Neuraminidase production by Bacteroides species

Abstract 128 strains of Bacteroides isolated from clinical specimens were surveyed for their ability to produce neuraminidase. All strains of Bacteroides fragilis and the B. fragilis group were neuraminidase-positive, as were strains of B. oralis and B. bivius . All strains of B. capillosus, B. ruminicola, B. disiens, B. multiacidus and B. uniformis did not produce a detectable neuraminidase. When human erythrocytes were exposed to cell extracts of neuraminidase-producing Bacteroides , and then tested with peanut ( Arachis hypogeae ) lectin, agglutination occurred. It was concluded that the production of neuraminidase by clinical isolates of Bacteroides may be associated with the pathophysiology of severe Bacteroides infections.     

A rapid assay of the sialidase activity in species of the Bacteroides fragilis group by using peanut lectin hemagglutination

In this study, a novel, simple and rapid hemagglutination assay by using a peanut lectin to detect a neuraminidase activity in strains of the Bacteroides fragilis group was developed. One hundred and fourteen species of the B. fragilis group isolated from children with and without diarrhea and 15 reference strains were evaluated. Neuraminidase production was determined by using the method above described and its inhibition was observed by using galactose. The neuraminidase production was observed in 54 (84.37%) diarrhea and in 43 (86%) non-diarrhea strains. HA titers were ranged from 2 to 32. This neuraminidase assays based on PNA hemagglutination is highly sensitive, reproducible and could be used as a tool to detect the sialidase activity in anaerobic bacteria, particularly, in species of the B. fragilis group.     

A study of the candidate virulence factors of Bacteroides fragilis

Bacteroides fragilis strains were classified as virulent or avirulent on the basis of their clearance from the subcutaneous tissues of mice. To determine the factors which may contribute to the virulence of B. fragilis strains, we studied encapsulation, hydrophobicity, growth rate, serum sensitivity, agglutination with erythrocytes of different origin, and neuraminidase production. The strains of the virulent group displayed a higher growth rate in broth and a lower sensitivity to the bactericidal activity of serum than the strains of the avirulent group. They also agglutinated different types of erythrocytes more strongly than did the avirulent strains. No significant differences were found between the two groups of strains as regards encapsulation, hydrophobicity and neuraminidase activity.     

The human commensal Bacteroides fragilis binds intestinal mucin

The mammalian gastrointestinal tract harbors a vast microbial ecosystem, known as the microbiota, which benefits host biology. Bacteroides fragilis is an important anaerobic gut commensal of humans that prevents and cures intestinal inflammation. We wished to elucidate aspects of gut colonization employed by B. fragilis. Fluorescence in situ hybridization was performed on colonic tissue sections from B. fragilis and Escherichia coli dual-colonized gnotobiotic mice. Epifluorescence imaging reveals that both E. coli and B. fragilis are found in the lumen of the colon, but only B. fragilis is found in the mucosal layer. This observation suggests that physical association with intestinal mucus could be a possible mechanism of gut colonization by B. fragilis. We investigated this potential interaction using an in vitro mucus binding assay and show here that B. fragilis binds to murine colonic mucus. We further demonstrate that B. fragilis specifically and quantitatively binds to highly purified mucins (the major constituent in intestinal mucus) using flow cytometry analysis of fluorescently labeled purified murine and porcine mucins. These results suggest that interactions between B. fragilis and intestinal mucin may play a critical role during host-bacterial symbiosis.     

An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system

The mammalian gastrointestinal tract harbors a complex ecosystem consisting of countless bacteria in homeostasis with the host immune system. Shaped by evolution, this partnership has potential for symbiotic benefit. However, the identities of bacterial molecules mediating symbiosis remain undefined. Here we show that, during colonization of animals with the ubiquitous gut microorganism Bacteroides fragilis, a bacterial polysaccharide (PSA) directs the cellular and physical maturation of the developing immune system. Comparison with germ-free animals reveals that the immunomodulatory activities of PSA during B. fragilis colonization include correcting systemic T cell deficiencies and T(H)1/T(H)2 imbalances and directing lymphoid organogenesis. A PSA mutant of B. fragilis does not restore these immunologic functions. PSA presented by intestinal dendritic cells activates CD4+ T cells and elicits appropriate cytokine production. These findings provide a molecular basis for host-bacterial symbiosis and reveal the archetypal molecule of commensal bacteria that mediates development of the host immune system.     

