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Bacteroides fragilis is the most commonly isolated anaerobic bacteria from infectious processes. Several virulence traits contribute to the pathogenic nature of this bacterium, including the ability to tolerate the high concentrations of bile found in the gastrointestinal tract (GIT). The activity of bile salts is similar to detergents and may lead to membrane permeabilization and cell death. Modulation of outer membrane proteins (OMPs) is considered a crucial event to bile salts resistance. The primary objective of the current work was to identify B. fragilis proteins associated with the stress induced by high concentration of bile salts. The outer membrane of B. fragilis strain 638R was isolated after growth either in the presence of 2% conjugated bile salts or without bile salts. The membrane fractions were separated on SDS-PAGE and analyzed by ESI-Q/TOF tandem mass spectrometry. A total of 37 proteins were identified; among them nine were found to be expressed exclusively in the absence of bile salts whereas eight proteins were expressed only in the presence of bile salts. These proteins are related to cellular functions such as transport through membrane, nutrient uptake, and protein-protein interactions. This study demonstrates the alteration of OMPs composition in B. fragilis during bile salts stress resistance and adaptation to environmental changes. Proteomics of OMPs was also shown to be a useful approach in the identification of new targets for functional analyses., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The binding of Bacteroides fragilis to plasmatic fibronectin was investigated using strains isolated from healthy subjects and from patients with bacteremia. They were cultivated in a synthetic media in which variations in cysteine concentrations determined alterations in the oxidation-reduction potential (Eh). All the strains assayed were capable of adhering to plasmatic fibronectin when cultivated under oxidizing and reducing conditions. Bacteroides fragilis 1405 showed the greatest difference when the results under these conditions were compared and it was selected for further investigations. Chemical treatments suggested the involvement of a protein in the interaction between B. fragilis and plasmatic fibronectin. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of outer membrane proteins (OMPs) revealed differences between the extracts obtained from cultures grown under the two conditions. Protein bands of c. 102, 100, 77, 73, 50 and 40 kDa were more highly expressed under oxidizing than reducing conditions. Dot blot analysis showed a stronger recognition of plasmatic fibronectin by OMPs obtained from cultures grown under higher Eh, and Western blot assays confirmed a band of c. 102 kDa as fibronectin-binding protein. This protein was sequenced and revealed to be a putative TonB-dependent OMPs. PCR analysis confirmed the presence of this gene in all the studied strains.

The adherence of Bacteroides fragilis strains to immobilized laminin-1 (LMN-1) was investigated using this protein adsorbed onto glass. Among the 27 strains isolated from infectious processes and assayed, 13 presented strong adherence to LMN-1. Among them, two strains, MC2 and 1081, showed the strongest association, and for that reason they were selected for further studies in which adherence to this protein was confronted with both physical-chemical and enzymatic treatments, along with concurrence assays with the LMN-1 molecule itself and the LMN-1-residing amino acid sequences (RGD, IKVAV, YIGSR, AG73, A13 and C16). The chemical and enzymatic treatments resulted in sharp decreases in binding rates of those strains, and competition experiments with LMN-1- residing amino acids revealed that, except for RGD and A13, all the others were effective at reducing bacterial binding of the bacteria. The outer membrane proteins (OMPs) of B. fragilis were extracted and assayed onto dot-blotted LMN-1, and when the extracts were chemically treated, especially with metasodium periodate, a drastic reduction in bacterial binding occurred. Results of the latter assays clearly indicate that bacterial molecules involved in both recognition and binding of B. fragilis to LMN-1 are present in OMP extracts. Taken together, our results strongly indicate that a B. fragilis surface glycoprotein may play a key role in bacterial association with LMN-1.

Bacteriodes fragilis isolated from aquatic environment, from infectious process and from human feces were compared as to their outer membrane protein electrophoretic profiles after staining with Coomassie blue and reacting with antibodies prepared against whole-cell antigens of a reference strain from a clinical source. A marked homogeneity was found among the strains with these methodologies. The profiles of all strains obtained after radio-iodination of the intact cell showed qualitative similarity when compared with the profiles obtained by the other methods. Thus, these data allow us to suggest the designation of the peptides observed in the autoradiograms as surface-exposed proteins. Differences observed in the autoradiograms in the expression of bands mainly detected at a molecular weight of 28 in the commensal strain 118,310 defined previously as avirulent, in addition to a distinction in the titres of agglutination with the sera tested and lower reactivity in the immunoblotting assays, suggest a relationship of the B. fragilis surface architecture with the virulence potential as well as with the origin of the strain.

Surface vesicles (SV) defined by electron microscopy as outer membrane (OM) extrusions were detected in Bacteroides fragilis strains from distinct sources. A partial identity between SV and OM electrophoretic protein profiles, in addition to the microscopic analysis, may suggest the designation of OMSV. Sialidase activity, a virulence determinant, was associated with these sub-cellular structures in all the strains, but in an inverse relation to the vesicle quantity per cell. A commensal strain, previously defined as avirulent in an animal model, presented the lowest vesicle-associated sialidase activity and the greatest SV expression as opposed to what happened with clinical and environmental strains. These results seem to suggest that these surface components have a function in commensal stages of B. fragilis.

An iron-repressible 44-kDa outer membrane protein plays a crucial role in the acquisition of heme by the anaerobic bacterium Bacteroides fragilis. The DNA sequence of the gene encoding the 44-kDa protein (hupA) was determined. The hupA gene encodes a protein of 431 amino acid residues with a calculated molecular mass of 48,189 Da. The hupA gene is preceded by an open reading frame of 480 bp that probably encodes a protein with a calculated molecular mass of 18,073 Da. hupA and this open reading frame are likely organized in an operon, and a sequence homologous to the Escherichia coli consensus Fur box was present in the putative promoter region of the operon. Heme-binding studies showed that HupA binds heme. Analysis of the deduced amino acid sequence revealed signature heme-binding consensus motifs, characteristic of heme lyases. Subcellular localization studies in E. coli revealed that HupA was mainly found in the cytoplasmic membrane but not in the outer membrane of E. coli. This suggested that B. fragilis uses another strategy for the translocation of this outer membrane protein across its cell envelope than E. coli does. HupA did not have significant homology with other putative bacterial heme receptors.