A Distinct Gut Microbiota Exists Within Crohn's Disease-Related Perianal Fistulae.

BACKGROUND: Gut bacteria are strongly suspected to play a key role in the pathogenesis of Crohn's disease (CD). Studies have demonstrated alterations in the gut microbiota in this patient population. The purpose of this study was to characterize the gut microbiota of fistulizing perianal CD. MATERIALS AND METHODS: Stool and fistula samples were obtained from patients undergoing surgery for CD-related anorectal fistulae. Microbial compositions of matched stool and fistula samples were characterized using 16S rRNA gene profiling. The effect of sample type, patient gender, disease classification (Montreal A/B), disease activity (Harvey Bradshaw Index), antibiotic use, and presence of active proctitis on microbial composition was assessed. RESULTS: Samples were obtained from 18 patients. Bacteroides was the most abundant genera across all samples collected, followed by Streptococcus and Bifidobacterium. Bifidobacterium was present at significantly higher levels in fecal samples than fistula samples, whereas Achromobacter and Corynebacterium were present at significantly higher levels in fistula samples. Antibiotic, but not thiopurine or antitumor necrosis factor medication, exposure affected the gut microbial composition. Patient gender, disease classification, disease activity, and presence of active proctitis did not alter stool or fistula microbiota. CONCLUSIONS: Our data show that the gut microbiota within CD-related anorectal fistulae is distinct from that in stool samples obtained from the same patients. We also observe a dysbiosis in patients treated with antibiotics compared with those not treated with antibiotics.

Genetic analysis of signal integration by the Sinorhizobium meliloti sensor kinase FeuQ.

Two-component signalling systems allow bacteria to recognize and respond to diverse environmental stimuli. Auxiliary proteins can provide an additional layer of control to these systems. The Sinorhizobium meliloti FeuPQ two-component system is required for symbiotic development and is negatively regulated by the auxiliary small periplasmic protein FeuN. This study explores the mechanistic basis of this regulation. We provide evidence that FeuN directly interacts with the sensor kinase FeuQ. Isolation and characterization of an extensive set of FeuN-insensitive and FeuN-mimicking variants of FeuQ reveal specific FeuQ residues (periplasmic and intracellular) that control the transmission of FeuN-specific signalling information. Similar analysis of the FeuN protein highlights short patches of compatibly charged residues on each protein that probably engage one another, giving rise to the downstream effects on target gene expression. The accumulated evidence suggests that the periplasmic interaction between FeuN and FeuQ introduces an intracellular conformational change in FeuQ, resulting in an increase in its ability to remove phosphate from its cognate response regulator FeuP. These observations underline the complex manner in which membrane-spanning sensor kinases interface with the extracytoplasmic environment and convert that information to changes in intracellular processes.

FeuN, a novel modulator of two-component signalling identified in Sinorhizobium meliloti.

Sinorhizobium meliloti is a nitrogen-fixing bacterial symbiont of alfalfa and related legumes. Symbiotic infection by S. meliloti requires an osmosensory two-component system composed of the response regulator FeuP and the sensor kinase FeuQ. The FeuPQ pathway positively regulates transcription of multiple genes including ndvA, which encodes the cyclic glucan exporter. Here we show that proper regulation of this signalling pathway is essential for cell viability. Without the small 83 amino acid protein FeuN, S. meliloti cells are unable to grow, and this phenotype is dependent on the FeuPQ pathway. Using Escherichia coli as a heterologous system, we show that expression of feuP and feuQ leads to a dramatic increase in ndvA promoter activity, but that simultaneous expression of feuN abrogates this effect. Random mutagenesis of the feuPQ bicistron revealed a defined region of the FeuQ protein in and around its two predicted transmembrane domains that are required for FeuN-dependent signalling modulation. Marker enzyme fusion experiments indicate that most of the FeuN polypeptide is localized to the periplasm. Our data support a model in which FeuN interacts directly with FeuQ to attenuate phosphorylation of FeuP, and that without this activity, hyperactive signalling through FeuPQ results in cessation of growth or death.