Transmembrane diffusion of hydrophobic antimicrobial agents and cell surface hydrophobicity in Bacteroides fragilis

The transmembrane diffusion of hydrophobic antimicrobial agents, e.g. lincomycin and clindamycin, was examined in Bacteroides fragilis which is sensitive to these agents. The results showed that these agents penetrate efficiently through the outer membrane. Cell surface hydrophobicity measured by the partition assay between water and p-xylene revealed that the cell surface of B. fragilis is more hydrophobic than that of Salmonella typhimurium or Pseudomonas aeruginosa. Furthermore, treatment with low concentrations of surfactant caused cell lysis. These results suggest that the cell surface hydrophobicity in B. fragilis plays an important role in the efficient transmembrane penetration of hydrophobic compounds. This efficiency explains the susceptibility of B. fragilis to hydrophobic antimicrobial agents.     

Cloning and expression of the Bacteroides fragilis TAL2480 neuraminidase gene, nanH, in Escherichia coli.

We have cloned the Bacteroides fragilis TAL2480 neuraminidase (NANase) structural gene, nanH, in Escherichia coli. This was accomplished by using the cloning shuttle vector pJST61 and a partial Sau3A library of TAL2480 chromosomal inserts created in E. coli. The library was mobilized into the NANase-deficient B. fragilis TM4000 derivative TC2. NANase-producing colonies were enriched by taking advantage of the inability of TC2, but not the wild-type of NANase+ revertant, to grow in vitro in fluid aspirated from the rat granuloma pouch. Plasmids pJST61-TCN1 and pJST61-TCN3, containing inserts of 9.1 and 4.5 kilobases (kb), respectively, were found in the TC2 derivatives that grew in the rat pouch medium. In B. fragilis, NANase production from the two plasmids was inducible by free N-acetylneuraminic acid or sialic acid-containing substrates, just as in the parental TAL2480 strain. However, when these plasmids were transferred back to E. coli, NANase activity was barely detectable. A 3.5-kb portion of the insert in pJST61-TCN3 was subcloned in pJST61 to give plasmid pJST61-SC3C; NANase was produced from this plasmid both in E. coli and in B. fragilis. In E. coli, NANase expression was under the control of the vector promoter lambda pR and was therefore completely abolished by the presence of a lambda prophage. In B. fragilis, NANase production was inducible by free N-acetylneuraminic acid or sialic acid-containing substrates. By using deletion analysis and Tn1000 mutagenesis, the NANase structural gene and control region that functions in B. fragilis were localized to a 1.5- to 2.0-kb region of the insert. A partial nucleotide sequence of the NANase-deficient Tn1000 insertion mutants allowed us to identify the nanH gene and deduce the amino acid sequence of a portion of the NANase protein. We identified five regions showing great similarity to the Asp boxes, -Ser-X-Asp-X-Gly-X-Thr-Trp-, of other bacterial and viral NANase proteins.     

