Cloning and expression of the phospholipase D gene from Corynebacterium pseudotuberculosis in Escherichia coli.

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.     

Mechanism of hemolysis by Renalin, a CAMP-like protein from Corynebacterium renale.

Synergistic hemolysis of sheep erythrocytes caused by Staphylococcus aureus and Corynebacterium renale resulted from the combined action of extracellular staphylococcal sphingomyelinase C and a newly described extracellular agent of C. renale (renalin). The affinity of renalin for ceramide was considered to play a key role in causing hemolysis in erythrocytes in which ceramide had been generated through the action of sphingomyelinase C.     

Rational attenuation of Corynebacterium pseudotuberculosis: potential cheesy gland vaccine and live delivery vehicle.

The phospholipase D (PLD) gene (pld) has been deleted from the Corynebacterium pseudotuberculosis chromosome by using site-specific mutagenesis. Sheep infection trials indicate that the PLD-negative C. pseudotuberculosis strain (Toxminus) is incapable of inducing caseous lymphadentis (cheesy gland) even at doses two logs higher than that at which the wild-type strain produces the disease. This clearly establishes PLD as a major C. pseudotuberculosis virulence factor. Vaccination of sheep with live Toxminus C. pseudotuberculosis elicits strong humoral and cell-mediated immune responses and protects the animals from wild-type challenge.     

Corynebacterium group D2 ("Corynebacterium urealyticum") constitutes a new genomic species.

Twenty-one Corynebacterium group D2 ("C. urealyticum") strains were found to constitute a tight DNA hybridization group distinct from named Corynebacterium species. The strains of Corynebacterium group D2 had cell wall component type IV, short chain mycolic acids and G+C content of DNA of 65-66 mol %. Corynebacterium group D2 constitutes a genomic species which can be identified by phenotypic tests.     

Characterization of United Kingdom isolates of Corynebacterium pseudotuberculosis using pulsed-field gel electrophoresis

Caseous lymphadenitis is a chronic suppurative disease caused by Corynebacterium pseudotuberculosis and is responsible for serious economic losses to the sheep and goat industry. Caseous lymphadenitis was first reported for goats in the United Kingdom in 1990 and for sheep in 1991. Recent evidence suggests that the prevalence of the disease within the national flock is increasing. Fifty isolates of C. pseudotuberculosis from the United Kingdom comprising sheep and horse isolates, the original goat outbreak strain, and the type strain were characterized by biotyping, antimicrobial susceptibility, production of phospholipase D, and genotyping by pulsed-field gel electrophoresis using SfiI and SmaI. All of the isolates were confirmed as C. pseudotuberculosis, and all produced phospholipase D but none reduced nitrate. Restriction with SfiI generated 16 to 18 bands between 48.5 and 290 kb and differentiated six pulsotypes. We conclude that 80% of the strains tested were epidemiologically related to the outbreak strain and that the equine profile was distinct both phenotypically and genotypically.     

Targeting improves the efficacy of a DNA vaccine against Corynebacterium pseudotuberculosis in sheep

A large-scale DNA vaccination trial was performed with sheep to investigate whether an antigen targeted by CTLA-4 enhanced and accelerated the humoral immune response. Vaccination with genetically detoxified phospholipase D (DeltaPLD) has been shown to be effective, at least partially, against Corynebacterium pseudotuberculosis, the causal agent of caseous lymphadenitis in sheep. CTLA-4 binds to B7 on antigen-presenting cells and thus was used to direct the fusion antigens to sites of immune induction. Here we demonstrated that targeting DeltaPLD as a CTLA-4 fusion protein significantly enhanced the speed, magnitude, and longevity of the antibody response compared to that obtained with DNA encoding DeltaPLD. While all groups of sheep vaccinated with DNA encoding DeltaPLD were afforded better protection against an experimental challenge with C. pseudotuberculosis than those immunized with an irrelevant plasmid or those left unimmunized, the best protection was provided by the targeted DNA vaccine. We propose that targeting antigens to antigen-presenting cells offers a generic strategy for enhancing the efficacy of DNA vaccines.     

