[Features of biofilm morphology, formed by Corynebacterium diphtheriae gravis tox+ strains].

AIM: Study the structure of homogenous microbial communities of Corynebacterium diphtheriae gravis tox+ strains during formation of biofilms in vitro. MATERIALS AND METHODS: Object of study--typical and biofilm cultures of C. diphtheriae gravis tox+ museum and circulating strains. Intensity of biofilm formation was evaluated by OD on microplate reader at 540 nm wave length studying 120 and 720 hour cultures. S-450 (Hitachi, Japan) scanning electron microscope was used. RESULTS: The peak of exopolysaccharide matrix (EPS) formation, that is formed in the process of biofilm formation, by museum strain takes place at earlier terms of cultivation (120 hours) than circulating (720 hours). An inverse correlation was established during analysis of bacterial cells of museum and circulating strains of C. diphtheriae during biofilm formation between them and intensity of EPS formation. At maximum EPS content, that took place at various terms of cultivation of the 2 studied strains of diphtheria causative agent, a reduction of corynebacteria cells was observed. CONCLUSION: Bacterial biofilms of museum and circulating strains of C. diphtheriae and patterns of dynamics of EPS reflect, probably, adaptive abilities of the causative agent, that determine its competitiveness in the fight for adhesion sites, resistance to factors of natural immunity and as a result--prolonged persistence in the organism of bacterial carriers.

[THE COMPARATIVE ANALYSIS OF TECHNIQUES OF IDENTIFICATION OF CORYNEBACTERIUM NON DIPHTHERIAE].

The comparative analysis was carried out concerning effectiveness of three techniques of identification of Corynebacterium non diphtheriae: bacteriological, molecular genetic (sequenation on 16SpRNA) andmass-spectrometric (MALDI-ToFMS). The analysis covered 49 strains of Corynebacterium non diphtheriae (C.pseudodiphheriticum, C.amycolatum, C.propinquum, C.falsenii) and 2 strains of Corynebacterium diphtheriae isolated under various pathology form urogenital tract and upper respiratory ways. The corinbacteria were identified using bacteriologic technique, sequenation on 16SpRNA and mass-spectrometric technique (MALDIToF MS). The full concordance of results of species' identification was marked in 26 (51%) of strains of Corynebacterium non diphtheriae at using three analysis techniques; in 43 (84.3%) strains--at comparison of bacteriologic technique with sequenation on 16S pRNA and in 29 (57%)--at mass-spectrometric analysis and sequenation on 16S pRNA. The bacteriologic technique is effective for identification of Corynebacterium diphtheriae. The precise establishment of species belonging of corynebacteria with variable biochemical characteristics the molecular genetic technique of analysis is to be applied. The mass-spectrometric technique (MALDI-ToF MS) requires further renewal of data bases for identifying larger spectrum of representatives of genus Corynebacterium.

[Adhesion of corynebacterium diphtheriae: the role of surface structures and formation mechanism].

The paper is devoted to the study of surface structures including pili (fimbriae) 67-72p surface protein, DIP 1281 surface protein, lipoarabinomannan CdiLAM and their role in the adhesion and colonization of the mucous membrane of the throat by Corynebacterium diphtheriae. A description is offered for the main stages in the adhesion process of diphtheria causative agent and the ability of its adhesins to stimulate the effect of innate and acquired immunity factors. The paper stresses prospectiveness of the development of vaccines forming immunoprotection of the organism against adhesive activity of C. diphtheriae and also preventing their colonization and reproduction. That would facilitate a solution for the problem of diphtheria carrier state, which cannot be solved using the existing means of preventive vaccination.

SubMICs of penicillin and erythromycin enhance biofilm formation and hydrophobicity of Corynebacterium diphtheriae strains.