Discerning the Role of Bacteroides fragilis in Celiac Disease Pathogenesis

Celiac disease (CD) is associated with intestinal dysbiosis, which can theoretically lead to dysfunctions in host-microbe interactions and contribute to the disease. In the present study, possible differences in Bacteroides spp. and their pathogenic features between CD patients and controls were investigated. Bacteroides clones (n = 274) were isolated, identified, and screened for the presence of the virulence genes (bft and mpII) coding for metalloproteases. The proteolytic activity of selected Bacteroides fragilis strains was evaluated by zymography and, after gastrointestinal digestion of gliadin, by high-pressure liquid chromatography/electrospray ionization/tandem mass spectrometry. The effects of B. fragilis strains on Caco-2 cell culture permeability and inflammatory response to digested gliadin were determined. B. fragilis was more frequently identified in CD patients than in healthy controls, in contrast to Bacteroides ovatus. B. fragilis clones carrying virulence genes coding for metalloproteases were more abundant in CD patients than in controls. B. fragilis strains, representing the isolated clones and carrying metalloprotease genes, showed gelatinase activity and exerted the strongest adverse effects on the integrity of the Caco-2 cell monolayer. All B. fragilis strains also showed gliadin-hydrolyzing activity, and some of them generated immunogenic peptides that preserved or increased inflammatory cytokine production (tumor necrosis factor alpha) and showed increased ability to permeate through Caco-2 cell cultures. These findings suggest that increased abundance of B. fragilis strains with metalloprotease activities could play a role in CD pathogenesis, although further in vivo studies are required to support this hypothesis.     

Pbp, a cell-surface exposed plasminogen binding protein of Bacteroides fragilis

The Gram-negative anaerobic bacterium B. fragilis is a member of the commensal flora of the human intestine, but is also frequently found in severe intra-abdominal infections. Several B. fragilis virulence factors have been implicated in the development of these infections. A B. fragilis protein of circa 60-kDa was identified as a putative plasminogen binding protein (Pbp). The corresponding gene was located, cloned, sequenced and the subcellular localization of the protein was investigated. Pbp was both determined in the outer membrane of B. fragilis and of E. coli that expressed the cloned protein. Protease accessibility studies showed that the protein is expressed at the cell surface. Importantly, we demonstrated that Pbp is sufficient and required for plasminogen binding to whole cells in both E. coli and B. fragilis. Pbp-like proteins were also detected in some other Bacteroides subspecies. The role of this potential B. fragilis virulence factor in pathogenicity is discussed.     

The interaction of Bacteroides fragilis with components of the human fibrinolytic system

Bacteroides fragilis is a minor component of the intestinal microbiota and the most frequently isolated from intra-abdominal infections and bacteremia. Previously, our group has shown that molecules involved in laminin-1 (LMN-1) recognition were present in outer membrane protein extracts of B. fragilis MC2 strain. One of these proteins was identified and showed 98% similarity to a putative B. fragilis plasminogen-binding protein precursor, deposited in the public database. Thus, the objective of this work was to overexpress and further characterize this novel adhesin. The ability of B. fragilis MC2 strain and purified protein to convert plasminogen into plasmin was tested. Our results showed that B. fragilis strain MC2 strain adhered to both LMN-1 and plasminogen and this adhesion was inhibited by either LMN-1 or plasminogen. Regarding the plasminogen activation activity, both the whole bacterial cell and the purified protein converted plasminogen into plasmin similar to streptokinase used as a positive control. Bacterial receptors that recognize plasminogen bind to it and enhance its activation, transforming a nonproteolytic bacterium into a proteolytic one. We present in vitro evidence for a pathogenic function of the plasminogen receptor in promoting adherence to laminin and also the formation of plasmin by B. fragilis .     

Formation of glycoprotein degrading enzymes by Bacteroides fragilis

Bacteroides fragilis NCDO 2217 produced a wide range of cell-associated hydrolytic enzymes (neuraminidase, alpha-fucosidase, alpha-N-acetylgalactosaminidase, beta-galactosidase, beta-N-acetylglucosaminidase) that could potentially degrade the carbohydrate moieties of mucin, a complex glycoprotein. The type of substrate used for growth markedly influenced their formation in batch cultures. Synthesis of neuraminidase, alpha-fucosidase, alpha-N-acetylgalactosaminidase and to a lesser extent, beta-N-acetylglucosaminidase, was inversely related to growth rate in continuous cultures (D = 0.03 h-1-0.23 h-1) in which porcine gastric mucin provided the sole source of carbon and nitrogen.     

