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Frontiers in Cellular and Infection... 2017Human gut microbiota is believed to be directly or indirectly involved in cardiovascular diseases and hypertension. However, the identification and functional status of...
Human gut microbiota is believed to be directly or indirectly involved in cardiovascular diseases and hypertension. However, the identification and functional status of the hypertension-related gut microbe(s) have not yet been surveyed in a comprehensive manner. Here we characterized the gut microbiome in hypertension status by comparing fecal samples of 60 patients with primary hypertension and 60 gender-, age-, and body weight-matched healthy controls based on whole-metagenome shotgun sequencing. Hypertension implicated a remarkable gut dysbiosis with significant reduction in within-sample diversity and shift in microbial composition. Metagenome-wide association study (MGWAS) revealed 53,953 microbial genes that differ in distribution between the patients and healthy controls (false discovery rate, 0.05) and can be grouped into 68 clusters representing bacterial species. Opportunistic pathogenic taxa, such as, spp., spp., and were frequently distributed in hypertensive gut microbiome, whereas the short-chain fatty acid producer, such as, spp. and , were higher in controls. The number of hypertension-associated species also showed stronger correlation to the severity of disease. Functionally, the hypertensive gut microbiome exhibited higher membrane transport, lipopolysaccharide biosynthesis and steroid degradation, while in controls the metabolism of amino acid, cofactors and vitamins was found to be higher. We further provided the microbial markers for disease discrimination and achieved an area under the receiver operator characteristic curve (AUC) of 0.78, demonstrating the potential of gut microbiota in prediction of hypertension. These findings represent specific alterations in microbial diversity, genes, species and functions of the hypertensive gut microbiome. Further studies on the causality relationship between hypertension and gut microbiota will offer new prospects for treating and preventing the hypertension and its associated diseases.
Topics: Bacteria; Dysbiosis; Feces; Female; Gastrointestinal Microbiome; Gastrointestinal Tract; Humans; Hypertension; Male; Metagenome; Middle Aged; Whole Genome Sequencing
PubMed: 28884091
DOI: 10.3389/fcimb.2017.00381 -
ACS Chemical Biology Jun 2017Cysteine proteases are among the most abundant hydrolytic enzymes produced by bacteria, and this diverse family of proteins have significant biological roles in...
Cysteine proteases are among the most abundant hydrolytic enzymes produced by bacteria, and this diverse family of proteins have significant biological roles in bacterial viability and environmental interactions. Members of the clostripain-like (C11) family of cysteine proteases from commensal gut bacterial strains have recently been shown to mediate immune responses by inducing neutrophil phagocytosis and activating bacterial pathogenic toxins. Development of substrates, inhibitors, and probes that target C11 proteases from enteric bacteria will help to establish the role of these proteins at the interface of the host and microbiome in health and disease. We employed a mass spectrometry-based substrate profiling method to identify an optimal peptide substrate of PmC11, a C11 protease secreted by the commensal bacterium Parabacteroides merdae. Using this substrate sequence information, we synthesized a panel of fluorogenic substrates to calculate k and K and to evaluate the importance of the P2 amino acid for substrate turnover. A potent and irreversible tetrapeptide inhibitor with a C-terminal acyloxymethyl ketone warhead, Ac-VLTK-AOMK, was then synthesized. We determined the crystal structure of PmC11 in complex with this inhibitor and uncovered key active-site interactions that govern PmC11 substrate recognition and specificity. This is the first C11 protease structure in complex with a substrate mimetic and is also the highest resolution crystal structure of a C11 protease to date at 1.12 Å resolution. Importantly, subjecting human epithelial cell lysates to PmC11 hydrolysis in combination with subtiligase-based N-terminal labeling and tandem mass spectrometry proteomics complemented the stringent substrate specificity observed in the in vitro substrate profiling experiment. The combination of chemical biological, biophysical, and biochemical techniques presented here to elucidate and characterize PmC11 substrate selectivity can be expanded to other proteases and the development of chemical tools to study these essential proteins in biologically relevant samples, such as the highly complex distal gut microbiome.
Topics: Bacterial Proteins; Crystallography, X-Ray; Cysteine Endopeptidases; Cysteine Proteases; Enterobacteriaceae; Epithelial Cells; Humans; Molecular Structure; Substrate Specificity; Symbiosis
PubMed: 28414448
DOI: 10.1021/acschembio.7b00143 -
The Journal of Biological Chemistry Apr 2016The structure of a C11 peptidase PmC11 from the gut bacterium, Parabacteroides merdae, has recently been determined, enabling the identification and characterization of...
