Cross-reacting antigens of human thymus myoid cells and stable L-forms of group A streptococci

Indirect immunofluorescence has shown a similarity between the antigen components of group A streptococcus L-forms and human thymus myoid cells. An analogous antigen (or antigens) is present in the cytoplasmic membrane of human myocardial cell fibers. The depletion of antiserum to the streptococcal L-forms both by the culture of L-forms grown in meat or casein media and by the homogenate of the cardiac muscle leads to the inhibition of immunofluorescence. The depletion of serum by the homogenate of other tissues (liver) or by L-form culture does not virtually affect the immunofluorescence intensity. According to the authors' opinion, the similarity of antigens of group A streptococcus L-forms to the antigenic components of organ tissues is likely to be responsible for long-term persistence of the microorganisms under consideration and to favour, in some cases, the occurrence of autoantibodies. The latter circumstance might lead to pathological changes in organs containing cross-reacting antigens.     

Common antigens of stable group A streptococcal L forms and muscle fibers of human myocardium]

The immunofluorescence method was used to demonstrate that the antigens of stable streptococcus L-forms and of the cytoplasmic membrane of human myocardium muscle fibers were common. The common antigen was included into the composition of the surface membrane of the muscle cell adjacent to the sarcolemma, and of the transverse tubule membranes of the sarcoplasma reticulum passing in the Z-band region of the muscle fiber sarcomeres. The reactions is completely prevented by the exhaustion of the anti-serum to the antigen of the L-forms with the homogenate of human myocardium tissues or a suspension of the L-form cultures grown both on meat and on casein media. Exhaustion with tissue homogenate of other organs (the liver) or with concentrated nutrient medium practically failed to influence the extent of this reaction. The authors believe that the commonnes of the antigens of stable L-forms of streptococcus, group A, cultures and of the myocardium could serve as one of the causes of prolonged persistence of L-forms in human and animal organism.     

Identification with the aid of various serological reactions of L forms of streptococci isolated from the live organism]

Growth inhibition, agglutination, precipitation, and passive hemagglutination tests were used for the identification of the L-forms of streptococci isolated from the organism of experimental rabbits both after the infection with the L-forms of streptococci and with the streptococci of group A. The tests were positive not only with the antiserum of homologous, but also of heterologous strains of the L-form of streptococcus, group A. The L-form cultures isolated from the experimental animals failed to differ from the laboratory strain of the L-forms of streptococcus, group A, by serological properties.     

Further study of the L forms of Streptococci in blood cultures from the patients with rheumatic fever depending on the course of the disease and the methods of cultivation]

Cultures of blood from patients with rheumatism during the active and inactive phases of the disease, treated and untreated with antibiotics were studied. The following results were obtained in blood cultures (on Klodnisky's medium with a 0.01% cystine) of 105 patients with rheumatism: L-forms of streptococcus were isolated in the 88 cases, irrespective of the severity of the disease or the treatment, streptococcus--in 8, and the result proved to be negative in 9 cases. Hemoculture on broth with a 1% glucose solution proved to be positive (streptococcus) in one patient only. The constancy with which the L-forms of streptococcus were isolated in hemocultures from the patients with rheumatism and also the peculiarity of their pathogenic properties modelling the rheumatism under experimental conditions pointed to their role in the pathogenesis of rheumatism. A method of isolation of hemocultures on Klodnitsky's medium with a 0.01% cystine can be recommended for the diagnosis of a septic condition caused by the L-forms of streptococcus in patients suffering from rheumatism.     

Ultrastructure of LLR-mutants of E. coli K12]

The author studied the ultrastructure of two spherical E. coli K12 mutants (llr) obtained under the effect of N-nitroso-N-methylurea. Seven morphological types of cells differing from one another by shape, size and cytoarchitectonics were distinguished. Superficial structures of the majority of the cells were represented by the membranes of the cell wall and the cytoplasmic membrane of common structure. Some of the cells had only one membrane coat and a high electron optic density of the cytoplasm. Transitional forms of cells were also encountered. The ultrastructure of each morphological type in the population of the llr-mutants was described in detail. The capacity of the mutants to vacuolization, to the intra- and extracellular budding, and also the ability to form multiple membrane structures resembled analogous structures of stable L-forms of the Gram-negative microbes. The problems of morphological differentiation of the L-forms and of the llr-mutants, and also problems connected with the formation of the multiple membrane structures and small elemental bodies in the cells of the llr-mutants are discussed.     

