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PubMed1964

CULTURE OF THE CYTOPATHIC AREA SURROUNDING VIRULENT CELLS OF BACILLUS ANTHRACIS IN MOUSE SPLEEN.

Roth, Ivan L. (Baylor University College of Medicine, Houston, Tex.), and Robert P. Williams. Nature of the cytopathic area surrounding virulent cells of Bacillus anthracis in mouse spleen. J. Bacteriol. 88:523-530. 1964.-Virulent anthrax bacilli in splenic tissue of mice were seen by electron microscopy to be surrounded by a clear zone that was designated the cytopathic area. Previous experiments did not establish the nature of the area. In the present experiments, virulent anthrax bacilli were grown in a system enriched with CO(2). The bacilli were capsulated and retained the capsule after they were killed by autoclaving. When grown without CO(2), these virulent bacilli were not capsulated. Also used was an avirulent strain of Bacillus anthracis that did not form capsules in either the presence or absence of CO(2). Mice were inoculated intravenously with suspensions containing 5 x 10(8) per 0.5 ml of (i) living or (ii) killed, capsulated virulent bacilli; (iii) killed, noncapsulated virulent bacilli; and (iv) living, avirulent bacilli. Bacteria were found in the spleen 1 hr after injection. Electron microscopy revealed that both living and dead, capsulated virulent bacilli were surrounded by a clear area. No clear area was found around killed, noncapsulated virulent bacilli. Clear areas were never seen around avirulent bacilli. Light microscopy of smears prepared from splenic tissue and stained for the presence of bacterial capsules corroborated the electron microscopic findings. These results established that the cytopathic area surrounding virulent bacilli was capsule.

Animals↗
PubMed1999

Capsular sialic acid limits C5a production on type III group B streptococci.

The majority of type III group B streptococcus (GBS) human neonatal infections are caused by a genetically related subgroup called III-3. We have proposed that a bacterial enzyme, C5a-ase, contributes to the pathogenesis of neonatal infections with GBS by rapidly inactivating C5a, a potent pro-inflammatory molecule, but many III-3 strains do not express C5a-ase. The amount of C5a produced in serum following incubation with representative type III strains was quantitated in order to better understand the relationship between C5a production and C5a-ase expression. C5a production following incubation of bacteria with serum depleted of antibody to the bacterial surface was inversely proportional to the sialic acid content of the bacterial capsule, with the more heavily sialylated III-3 strains generating less C5a than the less-virulent, less-sialylated III-2 strains. The amount of C5a produced correlated significantly with C3 deposition on each bacterial strain. Repletion with type-specific antibody caused increased C3b deposition and C5a production through alternative pathway activation, but C5a was functionally inactivated by strains that expressed C5a-ase. The increased virulence of III-3 strains compared to that of III-2 strains results at least partially from the higher sialic acid content of III-3 strains, which inhibits both opsonophagocytic killing and C5a production in the absence of type-specific antibody. We propose that C5a-ase is not necessary for III-3 strains to cause invasive disease because the high sialic acid content of III-3 strains inhibits C5a production.

Adhesins, Bacterial↗
PubMed1995

The pentameric structure of IgM is necessary to enhance opsonization of Bacteroides thetaiotaomicron and Bacteroides fragilis via the alternative complement pathway.

Studies were conducted to investigate the mechanisms by which natural IgM antibodies act together with the alternative complement pathway to promote opsonization and adherence of encapsulated Bacteroides thetaiotaomicron and Bacteroides fragilis to polymorphonuclear leukocytes (PMN). A model system consisting of the six isolated proteins of the alternative pathway was used. A comparison of the opsonic effects of pentameric and monomeric forms of isolated normal IgM demonstrated that, although the monomeric form bound to Bacteroides as effectively as the pentameric form and promoted complement deposition to the same extent, it was unable to enhance alternative pathway-dependent opsonization and adherence of Bacteroides to PMN. When opsonization was performed in two steps with pentameric IgM added either before or after alternative pathway components, a marked enhancement of adherence to PMN was observed only in the former case, suggesting IgM must act prior to complement to be effective. Electron microscopic studies demonstrated that, when added with complement, pentameric IgM, but not monomeric IgM, stabilized the bacterial capsule to the dehydration in dimethylformamide used for embedding in Lowicryl K4M. A strong correlation was observed between capsular stability and ability to be bound by PMN. The results suggest that pentameric IgM alters the structure of capsular components, perhaps through crosslinking, and this is in turn facilitates interaction of C3bi and C3b with CR3 and CR1, their respective receptors on PMN.

