Biology subjects

Schmitt, L.

Publications and source records attributed to Schmitt, L..

32 records · Page 2Linked to original sources

bioRxiv2023

Monocyte production of C1q potentiates CD8+ T cell effector function following respiratory viral infection

Respiratory viral infections remain a leading cause of morbidity and mortality. Using a murine model of human metapneumovirus (HMPV), we identified recruitment of a C1q-producing inflammatory monocyte population concomitant with viral clearance by adaptive immune cells. Genetic ablation of C1q led to reduced CD8+ T cell function. Production of C1q by a myeloid lineage was sufficient to enhance CD8+ T cell function. Activated and dividing CD8+ T cells expressed a putative C1q receptor, gC1qR. Perturbation of gC1qR signaling led to altered CD8+ T cell IFN-{gamma} production and metabolic capacity. Autopsy specimens from fatal respiratory viral infections in children demonstrated diffuse production of C1q by an interstitial population. Humans with severe COVID-19 infection also demonstrated upregulation of gC1qR on activated and rapidly dividing CD8+ T cells. Collectively, these studies implicate C1q production from monocytes as a critical regulator of CD8+ T cell function following respiratory viral infection.

immunology↗

bioRxiv2023

Probing macromolecular crowding at the lipid membrane interface with genetically-encoded sensors

Biochemical processes within the living cell occur in a highly crowded environment. The phenomenon of macromolecular crowding is not an exclusive feature of the cytoplasm and can be observed in the densely protein-packed, nonhomogeneous cellular membranes and at the membrane interfaces. Crowding affects diffusional and conformational dynamics of proteins within the lipid bilayer, and modulates the membrane organization. However, the non-invasive quantification of the membrane crowding is not trivial. Here, we developed the genetically- encoded fluorescence-based sensor for probing the macromolecular crowding at the membrane interfaces. Two sensor variants, both composed of fluorescent proteins and a membrane anchor, but differing by the flexible linker domains were characterized in vitro, and the procedures for the membrane reconstitution were established. Lateral pressure induced by membrane-tethered synthetic and protein crowders altered the sensors conformation, causing increase in the intramolecular Forsters resonance energy transfer. The effect of protein crowders only weakly correlated with their molecular weight, suggesting that other factors, such as shape and charge play role in the quinary interactions. Upon their expression, the designed sensors were localized to the inner membrane of E. coli, and measurements performed in extracted membrane vesicles revealed low level of interfacial crowding. The sensors offer broad opportunities to study interfacial crowding in a complex environment of native membranes, and thus add to the toolbox of methods for studying membrane dynamics and proteostasis.

biophysics↗

bioRxiv2023

Novel orthotopic model of neuroblastoma in RNU homozygous rats

Neuroblastoma, the most common extracranial solid malignancy in children, accounts for 15% of pediatric cancer deaths despite multimodal therapy including surgical resection. Unfortunately, complete surgical resection remains challenging due to encasement of major neurovascular structures, unclear tumor margins, and remote nodal disease. While mouse models of neuroblastoma are extremely valuable for studying tumor biology and medical treatments, the small size renders the mouse model insufficient to evaluate novel surgical therapy. Here, we have developed a novel rat model of neuroblastoma to facilitate further development of surgical treatment. Human neuroblastoma cells (SK-N-BE(2)) were injected into the adrenal gland of RNU nude rats. They developed 2 cm xenograft tumors at 5 weeks which were easily identifiable on MRI imaging and on visual inspection. The rats began losing weight and neared end stage at 7 weeks, at which point surgical resection was attempted. While surgical resection was technically feasible, the rats were too frail to survive surgery at the late stage. The pathology of the tumors was consistent with neuroblastoma: small round blue cells with strong PHOX2B staining. Thus, we present a novel rat neuroblastoma model that can be used for development of surgical techniques, such as the use of intraoperative contrast agents.

cancer biology↗

bioRxiv2023

Hypersensitivity to distractors in Fragile X syndrome from loss of modulation of cortical VIP interneurons.

