Neutrophil activation by Escherichia coli isolates from human intestine: effects of bacterial hydroperoxidase activity and surface hydrophobicity.

Successful colonization of the intestine requires that bacteria interact with the innate immune system and, in particular, neutrophils. Progression of inflammatory bowel diseases (IBD) is associated with alterations in gut microbiota, and dysbiosis in Crohn's disease (CD) patients is often associated with an expansion of Escherichia coli. Here, we investigated the ability of such E. coli isolates to avoid neutrophil activation and to utilize reactive oxygen species. Neutrophil activation was detected in vitro in normal human blood via luminol chemiluminescence (CL) induced by reactive oxygen and halogen species generated by neutrophils. No significant difference in neutrophil activation in vitro was detected between isolates from inflamed (23 isolates) vs healthy intestines (5 isolates), with 10-fold variation within both groups (2.9-61.2 mV). CL activity of isolates from the same patient differed by 1.5-5 times. Twenty-four isolates from ileal aspirate, biopsy, and feces of seven patients with CD and one patient with no intestine inflammation were tested for extracellular peroxidase and catalase activity and cell surface hydrophobicity. Average values between patients varied from 26 ± 3 to 73 ± 18 µmol·g-1 of air dry weight for peroxidase activity, from 15 ± 2 to 189 ± 56 mmol·g-1 of air dry weight for catalase activity, and from 5 ± 3 to 105 ± 9 a.u. for the hydrophobic probe fluorescence. Extracellular peroxidase activity and hydrophobicity of bacterial cell surface correlated negatively with stimulated neutrophil CL. The ability of some isolates to avoid neutrophil activation and to utilize reactive oxygen species may provide a strategy to survive assault by the innate immune system.

Mucin adsorbed by E. coli can affect neutrophil activation in vitro.

Bacteria colonizing human intestine adhere to the gut mucosa and avoid the innate immune system. We previously demonstrated that Escherichia coli isolates can adsorb mucin from a diluted solution in vitro. Here, we evaluated the effect of mucin adsorption by E. coli cells on neutrophil activation in vitro. Activation was evaluated based on the detection of reactive oxygen species production by a chemiluminescent reaction (ChL), observation of morphological alterations in neutrophils and detection of exocytosis of myeloperoxidase and lactoferrin. We report that mucin adsorbed by cells of SharL1 isolate from Crohn's disease patient's inflamed ileum suppressed the potential for the activation of neutrophils in whole blood. Also, the binding of plasma complement proteins and immunoglobulins to the bacteria was reduced. Desialylated mucin, despite having the same adsorption efficiency to bacteria, had no effect on the blood ChL response. The effect of mucin suggests that it shields epitopes that interact with neutrophils and plasma proteins on the bacterial outer membrane. Potential candidates for these epitopes were identified among the proteins within the bacterial outer membrane fraction by 2D-PAGE, fluorescent mucin binding on a blot and HPLC-MS/MS. In vitro, the following proteins demonstrated mucin adsorption: outer membrane porins (OmpA, OmpC, OmpD and OmpF), adhesin OmpX, the membrane assembly factor OmpW, cobalamine transporter, ferrum uptake protein and the elongation factor Ef Tu-1. In addition to their other functions, these proteins are known to be bacterial surface antigens. Therefore, the shielding of epitopes by mucin may affect the dynamics and intensity of an immune response.

Proteomic dataset: Profiling of cultivated Echerichia coli isolates from Crohn's disease patients and healthy individuals.

One of the dysbioses often observed in Crohn's disease (CD) patients is an increased abundance of Escherichia coli (10-100 fold compared to healthy individuals) (Gevers et al., 2014). The data reported is a large-scale proteome profile for E. coli isolates collected from CD patients and healthy individuals. 43 isolates were achieved from 30 CD patients (17 male, 12 female, median age 30) and 19 isolates from 7 healthy individuals (7 male, median age 19). Isolates were cultivated on LB medium at aerobic conditions up to medium log phase. Protein extraction was performed with sodium deoxycholate (DCNa) and urea, alcylation with tris(2-carboxyethyl)phosphine and iodacetamide. Protein trypsinolysis was performed as described in (Matyushkina et al., 2016). Total cell proteomes were analysed by shotgun proteomics with HPLC-MS/MS on a maXis qTOF mass-spectrometer. The data including HPLC-MS/MS raw files and exported Mascot search results was deposited to the PRIDE repository project accession: PXD010920, project https://doi.org/10.6019/PXD010920.

Genome analysis of E. coli isolated from Crohn's disease patients.

BACKGROUND: Escherichia coli (E. coli) has been increasingly implicated in the pathogenesis of Crohn's disease (CD). The phylogeny of E. coli isolated from Crohn's disease patients (CDEC) was controversial, and while genotyping results suggested heterogeneity, the sequenced strains of E. coli from CD patients were closely related. RESULTS: We performed the shotgun genome sequencing of 28 E. coli isolates from ten CD patients and compared genomes from these isolates with already published genomes of CD strains and other pathogenic and non-pathogenic strains. CDEC was shown to belong to A, B1, B2 and D phylogenetic groups. The plasmid and several operons from the reference CD-associated E. coli strain LF82 were demonstrated to be more often present in CDEC genomes belonging to different phylogenetic groups than in genomes of commensal strains. The operons include carbon-source induced invasion GimA island, prophage I, iron uptake operons I and II, capsular assembly pathogenetic island IV and propanediol and galactitol utilization operons. CONCLUSIONS: Our findings suggest that CDEC are phylogenetically diverse. However, some strains isolated from independent sources possess highly similar chromosome or plasmids. Though no CD-specific genes or functional domains were present in all CD-associated strains, some genes and operons are more often found in the genomes of CDEC than in commensal E. coli. They are principally linked to gut colonization and utilization of propanediol and other sugar alcohols.