Adult↗
PubMed2004

Correlation of antigenic epitope and antibody gene usage in the human immune response to Streptococcus pneumoniae type 23F capsular polysaccharide.

The human antibody response to the capsular polysaccharide of Streptococcus pneumoniae type 23F is predominated by antibodies which express either VkappaL6 or VkappaA23 light chain genes. In this report, we demonstrate that the antigenic epitopes recognized by these two families can be differentiated based on their specificity for the hapten l-rhamnose, a constituent sugar of the bacterial capsule polymer. The VkappaL6 family utilizes light chains with an unusually long third complementarity-determining region. These VkappaL6-encoded Fabs are 100-fold more sensitive to inhibition with l-rhamnose than VkappaA23 Fabs. VkappaL6 Fabs preferentially recognize the l-isomer of the hapten and can distinguish l-rhamnose from the structurally similar sugar l-mannose. We also demonstrate that the VkappaL6 family component of the antibody response recognizes the polysaccharide antigens of Group B streptococci (GBS), thereby accounting for the previously reported cross-reaction between PPS 23F-specific antisera and Group B streptococci.

Animals↗
PubMed2006

Evolution of bacteriophages infecting encapsulated bacteria: lessons from Escherichia coli K1-specific phages.

Bacterial capsules are not only important virulence factors, but also provide attachment sites for bacteriophages that possess capsule degrading enzymes as tailspike proteins. To gain insight into the evolution of these specialized viruses, we studied a panel of tailed phages specific for Escherichia coli K1, a neuroinvasive pathogen with a polysialic acid capsule. Genome sequencing of two lytic K1-phages and comparative analyses including a K1-prophage revealed that K1-phages did not evolve from a common ancestor. By contrast, each phage is related to a different progenitor type, namely T7-, SP6-, and P22-like phages, and gained new host specificity by horizontal uptake of an endosialidase gene. The new tailspikes emerged by combining endosialidase domains with the capsid binding module of the respective ancestor. For SP6-like phages, we identified a degenerated tailspike protein which now acts as versatile adaptor protein interconnecting tail and newly acquired tailspikes and demonstrate that this adapter utilizes an N-terminal undecapeptide interface to bind otherwise unrelated tailspikes. Combining biochemical and sequence analyses with available structural data, we provide new molecular insight into basic mechanisms that allow changes in host specificity while a conserved head and tail architecture is maintained. Thereby, the present study contributes not only to an improved understanding of phage evolution and host-range extension but may also facilitate the on purpose design of therapeutic phages based on well-characterized template phages.

Bacterial Capsules↗
PubMed1998

Inadequate anti-polysaccharide antibody responses in the chicken.

Chickens are notorious for the fact that they carry bacteria such as Salmonellae and Campylobacter, which can cause zoonoses by contamination of the end product, without hampering growth and development of the chicken itself. This carrier status can only been explained by the inability of the chickens immune system to eliminate the pathogen, this in turn being due to insufficient humoral responses towards the polysaccharides of the bacterial capsule. In a previous study, we demonstrated that in chickens a model thymus-independent type 2 (TI-2) polysaccharide antigen, trinitrophenylated Ficoll (TNP-Ficoll), hardly evokes a humoral immune response. Furthermore this TI-2 antigen was shown to exhibit a very specific initial localization pattern after intravenous injection, i.e. in the periellipsoidal lymphocyte sheaths (PELS) and the surrounding ring of macrophages. The functional equivalent of these macrophages in mammals, the marginal zone macrophages, were shown to suppress the humoral responses against TI-2 antigens. Therefore we investigated whether other standard TI-2 antigen models also induce low antibody responses, whether this low response is dose-dependent, and whether macrophages are responsible for this low response. It was found that other TI-2 antigens, such as hydroxyethyl starch and detoxified lipopolysaccharides, also induced very low IgM and IgG responses, indicating a general phenomenon that could not be overcome by using a higher dose of antigen. In addition, selective depletion of splenic macrophages with liposomes containing dichloromethylene diphosphonate prior to immunization increased the specific humoral response to TD and TI-1 antigens, but failed to do so for TI-2 antigen. This result indicates that the low humoral responses are not (only) due to a macrophage suppressive activity but also to other yet unknown mechanisms, for example the lack of responsive B cells in the splenic PELS.

