<Emphasis Type="Italic">Corynebacterium diphtheriae</Emphasis> invasion-associated protein (DIP1281) is involved in cell surface organization,adhesion and internalization in epithelial cells

Background  

Corynebacterium diphtheriae, the causative agent of diphtheria, is well-investigated in respect to toxin production, while little is known about C. diphtheriae factors crucial for colonization of the host. In this study, we investigated the function of surface-associated protein DIP1281, previously annotated as hypothetical invasion-associated protein.     

Neuraminidase ofCorynebacterium diphtheriae: Isolation,purification and some characteristics

A method is described for neuraminidase isolation from a nontoxicogenic C₇ strain ofCorynebacterium diphtheriae by means of a precipitation from cultivation liquid using ZnCl₂ and a subsequent fractionation on a P-60 biogel column. The method yields a neuraminidase preparation homogeneous with respect to antigenity and exhibiting also homogeneity during studies with free electrophoresis.     

Optimization of diphtheria toxin production by <Emphasis Type="Italic">Corynebacterium diphtheriae</Emphasis> using a casein-based medium in a fermenter

The Td-based combined vaccine contains only small amounts of the diphtheria toxoid antigen. However, a high level of purity is necessary for this antigen. The diphtheria toxin is produced by growing Corynebacterium diphtheriae in a semisynthetic, casein-based medium in a fermenter. In order to obtain a highly pure diphtheria toxoid, the optimal conditions to express the toxin at 300 Lf/mL in a fermenter culture were determined. When C. diphtheriae was cultivated in a fermenter and a high concentration of toxin was obtained, specific patterns for the pH and dissolved oxygen levels identified. Overall, the fermenter cultivation process was divided into four stages according to variations in the pH. A specific range of K _La in the fermenter (0.0092 ~ 0.0093/sec) was required to produce high level expression of diphtheria toxin. The amount of toxin expression varied significantly according to culture conditions. Agitation and aeration in the fermenter affected toxin expression, even when the optimal K _La value for toxin production was maintained. A previous study has reported that the amounts of agitation and aeration are important factors when cultivating fungus in the fermenter to produce chitinolytic enzyme. A mass production of diphtheria toxoid with a purity level greater than 2,500 Lf/ mgPN was obtained through purification and detoxification from this optimized toxin production.     

Strain-specific differences in pili formation and the interaction of <Emphasis Type="Italic">Corynebacterium diphtheriae</Emphasis> with host cells

Background  

Corynebacterium diphtheriae, the causative agent of diphtheria, is well-investigated in respect to toxin production, while little is known about C. diphtheriae factors crucial for colonization of the host. In this study, we investigated strain-specific differences in adhesion, invasion and intracellular survival and analyzed formation of pili in different isolates.     

Molecular composition of diphtheria toxoid produced using semi-synthetic and meat extract-based broths

The use of semi-synthetic broths for cultivation of Corynebacterium diphtheriae instead of a meat extract-based broth avoids the presence of highly undesirable bovine meat antigens in the diphtheria toxoid. As information on the composition of casein digest-based broths used for the production of diphtheria toxoid is scarce, we have now developed one. The composition of a casein-based medium that supports vigorous bacterial growth as well as high toxin production is described below. The comparative analysis of the toxoids, produced using the meat-based Pope–Lingood and the casein digest-based broths, showed considerable differences in their molecular composition. The variance of weight distribution of toxoid-containing molecular complexes was smaller when the semi-synthetic broth was used. Normal human therapeutic IgG recognizes some of the proteins in the meat-based medium but does not react with any components of the semi-synthetic medium. While precipitation at the isoelectric point of the diphtheria toxoid produced by culturing the C. diphtheriae strain in the semi-synthetic medium resulted in a preparation meeting the requirement for purity (more than 1500 limit floculation Lf/mg protein nitrogen PN), the toxoid produced in the Pope–Lingood broth failed to meet this requirement in some cases, even after a second purification step using ultrafiltration.     

Specificity of the precipitation reaction of one-zone diphtheria antitoxin for toxicity determination ofCorynebacterium diphtheriae strains

In immunodiffusion analysis of crude diphtheria toxin, one-zone diphtheria antitoxin may give one or two subsidiary lines in addition to the main precipitation line. The subsidiary lines belong to antigenic fragments of the toxin molecule. These fragments are formed from the complete molecule, probably by proteolytic degradation by bacterial enzymes. Other forms of fragment production were not demonstrated. When testing the toxicity of strains ofCorynebacterium diphtheriae by means of one-zone antitoxin, any precipitation reaction observed can thus be regarded as specific evidence of the toxicity of the test strain.     

