The N-terminal domain of RTX toxin ApxI of Actinobacillus pleuropneumoniae elicits protective immunity in mice.
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ABSTRACT:
SUBMITTER: Seah JN
PROVIDER: S-EPMC130407 | biostudies-literature | 2002 Nov
REPOSITORIES: biostudies-literature
Publications
The N-terminal domain of RTX toxin ApxI of Actinobacillus pleuropneumoniae elicits protective immunity in mice.
Seah J N JN Frey J J Kwang J J
Infection and immunity 20021101 11
We expressed three Actinobacillus pleuropneumoniae ApxI deletion derivatives to map the domain that could induce protective immunity. Antiserum to ApxI N-terminal covered by residues 40 to 380 was found to neutralize ApxI hemolytic activity but not ApxIII cytotoxicity. When used as a subunit vaccine in mice, this recombinant N-terminal fragment elicited protection against lethal infection with heterologous A. pleuropneumoniae serovars. ...[more]
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Project description:Actinobacillus pleuropneumoniae-RTX-toxin I (ApxI), an important virulence factor, is secreted by serotypes 1, 5, 9, 10, and 11 of A. pleuropneumoniae. However, sequences homologous to the secretion genes apxIBD of the ApxI operon are present in all 12 serotypes except serotype 3. The purpose of this study was to determine and compare the structures of the ApxI operons of the 12 A. pleuropneumoniae serotypes. We focused on the nucleotide sequence comparison of the ApxI-coding genes, the structures of the ApxI operons, and the transcription of the ApxI operons. We determined the nucleotide sequences of the toxin-encoding apxICA genes of serotype 9 and found that the gene for the structural toxin, apxIA, was almost identical to the apxIA gene of serotype 1. The toxin-encoding genes of the other serotypes are also similar for the main part; nevertheless, two variants were identified, one in serotypes 1, 9, and 11 and one in serotypes 5 and 10. The two apxIA variants differ mainly within the distal 110 nucleotides. Structural analysis demonstrated that intact ApxI operons, consisting of the four contiguous genes apxICABD, are present in serotypes 1, 5, 9, 10, and 11. ApxI operons with a major deletion in the apxICA genes are present in serotypes 2, 4, 6, 7, 8, and 12. Serotype 3 does not contain ApxI operon sequences. We found that all ApxI operons are transcriptionally active despite the partial deletion of the operon in some serotypes. The implications of these data for the expression and secretion of ApxI and the other Apx-toxins, ApxII and ApxIII, as well as for the development of a subunit vaccine against A. pleuropneumoniae will be discussed.
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Project description:Actinobacillus pleuropneumoniae RTX-toxin III (ApxIII) is implicated as an important virulence factor of A. pleuropneumoniae, the causative agent of porcine pleuropneumonia. Recently, the genes coding for ApxIII (apxIIICA) of serotype 8 were cloned and characterized. The toxin appeared to be a member of the RTX-toxin family, as are the other two secreted toxins of A. pleuropneumoniae, i.e., ApxI and ApxII. In this report, we describe the cloning and sequencing of the remaining part of the ApxIII operon of serotype 8. This sequence coded for the RTX secretion proteins ApxIIIB and ApxIIID, which showed 86 and 63% similarity to ApxIB and ApxID, respectively, and 83 and 63% similarity to HlyB and HlyD of Escherichia coli, respectively. Potential functional domains, such as eight transmembrane regions and an ATP-binding cassette, were present in ApxIIIB. We examined the presence of apxIIICABD sequences in the 12 serotypes of A. pleuropneumoniae and found that these sequences were present only in serotypes 2, 3, 4, 6, and 8, the serotypes that secrete ApxIII. Comparison of the apxIIICABD gene sequences of the serotypes revealed very few serotype-specific differences. Only the C terminus of ApxIIIA of serotype 2 differed from ApxIIIA of the other serotypes. The differences were located between the glycine-rich repeats and the secretion signal. The analysis of the apxIIICABD genes completed our efforts to characterize the ApxI, ApxII, and ApxIII operons of the reference strains of the 12 serotypes of A. pleuropneumoniae. We present a complete map of the ApxI, ApxII, and ApxIII operons and discuss this in terms of gene expression and complementation and the role of the toxins in pathogenesis.
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Project description:RTX cytolysins are a family of calcium-dependent, pore-forming, secreted toxins found in a variety of gram-negative bacteria. The prototypical member of this family is the alpha-hemolysin of Escherichia coli. The RTX genetic determinants from seven members of the family Pasteurellaceae, Pasteurella haemolytica, Actinobacillus actinomycetemcomitans, and A. pleuropneumoniae serotypes 1,5,7, and 9 were previously cloned and sequenced. Using the leukotoxin determinant from P. haemolytica serotype A1 as a probe, we detected the presence of RTX-type determinants in Actinobacillus suis, A. equuli, and A. lignieresii of the family Pasteurellaceae. All three species elaborate proteins of approximately 104 to 110 kDa that are recognized by polyclonal antisera against the 104-kDa hemolysin of A. pleuropneumoniae serotype 1. An RTX determinant of A. suis isolate 3714 was cloned and sequenced and was found to be almost identical to the RTX determinant of A. pleuropneumoniae serotypes 5 and 9. In addition, the determinant is not composed of four contiguous genes, as had been reported for most other RTX determinants; instead, the genes encoding the two proteins responsible for secretion of the toxin are at a locus distinct from that containing the toxin structural and activation genes.
| S-EPMC257139 | biostudies-other
Project description:BackgroundActinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia and represents a major burden to the livestock industry. Virulence can largely be attributed to the secretion of a series of haemolytic toxins, which are highly immunogenic. A. pleuropneumoniae also encodes a cytoplasmic N-glycosylation system, which involves the modification of high molecular weight adhesins with glucose residues. Central to this process is the soluble N-glycosyl transferase, ngt, which is encoded in an operon with a subsequent glycosyl transferase, agt. Plasmid-borne recombinant expression of these genes in E. coli results in the production of a glucose polymer on peptides containing the appropriate acceptor sequon, NX(S/T). However to date, there is little evidence to suggest that such a glucose polymer is formed on its target peptides in A. pleuropneumoniae. Both the toxins and glycosylation system represent potential targets for the basis of a vaccine against A. pleuropneumoniae infection.ResultsIn this study, we developed cytoplasmic glycoengineering to construct glycoconjugate vaccine candidates composed of soluble toxin fragments modified by glucose. We transferred ngt and agt to the chromosome of Escherichia coli in order to generate a native-like operon for glycoengineering. A single chromosomal copy of ngt and agt resulted in the glucosylation of toxin fragments by a short glycan, rather than a polymer.ConclusionsA vaccine candidate that combines toxin fragment with a conserved glycan offers a novel approach to generating epitopes important for both colonisation and disease progression.
| S-EPMC6318927 | biostudies-literature