A complementary metaproteomic approach to interrogate microbiome cultivated from clinical colon biopsies.

The human gut microbiome plays a vital role in preserving individual health and is intricately involved in essential functions. Imbalances or dysbiosis within the microbiome can significantly impact human health and are associated with many diseases. Several metaproteomics platforms are currently available to study microbial proteins within complex microbial communities. In this study, we attempted to develop an integrated pipeline to provide deeper insights into both the taxonomic and functional aspects of the cultivated human gut microbiomes derived from clinical colon biopsies. We combined a rapid peptide search by MSFragger against the Unified Human Gastrointestinal Protein database and the taxonomic and functional analyses with Unipept Desktop and MetaLab-MAG. Across seven samples, we identified and matched nearly 36,000 unique peptides to approximately 300 species and 11 phyla. Unipept Desktop provided gene ontology, InterPro entries, and enzyme commission number annotations, facilitating the identification of relevant metabolic pathways. MetaLab-MAG contributed functional annotations through Clusters of Orthologous Genes and Non-supervised Orthologous Groups categories. These results unveiled functional similarities and differences among the samples. This integrated pipeline holds the potential to provide deeper insights into the taxonomy and functions of the human gut microbiome for interrogating the intricate connections between microbiome balance and diseases.

RapidAIM 2.0: a high-throughput assay to study functional response of human gut microbiome to xenobiotics.

Aim: Our gut microbiome has its own functionalities which can be modulated by various xenobiotic and biotic components. The development and application of a high-throughput functional screening approach of individual gut microbiomes accelerates drug discovery and our understanding of microbiome-drug interactions. We previously developed the rapid assay of individual microbiome (RapidAIM), which combined an optimized culturing model with metaproteomics to study gut microbiome responses to xenobiotics. In this study, we aim to incorporate automation and multiplexing techniques into RapidAIM to develop a high-throughput protocol. Methods: To develop a 2.0 version of RapidAIM, we automated the protein analysis protocol, and introduced a tandem mass tag (TMT) multiplexing technique. To demonstrate the typical outcome of the protocol, we used RapidAIM 2.0 to evaluate the effect of prebiotic kestose on ex vivo individual human gut microbiomes biobanked with five different workflows. Results: We describe the protocol of RapidAIM 2.0 with extensive details on stool sample collection, biobanking, in vitro culturing and stimulation, sample processing, metaproteomics measurement, and data analysis. The analysis depth of 5,014 ± 142 protein groups per multiplexed sample was achieved. A test on five biobanking methods using RapidAIM 2.0 showed the minimal effect of sample processing on live microbiota functional responses to kestose. Conclusions: Depth and reproducibility of RapidAIM 2.0 are comparable to previous manual label-free metaproteomic analyses. In the meantime, the protocol realizes culturing and sample preparation of 320 samples in six days, opening the door to extensively understanding the effects of xenobiotic and biotic factors on our internal ecology.

Pairing metagenomics and metaproteomics to characterize ecological niches and metabolic essentiality of gut microbiomes.

The genome of a microorganism encodes its potential functions that can be implemented through expressed proteins. It remains elusive how a protein's selective expression depends on its metabolic essentiality to microbial growth or its ability to claim resources as ecological niches. To reveal a protein's metabolic or ecological role, we developed a computational pipeline, which pairs metagenomics and metaproteomics data to quantify each protein's gene-level and protein-level functional redundancy simultaneously. We first illustrated the idea behind the pipeline using simulated data of a consumer-resource model. We then validated it using real data from human and mouse gut microbiome samples. In particular, we analyzed ABC-type transporters and ribosomal proteins, confirming that the metabolic and ecological roles predicted by our pipeline agree well with prior knowledge. Finally, we performed in vitro cultures of a human gut microbiome sample and investigated how oversupplying various sugars involved in ecological niches influences the community structure and protein abundance. The presented results demonstrate the performance of our pipeline in identifying proteins' metabolic and ecological roles, as well as its potential to help us design nutrient interventions to modulate the human microbiome.

Multilevel Proteomic Profiling of Colorectal Adenocarcinoma Caco-2 Cell Differentiation to Characterize an Intestinal Epithelial Model.

