The regulatory mechanism of Hsp90alpha secretion and its function in tumor malignancy.

Heat shock protein 90-alpha (Hsp90alpha) is an intracellular molecular chaperone. However, it can also be secreted with the underlying regulatory mechanism remaining far from clear. Here we show that the secreted Hsp90alpha is a C-terminal truncated form and its secretion is regulated by the C-terminal EEVD motif via interacting with proteins containing tetratricopeptide repeat domains. We also demonstrate that secretion of Hsp90alpha is determined by the phosphorylation status at residue Thr-90, regulated by protein kinase A and protein phosphatase 5. We further demonstrate that the secretion of Hsp90alpha is a prerequisite for its proinvasiveness function and blocking the secreted Hsp90alpha results in significant inhibition of tumor metastasis. Meanwhile, the level of plasma Hsp90alpha is positively correlated with tumor malignancy in clinical cancer patients. In sum, our results reveal the regulatory mechanism of Hsp90alpha secretion, and its function in tumor invasiveness, indicating it can be a promising diagnostic marker for tumor malignancy in clinical application.

Crohn's disease in endoscopic remission, obesity, and cases of high genetic risk demonstrates overlapping shifts in the colonic mucosal-luminal interface microbiome.

BACKGROUND: Crohn's disease (CD) patients demonstrate distinct intestinal microbial compositions and metabolic characteristics compared to unaffected controls. However, the impact of inflammation and underlying genetic risk on these microbial profiles and their relationship to disease phenotype are unclear. We used lavage sampling to characterize the colonic mucosal-luminal interface (MLI) microbiome of CD patients in endoscopic remission and unaffected controls relative to obesity, disease genetics, and phenotype. METHODS: Cecum and sigmoid colon were sampled from 110 non-CD controls undergoing screening colonoscopy who were stratified by body mass index and 88 CD patients in endoscopic remission (396 total samples). CD polygenic risk score (GRS) was calculated using 186 known CD variants. MLI pellets were analyzed by 16S ribosomal RNA gene sequencing, and supernatants by untargeted liquid chromatography-mass spectrometry. RESULTS: CD and obesity were each associated with decreased cecal and sigmoid MLI bacterial diversity and distinct bacterial composition compared to controls, including expansion of Escherichia/Shigella. Cecal and sigmoid dysbiosis indices for CD were significantly greater in obese controls than non-overweight controls. CD, but not obesity, was characterized by altered biogeographic relationship between the sigmoid and cecum. GRS was associated with select taxonomic shifts that overlapped with changes seen in CD compared to controls including Fusobacterium enrichment. Stricturing or penetrating Crohn's disease behavior was characterized by lower MLI bacterial diversity and altered composition, including reduced Faecalibacterium, compared to uncomplicated CD. Taxonomic profiles including reduced Parasutterella were associated with clinical disease progression over a mean follow-up of 3.7 years. Random forest classifiers using MLI bacterial abundances could distinguish disease state (area under the curve (AUC) 0.93), stricturing or penetrating Crohn's disease behavior (AUC 0.82), and future clinical disease progression (AUC 0.74). CD patients showed alterations in the MLI metabolome including increased cholate:deoxycholate ratio compared to controls. CONCLUSIONS: Obesity, CD in endoscopic remission, and high CD genetic risk have overlapping colonic mucosal-luminal interface (MLI) microbiome features, suggesting a shared microbiome contribution to CD and obesity which may be influenced by genetic factors. Microbial profiling during endoscopic remission predicted Crohn's disease behavior and progression, supporting that MLI sampling could offer unique insight into CD pathogenesis and provide novel prognostic biomarkers.

Microgeographic Proteomic Networks of the Human Colonic Mucosa and Their Association With Inflammatory Bowel Disease.