DNase test as a novel approach for the routine screening of Corynebacterium diphtheriae

Aims: To examine the value of the DNase test as an alternative procedure for differentiating Corynebacterium diphtheriae from Corynebacterium‐like colonies. Methods and Results: DNase test medium was inoculated by spotting a loopful of bacterial growth and incubated aerobically at 37°C. The DNase production was detectable following both 24 and 48 h incubation periods. The DNase activity was detected in all 91 C. diphtheriae (37 toxigenic and 54 nontoxigenic) strains examined, previously identified by both conventional biochemical methods and API Coryne System. Conversely, DNase test results were negative in 93·9% of the 564 nondiphtherial Gram‐positive rod clinical strains. Conclusions: The DNase test emerged as an easily interpretable and cost‐effective alternative screening procedure for C. diphtheriae laboratory identification. Significance and Impact of the Study: The method should facilitate routine laboratory diagnosis of toxigenic and nontoxigenic C. diphtheriae.     

Characterization of the starch-degrading enzyme ofCorynebacterium diphtheriae typegravis

An inducible cell-surface-bound starch-degrading enzyme produced byCorynebacterium diphtheriae typegravis in tryptose-sodium chloride-maltose broth was partially purified by diethylaminoethylcellulose (DEAE) column chromatography. The partially purified enzyme had approximately six times the specific activity of the crude enzyme. Forty-five percent of the enzyme was recovered in the partially purified form after DEAE chromatography. A molecular weight for the enzyme of approximately 61 200 was obtained by glycerol density gradient centrifugation. The enzyme was inhibited by mercuric ions,p-chloromercuribenzoate, and iodoacetic acid. It was inhibited slightly by EDTA and 1,10-phenanthroline. The enzyme was not inhibited by mercaptoethanol, dithiothreitol, fluoride, bromide, or chloride ions. Activity was lost after precipitation with ethanol, and after concentration with Lyphogel, Sephadex G-15, or lyophilization, but no activity was lost after precipitation with ammonium sulfate. Calcium ions did not activate the enzyme. Maltose was the best inducer of the enzyme. All the saccharides tested induced the enzyme slightly. No phosphorylase was found associated with the organism. However, a phosphatase and possibly an α-glucosidase were produced by the organism. The properties of the starch-degrading enzyme(s) ofCorynebacterium diphtheriae typegravis resemble those of either an amylase, an α-glucosidase, or both. This investigation was supported by Public Health Service grant A102924-10 from the National Institute of Allergies and Infectious Diseases.     

Expression of a biologically active diphtheria toxin fragment B in Escherichia coli

The toxB gene of Corynebacterium diphtheriae bacteriophage β encoding the B fragment of diphtheria toxin was cloned into an inducible expression vector. When expressed In Escherichia coli, fragment B was not proteolysed and was indistinguishable, by immunological criteria, from wild-type C. diphthsriae derived fragment B. Soluble fragment B was partially purified from the cytoplasm by saline precipitation steps and was shown to compete with the wild-type diphtheria toxin for binding to receptors of sensitive eukaryotic cells. A complete diphtheria toxin was reconstituted by formation of the disulphide bridge between purified fragment A and recombinant fragment B, which migrates at the expected Mr on Western blots and which was able to block protein synthesis by ADP-ribosylation of elongation factor–2, thereby indicating that the recombinant fragment B had retained its biological activity.     