流感病毒神经氨酸酶的表达及其在药物筛选中的应用

A型流感病毒H5N1神经氨酸酶奥司他韦敏感型及耐药突变型基因经优化后,克隆于pcDNA4/TO 表达载体,并转染T-REx293 建立稳定细胞株,经四环素诱导能特异表达神经氨酸酶,其活性被特异性抑制剂奥司他韦所抑制。利用该稳定细胞株制备的神经氨酸酶,对3000多种天然产物和中药提取物进行了筛选,结果显示黄芩甙和黄芩素对奥司他韦敏感型神经氨酸酶和耐药型神经氨酸酶具有相似的抑制作用。该神经氨酸酶制备方法安全、简便、稳定,有利于建立神经氨酸酶抑制剂的高通量筛选方法。A型流感病毒H5N1神经氨酸酶奥司他韦敏感型及耐药突变型基因经优化后,克隆于pcDNA4/TO 表达载体,并转染T-REx293 建立稳定细胞株,经四环素诱导能特异表达神经氨酸酶,其活性被特异性抑制剂奥司他韦所抑制。利用该稳定细胞株制备的神经氨酸酶,对3000多种天然产物和中药提取物进行了筛选,结果显示黄芩甙和黄芩素对奥司他韦敏感型神经氨酸酶和耐药型神经氨酸酶具有相似的抑制作用。该神经氨酸酶制备方法安全、简便、稳定,有利于建立神经氨酸酶抑制剂的高通量筛选方法。     

Adhesion of human granulocytes and T lymphocytes to vascular endothelial cells after stimulation with Bacteroides fragilis endotoxin and enterotoxin]

The aim of presented study was to estimates the number of human granulocytes and T lymphocytes adhering to 1 mm2 of vascular endothelial cell culture stimulated by Bacteroides fragilis endotoxins (LPS) and enterotoxin (BFT). HMEC-1 cells were activated with bacterial preparations at the concentration of 10 (micrograms/ml for 4 and 24 hours. Granulocytes and T lymphocytes were isolated from peripheral blood of healthy blood donors. The adhesion tests of granulocytes and adhesion tests of resting and activated with PMA (at the concentration of 10 ng/ml) T lymphocytes to the non-stimulated and stimulated by B. fragilis compounds (LPS and BFT) vascular endothelium were performed. The number of viable leukocytes, which adhered to the endothelium, was determined using inverted microscope (magnification 200x). The results were presented as the number of viable cells adhering to 1 mm2 of the endothelial cell culture. The results of experiments indicate that granulocytes and T lymphocytes (resting and after activation with PMA even in greater number) adhere to the endothelial cells stimulated by B. fragilis endotoxins and enterotoxin. B. fragilis toxins are weaker stimulants of human leukocyte adhesion to the HMEC-1 cells than E. coli O55:B5 LPS. B. fragilis LPS and BFT preparations stimulate endothelial cells to the adhesion of granulocytes in similar manner, whereas the activation of vascular endothelium to the adhesion of T lymphocytes is differentiated.     

Biochemical and genetic analyses of a catalase from the anaerobic bacterium Bacteroides fragilis.

A single catalase enzyme was produced by the anaerobic bacterium Bacteroides fragilis when cultures at late log phase were shifted to aerobic conditions. In anaerobic conditions, catalase activity was detected in stationary-phase cultures, indicating that not only oxygen exposure but also starvation may affect the production of this antioxidant enzyme. The purified enzyme showed a peroxidatic activity when pyrogallol was used as an electron donor. It is a hemoprotein containing one heme molecule per holomer and has an estimated molecular weight of 124,000 to 130,000. The catalase gene was cloned by screening a B. fragilis library for complementation of catalase activity in an Escherichia coli catalase mutant (katE katG) strain. The cloned gene, designated katB, encoded a catalase enzyme with electrophoretic mobility identical to that of the purified protein from the B. fragilis parental strain. The nucleotide sequence of katB revealed a 1,461-bp open reading frame for a protein with 486 amino acids and a predicted molecular weight of 55,905. This result was very close to the 60,000 Da determined by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified catalase and indicates that the native enzyme is composed of two identical subunits. The N-terminal amino acid sequence of the purified catalase obtained by Edman degradation confirmed that it is a product of katB. The amino acid sequence of KatB showed high similarity to Haemophilus influenzae HktE (71.6% identity, 66% nucleotide identity), as well as to gram-positive bacterial and mammalian catalases. No similarities to bacterial catalase-peroxidase-type enzymes were found. The active-site residues, proximal and distal hemebinding ligands, and NADPH-binding residues of the bovine liver catalase-type enzyme were highly conserved in B. fragilis KatB.     

