Bacteroides fragilis expresses three proteins similar to Porphyromonas gingivalis HmuY: Hemophore-like proteins differentially evolved to participate in heme acquisition in oral and gut microbiomes.

Oral and gut microbiomes are important for the maintenance of homeostasis in the human body. Altered or disturbed mutualism between their members results in dysbiosis with local injury and subsequent systemic diseases. The high bacterial density causes intense competition among microbiome residents to acquire nutrients, including iron and heme, the latter of high importance for heme auxotrophic members of the Bacteroidetes phylum. Our main hypothesis is that the heme acquisition mechanism, with the leading role played by a novel HmuY family of hemophore-like proteins, can be used to fulfill nutritional requirements and increase virulence. We characterized HmuY homologs expressed by Bacteroides fragilis and compared their properties with the first representative of this family, the HmuY protein of Porphyromonas gingivalis. In contrast to other Bacteroidetes members, B. fragilis produces three HmuY homologs (Bfr proteins). All bfr transcripts were produced at higher levels in bacteria starved of iron and heme (fold change increase ~60, ~90, and ~70 for bfrA, bfrB, and bfrC, respectively). X-ray protein crystallography showed that B. fragilis Bfr proteins are structurally similar to P. gingivalis HmuY and to other homologs, except for differences in the potential heme-binding pockets. BfrA binds heme, mesoheme, and deuteroheme, but preferentially under reducing conditions, using Met175 and Met146 to coordinate heme iron. BfrB binds iron-free protoporphyrin IX and coproporphyrin III, whereas BfrC does not bind porphyrins. HmuY is capable of heme sequestration from BfrA, which might increase the ability of P. gingivalis to cause dysbiosis also in the gut microbiome.

Synthesis, characterisation, and evaluation of a cross-linked disulphide amide-anhydride-containing polymer based on cysteine for colonic drug delivery.

The use of disulphide polymers, a low redox potential responsive delivery, is one strategy for targeting drugs to the colon so that they are specifically released there. The objective of this study was to synthesise a new cross-linked disulphide-containing polymer based on the amino acid cysteine as a colon drug delivery system and to evaluate the efficiency of the polymers for colon targeted drug delivery under the condition of a low redox potential. The disulphide cross-linked polymers were synthesised via air oxidation of 1,2-ethanedithiol and 3-mercapto-N-2-(3-mercaptopropionamide)-3-mercapto propionic anhydride (trithiol monomers) using different ratio combinations. Four types of polymers were synthesised: P10, P11, P151, and P15. All compounds synthesised were characterised by NMR, IR, LC-MS, CHNS analysis, Raman spectrometry, SEM-EDX, and elemental mapping. The synthesised polymers were evaluated in chemical reduction studies that were performed in zinc/acetic acid solution. The suitability of each polymer for use in colon-targeted drug delivery was investigated in vitro using simulated conditions. Chemical reduction studies showed that all polymers were reduced after 0.5-1.0 h, but different polymers had different thiol concentrations. The bacterial degradation studies showed that the polymers were biodegraded in the anaerobic colonic bacterial medium. Degradation was most pronounced for polymer P15. This result complements the general consensus that biodegradability depends on the swellability of polymers in an aqueous environment. Overall, these results suggest that the cross-linked disulphide-containing polymers described herein could be used as coatings for drugs delivered to the colon.

The strict anaerobe Bacteroides fragilis grows in and benefits from nanomolar concentrations of oxygen.

Strict anaerobes cannot grow in the presence of greater than 5 micro M dissolved oxygen. Despite this growth inhibition, many strict anaerobes of the Bacteroides class of eubacteria can survive in oxygenated environments until the partial pressure of O2 (PO2) is sufficiently reduced. For example, the periodontal pathogens Porphyromonas gingivalis and Tannerella forsythensis colonize subgingival plaques of mammals, whereas several other Bacteroides species colonize the gastrointestinal tract of animals. It has been suggested that pre-colonization of these sites by facultative anaerobes is essential for reduction of the PO2 and subsequent colonization by strict anaerobes. However, this model is inconsistent with the observation that Bacteroides fragilis can colonize the colon in the absence of facultative anaerobes. Thus, this strict anaerobe may have a role in reduction of the environmental PO2. Although some strictly anaerobic bacteria can consume oxygen through an integral membrane electron transport system, the physiological role of this system has not been established in these organisms. Here we demonstrate that B. fragilis encodes a cytochrome bd oxidase that is essential for O2 consumption and is required, under some conditions, for the stimulation of growth in the presence of nanomolar concentrations of O2. Furthermore, our data suggest that this property is conserved in many other organisms that have been described as strict anaerobes.