Emergent advancements on the role of the intestinal microbiome for human health and disease necessitate well-defined intestinal cellular models to study and rapidly assess host, microbiome, and drug interactions. Differentiated Caco-2 cell line is commonly utilized as an epithelial model for drug permeability studies and has more recently been utilized for investigating host-microbiome interactions. However, its suitability to study such interactions remains to be characterized. Here, we employed multilevel proteomics to demonstrate that both spontaneous and butyrate-induced Caco-2 differentiations displayed similar protein and pathway changes, including the downregulation of proteins related to translation and proliferation and upregulation of functions implicated in host-microbiome interactions, such as cell adhesion, tight junction, extracellular vesicles, and responses to stimuli. Lysine acetylomics revealed that histone protein acetylation levels were decreased along with cell differentiation, while the acetylation in proteins associated with mitochondrial functions was increased. This study also demonstrates that, compared to spontaneous differentiation methods, butyrate-containing medium accelerates Caco-2 differentiation, with earlier upregulation of proteins related to host-microbiome interactions, suggesting its superiority for assay development using this intestinal model. Altogether, this multiomics study emphasizes the controlled progression of Caco-2 differentiation toward a specialized intestinal epithelial-like cell and establishes its suitability for investigating the host-microbiome interactions.

Spectral entropy as a measure of the metaproteome complexity.

The diversity and complexity of the microbiome's genomic landscape are not always mirrored in its proteomic profile. Despite the anticipated proteomic diversity, observed complexities of microbiome samples are often lower than expected. Two main factors contribute to this discrepancy: limitations in mass spectrometry's detection sensitivity and bioinformatics challenges in metaproteomics identification. This study introduces a novel approach to evaluating sample complexity directly at the full mass spectrum (MS1) level rather than relying on peptide identifications. When analyzing under identical mass spectrometry conditions, microbiome samples displayed significantly higher complexity, as evidenced by the spectral entropy and peptide candidate entropy, compared to single-species samples. The research provides solid evidence for the complexity of microbiome in proteomics indicating the optimization potential of the bioinformatics workflow.

The Landscape and Perspectives of the Human Gut Metaproteomics.

The human gut microbiome is closely associated with human health and diseases. Metaproteomics has emerged as a valuable tool for studying the functionality of the gut microbiome by analyzing the entire proteins present in microbial communities. Recent advancements in liquid chromatography and tandem mass spectrometry (LC-MS/MS) techniques have expanded the detection range of metaproteomics. However, the overall coverage of the proteome in metaproteomics is still limited. While metagenomics studies have revealed substantial microbial diversity and functional potential of the human gut microbiome, few studies have summarized and studied the human gut microbiome landscape revealed with metaproteomics. In this article, we present the current landscape of human gut metaproteomics studies by re-analyzing the identification results from 15 published studies. We quantified the limited proteome coverage in metaproteomics and revealed a high proportion of annotation coverage of metaproteomics-identified proteins. We conducted a preliminary comparison between the metaproteomics view and the metagenomics view of the human gut microbiome, identifying key areas of consistency and divergence. Based on the current landscape of human gut metaproteomics, we discuss the feasibility of using metaproteomics to study functionally unknown proteins and propose a whole workflow peptide-centric analysis. Additionally, we suggest enhancing metaproteomics analysis by refining taxonomic classification and calculating confidence scores, as well as developing tools for analyzing the interaction between taxonomy and function.

Glycomacropeptide as an Efficient Agent to Fight Pathophysiological Mechanisms of Metabolic Syndrome.

There is currently a growing interest in the use of nutraceuticals as a means of preventing the development of complex diseases. Given the considerable health potential of milk-derived peptides, the aim of this study was to investigate the protective effects of glycomacropeptide (GMP) on metabolic syndrome. Particular emphasis was placed on the potential mechanisms mitigating cardiometabolic disorders in high-fat, high-fructose diet-fed mice in the presence of GMP or Bipro, an isocaloric control. The administration of GMP for 12 weeks reduced obesity, hyperglycemia and hyperinsulinemia caused by a high-fat, high-fructose diet, resulting in a decline in insulin resistance. GMP also lessened systemic inflammation, as indicated by decreased circulating inflammatory cytokines. In the intestinal and hepatic tissues, GMP improved homeostasis by increasing insulin sensitivity and attenuating high-fat, high-fructose-induced inflammation, oxidative stress and endoplasmic reticulum stress. Biochemical and histological analyses revealed improved hepatic steatosis and fatty acid composition in the livers of high-fat, high-fructose diet-fed mice treated with GMP compared to Bipro. A trend toward a decrease in bile acids without any marked changes in intestinal microbiota composition characterized GMP-treated animals compared to those administered Bipro. GMP offers considerable potential for fighting metabolic syndrome-related components and complications given its beneficial effects on risk factors such as inflammation, oxidative stress and endoplasmic reticulum stress without involving the intestinal microbiota.