Immunological characterization of diphtheria toxin recovered from Corynebacterium pseudotuberculosis

Diphtheria toxin (DT) is a potent toxin produced by the so-called diphtheria group which includes Corynebacterium diphtheriae (C. diphtheriae), Corynebacterium ulcerans (C. ulcerans), and Corynebacterium pseudotuberculosis (C. pseudotuberculosis). The present investigation is aimed to study in detail the production of DT by C. pseudotuberculosis. Twenty isolates were obtained from sheep diseased with caseous lymphadenitis (CLA) and twenty-six isolates were obtained from 26 buffaloes diseased with oedematous skin disease (OSD). All isolates were identified by standard microbiological and DT production was assayed serologically by modified Elek test and immunoblotting. All sheep isolates were nitrate negative, failed to hydrolyze starch and could not produce DT, while all buffalo isolates (biotype II) revealed positive results and a specific band of 62 kDa, specific to DT, was resulted in all concentrated cell fractions (CF), but was absent from non-toxigenic biotype I isolates. At the same time, another band of 31 kDa specific to the PLD gene was obtained with all isolates of biotype I and II. Moreover, all isolates showed positive synergistic hemolytic activity and antagonistic hemolysis with β-hemolytic Staphylococci. The obtained results also indicated that C. pseudotuberculosis could be classified into two strains; non-toxigenic biotype I strain, which failed to produce DT as well as being negative to nitrate and starch hydrolysis, and toxigenic biotype II strain, which can reduce nitrate, hydrolyze starch as well as produce DT.     

Mutation in the Structural Gene for Diphtheria Toxin carried by Temperate Phage β

Corynebacterium diphtheriae strains lyso-genic for phage β are able to produce diphtheria toxin. This article describes evidence suggesting that the toxin structural gene is part of the phage genome.     

Use of whey for production of exocellular polysaccharide by a mutant strain ofXanthomonas campestris

Growth and kinetics of the production of exocellular polysaccharide was studied in a mutant strain ofXanthomonas campestris lac ⁺ during cultivation in a submerged culture in a medium containing whey. The maximum production of the polymer was observed at the initial stage of the stationary growth phase of the culture. The mean production yield was about 1.4%. The results were comparable with those obtained during cultivation on a lactose medium. Translated by Č. Novotny     

Overexpression and purification of the recombinant diphtheria toxin variant CRM197 in Escherichia coli

The expression of the recombinant diphtheria toxin mutant CRM197 in bacteria other than Corynebacterium diphtheriae has proven to be difficult. Here we propose a new and alternative procedure for the production of full-length CRM197 in Escherichia coli. The present study relates specifically to the expression of an artificial sequence and to a method for the isolation and purification of the corresponding protein. In particular, a synthetic gene coding for CRM197, bearing a short histidine tag and optimized for E. coli codon usage, was cloned in the pET9a vector. Accordingly, the over-expression of the protein was simply induced with arabinose in E. coli BL21AI. The recombinant protein was insoluble and always found inside protein aggregates, which were solubilised using urea. Surprisingly, the expression of CRM197, devoid of the short tag, always failed. Following a refolding step, the his-tagged CRM197 was purified by affinity and gel-filtration chromatography and the purity of the final preparation reached 95%. Interestingly, the recombinant protein features DNase activity, indicating that the presence of the tag is not affecting its biochemical properties. However, the removal of the synthetic tag could be easily obtained by incubating the target protein with a proper quantity of a commercial enterokinase.     

A comparative structure‐function analysis of active‐site inhibitors of Vibrio cholerae cholix toxin

Cholix toxin from Vibrio cholerae is a novel mono‐ADP‐ribosyltransferase (mART) toxin that shares structural and functional properties with Pseudomonas aeruginosa exotoxin A and Corynebacterium diphtheriae diphtheria toxin. Herein, we have used the high‐resolution X‐ray structure of full‐length cholix toxin in the apo form, NAD⁺ bound, and 10 structures of the cholix catalytic domain (C‐domain) complexed with several strong inhibitors of toxin enzyme activity (NAP, PJ34, and the P‐series) to study the binding mode of the ligands. A pharmacophore model based on the active pose of NAD⁺ was compared with the active conformation of the inhibitors, which revealed a cationic feature in the side chain of the inhibitors that may determine the active pose. Moreover, a conformational search was conducted for the missing coordinates of one of the main active‐site loops (R‐loop). The resulting structural models were used to evaluate the interaction energies and for 3D‐QSAR modeling. Implications for a rational drug design approach for mART toxins were derived. Copyright © 2015 John Wiley & Sons, Ltd.     