A pyrolysis-mass spectrometry study of Corynebacterium spp

Clinical (66) and collection (38) strains of Corynebacterium spp., including C. jeikeium and CDC group D2, and of Listeria monocytogenes were examined. Conventional characters used in species identification were assessed by a microbiochemical method, and pyrolysis-mass spectrometry (Py-MS) was performed with a Horizon Instruments PYMS 200X. Classification based on Py-MS data yielded clusters that corresponded with species identification and classification groups from conventional data. One small group of clinical strains, homogeneous in conventional tests and Py-MS, comprised isolates from sputum samples from patients undergoing ventilation; they were similar to collection strains of C. renale and C. striatum; the latter species has been implicated in chest infection. Another group, similar to C. minutissimum in both systems, comprised clinical strains isolated from urogenital specimens. L. monocytogenes strains were clearly distinct from Corynebacterium spp. Groups comprising CDC D2 strains and C. jeikeium were resolved, and were similar to other Corynebacterium spp. Two collection strains of C. xerosis were distinct in conventional tests and Py-MS.     

Prevalence of Corynebacterium urealyticum in urine specimens collected at a university-affiliated medical center.

Corynebacterium urealyticum (formerly Corynebacterium group D2) has been implicated as a cause of alkaline-encrusted cystitis and urinary tract struvite calculi. Despite preselecting urine specimens with neutral and alkaline pHs and using prolonged incubation on a selective medium, isolation of this organism was rarely observed in a population of hospitalized patients. We do not recommend routine cultures for this organism unless the urine is alkaline and struvite crystals, leukocytes, and erythrocytes are present.     

Probing the heat shock response of Corynebacterium pseudotuberculosis: the major virulence factor, phospholipase D, is downregulated at 43 degrees C

Heat shock response genes have been characterised in many organisms. Such genes are often induced not only following heat stress but also following a range of other stresses. In pathogenic bacteria, the common heat shock genes are usually induced during the initial infection process. The identification of other genes regulated during heat shock, besides the classical heat shock genes such as those of the dnaK and groEL operons, may provide information about other cellular responses such as membrane remodelling and nutrient scavenging that may be important in the early stages of infection. In this study, macroarray analysis has been used to identify a number of genes of Corynebacterium pseudotuberculosis that are either upregulated (e.g. clpB, dnaK) or downregulated (e.g. fagC, fas) in vitro following a heat shock. The major virulence factor, phospholipase D, was found to be highly downregulated.     

Protection of sheep against caseous lymphadenitis by use of a single oral dose of live recombinant Corynebacterium pseudotuberculosis.

An inactive form of the Corynebacterium pseudotuberculosis phospholipase D (PLD) gene was constructed and expressed in a PLD-negative strain (designated Toxminus) of C. pseudotuberculosis. Antibody responses specific to Toxminus and both Toxminus and PLD proteins were detected in sheep following oral administration of Toxminus or Toxminus expressing the PLD toxoid, respectively. However, only those sheep vaccinated with Toxminus expressing PLD toxoid were protected against wild-type challenge. These results confirm the importance of PLD as a protective antigen and demonstrate both the potential for developing an oral caseous lymphadenitis vaccine and C. pseudotuberculosis Toxminus as a live vaccine vector.     

Fatal sepsis caused by Corynebacterium amycolatum in a premature infant.

Corynebacterium amycolatum has not been reported as a cause of human infections up to now, but usually the bacterium is misidentified in clinical specimens as Corynebacterium xerosis. We report the first case of neonatal sepsis due to Corynebacterium amycolatum with a fatal outcome in a premature infant.     

In vitro and in vivo study of stone formation by Corynebacterium group D2 (Corynebacterium urealyticum).

