Ultrastructural localization of the fibrinogen-binding domain of streptococcal M protein.

Binding of fibrinogen to the M protein located on the surface fibrillae of group A streptococci impedes deposition of complement and thus contributes to the virulence of these organisms. We investigated this binding by electron microscopy using postembedding immunogold labeling. Both fibrinogen and its D fragment formed a distinct dense layer in the surface fibrillae, separated by 10 nm from the compact part of the cell wall. Labeling the sections with anti-fibrinogen or anti-fragment D showed that the fibrinogen-binding region lay within a 25-nm segment of the fibrillae beginning approximately 30 nm from the inner surface of the cell wall. The outer surface of the fibrinogen layer could be labeled with antibody to the amino-terminal half of type 24 M protein, indicating that the fibrillar tips remained exposed after fibrinogen binding. The degree of labeling with anti-fibrinogen, determined by gold particle counting, was the same whether the bacterial cells had been incubated with purified fibrinogen or whole plasma. These results indicate that the fibrinogen-binding region lies in the distal (amino-terminal) half of the M protein molecule but excludes the most distal portion, which is the site of epitopes that interact with opsonic anti-M antibody, and that plasma proteins other than fibrinogen, a number of which are known to bind to group A streptococci, do not interfere with fibrinogen binding.

[Biological properties of Streptococcus group B: the interrelations of the virulence in mice, the adhesion to human epithelial cells and the production of type-specific antigen]. / Biologicheskie svoistva streptokokkov gruppy B: vzaimootnosheniia mezhdu virulentnost'iu dlia myshei, adgeziei k épitelial'nym kletkam cheloveka i produktsiei tipospetsificheskogo antigena.

The results presented in this work confirm the possibility of selecting the subpopulations of group B streptococci by the passage of these microorganisms through mice. This process was accompanied by the accumulation of cells with a high level of type-specific antigen (TSA). The passage of group B streptococci in the presence of type-specific antibodies led to the selection of avirulent microorganisms with low TSA production and high adhesiveness. These data may be considered to be the indirect evidence of the screening effect of TSA contained in the capsule of group B streptococci with respect to the ligand structures of these microbes. This suggestion is confirmed by the behavior of the variants of group B streptococci, obtained in the course of this investigation, on virus-infected tissue when TSA+ strains lost their ability to recognize viral polypeptides serving as receptors for TSA- variants of the streptococci.

Electron microscopic localization of lipoteichoic acid on group A streptococci.

The location of lipoteichoic acid (LTA) on the surface of group A streptococci was studied by immunoelectron microscopic and ultrastructural cytochemical methods, i.e. by means of LTA antibodies labelled with ferritin, or concanavalin A labelled with ferritin or colloidal gold. All these methods proved the LTA to be located on the outer cell surface of most group A streptococcus strains. The differences in the intensity of labelling paralleled the hydrophobicity of the strains, being substantially higher in the strains exhibiting a high degree of hydrophobicity. Treatment of streptococci with pronase or trypsin led to a complete loss of surface-located LTA. On the other hand, pepsin treatment of streptococci under mild conditions resulted in an increased amount of surface-located LTA in some strains. On the isolated cell walls, LTA could be demonstrated only on the outer surface of the walls. These findings correlated well with the presumed role of group A streptococcus LTA in the adherence of streptococci to the epithelial cells which is accomplished with the aid of surface-located LTA molecules.

Immuno-electronmicroscopic demonstration of capsules on group-B streptococci of new serotypes and type candidates.

The location of type polysaccharides on the cells of reference strains of group-B streptococci of serotypes IV and V and new type candidates NT6 and 7271 was investigated by electronmicroscopy of the bacteria after incubation with homologous type-specific antiserum. A distinct capsular layer was found on the surface of the cells of all these strains. Sialic acid, an integral part of all the conventional type polysaccharides of group-B streptococci, was also detected in all the strains examined.

Adherence of group B streptococci to human buccal epithelial cells, its dependence on the biological state of the culture.

The adherence activity of Streptococcus agalactiae (group B) strains is directly dependent on the biological state of cultures. The aim of the present paper was to consider the effect of repeated transfers of cultures alternately on solid and liquid media and the effect of the growth phase. The strains, adhering weakly, strongly and variably to epithelial cells were employed in studies on the effect of repeated transfer. The percentage of adherent epithelial cells differed significantly after the first or the second transfer. On storage of the strains after the 3rd transfer at 4 degrees C for 4 d, the adherence activity decreased to the level of non-transferred strains. Ultrastructural analysis revealed in all strains the presence of capsular material, its character being similar both in strongly and in weakly adhering cultures. In weakly adherent strains, the structure of the capsular layer has not changed during transfer whereas the adherence of the strain increased considerably. The effect of the growth phase was studied during 3-48 h of incubation. The growth phase did not influence the adherence activity in strains that had been allowed to multiply for 3-24 h. After a long-term multiplication beyond 24 h, the adherence activity gradually decreased.

Adherence to epithelial cells and ultrastructure of fosfomycin-resistant mutants of group A streptococci.