Subinhibitory concentrations (subMICs) of antibiotics may alter bacterial surface properties and change microbial physiology. This study aimed to investigate the effect of a subMIC (⅛ MIC) of penicillin (PEN) and erythromycin (ERY) on bacterial morphology, haemagglutinating activity, cell-surface hydrophobicity (CSH) and biofilm formation on glass and polystyrene surfaces, as well as the distribution of cell-surface acidic anionic residues of Corynebacterium diphtheriae strains (HC01 tox(-) strain; CDC-E8392 and 241 tox(+) strains). All micro-organisms tested were susceptible to PEN and ERY. Growth in the presence of PEN induced bacterial filamentation, whereas subMIC of ERY caused cell-size reduction of strains 241 and CDC-E8392. Adherence to human erythrocytes was reduced after growth in the presence of ERY, while CSH was increased by a subMIC of both antibiotics in bacterial adherence to n-hexadecane assays. Conversely, antibiotic inhibition of biofilm formation was not observed. All strains enhanced biofilm formation on glass after treatment with ERY, while only strain 241 increased glass adherence after cultivation in the presence of PEN. Biofilm production on polystyrene surfaces was improved by ⅛ MIC of ERY. After growth in the presence of both antimicrobial agents, strains 241 and CDC-E8392 exhibited anionic surface charges with focal distribution. In conclusion, subMICs of PEN and ERY modified bacterial surface properties and enhanced not only biofilm formation but also cell-surface hydrophobicity. Antibiotic-induced biofilm formation may contribute to the inconsistent success of antimicrobial therapy for C. diphtheriae infections.

Contour and persistence length of Corynebacterium diphtheriae pili by atomic force microscopy.

Many bacteria are characterized by nanoscale ultrastructures, for example S-layers, flagella, fimbriae, or pili. The last two are especially important for attachment to different abiotic and biotic surfaces and for host-pathogen interactions. In this study, we investigated the geometric and elastic properties of pili of different Corynebacterium diphtheriae strains by atomic force microscopy (AFM). We performed quantitative contour-length analysis of bacterial pili and found that the visible contour length of the pili can be described by a log-normal distribution. Our data revealed significant strain-specific variations in the mean visible contour length of the pili, ranging from 260 to 1,590 nm. To estimate their full contour length, which is not directly accessible from the AFM images, we developed a simple correction model. Using this model, we determined the mean full contour length as 510-2,060 nm. To obtain the persistence length we used two different methods of analysis, one based on the end-to-end distance of the pili and one based on the bending angles of short segments. In comparison, the bending angle analysis proved to be more precise and resulted in persistence lengths in the narrow range of 220-280 nm, with no significant strain-specific variations. This is small compared with some other bacterial polymers, for example type IV pili, F-pili, or flagella.

Strain-specific differences in pili formation and the interaction of Corynebacterium diphtheriae 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. RESULTS: Adhesion of different C. diphtheriae strains to epithelial cells and invasion of these cells are not strictly coupled processes. Using ultrastructure analyses by atomic force microscopy, significant differences in macromolecular surface structures were found between the investigated C. diphtheriae strains in respect to number and length of pili. Interestingly, adhesion and pili formation are not coupled processes and also no correlation between invasion and pili formation was found. Using RNA hybridization and Western blotting experiments, strain-specific pili expression patterns were observed. None of the studied C. diphtheriae strains had a dramatic detrimental effect on host cell viability as indicated by measurements of transepithelial resistance of Detroit 562 cell monolayers and fluorescence microscopy, leading to the assumption that C. diphtheriae strains might use epithelial cells as an environmental niche supplying protection against antibodies and macrophages. CONCLUSIONS: The results obtained suggest that it is necessary to investigate various isolates on a molecular level to understand and to predict the colonization process of different C. diphtheriae strains.

Acyl enzyme intermediates in sortase-catalyzed pilus morphogenesis in gram-positive bacteria.

