Biology subjects

Roy, C.

Publications and source records attributed to Roy, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Dynamics and activation of the Legionella Type IV secretion system

Legionella pneumophila uses the Dot/Icm type IV secretion system (T4SS) to translocate hundreds of effector proteins into host cells, yet how this transenvelope machine assembles and becomes secretion competent remains poorly understood. Here, using cryo-electron tomography and in situ single-particle cryo-electron microscopy, we resolve distinct structural and functional states of the native Dot/Icm T4SS. Unexpectedly, the T4SS predominantly occupies a resting state containing the outer membrane core complex but lacking the inner-membrane complex, including DotA. Recruitment of inner-membrane components establishes a fully assembled but secretion-inactive primed state. A 3.6 [A] cryo-EM structure of the DotO-DotI-DotJ-IcmT complex, integrated with in situ reconstructions, reveals how membrane-associated components organize DotO into a hexamer of dimers ATPase platform. Upon host contact, the T4SS undergoes extensive conformational remodeling while preserving its transenvelope scaffold. Activation involves dilation of the DotG outer membrane gate and transformation of the DotA-IcmX protochannel into an open translocation conduit that extends beyond the bacterial surface. Substrate-associated state exhibit further channel expansion, revealing structural plasticity associated with effector translocation. During macrophage infection, the extracellular conduit bridges the bacterial and host membranes, establishing a direct pathway for effector delivery. Together, our findings reveal a stepwise mechanism of Dot/Icm T4SS channel assembly and activation, in which coordinated remodeling of the secretion channel converts a resting apparatus into a host-engaged translocation machine.

microbiology↗

bioRxiv2026

A nuclear role for the contractile protein troponin I/UNC-27 in regulating muscle aging in C. elegans

Muscle ageing is characterized by evolutionarily conserved subcellular alterations across diverse organisms. In Caenorhabditis elegans, the decline in sarcomeric gene expression is among the earliest detectable ageing-associated changes, emerging at the onset of adulthood. To identify causal regulators of muscle ageing in an unbiased manner, we developed a genetic screening strategy that enables visual monitoring of muscle ageing at both cellular and organismal scales. Using this approach, we identified a mutation that delays the age-associated loss of sarcomeric transcripts. Unexpectedly, the mutation maps to the troponin I gene unc-27, which encodes a conserved regulator of muscle contraction not previously implicated in gene regulation. The mutation alters a single amino acid within a predicted nuclear localization signal (NLS). We found that multiple NLS motifs mediate the active transport of UNC-27 into muscle nuclei from early adulthood onward. Disruption of UNC-27 nuclear localization preserves sarcomeric gene expression during ageing and delays early hallmarks of muscle decline, including proteostatic imbalance and mitochondrial fragmentation. Transcriptomic analyses further revealed that nuclear UNC-27 selectively regulates the expression of genes encoding structural components of the muscle apparatus in adult animals. These results support the existence of a homeostatic sarcomere surveillance pathway, in which a structural protein unexpectedly acquires a transcriptional regulatory role in response to age-associated physiological state. The conservation of NLS motifs in mammalian UNC-27 orthologues suggests that this mechanism may be evolutionarily conserved, with potential relevance to human muscle physiology and disease.

cell biology↗

bioRxiv2026

Danish profile of soft rot Pectobacteriaceae; A three-year field sampling study proving several clonal clades of soft rot isolates across diverse locations implicating a common origin

Soft rot Pectobacteriaceae (SRP) are among the most economically important plant pathogenic bacteria and are especially known to be problematic in potato production. The epidemiology of disease transmission has been investigated for almost a century, and several aspects have been highlighted as plausible infection routes. However, it is generally accepted that the major source of disease is the latently infected mother tuber, but several parameters are still influencing disease prevalence including contaminated equipment, soil water status as well as temperature. Management of the disease is limited to hygiene practices, dry storage and seed certification systems but several studies have also proven biocontrol agents such as bacteriophages (phages) as promising tools. Despite the severity of SRP on potato production, little is known about the genetic diversity of SRPs in Denmark, and since only few isolates are available, the possibility to design a broadly effective phage cocktail is limited. Here we describe a three-year field study utilizing an agri-citizen science approach where Danish farmers provided symptomatic potato plants or tubers, together with metadata such as date, location, potato variety and origin. By using whole genome sequencing (Illumina and Nanopore) together with metadata we were able to investigate and monitor the epidemiological disease spread across the country using 103 complete genomes, sampled across all three years. In this study we provide epidemiological evidence of disease origins and a suite of phages that could be used as a biocontrol tool for early disease intervention. Our results revealed several clonal clades across diverse locations (SNPs < 20) which strongly indicate common origin. A total of 17 Pectobacterium phages were tested and did target > 80% of clonal clades. Based on the clonality across the soft rot isolates we propose the possibility to set in early on using phages targeting strains relevant for soft rot development, with the possibility of a surveillance program together with customizing the phage preference.

