Biology subjects

Peltier, J.

Publications and source records attributed to Peltier, J..

16 recordsLinked to original sources

bioRxiv2025

Deciphering the RNA-based regulation mechanism of the phage-encoded AbiF system in Clostridioides difficile

Clostridioides difficile is the major cause of nosocomial infections associated with antibiotic therapy. The severity of C. difficile infections increased worldwide with the emergence of hypervirulent strains, including 027 ribotype epidemic strains. Many aspects of C. difficiles adaptation strategies during pathogenesis remain poorly understood. This pathogen thrives in gut communities that are rich in microbes and phages. To regulate horizontal transfer of genetic material during its infection cycle, C. difficile relies on diverse mechanisms. More specifically, CRISPR (clustered regularly interspaced short palindromic repeats)-Cas and Toxin-Antitoxin (TA) systems contribute to prophage maintenance, prevention of phage infection, and stress response. Abortive infection (Abi) systems can provide additional lines of anti-phage defense. RNAs have emerged as key components of these systems including CRISPR RNAs and antitoxin RNAs within type I and type III TA. We report here the identification of a new AbiF-like system within a prophage of the hypervirulent C. difficile strain R20291. It is associated with an Abi_2/AbiD/F protein family largely distributed in Bacillota and Pseudomonadota with structural links to ancestral Cas13 proteins at the origin of the RNA-targeting CRISPR-Cas13 systems. We demonstrated toxic activity of the AbiFCd protein in C. difficile and in Escherichia coli and negative regulation of the abiFCd expression by an associated non-coding RNA RCd22. RCd22 contains two conserved abiF motifs and is active both in cis and in trans to neutralize the toxin by direct RNA-protein interaction, similar to RNA antitoxin in type III TA. A mass spectrometry interactomics analysis of protein fractions from MS2-Affinity Purification coupled with RNA sequencing (MAPS) revealed the AbiFCd protein among the most enriched RCd22 partners in C. difficile. Structural modeling of the RNA-protein complex and mutagenesis analysis revealed key positions on both protein and RNA partners for this interaction and toxic activity. In summary, these findings provide valuable insights into the mechanisms of interaction between bacteria and phages, which are pertinent to the advancement of phage therapy, genome editing, epidemiological surveillance, and the formulation of novel therapeutic approaches.

microbiology↗

bioRxiv2024

The L,D-transpeptidation pathway is inhibited by antibiotics of the β-lactam class in Clostridioides difficile

The resistance of Clostridioides difficile to the {beta}-lactam antibiotics cephalosporins, which target the peptidoglycan (PG) assembly, is a leading contributor to the development of C. difficile infections. C. difficile has an original PG structure with a predominance of 3[->]3 cross-links generated by L,D-transpeptidases (LDTs). C. difficile forms spores and we show that the spore cortex PG contains exclusively 3[->]3 cross-links. PG and spore cortex of C. difficile cells were largely unaffected by the deletion of the three predicted LDTs, revealing the implication of a new family of LDTS. The D,D-carboxypeptidases producing the essential LDT substrate were inactivated by cephalosporins, resulting in the inhibition of the L,D-transpeptidation pathway. In contrast, the participation of penicillin-binding proteins (PBPs) to PG cross-linking increased in the presence of the antibiotics. Our findings highlight that cephalosporin resistance is not primarily mediated by LDTs and illustrate the plasticity of the PG biosynthesis machinery in C. difficile.

microbiology↗

bioRxiv2024

Clostridioides difficile binary toxin CDT induces biofilm-like persisting microcolonies

Clinical symptoms of Clostridioides difficile infection (CDI) range from diarrhea to pseudomembranous colitis. A major challenge in managing CDI is the high rate of relapse. Several studies correlate production of CDT binary toxin by clinical strains of Clostridioides difficile with higher relapse rates. Although the mechanism of action of CDT on host cells is known, its exact contribution to CDI is still unclear. To understand the physiological role of CDT during CDI, we established two hypoxic relevant intestinal models, Transwell and Microfluidic Intestine-on-Chip systems. Both were challenged with the epidemic strain UK1 CDT+ and its isogenic CDT- mutant. We report that CDT binary toxin induces mucin-associated microcolonies that increase C. difficile colonization and display biofilm-like properties by enhancing C. difficile resistance to vancomycin but not to fidaxomicin, a biofilm disrupting antibiotic. Importantly, biofilm- like CDT-dependent microcolonies were also observed in the caecum and colon of infected mice. Hence, our study shows that CDT toxin induces biofilm-like microcolonies, increasing C. difficile colonization and persistence.

microbiology↗

bioRxiv2023

The TcdE holin drives toxin secretion and virulence in Clostridioides difficile.