In gram-positive bacteria, covalently linked pilus polymers are assembled by a specific transpeptidase enzyme called pilus-specific sortase. This sortase is postulated to cleave the LPXTG motif of a pilin precursor between threonine and glycine and to form an acyl enzyme intermediate with the substrate. Pilus polymerization is believed to occur through the resolution of this intermediate upon specific nucleophilic attack by the conserved lysine located within the pilin motif of another pilin monomer, which joins two pilins with an isopeptide bond formed between threonine and lysine. Here, we present evidence for sortase reaction intermediates in Corynebacterium diphtheriae. We show that truncated SrtA mutants that are loosely bound to the cytoplasmic membrane form high-molecular-weight complexes with SpaA polymers secreted into the extracellular milieu. These complexes are not formed with SpaA pilin mutants that have alanine substitutions in place of threonine in the LPXTG motif or lysine in the pilin motif. The same phenotype is observed with alanine substitutions of either the conserved cysteine or histidine residue of SrtA known to be required for catalysis. Remarkably, the assembly of SpaA pili, or the formation of intermediates, is abolished with a SrtA mutant missing the membrane-anchoring domain. We infer that pilus polymerization involves the formation of covalent pilin-sortase intermediates, which occurs within a molecular platform on the exoplasmic face of the cytoplasmic membrane that brings together both sortase and its cognate substrates in close proximity to each other, likely surrounding a secretion apparatus. We present electron microscopic data in support of this picture.

Sortases and pilin elements involved in pilus assembly of Corynebacterium diphtheriae.

Corynebacterium diphtheriae SpaA pili are composed of three pilin subunits, SpaA, SpaB and SpaC. SpaA, the major pilin protein, is distributed uniformly along the pilus shaft, whereas SpaB is observed at regular intervals, and SpaC seems to be positioned at the pilus tip. Pilus assembly in C. diphtheriae requires the pilin motif and the C-terminal sorting signal of SpaA, and is proposed to occur by a mechanism of ordered cross-linking, whereby pilin-specific sortase enzymes cleave precursor proteins at sorting signals and involve the side-chain amino groups of pilin motif sequences to generate covalent linkages between pilin subunits. We show here that two elements of SpaA pilin precursor, the pilin motif and the sorting signal, are together sufficient to promote the polymerization of an otherwise secreted protein by a process requiring the function of the sortase A gene (srtA). Five other sortase genes are dispensable for SpaA pilus assembly. Further, the incorporation of SpaB into SpaA pili requires a glutamic acid residue within the E box motif of SpaA, a feature that is found to be conserved in other Gram-positive pathogens that encode sortase and pilin subunit genes with sorting signals and pilin motifs. When the main fimbrial subunit of Actinomyces naeslundii type I fimbriae, FimA, is expressed in corynebacteria, C. diphtheriae strain NCTC13129 polymerized FimA to form short fibres. Although C. diphtheriae does not depend on other actinomycetal genes for FimA polymerization, this process involves the pilin motif and the sorting signal of FimA as well as corynebacterial sortase D (SrtD). Thus, pilus assembly in Gram-positive bacteria seems to occur by a universal mechanism of ordered cross-linking of precursor proteins, the multiple conserved features of which are recognized by designated sortase enzymes.

Comparative cell wall core biosynthesis in the mycolated pathogens, Mycobacterium tuberculosis and Corynebacterium diphtheriae.