Rosette formation by insect macrophages inhibition by cytochalasin B.

Macrophages from the insect Spodoptera eridania possess membrane receptors for unmodified avian and mammalian erythrocytes, with which they form spontaneous rosettes. Rosette formation occurs in the absence of serum proteins and divalent cations. Individual macrophages bear receptors for several types of red cells. The level of naturally-occurring hemagglutinins against a particular test erythrocyte is not correlated with macrophage reactivity against that red cell. In contrast with mammalian macrophages, neuraminidase treatment of either hemocytes or erythrocytes does not cause a marked enhancement of binding. Pretreatment of macrophages or erythrocytes with cytochalasin B causes reversible inhibition of resetting probably by interfering with normal microfilament function, suggesting that optimal binding occurs when membranes are functioning normally on both macrophages and red cells. Colchicine and vinblastine do not influence resetting; therefore, microtubules are probably not involved in erythrocyte binding.     

The bactericidal mechanisms of polymorphonuclear leukocytes against Bacteroides fragilis: significance of the oxygen-dependent system

The mechanism by which polymorphonuclear leukocytes (PMNs) kill ingested Bacteroides fragilis was examined using PMNs from patients with chronic granulomatous disease (CGD) which is an inherited disease characterized by the defect of their PMNs in oxygen-radical generation. The phagocytosis of B. fragilis by PMNs from CGD patients was comparable to that by normal PMNs. Although CGD cells killed B. fragilis to some extent, they did so less effectively than the normal PMNS. B. fragilis was killed by a xanthine oxidase system that generates oxygen radicals. When PMNs were incubated with opsonized B. fragilis, B. fragilis triggered the release of O2- and H2O2 from normal PMNs. Thus, normal PMNs appear to kill B. fragilis by both oxygen-dependent and oxygen-independent mechanisms.     

Human endothelial cell adhesiveness for neutrophils, induced by Escherichia coli lipopolysaccharide in vitro, is inhibited by Bacteroides fragilis lipopolysaccharide

Recent studies in vitro have demonstrated that LPS from Gram-negative bacteria are capable of inducing endothelial cells to express a cell surface property that promotes the adherence of neutrophils (polymorphonuclear cells, PMN). We have investigated the effects of LPS from Bacteroides fragilis, an organism documented to have little toxicity in vivo, on the induction of this property in human endothelial cells. Monolayers of cultured human umbilical vein endothelial cells (HUVE) exhibited no increase in adhesiveness for 51Cr-radiolabeled PMN after 4 h of exposure to B. fragilis LPS from 1 ng to 10 micrograms/ml. Escherichia coli LPS elicited a dose-dependent enhancement of HUVE adhesiveness for PMN over the same concentration range, reaching a maximum of 49.4 +/- 6.6% at 10 micrograms/ml. Like E. coli LPS, B. fragilis LPS converted chromogenic substrate in the Limulus amebocyte lysate assay, and was directly cytotoxic to bovine aortic endothelial cells. Both B. fragilis LPS activities required doses two-to-three log-fold higher than for E. coli LPS. In addition, we found that B. fragilis LPS inhibited the induction of HUVE adhesiveness for PMN by E. coli LPS. This inhibition was also dose-dependent, becoming maximal (greater than 80%) when B. fragilis LPS was in 10- to 20-fold excess. Tumor necrosis factor and IL-1, two monokines which also elicit HUVE adhesiveness for PMN, were not inhibited by B. fragilis LPS, suggesting a mechanism of HUVE activation by LPS which is signal-specific, and which recognizes specificities of LPS structure.     

