Identification of the alpha-enolase P46 in the extracellular membrane vesicles of Bacteroides fragilis.

BACKGROUND: Members of the Bacteroides fragilis group are the most important components of the normal human gut microbiome, but are also major opportunistic pathogens that are responsible for significant mortality, especially in the case of bacteraemia and other severe infections, such as intra-abdominal abscesses. Up to now, several virulence factors have been described that might explain the involvement of B. fragilis in these infections. The secretion of extracellular membrane vesicles (EMVs) has been proposed to play a role in pathogenesis and symbiosis in gram-negative bacteria, by releasing soluble proteins and other molecules. In B. fragilis, these vesicles are known to have haemagglutination and sialidosis activities, and also contain a capsular polysaccharide (PSA), although their involvement in virulence is still not clear. OBJECTIVE: The aim of this study was to identify proteins in the EMV of the 638R B. fragilis strain by mass spectrometry, and also to assess for the presence of Bfp60, a surface plasminogen (Plg) activator, previously shown in B. fragilis to be responsible for the conversion of inactive Plg to active plasmin, which can also bind to laminin-1. METHODS: B. fragilis was cultured in a minimum defined media and EMVs were obtained by differential centrifugation, ultracentrifugation, and filtration. The purified EMVs were observed by both transmission electron microscopy (TEM) and immunoelectron microscopy (IM). To identify EMV constituent proteins, EMVs were separated by 1D SDS-PAGE and proteomic analysis of proteins sized 35 kDa to approximately 65 kDa was performed using mass spectrometry (MALDI-TOF MS). FINDINGS: TEM micrographs proved the presence of spherical vesicles and IM confirmed the presence of Bfp60 protein on their surface. Mass spectrometry identified 23 proteins with high confidence. One of the proteins from the B. fragilis EMVs was identified as an enolase P46 with a possible lyase activity. MAIN CONCLUSIONS: Although the Bfp60 protein was not detected by proteomics, α-enolase P46 was found to be present in the EMVs of B. fragilis. The P46 protein has been previously described to be present in the outer membrane of B. fragilis as an iron-regulated protein.

The redox potential interferes with the expression of laminin binding molecules in Bacteroides fragilis.

The Bacteroides fragilis ATCC strain was grown in a synthetic media with contrasting redox potential (Eh) levels [reduced (-60 mV) or oxidised (+100 mV)] and their adhesion capacity to extracellular matrix components was evaluated. The strain was capable of adhering to laminin, fibronectin, fibronectin + heparan sulphate and heparan sulphate. A stronger adherence to laminin after growing the strain under oxidising conditions was verified. Electron microscopy using ruthenium red showed a heterogeneous population under this condition. Dot-blotting analyses confirmed stronger laminin recognition by outer membrane proteins of cells cultured at a higher Eh. Using a laminin affinity column, several putative laminin binding proteins obtained from the cultures kept under oxidising (60 kDa, 36 kDa, 25 kDa and 15 kDa) and reducing (60 kDa) conditions could be detected. Our results show that the expression of B. fragilis surface components that recognise laminin are influenced by Eh variations.

Gut microbiota utilize immunoglobulin A for mucosal colonization.

The immune system responds vigorously to microbial infection while permitting lifelong colonization by the microbiome. Mechanisms that facilitate the establishment and stability of the gut microbiota remain poorly described. We found that a regulatory system in the prominent human commensal Bacteroides fragilis modulates its surface architecture to invite binding of immunoglobulin A (IgA) in mice. Specific immune recognition facilitated bacterial adherence to cultured intestinal epithelial cells and intimate association with the gut mucosal surface in vivo. The IgA response was required for B. fragilis (and other commensal species) to occupy a defined mucosal niche that mediates stable colonization of the gut through exclusion of exogenous competitors. Therefore, in addition to its role in pathogen clearance, we propose that IgA responses can be co-opted by the microbiome to engender robust host-microbial symbiosis.

Finding a needle in a haystack: Bacteroides fragilis polysaccharide A as the archetypical symbiosis factor.

Starting from birth, all animals develop a symbiotic relationship with their resident microorganisms that benefits both the microbe and the host. Recent advances in technology have substantially improved our ability to direct research toward the identification of important microbial species that affect host physiology. The identification of specific commensal molecules from these microbes and their mechanisms of action is still in its early stages. Polysaccharide A (PSA) of Bacteroides fragilis is the archetypical example of a commensal molecule that can modulate the host immune system in health and disease. This zwitterionic polysaccharide has a critical impact on the development of the mammalian immune system and also on the stimulation of interleukin 10-producing CD4+ T cells; consequently, PSA confers benefits to the host with regard to experimental autoimmune, inflammatory, and infectious diseases. In this review, we summarize the current understanding of the immunomodulatory effects of B. fragilis PSA and discuss these effects as a novel immunological paradigm. In particular, we discuss recent advances in our understanding of the unique functional mechanisms of this molecule and its therapeutic potential, and we review the recent literature in the field of microbiome research aimed at discovering new commensal products and their immunomodulatory potential.