Adherence of three strains of group A streptococci and their fosfomycin-resistant mutants to HEp-2 tissue culture cells was compared with some cell-surface characteristics, i.e. ultrastructure and hydrophobicity. Among Fosr mutants, both well-adhering and weakly adhering mutants were found. Clonal analysis of the mutants proved their greater stability in the adherence. Well-adhering parent strains of streptococci and Fosr mutants exhibited surface fibrillae in contrast to weakly adhering Fosr mutants which were devoid of fibrillae or contined fibrillae of lower electron density. Decrease of adherence of Fosr mutants of two strains was accompanied by a decrease of their hydrophobicity.

Blood-platelets-damaging activity of some synthetic Streptococcus peptidoglycan subunits and analogues.

A series of 25 synthetic subunits or analogues of streptococcal peptidoglycan was tested for their ability to damage rabbit blood platelets. The morphological changes of the platelets were studied on an ultrastructural level. Minimal subunit structure able to produce a complete lysis of the platelets was found to be muramyldipeptide (MDP). Comparable lysis of platelets was also caused by muramyltetrapeptide and MDP containing D-Ala instead of L-Ala. The lytic effect was dose-dependent and was exhibited rather after high doses of the substances used (up to 500 micrograms/ml). For the lytic reaction, the configuration of C3 and C4 in the muramyl residue of MDP was essential. Many substances produced only degranulation without lysis of the platelets and some of them did not influence the platelet ultrastructure at all. The paper presents some structure-to-function relationships of the compounds and shows that the platelet-damaging activity of peptidoglycan may be related to certain portions of the peptidoglycan molecule. This activity should be tested when the immunomodulatory substances derived from the bacterial peptidoglycan are searched for.

Electron microscopic study of the effect of gentamicin and oxacillin on Staphylococcus aureus cells: significance of time and sequence of application in combined effect.

The ultrastructure of Staphylococcus aureus cells was studied after their treatment with gentamicin and oxacillin, each used alone or in a simultaneous or successive combination. Gentamicin (concentrations 1 or 2 micrograms/ml, incubation time 1-24 h) did not produce any ultrastructural changes in staphylococcus cells. Oxacillin in the same concentrations caused derangement of cell division accompanied by an increase in cross-wall and decrease of peripheral cell wall thicknesses. Simultaneous incubation of staphylococcus cells with gentamicin and oxacillin (both 1 microgram/ml) led to morphological changes corresponding to those induced by oxacillin. On the other hand, successive incubation with oxacillin (2-5 h), followed by gentamicin (5-24 h) caused complete destruction of staphylococci. This effect was not observed when cells were first incubated with gentamicin and then oxacillin. The study shows the importance of time and sequence factors in the combined effect of tested antimicrobials.

Adherence of vaginal and pharyngeal strains of group B streptococci to human vaginal and pharyngeal epithelial cells.

In vitro tests for adherence to human vaginal and pharyngeal epithelial cells were used to study the problem of tissue-specific tropism in group B streptococci (GBS). Twenty-two vaginal or pharyngeal clinical isolates of GBS (serotypes Ia, Ib, II, and III) were used. No significant differences in adherence to vaginal and pharyngeal epithelial cells were found between GBS from both sources: statistical analysis furnished no evidence for tissue-specific tropism. Serotype III vaginal GBS adhered better to vaginal and pharyngeal epithelial cells than did serotype III GBS strains isolated from the pharynx. However, pronounced differences in the level of adherence were found among strains of the same serotypes and from the same sources. Thus, the results obtained suggest that differences in adherence may rather be strain-dependent that type-dependent.

The adhesin structures involved in the adherence of group B streptococci to human vaginal cells.

The adherence of group B streptococci (GBS) of serotypes Ia, II and III to human vaginal cells was studied in vitro. The adherence was not dependent on the viability of bacteria; killing of GBS by UV irradiation or glutaraldehyde treatment did not inhibit the adherence. Killing of GBS by heating to 56 degrees C for 1 h led to a pronounced decrease of adherence, demonstrating the thermosensitivity of the GBS structures involved. The protein nature of these structures was proved by a significant reduction of adherence after pretreatment of GBS with trypsin or pepsin. Pretreatment of GBS with sialidase had no influence on the adherence. Such a pretreatment of vaginal cells caused an increase of adherence showing that the receptors on epithelial cells may be partly masked by sialic acid.

Binding of horse-spleen ferritin to group A streptococci.

Receptors for horse-spleen ferritin were found on group A streptococci. Both electron microscopic and chemical investigation of Streptococcus cells treated with ferritin showed that M + variants of group A streptococci were able to bind substantially more ferritin than M - variants of the same serotypes. Ferritin receptors were located on the tops of filamentous protrusions of Streptococcus cell walls and only on the outer surface of isolated cell walls. Trypsin treatment destroyed the ferritin-binding capacity of streptococci completely, while mild pepsin treatment left the ferritin receptors undisturbed, or uncovered additional ones. The ferritin receptors were not identical with receptors for the Fc-portion of swine IgG. The finding of ferritin receptors on bacteria necessitates careful interpretation of results obtained by immunoferritin localization techniques.