Corynebacterium group D2 inoculated into normal human urine formed struvite crystals and an increase in pH and ammonium concentration after 24 h of incubation. Zinc disks dipped into a broth culture of this microorganism and inserted into the bladders of rats produced stones with a mean weight of 12.5 mg (ranging from 1 to 57.7 mg) after 12 days. Analysis of the infrared spectrum determined the stones to be composed of struvite. From these results its seems that stone formation by Corynebacterium group D2 may be possible both in vitro and in vivo, which may confirm a previous report involving these bacteria in human clinical encrusted cystitis.     

Selective killing of human monocytes and cytokine release provoked by sphingomyelinase (beta-toxin) of Staphylococcus aureus.

The best-known activity of Staphylococcus aureus sphingomyelinase C, alias beta-toxin, is as a hemolysin that provokes hot-cold lysis of erythrocytes which contain substantial amounts of sphingomyelin in the plasma membrane. Sheep erythrocytes are most susceptible, and we found that one hemolytic unit, representing the toxin concentration that elicits 50% hemolysis of 2.5 X 10(8) erythrocytes per ml, corresponds to 0.05 enzyme units or to approximately 0.25 microg of sphingomyelinase per ml. The cytotoxic action of beta-toxin on nucleated cells has not been described in any detail before, and the present investigation was undertaken to fill this information gap. We now identify beta-toxin as a remarkably potent monocytocidal agent. At a concentration of 0.001 U/ml, corresponding to approximately 5 ng/ml, beta-toxin killed over 50% of human monocytes (10(6) cells per ml) within 60 min. By contrast, 1 to 5 microg of beta-toxin per ml had no cytocidal effects on human granulocytes, fibroblasts, lymphocytes, or erythrocytes. A selective monocytocidal action was also observed with sphingomyelinase C from Bacillus cereus and a Streptomyces sp., whereas phospholipase A2 and phospholipase D at 100 U/ml were without effect. Monocytes succumbing to the action of beta-toxin processed and released interleukin-1beta, soluble interleukin-6 receptor, and soluble CD14 into the supernatant. Thus, monocyte killing by beta-toxin is associated with cytokine-related events that are important for the initiation and progression of infectious disease. These findings uncover a potentially important role for sphingomyelinase as a determinant of microbial pathogenicity.     

Selective medium for Corynebacterium equi isolation.

The development of a selective medium for the isolation of Corynebacterium equi is described. The medium has been used to examine fecal samples from 127 horses of which 90 have been found to carry the organism.     

Skin colonization by Corynebacterium groups D2 and JK in hospitalized patients.

To determine the prevalence of Corynebacterium group D2 and JK organisms on the skin of different types of patients, 200 hospitalized subjects, half of them admitted to a university hospital and the others in a chronic care institution, were surveyed. Samples were taken from the axilla, groin, and abdominal wall. Corynebacterium group D2 and JK organisms were isolated from at least one of the three skin sites in both groups of patients. Only five patients harbored groups D2 and JK at the same time but a different skin sites. The rate of colonization by group D2 organisms was higher in females (43.3%) than in males (17.7%); on the contrary, group JK organisms were isolated more frequently from males (32.1%) than from females (13.5%). All these differences were statistically significant. Corynebacterium group D2 and JK organisms are widely distributed on the skin of hospitalized patients, and the prevalence is sex related.     

Changes in ovine erythrocyte morphology due to sphingomyelin degradation by Corynebacterium pseudotuberculosis phospholipase D

Corynebacterium pseudotuberculosis produces a sphingomyelin-specific phospholipase D exotoxin that is a major determinant in the pathogenesis of caseous lymphadenitis. We assessed the changes induced in the morphology and sphingomyelin concentration of ovine erythrocytes after incubation with broth culture filtrates or purified exotoxin. Sphingomyelin was hydrolysed nearly in parallel with shape change. Morphologically, spherostomatocytes were seen initially, and later these contained numerous vacuoles at the periphery. Vacuoles seen in thin sections with transmission electron microscopy appeared as pits with scanning electron microscopy. Pitting became progressively worse with time, leading to extensive scalloping of the membrane surface. Chemically, significant decreases (P less than or equal to 0.05) in erythrocyte membrane sphingomyelin content and significant increases (P less than or equal to 0.05) in the content of glycerophospholipids (i.e. phosphatidylglycerol) were observed in erythrocytes incubated with broth culture filtrates or purified exotoxin. These alterations may contribute to the pathophysiology of acute infections induced by C. pseudotuberculosis.     