Bacteroides fragilis lipopolysaccharide and group B streptococcus serotype II glycocalyx have a common major antigenic determinant.

A monoclonal antibody (MAB) to the beta-1-6-linked digalactose structure in the lipopolysaccharide (LPS) of Bacteroides fragilis reacted with 47 of 416 group B Streptococcus (GBS) strains tested by an immunofluorescence technique (IF). The reactivity of MAB was, with a few exceptions, limited to type II GBS. Gas chromatography-mass spectrometry analysis demonstrated that an antigen purified by immunoaffinity chromatography using MAB from type II GBS contained galactose, glucose and fatty acids. This confirmed that MAB is directed to the digalactose (which in earlier studies was found to occur) in the capsular lipocarbohydrate specific to type II GBS. The positive strains yielded a strong, apple-green surface stain by means of the IF using MAB. Various immuno-electron microscopic (IEM) methods showed that the determinant was located in the glycocalyx layer of GBS at a distance of about 15 nm from the streptococcal cell wall. The structure harbouring the determinant was found to be very loosely attached to the bacteria. However the cross-reactive determinant seemed to maintain its immunoreactivity whether it was extracted by gentle washing with saline or with harsher treatments usually reserved for preparing streptococcal polysaccharide antigens. In conclusion, the study shows that the determinant is an integral part of the type-specific antigen of type II GBS and that MAB has a potential use as a serotyping reagent.

Demonstration of bacteroides capsules by light microscopy and ultrastructural cytochemistry.

Forty-six anaerobic gram-negative bacilli, including 26 members of the Bacteroides fragilis group (BFG), were examined for capsules by the India ink technic. Thirty-five were encapsulated, including all the BFG strains. As a follow-up, seven of these isolates and two previously studied reference strains (B. fragilis ATCC 23745 and Bacteroides vulgatus ATCC 8482) were examined for capsules by ultrastructural cytochemistry. Using the periodic acid thiocarbohydrazide silver proteinate (PATCSP) method of Thiéry, all the BFG examined were encapsulated. In addition to the reference strains, this included one strain of B. fragilis and four of Bacteroides thetaiotaomicron. One non-BFG strain showed no capsular material. Differences between these results and those reported previously with the ruthenium red technic may reflect species differences in the chemical composition of Bacteroides capsules.

An intracellular structure in Bacteroides fragilis.

An unusual intracellular circular or oval structure was discovered during a morphological study of Bacteroides fragilis by electronmicroscopy. The structure is complex, composed of multiple concentric layers surrounding a central region. The dimensions and profile are consistent in all the sections observed. The sections illustrated may represent various stages in the morphogenesis of the complete structure.

Phagocytic and serum killing of capsulate and non-capsulate Bacteroides fragilis.

The relative susceptibilities of capsulate and non- capsulate variants of Bacteroides fragilis to serum and phagocytic killing were investigated. The capsule of B. fragilis did not confer resistance to serum killing. Phagocytic killing of non- capsulate B. fragilis occurred at bacterial concentrations of 1 X 10(6) and 1 X 10(7) cfu/ml. Capsulate B. fragilis organisms were also phagocytosed and killed at a concentration of 1 X 10(6) cfu/ml, but phagocytosis and killing were impaired at a concentration of 1 X 10(7) cfu/ml.

Flow cytometric analysis of within-strain variation in polysaccharide expression by Bacteroides fragilis by use of murine monoclonal antibodies.

The reactivity of four different monoclonal antibodies (MAbs) with populations of Bacteroides fragilis NCTC 9343, enriched by density gradient centrifugation for a large capsule, small capsule and electron-dense layer (EDL) only visible by electronmicroscopy, was examined. The MAbs reacted strongly with polysaccharides present in both the large capsule- and EDL-enriched populations but not in the small capsule-enriched populations. The pattern of labelling was determined by immunoblotting, immunofluorescence and immuno-electronmicroscopy, and flow cytometry. The MAbs labelled cell membrane-associated epitopes in the large capsule- and EDL-enriched populations and cell-free material in the EDL population. By immunoblotting, ladders of repeating polysaccharide subunits were evident in the EDL population but not in the large capsule population. The proportion of cells labelled within each population was determined by flow cytometry. The reactivity of another MAb with the small capsule population was confirmed by flow cytometry. A qualitative indication of epitope expression was obtained by examination of the flow cytometric profiles. Differential expression of the same saccharide epitope was observed both between and within structurally distinct B. fragilis populations. The MAbs were species-specific and cross-reacted with several recent clinical isolates. These polysaccharides may be relevant to the virulence of B. fragilis.