microbiology↗

bioRxiv2026

Comparison of Four Assays That Measure Antibodies to Ebola Virus Glycoprotein

The accurate measurement of Ebola virus (EBOV)-specific antibody responses is crucial to assessing immunity induced by EBOV infection or vaccination. For this purpose, the Filovirus Animal Nonclinical Group (FANG) anti-EBOV glycoprotein (GP1,2) ELISA is considered the "gold-standard". However, it has limitations such as high repeat-rates and variability, and low throughput. Here, we describe two new alternative assays: a Single-Molecule Assay Planar EBOV GP1,2 ELISA and a multiplexed EBOV GP1,2, EBOV nucleoprotein, and EBOV Viral Protein 40 Luminex assay, and compare these with two versions of the FANG ELISA. Samples were selected from participants receiving vaccine or placebo in a randomized, placebo-controlled, double-blinded study of two EBOV vaccines (PREVAIL 1), and a longitudinal cohort study of Ebola virus disease (EVD) survivors and their close contacts (PREVAIL 3). All four assays were concordant in their measurements of anti-EBOV GP1,2-specific immunoglobulin G responses, allowing for the determination of conversion equations for antibody measurements across assays. In addition, all four showed a similar ability to distinguish vaccine recipients from placebo recipients and EVD survivors from their close contacts. Compared to the FANG assays, the Quanterix and Luminex assays had lower variability, lower repeat rates, and higher throughput, making them good alternatives for future studies.

immunology↗

bioRxiv2026

Type IV Secretion System Drives Lipid Mixing

Type IV secretion systems (T4SSs) are versatile molecular machines used by bacteria to secrete protein effectors into host cells, promoting pathogenesis, and to transfer DNA between bacteria through conjugation, driving horizontal gene transfer. Most, like Dot/Icm of the pathogen Legionella pneumophila (L. pneumophila) or Escherichia coli (E. coli) RK2, are primed for substrate delivery only upon contact with a target membrane, but mechanisms are unknown. A pilus could bind a receptor to initiate priming, but many T4SSs, especially those that deliver effectors, lack a pilus. Here, we present evidence that T4SSs are primed by direct contact with target membrane lipids. Combining fluorescence assays with genetics and biochemistry, we found that Dot/Icm drives lipid exchange between bacterial cells and between bacteria and synthetic membranes containing only lipids. Lipid exchange requires membrane contact but does not require ATP hydrolysis or even full complex assembly. Minimally, the outer membrane core complex protein DotG needs to be present in at least one of the apposed membranes. We similarly observed lipid mixing with the simpler E. coli RK2 T4SS, where we could follow lipid mixing and plasmid transfer simultaneously. We found that lipid mixing always preceded or accompanied plasmid transfer, suggesting it may be part of the contact-dependent priming mechanism. Lipid mixing was inhibited or promoted by lipids that inhibit or promote membrane fusion, respectively. Lipids inhibiting lipid mixing also inhibited substrate transfer. Together, our results suggest that initial contact between DotG outer segments and target membrane lipids promotes lipid mixing as part of the mechanism that primes T4SS for substrate translocation. HighlightsO_LIThe Legionella pneumophila Dot/Icm secretion system requires contact with a target membrane for effector translocation, but how membrane contact primes the machinery for this is unknown. C_LIO_LIWe found that Dot/Icm drives contact-dependent lipid mixing between bacteria or between bacteria and liposomes, showing that Dot/Icm priming does not require a protein receptor. C_LIO_LILipid mixing also occurs with the simpler Type IVA system (Escherichia coli RK2) during conjugation, suggesting lipid mixing is a general feature of Type IV Secretion System function. C_LIO_LIThe core complex protein DotG is sufficient to drive lipid mixing, suggesting DotG-target membrane interactions may destabilize the target membrane. C_LI

microbiology↗

bioRxiv2025

Legal Harvest of Shorebirds and Resident Game Birds on a Caribbean island: A Martinique Case Study

