Sphingomyelinases D induce direct association of C1q to the erythrocyte membrane causing complement mediated autologous haemolysis.

Bites by Loxosceles spiders can induce severe clinical symptoms, including dermonecrosis, thrombosis, vascular leakage, haemolysis and persistent inflammation. The causative toxin is a sphingomyelinase D (SMase D) that cleaves sphingomyelin into choline and ceramide-1-phosphate. A similar enzyme, showing comparable bioactivity, is secreted by certain pathogenic corynebacteria and acts as a potent virulence factor. We have previously found that SMase D toxins led to an increased susceptibility of human erythrocytes (E) to activation of complement (C) via the classical pathway (CP) in the absence of antibodies. In the present study we have investigated the CP initiating components involved in the haemolysis induced by SMases from Corynebacterium pseudotuberculosis (PLD) and from Loxosceles intermedia venom (P1). When P1 or PLD treated E were incubated with C8-depleted human serum, an increase in C1q, serum amyloid protein (SAP) and C-reactive protein (CRP) binding was observed. While purified C1q, SAP and CRP were found to bind to P1 or PLD treated E, depletion of SAP or CRP from human serum did not prevent C-mediated lysis, suggesting that pentraxins are not involved in the initiation of C-activation. However depletion of C1 lead to a greatly reduced haemolysis, demonstrating that the activation of the CP is caused by direct binding of C1q to the SMase treated cells. Binding of fluid phase C-regulators C4b-binding protein and factor H was also observed, however these C-regulators in conjunction with the membrane bound C-regulators were unable to prevent haemolysis, demonstrating the potency of SMase D facilitated binding of C1 and activation of C.

Isolation of Arcanobacterium pyogenes from the porcine gastric mucosa.

Arcanobacterium (Actinomyces) pyogenes is an inhabitant of the mucous membranes of the respiratory and genital tracts of a number of domestic animal species. However, following a precipitating physical or microbial insult, A. pyogenes can become an opportunistic pathogen, associated with suppurative infections. The isolation of A. pyogenes from the bovine ruminal wall indicated that this organism may also inhabit the gastrointestinal tract of, at least, cattle. To determine whether A. pyogenes was also present on the gastric mucosa of a monogastric animal, porcine stomachs were cultured for the presence of this organism. Of 13 stomachs sampled, A. pyogenes was isolated from 5 (39%). The identity of the organism was confirmed by PCR with primers specific to the plo gene, which encodes the A. pyogenes haemolytic exotoxin pyolysin. In addition, an isolate from each positive stomach was subjected to 16S rRNA gene sequencing and the identification as A. pyogenes was confirmed. These data indicate that A. pyogenes may be resident on the gastric mucosa of pigs.

Isolation of Arcanobacterium (Actinomyces) pyogenes from cases of feline otitis externa and canine cystitis.

Arcanobacterium pyogenes is a normal inhabitant of the mucous membranes of domestic animals, such as cattle, sheep, swine, and goats. It is also an opportunistic pathogen in these animals, where it causes a variety of purulent infections involving the skin, joints, and visceral organs. Two recent cases of isolation of A. pyogenes from companion animals are reported. In the first case, a cat presented with a chronic otitis externa, from which A. pyogenes was isolated in pure culture. The second case involved a dog with a urinary tract infection, where A. pyogenes was isolated from urine as the predominant bacterial species. In both cases, the A. pyogenes isolates were presumptively identified by macrobiochemical tests, and then their identities were confirmed by polymerase chain reaction analysis and 16S rRNA gene sequencing.

Molecular characterization of the pore-forming toxin, pyolysin, a major virulence determinant of Arcanobacterium pyogenes.

Arcanobacterium pyogenes is a common inhabitant and opportunistic pathogen of domestic animals. The pathogenesis of this organism in a range of suppurative diseases is not well understood. However, the development of genetic techniques to study this organism has allowed advances in the analysis of A. pyogenes virulence factors. A major step in this analysis was the identification and cloning of the A. pyogenes hemolytic exotoxin, pyolysin (PLO). PLO is the most divergent member of the cholesterol-binding pore-forming family of toxins. PLO is also divergent in a C-terminal undecapeptide motif which is almost invariant among other members of the family. This divergent undecapeptide motif is required for the full cytolytic activity of PLO and is also responsible for its oxygen-resistant nature. Insertional inactivation of the plo gene results in a significant reduction in virulence in an intraperitoneal mouse model of infection. The virulence of the plo mutant can be restored by providing PLO in trans, suggesting that PLO is a major virulence factor in A. pyogenes pathogenesis in mice. Results of previous vaccination trials with crude antigens against A. pyogenes infection in domestic animals and mice have been equivocal at best. However, a recombinant PLO-based subunit vaccine protected mice from experimental A. pyogenes infection, indicating that PLO is also an important host protective antigen. These results provide promise that the dogma that domestic animals are recalcitrant to vaccination against A. pyogenes infection may prove false.