Post-antibiotic effect in Bacteroides fragilis group.

Post-antibiotic effect (PAE) is the transient suppression of bacterial growth after brief antimicrobial exposure. While numerous reports have described PAE with aerobic and facultative microorganisms, virtually no studies have been conducted with anaerobic isolates. Intraabdominal isolates of the Bacteroides fragilis group were exposed for one hour to antibiotic (cefoxitin, cefotetan, and imipenem) concentrations two to four times the minimal inhibitory concentration (MIC). Post-antibiotic effect was described as the difference between the time required for microbial growth in the test versus the control to increase one Log10 above the quantitation observed immediately after drug removal. Bacteroides fragilis, B. ovatus, B. thetaiotaomicron and B. vulgatus exhibit PAEs for all test compounds. The time intervals for the PAEs were strain variable and ranged from six to 50 hours. No PAE was demonstrated with B. distasonis strains by the broth dilution technique. The results suggest that brief high dose exposure of some members of the B. fragilis group to anaerobe active beta-lactams produces a prolonged suppression in growth. In theory, a prolonged PAE could influence the dosage regimentation of selective antibiotics.

Pathogenicity of the Bacteroides fragilis group.

The Bacteroides fragilis group is one of the most important pathogens in polymicrobial infections. The distribution of the different members of the B. fragilis group in clinical infections varies. Bacteroides fragilis accounts for 63 percent of all the group isolates, Bacteroides thetaiotaomicron for 14 percent, Bacteroides vulgatus and Bacteroides ovatus for seven percent each, Bacteroides distasonis for six percent and Bacteroides uniformis for two percent. All members of the group induced bacteremia that was associated with an average mortality of 27 percent. The B. fragilis group resist beta lactam antibiotics by producing the enzyme beta-lactamase. This enzyme can be detected in abscess fluid, and can interfere with the eradication of other bacteria that are susceptible to penicillins and cephalosporins. All members of the B. fragilis group can become encapsulated during an inflammatory process as was demonstrated in a subcutaneous abscess model in mice. Non-encapsulated strains can become encapsulated with the assistance of their aerobic counterparts. These encapsulated strains are more virulent to the host than non-encapsulated strains. This increased virulence can be demonstrated by a higher rate of induction of bacteremia, and a greater enhancement of growth of other bacteria in mixed infection. Antimicrobial therapy directed only at the eradication of the aerobic bacteria did not prevent encapsulation, or reduce the number of Bacteroides species. The virulence of all members of the B. fragilis group highlights the need to direct antimicrobial therapy against all members of this group.

Peritoneal infection: a possible redox disease.

In a recent report, pertaining to Bacteroidesfragilis peritonitis, the influence of oxidation-reduction (redox) potential provided experimental evidence for B. fragilis penetration into Hela cell monolayers (using 3D imaging techniques). Bacteria grown under oxidizing conditions (+mV redox) penetrated into tissue cells unlike that of reducing conditions (-mV redox). The present results emphasise the significance of the level of redox potential during infection with an interpretation based on anaerobe/aerobe environmental flux, triggering the invasive mechanism.

Identification of the alpha-enolase P46 in the extracellular membrane vesicles of Bacteroides fragilis