BACKGROUND & AIMS: Interactions between mucosal cell types, environmental stressors, and intestinal microbiota contribute to pathogenesis in inflammatory bowel disease (IBD). Here, we applied metaproteomics of the mucosal-luminal interface to study the disease-related biology of the human colonic mucosa. METHODS: We recruited a discovery cohort of 51 IBD and non-IBD subjects endoscopically sampled by mucosal lavage at 6 colonic regions, and a validation cohort of 38 no-IBD subjects. Metaproteome data sets were produced for each sample and analyzed for association with colonic site and disease state using a suite of bioinformatic approaches. Localization of select proteins was determined by immunoblot analysis and immunohistochemistry of human endoscopic biopsy samples. RESULTS: Co-occurrence analysis of the discovery cohort metaproteome showed that proteins at the mucosal surface clustered into modules with evidence of differential functional specialization (eg, iron regulation, microbial defense) and cellular origin (eg, epithelial or hemopoietic). These modules, validated in an independent cohort, were differentially associated spatially along the gastrointestinal tract, and 7 modules were associated selectively with non-IBD, ulcerative colitis, and/or Crohn's disease states. In addition, the detailed composition of certain modules was altered in disease vs healthy states. We confirmed the predicted spatial and disease-associated localization of 28 proteins representing 4 different disease-related modules by immunoblot and immunohistochemistry visualization, with evidence for their distribution as millimeter-scale microgeographic mosaic. CONCLUSIONS: These findings suggest that the mucosal surface is a microgeographic mosaic of functional networks reflecting the local mucosal ecology, whose compositional differences in disease and healthy samples may provide a unique readout of physiologic and pathologic mucosal states.

A Disease-Associated Microbial and Metabolomics State in Relatives of Pediatric Inflammatory Bowel Disease Patients.

BACKGROUND & AIMS: Microbes may increase susceptibility to inflammatory bowel disease (IBD) by producing bioactive metabolites that affect immune activity and epithelial function. We undertook a family based study to identify microbial and metabolic features of IBD that may represent a predisease risk state when found in healthy first-degree relatives. METHODS: Twenty-one families with pediatric IBD were recruited, comprising 26 Crohn's disease patients in clinical remission, 10 ulcerative colitis patients in clinical remission, and 54 healthy siblings/parents. Fecal samples were collected for 16S ribosomal RNA gene sequencing, untargeted liquid chromatography-mass spectrometry metabolomics, and calprotectin measurement. Individuals were grouped into microbial and metabolomics states using Dirichlet multinomial models. Multivariate models were used to identify microbes and metabolites associated with these states. RESULTS: Individuals were classified into 2 microbial community types. One was associated with IBD but irrespective of disease status, had lower microbial diversity, and characteristic shifts in microbial composition including increased Enterobacteriaceae, consistent with dysbiosis. This microbial community type was associated similarly with IBD and reduced microbial diversity in an independent pediatric cohort. Individuals also clustered bioinformatically into 2 subsets with shared fecal metabolomics signatures. One metabotype was associated with IBD and was characterized by increased bile acids, taurine, and tryptophan. The IBD-associated microbial and metabolomics states were highly correlated, suggesting that they represented an integrated ecosystem. Healthy relatives with the IBD-associated microbial community type had an increased incidence of elevated fecal calprotectin. CONCLUSIONS: Healthy first-degree relatives can have dysbiosis associated with an altered intestinal metabolome that may signify a predisease microbial susceptibility state or subclinical inflammation. Longitudinal prospective studies are required to determine whether these individuals have a clinically significant increased risk for developing IBD.

Association of Systemic Sclerosis With a Unique Colonic Microbial Consortium.

OBJECTIVE: To compare colonic microbial composition in systemic sclerosis (SSc) patients and healthy controls and to determine whether certain microbial genera are associated with gastrointestinal (GI) tract symptoms in patients with SSc. METHODS: Healthy controls were age- and sex-matched (1:1) with adult SSc patients. Cecum and sigmoid mucosal lavage samples were obtained during colonoscopy. The microbiota in these samples were determined by Illumina HiSeq 2000 16S sequencing, and operational taxonomic units were selected. Linear discriminant analysis effect size was used to identify the genera that showed differential expression in SSc patients versus controls. Differential expression analysis for sequence count data was used to identify specific genera associated with GI tract symptoms. RESULTS: Among 17 patients with SSc (88% female; median age 52.1 years), the mean ± SD total GI Tract 2.0 score was 0.7 ± 0.6. Principal coordinate analysis illustrated significant differences in microbial communities in the cecum and sigmoid regions in SSc patients versus healthy controls (both P = 0.001). Similar to the findings in inflammatory disease states, SSc patients had decreased levels of commensal bacteria, such as Faecalibacterium and Clostridium, and increased levels of pathobiont bacteria, such as Fusobacterium and γ-Proteobacteria, compared with healthy controls. Bifidobacterium and Lactobacillus, which are typically reduced under conditions of inflammation, were also increased in abundance in patients with SSc. In SSc patients with moderate/severe GI tract symptoms, the abundance of Bacteroides fragilis was decreased, and that of Fusobacterium was increased, compared with patients who had no or mild symptoms. CONCLUSION: This study demonstrates a distinct colonic microbial signature in SSc patients compared with healthy controls. This unique ecologic change may perpetuate immunologic aberrations and contribute to clinical manifestations of SSc.

