Mapping and comprehensive analysis of the extracellular and cell surface proteome of the human pathogen Corynebacterium diphtheriae

Secreted proteins of the human pathogen Corynebacterium diphtheriae might be involved in important pathogen-host cell interactions. Here, we present the first systematic reference map of the extracellular and cell surface proteome fractions of the type strain C. diphtheriae C7s(-)tox-. The analysis window of 2-DE covered the pI range from 3 to 10 along with a MW range from 8 to 150 kDa. Computational analysis of the 2-D gels detected almost 150 protein spots in the extracellular proteome fraction and about 80 protein spots of the cell surface proteome. MALDI-TOF-MS and PMF with trypsin unambiguously identified 107 extracellular protein spots and 53 protein spots of the cell surface, representing in total 85 different proteins of C. diphtheriae C7s(-)tox-. Several of the identified proteins are encoded by pathogenicity islands and might represent virulence factors of C. diphtheriae. Additionally, four solute-binding proteins (HmuT, Irp6A, CiuA, and FrgD) of different iron ABC transporters were identified, with the hitherto uncharacterized FrgD protein being the most abundant one of the cell surface proteome of C. diphtheriae C7s(-)tox-.     

Use of immunoenzyme analysis for determining antibacterial antibodies in diphtheria infection

A diagnostic EIA system for the detection of antibacterial antibodies in diphtheria infection has been developed. As antigen, homogeneous membrane protein (mol. wt. 64 KD) obtained from Corynebacterium diphtheriae cell walls has been used. This protein antigen has been prepared with the use of nonionic detergent NP-40.     

Immunoadjuvant activities of cell walls and their water-soluble fractions prepared from various gram-positive bacteria.

The cell walls from all 21 species of gram-positive bacteria examined, except lysozyme-susceptible Micrococcus lysodeikticus (NCTC 2665) and lysozyme-resistant Staphylococcus epidermidis (ATCC 155), were found to be definitely adjuvant-active in both stimulation of increased serum antibody levels and induction of delayed-type hypersensitivity to ovalbumin when administered to guinea-pigs as water-in-oil emulsions. Using various cell wall lytic enzymes, the immunoadjuvant principles were solubilized with full retention of the adjuvant activities from walls of Staphylococcus aureus (Copenhagen), Streptococcus pyogens (group A, type 6; S43/100), Streptococcus salivarius (IFO 3350), Streptococcus faecalis (IFO 12580), Streptococcus mutans (BHT), Lactobacillus plantarum (ATCC 8014), Bacillus megaterium (IFO 12068), Corynebacterium diphtheriae (Park-Williams No. 8), Mycobacterium smegmatis, and Actinomyces viscous (ATCC 15987). Evidence was obtained that the non-peptidoglycan portion of the cell walls is not essential for manifestation of immunoadjuvancy.     

Construction and consequences of directed mutations affecting the hemin receptor in pathogenic Corynebacterium species

Genes encoding an ATP-binding cassette transporter system involved in hemin iron utilization from Corynebacterium ulcerans were cloned and characterized. The genes are homologous to a hemin transport system previously identified in Corynebacterium diphtheriae. Disruption of the hmuT gene, which encodes the putative hemin receptor, resulted in greatly reduced ability of C. ulcerans to use hemin or hemoglobin as an iron source. Inactivation of hmuT in C. diphtheriae by site-specific recombination had no effect on hemin utilization, which suggests that C. diphtheriae has an additional system for transporting hemin.     

Feasibility of decreasing the adhesive activity of Corynebacterium diphtheriae by biopolymers of natural origin]

Possible decreasing of the Corynebacterium diphtheriae adhesive activity by natural biopolymers was studied. It was shown that the strains of C.diphtheriae circulating on the Primorye Territory had middle, low or minimal adhesive activity. Natural biopolymers were found to decrease the adhesive properties of C.diphtheriae. The results of the study are promising for further investigation of natural biopolymers as agents preventing C.diphtheriae colonization on the stomatopharynx mucosa.     

Utilization of host iron sources by Corynebacterium diphtheriae: identification of a gene whose product is homologous to eukaryotic heme oxygenases and is required for acquisition of iron from heme and hemoglobin.