Ultrastructure of Brucella abortus L-forms induced by penicillin in a liquid and in a semisolid medium

Brucella abortus L-forms were induced by 5.0 or 10.0 mug of penicillin/ml in a broth medium containing 0.3 m sucrose, and in a semisolid medium containing 10% calf serum and 20.0, 40.0, or 60.0 mug of penicillin/ml. After 96 hr of incubation, L-forms of various sizes and shapes were observed. Basic structures of the L-forms were similar whether induced in liquid or semisolid medium. L-forms had two "unit" membranes, each consisting of two outer dense layers separated by a lucent layer. A few large, irregularly shaped organisms in penicillin-treated broth cultures had additional surface material and were referred to as "transitional" forms. In contrast with L-forms, the bacterial cells were fairly uniform in size and shape, were smaller, and had a more complex cell wall structure. Small bodies limited by a "unit" membrane were present within and around numerous L-forms from liquid and semisolid medium cultures. Other internal membranous structures were also seen in some L-forms. Most Brucella L-forms described in this paper reverted to bacteria in the absence of penicillin and were structurally characteristic of unstable L-forms.     

Ultrastructure of L forms. IV. L forms of nonagglutinating vibrios]

A study was made of the ultrastructure of stable L-forms of Nag vibrios aged 24 hours. Cells of all types of the L-forms had cytoplasmic membranes, and a three-layered structure, which was found not everywhere. Externally of the cytoplasmic membrane, in some areas of the individual cells there were revealed a plastic layer of cell wall and a basal membrane. However, in difference to bacterial forms of the vibryos, rigidity of the cell wall was disturbed, and the links between the cell wall and the cytoplasmic membrane were indetectable. There were regularly revealed lamellar of myelin-like membranous structures in the cytoplasm, which did not occur in bacterial forms, and also lamellar mesosomes. The latter were found in the sites of cell division. Viability of small bodies as the minimal reproductive forms of the L-cultures is confirmed by the presence in them of a nucleoid and of the binary division.     

Dynamics of formation of antibodies of different immunochemical types to several L-forms and mycoplasmae]

Both IgM- and IgG-immunoglobulins were revealed in the sera of rabbits immunized with L-forms of hemolytic streptococcus, group A, and Mycoplasma fermentans. In the process of immune response the hemagglutinin titres proved to increase, with IgG antibodies prevalence in the hyperimmune sera.     

Lipid composition of Staphylococcus aureus and its derived L-forms.

Two strains of Staphylococcus aureus (Newman and Tazaki) and their derived L-forms were cultured in serum-containing broth and the differences in their lipid compositions were analyzed. Cardiolipin accounted for more than 50% of the total phospholipid phosphorus in L-forms, but for less than 25% in parent bacteria. The cardiolipin content of L-forms was very high through all growth phases, although it increased gradually as growth proceeded. Significant amounts of cholesterol and its esters were present in parent strains and L-forms, all of which incorporated serum cholesterol into the cell membrane. On the other hand, they could be detected in the L-forms but not in the parent strains when they were cultured in serum-free broth. To examine the ability of L-forms to synthesize cholesterol, the cholesterol content of L-forms cultured in serum-free broth was compared with that of the medium. The results indicated that staphylococcal L-forms could synthesize cholesterol and its esters. These differences in lipid composition suggested that modification of membrane lipids may occur as an adaptational change in response to the disappearance of the cell wall.     

Localization and possible development cycle of mycoplasmas and bacterial L-forms in cell cultures]

Using vital stain chlorazol-black E the authors studied the localization and behaviour of 5 mycoplasma species and the stable L-form of beta-hemolytic streptococcus of group A in various continuous cell lines. Mycoplasmas and L-forms had a definite evolution cycle in the cells. At first they are arranged extracellularly and on the surface of the cells, and then intracellularly where they multiply intensively; later they are again localized on the surface of the cells and extracellularly. This cycle proved to depend on the type of infection. The character of the localization depended on the type of the causative agent and of the culture.     

Studies on the Minimum Reproducible Unit of Staphylococcal L-Forms

The minimum size of a reproducible unit of staphylococcal L-forms was determined by filtration and electron microscopic methods. Ultrathin sections of an induced strain of Staphylococcal L-forms (STA-EMT-1) in liquid medium revealed several types of structures, all of which were bound by a single membrane and most of which possessed ribosome-like granules. Many of the small granules were less than 0.3 μm and were attached to the membrane of the large bodies. Using a serial filtration method, it was observed that viable L-forms were still detected in 0.22 μm filtrate, but the viable cell count of L-forms decreased in number with the decrease in pore size of membrane filters. A fractionation technique, using L-forms filtered through a membrane filter with a 0.45 μm pore size, revealed that there were three classes of small bodies but only the first class with ribosome-like granules over approximately 0.2 μm in diameter seems to be able to reproduce.     