Attention deficit is one of the most prominent and disabling symptoms in Fragile X Syndrome (FXS). Hypersensitivity to sensory stimuli contributes to attention difficulties by overwhelming and/or distracting affected individuals, which disrupts activities of daily living at home and learning at school. We find that auditory or visual distractors selectively impair visual discrimination performance in both humans and mice with FXS, but not their typically developing controls. Vasoactive intestinal polypeptide (VIP) neurons were significantly modulated by incorrect responses in the post-stimulus period during early distractor trials in WT mice, consistent with their known role as error signals. Strikingly, however, VIP cells from Fmr1-/- mice showed little modulation in error trials, and this correlated with their poor performance on the distractor task. Thus, VIP interneurons and their reduced modulatory influence on pyramidal cells, could be a potential therapeutic target for attentional difficulties in FXS.

neuroscience↗

bioRxiv2022

Heterologously secreted MbxA from Moraxella bovis induces a membrane blebbing response of the human host cell

Many proteins of the Repeats in Toxins (RTX) protein family are toxins of Gram-negative pathogens including hemolysin A (HlyA) of uropathogenic E. coli. RTX proteins are secreted via Type I secretion systems (T1SS) and adopt their native conformation in the Ca2+-rich extracellular environment. Here we employed the E. coli HlyA T1SS as a heterologous surrogate system for the RTX toxin MbxA from the bovine pathogen Moraxella bovis. In E. coli the HlyA system successfully activates the heterologous MbxA substrate by acylation and secretes the precursor proMbxA and active MbxA allowing purification of both species in quantities sufficient for a variety of investigations. The activating E. coli acyltransferase HlyC recognizes the acylation sites in MbxA, but unexpectedly in a different acylation pattern as for its endogenous substrate HlyA. HlyC-activated MbxA shows host species-independent activity including a so-far unknown toxicity against human lymphocytes and epithelial cells. Using live- cell imaging, we show an immediate MbxA-mediated permeabilization and a rapidly developing blebbing of the plasma membrane in epithelial cells, which is associated with immediate cell death.

biochemistry↗

bioRxiv2022

The periplasmic chaperone Skp prevents misfolding of the secretory lipase A from Pseudomonas aeruginosa

Pseudomonas aeruginosa is a wide-spread opportunistic human pathogen and a high-risk factor for immunodeficient people and patients with cystic fibrosis. The extracellular lipase A belongs to the virulence factors of P. aeruginosa. The lipase undergoes folding and activation in the periplasm prior the secretion. Here, we demonstrate that the ubiquitous periplasmic chaperone Skp of P. aeruginosa, but not SurA, FkpA, PpiD or YfgM, efficiently prevents misfolding of the aggregation-prone lipase A and facilitates its activation by a specific foldase LipH. Small-angle X-ray scattering visualizes the trimeric architecture of P. aeruginosa Skp and identifies two primary conformations of the chaperone, a compact and a widely open. We describe two binding modes of Skp to the lipase, with affinities of 20 nM and 2 M, which correspond to 1:1 and 1:2 stoichiometry of the lipase:Skp complex. Two Skp trimers are required to stabilize the lipase via the apolar interactions, which are not affected by high salt concentrations typical for the sputum of cystic fibrosis patients. The chaperoning effect of Skp points to its potent role in maturation and secretion of the lipase in Pseudomonas species.

biochemistry↗

bioRxiv2021

A MademoiseLLE domain binding platform links the key RNA transporter to endosomes

Spatiotemporal expression can be achieved by transport and translation of mRNAs at defined subcellular sites. An emerging mechanism mediating mRNA trafficking is microtubule- dependent co-transport on shuttling endosomes. Although progress has been made in identifying various components of the endosomal mRNA transport machinery, a mechanistic understanding of how these RNA-binding proteins are connected to endosomes is still lacking. Here, we demonstrate that a flexible MademoiseLLE (MLLE) domain platform within RNA- binding protein Rrm4 of Ustilago maydis is crucial for endosomal attachment. Our structure/function analysis uncovered three MLLE domains at the C-terminus of Rrm4 with a functionally defined hierarchy. MLLE3 recognises two PAM2-like sequences of the adaptor protein Upa1 and is essential for endosomal shuttling of Rrm4. MLLE1 and MLLE2 are most likely accessory domains exhibiting a variable binding mode for interaction with currently unknown partners. Thus, endosomal attachment of the mRNA transporter is orchestrated by a sophisticated MLLE domain binding platform.

