Project description:Metaproteomic analysis of air particulate matter provides information about the properties of bioaerosols in the atmosphere and their influence on climate and public health. In this work, a new method for the extraction and analysis of proteins in airborne particulate matter from quartz microfiber filters was developed. Different protein extraction procedures were tested in order to select the best extraction protocol in terms of protein recovery. The optimized method was tested for extraction of proteins from spores of ubiquitous bacteria species and used for the first time for the metaproteomics characterization of filters from work environment. In particular, ambient aerosol samples were collected in different working environments, i.e. a composting plant, wastewater treatment plant and agricultural holding. One-hundred seventy-nine, 15, 205 and 444 proteins were successfully identified in composting plant, wastewater treatment plant, and agricultural holding, respectively. All identified proteins were mainly originate from fungi, bacteria and plants which is in line with the major categories of primary biological aerosol particles. The paper is the first metaproteomic study applied to bioaereosol samples collected in occupationally relevant environmental sites providing interesting information on the composting, wastewater treatment and feed blending processes. Significance This manuscript describes the metaproteomic analysis of aerosol samples collected in work enviroments. This is a novel use of aereosol samples and is needed as there is no really comprehensive way of analysing aereosol samples from a metaproteomic point of view. This paper could help to advance methods for metaproteomic analysis of bioaersols, specifically by comparing protein extraction protocols and pairing the best performing extraction protocol with a gel-free protein separation procedure applied for the first time for analysis of bioaerosol samples. The obtained data showed as bioaerosol was essentially made of fungi, bacteria and plant proteins, many of which could be associated to possible aerosolisation and could be a major health concern for workers on site and to the populations residing in neighbouring area.
Project description:Cable bacteria of the family Desulfobulbaceae form centimeter-long filaments comprising thousands of cells. They occur worldwide in the surface of aquatic sediments, where they connect sulfide oxidation with oxygen or nitrate reduction via long-distance electron transport. In the absence of pure cultures, we used single-filament genome amplification and metagenomics to retrieve draft genomes of three marine Candidatus Electrothrix and one freshwater Ca. Electronema species. These genomes contain >50% of unknown genes but still largely share their core genomic makeup with sulfate-reducing and sulfur-disproportionating Desulfobulbaceae, with few genes lost and 212 unique genes conserved among cable bacteria. Last common ancestor analysis indicated gene divergence and lateral gene transfer as equally important origins of these unique genes. With support from metaproteomic data of Ca. Electronema, the genomes suggest that cable bacteria oxidize sulfide by inversing the canonical sulfate reduction pathway and fix CO2 using the Wood-Ljungdahl pathway. Cable bacteria show limited organotrophic potential, may assimilate smaller organic acids and alcohols, fix N2, and synthesize polyphosphates and polyglucose as storage compounds; several of these traits were confirmed by cell-level experimental analyses. We propose a model for electron flow from sulfide to oxygen that involves periplasmic cytochromes, type IV pili as integral components of conductive periplasmic fibers, and periplasmic oxygen reduction. This model proposes that an active cable bacterium gains energy in the anodic, sulfide-oxidizing cells, while cells in the oxic zone flare off electrons through intense cathodic oxygen respiration without energy conservation; this peculiar form of multicellularity seems unparalleled in the microbial world.
Project description:Bacterial vaginosis (BV) is the most common cause of vaginal discharge among women worldwide. BV is characterized by an imbalance in the vaginal microbiota with depletion of protective Lactobacillus species and overgrowth of facultative and strictly anaerobic bacteria. Although the development of a polymicrobial biofilm on the vaginal epithelium is a hallmark of BV, interactions between key BV-associated bacteria (BVAB) [i.e. Gardnerella vaginalis, Fannyhessea vaginae, and Prevotella bivia] present in the biofilm are still not completely understood. In this study, we aimed to analyse the transcriptome of single and triple-species biofilms growing in the rich medium, New York City III (NYCIII). A previous analysis of triple-species biofilms composition by qPCR showed that the biofilms were mainly composed of G. vaginalis, followed by F. vaginae and P. bivia. The transcriptomic analysis revealed a total of 431 (34 upregulated and 397 downregulated), 126 (36 upregulated and 90 downregulated), and 39 (31 upregulated and 8 downregulated) differentially expressed genes for G. vaginalis, F. vaginae, and P. bivia, respectively. Gene ontology only detected enrichment for the downregulated genes of G. vaginalis and 47 GO terms were associated with molecular functions, cellular components and biological processes, mainly metabolism. Hence, this work showed the adaptation of 3 BVAB when growing in a triple-species biofilm, with several genes being differentially expressed in all the species growing in a polymicrobial biofilm.
Project description:Metaproteomic and Metagenomic Coupled Approach to Investigate Microbial Response to Electrochemical Conditions in Microbial Fuel Cell
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: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: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.