On the relation between toxin production and protein synthesis byCorynebacterium diphtheriae and the iron content of the culture medium

Summary An investigation of the quantitative relationship between toxin production and protein synthesis byC. diphtheriae and the iron content of the medium has been reported on. Both functions can be expressed quantitatively,viz., Lf/ml and Lf/mg P.N.Both functions appeared to be narrowly related to the iron concentration of the medium. The optimal Fe-concentration for the greatest purity of the toxin (Lf/mg P.N.) lies, however, lower than that for the maximal yield of toxin (Lf/ml).In connection with the fact that for the subsequent purification of the toxoid to be obtained from the diphtheria toxin, starting from as pure as possible raw material is essential, a somewhat lower production of toxin than an optimal iron concentration might yield, has to be accepted.     

New Method for the Large-Scale Preparation of Diptheria Toxoid: Purification of Toxin

Diptheria toxin of high purity was prepared in batch cultures of 1 to 40 liters by procedures capable of processing 200-liter batches without modification. The procedure incorporates preliminary purification of the growth medium and, after deep fermentation or surface culture of Corynebacterium diphtheriae, both concentration and partial purification of the toxin by membrane ultrafiltration. Final purification is achieved by Sephadex G-100 gel filtration. Purities of 2,000 to 2,500 flocculation units per mg of protein nitrogen (260 to 410 flocculation units per unit of absorbance at 280 nm) were routinely obtained with only a 10% loss of toxin. The toxin appeared pure on immunoelectrophoresis and ultracentrifugation, and only minor amounts of lower-molecular-weight impurities were revealed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Significant advantages of the procedure are its rapidity and reproducibility and the fact that all stages are performed at 4 C in neutral isotonic buffer.     

Bacteriological Properties of a Sucrose-Fermenting Corynebacterium diphtheriae Strain Isolated from a Case of Endocarditis

An atypical sucrose-fermenting Corynebacterium diphtheriae strain was isolated from three blood cultures of a 14-year-old girl presented to a university teaching hospital in Rio de Janeiro, Brazil. She had mitral endocarditis that proved to be fatal despite intensive antibiotic therapy. Blood cultures showed a fluorescent Gram-positive, aerobic, coryneform-like bacillus presenting pyrazinamidase and CAMP reaction negative. The isolate was identified as a toxigenic strain of C. diphtheriae var. mitis by both Elek and radial immunodiffusion (RID) tests. The invasive C. diphtheriae sucrose-fermenting biotype strain adhered to glass surfaces and expressed pronounced hemagglutinating activity (titer 8), a property common among the nonfermenting biotype strains. Laboratories should be alert to the possibility of the isolation of C. diphtheriae with a positive sucrose fermentation test, especially when nontoxigenic strains are isolated from uncommon anatomic sites. Received: 24 November 1997 / Accepted: 5 March 1998     

Strengthening the laboratory diagnosis of pathogenic Corynebacterium species in the Vaccine era

Over the last three decades, successful implementation of the diphtheria vaccination in the developed and developing countries has reduced the infections caused by the toxigenic strains of Corynebacterium diphtheriae, but a concomitant increase in the invasive infections due to the nontoxigenic strains was seen. In addition, the recent reports on the emergence of nontoxigenic toxin gene‐bearing strains, having the potential to revert back to toxigenic form poses a significant threat to human beings. Besides infections caused by C. diphtheriae, the emergence of the respiratory, cutaneous and invasive infections by related pathogenic Corynebacterium species like C. ulcerans and C. pseudotuberculosis, complicate the diagnosis and management of infection. These observations together with the widespread prevalence of diphtheria in the vaccine era, necessitates the strengthening of the epidemiological surveillance and laboratory diagnosis of the pathogen. This review provides the overview of the advantages and limitations of different molecular methods and the role of MALDI‐TOF in the laboratory diagnosis of Diphtheria. The contribution of next generation sequencing technology and different genotyping techniques in understanding the pathogenicity, transmission dynamics and epidemiology of the C. diphtheriae is discussed.     

Submerged cultivation of a strain ofHumicola lutea 72 producing acid protease

Summary It is demonstrated for the first time that a species from the genusHumicola is a potential source of acid protease. A strain was classified by morphological investigations asHumicola lutea. The influence of constituents of the culture medium on the growth and acid protease production ofH. lutea 72 in submerged cultivation in flasks was investigated. An improved medium was devised for future studies. The optimal aeration rate, inoculum level and cultivation time were determined. A maximal proteolytic activity of 670 g tyrosine liberated from casein ml^–1 culture filtrate min^–1 at pH 3.0 was obtained.