The recent determination of the complete genome sequence of Corynebacterium diphtheriae, the aetiological agent of diphtheria, has allowed a detailed comparison of its physiology with that of its closest sequenced pathogenic relative Mycobacterium tuberculosis. Of major importance to the pathogenicity and resilience of the latter is its particularly complex cell envelope. The corynebacteria share many of the features of this extraordinary structure although to a lesser level of complexity. The cell envelope of M. tuberculosis has provided the molecular targets for several of the major anti-tubercular drugs. Given a backdrop of emerging multi-drug resistant strains of the organism (MDR-TB) and its continuing global threat to human health, the search for novel anti-tubercular agents is of paramount importance. The unique structure of this cell wall and the importance of its integrity to the viability of the organism suggest that the search for novel drug targets within the array of enzymes responsible for its construction may prove fruitful. Although the application of modern bioinformatics techniques to the 'mining' of the M. tuberculosis genome has already increased our knowledge of the biosynthesis and assembly of the mycobacterial cell wall, several issues remain uncertain. Further analysis by comparison with its relatives may bring clarity and aid the early identification of novel cellular targets for new anti-tuberculosis drugs. In order to facilitate this aim, this review intends to illustrate the broad similarities and highlight the structural differences between the two bacterial envelopes and discuss the genetics of their biosynthesis.

Assembly of pili on the surface of Corynebacterium diphtheriae.

Pili of Gram-negative pathogens are formed from pilin precursor molecules by non-covalent association within the outer membrane envelope. Gram-positive microbes employ the cell wall peptidoglycan as a surface organelle for the covalent attachment of proteins, however, an assembly pathway for pili has not yet been revealed. We show here that pili of Corynebacterium diphtheriae are composed of three pilin subunits, SpaA, SpaB and SpaC. SpaA, the major pilin protein, is distributed uniformly along the pilus shaft, whereas SpaB is observed at regular intervals and SpaC seems positioned at the pilus tip. Assembled pili are released from the bacterial surface by treatment with murein hydrolase, suggesting that the pilus fibres may be anchored to the cell wall envelope. All three pilin subunit proteins are synthesized as precursors carrying N-terminal signal peptides and C-terminal sorting signals. Some, but not all, of the six sortase genes encoded in the genome of C. diphtheriae are required for precursor processing, pilus assembly or cell wall envelope attachment. Pilus assembly is proposed to occur by a mechanism of ordered cross-linking, whereby pilin-specific sortase enzymes cleave precursor proteins at sorting signals and involve the side chain amino groups of pilin motif sequences to generate links between pilin subunits. This covalent tethering of adjacent pilin subunits appears to have evolved in many Gram-positive pathogens that encode sortase and pilin subunit genes with sorting signals and pilin motifs.

[The use of DNA fingerprint analysis for the differentiation of populations of toxigenic Corynebacterium diphtheriae]. / Primenenie fingerprintnogo analiza DNK dlia differentsiatsii populiatsii toksigennykh Corynebacterium diphtheriae.

13 C. diphtheriae strains were used as a model to establish the conditions of making the fingerprint analysis of chromosomal DNA. These strains, subdivided into 7 groups in accordance with the character of their restriction splitting, were mostly isolated from territorially close sources and belonged to the same phagotype. Probably, C. diphtheriae DNA has strain variations manifested by an unequal number and location of the sites of the recognition of specific endonucleases, which may be used for the intraspecific differentiation of C. diphtheriae.

[Adhesion in Corynebacterium diphtheriae]. / Adgeziia u Corynebacterium diphtheriae.

The study of 8 C. diphtheriae strains of different origin revealed that these strains were capable of inducing the agglutination of trypsinized sheep red blood cells (SRBC). Toxigenic strains gravis isolated from diphtheria patients were more active in their adhesion to SRBC than toxigenic strains gravis isolated from carriers. The latter were, in their turn, more active than nontoxigenic strains mitis. No fimbriae were detected on the cell surface by electron microscopy.

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 corynebacteria. I. Macrocells]. / Geteromorfizm korinebakterii. Soobshchenie I. Makrokletki.