Corynebacterium prosthetic joint infection

Identification of Corynebacterium species may be challenging. Corynebacterium species are occasional causes of prosthetic joint infection (PJI), but few data are available on the subject. Based on the literature, C. amycolatum, C. aurimucosum, C. jeikeium, and C. striatum are the most common Corynebacterium species that cause PJI. We designed a rapid PCR assay to detect the most common human Corynebacterium species, with a specific focus on PJI. A polyphosphate kinase gene identified using whole-genome sequence was targeted. The assay differentiates the antibiotic-resistant species C. jeikeium and C. urealyticum from other species in a single assay. The assay was applied to a collection of human Corynebacterium isolates from multiple clinical sources, and clinically relevant species were detected. The assay was then tested on Corynebacterium isolates specifically associated with PJI; all were detected. We also describe the first case of C. simulans PJI.     

Rapid detection and molecular differentiation of toxigenic Corynebacterium diphtheriae and Corynebacterium ulcerans strains by LightCycler PCR

The systemic symptoms of diphtheria are caused by the tox-encoded diphtheria toxin (DT) which is produced by toxigenic Corynebacterium spp. Besides the classical agent C. diphtheriae, the zoonotic pathogen C. ulcerans has increasingly been reported as an emerging pathogen for diphtheria. The reliable detection of toxigenic Corynebacterium spp. is of substantial importance for both diphtheria surveillance in the public health sector and the clinical workup of a patient with diphtherialike symptoms. Since the respective tox genes of C. diphtheriae and C. ulcerans differ from each other in both DNA and amino acid sequence, both tox genes should be covered by novel real-time PCR methods. We describe the development and validation of a LightCycler PCR assay which reliably recognizes tox genes from both C. diphtheriae and C. ulcerans and differentiates the respective target genes by fluorescence resonance energy transfer (FRET) hybridization probe melting curve analysis.     

Phospholipase D activity of Vibrio damsela cytolysin and its interaction with sheep erythrocytes.

Exposure of sheep erythrocytes to sublytic amounts of Vibrio damsela cytolysin markedly reduced their membrane sphingomyelin content and their sensitivity to lysis by the sphingomyelin-dependent cytolysins staphylococcal sphingomyelinase C (beta-toxin) and helianthin. The toxin was found to be a phospholipase D active against sphingomyelin.     

Corynebacterium ulcerans and Corynebacterium pseudotuberculosis responses to DNA probes derived from corynephage beta and Corynebacterium diphtheriae.

Strains of Corynebacterium ulcerans and Corynebacterium pseudotuberculosis (Corynebacterium ovis) were examined for the production of diphtheria toxin. A majority of C. ulcerans strains (25 of 37) and 1 C. pseudotuberculosis strain (1 of 14) gave a positive Elek test for diphtheria toxin, and for all strains but 1, production of diphtheria toxin was inhibited at the same level of Fe2+ as was the Corynebacterium diphtheriae control. All Elek-positive cultures as well as two Elek-negative isolates of C. ulcerans gave a positive signal when hybridized with a DNA probe unambiguous for the diphtheria toxin gene (tox) under conditions of high stringency. The majority of probe-positive C. ulcerans strains contained three or more DNA restriction fragments that hybridized with converting corynephage beta, suggesting that in C. ulcerans as in C. diphtheriae there may be a relationship between toxinogeny and carriage of beta-related phage. Selected strains of C. diphtheriae, C. ulcerans, and C. pseudotuberculosis were examined for DNA homology by a semiquantitative technique. There was very little homology between C. diphtheriae and members of the other two species. Strains of C. ulcerans and C. pseudotuberculosis, although more closely related, appeared to belong to distinct species as well.