antibody to serotype 5 capsule was investigated. Encapsulated H. pleuropneumoniae serotype 5 were resistant to killing by complement and antibody to capsule or somatic antigens, whereas a noncapsulated mutant was sensitive to killing by the alternative complement pathway alone. Antiserum to whole H. pleuropneumoniae serotype 5 bacteria or monospecific antiserum to capsule was capable of opsonizing bacteria of the homologous serotype for phagocytosis by swine polymorphonuclear leukocytes but was not opsonic for a heterologous serotype. An immunoglobulin M monoclonal antibody to the serotype 5 capsule was not opsonic for any serotype. Mice were protected against lethal, intranasal challenge with the homologous or heterologous serotype after immunization with live encapsulated or noncapsulated bacteria, but not after immunization with killed bacteria, lipopolysaccharide, or a capsule-protein conjugate vaccine. The protection induced by immunization with live bacteria was transferred to nonimmune, syngeneic mice by serum but not by spleen cells. Nonimmune pigs passively immunized with monospecific swine serum to capsule were protected from lethal infection but not from development of hemorrhagic lung lesions, whereas pigs passively immunized with swine antiserum to live bacteria did not develop severe respiratory lesions. Thus, the capsule of H. pleuropneumoniae serotype 5 was inhibitory to the bactericidal activity of serum and was antiphagocytic. Antibody to the capsule was opsonic but was not fully protective.     

Efficacy of a cell extract from Actinobacillus (Haemophilus) pleuropneumoniae serotype 1 against disease in swine.

We partially characterized a cell extract (CE) from Actinobacillus pleuropneumoniae serotype 1 and used the CE to test the efficacy of secreted proteins against disease. Secreted products from 4-h culture supernatants were precipitated with 20% polyethylene glycol. Analysis of the CE indicated the presence of protein, endotoxin, and carbohydrate. Hemolytic activity to bovine erythrocytes and cytotoxic activity to porcine mononuclear leukocytes was also demonstrated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the CE from a 4-h culture showed a major band at 110 kilodaltons (kDa), while a CE of a 26-h culture indicated the presence of a number of additional proteins, including the 110-kDa protein. The 110-kDa protein was also identified as a glycoprotein by periodic acid-Schiff and silver staining. A single band precipitated against convalescent-phase pig antiserum when the polyethylene glycol precipitate was used in an Ouchterlony plate. Vaccination with CE conferred greater protection against challenge with the homologous serotype than either a commercial bacterin or an outer membrane protein vaccine. Hemolysin-neutralizing titers were higher both pre- and postchallenge in the group vaccinated with the CE compared with in all other groups. We believe that this demonstrates the importance of secreted factors in protection against disease and suggests that the 110-kDa protein is an important immunogen.     

Identification of a second hemolysin (HlyII) in Actinobacillus pleuropneumoniae serotype 1 and expression of the gene in Escherichia coli.

Hemolysin genes of the reference strains of Actinobacillus pleuropneumoniae serotypes 1 and 2 were identified, cloned, and expressed in Escherichia coli by using polymerase chain reaction amplification with oligonucleotides derived from the DNA sequence of the corresponding appA gene from A. pleuropneumoniae serotype 5. The three genes from serotypes 1, 2, and 5 have identical restriction maps and appear to encode a hemolysin which was previously identified in serotype 2 and designated HlyII. Gene appA is different from hlyIA encoding the major hemolysin type I (HlyI) which was identified earlier in serotype 1. Polymerase chain reaction amplification with oligonucleotides derived from the DNA sequence of hlyIA of serotype 1 showed that the gene encoding HlyI is present in serotype 1 but not in serotype 2, in contrast to the gene encoding HlyII that was present in both serotypes. This was confirmed by Western blot (immunoblot) experiments using monoclonal antibodies specific for either recombinant HlyI or recombinant HlyII, which showed that A. pleuropneumoniae serotype 1 strain 4074 produces both HlyI and HlyII, whereas serotype 2 strain S1536 produces only HlyII. The expression of both hemolysins was investigated in all serotypes by the use of monoclonal antibodies. HlyI was shown to be expressed by the reference strains of serotypes 1, 5a, 5b, 9, 10, and 11, whereas HlyII was shown to be expressed by the reference strains of all 12 serotypes tested except serotype 10. A. pleuropneumoniae serotype 1 strain 4074 is the first bacterium which has been shown to contain two different actively expressed RTX toxin genes. Comparison of our data with those from other groups shows that the originally described strongly hemolytic hemolysin type I (HlyI) corresponds to cytolysin I (ClyI) which was recently described by others, while the weakly hemolytic hemolysin type II (HlyII) seems to be identical to ClyII and AppA.     

Prevalence of seroreactors to the 104-kilodalton hemolysin of Actinobacillus pleuropneumoniae in swine herds.

