The three different pore-forming RTX-toxins of Actinobacillus pleuropneumoniae are reviewed, and new and uniform designations for these toxins and their genes are proposed. The designation ApxI (for &tinobacillus pZeuropneumoniae RTX-toxin I) is proposed for the RTX-toxin produced by the reference strains for serotypes 1, 5a, 5b, 9,lO and 11, which was previously named haemolysin I (HlyI) or cytolysin I (ClyI). This protein is strongly haemolytic and shows strong cytotoxic activity towards pig alveolar macrophages and neutrophils; it has an apparent molecular mass in the range 105 to 110 kDa. The genes of the apxZ operon will have the designations apxZC, apxZA, apxZB, and apxZD for the activator, the structural gene and the two secretion genes respectively. The designation ApxII is proposed for the RTX-toxin which is produced by all serotype reference strains except serotype 10 and which was previously named App, HlyII, ClyII or Cyt. This protein is weakly haemolytic and moderately cytotoxic and has an apparent molecular m a s between 103 and 105 kDa. The genes of the apxZZ operon will have the designations apxZZC for the activator gene and apxZZA for the structural toxin gene. In the apxZZ operon, no genes for secretion proteins have been found. Secretion of ApxII seems to occur via the products of the secretion genes apxZB and apxZD of the apxZ operon. The designation ApxIII is proposed for the nonhaemolytic RTX-toxin of the reference strains for serotypes 2, 3, 4, 6 and 8, which was previously named cytolysin 111 (ClyIII), pleurotoxin (Ptx), or macrophage toxin (Mat). This protein is strongly cytotoxic and has an apparent molecular mass of 120 kDa. The genes of the apxZZZ operon have the designations apxZZZC, apxZZZA, apxZZZB and apxZZZD for the activator gene, the structural gene and the two secretion genes respectively.

No abstract

Characterization of a series of urease-negative transposon mutations of Actinobacillus pleuropneumoniae revealed that many of the mutants had insertions 2 to 4 kbp upstream of the urease gene cluster. A 5-kbp upstream region of DNA was sequenced and found to contain six open reading frames (ORFs) transcribed in the same orientation as the urease genes. As well, a partial ORF, apuR, 202 bp upstream of these six ORFs, is transcribed in the opposite orientation. The predicted product of this partial ORF shows homology with many members of the LysR family of transcriptional regulators. Five of the ORFs (cbiKLMQO) appear to form an operon encoding a putative nickel and cobalt periplasmic permease system. The cbiM and cbiQ genes encode proteins that have sequence similarity with known cobalt transport membrane proteins, and the cbiO gene encodes a cobalt transport ATP-binding protein homologue. The product of the cbiK gene is predicted to be the periplasmic-binding-protein component of the system, though it does not show any sequence similarity with CbiN, the cobalt-binding periplasmic protein. Escherichia coli clones containing this putative transport operon together with the urease genes of A. pleuropneumoniae were urease positive when grown in unsupplemented Luria-Bertani broth, whereas a clone containing only the minimal urease gene cluster required the addition of high concentrations of NiCl 2 for full urease activity. This result supports the hypothesis that nickel is a substrate for this permease system. The sixth ORF, utp, appears to encode an integral membrane protein which has significant sequence identity with mammalian urea transport proteins, though its function in A. pleuropneumoniae remains to be determined.The genes required for urease activity in various bacterial species typically include those encoding the structural subunits and those encoding the accessory proteins involved in insertion of two nickel ions within the catalytic site (24). In bacteria with urea-inducible gene clusters, the regulatory gene, ureR, is also present (24). Because of the requirement for nickel as a cofactor, genes such as Helicobacter pylori nixA and Alcaligenes eutrophus hoxN that encode nickel uptake systems have also been shown to affect urease activity (23,41).We recently identified the urease gene cluster of the gramnegative swine pathogen Actinobacillus pleuropneumoniae (4). The organization of the A. pleuropneumoniae gene cluster was found to be similar to that of other bacterial ureases, with the first three genes encoding the structural subunits (UreABC), and the accessory proteins (UreEFGD) encoded by four contiguous genes downstream. There was no evidence of a regulatory gene (ureR) upstream of the cluster.In order to further characterize the urease activity of A. pleuropneumoniae, a bank of transposon mutants of strain CM5 Nal r was generated and screened for urease-negative isolates. A large number of insertions in the urease-negative mutants mapped upstream of the urease gene cluster. Therefore, the 5-kbp reg...

The tonsil of the soft palate in pigs is a secondary lymphoid tissue that provides a first line of defense against foreign antigens entering by the mouth or nares. It has been known for a long time to be the site of colonization of important swine and zoonotic bacterial pathogens. Initially our understanding of microbes present at this site came from culture-based studies. Very recently, sequence-based approaches have been used to identify the core microbiome of the swine tonsil. Although animal to animal and herd to herd variation was detected in these studies, >90 of the organisms detected belonged to the phyla Proteobacteria and Firmicutes. Members of the family Pasteurellaceae appeared to be predominate in the tonsil; however, the relative proportions of Actinobacillus, Haemophilus, and Pasteurella varied. Members of the families Moraxellaceae, Fusobacteriaceae, Veillonellaceae, and Neisseriaceae were also seen as frequent residents of the tonsil.

Background Actinobacillus pleuropneumoniae, the causative agent of porcine contagious pleuropneumonia, is an important pathogen of swine throughout the world. It must rapidly overcome the innate pulmonary immune defenses of the pig to cause disease. To better understand this process, the objective of this study was to identify genes that are differentially expressed in a medium that mimics the lung environment early in the infection process.Methods and Principal FindingsSince bronchoalveolar lavage fluid (BALF) contains innate immune and other components found in the lungs, we examined gene expression of a virulent serovar 1 strain of A. pleuropneumoniae after a 30 min exposure to BALF, using DNA microarrays and real-time PCR. The functional classes of genes found to be up-regulated most often in BALF were those encoding proteins involved in energy metabolism, especially anaerobic metabolism, and in cell envelope, DNA, and protein biosynthesis. Transcription of a number of known virulence genes including apxIVA and the gene for SapF, a protein which is involved in resistance to antimicrobial peptides, was also up-regulated in BALF. Seventy-nine percent of the genes that were up-regulated in BALF encoded a known protein product, and of these, 44% had been reported to be either expressed in vivo and/or involved in virulence.ConclusionsThe results of this study suggest that in early stages of infection, A. pleuropneumoniae may modulate expression of genes involved in anaerobic energy generation and in the synthesis of proteins involved in cell wall biogenesis, as well as established virulence factors. Given that many of these genes are thought to be expressed in vivo or involved in virulence, incubation in BALF appears, at least partially, to simulate in vivo conditions and may provide a useful medium for the discovery of novel vaccine or therapeutic targets.