Cloning, expression, and characterization of a neuraminidase gene from Arcanobacterium pyogenes.

Arcanobacterium pyogenes is an opportunistic pathogen, associated with suppurative infections in domestic animals. In addition to pyolysin, a pore-forming, cholesterol-binding toxin, A. pyogenes expresses a number of putative virulence factors, including several proteases and neuraminidase activity. A 3,009-bp gene, nanH, was cloned and sequenced and conferred neuraminidase activity on an Escherichia coli host strain. The predicted 107-kDa NanH protein displayed similarity to a number of bacterial neuraminidases and contained the RIP/RLP motif and five copies of the Asp box motif found in all bacterial neuraminidases. Recombinant His-tagged NanH was found to have pH and temperature optima of 5.5 to 6.0 and 55 degrees C, respectively. Insertional deletion of the nanH gene resulted in the reduction, but not absence, of neuraminidase activity, indicating the presence of a second neuraminidase gene in A. pyogenes. NanH was localized to the A. pyogenes cell wall. A. pyogenes adhered to HeLa, CHO, and MDBK cells in a washing-resistant manner. However, the nanH mutant was not defective for adherence to epithelial cells. The role of NanH in host epithelial cell adherence may be masked by the presence of a second neuraminidase in A. pyogenes.

Thiol-activated cytolysins: structure, function and role in pathogenesis.

Members of the thiol-activated family of cytolysins are involved in the mechanism of pathogenesis of a number of Gram-positive species. While they are pore-forming toxins, their major pathogenic effects may be more subtle than simple lysis of host cells, and may include interference with immune cell function and cytokine induction. Crystal structure, electron microscopy, mutagenesis and antibody binding studies have led to the modeling of a novel mechanism of pore formation, encompassing membrane-binding, membrane insertion and oligomerization. Despite their designation as thiol-activated cytolysins, it is now clear that thiol activation is not an important property of this group of toxins.

An Arcanobacterium (Actinomyces) pyogenes mutant deficient in production of the pore-forming cytolysin pyolysin has reduced virulence.

Pyolysin (PLO), the hemolytic exotoxin expressed by Arcanobacterium (Actinomyces) pyogenes, is a member of the thiol-activated cytolysin family of bacterial toxins. Insertional inactivation of the plo gene results in loss of expression of PLO with a concomitant loss in hemolytic activity. The plo mutant, PLO-1, has an approximately 1. 8-log10 reduction in the 50% infectious dose compared to that for wild-type A. pyogenes in a mouse intraperitoneal infection model. Studies involving cochallenge of wild-type and PLO-1 bacteria resulted in recovery of similar numbers of both strains, suggesting that PLO production is required for survival in vivo. Recombinant, His-tagged PLO (His-PLO) is cytotoxic for mouse peritoneal macrophages and J774 cells in a dose-dependent manner. Protection against challenge with A. pyogenes could be afforded by vaccination with formalin-inactivated His-PLO, suggesting that PLO is a host-protective antigen, as well as a virulence determinant.

Complete nucleotide sequence of the 27-kilobase virulence related locus (vrl) of Dichelobacter nodosus: evidence for extrachromosomal origin.

The vrl locus is preferentially associated with virulent isolates of the ovine footrot pathogen, Dichelobacter nodosus. The complete nucleotide sequence of this 27.1-kb region has now been determined. The data reveal that the locus has a G+C content much higher than the rest of the D. nodosus chromosome and contains 22 open reading frames (ORFs) encoding products including a putative adenine-specific methylase, two potential DEAH ATP-dependent helicases, and two products with sequence similarity to a bacteriophage resistance system. These ORFs are all in the same orientation, and most are either overlapping or separated by only a few nucleotides, suggesting that they comprise an operon and are translationally coupled. Expression vector studies have led to the identification of proteins that correspond to many of these ORFs. These data, in combination with evidence of insertion of vrl into the 3' end of an ssrA gene, are consistent with the hypothesis that the vrl locus was derived from the insertion of a bacteriophage or plasmid into the D. nodosus genome.