A possibility of formation of macrocells (MaC) of diphtheria bacilli was expressed to a different degree; the extreme expression are giant and supergiant forms. Giant forms with section dimension of 3--5micron retained their capacity to disorderly septation with the resultant formation of microcells (MiC). Apparently some of the septa were not realized. Supergiant forms were revealed as a layer and its transverse section. The length of the latter reached 30micron with the transverse section of 2--3micron. The layer has festooned contours with shallow invaginations, but marked day-like cuttings into the body (in case of transverse layer section). Septation of supergiant form was abortive in character. Both forms had homogeneous cytoplasm surrounded by a cell wall. In the supergiant form the latter was morphologically defective in the deepest part of the invaginates. The ultrastructure of both bacterial forms pointed to their rapid growth; however, the supergiant form was likely to be doomed to degeneration and chaotic disintegration.

Studies on the fine structure of corynebacterium diphtheriae-granules.

The seemingly homogeneous metachromatic and electron-scattering granules of Corynebacterium diphtheriae have two structural components as revealed under conditions of low beam load and by use of ribonuclease. They contain high percentages of P and Ca. Findings point to participation of ribosomes and intracytoplasmic membranes in the formation of the granules.

[Extracellular material of some representatives of the genus Corynebacterium (the electron microscopic aspect)]. / K voprosu ob éktratselliuliarnom materiale nekotorykh predstavitelei roda Corynebacterium (élektronno-mikroskopicheskii aspekt).

At the active developmental phases (up to 2 days) cells of toxigenic and nontoxigenic corynebacteria form extracellular vesicle-like material of two types which can be revealed both on whole cells set off by metal and stained negatively, and in ultrathin sections. Extracellular material of the first type is a derivative of an extensive membranous coat of corynebacteria and is formed as a result of its fragmentation. Vesicles of this type are devoid of electron microscope-dense content, have no tendency to coalescence, and fail to promote cell agglutination. Extracellular material of the second type is primarily formed by local thickening of the surface wall layer limited by its external dense layer and the main massif. These thickenings are filled with a microgranular substance of medium electron optic density; after accumulation this substance is released into the external environment. Vesicles of this type promote cell agglutination. Undoubtedly, extracellular material of the second type has a direct relation to the cell metabolic processes. Extracellular material of both types is encountered in all the cultures grown both on hard and in fluid nutrient media. However, in the latter case the process is apparently much more intensive. There is strict correlation between the morphology of the extracellular material and strain signs of the culture, although material of the second type is found to prevail in the cells from cultures possessing toxigenic activity.

[Comparative electron-microscopic study of 8 representatives of the genus Corynebacterium grown on solid nutrient medium during the stationary phase of development]. / Sravnitel'noe elektronno-mikroskopicheskoe izuchenie 8 predstavitelei roda Corynebacterium, vyrashchennykh na tverdoi pitatel'noi srede v statsionarnoi faze razvitiia

After 18 hours of growth on selective serum-agar medium C diphtheriae cultures with different toxicogenic activity, and also diphtheroid and Hoffmann's baccillus cultures were removed, washed of the remnants of the nutrient medium and fixed under cold conditions by two combined methods (with glutaric aldehyde-osmic acid--uranyl acetate, and potassium permeanganate--uranyl acetate). The preparations were studied in ultrathin sections. It appeared that corynebacteria had during the stationary phase of development a general structural plan characteristic of Gram positive microorganisms and for all the corynebacterium genus. Cells of diphtheria toxicognic strains had signs of the accelerated (in comparison with other strains) rate of development; the principal mass of toxicogenic cells after 18 hours of growth had morphological signs of the stage of rest. The majority of cells whose toxicogenicity was inconstant had an extensive microcapsule which was also a characteristic element of the diphtheroid and Hoffmann's bacillus ultrastructure. The total thickness of the walls in the cells of toxicogenic strains and of the strains whose toxicogenicity was inconstant constituted 190-200 A; in nontoxicogenic strains, diphtheroid and Hoffmann's bacillus it was from 230 to 320 A. Surface structures of corynebacteria were differentiated better in the cells with toxicogenic activity. In the majority of cells of nontoxicogenic strains and also diphtheroid and Hoffmann's bacillus individual wall layers were differentiated with difficulty.