A rabbit homologous polyclonal antiserum to the 104-kilodalton hemolysin of Actinobacillus pleuropneumoniae serotype 1 strain CM-5 was specifically produced and used in an antigen capture enzyme-linked immunosorbent assay (ELISA) to detect swine serum antibodies to this potentially important virulence factor. Sera from pigs experimentally infected with the most common disease-producing serotypes (serotypes 1, 2, 5, and 7) of A. pleuropneumoniae produced positive results in this ELISA. Of 144 serum samples collected from 10 herds free of pleuropneumonia and 155 serum samples from 11 herds with a history of the disease, 68 (47%) and 148 (95%), respectively, were found positive by the ELISA. In addition, pigs naturally infected with Actinobacillus suis produced antibodies which seroreacted in this ELISA. The results indicated that a high proportion of swine have antibodies seroreactive with the 104-kilodalton hemolysin produced by A. pleuropneumoniae.     

Chemical differences in lipopolysaccharides from Actinobacillus (Haemophilus) actinomycetemcomitans and Haemophilus aphrophilus: clues to differences in periodontopathogenic potential and taxonomic distinction.

While Actinobacillus actinomycetemcomitans has been associated with rapidly progressive periodontal destruction in man, the closely related Haemophilus aphrophilus has not been related to periodontal disease. This may be due to differences in composition and structure of the lipopolysaccharides (LPS) of these dental-plaque bacteria, since LPS probably exerts a series of detrimental effects on the periodontium. LPS was prepared by the phenol-water procedure from the type strains of A. actinomycetemcomitans and H. aphrophilus, purified by hexane extraction and ultracentrifugation, and analyzed with gas chromatography and gas chromatography-mass spectrometry. While the lipid content of LPS from A. actinomycetemcomitans constituted 35.4%, it was only 18.4% in H. aphrophilus: 3-hydroxytetradecanoic and tetradecanoic acids were 21.1 and 14.3% in A. actinomycetemcomitans and 10.9 and 7.5% in H. aphrophilus. There were qualitative and quantitative differences in the polysaccharide portions of their LPS. A actinomycetemcomitans contained both D-glycero-D-mannoheptose and L-glycero-D-mannoheptose (7.8 and 11.3%); H. aphrophilus contained only L-glycero-D-mannoheptose (17.4%). The rhamnose, fucose, galactose, glucose, and glucosamine/galactosamine contents in A. actinomycetemcomitans were 2.6, 5.2, 10.1, 22.4, and 5.2%, respectively; in H. aphrophilus, they were 2.1, 2.6, 19.4, 36.4, and 3.7%. Chemical differences in LPS from A. actinomycetemcomitans and H. aphrophilus may contribute to the divergence in periodontopathogenic potential of these organisms and help taxonomic differentiation.     

Studies of phospholipase C (heat-labile hemolysin) in Pseudomonas aeruginosa.

The chromogenic substrate p-nitrophenylphosphorylcholine was used to measure phospholipase C (heat-labile hemolysin) production by clinical strains of Pseudomonas aeruginosa. Analysis of strains from various types of infection showed that phospholipase C production by urinary tract isolates was significantly greater than that by lung, blood, or other isolates (e.g., wounds, etc.). The above method was also found to be effective for the isolation of several classes of phospholipase C mutants.     

Functional analysis of the Ca(2+)-regulated hemolysin I operon of Actinobacillus pleuropneumoniae serotype 1.

The genetic determinant encoding the synthesis and secretion of hemolysin I (HlyI; gene designation, hlyI) by Actinobacillus pleuropneumoniae serotype 1 4074T was cloned in the lambda vector EMBL4. A 10.2-kb fragment that encoded hemolytic activity in the phage lysate was aligned by Southern blot hybridization to genes hlyC, hlyA, hlyB, and hlyD of the Escherichia coli hemolysin operon, and expression of the A. pleuropneumoniae genes in E. coli revealed that they have the same functions as their E. coli analogs: hlyIC encodes a protein that activates inactive 105-kDa prohemolysin I (encoded by hlyIA) to active hemolysin I, while hlyIB and hlyID are necessary for HlyIA secretion. Northern (RNA) hybridization of A. pleuropneumoniae RNA revealed that the gene cluster is transcribed as two RNA species, a major one of 3.5 kb, corresponding to hlyICA, and a second, minor one of 7.5 kb, corresponding to the whole operon, hlyICABD. The level of hlyI mRNA was substantially higher in A. pleuropneumoniae 4074T cells grown in the presence of Ca2+, supporting the view that the expression of the hlyI determinant is Ca2+ regulated. Parallel RNA hybridization with random gene probes suggested that this Ca2+ regulation is specific for the hlyI determinant.     

Haemophilus ducreyi hemolysin acts as a contact cytotoxin and damages human foreskin fibroblasts in cell culture.

Haemophilus ducreyi, which causes the sexually transmitted disease chancroid, produces several factors that damage human cells. We used isogenic mutants of H. ducreyi 35000 to demonstrate that the hemolytic activity and the cytotoxic effect of H. ducreyi on human foreskin fibroblasts are due to the same toxin.     

