Mother-to-newborn transmission of mycobacterial L-forms and Vδ2 T-cell response in placentobiome of BCG-vaccinated pregnant women.

The ability of bacteria to exist as a population of self-replicating forms with defective or entirely missing cell wall (L-forms) is an adaptive mechanism for their survival and reproduction under unfavorable conditions. Bacterial mother-to-fetus transfer is a universal phenomenon in the animal kingdom. However, data about vertical transfer of L bacterial forms are extremely scarce. Bacille Calmette-Guérin is an attenuated strain of M. bovis and the only licensed vaccine used for tuberculosis prevention. We already have shown that filterable L-forms of BCG exist freely in the vaccine and are able to reproduce and to form colonies. The present study was focused on the placental microbiome in the context of mother's BCG vaccination. Here we report an isolation of filterable mycobacterial L-form cultures from gestational tissues and blood of healthy newborns delivered by healthy BCG-vaccinated mothers after normal pregnancy. Of note, vertically transmitted mycobacterial L-forms as a part of placentobiome of the pregnant women didn't influence the number of resident pathogen-reactive Vδ2 cells. Placenta colonization with mycobacterial L-forms occurs by maternal blood-to-decidua transfer very early in gestation. Together, these data showed that BCG L-forms have the capacity to pass trans-placental barrier and that maternal BCG vaccination affects the placentobiome.

Cell-wall deficient L. monocytogenes L-forms feature abrogated pathogenicity.

Stable L-forms are cell wall-deficient bacteria which are able to multiply and propagate indefinitely, despite the absence of a rigid peptidoglycan cell wall. We investigated whether L-forms of the intracellular pathogen L. monocytogenes possibly retain pathogenicity, and if they could trigger an innate immune response. While phagocytosis of L. monocytogenes L-forms by non-activated macrophages sometimes resulted in an unexpected persistence of the bacteria in the phagocytes, they were effectively eliminated by IFN-γ preactivated or bone marrow-derived macrophages (BMM). These findings were in line with the observed down-regulation of virulence factors in the cell-wall deficient L. monocytogenes. Absence of Interferon-ß (IFN-ß) triggering indicated inability of L-forms to escape from the phagosome into the cytosol. Moreover, abrogated cytokine response in MyD88-deficient dendritic cells (DC) challenged with L. monocytogenes L-forms suggested an exclusive TLR-dependent host response. Taken together, our data demonstrate a strong attenuation of Listeria monocytogenes L-form pathogenicity, due to diminished expression of virulence factors and innate immunity recognition, eventually resulting in elimination of L-form bacteria from phagocytes.

Filterable forms and L-forms of Mycobacterium bovis BCG: impact for live vaccine features.

Bacterial L-form conversion, or existence without cell walls, is assumed a universal phenomenon in nature. An interesting aspect of this phenomenon is occurrence of L-forms in vaccine strains. Since BCG is currently a widely used and extensively studied live vaccine for tuberculosis, understanding L-form conversion of M. bovis BCG bacilli can provide new insight into behavior of BCG vaccine. In this respect, specific features, concerning the ability of BCG vaccine to produce viable filterable forms and L-forms, were studied by filtration and starvation stress experiments in vitro. The filterable forms obtained after filtration of BCG suspension, grew on Middlebrook 7H9 semisolid agar and formed typical "fried eggs" L-form colonies. Electron microscopy clearly demonstrated presence of L-form elements with size smaller than the size of bacterial filter pores of 0.2 µm in M. bovis BCG strains. Development of L-form subpopulation with typical morphological appearance of self-replicating cell wall-defective forms was observed after filtration, as well as after starvation stress. Specific DNA detection of pncA gene in derived L-form cultures from filterable and stressed BCG strains verified their identity as M. bovis BCG. In conclusion, the results confirm existence of filterable forms in commercial BCG vaccine, which are able to develop L-form population under appropriate conditions. L-form transformation of BCG bacilli displays a new intriguing aspect concerning exhibition of unusual features and atypical behavior of live BCG vaccine. Further research is requested to explore the influence of L-form phenomenon on BCG vaccine effects in vivo.

[Microorganism persistence in hematopoietic tissue: means for detecting it and its significance]. / Persistentsiia mirkoorganizmov v krovetvornoi tkani: puti vyiavleniia i znachimost'.

