Bifidobacterium infantis associates with T cell immunity in human infants and is sufficient to enhance antigen-specific T cells in mice.

Bacille Calmette-Guerin (BCG) vaccine can elicit good TH1 responses in neonates. We hypothesized that the pioneer gut microbiota affects vaccine T cell responses. Infants who are HIV exposed but uninfected (iHEU) display an altered immunity to vaccination. BCG-specific immune responses were analyzed at 7 weeks of age in iHEU, and responses were categorized as high or low. Bifidobacterium longum subsp. infantis was enriched in the stools of high responders, while Bacteroides thetaiotaomicron was enriched in low responders at time of BCG vaccination. Neonatal germ-free or SPF mice orally gavaged with live B. infantis exhibited significantly higher BCG-specific T cells compared with pups gavaged with B. thetaiotaomicron. B. infantis and B. thetaiotaomicron differentially affected stool metabolome and colonic transcriptome. Human colonic epithelial cells stimulated with B. infantis induced a unique gene expression profile versus B. thetaiotaomicron. We thus identified a causal role of B. infantis in early-life antigen-specific immunity.

Proteomic Profiling of Urine From Hospitalized Patients With Severe Pneumonia due to SARS-CoV-2 vs Other Causes: A Preliminary Report.

The pathogenesis of coronavirus disease 2019 (COVID-19) pneumonia remains poorly understood. The urine proteome of hospitalized patients with severe COVID-19 pneumonia, compared with severe non-COVID-19 pneumonia controls, was distinct and associated with lower abundance of several host proteins. Protein-specific machine learning analysis outlined biomarker combinations able to differentiate COVID-19 pneumonia from non-COVID-19 pneumonia controls.

DRP1 interacts directly with BAX to induce its activation and apoptosis.

The apoptotic executioner protein BAX and the dynamin-like protein DRP1 co-localize at mitochondria during apoptosis to mediate mitochondrial permeabilization and fragmentation. However, the molecular basis and functional consequences of this interplay remain unknown. Here, we show that BAX and DRP1 physically interact, and that this interaction is enhanced during apoptosis. Complex formation between BAX and DRP1 occurs exclusively in the membrane environment and requires the BAX N-terminal region, but also involves several other BAX surfaces. Furthermore, the association between BAX and DRP1 enhances the membrane activity of both proteins. Forced dimerization of BAX and DRP1 triggers their activation and translocation to mitochondria, where they induce mitochondrial remodeling and permeabilization to cause apoptosis even in the absence of apoptotic triggers. Based on this, we propose that DRP1 can promote apoptosis by acting as noncanonical direct activator of BAX through physical contacts with its N-terminal region.

An integrated workflow for enhanced taxonomic and functional coverage of the mouse fecal metaproteome.

Intestinal microbiota plays a key role in shaping host homeostasis by regulating metabolism, immune responses and behavior. Its dysregulation has been associated with metabolic, immune and neuropsychiatric disorders and is accompanied by changes in bacterial metabolic regulation. Although proteomics is well suited for analysis of individual microbes, metaproteomics of fecal samples is challenging due to the physical structure of the sample, presence of contaminating host proteins and coexistence of hundreds of taxa. Furthermore, there is a lack of consensus regarding preparation of fecal samples, as well as downstream bioinformatic analyses following metaproteomics data acquisition. Here we assess sample preparation and data analysis strategies applied to mouse feces in a typical mass spectrometry-based metaproteomic experiment. We show that subtle changes in sample preparation protocols may influence interpretation of biological findings. Two-step database search strategies led to significant underestimation of false positive protein identifications. Unipept software provided the highest sensitivity and specificity in taxonomic annotation of the identified peptides of unknown origin. 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 metaproteomics when studying complex microbiome samples.

Cell Wall Proteomics Reveal Phenotypic Adaption of Drug-Resistant Mycobacterium smegmatis to Subinhibitory Rifampicin Exposure.

Despite the availability of effective drug treatment, Mycobacterium tuberculosis (Mtb), the causative agent of TB disease, kills ~1. 5 million people annually, and the rising prevalence of drug resistance increasingly threatens to worsen this plight. We previously showed that sublethal exposure to the frontline anti-TB drug, rifampicin, resulted in substantial adaptive remodeling of the proteome of the model organism, Mycobacterium smegmatis, in the drug-sensitive mc2155 strain [wild type (WT)]. In this study, we investigate whether these responses are conserved in an engineered, isogenic mutant harboring the clinically relevant S531L rifampicin resistance-conferring mutation (SL) and distinguish the responses that are specific to RNA polymerase ß subunit- (RpoB-) binding activity of rifampicin from those that are dependent on the presence of rifampicin alone. We verified the drug resistance status of this strain and observed no phenotypic indications of rifampicin-induced stress upon treatment with the same concentration as used in WT (2.5 µg/ml). Thereafter, we used a cell wall-enrichment strategy to focus attention on the cell wall proteome and observed 253 proteins to be dysregulated in SL bacteria in comparison with 716 proteins in WT. We observed that decreased abundance of ATP-binding cassette (ABC) transporters and increased abundance of ribosomal machinery were conserved in the SL strain, whereas the upregulation of transcriptional machinery and the downregulation of numerous two-component systems were not. We conclude that the drug-resistant M. smegmatis strain displays some of the same proteomic responses observed in WT and suggest that this evidence supports the hypothesis that rifampicin exercises effects beyond RpoB-interaction alone and that mycobacteria recognise rifampicin as a signaling molecule in an RpoB-independent manner at sublethal doses. Taken together, our data indicates mixed RpoB-independent and RpoB-dependent proteomic remodeling in WT mycobacteria, with evidence for RpoB-independent ABC transporter downregulation, but drug activity-based transcriptional upregulation and two-component system downregulation.

