The genus Corynebacterium is a heterogeneous group of species comprising human and animal pathogens and environmental bacteria. It is defined on the basis of several phenotypic characters and the results of DNA-DNA relatedness and, more recently, 16S rRNA gene sequencing. However, the 16S rRNA gene is not polymorphic enough to ensure reliable phylogenetic studies and needs to be completely sequenced for accurate identification. The almost complete rpoB sequences of 56 Corynebacterium species were determined by both PCR and genome walking methods. In all cases the percent similarities between different species were lower than those observed by 16S rRNA gene sequencing, even for those species with degrees of high similarity. Several clusters supported by high bootstrap values were identified. In order to propose a method for strain identification which does not require sequencing of the complete rpoB sequence (approximately 3,500 bp), we identified an area with a high degree of polymorphism, bordered by conserved sequences that can be used as universal primers for PCR amplification and sequencing. The sequence of this fragment (434 to 452 bp) allows accurate species identification and may be used in the future for routine sequence-based identification of Corynebacterium species.

Bites by Loxosceles spiders can produce severe clinical symptoms, including dermonecrosis, thrombosis, vascular leakage, hemolysis, and persistent inflammation. The causative factor is a sphingomyelinase D (SMaseD) 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. However, the molecular basis for SMaseD toxicity is not well understood, which hampers effective therapy. Here we show that the spider and bacterial SMases D hydrolyze albumin-bound lysophosphatidylcholine (LPC), but not sphingosylphosphorylcholine, with K(m) values (approximately 20-40 microm) well below the normal LPC levels in blood. Thus, toxic SMases D have intrinsic lysophospholipase D activity toward LPC. LPC hydrolysis yields the lipid mediator lysophosphatidic acid (LPA), a known inducer of platelet aggregation, endothelial hyperpermeability, and pro-inflammatory responses. Introduction of LPA(1) receptor cDNA into LPA receptor-negative cells renders non-susceptible cells susceptible to SmaseD, but only in LPC-containing media. Degradation of circulating LPC to LPA with consequent activation of LPA receptors may have a previously unappreciated role in the pathophysiology of secreted SMases D.

Corynebacterium pseudotuberculosis is a widespread facultative intracellular pathogen that causes caseous lymphadenitis disease in sheep and goats, and generates cutaneous abscesses and granulomas in horses and cattle. Although some genes have been studied for diagnostic and phylogenetic analysis within the genus Corynebacterium, at subspecies level the pathogen has been poorly analyzed. The aim of this study was to characterize C. pseudotuberculosis strains isolated from domestic animals, through the sequencing of a hypervariable rpoB gene segment. As result, there were identified host associated rpoB polymorphisms in strains infecting sheep, goats and horses from Chile. These differences suggest the existence of bacterial genotypes, in which the nucleotide similarity values were ranging from 98.8 to 99.8%. In conclusion, the analysis of polymorphisms in the partial rpoB sequence can be used as a diagnostic tool that differentiates C. pseudotuberculosis strains at subspecies level.

Two Corynebacterium strains were isolated from lymph nodes of wild boars showing severe alterations caused by caseous lymphadenitis. The wild boars came from different districts in southern Germany; one was found dead, the other had been shot. The two Corynebacterium strains obtained were both positive for phospholipase D. Further analysis of biochemical profiles did not allow unambiguous differentiation between Corynebacterium ulcerans and Corynebacterium pseudotuberculosis. Fourier-transformed infrared spectroscopy as well as partial sequencing of the genes for 16S rRNA and RNA polymerase beta subunit (rpoB) clearly identified both strains as Corynebacterium ulcerans. The tox gene for diphtheria toxin (DT) could be detected in both porcine isolates by PCR. Partial DNA sequencing of this tox gene showed significant differences from sequences described for other Corynebacterium ulcerans strains and a higher degree of similarity to that of Corynebacterium diphtheria. Production of diphtheria toxin could not be detected. These results indicate that wild game could be a reservoir for zoonotic Corynebacterium ulcerans.

A two-step allele replacement mutagenesis procedure, using a conditionally replicating plasmid, was developed to allow the creation of targeted, marker-free mutations in Corynebacterium pseudotuberculosis. The relationship between homologous sequence length and recombination frequency was determined, and enhanced plasmid excision was observed due to the rolling-circle replication of the mutagenesis vector. Furthermore, an antibiotic enrichment procedure was applied to improve the recovery of mutants. Subsequently, as proof of concept, a marker-free, cp40-deficient mutant of C. pseudotuberculosis was constructed.

C. pseudotuberculosis is an important animal pathogen that causes substantial economical loss in sheep and goat farming. Zoonotic infections in humans are rare, but when they occur they are often severe and difficult to treat. One of the most studied proteins from this bacterium, the secreted protein CP40 is being developed as a promising vaccine candidate and has been characterized as a serine protease. In this study we have investigated if CP40 is an endoglycosidase rather than a protease. CP40 does not show any protease activity and contains an EndoS-like family 18 of glycoside hydrolase (chitinase) motif. It hydrolyzes biantennary glycans on both human and ovine IgGs. CP40 is not a general chitinase and cannot hydrolyze bisecting GlcNAc. Taken together we present solid evidence for re-annotating CP40 as an EndoS-like endoglycosidase. Redefining the activity of this enzyme will facilitate subsequent studies that could give further insight into immune evasion mechanisms underlying corynebacterial infections in animals and humans.