Staufen1 controls mitochondrial metabolism via HIF2α in embryonal rhabdomyosarcoma and promotes tumorigenesis.

Elevated mitochondrial metabolism promotes tumorigenesis of Embryonal Rhabdomyosarcomas (ERMS). Accordingly, targeting oxidative phosphorylation (OXPHOS) could represent a therapeutic strategy for ERMS. We previously demonstrated that genetic reduction of Staufen1 (STAU1) levels results in the inhibition of ERMS tumorigenicity. Here, we examined STAU1-mediated mechanisms in ERMS and focused on its potential involvement in regulating OXPHOS. We report the novel and differential role of STAU1 in mitochondrial metabolism in cancerous versus non-malignant skeletal muscle cells (NMSkMCs). Specifically, our data show that STAU1 depletion reduces OXPHOS and inhibits proliferation of ERMS cells. Our findings further reveal the binding of STAU1 to several OXPHOS mRNAs which affects their stability. Indeed, STAU1 depletion reduced the stability of OXPHOS mRNAs, causing inhibition of mitochondrial metabolism. In parallel, STAU1 depletion impacted negatively the HIF2α pathway which further modulates mitochondrial metabolism. Exogenous expression of HIF2α in STAU1-depleted cells reversed the mitochondrial inhibition and induced cell proliferation. However, opposite effects were observed in NMSkMCs. Altogether, these findings revealed the impact of STAU1 in the regulation of mitochondrial OXPHOS in cancer cells as well as its differential role in NMSkMCs. Overall, our results highlight the therapeutic potential of targeting STAU1 as a novel approach for inhibiting mitochondrial metabolism in ERMS.

MetaPep: A core peptide database for faster human gut metaproteomics database searches.

Metaproteomics has increasingly been applied to study functional changes in the human gut microbiome. Peptide identification is an important step in metaproteomics research, with sequence database search (SDS) and spectral library search (SLS) as the two main methods to identify peptides. However, the large search space in metaproteomics studies causes significant challenges for both identification methods. Moreover, with the development of mass spectrometry, it is now feasible to perform metaproteomic projects involving 100-1000 individual microbiomes. These large-scale projects create a conundrum for searching large databases. In this study, we constructed MetaPep, a core peptide database (including both collections of peptide sequences and tandem MS spectra) greatly accelerating the peptide identifications. Raw files from fifteen metaproteomics projects were re-analyzed and the identified peptide-spectrum matches (PSMs) were used to construct the MetaPep database. The constructed MetaPep database achieved rapid and accurate identification of peptides for human gut metaproteomics. MetaPep has a large collection of peptides and spectra that have been identified in published human gut metaproteomics datasets. MetaPep database can be used as an important resource in the current stage of human gut metaproteomics research. This study showed the possibility of applying a core peptide database as a generic metaproteomics workflow. MetaPep could also be an important resource for future human gut metaproteomics research, such as DIA (data-independent acquisition) analysis.

The Influence of Protein Secretomes of Enterococcus durans on ex vivo Human Gut Microbiome.

The gut microbiome plays a critical role to all animals and humans health. Methods based on ex vivo cultures are time and cost-effective solutions for rapid evaluation of probiotic effects on microbiomes. In this study, we assessed whether the protein secretome from the potential probiotic Enterococcus durans LAB18S grown on fructoligosaccharides (FOS) and galactoligosaccharides (GOS) had specific effects on ex vivo cultured intestinal microbiome obtained from a healthy individual. Metaproteomics was used to evaluate changes in microbial communities of the human intestinal microbiome. Hierarchical clustering analysis revealed 654 differentially abundant proteins from the metaproteome samples, showing that gut microbial protein expression varied on the presence of different E. durans secretomes. Increased amount of Bacteroidetes phylum was observed in treatments with secretomes from E. durans cultures on FOS, GOS and albumin, resulting in a decrease of the Firmicutes to Bacteroidetes (F/B) ratio. The most functionally abundant bacterial taxa were Roseburia, Bacteroides, Alistipes and Faecalibacterium. The results suggest that the secretome of E. durans may have favorable effects on the intestinal microbial composition, stimulating growth and different protein expression of beneficial bacteria. These findings suggest that proteins secreted by E. durans growing on FOS and GOS have different effects on the modulation of gut microbiota functional activities during cultivation.