Electroporation-mediated transformation of the ovine footrot pathogen Dichelobacter nodosus.

Studies on the potential virulence genes of the ovine footrot pathogen Dichelobacter nodosus have been hindered by the lack of a genetic system for this organism. In an attempt to accomplish the transformation of D. nodosus cells, we constructed a plasmid that contained part of a native D. nodosus plasmid and carried a tetracycline resistance gene that was located between the D. nodosus rrnA promoter and terminator. This plasmid was used to transform several D. nodosus strains to tetracycline resistance. Analysis of two independent transformants from each parental strain showed that in nearly all of these derivatives, the plasmid was not replicating independently, but that the tetracycline resistance gene had inserted by homologous recombination into one of the three rrn operons located on the chromosome. In most of the transformants, double reciprocal crossover events had occurred. These results are highly significant for genetic studies in D. nodosus and for footrot pathogenesis studies, since by using reverse genetics it will now be possible to examine the role of putative D. nodosus-encoded virulence genes in the disease process.

Clostridium perfringens type E animal enteritis isolates with highly conserved, silent enterotoxin gene sequences.

Several Clostridium perfringens genotype E isolates, all associated with hemorrhagic enteritis of neonatal calves, were identified by multiplex PCR. These genotype E isolates were demonstrated to express alpha and iota toxins, but, despite carrying sequences for the gene (cpe) encoding C. perfringens enterotoxin (CPE), were unable to express CPE. These silent cpe sequences were shown to be highly conserved among type E isolates. However, relative to the functional cpe gene of type A isolates, these silent type E cpe sequences were found to contain nine nonsense and two frameshift mutations and to lack the initiation codon, promoters, and ribosome binding site. The type E animal enteritis isolates carrying these silent cpe sequences do not appear to be clonally related, and their silent type E cpe sequences are always located, near the iota toxin genes, on episomal DNA. These findings suggest that the highly conserved, silent cpe sequences present in most or all type E isolates may have resulted from the recent horizontal transfer of an episome, which also carries iota toxin genes, to several different type A C. perfringens isolates.

The Arcanobacterium (Actinomyces) pyogenes plasmid pAP1 is a member of the pIJ101/pJV1 family of rolling circle replication plasmids.

The 2.4-kb plasmid pAP1 from Arcanobacterium (Actinomyces) pyogenes had sequence similarity within the putative replication protein and double-stranded origin with the pIJ101/pJV1 family of plasmids. pJGS84, a derivative of pAP1 containing a kanamycin resistance gene, was able to replicate in Escherichia coli and Corynebacterium pseudotuberculosis, as well as in A. pyogenes. Detection of single-stranded DNA intermediates of pJGS84 replication suggested that this plasmid replicates by the rolling circle mechanism.

Complementation analysis of the Dichelobacter nodosus fimN, fimO, and fimP genes in Pseudomonas aeruginosa and transcriptional analysis of the fimNOP gene region.

The causative agent of ovine footrot, the gram-negative anaerobe Dichelobacter nodosus, produces polar type IV fimbriae, which are the major protective antigens. The D. nodosus genes fimN, fimO, and fimP are homologs of the Pseudomonas aeruginosa fimbrial assembly genes, pilB, pilC, and pilD, respectively. Both the pilD and fimP genes encode prepilin peptidases that are responsible for cleavage of the leader sequence from the immature fimbrial subunit. To investigate the functional similarity of the fimbrial biogenesis systems from these organisms, the D. nodosus genes were introduced into P. aeruginosa strains carrying mutations in the homologous genes. Analysis of the resultant derivatives showed that the fimP gene complemented a pilD mutant of P. aeruginosa for both fimbrial assembly and protein secretion. However, the fimN and fimO genes did not complement pilB or pilC mutants, respectively. These results suggest that although the PilD prepilin peptidase can be functionally replaced by the heterologous FimP protein, the function of the PilB and PilC proteins may require binding or catalytic domains specific for the P. aeruginosa fimbrial assembly system. The transcriptional organization and regulation of the fimNOP gene region were also examined. The results of reverse transcriptase PCR and primer extension analysis suggested that these genes form an operon transcribed from two sigma70-type promoters located upstream of ORFM, an open reading frame proximal to fimN. Transcription of the D. nodosus fimbrial subunit was found to increase in cells grown on solid media, and it was postulated that this regulatory effect may be of significance in the infected footrot lesion.