Many microorganisms are capable of prolonged persistence in the marrow. In this study, carried out by the method of negative selection based on the treatment of mouse marrow cells with specific antimicrobial sera and complement, Mycoplasma arthritidis and L-forms of group B streptococci were found to be capable of persisting in the marrow in close association with the late category of clonogenic precursor cells, CFU-7, as well as, to a lesser extent, with late erythroid precursors, CFUe. Early colony-forming cells, CFUs-12 and PFUe, as well as granulocyto-macrophagal precursors, CFUgm, did not practically express antigens to the given infective agents on their surface.

Effect of Escherichia coli L-form cytoplasmic membranes on the interaction between macrophages and Lewis lung carcinoma cells: scanning electron microscopy.

Scanning electron microscopy (SEM) investigations on the interactions between peritoneal macrophages from Lewis lung carcinoma (LLC)-bearing mice and LLC tumour cells during 21 days after tumour implantation were carried out. The action of lipopolysaccharide (LPS)-containing cytoplasmic membranes (CM), from the stable protoplast type L-form of Escherichia coli, on the activity of in vitro phagocytosis was studied; CM induced a continuous increase in macrophage numbers. Activation of macrophage surfaces in healthy and tumour-bearing mice was established. Lamelipods, pseudopods and migration fringes 14 days after CM application were seen. Crater-like cavities deeply in the macrophage cells as well as adherent or prominent engulfed tumour cells within macrophages were observed during in vitro interaction with LLC cells. Macrophages from tumour-bearing mice without CM treatment showed less activation evaluated by SEM during earlier stages of tumour growth. The SEM investigation proved the temporary stimulating effect of E. coli L-form CM on the cell surface activation of peritoneal macrophages in healthy and LLC-bearing mice.

Characterization of a stable L-form of the fish pathogen Aeromonas salmonicida.

A stable L-form of Aeromonas salmonicida, which resulted from induction with benzylpenicillin, contained more of an outer membrane protein, with an estimated molecular weight of 40 kDa but less of 47.9 and 38 kDa proteins, than did parental walled cells. In addition, from Western blots, two protein bands reacted strongly with a polyclonal antiserum. The antiserum did not react demonstrably with the band detected in the L-forms on the gels.

Conversion of Salmonella typhimurium to L-forms contributes to the maintenance of acquired immunity against murine typhoid.

Conversion of Salmonella typhimurium to L-forms, both in vitro and in vivo, resulted in the expression of proteins cross-reacting to the mycobacterial 65,000 MW heat-shock protein (hsp). Immunization of C3H/HeJ mice with a protective dose of stable L-form S. typhimurium induced gamma delta T cells in the liver, in accordance with the multiplication of L-form Salmonella in Kupffer cells. The number of gamma delta T cells decreased after the intracellular growth of L-form Salmonella plateaued. Persistance of the L-forms in Kupffer cells, however, allowed hepatic gamma delta T cells to increase within 48 hr of infection with virulent S. typhimurium. Thus, the intrahepatic colonization of L-form Salmonella seems to keep gamma delta T cells on standby, but the emergence of these T cells does not correlate with the expression of L-form hsp. In addition, Kupffer cells colonized by L-forms constitutively synthesized mRNA for interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha). These results suggest that conversion of S. typhimurium to L-forms in phagocytic cells builds up and maintains acquired resistance, conferred by live-cell vaccines of S. typhimurium, against murine typhoid.

[The biomedical aspects of the persistence of bacteria]. / Biomeditsinskie aspekty persistentsii bakterii.

A correlation between the structure and function of bacteria was analyzed in the process of their persistence in the host body. A variety of persistent forms of bacteria was shown to be based on the isolation of their morphological substrate, peptidoglycane, which a cell "masked" (screening by surface bacterial structures, antigenic mimicry), "lost" (L-forms of bacteria, mycoplasmas) or protected against the system of host immunity by secreted factors. A new group of secreted bacterial (antilysozyme, anti-interferon, anti-immunoglobulin, anticomplement) factors, permitting microbial persistence in the host body, was described. Different methodological approaches to their determination were developed on the basis of the principle of "delayed antagonism". Applied aspects of the problem of persistence of bacteria were reviewed. The efficacy of new methods developed for the isolation and identification of a causative agent under the control of persistence markers was demonstrated on facultative microflora in different surgical, obstetrical, gynecological, urological diseases and diseases of internal organs. The facts concerning the use of the factors of bacterial persistence were presented for the solution of therapeutic (selection of means for controlling cell parasites), prognostic (development of carrier state in convalescents) and ecological (microbiological monitoring of the environment) problems.