Animals↗
PubMedDate not supplied

[Study of protective role of the polysaccharide-containing components of capsules of Azospirillum brasilense].

The involvement of the carbohydrate components of the Azospirillum brasilense Sp245 capsules in bacterial protection from the action of extreme factors was investigated. The survival of encapsulated and non-encapsulated azospirilla exposed to elevated (46-48 degrees C) and below-freezing (-20 and -70 degrees C) temperatures, extreme pH values (2 and 10), and to drying was studied. High-molecular-weight carbohydrate-containing complexes (lipopolysaccharide-protein complex and polysaccharide-lipid complex) were isolated from the capsular material of azospirilla. It was shown that the addition of these complexes to the suspension of decapsulated cells before exposing them to extreme factors enhanced their survival rates by 15 to 51%.

Adaptation, Biological↗
PubMedDate not supplied

Capsular hyaluronic acid-mediated adhesion of Pasteurella multocida to turkey air sac macrophages.

Serogroup A strains of Pasteurella multocida, the major cause of fowl cholera, are resistant to phagocytosis in nonimmunized birds. Adherence studies with a capsulated strain of P. multocida (serotype A:3) and turkey air sac macrophages in culture showed that the bacteria were capable of adhering in large numbers to the macrophages but were not internalized. A noncapsulated variant of the bacteria (serotype -:3) showed little or no adherence and was not internalized. These data indicated that the adhesive properties were caused by the presence of a capsule on the bacteria. The role of capsular hyaluronic acid in adherence to macrophages was investigated. Depolymerization of the bacterial capsule with hyaluronidase increased phagocytosis by macrophage cultures, and addition of hyaluronic acid to the macrophages inhibited bacterial adherence. Additionally, exposure of macrophages to chondroitin sulfate B, an anionic polysaccharide similar to hyaluronic acid, did not affect the adhesive properties and resistance to phagocytosis of capsulated organisms. Treatment of macrophages with sodium metaperiodate or trypsin suppressed bacterial binding. Collectively, these data indicate that P. multocida adhesion to air sac macrophages, but not internalization, is mediated by capsular hyaluronic acid and suggest that recognition of this bacterial polysaccharide is a result of a specific glycoprotein receptor.

Animals↗
PubMed1992

Intracellular growth of Mycobacterium avium in human macrophages is linked to the increased synthesis of prostaglandin E2 and inhibition of the phagosome-lysosome fusions.

A virulent strain of Mycobacterium avium grew actively inside human adherent peripheral blood monocyte-derived macrophages. Bacteria were always confined to the phagosome compartment and were encapsulated. Cytochemical labeling of acid phosphatase using transmission electron microscopy showed a strong inhibition of the phagosome-lysosome fusions (PLF) in macrophages as not more than 25-30% bacteria containing phagosome at any time effectively fused with lysosomes. In case of a positive fusion event, the bacterial capsule prevented the diffusion of the lysosomal contents to the bacterial surface. Moreover, the infection of macrophages both by living and gamma-killed M. avium was linked to an increased synthesis of prostaglandin E2 (PGE2); however the total amount of PGE2 synthesized in the latter case was significantly lower than that observed with viable organisms. Our results suggest that the inability of human macrophages to control M. avium infection is linked to immunosuppressive pathways, e.g. enhanced synthesis of PGE2 and also to an impairment of normal microbicidal functions of the infected macrophages.

Acid Phosphatase↗
PubMed1988

[Structure and distribution of RU 41740].

