The Presence of Genotoxic and/or Pro-inflammatory Bacterial Genes in Gut Metagenomic Databases and Their Possible Link With Inflammatory Bowel Diseases.

Background: The human gut microbiota is a dynamic community of microorganisms that mediate important biochemical processes. Differences in the gut microbial composition have been associated with inflammatory bowel diseases (IBD) and other intestinal disorders. In this study, we quantified and compared the frequencies of eight genotoxic and/or pro-inflammatory bacterial genes found in metagenomic Whole Genome Sequences (mWGSs) of samples from individuals with IBD vs. a cohort of healthy human subjects. Methods: The eight selected gene sequences were clbN, clbB, cif, cnf-1, usp, tcpC from Escherichia coli, gelE from Enterococcus faecalis and murB from Akkermansia muciniphila. We also included the sequences for the conserved murB genes from E. coli and E. faecalis as markers for the presence of Enterobacteriaceae or Enterococci in the samples. The gene sequences were chosen based on their previously reported ability to disrupt normal cellular processes to either promote inflammation or to cause DNA damage in cultured cells or animal models, which could be linked to a role in IBD. The selected sequences were searched in three different mWGS datasets accessed through the Human Microbiome Project (HMP): a healthy cohort (N = 251), a Crohn's disease cohort (N = 60) and an ulcerative colitis cohort (N = 17). Results: Firstly, the sequences for the murB housekeeping genes from Enterobacteriaceae and Enterococci were more frequently found in the IBD cohorts (32% E. coli in IBD vs. 12% in healthy; 13% E. faecalis in IBD vs. 3% in healthy) than in the healthy cohort, confirming earlier reports of a higher presence of both of these taxa in IBD. For some of the sequences in our study, especially usp and gelE, their frequency was even more sharply increased in the IBD cohorts than in the healthy cohort, suggesting an association with IBD that is not easily explained by the increased presence of E. coli or E. faecalis in those samples. Conclusion: Our results suggest a significant association between the presence of some of these genotoxic or pro-inflammatory gene sequences and IBDs. In addition, these results illustrate the power and limitations of the HMP database in the detection of possible clinical correlations for individual bacterial genes.

Acidic-store depletion is required for human platelet aggregation.

Platelet stimulation with thrombin induces an elevation in cytoplasmic free Ca(2+) concentration ([Ca(2+)]c) due to Ca(2+) release from intracellular stores and entry from the extracellular medium. Two different intracellular Ca(2+) stores have been described in human platelets: the dense tubular system and the lysosomal-like acidic stores. In the present study, we investigated the contribution of the acidic stores in thrombin-induced platelet aggregation. We have found that platelet aggregation induced by thrombin is reduced in a Ca(2+)-free medium. Discharge of the acidic Ca(2+) stores by treatment with the sarcoendoplasmic Ca(2+)-ATPase (SERCA)3 selective inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone reduced thrombin-evoked platelet aggregation. In the presence of 2,5-di-(tert-butyl)-1,4-hydroquinone, platelet aggregation induced by the protease-activated receptor (PAR)-1 and PAR-4 agonist peptides, SFLLRN and AYPGKF, respectively, was significantly reduced. In cells with depleted acidic stores, activation of GPIb-IX-V by thrombin resulted in reduced or no platelet aggregation in a medium containing 1 mmol/l Caor in a Ca(2+)-free medium, respectively. This finding suggests that Ca(2+) accumulation in the acidic Ca(2+) compartments is required for platelet aggregation induced by activation of the G-coupled PAR-1 and PAR-4 thrombin receptors and, by the occupation of the leucine-rich glycoprotein GPIb-IX-V and provide evidence supporting a functional role of the lysosomal-like acidic Ca(2+) stores in human platelets.

Involvement of SNARE proteins in thrombin-induced platelet aggregation: evidence for the relevance of Ca2+ entry.

Thrombin induces platelet activation through a variety of intracellular mechanisms, including Ca(2+) mobilization. The protein of the exocytotic machinery SNAP-25, but not VAMPs, is required for store-operated Ca(2+) entry, the main mechanism for Ca(2+) influx in platelets. Hence, we have investigated the role of the SNAP-25 and VAMPs in thrombin-induced platelet aggregation. Platelet stimulation with thrombin or selective activation of thrombin receptors PAR-1, PAR-4 or GPIb-IX-V results in platelet aggregation that, except for GPIb-IX-V receptor, requires Ca(2+) entry for full activation. Depletion of the intracellular Ca(2+) stores using pharmacological tools was unable to induce aggregation except when cytosolic Ca(2+) concentration reached a critical level (around 1.5 microM). Electrotransjection of cells with anti-SNAP-25 antibody reduced thrombin-evoked platelet aggregation, while electrotransjection of anti-VAMP-1, -2 and -3 antibody had no effect. These findings support a role for SNAP-25 but not VAMP-1, -2 and -3 in platelet aggregation, which is likely mediated by the regulation of Ca(2+) mobilization in human platelets.

Hydrogen peroxide and peroxynitrite enhance Ca2+ mobilization and aggregation in platelets from type 2 diabetic patients.

Cytosolic Ca2+ mobilization, especially Ca2+ entry, is enhanced in platelets from type 2 diabetic individuals, which might result in platelet hyperaggregability. In the present study, we report an increased oxidant production in resting and stimulated platelets from diabetic donors. Pretreatment of platelets with catalase or trolox, an analog of vitamin E, reversed the enhanced Ca2+ entry, evoked by thapsigargin plus ionomycin or thrombin, observed in platelets from diabetic subjects, so that in the presence of these scavengers Ca2+ entry was similar in platelets from healthy and diabetic subjects. In contrast, mannitol was without effect on Ca2+ mobilization. Catalase and trolox reduced thrombin-induced aggregation in platelets from type 2 diabetic subjects, while mannitol did not modify thrombin-induced platelet hyperaggregability. We conclude that H2O2 and ONOO- are likely involved in the enhanced Ca2+ mobilization observed in platelets from type 2 diabetic patients, which might lead to platelet hyperactivity and hyperaggregability.

Store-operated Ca(2+) entry and tyrosine kinase pp60(src) hyperactivity are modulated by hyperglycemia in platelets from patients with non insulin-dependent diabetes mellitus.

We have investigated the involvement of store-operated Ca(2+) entry (SOCE) in the abnormal platelet Ca(2+) homeostasis in patients with non insulin-dependent diabetes mellitus (NIDDM). In a medium containing 180 mg/dL glucose, platelets from NIDDM patients showed an increased SOCE compared to controls. We found that tyrosine phosphorylation was elevated in platelets from NIDDM patients. Consistent with this, the activity of the tyrosine kinase pp60(src) is enhanced in platelets from diabetic patients. When the experiments were performed in a medium containing 90 mg/dL both, SOCE and pp60(src) activity, were similar to those found in control platelets. Our results indicate that SOCE is altered in platelets from NIDDM patients probably due to the increased activity of the tyrosine kinase pp60(src). Both, SOCE and pp60(src) activity in platelets from NIDDM patients are more susceptible to the extracellular glucose concentration, which seems to be involved in the dysfunction of these mechanisms.