Clostridioides difficile is the leading cause of healthcare associated infections. The Pathogenicity Locus (PaLoc) toxins TcdA and TcdB promote host disease. These toxins lack canonical N-terminal signal sequences for translocation across the bacterial membrane, suggesting alternate mechanisms of release, which have included targeted secretion and passive release from cell lysis. While the holin TcdE has been implicated in TcdA and TcdB release, its role in vivo remains unknown. Here, we show profound reductions in toxin secretion in tcdE mutants in the highly virulent strains UK1 (epidemic ribotype 027, Clade 3) and VPI10463 (ribotype 087, Clade 1). Notably, tcdE deletion in either strain rescued highly susceptible gnotobiotic mice from lethal infection by reducing acute extracellular toxin to undetectable levels, limiting mucosal damage, and enabling long-term survival, in spite of continued toxin gene expression in tcdE mutants. Our findings confirm TcdEs critical functions in vivo for toxin secretion and C. difficile virulence.

microbiology↗

bioRxiv2022

Elucidating dynamic anaerobe metabolism with HRMAS 13C NMR and genome-scale metabolic modeling.

Anaerobic microbial metabolism drives critical functions within global ecosystems, host-microbiota interactions, and industrial applications, yet remains ill-defined. Here we advance versatile approaches to elaborate dynamic metabolism in living cells of obligate anaerobes, using the pathogen Clostridioides difficile, an amino acid and carbohydrate-fermenting Clostridia. High-Resolution Magic Angle Spinning (HRMAS) Nuclear Magnetic Resonance (NMR) spectroscopy of C. difficile grown with uniformly labeled 13C substrates informed dynamic flux balance analysis (dFBA) of the pathogens genome-scale metabolism. Predictions identified metabolic integration of glycolytic and amino acid fermentation pathways at alanines biosynthesis, to support efficient energy generation, maintenance of redox balance, nitrogen handling, and biomass generation. Model predictions advanced an approach using the sensitivity of 13C NMR spectroscopy to simultaneously track cellular carbon and nitrogen flow, from [U-13C]glucose and [15N] leucine, confirming the formation of [13C,15N]alanine. We illustrate experimental and computational approaches to elaborate complex anaerobic metabolism for diverse applications.

microbiology↗

bioRxiv2021

The E3 ligase RNF115 regulates phagosome maturation and host response to bacterial infection

Phagocytosis is a key process in innate immunity and homeostasis. After uptake, newly formed phagosomes mature by acquisition of endo-lysosomal enzymes. Macrophage activation by interferon-gamma (IFN-{gamma}) increases microbicidal activity, but delays phagosomal maturation by an unknown mechanism. Using quantitative proteomics, we show that phagosomal proteins harbour high levels of typical and atypical ubiquitin chain types. Moreover, phagosomal ubiquitylation of vesicle trafficking proteins is substantially enhanced upon IFN-{gamma} activation of macrophages, suggesting a role in regulating phagosomal functions. We identified the E3 ubiquitin ligase RNF115, which is enriched on phagosomes of IFN-{gamma} activated macrophages, as an important regulator of phagosomal maturation. Loss of RNF115 protein or ligase activity enhanced phagosomal maturation and increased cytokine responses to bacterial infection, suggesting that both innate immune signalling from the phagosome and phagolysosomal trafficking are controlled through ubiquitylation. RNF115 knock-out mice show less tissue damage in response to S. aureus infection, indicating a role of RNF115 in inflammatory responses in vivo. In conclusion, RNF115 and phagosomal ubiquitylation are important regulators of innate immune functions during bacterial infections.

biochemistry↗

bioRxiv2021

Real-time HRMAS 13C NMR of obligately anaerobic cells identifies new metabolic targets in the pathogen Clostridioides difficile