Indirect enzyme-linked immunosorbent assay for detection of antibody to a 110,000-molecular-weight hemolysin of Actinobacillus pleuropneumoniae.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect swine antibody to a 110,000-molecular-weight hemolysin (110K hemolysin) of Actinobacillus pleuropneumoniae. Affinity-purified rabbit polyclonal or mouse monoclonal immunoglobulin G to the hemolysin of A. pleuropneumoniae serotype 5 strain J45, followed by hemolysin-rich concentrated culture supernatant, was used to bind swine antibody to hemolysin to microdilution plates. Sixty-nine serum samples from swine that were clinically normal, presented with clinical evidence of pleuropneumonia, were experimentally immunized or challenged, or were free of pleuropneumonia were tested, and their ELISA titers were compared with complement fixation (CF) titers. On the basis of serum samples from swine that were clinically normal and negative by CF, an ELISA titer of 1:320 or greater was considered positive. In comparison with CF, the sensitivity of the ELISA was 98.1% and the specificity was 90%. The two samples negative by CF and positive by indirect ELISA were, however, also positive for antibody to serotype 5 capsule by ELISA. Immunization of normal pigs with whole cells or purified hemolysin boosted titers 4- to 128-fold within 4 weeks. Immunoblotting demonstrated that the affinity-purified immunoglobulin G to hemolysin used for capture in the assay recognized only a 110K protein of A. pleuropneumoniae serotypes 1 to 7, although the reactivity was quantitatively variable between serotypes. Therefore, the indirect ELISA is capable of identifying animals infected with or exposed to most, if not all, serotypes of A. pleuropneumoniae. If an indirect ELISA titer of 1:320 or greater is considered positive, the assay can be a valuable diagnostic tool in both clinical and research laboratories.     

Hemolysin patterns of Actinobacillus pleuropneumoniae.

The secreted hemolytic activities produced by the reference strains and field isolates of the 12 serotypes and 2 subtypes of Actinobacillus pleuropneumoniae were analyzed. Serotype 1 produced a Ca2(+)-inducible hemolysin, which was previously characterized as a 105-kilodalton protein and was named hemolysin I (HlyI). Serotypes 2, 4, 6, 7, and 8 produced a different hemolytic activity that was not inducible by Ca2+ but required this ion for its activity. The hemolytic activity produced by these serotypes was much weaker than that found in serotype 1 and was not neutralized by rabbit antibodies against HlyI. It was, however, neutralized by serum from pigs that were experimentally infected with a serotype 2 strain and was called hemolysin II (HlyII). Serotypes 5a, 5b, 9, 10, and 11 produced both HlyI and HlyII. In these strains, HlyI was the major contributor to the hemolytic activity. The remaining serotypes, 3 and 12, produced a very weak hemolytic activity, which was not further analyzed. Immunoblot analysis of the culture supernatants from all 12 serotypes with rabbit polyclonal antibodies directed against HlyI revealed reactions with a protein in the 105-kilodalton size range for all serotypes, indicating that HlyI and HlyII might be serologically related. Strains producing active HlyI seem to belong to serotypes that are generally considered to be virulent types and that are frequently isolated from pigs in severe pleuropneumonia outbreaks.     

Humoral antibody response and protective immunity in swine following immunization with the 104-kilodalton hemolysin of Actinobacillus pleuropneumoniae.

Five cesarean-derived, colostrum-deprived pigs were given three adjuvant-supplemented subcutaneous and one intravenous injection of the purified 104-kDa hemolysin from serotype 1 Actinobacillus pleuropneumoniae CM-5. Six control animals received phosphate-buffered saline only. Five of six control pigs died within 24 h after challenge. The sixth control pig was moribund and euthanized after 48 h. All six pigs had pleuropneumonia, and A. pleuropneumoniae was isolated from all six lungs. None of the vaccinated pigs died as a result of challenge. After being euthanized, two pigs in this group had no lung lesions but three had chronic pleuropneumonia involving 10, 20, and 40% of the lung tissue. A. pleuropneumoniae was isolated from lung lesions of these three animals but not from the two pigs without lesions. The prechallenge hemolysin-neutralizing antibody titers in the vaccinated pigs were 1:10,900, 1:10,600, 1:4,800, 1:3,900, and 1:3,000, in order of increasing lung involvement. None of the control pigs had neutralizing antibodies. Enzyme-linked immunosorbent assay (ELISA) antibodies to capsule, lipopolysaccharide, and hemolysin were not detected in serum samples collected from the control pigs. In the vaccinated group, prechallenge sera did not contain ELISA antibodies to capsule or lipopolysaccharide. ELISA antibodies to the hemolysin were detected only in the prechallenge and postchallenge serum samples. These results indicate that pigs immunized with the 104-kDa hemolysin of serotype 1 A. pleuropneumoniae are protected against challenge with virulent bacteria. The association between neutralizing antibodies and protection indicates indirectly that the hemolysin is an important virulence factor.