The phospholipase D (PLD) gene from Corynebacterium pseudotuberculosis has been cloned, sequenced, and expressed in Escherichia coli. Analysis of DNA sequence data reveals a major open reading frame encoding a 31.4-kilodalton protein, a size consistent with that estimated for the PLD protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of these data with the amino-terminal protein sequence indicates that the mature PLD protein is preceded by a 24-residue signal sequence. Expression of the PLD gene in E. coli is initiated from the corynebacterial promoter, and the resulting protein has sphingomyelinase activity. Primer extension mapping localized the 5' end of the PLD gene mRNA to a site 5 to 7 base pairs downstream of a region similar to the consensus sequence for E. coli promoters. Northern and Southern blot analyses suggest that the gene is transcribed from mRNA approximately 1.1 kilobases in length and that it is present in a single copy within the C. pseudotuberculosis genome.

A toxic phospholipase D (PLD) is putatively involved in pathogenesis of Corynebacterium pseudotuberculosis infections. We report here the cloning and expression of the PLD gene (pld) in Escherichia coli. A cosmid library of DNA from C. pseudotuberculosis biovar ovis isolate Whetten 1 was constructed and screened for PLD-producing recombinants by plating them on LB agar containing sheep erythrocytes and equi factors. One recombinant, designated pCpO1, yielded a gene product which displayed synergistic hemolytic and sphingomyelinase D activities, both of which are characteristic of PLD. Subcloning into pUC19 yielded a recombinant, pCpO50, which contained a 1.8-kilobase insert. Analysis of supernatant fluids and cell extracts of cultures of E. coli(pCpO50) revealed sphingomyelinase activity and a protein of about 31,000 Mr, neither of which were detected in E. coli(pUC19). The 31-kilodalton protein also reacted with antibodies in serum from a sheep naturally infected with C. pseudotuberculosis, serum which also contained PLD-neutralizing antibodies. When Southern blots of BamHI digests of DNA from biovar ovis and biovar equi isolates of C. pseudotuberculosis were probed with pCpO50, bands of 4.8 and 1.9 kilobases, respectively, were seen, suggesting that the genome organization of pld is different for isolates from the two biovars.

With the recognition of several diphtheria outbreaks and the emergence of nontoxigenic corynebacteria strains, there has been renewed interest in the development of laboratory diagnostic methods. Previously reported polymerase chain reaction (PCR) assays can have low diagnostic sensitivity or give species misidentifications among clinical isolates. The aim of the present study was the development of combined real-time PCR assays, based on the tox and rpoB genes, for the detection and differentiation of toxigenic and nontoxigenic corynebacteria. By the PCR tox assay, it was possible to perform the direct identification of DT tox gene of Corynebacterium diphtheriae and Corynebacterium ulcerans, while the PCR rpoB assay differentiated C. diphtheriae from C. ulcerans, irrespective of their toxigenic status. In addition, we detected the DT toxin of Corynebacterium pseudotuberculosis for the first time. These assays revealed high sensitivity, specificity, and reproducibility, and the availability of plasmid controls will facilitate further research into the diagnostics of diphtheria corynebacteria.

The chromosomal gene encoding the phospholipase D from Corynebacterium pseudotuberculosis (biovar ovis) isolate Whetten 1 was replaced with an allele containing a nonsense mutation. The virulence of the mutant strain (W1.31r1) and the isogenic parental strain were then compared by inoculation of goats. The wild-type strain caused abscessation at the site of infection, which then spread to the regional lymph node, while W1.31r1 had a reduced ability to establish a primary infection and was incapable of dissemination. Our results confirm that phospholipase D is a virulence determinant of C. pseudotuberculosis that increases the persistence and spread of the bacteria within the host.

A 40-kDa protein from Corynebacterium pseudotuberculosis has been previously identified as a protective antigen against ovine caseous lymphadenitis. From genomic DNA libraries of C. pseudotuberculosis, we have cloned and sequenced the 40-kDa protein gene, which was found to contain an open reading frame of 1,137 bp encoding a protein of 379 amino acids. No significant homology with previously published DNA or amino acid sequence data was found in databases, suggesting that this is a novel protein. Recombinant 40-kDa protein was overexpressed as a fusion protein to 15% of total cell proteins in Escherichia coli. Biochemical analysis of native and recombinant 40-kDa proteins has revealed associated proteolytic activity, which was shown to be of the serine protease type through the use of specific inhibitors. We suggest that this novel protective antigen be termed corynebacterial protease 40 (CP40).

The genes encoding toxic phospholipases D (PLD) from Corynebacterium pseudotuberculosis (Cp)biovar equi and C. ulcerans (Cu) have been cloned and sequenced. The deduced proteins are 307 amino acids (aa) in length and include a putative signal sequences of 26-aa. A molecular mass of 31.2 and 31.0 kDa and pI values of 8.84 and 6.73 are predicted for the secreted (mature) proteins from Cp and Cu, respectively. Comparison of the deduced primary structure of the two proteins to those of the PLD produced by Cp biovar ovis and Arcanobacterium haemolyticum (Ah) revealed that the four enzymes share 64-97% identity. The aa sequences of this group of proteins were unique when compared to the sequences of other phospholipases in GenBank and were found to share only small regions of homology with other proteins, including two conserved domains of glyceraldehyde-3-phosphate dehydrogenase (G3PD). The similarity of PLD from Cp biovar equi, Cu and Ah to the PLD of Cp biovar ovis suggests that these enzymes may act as virulence determinants.