microbiology↗

bioRxiv2021

Novel intracellular phospholipase B from Pseudomonas aeruginosa with activity towards endogenous phospholipids affects biofilm assembly

Pseudomonas aeruginosa is a severe threat to immunocompromised patients due to its numerous virulence factors and multiresistance against antibiotics. This bacterium produces and secretes various toxins with hydrolytic activities including phospholipases A, C and D. However, the function of intracellular phospholipases for bacterial virulence has still not been established. Here we demonstrate that the hypothetical gene pa2927 of P. aeruginosa encodes a novel phospholipase B named PaPlaB. PaPlaB isolated from detergent-solubilized membranes of E. coli rapidly degraded various GPLs including endogenous GPLs isolated from P. aeruginosa cells. Cellular localization studies suggest that PaPlaB is peripherally bound to the inner and outer membrane of E. coli, yet the active form was predominantly associated with the cytoplasmic membrane. In vitro activity of purified and detergent-stabilized PaPlaB increases at lower protein concentrations. The size distribution profile of PaPlaB oligomers revealed that decreasing protein concentration triggers oligomer dissociation. These results indicate that homooligomerisation regulates PaPlaB activity by a yet unknown mechanism, which might be required for preventing bacteria from self-disrupting the membrane. We demonstrated that PaPlaB is an important determinant of the biofilm lifestyle of P. aeruginosa, as shown by biofilm quantification assay and confocal laser scanning microscopic analysis of biofilm architecture. This novel intracellular phospholipase B with a putative virulence role contributes to our understanding of membrane GPL degrading enzymes and may provide a target for new therapeutics against P. aeruginosa biofilms.

biochemistry↗

bioRxiv2021

A new twist in ABC transporter mediated multidrug resistance - Pdr5 is a drug/proton co-transporter

The two major efflux pump systems are involved in multidrug resistance (MDR): (i) ATP binding cassette (ABC) transporters and (ii) secondary transporters. While the former use binding and hydrolysis of ATP to facilitate export of cytotoxic compounds, the latter utilize electrochemical gradients to expel their substrates. Pdr5 from Saccharomyces cerevisiae is a prominent member of eukaryotic ABC transporters that are involved in MDR and used as a frequently studied model system. Although investigated for decades, the underlying molecular mechanisms of transport and specificity remain elusive. Here, we provide electrophysiological data on reconstituted Pdr5 demonstrating that this MDR efflux pump does not only actively translocate its substrates across the lipid bilayer, but generates a proton motif force in the presence of Mg2+-ATP and substrates by acting as a proton/drug co-transporter. Importantly, a strictly substrate dependent co-transport of protons was also observed in in vitro transport studies using Pdr5-enriched plasma membranes. Similar observations have not yet been reported for any other MDR efflux pump. We conclude from these results that the mechanism of MDR conferred by Pdr5 and likely other transporters is more complex than the sole extrusion of cytotoxic compounds and involves secondary coupled processes suitable to increase the effectiveness.

biochemistry↗

bioRxiv2021

Cumulative growth and stress responses to the 2018-2019 drought in a European floodplain forest

Droughts increasingly threaten the worlds forests and their potential to mitigate climate change. In 2018-2019, Central European forests were hit by two consecutive hotter drought years, an unprecedented phenomenon that is likely to occur more frequently with climate change. Here, we examine tree growth and physiological stress responses (increase in carbon isotope composition; {Delta}{delta}13C) to this consecutive drought based on tree-rings of dominant tree species in a Central European floodplain forest. Tree growth was not reduced for most species in 2018, indicating that water supply in floodplain forests can partly buffer meteorological water deficits. Drought stress responses in 2018 were comparable to former single drought years but the hotter drought in 2018 induced drought legacies in tree growth while former droughts did not. We observed strong decreases in tree growth and increases in {Delta}{delta}13C across all tree species in 2019, which are likely driven by the cumulative stress both consecutive hotter droughts exerted. Our results show that consecutive hotter droughts pose a novel threat to forests under climate change, even in forest ecosystems with comparably high levels of water supply.