RU 41740 is obtained from Klebsiella pneumoniae by organic extraction. It is mainly composed of carbohydrates with a small portion of proteins. Its active principle consists of two repetitive glyco-protein subunits: P1 which has a relative molecular mass of 95 kD and comes from the bacterial capsule, and F1 (relative molecular mass 350 kD) which is issued from the external bacterial membrane. Animal studies performed with tritiated RU 41740 have shown that the compound crosses the intestinal wall and that it rapidly and preferentially enters the lymphatic system. After oral administration to mice, the drug is detected by immunofluorescence in the basal end of the cells and chorion of the digestive tract epithelium.

Adjuvants, Immunologic↗
PubMed1979

An electron microscopic India ink technique for demonstrating capsules on microorganisms: studies with Streptococcus pneumoniae, Staphylococcus aureus, and Neisseria gonorrhoeae.

A technique using India ink in electron microscopic preparations was used to study bacterial capsules. Capsules were demonstrated on Streptococcus pneumoniae and Staphylococcus aureus (strain M and the Smith diffuse variant) from in vitro cultures. Two types of false capsules were observed on Neisseria gonorrhoeae grown in vitro or obtained from human urethral exudates. No true capsules were definitively identified on gonococci from these sources. The technique described does not require the use of specific anticapsular antibody or specific polysaccharide stains and permits the detection of artifacts that may, by the use of light microscopic methods, be misinterpreted as capsules.

Antigens, Bacterial↗
PubMed1997

Binding of Staphylococcus aureus to milk fat globules increases resistance to penicillin-G.

The susceptibility to penicillin-G of Staphylococcus aureus strains that cause mastitis was tested in milk and in Iso-sensitest broth (ISB): The minimal inhibitory concentrations (MIC) of beta-lactamase-positive strains in milk were 10-100-fold those in ISB, whereas the MIC of beta-lactamase-negative strains in milk were some 10-fold those in ISB; beta-lactamase production was induced by milk in beta-lactamase-positive strains. Much of the increase in resistance to penicillin-G caused by milk can be attributed to milk fat globules; the increase in resistance was related to the binding capacity of the bacteria to milk fat globules as well as to capsule formation by the bacteria. It appears that the binding of the staphylococci to the fat globules and bacterial capsule formation resulted in a biofilm type of growth. In this case, the staphylococci behaved differently from the planktonic type of growth in artificial broth medium in which antibiotic susceptibility testing is usually carried out.

Animals↗
PubMed1999

A brief history of the pneumococcus in biomedical research.

Because of its importance in human disease, Streptococcus pneumoniae has been the subject of intensive study at both the clinical and basic scientific levels for more than a century. In a number of instances, important advances in basic biology have resulted. Among these are the development of Gram's stain for identification of bacteria in patient specimens, investigations of the role of the bacterial capsule in resistance to phagocytosis by cells of the host immune system, demonstration that molecules other than proteins are capable of eliciting host humoral immune responses, development of safe and effective vaccines composed of isolated bacterial exopolysaccharides, confirmation of the efficacy of penicillin against serious gram-positive infections, and perhaps most important confirmation that DNA alone encodes genetic information.

History, 19th Century↗
PubMed1993

A brief history of the pneumococcus in biomedical research: a panoply of scientific discovery.

Because of its prominence as a cause of disease in humans, Streptococcus pneumoniae has been the subject of intensive investigation at both the clinical level and the basic scientific level during the past century. In a number of instances, these studies have resulted in important progress toward the comprehension of basic biological principles. The areas advanced by studies of the pneumococcus include an understanding of the concept of pathogenesis of infectious disease; the development of Gram's stain for identification of bacteria in specimens from patients; the elucidation of the role of the bacterial capsule in resistance to phagocytosis by cells of the host's immune system; the demonstration that molecules other than proteins are capable of eliciting the host's humoral immune responses and later, by extension, that isolated bacterial exopolysaccharides can be used safely and effectively as vaccines in humans; the documentation of the efficacy of penicillin; the collection of conclusive evidence that DNA encodes genetic information; and the investigation of putative proteinaceous virulence factors.

Animals↗
PubMed2000

Heterologous expression of an immunogenic pneumococcal type 3 capsular polysaccharide in Lactococcus lactis.