The Arcanobacterium (Actinomyces) pyogenes hemolysin, pyolysin, is a novel member of the thiol-activated cytolysin family.

Arcanobacterium (Actinomyces) pyogenes, an animal pathogen, produces a hemolytic exotoxin, pyolysin (PLO). The gene encoding PLO was cloned, and sequence analysis revealed an open reading frame of 1,605 bp encoding a protein of 57.9 kDa. PLO has 30 to 40% identity with the thiol-activated cytolysins (TACYs) of a number of gram-positive bacteria. The activity of PLO was found to be very similar to those of other TACYs, except that it was not thiol activated. The highly conserved TACY undecapeptide is divergent in PLO; in particular, the cysteine residue required for thiol activation has been replaced with alanine. However, mutagenesis of the alanine residue to cysteine did not confer thiol activation on PLO, suggesting a conformational difference in the undecapeptide region of this toxin. Specific antibodies against purified, recombinant PLO completely neutralized the hemolytic activity of A. pyogenes, suggesting that this organism produces a single hemolysin. Furthermore, these antibodies could passively protect mice against lethal challenge with A. pyogenes, suggesting that like other TACYs PLO is an important virulence factor in the pathogenesis of this organism.

Electroporation-mediated transformation of Arcanobacterium (Actinomyces) pyogenes.

Plasmids derived from pNG2 or RSF1010 were introduced into strains of Arcanobacterium (Actinomyces) pyogenes by electroporation. Electroporation conditions were varied systematically to give a maximum electroporation frequency of 3.7 x 10(5) CFU/microgram DNA at 1.5 kV/cm and 246 omega, resulting in a time constant of approximately 10 ms. The A. pyogenes transformants expressed plasmid-encoded resistance to chloramphenicol, erythromycin, kanamycin, and streptomycin. The source of incoming DNA affected the growth rate of transformants, but not the electroporation efficiency. This is the first report of genetic transformation of the veterinary pathogen A. pyogenes.

Virulence regions and virulence factors of the ovine footrot pathogen, Dichelobacter nodosus.

Ovine footrot is a debilitating and highly infectious disease that is primarily caused by the Gram-negative, anaerobic bacterium Dichelobacter nodosus. The major antigens implicated in virulence are the type IV fimbriae and extracellular proteases. The fimbriae show sequence and structural similarity to other type IV fimbriae, this similarity extends to genes that are involved in fimbrial biogenesis. Several acidic and basic extracellular serine proteases are produced by both virulent and benign isolates of D. nodosus. Subtle functional differences in these proteases appear to be important in virulence. In addition, there are two chromosomal regions that have a genotypic association with virulence. The partially duplicated and rearranged vap regions appear to have arisen from the insertion of a plasmid into a tRNA gene via an integrase-mediated site-specific insertion event. The 27 kb vrl region has several genes often found on bacteriophages and has inserted into an ssrA gene that may have a regulatory role in the cell. The determination of the precise role that each of these genes and gene regions has in virulence awaits the development of methods for the genetic analysis and manipulation of D. nodosus.

Comparison of gene probe and conventional methods for the differentiation of ovine footrot isolates of Dichelobacter nodosus.

In a collaborative study that involved four Australian veterinary diagnostic laboratories a gene probe test based on the recombinant plasmids pJIR318, pJIR314B, and pJIR313, which contain genomic vap or vrl regions, was compared with conventional tests used for the differential diagnosis of ovine footrot. A total of 771 clinical dichelobacter nodosus isolates were tested and designated as belonging to one of several gene probe categories. The results showed that 87% of the virulent isolates belonged to gene probe category 1, compared to only 6% of the benign isolates. It was concluded that there was good correlation between the gene probe test and the virulence designation of these isolates as well as the results of elastase, gelatin-gel and protease isoenzyme tests. Furthermore, the gene probe test was converted to a polymerase chain reaction (PCR)-based test. It is suggested that diagnostic laboratories consider carrying out both this PCR test and tests based on the extracellular proteases of D. nodosus.