Corynebacterium diphtheriae was examined for the ability to utilize various host compounds as iron sources. C. diphtheriae C7(-) acquired iron from heme, hemoglobin, and transferrin. A siderophore uptake mutant of strain C7 was unable to utilize transferrin but was unaffected in acquisition of iron from heme and hemoglobin, which suggests that C. diphtheriae possesses a novel mechanism for utilizing heme and hemoglobin as iron sources. Mutants of C. diphtheriae and Corynebacterium ulcerans that are defective in acquiring iron from heme and hemoglobin were isolated following chemical mutagenesis and streptonigrin enrichment. A recombinant clone, pCD293, obtained from a C7(-) genomic plasmid library complemented several of the C. ulcerans mutants and three of the C. diphtheriae mutants. The nucleotide sequence of the gene (hmuO) required for complementation was determined and shown to encode a protein with a predicted mass of 24,123 Da. Sequence analysis revealed that HmuO has 33% identity and 70% similarity with the human heme oxygenase enzyme HO-1. Heme oxygenases, which have been well characterized in eukaryotes but have not been identified in prokaryotes, are involved in the oxidation of heme and subsequent release of iron from the heme moiety. It is proposed that the HmuO protein is essential for the utilization of heme as an iron source by C. diphtheriae and that the heme oxygenase activity of HmuO is involved in the release of iron from heme. This is the first report of a bacterial gene whose product has homology to heme oxygenases.     

Growth of the surface of Corynebacterium diphtheriae

Surface structure and growth of the surface of Corynebacterium diphtheriae mitis strain were investigated by scanning electron microscopy and the immunofluorescence technique. The surface of the cell revealed by the scanning electron microscope showed a few elevated circular zones which encompassed the cell. The cell diameter increased at this zone and this gave the club-shaped appearance to this species. The cell surface labeled with specific antibodies against the whole bacterial cell and tagged with ferritin remained at a constant length during cell division cycles and the new cell surface emerged from the polar ends of the cell. This new wall surface was completely devoid of the ferritin particles indicating that the cell wall component on the old preexistent wall was completely conserved. A similar finding was obtained by immunofluorescence microscopy. C. diphtheriae, unlike Bacillus spp., showed apical growth as has been observed in fungal cells.     

Heteromorphism of the corynebacteria. III. D- (dark) and C- (clear) cell types and multiseptate specimens]

A population of Corynebacterium diphtheriae may consist of cells with accented (osmiophilic) cytoplasm (cells of the "dark" type, or D type) and cells with cytoplasm having no pronounced osmiophilic properties (cells of the "clear" type, or C type). The divergence of the population into cells of the D and C types occurs at the stage of cell division, the original mother cell being able to divide into 2 or more individual cells belonging to different types (elongated multiseptate cells). At the same time no morphological disturbances in septation may be observed. The ability of each type of cells for division into the corresponding individual daughter cells indicates their being biologically valid. The mixed (D--C) population of Corynebacterium diphtheriae is considered to be a sign of the dissociation of bacterial culture.     

Etiologic role of Corynebacterium non diphtheriae in patients with different pathology

Bacteriologic examination of 1589 patients showed that, aside from C. diphtheriae, 11% of acute upper respiratory tract infections were caused by other Corynebacterium species. Such bacteria can cause infections of various localizations (bronchitis, pyelonephritis, urethritis, colpitis, dermatitis, arthritis, etc.). C. pseudodiphtheriticum and C. xerosis were isolated from clinical specimens most frequently. Corynebacterium spp. have adhesive, hemolytic, hemagglutinating, and neuraminidase activity; some of them are highly pathogenic. The most virulent, were following species: C. diphtheriae, C. pseudotuberculosis, C. urealyticum, and C. ulcerans. Corynebacterium non diphtheriae were frequently isolated from clinical specimens in association with staphylococci and streptococci. In such cases, factors of pathogenicity and resistance to antibiotics were more pronounced. Strains isolated with association with other bacteria have lost susceptibility to tetracycline, oleandomycin, penicillin, and erythromycin. It is important to be vigilant about bacteria from Corynebacterium genus in clinical settings, and thoroughly study their biologic characteristics, especially in immunocompromised patients.     

Cloning in Escherichia coli of the mutant gene coding the diphtheria toxin

Bacteriophage beta 45 of Corynebacterium diphtheriae was harvested. The extracted DNA of the bacteriophage was digested by the restriction endonuclease BamHI and inserted into the BamHI cleavage site of pUC19 vector plasmid. Plasmid pNVY5 containing a mutant gene crm45 of diphtheriae toxin in a 3.9 bpn fragment was isolated from the hybrid plasmids obtained. Cell free extracts of E. coli strain TG1 (pVNY5) contain the nontoxic protein crm45 possessing the specific enzymatic activity of diphtheriae toxin (ADP ribosylation on wheat elongation factor two). According to orientation of BamHI fragment in pNVY5 plasmid it is concluded that the crm45 gene is expressed using its own promoter.     