The LDL receptor is regulated by membrane cholesterol as revealed by fluorescence fluctuation analysis.

Membrane cholesterol-rich domains have been shown to be important for regulating a range of membrane protein activities. Low-density lipoprotein receptor (LDLR)-mediated internalization of cholesterol-rich LDL particles is tightly regulated by feedback mechanisms involving intracellular sterol sensors. Since LDLR plays a role in maintaining cellular cholesterol homeostasis, we explore the role that membrane domains may have in regulating LDLR activity. We expressed a fluorescent LDLR-mEGFP construct in HEK293T cells and imaged the unligated receptor or bound to an LDL/DiI fluorescent ligand using total internal reflection fluorescence microscopy. We studied the receptor's spatiotemporal dynamics using fluorescence fluctuation analysis methods. Image cross correlation spectroscopy reveals a lower LDL-to-LDLR binding fraction when membrane cholesterol concentrations are augmented using cholesterol esterase, and a higher binding fraction when the cells are treated with methyl-ß-cyclodextrin) to lower membrane cholesterol. This suggests that LDLR's ability to metabolize LDL particles is negatively correlated to membrane cholesterol concentrations. We then tested if a change in activity is accompanied by a change in membrane localization. Image mean-square displacement analysis reveals that unligated LDLR-mEGFP and ligated LDLR-mEGFP/LDL-DiI constructs are transiently confined on the cell membrane, and the size of their confinement domains increases with augmented cholesterol concentrations. Receptor diffusion within the domains and their domain-escape probabilities decrease upon treatment with methyl-ß-cyclodextrin, consistent with a change in receptor populations to more confined domains, likely clathrin-coated pits. We propose a feedback model to account for regulation of LDLR within the cell membrane: when membrane cholesterol concentrations are high, LDLR is sequestered in cholesterol-rich domains. These LDLR populations are attenuated in their efficacy to bind and internalize LDL. However, when membrane cholesterol levels drop, LDL has a higher binding affinity to its receptor and the LDLR transits to nascent clathrin-coated domains, where it diffuses at a slower rate while awaiting internalization.

Revealing proteome-level functional redundancy in the human gut microbiome using ultra-deep metaproteomics.

Functional redundancy is a key ecosystem property representing the fact that different taxa contribute to an ecosystem in similar ways through the expression of redundant functions. The redundancy of potential functions (or genome-level functional redundancy [Formula: see text]) of human microbiomes has been recently quantified using metagenomics data. Yet, the redundancy of expressed functions in the human microbiome has never been quantitatively explored. Here, we present an approach to quantify the proteome-level functional redundancy [Formula: see text] in the human gut microbiome using metaproteomics. Ultra-deep metaproteomics reveals high proteome-level functional redundancy and high nestedness in the human gut proteomic content networks (i.e., the bipartite graphs connecting taxa to functions). We find that the nested topology of proteomic content networks and relatively small functional distances between proteomes of certain pairs of taxa together contribute to high [Formula: see text] in the human gut microbiome. As a metric comprehensively incorporating the factors of presence/absence of each function, protein abundances of each function and biomass of each taxon, [Formula: see text] outcompetes diversity indices in detecting significant microbiome responses to environmental factors, including individuality, biogeography, xenobiotics, and disease. We show that gut inflammation and exposure to specific xenobiotics can significantly diminish the [Formula: see text] with no significant change in taxonomic diversity.

Positive mood-related gut microbiota in a long-term closed environment: a multiomics study based on the "Lunar Palace 365" experiment.