In order to develop a new system for the analysis of capsular biosynthetic pathways we have explored the possibility of expressing type 3 capsular polysaccharide (CPS) from the pathogen Streptococcus pneumoniae in Lactococcus lactis, an unencapsulated lactic acid bacterium being developed as a vaccine delivery vehicle for mucosal immunization. Only three of the four type 3 CPS biosynthesis genes were found to be necessary for the abundant formation (120 mg liter(-1)) of an extracellular type 3 CPS in L. lactis, implying a role for the type 3-specific synthase in the extracellular transport of the CPS or implying the existence of an alternative export system in L. lactis. The authenticity of the expressed heterologous polysaccharide was established by chemical and immunological analyses. Proton and carbon nuclear magnetic resonance spectroscopy of CPSs purified from L. lactis and S. pneumoniae showed that the two CPS structures were identical. When mice were immunized intraperitoneally with 3.5 x 10(6) CFU of live recombinant lactococci expressing a total of approximately 0.5 microgram of type 3 CPS, the immune responses elicited appeared identical to those observed in mice inoculated with 0.5 microgram of type 3 CPS purified from S. pneumoniae. These findings show that L. lactis is a useful host in which to study the role and function of genes involved in the production of bacterial capsules. Additionally, L. lactis shows potential as a host for the safe production of capsule antigens and as a vaccine delivery vehicle for polysaccharide antigens.

Animals↗
PubMed1998

Cationized ferritin as a stain for electron microscopic observation of bacterial ultrastructure.

The electron microscope has proven very effective for visualization of various morphological features of bacteria. Cationized ferritin (CF) is a stain commonly used to increase the electron microscopic resolution of bacterial cells, thereby enabling detailed analysis of their morphological and structural features. CF has been useful for microscopic examination of the bacterial capsule, cell wall, S-layer, and various unique morphological structures. In addition, as a cation, CF binds only to negatively charged molecules. Thus, CF has been used to identify sites of anionic charge on the bacterial cell surface, which has led to insights concerning the formation and turnover of bacterial peptidoglycan and the S-layer proteins. As a cation, however, CF may also interact with certain cellular components, causing erroneous interpretation of microscopic results. This review provides a discussion of both the strengths and weaknesses of CF when used as a stain for electron microscopy.

Bacteria↗
PubMed1965

Structure and composition of the Bacillus anthracis capsule.

Avakyan, A. A. (Academy of Medical Sciences, Moscow, USSR), L. N. Katz, K. N. Levina, and I. B. Pavlova. Structure and composition of the Bacillus anthracis capsule. J. Bacteriol. 90:1082-1095. 1965.-Observations by various methods of light microscopy (phase contrast, dark-field, and fluorescence) revealed the complex structure of the Bacillus anthracis capsule, which changes regularly during the growth cycle of the culture. Special cytological methods of staining the capsule made it possible to study its fine structure, which is not revealed by negative staining with India ink. For example, the capsule shows a membranelike outline, fine transverse lines, and interruptions and transverse septa traversing the entire capsule. By using cytochemical methods, it was found that the capsule has a stratified structure and that the various layers of the capsule differ as to the value of the isoelectric point, metachromatic ability, sensitivity to various enzymes, and, consequently, chemical composition. It was thus shown that the membranelike outline of the capsule consists of peptides and neutral mucopolysaccharides. The middle part of the capsule consists of a complex of substances of both polysaccharide and protein nature, and the inner part consists of acid mucopolysaccharides. Observation of the capsular forms of B. anthracis by means of an electron microscope revealed differences in the osmiophilia and submicroscopic structure of the membranelike outline and the middle and inner parts of the capsule. Immunochemical studies conducted by the fluorescent-antibody method revealed localization of antigens in different parts of the capsule, and made it possible to differentiate the capsular antigens according to their serum-staining ability and according of their relations to enzymes, i.e., their chemical composition. This paper concerns the possibility of studying the fine structure of bacterial capsules in fixed preparations, and the differences and similarities of the antigens of the capsule and cell wall of B. anthracis and of the related species, B. megaterium.

Bacillus anthracis↗