Determination of antibodies to Streptococcus group A polysaccharide by an immunodiffusion method in human sera in various pathological processes of streptococcal etiology

A method for the assay of antibodies to the specific antigenic determinant of group A streptococcal polysaccharide (A-polysaccharide) in human sera was developed. The sera were tested in the precipitation test in agar gel with different doses of A-polysaccharide. The presence of a high level of the above-mentioned antibodies is indicative of infection caused by group A streptococcus, but not streptococci of other groups or by the L-forms of streptococci. In 87.5% of patients with primary rheumatism a high level of antibodies to the specific antigenic determinant of A-polysaccharide was detected during the first day of the disease, which confirms most convincingly the etiological role of group A streptococcus in rheumatism. Considerable differences in the level of antibodies to A-polysaccharide in the active and non-active phases of rheumatism have been established, which makes it possible to use the presence of a high level of these antibodies as an indicator of the rheumatic process activity. A considerable percentage of sera with a high level of antibodies to A-polysaccharide was also detected in erysipelas and acute glomerulonephritis patients.     

Intracellular vesicles as reproduction elements in cell wall-deficient L-form bacteria

Cell wall-deficient bacteria, or L-forms, represent an extreme example of bacterial plasticity. Stable L-forms can multiply and propagate indefinitely in the absence of a cell wall. Data presented here are consistent with the model that intracellular vesicles in Listeria monocytogenes L-form cells represent the actual viable reproductive elements. First, small intracellular vesicles are formed along the mother cell cytoplasmic membrane, originating from local phospholipid accumulation. During growth, daughter vesicles incorporate a small volume of the cellular cytoplasm, and accumulate within volume-expanding mother cells. Confocal Raman microspectroscopy demonstrated the presence of nucleic acids and proteins in all intracellular vesicles, but only a fraction of which reveals metabolic activity. Following collapse of the mother cell and release of the daughter vesicles, they can establish their own membrane potential required for respiratory and metabolic processes. Premature depolarization of the surrounding membrane promotes activation of daughter cell metabolism prior to release. Based on genome resequencing of L-forms and comparison to the parental strain, we found no evidence for predisposing mutations that might be required for L-form transition. Further investigations revealed that propagation by intracellular budding not only occurs in Listeria species, but also in L-form cells generated from different Enterococcus species. From a more general viewpoint, this type of multiplication mechanism seems reminiscent of the physicochemical self-reproducing properties of abiotic lipid vesicles used to study the primordial reproduction pathways of putative prokaryotic precursor cells.     

咽部活组织中细菌L型的检出及意义

应用病原微生物培养、电镜、组织切片细菌学检查及Ｌ型抗体免疫组化染色等方法,检测６４例慢性咽炎组织的细菌Ｌ型。结果有４２例培养出细菌Ｌ型,其阳性率为６５．６％;它与切片革兰氏染色Ｌ型检出阳性率（６７．２％）无显著性差异,Ｐ＞０．０５。透射电镜在慢性咽炎组织的间质及上皮细胞、巨噬细胞等细胞内见皮细菌Ｌ型;且Ｌ型抗体免疫组化染色亦证实组织中有细菌Ｌ型抗原。提示,细菌Ｌ型感染与慢性咽炎关系密切,Ｌ型侵入组织并在宿主细胞内生长的特征,可能是慢性咽炎反复发作,迁延不愈的重要原因。     

Electron Microscopy of Tissue Culture Cells Infected with Brucella abortus

Thin sections of hamster kidney tissue cultures were examined by electron microscopy over a 7-day period after infection with Brucella abortus 3183. Numerous bacteria and structures resembling L-forms were present both intracellularly and extracellularly after the first 24 hr of infection. Most intracellular microorganisms were enclosed by a cytoplasmic membrane, but in a few instances no limiting membrane was detected. After 4 to 7 days, fewer microorganisms were present, and most normal-appearing bacteria were intracellular, particularly in antibiotic-treated cultures. Structures typical of Brucella L-forms were extracellular at the latter time intervals. Several structures were observed in cells from infected cultures whose relationship to the infecting organisms is not known. These consisted of various membranous structures within cytoplasmic vacuoles, myelin-like structures surrounding occasional intracellular organisms, and small bodies present within vacuoles and extracellularly. The latter structures observed throughout the experimental period appeared to occur more frequently as the duration of the infection increased.     