The structure of a C11 peptidase PmC11 from the gut bacterium, Parabacteroides merdae, has recently been determined, enabling the identification and characterization of a C11 orthologue, PNT1, in the parasitic protozoon Trypanosoma brucei. A phylogenetic analysis identified PmC11 orthologues in bacteria, archaea, Chromerids, Coccidia, and Kinetoplastida, the latter being the most divergent. A primary sequence alignment of PNT1 with clostripain and PmC11 revealed the position of the characteristic His-Cys catalytic dyad (His(99) and Cys(136)), and an Asp (Asp(134)) in the potential S1 binding site. Immunofluorescence and cryoelectron microscopy revealed that PNT1 localizes to the kinetoplast, an organelle containing the mitochondrial genome of the parasite (kDNA), with an accumulation of the protein at or near the antipodal sites. Depletion of PNT1 by RNAi in the T. brucei bloodstream form was lethal both in in vitro culture and in vivo in mice and the induced population accumulated cells lacking a kinetoplast. In contrast, overexpression of PNT1 led to cells having mislocated kinetoplasts. RNAi depletion of PNT1 in a kDNA independent cell line resulted in kinetoplast loss but was viable, indicating that PNT1 is required exclusively for kinetoplast maintenance. Expression of a recoded wild-type PNT1 allele, but not of an active site mutant restored parasite viability after induction in vitro and in vivo confirming that the peptidase activity of PNT1 is essential for parasite survival. These data provide evidence that PNT1 is a cysteine peptidase that is required exclusively for maintenance of the trypanosome kinetoplast.
Topics: Alleles; Animals; Catalytic Domain; Cysteine Proteases; Gene Expression Regulation, Enzymologic; Mice; Protozoan Proteins; Trypanosoma brucei brucei
PubMed: 26940875
DOI: 10.1074/jbc.M116.714972 -
The Journal of Biological Chemistry Apr 2016Clan CD cysteine peptidases, a structurally related group of peptidases that include mammalian caspases, exhibit a wide range of important functions, along with a...
Clan CD cysteine peptidases, a structurally related group of peptidases that include mammalian caspases, exhibit a wide range of important functions, along with a variety of specificities and activation mechanisms. However, for the clostripain family (denoted C11), little is currently known. Here, we describe the first crystal structure of a C11 protein from the human gut bacterium, Parabacteroides merdae (PmC11), determined to 1.7-Å resolution. PmC11 is a monomeric cysteine peptidase that comprises an extended caspase-like α/β/α sandwich and an unusual C-terminal domain. It shares core structural elements with clan CD cysteine peptidases but otherwise structurally differs from the other families in the clan. These studies also revealed a well ordered break in the polypeptide chain at Lys(147), resulting in a large conformational rearrangement close to the active site. Biochemical and kinetic analysis revealed Lys(147) to be an intramolecular processing site at which cleavage is required for full activation of the enzyme, suggesting an autoinhibitory mechanism for self-preservation. PmC11 has an acidic binding pocket and a preference for basic substrates, and accepts substrates with Arg and Lys in P1 and does not require Ca(2+) for activity. Collectively, these data provide insights into the mechanism and activity of PmC11 and a detailed framework for studies on C11 peptidases from other phylogenetic kingdoms.
Topics: Bacterial Proteins; Bacteroidaceae; Crystallography, X-Ray; Cysteine Proteases; Gastrointestinal Microbiome; Humans; Protein Structure, Secondary; Protein Structure, Tertiary
PubMed: 26940874
DOI: 10.1074/jbc.M115.706143 -
Frontiers in Microbiology 2015Microviridae, a family of bacteria-infecting ssDNA viruses, is one of the still poorly characterized bacteriophage groups, even though it includes phage PhiX174, one of...
Microviridae, a family of bacteria-infecting ssDNA viruses, is one of the still poorly characterized bacteriophage groups, even though it includes phage PhiX174, one of the main models in virology for genomic and capsid structure studies. Recent studies suggest that they are diverse and well represented in marine and freshwater virioplankton as well as in human microbiomes. However, their diversity, abundance, and ecological role are completely unknown in soil ecosystems. Here we present the comparative analysis of 17 completely assembled Microviridae genomes from 12 viromes of a Sphagnum-dominated peatland. Phylogenetic analysis of the conserved major capsid protein sequences revealed the affiliation to Gokushovirinae and Pichovirinae as well as to two newly defined subfamilies, the Aravirinae and Stokavirinae. Additionally, two new distinct prophages were identified in the genomes of Parabacteroides merdae and Parabacteroides distasonis representing a potential new subfamily of Microviridae. The differentiation of the subfamilies was confirmed by gene order and similarity analysis. Relative abundance analysis using the affiliation of the major capsid protein (VP1) revealed that Gokushovirinae, followed by Aravirinae, are the most abundant Microviridae in 11 out of 12 peat viromes. Sequences matching the Gokushovirinae and Aravirinae VP1 matching sequences, respectively, accounted for up to 4.19 and 0.65% of the total number of sequences in the corresponding virome, respectively. In this study we provide new genome information of Microviridae and pave the way toward quantitative estimations of Microviridae subfamilies.
PubMed: 25972855
DOI: 10.3389/fmicb.2015.00375