Sampling of intestinal microbiota and targeted amplification of bacterial 16S rRNA genes for microbial ecologic analysis.

Dysbiosis of host-associated commensal microbiota is emerging as an important factor in risk and phenotype of immunologic, metabolic, and behavioral diseases. Accurate analysis of microbial composition and functional state in humans or mice requires appropriate collection and pre-processing of biospecimens. Methods to sample luminal and mucosal microbiota from human or mouse intestines and to profile microbial phylogenetic composition using 16S rRNA sequencing are presented here. Data generated using the methods in this unit can be used for downstream quantitative analysis of microbial ecology.

Reprograming of gut microbiome energy metabolism by the FUT2 Crohn's disease risk polymorphism.

Fucosyltransferase 2 (FUT2) is an enzyme that is responsible for the synthesis of the H antigen in body fluids and on the intestinal mucosa. The H antigen is an oligosaccharide moiety that acts as both an attachment site and carbon source for intestinal bacteria. Non-secretors, who are homozygous for the loss-of-function alleles of FUT2 gene (sese), have increased susceptibility to Crohn's disease (CD). To characterize the effect of FUT2 polymorphism on the mucosal ecosystem, we profiled the microbiome, meta-proteome and meta-metabolome of 75 endoscopic lavage samples from the cecum and sigmoid of 39 healthy subjects (12 SeSe, 18 Sese and 9 sese). Imputed metagenomic analysis revealed perturbations of energy metabolism in the microbiome of non-secretor and heterozygote individuals, notably the enrichment of carbohydrate and lipid metabolism, cofactor and vitamin metabolism and glycan biosynthesis and metabolism-related pathways, and the depletion of amino-acid biosynthesis and metabolism. Similar changes were observed in mice bearing the FUT2(-/-) genotype. Metabolomic analysis of human specimens revealed concordant as well as novel changes in the levels of several metabolites. Human metaproteomic analysis indicated that these functional changes were accompanied by sub-clinical levels of inflammation in the local intestinal mucosa. Therefore, the colonic microbiota of non-secretors is altered at both the compositional and functional levels, affecting the host mucosal state and potentially explaining the association of FUT2 genotype and CD susceptibility.

HIV Infection is associated with compositional and functional shifts in the rectal mucosal microbiota.

BACKGROUND: Regardless of infection route, the intestine is the primary site for HIV-1 infection establishment and results in significant mucosal CD4+ T lymphocyte depletion, induces an inflammatory state that propagates viral dissemination, facilitates microbial translocation, and fosters establishment of one of the largest HIV reservoirs. Here we test the prediction that HIV infection modifies the composition and function of the mucosal commensal microbiota. RESULTS: Rectal mucosal microbiota were collected from human subjects using a sponge-based sampling methodology. Samples were collected from 20 HIV-positive men not receiving combination anti-retroviral therapy (cART), 20 HIV-positive men on cART and 20 healthy, HIV-negative men. Microbial composition of samples was analyzed using barcoded 16S Illumina deep sequencing (85,900 reads per sample after processing). Microbial metagenomic information for the samples was imputed using the bioinformatic tools PICRUST and HUMAnN. Microbial composition and imputed function in HIV-positive individuals not receiving cART was significantly different from HIV-negative individuals. Genera including Roseburia, Coprococcus, Ruminococcus, Eubacterium, Alistipes and Lachnospira were depleted in HIV-infected subjects not receiving cART, while Fusobacteria, Anaerococcus, Peptostreptococcus and Porphyromonas were significantly enriched. HIV-positive subjects receiving cART exhibited similar depletion and enrichment for these genera, but were of intermediate magnitude and did not achieve statistical significance. Imputed metagenomic functions, including amino acid metabolism, vitamin biosynthesis, and siderophore biosynthesis differed significantly between healthy controls and HIV-infected subjects not receiving cART. CONCLUSIONS: HIV infection was associated with rectal mucosal changes in microbiota composition and imputed function that cART failed to completely reverse. HIV infection was associated with depletion of some commensal species and enrichment of a few opportunistic pathogens. Many imputed metagenomic functions differed between samples from HIV-negative and HIV-positive subjects not receiving cART, possibly reflecting mucosal metabolic changes associated with HIV infection. Such functional pathways may represent novel interventional targets for HIV therapy if normalizing the microbial composition or functional activity of the microbiota proves therapeutically useful.

A modular organization of the human intestinal mucosal microbiota and its association with inflammatory bowel disease.

