Project description:Metaproteomic and Metagenomic Coupled Approach to Investigate Microbial Response to Electrochemical Conditions in Microbial Fuel Cell
Project description:A metaproteomics analysis was conducted on the infant fecal microbiome to characterize global protein expression in 8 samples obtained from infants with a range of early-life experiences. Samples included breast-, formula- or mixed-fed, mode of delivery, and antibiotic treatment and one set of monozygotic twins. Although label-free mass spectrometry-based proteomics is routinely used for the identification and quantification of thousands of proteins in complex samples, the metaproteomic analysis of the gut microbiome presents particular technical challenges. Among them: the extreme complexity and dynamic range of member taxa/species, the need for matched, well-annotated metagenomics databases, and the high inter-protein sequence redundancy/similarity between related members. In this study, a metaproteomic approach was developed for assessment of the biological phenotype and functioning, as a complement to 16S rRNA sequencing analysis to identify constituent taxa. A sample preparation method was developed for recovery and lysis of bacterial cells, followed by trypsin digestion, and pre-fractionation using Strong Cation Exchange chromatography. Samples were then subjected to high performance LC-MS/MS. Data was searched against the Human Microbiome Project database, and a homology-based meta-clustering strategy was used to combine peptides from multiple species into representative proteins. Bacterial taxonomies were also identified, based on species-specific protein sequences, and protein metaclusters were assigned to pathways and functional groups. The results obtained demonstrate the applicability of this approach for performing qualitative comparisons of human fecal microbiome composition, physiology and metabolism, and also provided a more detailed assessment of microbial composition in comparison to 16S rRNA.
Project description:Improper use of antibiotics in swine could reduce commensal bacteria and possibly increase pathogen infections via the gut resistome. This study aimed to compare the metaproteomic profiles of gut resistome and related metabolism in the cecal microbiota of fattening pigs raised under antibiotic-free (ABF) conditions with those of ordinary industrial pigs (CTRL).
Project description:Detection of immunogenic proteins remains an important task for life sciences as it nourishes the understanding of pathogenicity, illuminates new potential vaccine candidates and broadens the spectrum of biomarkers applicable in diagnostic tools. Traditionally, immunoscreenings of expression libraries via polyclonal sera on nitrocellulose membranes or screenings of whole proteome lysates in 2-D gel electrophoresis are performed. However, these methods feature some rather inconvenient disadvantages. Screening of expression libraries to expose novel antigens from bacteria often lead to an abundance of false positive signals owing to the high cross reactivity of polyclonal antibodies towards the proteins of the expression host. A method is presented that overcomes many disadvantages of the old procedures. We incorporated a fusion tag prior to our genes of interest and attached the expressed fusion proteins covalently on microarrays. This enhances the specific binding of the proteins compared to nitrocellulose. Thus, it helps to reduce the number of false positives significantly. It enables us to screen for immunogenic proteins in a shorter time, with more samples and statistical reliability. We validated our method by employing several known genes from Campylobacter jejuni NCTC 11168. Four of proteins that have previously been described as immunogenic have successfully been assessed immunogenic abilities with our method. One protein with no known immunogenic behaviour before suggested potential immunogenicity. The method presented offers a new approach for screening of bacterial expression libraries to illuminate novel proteins with immunogenic features. It could provide a powerful and attractive alternative to existing methods and help to detect and identify bacterial virulence factors, vaccine candidates and potential biomarkers.
Project description:Coral reefs worldwide are facing rapid decline due to coral bleaching. However, knowledge of the physiological characteristics and molecular mechanisms of coral symbionts respond to stress is scarce. Here, metagenomic and metaproteomic approach were utilized to shed light on the changes in the composition and functions of coral symbionts during coral bleaching. The results demonstrated that coral bleaching significantly affected the composition of symbionts, with bacterial communities dominating in bleached corals. Difference analysis of gene and protein indicated that symbiont functional disturbances in response to heat stress, resulting in abnormal energy metabolism that could potentially compromise symbiont health and resilience. Furthermore, our findings highlighted the highly diverse microbial communities of coral symbionts, with beneficial bacteria provide critical services to corals in stress responses, while pathogenic bacteria drive coral bleaching. This study provides comprehensive insights into the complex response mechanisms of coral symbionts under thermal stress and offers fundamental data for future monitoring of coral health.
Project description:we applied a metaproteomic approach to investigate the global protein expression profiles of two co-existing phytoplankton species H. akashiwo and P. donghaiense during their bloom periods, and characterized the differentially expressed proteins. Our results indicated different light-harvesting ability and nutritional niche divergence in utilization of carbon, nitrogen and phosphorus drove bloom occurrence of the two phytoplankton species under different ambient conditions.
Project description:The intestinal microbiota plays a key role in shaping host homeostasis by regulating metabolism, immune responses and behaviour. Its dysregulation has been associated with metabolic, immune and neuropsychiatric disorders and is accompanied by changes in bacterial metabolic regulation. Although proteomic is well suited for analysis of individual microbes, metaproteomic of faecal samples is challenging due to the physical structure of the sample, presence of contaminating host proteins and coexistence of hundreds of species. Furthermore, there is a lack of consensus regarding preparation of faecal samples, as well as downstream bioinformatic analyses following metaproteomic data acquisition. Here we assess sample preparation and data analysis strategies applied to mouse faeces in a typical LC-MS/MS metaproteomic experiment. We show that low speed centrifugation (LSC) of faecal samples leads to high protein identification rates but possibly enriched for a subset of taxa. During database search, two-step search strategies led to dramatic and underestimated accumulation of false positive protein identifications. Regarding taxonomic annotation, the MS-identified peptides of unknown origin were annotated with highest sensitivity and specificity using the Unipept software. Comparison of matching metaproteome and metagenome data revealed a positive correlation between protein and gene abundances. Notably, nearly all functional categories of detected protein groups were differentially abundant in the metaproteome compared to what would be expected from the metagenome, highlighting the need to perform metaproteomic when studying complex microbiome samples.
Project description:Collimonas is a genus of soil bacteria which comprises three recognized species: C. fungivorans, C. pratensis and C. arenae. The bacteria belonging to this genus share the ability to lyse chitin (chitinolysis) and feed on living fungal hyphae (mycophagy), but they differ in colony morphology, physiological properties and antifungal activity. In order to gain a better insight into the genetic background underlying this phenotypic variability of collimonads, we investigated the variability in the genomic content of five strains representing the three formally recognized Collimonas species. The genomic content of four test strains was hybridized on an array representing the reference strain C. fungivorans Ter331.
Project description:The link between human gut microbiota (a complex group of microorganisms including not only bacteria but also fungi, viruses, etc.,) and the physiological state is nowadays unquestionable. Metaproteomic has emerged as a useful technique to characterize this microbial community, not just taxonomically, but also focusing on specific biological processes carried out by gut microbiota that may have an effect in the host health or pathological state. Cystic fibrosis is a genetic disease in which the microbiota of the respiratory tract determines the patient's survival and differences in composition of gut microbiota of cystic fibrosis patients respect to healthy infants have been reported. In order to characterize this host-microbiota inter-relation, we carried out the metaproteomic study of 30 stool samples from infants with cystic fibrosis.