Characterization of an iron-regulated alpha-enolase of Bacteroides fragilis

This study describes the identification, cloning and molecular characterization of the alpha-enolase P46 of Bacteroides fragilis. The gram-negative anaerobic bacterium B. fragilis is a member of the commensal flora of the human intestine but is also frequently found in severe intra-abdominal infections. Several virulence factors have been described that may be involved in the development of these infections. Many of these virulence factors are upregulated under conditions of iron- or heme-starvation. We found a major protein of 46 kDa (P46) that is upregulated under iron-depleted conditions. This protein was identified as an alpha-enolase. Alpha-enolases in several gram-positive bacteria and eukaryotic cells are located at the cell surface and function as plasminogen-binding proteins. Localization studies demonstrated that P46 is mainly located in the cytoplasm and partly associated with the inner membrane (IM). Under iron-restricted conditions, however, P46 is localized primarily in the IM fraction. Plasminogen-binding to B. fragilis cells did occur but was not P46 dependent. A 60-kDa protein was identified as a putative plasminogen-binding protein in B. fragilis.     

The capsular polysaccharide of Bacteroides fragilis comprises two ionically linked polysaccharides.

Recently, we have shown that the capsular polysaccharide of Bacteroides fragilis NCTC 9343 is composed of an aggregate of two discrete large molecular weight polysaccharides (designated polysaccharides A and B). Following disaggregation of this capsular complex by very mild acid treatment, high resolution NMR spectroscopy demonstrated that polysaccharides A and B consist of highly charged repeating unit structures with unusual substituent groups (Baumann, H., Tzianabos, A. O., Brisson, J.-R., Kasper, D.L., and Jennings, H.J. (1992) Biochemistry 31, 4081-4089). Presently, we report that the capsular polysaccharide of B. fragilis represents a complex structure that is formed as a result of ionic interactions between polysaccharides A and B. Electron microscopy of immunogold-labeled organisms (with monoclonal antibodies specific for polysaccharides A and B) demonstrated that the two polysaccharides are co-expressed on the cell surface of B. fragilis. We have shown that the purified capsule complex is made up exclusively of polysaccharide A and polysaccharide B (no other macromolecular structure was detected) in a 1:3.3 ratio and that disaggregation of this complex into the native forms of the constituent polysaccharides could be accomplished by preparative isoelectric focusing. Structural analyses of the native polysaccharides A and B showed that they possessed the same repeating unit structures as the respective acid-derived polysaccharides. The ionic nature of the linkage between polysaccharides A and B was demonstrated by reassociation of the native polysaccharides to form an aggregated polymer comparable to the original complex. The distinctive composition of this macromolecule may provide a rationale for the unusual biologic properties associated with the B. fragilis capsular polysaccharide.     

Inhibition of Clostridium perfringens sporulation by Bacteroides fragilis and short-chain fatty acids

The effect a common fecal organism, Bacteroides fragilis, has on the sporulation of Clostridium perfringens, an organism linked to some cases of antibiotic associated diarrhea, was examined. Established B. fragilis cultures significantly decreased the number of heat resistant spores formed by C. perfringens ATCC 12915 and increased the number of vegetative cells. To determine if short-chain fatty acids (SCFA), fermentation products of B. fragilis, inhibited sporulation, the SCFA were added to sporulation broth. Sporulation decreased in the presence of acetate, isobutyrate, isovalerate, and succinate. Vegetative cell number for C. perfringens decreased in the cultures with isobutyrate. Propionate did not affect sporulation or vegetative cell number. The data support the hypothesis that the decrease in short-chain fatty acid concentration following antibiotic therapy predisposes patients to diarrheas caused by C. perfringens.