BACKGROUND Members of the Bacteroides fragilis group are the most important components of the normal human gut microbiome, but are also major opportunistic pathogens that are responsible for significant mortality, especially in the case of bacteraemia and other severe infections, such as intra-abdominal abscesses. Up to now, several virulence factors have been described that might explain the involvement of B. fragilis in these infections. The secretion of extracellular membrane vesicles (EMVs) has been proposed to play a role in pathogenesis and symbiosis in gram-negative bacteria, by releasing soluble proteins and other molecules. In B. fragilis, these vesicles are known to have haemagglutination and sialidosis activities, and also contain a capsular polysaccharide (PSA), although their involvement in virulence is still not clear. OBJECTIVE The aim of this study was to identify proteins in the EMV of the 638R B. fragilis strain by mass spectrometry, and also to assess for the presence of Bfp60, a surface plasminogen (Plg) activator, previously shown in B. fragilis to be responsible for the conversion of inactive Plg to active plasmin, which can also bind to laminin-1. METHODS B. fragilis was cultured in a minimum defined media and EMVs were obtained by differential centrifugation, ultracentrifugation, and filtration. The purified EMVs were observed by both transmission electron microscopy (TEM) and immunoelectron microscopy (IM). To identify EMV constituent proteins, EMVs were separated by 1D SDS-PAGE and proteomic analysis of proteins sized 35 kDa to approximately 65 kDa was performed using mass spectrometry (MALDI-TOF MS). FINDINGS TEM micrographs proved the presence of spherical vesicles and IM confirmed the presence of Bfp60 protein on their surface. Mass spectrometry identified 23 proteins with high confidence. One of the proteins from the B. fragilis EMVs was identified as an enolase P46 with a possible lyase activity. MAIN CONCLUSIONS Although the Bfp60 protein was not detected by proteomics, α-enolase P46 was found to be present in the EMVs of B. fragilis. The P46 protein has been previously described to be present in the outer membrane of B. fragilis as an iron-regulated protein.

Impact of anatomic site on growth, efflux-pump expression, cell structure, and stress responsiveness of Bacteroides fragilis.

This study investigated whether B. fragilis from various human sites acquired stable traits enabling it to express certain efflux pumps (EPs), adopt a particular cell structure, and tolerate certain stressors. Isolates from blood, abscess, and stool (n = 11 each) were investigated. Bacteria from various sites portrayed different ultrastructres and EP expression. Blood isolates were tolerant to nutrient limitation and stool isolates to NaCl and bile salt stress. Stressors significantly increased EP expression. These data demonstrate that (1) B. fragilis acquires stable traits from various in vivo microenvironments; (2) that EPs are involved in stress responsiveness; and (3) that EP expression is tightly controlled and site dependent.

Bile salts enhance bacterial co-aggregation, bacterial-intestinal epithelial cell adhesion, biofilm formation and antimicrobial resistance of Bacteroides fragilis.

Bacteroides fragilis is the most common anaerobic bacterium isolated from human intestinal tract infections. Before B. fragilis interacts with the intestinal epithelial cells, it is exposed to bile salts at physiological concentrations of 0.1-1.3%. The aim of this study was to determine how pre-treatment with bile salts affected B. fragilis cells and their interaction with intestinal epithelial cells. B. fragilis NCTC9343 was treated with conjugated bile salts (BSC) or non-conjugated bile salts (BSM). Cellular ultrastructure was assessed by electron microscopy, gene expression was quantified by comparative quantitative real-time RT-PCR. Adhesion to the HT-29 human intestinal cell line and to PVC microtitre plates (biofilm formation) was determined. Exposure to 0.15% BSC or BSM resulted in overproduction of fimbria-like appendages and outer membrane vesicles, and increased expression of genes encoding RND-type efflux pumps and the major outer membrane protein, OmpA. Bile salt-treated bacteria had increased resistance to structurally unrelated antimicrobial agents and showed a significant increase in bacterial co-aggregation, adhesion to intestinal epithelial cells and biofilm formation. These data suggest that bile salts could enhance intestinal colonization by B. fragilis via several mechanisms, and could therefore be significant to host-pathogen interactions.

The polysaccharide capsule of Bacteroides fragilis subspecies fragilis: immunochemical and morphologic definition.

A large-molecular-weight capsular polysaccharide was isolated from strains of Bacteroides fragilis subspecies fragilis. By means of electron microscopy and staining with ruthenium red, the thick polysaccharide capsule was also visualized. With use of a radioactive antigen-binding assay, antibody to this capsular polysaccharide was demonstrated in antisera prepared in rabbits to each of eight strains of B. fragilis fragilis. Antibody of similar specificity was not found in antisera prepared to Bacteroides melaninogenicus or to strains of Bacteroides fragilis subspecies vulgatus and Bacteroides fragilis subspecies distasonis; such antibody was found in antisera to only one of two strains of Bacteroides fragilis subspecies thetaiotaomicron. The radioactive antigen-binding assay is a sensitive test for the detection of antibody to capsular polysaccharide. This polysaccharide antigen may form the basis of a serogrouping system for B. fragilis.

Surface structures, haemagglutination and cell surface hydrophobicity of Bacteroides fragilis strains.