Fatty Acid Composition of L-Forms of Streptococcus faecalis Cultured at Different Osmolalities

The fatty acid composition of the membranes of three different penicillin-produced L-forms of Streptococcus faecalis was determined: (i) a stable (nonreverting) L-form (T(53)) cultured in brain heart infusion (BHI) with 0.5 M sucrose; (ii) a stable L-form (T(531)) cultured in BHI without sucrose; and (iii) an unstable L-form (T(9)) cultured in BHI with 0.5 M sucrose and 1,000 U of penicillin per ml. L-forms were obtained by centrifugation and lysed by washing in 1 mM tris(hydroxymethyl)aminomethane-hydrochloride buffer. The parent S. faecalis was also cultured in BHI and BHI containing 0.5 M sucrose, and washed with buffer. The fatty acid composition of L-forms of S. faecalis cultured in BHI without sucrose (370 mosmol) had higher C(18:1) and lower C(18) than L-forms cultured in the same media with added 0.5 M sucrose (950 mosmol) in both exponential and stationary cultures. In the stationary phase of growth, C(19) was reduced in the L-forms cultured without sucrose. Similar changes were seen in the parent S. faecalis cultured in the two types of media. These changes in membrane fatty acids may relate to osmo-regulation of the L-forms.     

Polyacrylamide Gel Identification of Bacterial L-Forms and Mycoplasma Species of Human Origin

Polyacrylamide gel electrophoretic patterns of acidified phenol extracts prepared from whole cells can be used for the identification of bacterial L-forms and Mycoplasma species of human origin. Ten human Mycoplasma serotypes and eight L-forms belonging to five different genera were studied. The gel patterns were sufficiently distinct and reproducible that it was possible not only to identify L-forms at the genus level (group with streptococci) and different Mycoplasma serotypes but also to differentiate between the two of them. The parentage of L-forms of Streptobacillus moniliformis L1, Listeria monocytogenes, Streptococcus MG, and Staphylococcus aureus Smith strain was established by relating their gel patterns directly to parent bacteria. It was found that an L-form designated S. moniliformis An (ATCC 14220) was actually an L-form of Proteus. In addition, it was shown electrophoretically that no relationship existed between the Streptococcus MG L-form and M. pneumoniae. The applicability of this method as a diagnostic and taxonomic tool for the differentiation of L-forms and mycoplasmas is discussed.     

Microbial associations in chronic respiratory tract diseases

In the peripheral blood of patients with chronic bronchitis the presence of Mycobacterium pneumoniae antigens has been registered in 20.0% of cases and the presence of group A Streptococcus haemolyticus antigens, in 24.0% of cases, the transformation of streptococci into the L-form being observed in 12.0% of cases. The presence of M. pneumoniae, streptococci and their L-forms, as well as associations of these microorganisms, is characteristic of patients with chronic obstructive bronchitis, which is, probably, one of the reasons for the maintenance of bronchial obstruction. In patients with chronic pneumonia and fibrosing alveolitis the antigens of the bacterial form of streptococcus and its L-forms have been detected only in a few cases.     

Ultrastructure of L-forms. II. L-forms of Listeria monocytogenes]

A study was made of ultrathin sections of the stable l-forms of listeria obtained under the action of penicillin in meat-peptone-liver broth. A marked cellular polymorphism was found in the L-form culture: within the same colony cells differed in size, shape and fine structure. It is supposed that polymorphism could be partially explained by a different plasticity and premeability of cytoplasmic membrane in different types of cells of the same L-colony. The three-layer structure of the membrane does not always display the same distinctness in various L-colony cells and also in different areas of the cell surface. Structureless material of low electron density, possibly a defective murein or its precursor, was revealed on the membrane surface. Electrondense inclusion bodies, mesosomes of ring-shaped or more complicated structure and two-contour vesicles were found in the cytoplasm. The cells multiplied by budding, by binary and anomalies division participation of mesosomes in this process was not proved by the L-forms.     

Antigenic structure of Salmonella L forms studied by an immunoferritin method

The antigenic structure of the L-forms of salmonellae (S. typhimurium and S. typhi) in comparison with that of the initial bacterial cultures and revertant cultures was studied with the use of the immunoferritin method. The L-forms of salmonellae were found to retain an insignificant amount of O-antigen, as well as to have K-antigen on the surface of the cytoplasmic membrane, but in a lesser amount than the initial strains. In the cultures reverting from the L-forms of S. typhimurium and S. typhi the quantitative and qualitative characteristics of O- and K-antigens were completely restored.