[An immunological analysis of bacterial persistence in the bone marrow]. / Immunologicheskii analiz bakterial'noi persistentsii v kostnom mozge.

A method for the evaluation of bacterial persistence in the bone marrow in association with particular clonogenic target cells was developed. The method was based on the negative selection of cells expressing microbial antigens after treatment with hyperimmune antiserum specific to a given infective agent and the subsequent quantitation of target cells thus eliminated in appropriate assays. Using this approach, we demonstrated that Mycoplasma arthritidis and L-forms of Streptococcus strain L-406 were capable of persisting in murine bone marrow in close association with CFUs-7 (a subpopulation of hematopoietic stem cells) for at least several months after experimental infection. Francisella tularensis was also found to be capable to express on the CFUs-7 membranes. Persisting microorganisms enhanced both proliferation and migration of CFUs-7.

[The results of a 15-year study of the persistence of Yersinia pestis L-forms]. / Itogi 15-letnego izucheniia persistentsii L-form Yersinia pestis.

New diagnostic preparations for the detection of L-forms, immunoglobulin erythrocyte diagnosticum and luminescent antimembrane immunoglobulins, are described. Proofs indicating that Y.pestis L-forms are constantly isolated from wild rodents and their ectoparasites in the natural foci of plague are given. To control the effectiveness of the sanitation of such natural foci, the use of the radioimmunoassay and the enzyme immunoassay, known to be the most sensitive techniques, are recommended. The facts indicating that Y.pestis contaminating ticks and fleas are converted into L-forms, capable of persisting for several years, are presented. After their reversion to back the initial rod-shaped form the transmission of infection is possible. A suggestion is put forward that the persisting forms of Y.pestis play an important role in the preservation of this infective agent in the natural focus of infection.

Chemiluminescent activity of macrophages during phagocytosis of different bacterial forms.

Studies on the luminol-dependent chemiluminescent activity of rat peritoneal macrophages after in vitro interactions with E. coli WF+ L-form cells, their cytoplasmic membranes (CM) or parent bacterial cells were carried out. It was established that the phagocytosis of CM induce 20 time stronger light emission as compared to the L-form and 40 times compared to the parent bacterium cells respectively. Electron microscopical investigation of ultrathin sections of rat peritoneal macrophages after 24 h interaction in vitro with CM showed activation of the cell surfaces and vacuolisation of the cytoplasm. Inhibition of the phagolysosome fusion during phagocytosis of CM was observed. The mechanism of the immunostimulating activity of CM is discussed.

Adjuvant activity of the Escherichia coli WF table L-form cytoplasmic membranes.

It was established that the stable E. coli WF+ L-form cytoplasmic membranes (CM) increase the antibody response in rabbit during experimental hyperimmunization with cells of Streptococcus pyogenes A49 and Proteus mirabilis D52. Using the skin-induration test and the reaction for aggregation of macrophages in presence of homologous antigens it was established that CM increase the cell-mediated immunity of guinea-pigs to protein antigens of the same bacterial strains.

[Antibody titer to streptococcal and staphylococcal L-form in Behçet's disease and other uveitis].

The antibody titer in serum to Streptococcus pyogenes L-form and Staphylococcus aureus L-form were determined by enzyme-linked immunosorbent assay in 28 patients with Behçet's disease, 31 patients with other uveitis (sarcoidosis: 10, Harada's disease: 5, tuberculosis: 4, rheumatoid arthritis: 4, lues: 2, juvenile rheumatoid arthritis: 2, herpes simplex: 2, trauma: 2) and 16 healthy normal controls. All L-forms were induced by the penicillin disk method. The antibody titer to Streptococcus pyogenes L-form in Behçet's disease was lower than that of other cases of uveitis and controls, and showed significant differences between controls, rheumatoid arthritis, sarcoidosis, tuberculosis and Harada's disease by the Student's t-test. The antibody titer to Staphylococcus aureus L-form in Behçet's disease showed no difference between controls and other cases of uveitis. In each uveitis and controls, and between active and inactive stages of all uveitis, there were no differences between titers. In Behçet's disease, antibody formation to Streptococcus pyogenes L-form may be specifically disturbed.