Phosphoproteome Study of Escherichia coli Devoid of Ser/Thr Kinase YeaG During the Metabolic Shift From Glucose to Malate.

Understanding phosphorylation-mediated regulation of metabolic enzymes, pathways, and cell phenotypes under metabolic shifts represents a major challenge. The kinases associated with most phosphorylation sites and the link between phosphorylation and enzyme activity remain unknown. In this study, we performed stable isotope labeling by amino acids in cell culture (SILAC)-based proteome and phosphoproteome analysis of Escherichia coli ΔyeaG, a strain lacking a poorly characterized serine/threonine kinase YeaG, to decipher kinase-substrate interactions and the effects on metabolic phenotype during shifts from glucose to malate. The starting point of our analysis was the identification of physiological conditions under which ΔyeaG exhibits a clear phenotype. By metabolic profiling, we discovered that ΔyeaG strain has a significantly shorter lag phase than the wild type during metabolic shift from glucose to malate. Under those conditions, our SILAC analysis revealed several proteins that were differentially phosphorylated in the ΔyeaG strain. By focusing on metabolic enzymes potentially involved in central carbon metabolism, we narrowed down our search for putative YeaG substrates and identified isocitrate lyase AceA as the direct substrate of YeaG. YeaG was capable of phosphorylating AceA in vitro only in the presence of malate, suggesting that this phosphorylation event is indeed relevant for glucose to malate shift. There is currently not enough evidence to firmly establish the exact mechanism of this newly observed regulatory phenomenon. However, our study clearly exemplifies the usefulness of SILAC-based approaches in identifying proteins kinase substrates, when applied in physiological conditions relevant for the activity of the protein kinase in question.

Mass Spectrometry-Based Analysis of Mycobacterial Single-Colony Proteome.

Mass spectrometry-based single-cell proteomic analysis has recently gained momentum and is now an emerging area with established protocols and promising results. Traditional proteomic studies, especially involving bacteria, have been limited to suspension cultures with large protein yields. Such studies, however, remain population centered with the uniqueness of individual responses to environmental challenges becoming diluted. To enable bacterial single-colony proteomics, we describe a quantitative mass spectrometry-based protocol to isolate and analyze the proteome of a single mycobacterial colony from 7H10 media, with growth supplements for optimal growth. Following protein purification and digestion, tryptic peptides are analyzed by UHPLC coupled to a hybrid Q Exactive mass spectrometer. Raw data were analyzed using the MaxQuant Suite, and downstream statistical analysis was performed using Perseus software. A total of 7805 unique peptides and 1387 proteins were identified. Data are available via ProteomeXchange with identifier PXD018168. In this chapter, we identify steps most prone to sample loss and describe measures of alleviation that allows the preservation of protein yield and boosts quantitative power while increasing reproducibility, of "very limiting samples."

Protocols for Preparation and Mass Spectrometry Analysis of Clinical Urine Samples to Identify Candidate Biomarkers of Schistosoma-Associated Bladder Cancer.

Advances in mass spectrometry instrumentation have revolutionized analytical capability in clinical proteomics. In parallel, various sample preparation methods have been developed to try to address the inherent complexity and dynamic range of clinical samples, typically involving a combination of depletion of abundant proteins followed by extensive prefractionation. However, the depth of coverage routinely achieved in discovery proteomics experiments on peripheral fluids such as serum, still leaves something to be desired, especially if no depletion or prefractionation is done in order to increase the throughput of clinical samples. Remarkably, despite being an easily accessible, typically sterile and diagnostically rich clinical sample, urine is often overlooked and as such has received less development effort. As an ultrafiltrate of blood, urine contains proteins and protein fragments originating from all parts of the body which may have diagnostic or prognostic potential if accurately and reproducibly quantified. Here, we describe an efficient and simple method for the concentration of urine samples by methanol-chloroform precipitation and subsequent in-solution tryptic digestion prior to discovery or targeted mass spectrometry analysis. We exemplify this method by reference to the discovery of novel candidate urinary biomarkers of schistosomiasis. Importantly, the methods described here have been used to identify >1900 protein groups in human urine by label-free discovery proteomics, without requiring any prior depletion or prefractionation, making this approach amenable to high throughput clinical biomarker studies in many diseases.

The translational regulator FMRP controls lipid and glucose metabolism in mice and humans.