Abnormalities of the intestinal microbiota are implicated in the pathogenesis of Crohn's disease (CD) and ulcerative colitis (UC), two spectra of inflammatory bowel disease (IBD). However, the high complexity and low inter-individual overlap of intestinal microbial composition are formidable barriers to identifying microbial taxa representing this dysbiosis. These difficulties might be overcome by an ecologic analytic strategy to identify modules of interacting bacteria (rather than individual bacteria) as quantitative reproducible features of microbial composition in normal and IBD mucosa. We sequenced 16S ribosomal RNA genes from 179 endoscopic lavage samples from different intestinal regions in 64 subjects (32 controls, 16 CD and 16 UC patients in clinical remission). CD and UC patients showed a reduction in phylogenetic diversity and shifts in microbial composition, comparable to previous studies using conventional mucosal biopsies. Analysis of weighted co-occurrence network revealed 5 microbial modules. These modules were unprecedented, as they were detectable in all individuals, and their composition and abundance was recapitulated in an independent, biopsy-based mucosal dataset 2 modules were associated with healthy, CD, or UC disease states. Imputed metagenome analysis indicated that these modules displayed distinct metabolic functionality, specifically the enrichment of oxidative response and glycan metabolism pathways relevant to host-pathogen interaction in the disease-associated modules. The highly preserved microbial modules accurately classified IBD status of individual patients during disease quiescence, suggesting that microbial dysbiosis in IBD may be an underlying disorder independent of disease activity. Microbial modules thus provide an integrative view of microbial ecology relevant to IBD.

Integrative analysis of the microbiome and metabolome of the human intestinal mucosal surface reveals exquisite inter-relationships.

BACKGROUND: Consistent compositional shifts in the gut microbiota are observed in IBD and other chronic intestinal disorders and may contribute to pathogenesis. The identities of microbial biomolecular mechanisms and metabolic products responsible for disease phenotypes remain to be determined, as do the means by which such microbial functions may be therapeutically modified. RESULTS: The composition of the microbiota and metabolites in gut microbiome samples in 47 subjects were determined. Samples were obtained by endoscopic mucosal lavage from the cecum and sigmoid colon regions, and each sample was sequenced using the 16S rRNA gene V4 region (Illumina-HiSeq 2000 platform) and assessed by UPLC mass spectroscopy. Spearman correlations were used to identify widespread, statistically significant microbial-metabolite relationships. Metagenomes for identified microbial OTUs were imputed using PICRUSt, and KEGG metabolic pathway modules for imputed genes were assigned using HUMAnN. The resulting metabolic pathway abundances were mostly concordant with metabolite data. Analysis of the metabolome-driven distribution of OTU phylogeny and function revealed clusters of clades that were both metabolically and metagenomically similar. CONCLUSIONS: The results suggest that microbes are syntropic with mucosal metabolome composition and therefore may be the source of and/or dependent upon gut epithelial metabolites. The consistent relationship between inferred metagenomic function and assayed metabolites suggests that metagenomic composition is predictive to a reasonable degree of microbial community metabolite pools. The finding that certain metabolites strongly correlate with microbial community structure raises the possibility of targeting metabolites for monitoring and/or therapeutically manipulating microbial community function in IBD and other chronic diseases.

Loss of intestinal core 1-derived O-glycans causes spontaneous colitis in mice.

Mucin-type O-linked oligosaccharides (O-glycans) are primary components of the intestinal mucins that form the mucus gel layer overlying the gut epithelium. Impaired expression of intestinal O-glycans has been observed in patients with ulcerative colitis (UC), but its role in the etiology of this disease is unknown. Here, we report that mice with intestinal epithelial cell-specific deficiency of core 1-derived O-glycans, the predominant form of O-glycans, developed spontaneous colitis that resembled human UC, including massive myeloid infiltrates and crypt abscesses. The colitis manifested in these mice was also characterized by TNF-producing myeloid infiltrates in colon mucosa in the absence of lymphocytes, supporting an essential role for myeloid cells in colitis initiation. Furthermore, induced deletion of intestinal core 1-derived O-glycans caused spontaneous colitis in adult mice. These data indicate a causal role for the loss of core 1-derived O-glycans in colitis. Finally, we detected a biosynthetic intermediate typically exposed in the absence of core 1 O-glycan, Tn antigen, in the colon epithelium of a subset of UC patients. Somatic mutations in the X-linked gene that encodes core 1 ß1,3-galactosyltransferase-specific chaperone 1 (C1GALT1C1, also known as Cosmc), which is essential for core 1 O-glycosylation, were found in Tn-positive epithelia. These data suggest what we believe to be a new molecular mechanism for the pathogenesis of UC.