Anaerobic microbial metabolism drives critical aspects of host-microbiome interactions and supports many economically important industrial applications. Yet, the metabolic pathways of anaerobic bacteria and their associated constraints for maintaining energy and redox balance are often poorly described. We employ High-Resolution Magic Angle Spinning Carbon-13 (13C) Nuclear Magnetic Resonance spectroscopy with dynamic flux balance analysis to resolve real-time metabolism in living cells of the obligately anaerobic pathogen Clostridioides difficile. Using 13C-labeled carbon sources, we elaborate the time-dependent progression of reductive and oxidative anaerobic fermentation pathways. Analyses identified new integration points for redox and nitrogen coupling between carbohydrate and amino acid metabolism, particularly in the production of 13C-alanine from 13C-glucose to provide an ammonia sink from co-occurring amino acid fermentation. Analyses conducted in the presence or absence of selenium, a required co-factor for the proline Stickland reductase, demonstrate further capacity to modulate cellular metabolism and resulting metabolites. Findings informed a genome-scale metabolic model of C. difficile, identifying alanine and associated electron carrier pools as critical metabolic integration points in energy flow and biomass expansion. We illustrate use of HRMAS NMR as a new analytical platform to resolve complex interactions in anaerobic metabolism and inform new metabolic targets to counter C. difficile infections.

microbiology↗

bioRxiv2021

An ERK5-KLF2 signalling module regulates early embryonic gene expression dynamics and stem cell rejuvenation

The ERK5 MAP kinase signalling pathway drives transcription of naive pluripotency genes in mouse Embryonic Stem Cells (mESCs). However, how ERK5 impacts on other aspects of mESC biology has not been investigated. Here, we employ quantitative proteomic profiling to identify proteins whose expression is regulated by the ERK5 pathway in mESCs. This reveals a function for ERK5 signalling in regulating dynamically expressed early embryonic 2-cell stage (2C) genes including the mESC rejuvenation factor ZSCAN4. ERK5-dependent ZSCAN4 induction in mESCs increases telomere length, a key rejuvenative process required for prolonged culture. Mechanistically, ERK5 promotes ZSCAN4 and 2C gene expression via transcription of the KLF2 pluripotency transcription factor. Surprisingly, ERK5 also directly phosphorylates KLF2 to drive ubiquitin-dependent degradation, encoding negative-feedback regulation of 2C gene expression. In summary, our data identify a regulatory module whereby ERK5 kinase and transcriptional activities bi-directionally control KLF2 levels to pattern dynamic 2C gene transcription and mESC rejuvenation.

developmental biology↗

bioRxiv2020

Identification of RNAs bound by Hfq reveals widespread RNA partners and a sporulation regulator in the human pathogen Clostridioides difficile

Noncoding RNAs (ncRNA) have emerged as important components of regulatory networks governing bacterial physiology and virulence. Previous deep-sequencing analysis identified a large diversity of ncRNAs in the human enteropathogen Clostridioides (Clostridium) difficile. Some of them are trans-encoded RNAs that could require the RNA chaperone protein Hfq for their action. Recent analysis suggested a pleiotropic role of Hfq in C. difficile with the most pronounced effect on sporulation, a key process during the infectious cycle of this pathogen. However, a global view of RNAs interacting with C. difficile Hfq is missing. In the present study, we performed RNA immunoprecipitation high-throughput sequencing (RIP-Seq) to identify Hfq-associated RNAs in C. difficile. Our work revealed a large set of Hfq-interacting mRNAs and ncRNAs, including mRNA leaders and coding regions, known and potential new ncRNAs. In addition to trans-encoded RNAs, new categories of Hfq ligands were found including cis-antisense RNAs, riboswitches and CRISPR RNAs. ncRNA-mRNA and ncRNA-ncRNA pairings were postulated through computational predictions. Investigation of one of the Hfq-associated ncRNAs, RCd1, suggests that this RNA contributes to the control of late stages of sporulation in C. difficile. Altogether, these data provide essential molecular basis for further studies of post-transcriptional regulatory network in this enteropathogen.

microbiology↗

bioRxiv2020

Predictive regulatory and metabolic network models for systems analysis of Clostridioides difficile

Though Clostridioides difficile is among the most studied anaerobes, the interplay of metabolism and regulation that underlies its ability to colonize the human gut is unknown. We have compiled public resources into three models and a portal to support comprehensive systems analysis of C. difficile. First, by leveraging 151 transcriptomes from 11 studies we generated a regulatory model (EGRIN) that organizes 90% of C. difficile genes into 297 high quality conditional co-regulation modules. EGRIN predictions, validated with independent datasets, recapitulated and extended regulons of key transcription factors, implicating new genes for sporulation, carbohydrate transport and metabolism. Second, by advancing a metabolic model, we discovered that 15 amino acids, diverse carbohydrates, and 10 metabolic genes are essential for C. difficile growth within an intestinal environment. Finally, by integrating EGRIN with the metabolic model, we developed a PRIME model that revealed unprecedented insights into combinatorial control of essential processes for in vivo colonization of C. difficile and its interactions with commensals. We have developed an interactive web portal (http://networks.systemsbiology.net/cdiff-portal/) to disseminate all data, algorithms, and models to support collaborative systems analyses of C. difficile.