OBJECTIVES: The Fragile X Mental Retardation Protein (FMRP) is a widely expressed RNA-binding protein involved in translation regulation. Since the absence of FMRP leads to Fragile X Syndrome (FXS) and autism, FMRP has been extensively studied in brain. The functions of FMRP in peripheral organs and on metabolic homeostasis remain elusive; therefore, we sought to investigate the systemic consequences of its absence. METHODS: Using metabolomics, in vivo metabolic phenotyping of the Fmr1-KO FXS mouse model and in vitro approaches, we show that the absence of FMRP induced a metabolic shift towards enhanced glucose tolerance and insulin sensitivity, reduced adiposity, and increased ß-adrenergic-driven lipolysis and lipid utilization. RESULTS: Combining proteomics and cellular assays, we highlight that FMRP loss increased hepatic protein synthesis and impacted pathways notably linked to lipid metabolism. Mapping metabolomic and proteomic phenotypes onto a signaling and metabolic network, we predicted that the coordinated metabolic response to FMRP loss was mediated by dysregulation in the abundances of specific hepatic proteins. We experimentally validated these predictions, demonstrating that the translational regulator FMRP associates with a subset of mRNAs involved in lipid metabolism. Finally, we highlight that FXS patients mirror metabolic variations observed in Fmr1-KO mice with reduced circulating glucose and insulin and increased free fatty acids. CONCLUSIONS: Loss of FMRP results in a widespread coordinated systemic response that notably involves upregulation of protein translation in the liver, increased utilization of lipids, and significant changes in metabolic homeostasis. Our study unravels metabolic phenotypes in FXS and further supports the importance of translational regulation in the homeostatic control of systemic metabolism.

Author Correction: Structure of the core of the type III secretion system export apparatus.

In the version of this article initially published, the PDB code associated with the study was given as 6F2E but should have been 6F2D in Table 1 and the data availability statement. The error has been corrected in the HTML and PDF versions of the article.

Structure of the core of the type III secretion system export apparatus.

Export of proteins through type III secretion systems is critical for motility and virulence of many major bacterial pathogens. Three putative integral membrane proteins (FliP, FliQ, FliR) are suggested to form the core of an export gate in the inner membrane, but their structure, assembly and location within the final nanomachine remain unclear. Here, we present the cryoelectron microscopy structure of the Salmonella Typhimurium FliP-FliQ-FliR complex at 4.2 Å. None of the subunits adopt canonical integral membrane protein topologies, and common helix-turn-helix structural elements allow them to form a helical assembly with 5:4:1 stoichiometry. Fitting of the structure into reconstructions of intact secretion systems, combined with cross-linking, localize the export gate as a core component of the periplasmic portion of the machinery. This study thereby identifies the export gate as a key element of the secretion channel and implies that it primes the helical architecture of the components assembling downstream.

Time-Dependent, HIV-Tat-Induced Perturbation of Human Neurons In Vitro: Towards a Model for the Molecular Pathology of HIV-Associated Neurocognitive Disorders.

A significant proportion of human immunodeficiency virus type 1 (HIV)-positive individuals are affected by the cognitive, motor and behavioral dysfunction that characterizes HIV-associated neurocognitive disorders (HAND). While the molecular etiology of HAND remains largely uncharacterized, HIV transactivator of transcription (HIV-Tat) is thought to be an important etiological cause. Here we have used mass spectrometry (MS)-based discovery proteomics to identify the quantitative, cell-wide changes that occur when non-transformed, differentiated human neurons are treated with HIV-Tat over time. We identified over 4000 protein groups (false discovery rate <0.01) in this system with 131, 118 and 45 protein groups differentially expressed at 6, 24 and 48 h post treatment, respectively. Alterations in the expression of proteins involved in gene expression and cytoskeletal maintenance were particularly evident. In tandem with proteomic evidence of cytoskeletal dysregulation we observed HIV-Tat induced functional alterations, including a reduction of neuronal intrinsic excitability as assessed by patch-clamp electrophysiology. Our findings may be relevant for understanding in vivo molecular mechanisms in HAND.

Quantitative proteomic analysis of HIV-1 Tat-induced dysregulation in SH-SY5Y neuroblastoma cells.

Despite affecting up to 70% of HIV-positive patients and being the leading cause of dementia in patients under 40 years, the molecular mechanisms involved in the onset of HIV-associated neurocognitive disorders (HAND) are not well understood. To address this, we performed SILAC-based quantitative proteomic analysis on HIV-Tat treated SH-SY5Y neuroblastoma cells. Isolated protein was fractionated by SDS-PAGE and analyzed by nLC-MS/MS on an Orbitrap Velos. Using MaxQuant, we identified and quantified 3077 unique protein groups, of which 407 were differentially regulated. After applying an additional standard deviation-based cutoff, 29 of these were identified as highly significantly and stably dysregulated. GO term analysis shows dysregulation in both protein translation machinery as well as cytoskeletal regulation that have both been implicated in other dementias. In addition, several key cytoskeletal regulatory proteins such as ARHGEF17, the Rho GTPase, SHROOM3, and CMRP1 are downregulated. Together, these data demonstrate that HIV-Tat can dysregulate neuronal cytoskeletal regulatory proteins that could lead to the major HAND clinical manifestation-synapse loss.