Biological activity of synthetic subunits of streptococcus peptidoglycan. III. Relationship of subunit and analogue structure to adjuvant activity in cell-mediated immunity.

Each of a series of synthetic peptidoglycan subunits and subunit analogues was injected in combination with streptococcus type M24 antigen extract. The substances tested were: (8a) N-acetylmuramyldipeptide (MDP) and the following derivatives thereof: MDP modified in positions C3 and C4, or with L-alanine substituted by L-2-aminobutyric acid or with the peptide chain prolonged (by three lysines or a polylysine); (b) some synthetically prepared peptides: a hexapeptide, a tridecapeptide and an octadecapeptide. Configurations in positions C3 and C4 were found essential for the adjuvant effect. Adjuvant activity, though somewhat lower than in MDP, was pronounced in the analogue containing the L-2-aminobutyryl residue. Surprisingly, potent adjuvant effect was displayed by the hexapeptide; prolongation of the peptidic chain was not effective. The use of a polymeric carrier for MDP increased the adjuvant effect. Contrary to expectation, streptococcal antigens used with immunoadjuvant materials showed that induced delayed hypersensitivity was type related.

[Adhesion of group B streptococci to vaginal epithelial cells]. / Adgeziia streptokokkov gruppy B na kletkakh vaginal'nogo épiteliia.

The work presents the results of studies on the optimum and standard conditions for the in vitro determination of the adhesiveness of group B streptococci with epithelial cell suspensions. Vaginal epithelium has proved to be the most convenient and adequate system for studying the adhesiveness of group B streptococci. The optimum infective dose of these bacteria has been found to range from 50 to 200 cocci per cell. The characteristics of the adhesion of group B streptococci to vaginal epithelium are highly reproducible and exhibit low dependence on the time of the incubation of the bacteria with epithelial cells; fluctuations in the adhesiveness of the cultures in the definite range of pH shifts are seemingly determined by the serotype of the strains.

[Submicroscopic aspects of the adherence of group B streptococci to vaginal epithelial cells]. / Submikroskopické aspekty adherence streptokoku skupiny B k vaginálním epiteliálním bunkám.

The submicroscopic structure of surfaces in the streptococci of group B, type III (strain 13/63) and the ultrastructure of the interaction of this streptococcus strain with human vaginal cells were studied. The surface of the majority of B streptococci was smooth after using the conventional fixing techniques of electron microscopy; however, about 25% of streptococcal cells had an additional layer of filamentous protrusions on their surface. A marked layer of capsular material was visualized by means of the preincubation of this streptococcus strain with a type-specific antiserum. The incubation of B streptococci with vaginal cells without any addition of antibody allowed for the demonstration of the contact of a part of the cells of the bacterial population with the epithelial cells through filamentous protrusions. However, the majority of the smooth-surface cells was separated from vaginal cells by a gap wide up to 150 nm. It was demonstrated by additional incubation with type-specific antiserum that the above mentioned gap corresponded to the capsular substance of bacteria. Hence the capsule of streptococci in group B is the basic component of their surface responsible for adherence to vaginal cells. Adherence of B streptococci to vaginal epithelia was accompanied neither by bacterium ingestion nor by the destruction of epithelial cells.

Isolation of varicella-zoster virus from pharyngeal and nasal swabs in varicella patients.

Thirteen children aged 5 months to 4 years were observed during a varicella epidemic in an Infants' Hospital; except for two normal individuals, the children had various forms of congenital defects. Eleven of the children developed varicella. During the first 3 days of exanthem, a total of 17 VZ virus strains were isolated: 12 from vesicular fluid, 3 from 23 nasal and 2 from 22 pharyngeal swabs. No strain was isolated during the incubation period despite 57 and 56 swabs having been collected from the throat and nose, respectively; nor was VZ virus isolated from 6 pharyngeal and 7 nasal swabs taken on the first day of exanthem. Isolation attempts performed from vesicular fluid to control quality of the isolation conditions gave a positivity rate of 100%. Under these optimal isolation conditions VZ virus was found in the nose or throat alongside skin vesicles in four of the 11 ill children. Besides VZ virus, the pharyngeal and nasal swabs yielded, respectively, four and four cytomegalovirus strains. The cytomegalovirus infections were inapparent.

Morphological evidence for different types of IgG-Fc receptors in group A streptococci.

The binding of immunoglobulin G (IgG) from man and 14 animal species to Group A streptococcal cells was studied by electron microscopy. Only the IgGs of rhesus monkey and guinea pig did not bind to any of the streptococcal strains tested. Fc receptors were seen located on filamentous protrusions of the streptococcal cell wall either regularly or in focal distribution. On the inner surface of isolated cell walls no Fc receptors were detected. Both the different labelling patterns for different IgG and the results of inhibition studies with homologous and heterologous IgGs suggested the existence of different types of Fc receptors in the same strain. Further characterization of the receptors, including their location, was performed by studying their susceptibility to several proteolytic enzymes.