Mechanism of the protective immunity against murine typhoid: persistence of Salmonella L forms in the liver after immunization with live-cell vaccines.

Live-cell vaccines of Salmonella typhimurium, either a sub-lethal dose of a wild-type (strain LT2) or a high dose of its two-heptose Rd1 mutant (strain SL1004), induced acquired resistance to murine typhoid, which remained 180 days after immunization. Growth of S. typhimurium as a bacillary form ceased between days 30 and 60 of immunization, but L forms of this bacterium colonized the liver (the mean number of L forms in the liver: 600 L-forming units) even at 180 days post-immunization. In contrast, a high inoculum of either a Ra mutant (strain TV148) of strain LT2 or S. schottmülleri 8006 sharing the same O antigenic components with those of S. typhimurium induced only a short-lived protection in proportion to the number of L forms in the liver, and the protective immunity was lost before day 180. However, there was no significant difference in the salmonella-specific T-cell responses among groups of immunized mice on day 180 of immunization. A lethal infection with strain LT2 in mice which had been immunized 75 days previously with living cells of strain SL1004 resulted in a rapid clearance of the challenge inoculum, together with a rapid elevation of anti-S. typhimurium antibody responses. Thus, the present data suggest that the long-lived immunity conferred upon live S. typhimurium vaccines is attributable to the colonization of this bacterium in the liver as L forms and the ability to colonize the liver as L forms is independent of the chain length of salmonella O-antigens.

[Cross reactive antigens of Acholeplasma laidlawii and L-form of Staphylococcus aureus].

We demonstrated that the membrane of Acholeplasma laidlawii PG8 and L-form of Staphylococcus aureus, both of which induce cellular immunity in BALB/c mice, were antigenically related each other. Foodpad responses of the mice immunized with a mixture of either antigen and Freund's complete adjuvant showed clearly a cross reaction when challenged with the other antigen. Cross responses to incorporate 3H-thymidine to the spleen lymphocytes of the mice immunized with either antigen occurred in the presence of the other antigen. Furthermore, the purified T cells, but not B cells, of the spleen were activated in the presence of antigen-presenting cells. These antigens existing in the membrane fractions of both microorganisms were purified by Razin's method. Finally, these membrane components of A. laidlawii and L-form of S. aureus were subjected to gel electrophoresis and transferring to nitrocellulose membrane and used to stimulate the spleen lymphocytes of the mice immunized with A. laidlawii or of non-immunized mice. The fractions representing molecular weights of approximately 45 kD, 25 kD, and 13 kD of both microorganisms consistently stimulated the lymphocytes of the immunized mice but not those of non-immunized mice.

[The immune status in experimental tuberculosis caused by altered forms of mycobacteria]. / Immunnyi status pri éksperimental'nom tuberkuleze, vyzvannom izmenennymi formami mikobakterii.

The immune status of guinea pigs in infection caused by M. tuberculosis L-forms and ultrafine forms has been studied. During the development of the infectious process the suppression of cell-mediated immunity factors has been found to occur. The indices of specific immunity factors correlate with the isolation rate of M. tuberculosis bacterial forms from the organs of infected animals and with the degree of their lesions. A change in the level of antibody formation has been established.

[Metabolic and growth inhibitions of Staphylococcus aureus L-form by immunoglobulin].

The coccal form as well as the L form of Staphylococcus aureus could produce the metabolic and growth inhibiting antibodies of the L form in the rabbit sera.

[Studies of antigens related to metabolic and growth inhibitions of Staphylococcus aureus L-form].

Metabolic and growth inhibiting activities in immunoglobulin (of anti-S. aureus L-form serum and anti-S. aureus coccal form serum) could be absorbed by cell membranes of S. aureus L-form and its coccal form, respectively. These activities could not be absorbed by cell membrane of Micrococcus luteus, Streptococcus pyogenes or Actinomyces viscosus. These findings suggested the existence of species-specific antigens of cell membrane. The membrane antigens of L-form related to the metabolic and growth inhibiting activities were stable to trypsin, heating and periodate, and were not solubilized by trypsin. A large part of the antigen in a typsin-insoluble membrane precipitate of L-form could be extracted by acetone and the subsequent use of chloroform-methanol (2: 1). A fractionation study of chloroform-methanol extract by using silicic acid calum indicated that more than two components were involved in metabolic and growth inhibiting activities.