systems biology↗

bioRxiv2020

Proteomics characterisation of the L929 cell supernatant and its role in BMDM differentiation

Bone marrow-derived macrophages (BMDMs) are a key model system to study macrophage biology in vitro. Commonly used methods to differentiate macrophages from bone marrow are treatment with either recombinant M-CSF or the supernatant of L929 cells, which secrete M-CSF. However, little is known about the composition of L929 cell conditioned media (LCCM) and how it affects BMDM phenotype. Here, we used quantitative mass spectrometry to characterise the kinetics of protein secretion from L929 cells over a two-week period, identifying 2,193 proteins. While M-CSF is very abundant in LCCM, we identified several other immune-regulatory proteins such as macrophage migration inhibitory factor (MIF), osteopontin and chemokines such as Ccl2 and Ccl7 at surprisingly high abundance levels. We therefore further characterised the proteomes of BMDMs after differentiation with M-CSF, M-CSF + MIF or LCCM, respectively. Interestingly, macrophages differentiated with LCCM induced a stronger anti-inflammatory M1 phenotype that those differentiated with M-CSF. This resource will be valuable to all researchers using LCCM for the differentiation of BMDMs.

immunology↗

bioRxiv2020

Type I toxin-antitoxin systems contribute to mobile genetic elements maintenance in Clostridioides difficile and can be used as a counter-selectable marker for chromosomal manipulation

Toxin-antitoxin (TA) systems are widespread on mobile genetic elements as well as in bacterial chromosomes. According to the nature of the antitoxin and its mode of action for toxin inhibition, TA systems are subdivided into different types. The first type I TA modules were recently identified in the human enteropathogen Clostridioides (formerly Clostridium) difficile. In type I TA, synthesis of the toxin protein is prevented by the transcription of an antitoxin RNA during normal growth. Here, we report the characterization of five additional type I TA systems present within phiCD630-1 and phiCD630-2 prophage regions of C. difficile 630. Toxin genes encode 34 to 47 amino acid peptides and their ectopic expression in C. difficile induces growth arrest. Growth is restored when the antitoxin RNAs, transcribed from the opposite strand, are co-expressed together with the toxin genes. In addition, we show that type I TA modules located within the phiCD630-1 prophage contribute to its stability and mediate phiCD630-1 heritability. Type I TA systems were found to be widespread in genomes of C. difficile phages, further suggesting their functional importance. We have made use of a toxin gene from one of type I TA modules of C. difficile as a counter-selectable marker to generate an efficient mutagenesis tool for this bacterium. This tool enabled us to delete all identified toxin genes within the phiCD630-1 prophage, thus allowing investigation of the role of TA in prophage maintenance. Furthermore, we were able to delete the large 49 kb phiCD630-2 prophage region using this improved procedure.

microbiology↗

bioRxiv2020

Macrophage Scavenger Receptor 1 mediates lipid-induced inflammation in non-alcoholic fatty liver disease

Obesity-associated inflammation is a key player in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). However, the exact mechanisms remain incompletely understood. Here we demonstrate that macrophage scavenger receptor 1 (MSR1, CD204) expression is associated with the occurrence of hepatic lipid-laden foamy macrophages and correlates with the degree of steatosis and steatohepatitis in a cohort of 170 NAFLD patients. Mice lacking Msr1 are protected against high fat-cholesterol diet (HFD)-induced metabolic disorder, showing fewer hepatic lipid-laden foamy macrophages, less hepatic inflammation, improved dyslipidemia and glucose tolerance, while showing a change in hepatic lipid metabolism. We show that MSR1 induces a pro-inflammatory response via the JNK signaling pathway upon triggering by saturated fatty acids. In vitro blockade of the receptor prevented the accumulation of lipids in primary macrophages which inhibited the switch towards a pro-inflammatory phenotype and the release of cytokines such as TNFa. Targeting MSR1 using monoclonal antibody therapy in an obesity-associated NAFLD mouse model and ex vivo human liver slices resulted in the prevention of foamy macrophage formation and liver inflammation. Moreover, we identified that rs41505344, a polymorphism in the upstream transcriptional region of MSR1, was associated with altered serum triglycerides and aspartate transaminase levels in a cohort of over 400,000 patients. Taken together, our data suggest a critical role for MSR1 in lipid homeostasis and a potential therapeutic target for the treatment of NAFLD. One Sentence SummaryThe immunometabolic role of MSR1 in human NAFLD.