Identification of a methylase gene for erythromycin resistance within the sequence of a spontaneously deleting fragment of Corynebacterium diphtheriae plasmid pNG2

Abstract A 9.6 Mda Corynebacterium diphtheriae plasmid, pNG2, mediates inducible resistance to erythromycin and other macrolide-lincosamide-streptogramin B (MLS) antibiotics. Spontaneous deletion of a 1.2 Mda fragment of pNG2 leaves the derivative plasmid incapable of conferring MLS-resistance on its host. The pNG2 region containing this fragment was sequenced as were the regions flanking it. The fragment contained 1606 bp, was flanked by almost perfect 23 bp inverted repeats, and appeared to be excised precisely. It contained an open reading frame encoding a leader protein plus a protein which was similar in its amino acid sequence to the rRNA methylases produced by other erythromycin-resistant, Gram-positive bacteria. The C. diphtheriae gene was named erm Cd.     

The draft genome sequence of Corynebacterium diphtheriae bv. mitis NCTC 3529 reveals significant diversity between the primary disease-causing biovars

We report the draft genome of the human pathogen Corynebacterium diphtheriae bv. mitis NCTC 3529. This is the first C. diphtheriae bv. mitis strain to be sequenced and reveals significant differences from the other primary biovar, C. diphtheriae bv. gravis.     

Draft Genome Sequence of Corynebacterium diphtheriae Biovar Intermedius NCTC 5011

We report an annotated draft genome of the human pathogen Corynebacterium diphtheriae bv. intermedius NCTC 5011. This strain is the first C. diphtheriae bv. intermedius strain to be sequenced, and our results provide a useful comparison to the other primary disease-causing biovars, C. diphtheriae bv. gravis and C. diphtheriae bv. mitis. The sequence has been deposited at DDBJ/EMBL/GenBank with the accession number AJVH01000000.     

Fatty and mycolic acid composition of Bacterionema matruchotii and related organisms.

Whole-organism methoanolysates of bacterionemae contained mycolic acids in addition to other long-chain fatty acids. These mycolic acids were similar in general structure and overall size to those found in strains of Corynebacterium diphtheriae and Corynebacterium xerosis. The long-chain fatty acids of bacterionemae, mainly straight-chain saturated and unsaturated acids, were similar to those of certain coryneform bacteria including C. diphtheriae. On the basis of these lipid data, and results of earlier studies, we recommend that the genus Bacterionema be transferred from the family Actinomycetaceae to the Coryneform Group of Bacteria.     

Indices of diphtheria (antitoxic) immunity in the dynamics of the disease and its treatment in adults

A total of 1,034 serum samples from 618 persons, including patients with different forms of diphtheria, carriers of the toxigenic forms of Corynebacterium diphtheriae, and angina patients, were studied. Analysis of the incidence of antibodies to C. diphtheriae toxin and their titers revealed that in more than half of all diphtheria patients no antibodies to C. diphtheriae toxin were detected upon admission to hospital. At the same time in 26% of the patients no antibodies were detected during the whole period of the disease; in such patients the toxic and subtoxic forms of diphtheria were registered twice as often as in seropositive patients. In 31% of the patients seronegative by the moment of hospitalization a rapid increase in the titers of antibodies C. diphtheriae toxin was observed in the course of the disease, which was indicative of the secondary character of immune response in patients who had been immunized earlier.     

Detection of homology to the beta bacteriophage integration site in a wide variety of Corynebacterium spp.

In toxigenic conversion of Corynebacterium diphtheriae C7, beta bacteriophage DNA integrates into either of two chromosomal attachment sites, attB1 or attB2. These attB sites share a 96-base-pair sequence with the attP sites of beta-related phages. The distribution of attB-related sites in other species of Corynebacterium was assessed by hybridization with a DNA probe containing both attB sites of the C7 strain and a second DNA probe containing the attP site of a beta-related phage. All but one of the 15 C. diphtheriae strains tested, regardless of origin or colonial type, contained at least two BamHI fragments that hybridized strongly to both of these probes under conditions of high stringency. Strains of C. ulcerans and C. pseudotuberculosis, species in which conversion to toxinogeny has also been demonstrated, also had one or two hybridizing BamHI fragments. The functionality of these sites as integration sites was demonstrated by isolating lysogens of all three species following single infection with one or more beta-related phages. As predicted, following lysogenization one of the DNA fragments that had exhibited homology with the attB1-attB2 probe was replaced by two hybridizing fragments. Other species of Corynebacterium, including pathogens and nonpathogens from animals, plant pathogens, and soil isolates also carried at least one BamHI fragment that hybridized with the attB1-attB2 and attP probes. The data indicate that sequences homologous to the beta phage integration sites in C. diphtheriae have been conserved in members of the genus Corynebacterium.