Characterization of apxlVA, a new RTX determinant of Actinobacillus pleuropneumoniae

Abstract

A fourth type of RTX determinant was identified in Actinobacillus pleuropneumoniae and was designated apxlVA. When expressed in Escherichia coli, recombinant ApxlVA showed a weak haemolytic activity and cohaemolytic synergy with the sphingomyelinase (beta-toxin) of Staphylococcus aureus. These activities required the presence of an additional gene, ORF1, that is located immediately upstream of apxlVA. The apxlVA gene product could not be detected in A. pleuropneumoniae cultures grown under various conditions in vitro; however, pigs experimentally infected with A. pleuropneumoniae serotypes 1,5 and 7 started to produce antibodies that reacted with recombinant ApxlVA 14 d post-infection, indicating that apxlVA is expressed in vivo. In addition, sera from pigs naturally and experimentally infected with any of the serotypes all reacted with recombinant ApxlVA. The apxlVA gene from the serotype 1 A. pleuropneumoniae type strain Shope 4074T encodes a protein with a predicted molecular mass of 202 kDa which has typical features of RTX proteins including hydrophobic domains in the Nterminal half and 24 glycine-rich nonapeptides in the C-terminal half that bind Ca2+. The glycine-rich nonapeptides are arranged in a modular structure and there is some variability in the number of modules in the ApxIVA proteins of different serotypes of A. pleuropneumoniae. The deduced amino acid sequences of the ApxIVA proteins have significant similarity with the Neisseria meningitidis iron-regulated RTX proteins FrpA and FrpC, and to a much lesser extent with other RTX proteins. The apxlVA gene could be detected in all A. pleuropneumoniae serotypes and seems to be species-specif ic. Although the precise role of this new RTX determinant in pathogenesis of porcine pleuropneumonia needs to be determined, apxlVA is the first in vivo induced toxin gene that has been described in A. pleuropneumoniae.

Genotypic Prevalence of apxIV in Actinobacillus Pleuropneumoniae Field Isolates

“…The PCR test can be completed rapidly, giving results such as the identification of the apxIV gene within 1 working day. The apxIV gene product could not be detected in A. pleuropneumoniae cultures grown under various conditions in vitro; however, pigs experimentally infected with serotypes 1, 5, and 7 produced antibodies that reacted with the ApxIV toxin, 13 suggesting that apxIV is induced in vivo. Therefore, PCR should be a valuable technique for detecting the apxIV gene in A. pleuropneumoniae.…”

“…Spontaneous deletion of the apxIV gene may not occur at hot spots of recombination or through the action of insertion sequence-like elements, as was the case with other apx genes. 1 Whether all 12 A. pleuropneumoniae serotypes carry the apxIV gene was not examined, but the combination of previous 13 and present results strongly suggests that all 12 A. pleuropneumoniae serotypes carry the apxIV gene and that the ApxIV toxin is species specific.…”

“…10,11 A total of 90 isolates were examined for the apxIV gene. The PCR for the apxIV gene was carried out as previously described, 13 with slight modification. The forward and reverse primers were 5Ј-TGGCACTGACGGTGATGAT-3Ј (nucleotides 6,018-6,036) and 5Ј-GGCCATCGACTCAACCAT-3Ј (nucleotides 6,459-6,442), respectively.…”

A Novel “Clip-and-link” Activity of Repeat in Toxin (RTX) Proteins from Gram-negative Pathogens

“…FrpC⌬RTX was tagged at the amino terminus by the FLAG peptide and at the carboxyl terminus by a His 6 affinity purification tag. pJFFapxIVA1HisCЈ was used for production of the recombinant carboxyl-terminally His-tailed ApxIVA protein in E. coli (12) and was generously provided by J. Frey from the University of Berne, Berne, Switzerland.…”

Aspectos fenotípicos, genotípicos e de diagnóstico da bactéria A. pleuropneumoniae

“…O App produz diferentes RTX (Apx), que apresentam efeitos citotóxicos e hemolíticos distintos (FREY, 1995;SCHALLER et al, 1999). Todos os sorotipos de App variam quanto à secreção de uma ou duas das toxinas ApxI, II e III (JANSEN et al, 1993).…”