BACKGROUND: Psychological health risk is one of the most severe and complex risks in manned deep-space exploration and long-term closed environments. Recently, with the in-depth research of the microbiota-gut-brain axis, gut microbiota has been considered a new approach to maintain and improve psychological health. However, the correlation between gut microbiota and psychological changes inside long-term closed environments is still poorly understood. Herein, we used the "Lunar Palace 365" mission, a 1-year-long isolation study in the Lunar Palace 1 (a closed manned Bioregenerative Life Support System facility with excellent performance), to investigate the correlation between gut microbiota and psychological changes, in order to find some new potential psychobiotics to maintain and improve the psychological health of crew members. RESULTS: We report some altered gut microbiota that were associated with psychological changes in the long-term closed environment. Four potential psychobiotics (Bacteroides uniformis, Roseburia inulinivorans, Eubacterium rectale, and Faecalibacterium prausnitzii) were identified. On the basis of metagenomic, metaproteomic, and metabolomic analyses, the four potential psychobiotics improved mood mainly through three pathways related to nervous system functions: first, by fermenting dietary fibers, they may produce short-chain fatty acids, such as butyric and propionic acids; second, they may regulate amino acid metabolism pathways of aspartic acid, glutamic acid, tryptophan, etc. (e.g., converting glutamic acid to gamma-aminobutyric acid; converting tryptophan to serotonin, kynurenic acid, or tryptamine); and third, they may regulate other pathways, such as taurine and cortisol metabolism. Furthermore, the results of animal experiments confirmed the positive regulatory effect and mechanism of these potential psychobiotics on mood. CONCLUSIONS: These observations reveal that gut microbiota contributed to a robust effect on the maintenance and improvement of mental health in a long-term closed environment. Our findings represent a key step towards a better understanding the role of the gut microbiome in mammalian mental health during space flight and provide a basis for future efforts to develop microbiota-based countermeasures that mitigate risks to crew mental health during future long-term human space expeditions on the moon or Mars. This study also provides an essential reference for future applications of psychobiotics to neuropsychiatric treatments. Video Abstract.

Microbiota-directed biotherapeutics: considerations for quality and functional assessment.

Mounting evidence points to causative or correlative roles of gut microbiome in the development of a myriad of diseases ranging from gastrointestinal diseases, metabolic diseases to neurological disorders and cancers. Consequently, efforts have been made to develop and apply therapeutics targeting the human microbiome, in particular the gut microbiota, for treating diseases and maintaining wellness. Here we summarize the current development of gut microbiota-directed therapeutics with a focus on novel biotherapeutics, elaborate the need of advanced -omics approaches for evaluating the microbiota-type biotherapeutics, and discuss the clinical and regulatory challenges. We also discuss the development and potential application of ex vivo microbiome assays and in vitro intestinal cellular models in this context. Altogether, this review aims to provide a broad view of promises and challenges of the emerging field of microbiome-directed human healthcare.

Early postnatal defects in neurogenesis in the 3xTg mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder leading to dementia. The hippocampus, which is one of the sites where neural stem cells reside and new neurons are born, exhibits the most significant neuronal loss in AD. A decline in adult neurogenesis has been described in several animal models of AD. However, the age at which this defect first appears remains unknown. To determine at which stage, from birth to adulthood, the neurogenic deficits are found in AD, we used the triple transgenic mouse model of AD (3xTg). We show that defects in neurogenesis are present as early as postnatal stages, well before the onset of any neuropathology or behavioral deficits. We also show that 3xTg mice have significantly fewer neural stem/progenitor cells, with reduced proliferation and decreased numbers of newborn neurons at postnatal stages, consistent with reduced volumes of hippocampal structures. To determine whether there are early changes in the molecular signatures of neural stem/progenitor cells, we perform bulk RNA-seq on cells sorted directly from the hippocampus. We show significant changes in the gene expression profiles at one month of age, including genes of the Notch and Wnt pathways. These findings reveal impairments in neurogenesis very early in the 3xTg AD model, which provides new opportunities for early diagnosis and therapeutic interventions to prevent neurodegeneration in AD.

MetaLab-MAG: A Metaproteomic Data Analysis Platform for Genome-Level Characterization of Microbiomes from the Metagenome-Assembled Genomes Database.

The studies of microbial communities have drawn increased attention in various research fields such as agriculture, environment, and human health. Recently, metaproteomics has become a powerful tool to interpret the roles of the community members by investigating the expressed proteins of the microbes. However, analyzing the metaproteomic data sets at genome resolution is still challenging because of the lack of efficient bioinformatics tools. Here we develop MetaLab-MAG, a specially designed tool for the characterization of microbiomes from metagenome-assembled genomes databases. MetaLab-MAG was evaluated by analyzing various human gut microbiota data sets and performed comparably or better than searching the gene catalog protein database directly. MetaLab-MAG can quantify the genome-level microbiota compositions and supports both label-free and isobaric labeling-based quantification strategies. MetaLab-MAG removes the obstacles of metaproteomic data analysis and provides the researchers with in-depth and comprehensive information from the microbiomes.

An economic and robust TMT labeling approach for high throughput proteomic and metaproteomic analysis.