Non-enterobacterial endotoxins stimulate human coronary artery but not venous endothelial cell activation via Toll-like receptor 2.

OBJECTIVE: To determine whether non-enterobacterial endotoxins, which are likely to constitute the majority of the circulating endotoxin pool, may stimulate coronary artery endothelial cell activation. METHODS AND RESULTS: Interleukin-8 secretion, monocyte adhesion, and E-selectin expression were measured in human umbilical vein endothelial cells (HUVECs) and coronary artery endothelial cells (HCAECs) challenged in vitro with highly purified endotoxins of common host colonisers Escherichia coli, Porphyromonas gingivalis, Pseudomonas aeruginosa, and Bacteroides fragilis. HCAECs but not HUVECs expressed Toll-like receptor (TLR)-2 and were responsive to non-enterobacterial endotoxins. Transfection of TLR-deficient HEK-293 cells with TLR2 or TLR4/MD2 revealed that while E. coli endotoxin utilised solely TLR4 to signal, the endotoxins, deglycosylated endotoxins (lipid-A), and whole heat-killed bacteria of the other species stimulated TLR2-but not TLR4-dependent cell-signalling. Blockade of TLR2 with neutralizing antibody prevented HCAEC activation by non-enterobacterial endotoxins. Comparison of each endotoxin with E. coli endotoxin in limulus amoebocyte lysate assay revealed that the non-enterobacterial endotoxins are greatly underestimated by this assay, which has been used in all previous studies to estimate plasma endotoxin concentrations. CONCLUSION: Circulating non-enterobacterial endotoxins may be an underestimated contributor to endothelial activation and atherosclerosis in individuals at risk of increased plasma endotoxin burden.

Postmortem drug metabolism by bacteria.

Studies were undertaken to determine the possible role of enteric bacteria in the postmortem bioconversion of the nitrobenzodiazepines flunitrazepam, clonazepam, and nitrazepam. Flunitrazepam, clonazepam, and nitrazepam were completely metabolized in blood in the presence of eight species of enteric bacteria to their respective 7-amino-metabolites. The rates of metabolism, at 37 degrees C, ranged from 0.1 ng/mL/min for Streptococcus faecalis to 8.8 ng/mL/min for Clostridium perfringens. The rate of conversion was reduced to 87% by a combination of 0.7% (w/v) sodium fluoride and potassium oxalate, and almost completely inhibited (96%) by 1% (w/v) sodium fluoride. pH had variable effects on the rate of metabolic bioconversion of nitrobenzodiazepines, while increasing temperatures were found to generally increase the rate of nitrobenzodiazepine bioconversion. These data support the proposal that bacteria may mediate postmortem bioconversion of the nitrobenzodiazepines.

Comparing chemistry to outcome: the development of a chemical distance metric, coupled with clustering and hierarchal visualization applied to macromolecular crystallography.

Many bioscience fields employ high-throughput methods to screen multiple biochemical conditions. The analysis of these becomes tedious without a degree of automation. Crystallization, a rate limiting step in biological X-ray crystallography, is one of these fields. Screening of multiple potential crystallization conditions (cocktails) is the most effective method of probing a proteins phase diagram and guiding crystallization but the interpretation of results can be time-consuming. To aid this empirical approach a cocktail distance coefficient was developed to quantitatively compare macromolecule crystallization conditions and outcome. These coefficients were evaluated against an existing similarity metric developed for crystallization, the C6 metric, using both virtual crystallization screens and by comparison of two related 1,536-cocktail high-throughput crystallization screens. Hierarchical clustering was employed to visualize one of these screens and the crystallization results from an exopolyphosphatase-related protein from Bacteroides fragilis, (BfR192) overlaid on this clustering. This demonstrated a strong correlation between certain chemically related clusters and crystal lead conditions. While this analysis was not used to guide the initial crystallization optimization, it led to the re-evaluation of unexplained peaks in the electron density map of the protein and to the insertion and correct placement of sodium, potassium and phosphate atoms in the structure. With these in place, the resulting structure of the putative active site demonstrated features consistent with active sites of other phosphatases which are involved in binding the phosphoryl moieties of nucleotide triphosphates. The new distance coefficient, CDcoeff, appears to be robust in this application, and coupled with hierarchical clustering and the overlay of crystallization outcome, reveals information of biological relevance. While tested with a single example the potential applications related to crystallography appear promising and the distance coefficient, clustering, and hierarchal visualization of results undoubtedly have applications in wider fields.