Nineteen strains of Bacteroides fragilis were examined by negative staining for surface structures. One strain (ATCC 23745) possessed peritrichous fibrils, 16 strains carried peritrichous fimbriae and two strains carried no surface structures. The fimbriae had a diameter of 2.1 +/- 0.25 nm and appeared to be 'curly'. Only a small proportion (4 to 41%, depending on the strain) of cells in a population carried fimbriae or fibrils. Strain A312 Showed phase variation of fimbriae as expression of fimbriae was repressed at 20 degrees C and in early exponential phase at 37 degrees C. The fibrils on strain ATCC 23745 did not exhibit phase variation in response to changes in incubation temperature, growth phase or growth in two different media. Capsules were demonstrated by the Indian ink method on 18 of the 19 strains, varying in size from strain to strain and within the same population. Cultures often contained both capsulate and noncapsulate cells. All strains possessed an electron dense ruthenium red staining layer between 7.9 and 23.9 nm in width attached to the outer membrane. Cell surface hydrophobicity quantified by the hexadecane partition assay gave low values ranging from 6.6 to 52.1%. Only a few strains were able to haemagglutinate and these were only weakly active. There was no correlation between cell surface hydrophobicity, haemagglutinating activity and surface structures.

Isolation and identification of encapsulated strains of Bacteroides fragilis.

One hundred three clinical isolates of Bacteroides fragilis were identified during a two-year period. Most of these isolates were strains of B. fragilis subspecies fragilis, which constitutes a minor component of the fecal flora in comparison with the other subspecies of B. fragilis. By use of several techniques for demonstration of capsules, it was found that only B. fragilis strains classified as subspecies fragilis were encapsulated. An indirect immunofluorescence assay was developed for identification of clinical isolates possessing capsular material that was immunologically similar to that found in the reference strain of B. fragilis subspecies fragilis. All strains examined that were classified as subspecies fragilis were positive in this assay for the capsular material, whereas strains of the other subspecies were negative. This tests represents a rapid and sensitive means of identifying the most prevalent anaerobic gram-negative bacillus involved in human infections. The capsular polysaccharide of B. fragilis subspecies fragilis is a unique factor associated with the predominant subspecies of B. fragilis isolated from clinical material.

Encapsulated strains of Bacteroides fragilis in clinical specimens.

Studies were made of the presence of capsules in 294 strains of the Bacteroides fragilis group of bacteria isolated from clinical infections and in 30 strains from the fecal flora using the Indian ink and ruthenium red staining methods. A total of 77% of the B. fragilis strains were encapsulated; higher figures were observed in isolates from blood and wound infections. Of the B. ovatus, B. distasonis, B. vulgatus, B. uniformis, and B. thetaiotaomicron isolates from clinical infections, 17% were encapsulated. The percentage of encapsulated Bacteroides strains isolated from feces was approximately the same as in the strains isolated from clinical infections.

Effect of growth of Bacteroides fragilis at different redox levels on potential pathogenicity in a HeLa cell system: demonstration by confocal laser scanning microscopy.

During trauma, the intestinal anaerobe, Bacteroides fragilis, may enter into a pathogenic state. The process coincides with changing environmental conditions particularly the redox level in situ. To gain insight into this phenomenon B. fragilis was grown at different redox levels, and the invasive potential was examined using an in vitro model consisting of HeLa cell monolayers. The clinical strain AIP 5-86 was taken from a small collection of B. fragilis strains able to penetrate into tissue cell monolayers when selected by an acridine orange-crystal violet fluorescent staining technique. Following preliminary investigation by confocal laser scanning microscopy (CLSM), this particular strain was regarded as representative for examining the invasive potential. After growth in a defined medium under oxidizing, reducing or intermediate Eh7 conditions, the washed mid-log phase bacteria were allowed to interact with HeLa cell monolayers for 45 min at 37 degrees C. The results were extensively monitored by CLSM to follow the reactions in a stereoscopic dimension. In addition, the bacteria were examined by transmission and scanning electron microscopy before interaction to distinguish characteristics in surface configuration. The growth of the bacteria at particular redox levels seemed to influence their potential for pathogenicity. After growth at relatively high Eh, the bacteria easily penetrated into the HeLa cells, but not at low Eh, as determined by the laser scanning studies. Examination of the bacteria alone by transmission and scanning electron microscopy revealed small vesicles and a tendency to aggregate after growth at the low redox level while there were rather few vesicles and an implied dispersion at the high redox level. This leaves it open whether the invasiveness was based on the alterations found during growth of the bacteria. Different redox levels as well as the respective changes of the bacterial surface may help to discern the commensal from the pathogenic state of B. fragilis.