immunology↗

bioRxiv2020

The mechanisms of in vivo commensal control of Clostridioides difficile virulence

We define multiple mechanisms by which commensals protect against or worsen Clostridioides difficile infection. Leveraging new systems-level models we show how metabolically distinct species of Clostridia modulate the pathogens colonization, growth, and virulence to impact host survival. Gnotobiotic mice colonized with the amino acid fermenter Paraclostridium bifermentans survived infection while mice colonized with the butyrate- producer, Clostridium sardiniense, more rapidly succumbed. Systematic in vivo analyses revealed how each commensal altered the gut nutrient environment, modulating the pathogens metabolism, regulatory networks, and toxin production. Oral administration of P. bifermentans rescued conventional mice from lethal C. difficile infection via mechanisms identified in specifically colonized mice. Our findings lay the foundation for mechanistically informed therapies to counter C. difficile infections using systems biologic approaches to define host-commensal-pathogen interactions in vivo. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/894915v2_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@15a6940org.highwire.dtl.DTLVardef@b4290borg.highwire.dtl.DTLVardef@1f2af49org.highwire.dtl.DTLVardef@1d1e6b9_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

microbiology↗

bioRxiv2019

Phosphoproteomics reveals that the hVPS34 regulated SGK3 kinase specifically phosphorylates endosomal proteins including Syntaxin-7, Syntaxin-12, RFIP4 and WDR44

The serum- and glucocorticoid-regulated kinase (SGK) isoforms contribute resistance to cancer therapies targeting the PI3K pathway. SGKs are homologous to Akt and these kinases display overlapping specificity and phosphorylate several substrates at the same residues, such as TSC2 to promote tumor growth by switching on the mTORC1 pathway. The SGK3 isoform is upregulated in breast cancer cells treated with PI3K or Akt inhibitors and recruited and activated at endosomes, through its phox homology domain binding to PtdIns(3)P. We undertook genetic and pharmacological phosphoproteomic screens to uncover novel SGK3 substrates. We identified 40 potential novel SGK3 substrates, including 4 endosomal proteins STX7 (Ser126) and STX12 (Ser139), RFIP4 (Ser527) and WDR44 (Ser346) that were efficiently phosphorylated in vitro by SGK3 at the sites identified in vivo, but poorly by Akt. We demonstrate that these substrates are poorly phosphorylated by Akt as they possess an n+1 residue from the phosphorylation site that is unfavorable for Akt phosphorylation. Phos-tag analysis revealed that stimulation of HEK293 cells with IGF1 to activate SGK3, promoted phosphorylation of a significant fraction of endogenous STX7 and STX12, in a manner that was blocked by knock-out of SGK3 or treatment with 14H inhibitor. SGK3 phosphorylation of STX12 enhanced interaction with the VAMP4/VTI1A/STX6 containing SNARE complex and promoted plasma membrane localization. Our data reveal novel substrates for SGK3 and suggest a mechanism by which STX7 and STX12 SNARE complexes are regulated by SGK3. They reveal new biomarkers for monitoring SGK3 pathway activity.

biochemistry↗

bioRxiv2017

Thermal proteome profiling of breast cancer cells reveals proteasomal activation by CDK4/6 inhibitor palbociclib

Palbociclib is a CDK4/6 inhibitor approved for metastatic estrogen receptor positive breast cancer. In addition to G1 cell cycle arrest, palbociclib treatment results in cell senescence, a phenotype that are not readily explained by CDK4/6 inhibition. In order to identify a molecular mechanism responsible for palbociclib-induced senescence, we performed thermal proteome profiling of MCF7 breast cancer cells. In addition to affecting known CDK4/6 targets, palbociclib induces a thermal stabilization of the 20S proteasome, despite not directly binding to it. We further show that palbociclib treatment increases proteasome activity independently of the ubiquitin pathway. This leads to cellular senescence which can be counteracted by proteasome inhibitors. Palbociclib-induced proteasome activation and senescence is mediated by reduced proteasomal association of ECM29. Loss of ECM29 activates the proteasome, blocks cell proliferation and induces senescence. Finally, we find that ECM29 mRNA levels are predictive of relapse-free survival in breast cancer patients treated with endocrine therapy. In conclusion, thermal proteome profiling identifies the proteasome and ECM29 protein as mediators of palbociclib activity in breast cancer cells.

cancer biology↗