Identification of a native Dichelobacter nodosus plasmid and implications for the evolution of the vap regions.

Studies on the role of various virulence factors of the ovine pathogen, Dichelobacter nodosus, have suffered from the absence of a mechanism for the introduction of DNA into this organism. As an initial step in the development of genetic methods, we have identified and cloned a native 10-kb plasmid, pJIR896, from a clinical isolate. This plasmid was found to be a circular form of vap region 1/3 that is found in the reference strain, A198. However, pJIR896 lacked the duplicated region present in the A198 sequence and instead contained a 1.7-kb putative insertion sequence, IS1253, which shared similarity to a number of unusual IS elements. A model is proposed for the evolution of vap region 1/3 which involves the integration of a plasmid, such as pJIR896, and subsequent rearrangements resulting from the deletion or transposition of IS1253.

Delineation of the virulence-related locus (vrl) of Dichelobacter nodosus.

Dichelobacter nodosus is the primary pathogen implicated in ovine footrot. In this paper we have delineated a 27 kb locus, termed the virulence-related locus (vrl), that was essentially specific for virulent D. nodosus isolates. The precise ends of this locus were mapped and the sequences of the junction regions from the virulent strain A198 were compared to corresponding sequences from the benign isolate C305. The left end of the vrl locus was located in a sequence similar to that of the small stable 10Sa RNA molecule of Escherichia coli, next to a phage-attachment-site-like sequence, which indicated that the vrl locus might have arisen by the integration of a phage. However, no attachment-like sequence could be found at the right end of the vrl locus. In the chromosome of the benign strain the sequences bordering vrl were not contiguous but were separated by about 3 kb. It was concluded that the divergence of the benign and virulent strains at this locus was a multi-step process. Several potential ORFs were identified at the junction regions but only one ORF, encoding a 126 kDa protein, was expressed in a T7 expression system in E. coli.

Identification of fimbrial assembly genes from Dichelobacter nodosus: evidence that fimP encodes the type-IV prepilin peptidase.

Dichelobacter nodosus (Dn) is the causative agent of footrot, an economically significant disease of sheep. One of the factors believed to be involved in the virulence of this organism is its ability to produce type-IV fimbriae, which are the major protective antigens. To investigate the process of fimbrial biogenesis in Dn, gene probes were constructed from pilus biogenesis genes of Pseudomonas aeruginosa (Pa) and used to isolate homologues from Dn. A homologue, designated fimP, of the Pa prepilin peptidase-encoding gene, pilD, was cloned using this approach. The fimP gene product was shown to possess endopeptidase activity when produced in Escherichia coli. Two other fimbrial biogenesis genes fimN and fimO, whose products show similarity to the Pa PilB and PilC proteins, respectively, were identified because of their linkage to fimP. The arrangement of fimN, fimO and fimP in Dn closely resembles the arrangement of pilB, pilC and pilD in Pa.

A multiple site-specific DNA-inversion model for the control of Omp1 phase and antigenic variation in Dichelobacter nodosus.

The molecular cloning and sequence analysis of four structurally variant linked genes (omp1A,B,C,D) that encode the major outer membrane protein of Dichelobacter nodosus strain VCS1001 are described. The isolation of rearranged copies of omp1A and omp1B, and the identification in the 5' regions of all four genes of short cross-over-site sequences that were similar to the Din family of cross-over-site sequences, suggested that site-specific DNA inversion was involved in omp1 rearrangement. Evidence for site-specific inversion of the 497 bp DNA fragment, which was located between the divergently orientated omp1A and omp1B genes, and which contained the promoter and 5' coding sequence of Omp1, was obtained by polymerase chain reaction-mediated amplification of inverted forms of these genes. However, to account for all of the omp1 gene copies cloned in this study, a more widespread inversion phenomenon must be involved in the rearrangement of these genes and a model for multiple site-specific DNA inversions at the omp1 locus is described. In this model the four structurally variant omp1 genes can be assembled from one of four structurally variant C-terminal coding regions and a conserved N-terminal coding region and can be expressed from a single promoter. It is postulated that this genetic capability endows D. nodosus with the ability to switch the antigenic specificity of one of its major surface proteins.