Multiplexed quantitative proteomics using tandem mass tag (TMT) is increasingly used in -omic study of complex samples. While TMT-based proteomics has the advantages of the higher quantitative accuracy, fewer missing values, and reduced instrument analysis time, it is limited by the additional reagent cost. In addition, current TMT labeling workflows involve repeated small volume pipetting of reagents in volatile solvents, which may increase the sample-to-sample variations and is not readily suitable for high throughput applications. In this study, we demonstrated that the TMT labeling procedures could be streamlined by using pre-aliquoted dry TMT reagents in a 96 well plate or 12-tube strip. As little as 50 µg dry TMT per channel was used to label 6-12 µg peptides, yielding high TMT labeling efficiency (∼99%) in both microbiome and mammalian cell line samples. We applied this workflow to analyze 97 samples in a study to evaluate whether ice recrystallization inhibitors improve the cultivability and activity of frozen microbiota. The results demonstrated tight sample clustering corresponding to groups and consistent microbiome responses to prebiotic treatments. This study supports the use of TMT reagents that are pre-aliquoted, dried, and stored for robust quantitative proteomics and metaproteomics in high throughput applications.

Butyrate's role in human health and the current progress towards its clinical application to treat gastrointestinal disease.

Butyrate is a key energy source for colonocytes and is produced by the gut microbiota through fermentation of dietary fiber. Butyrate is a histone deacetylase inhibitor and also signals through three G-protein coupled receptors. It is clear that butyrate has an important role in gastrointestinal health and that butyrate levels can impact both host and microbial functions that are intimately coupled with each other. Maintaining optimal butyrate levels improves gastrointestinal health in animal models by supporting colonocyte function, decreasing inflammation, maintaining the gut barrier, and promoting a healthy microbiome. Butyrate has also shown protective actions in the context of intestinal diseases such as inflammatory bowel disease, graft-versus-host disease of the gastrointestinal tract, and colon cancer, whereas lower levels of butyrate and/or the microbes which are responsible for producing this metabolite are associated with disease and poorer health outcomes. However, clinical efforts to increase butyrate levels in humans and reverse these negative outcomes have generated mixed results. This article discusses our current understanding of the molecular mechanisms of butyrate action with a focus on the gastrointestinal system, the links between host and microbial factors, and the efforts that are currently underway to apply the knowledge gained from the bench to bedside.

An emerging field: Post-translational modification in microbiome.

Post-translational modifications (PTMs) play an essential role in most biological processes. PTMs on human proteins have been extensively studied. Studies on bacterial PTMs are emerging, which demonstrate that bacterial PTMs are different from human PTMs in their types, mechanisms and functions. Few PTM studies have been done on the microbiome. Here, we reviewed several studied PTMs in bacteria including phosphorylation, acetylation, succinylation, glycosylation, and proteases. We discussed the enzymes responsible for each PTM and their functions. We also summarized the current methods used to study microbiome PTMs and the observations demonstrating the roles of PTM in the microbe-microbe interactions within the microbiome and their interactions with the environment or host. Although new methods and tools for PTM studies are still needed, the existing technologies have made great progress enabling a deeper understanding of the functional regulation of the microbiome. Large-scale application of these microbiome-wide PTM studies will provide a better understanding of the microbiome and its roles in the development of human diseases.

The Functional Characteristics of Goat Cheese Microbiota from a One-Health Perspective.

Goat cheese is an important element of the Mediterranean diet, appreciated for its health-promoting features and unique taste. A pivotal role in the development of these characteristics is attributed to the microbiota and its continuous remodeling over space and time. Nevertheless, no thorough study of the cheese-associated microbiota using two metaomics approaches has previously been conducted. Here, we employed 16S rRNA gene sequencing and metaproteomics to explore the microbiota of a typical raw goat milk cheese at various ripening timepoints and depths of the cheese wheel. The 16S rRNA gene-sequencing and metaproteomics results described a stable microbiota ecology across the selected ripening timepoints, providing evidence for the microbiologically driven fermentation of goat milk products. The important features of the microbiota harbored on the surface and in the core of the cheese mass were highlighted in both compositional and functional terms. We observed the rind microbiota struggling to maintain the biosafety of the cheese through competition mechanisms and/or by preventing the colonization of the cheese by pathobionts of animal or environmental origin. The core microbiota was focused on other biochemical processes, supporting its role in the development of both the health benefits and the pleasant gustatory nuances of goat cheese.