ecology↗

bioRxiv2021

Structure and efflux mechanism of the yeast pleiotropic drug resistance transporter Pdr5

Pdr5, a member of the extensive ABC transporter superfamily, is representative of a clinically relevant subgroup involved in pleiotropic drug resistance. Through the coupling of nucleotide hydrolysis with drug efflux, Pdr5 homologues enable pathogenic species to survive in the presence of chemically diverse antifungal agents. Our structural and functional results reveal details of an ATP-driven conformational cycle, which mechanically drives drug translocation through an amphipathic channel, and a clamping switch within a conserved linker loop that acts as a nucleotide sensor. One half of the transporter remains nearly invariant throughout the cycle, while its partner undergoes changes that are transmitted across interdomain interfaces to support a peristaltic motion of the pumped molecule. The efflux model proposed here rationalises the pleiotropic impact of Pdr5 and opens avenues for the development of effective antifungal compounds.

microbiology↗

bioRxiv2019

Mechanism of the secretion of the lanthipeptide nisin

Lanthipeptides are ribosomally synthesized and post-translationally modified peptides containing dehydrated amino acids and (methyl-)lanthionine rings. One of the best-studied example is nisin, which is synthesized as a precursor peptide comprising of an N-terminal leader peptide and a C-terminal core peptide. Amongst others, the leader peptide is crucial for enzyme recognition and acts as a secretion signal for the ABC transporter NisT which secrets nisin in a proposed channeling mechanism. Here, we present an in vivo secretion analysis of this process in the presence and absence of the maturation machinery composed of the dehydratase NisB and the cyclase NisC. The data clearly demonstrated that the function of NisC, but the mere presence of NisB modulated the apparent secretion rates. Additional in vitro studies of detergent-solubilized NisT revealed how the activity of this ABC transporter is again influenced by the enzymes of the maturation machinery, but not the substrate.

biochemistry↗

bioRxiv2019

A large-scale resource for tissue-specific CRISPR mutagenesis in Drosophila

Genetic screens are powerful tools for the functional annotation of genomes. In the context of multicellular organisms, interrogation of gene function is greatly facilitated by methods that allow spatial and temporal control of gene abrogation. Here, we describe a large-scale transgenic short guide (sg) RNA library for efficient CRISPR-based disruption of specific target genes in a constitutive or conditional manner. The library consists currently of more than 2600 plasmids and 1600 fly lines with a focus on targeting kinases, phosphatases and transcription factors, each expressing two sgRNAs under control of the Gal4/UAS system. We show that conditional CRISPR mutagenesis is robust across many target genes and can be efficiently employed in various somatic tissues, as well as the germline. In order to prevent artefacts commonly associated with excessive amounts of Cas9 protein, we have developed a series of novel UAS-Cas9 transgenes, which allow fine tuning of Cas9 expression to achieve high gene editing activity without detectable toxicity. Functional assays, as well as direct sequencing of genomic sgRNA target sites, indicates that the vast majority of transgenic sgRNA lines mediate efficient gene disruption. Furthermore, we conducted the so far largest fully transgenic CRISPR screen in any metazoan organism, which further supported the high efficiency and accuracy of our library and revealed many so far uncharacterized genes essential for development.

genetics↗

bioRxiv2018

From Metapopulation to Metacommunity and the Complications of Community Structure

The metacommunity, as it evolved from Levins metapopulation, provides a framework to consider the spatial organization of species interactions. Arguably the most fundamental feature of metapopu-lations and metacommunities are that demes are connected via migration. An important result from Levins metapopulation work--that increasing migration lowers regional extinction probability--is often incorporated into conceptions of metacommunities. We first use a toy model to show how this result from Levins metapopulation does not necessarily hold when considering community interactions in a metacommunity context. We also report results from a metacommunity field experiment conducted with a tropical terrestrial leaf litter community and show that migration induces the extinction of predators in the metacommunity. Our result corroborates the findings of a prior metacommunity experiment in a temperate terrestrial leaf litter community. The concordance between these experiments even with vastly different communities highlights the importance of considering trophic and non-trophic community structure to understand metacommunity dynamics.

ecology↗