Legal hunting practiced under regulatory frameworks is not always assessed for its sustainability. The lack of harvest data is of particular concern in the case of birds occurring on islands. Resident species on islands often exhibit limited geographic ranges and inherently small population sizes, rendering them particularly susceptible to environmental and anthropogenic threats. Islands also support seasonally fluctuating populations of migratory species, providing essential habitats for stopovers and non-breeding periods. Within this context, the sustainability of gamebird harvest practiced on islands within a regulatory framework needs to be evaluated to inform management and prevent negative impacts on populations. Here, we present a case study of an island-wide assessment of bird hunting estimates, based on data available from a legal hunting program, in Martinique, a tropical oceanic island part of the Caribbean biodiversity hotspot. Our objectives were to determine the magnitude of gamebird harvest, track changes in harvest levels over time, and assess harvest sustainability. Our assessment, which is based on hunting logbooks and hunter surveys between the 2012-2013 and 2021-2022 hunting seasons, provides harvest estimates for two groups of migratory species, shorebirds (Charadriidae, Scolopacidae) and teals (Anatidae), and two groups of resident species (Columbidae and Mimidae). In Martinique, harvest management measures, combined with increased hunters engagement and enforcement of regulations, has led more stringent harvest regulations, improved compliance from hunters, and a reduction of Martiniques national shorebird harvest. However, considering continued and rapid shorebirds population declines across the Western Atlantic flyway, shorebird harvest assessment at the flyway level remains a priority.

ecology↗

bioRxiv2025

A national assessment of waterbird hunting in coastal wetlands of Suriname, south America

The central northern coast of South America has extensive wetlands critical for waterbird conservation. While waterbird harvest occurs in the region, the impact on species population dynamics remains unclear. This study assesses waterbird hunting in the coastal wetlands of Suriname, addressing: (i) the extent of waterbird harvest, (ii) changes in harvest magnitude over time, and (iii) the motivations and methods used by hunters. We collected data via a national survey of licensed hunters in 2006 and 2016 using structured interviews. A Bayesian hierarchical model was used to analyze the data. We estimated harvest levels for 11 species and three groups (small herons, small shorebirds, large shorebirds). For most species, mean harvest per hunter significantly decreased from 2006 to 2016, except for blue-winged teal and migratory shorebirds. Most hunting was for non-commercial purposes (personal consumption and recreational). This is the first national assessment of waterbird hunting in Suriname. Harvest levels vary by species, and the sustainability of these levels remains uncertain. Managing hunting in Suriname requires addressing both legal and illegal hunting. Given Surinames importance for waterbirds, particularly species like the scarlet ibis and migratory shorebirds, it should be a priority for conservation efforts.

ecology↗

bioRxiv2025

The Legionella pneumophila type IVb secretion system effector BinA subverts amino acid transport to sensitize TORC1 signaling in macrophages

Legionella pneumophila is an environmental Gram-negative bacterium that parasitizes unicellular protozoa and can cause severe pulmonary infections when aerosolized bacteria are inhaled by humans. One critical aspect of Legionella pathogenesis is the establishment in the cytosol of infected macrophages of a unique ER-derived vacuole, that requires a sustained supply of host lipids during expansion. Subversion of pro-lipogenic pathways downstream of the metabolic checkpoint kinase mTOR (Mechanistic Target of Rapamycin) are critical for niche expansion. In eukaryotic cells, amino acids sufficiency and growth factor sensory signals converge on mTOR to ensure metabolic processes are coupled to nutrients/energy availability. Legionella can trigger mTOR signaling in infected cells by increasing the intracellular abundance of amino acids through inhibition of host translation. Here, we describe a novel mechanism by which Legionella sensitizes mTOR in infected macrophages. A forward genetic screen identified Lpg0393 protein as a putative bacterial mTOR regulator that contains a VPS9-domain typically found in eukaryotic GEFs (Guanine nucleotide exchange factors) for Rab5 GTPase family members (Rab5/Rab21/Rab22). We uncovered that Lpg0393 lowers the activation threshold for mTOR signaling upon stimulation with arginine or leucine through rewiring of processes upstream of mTOR by removing Rab5-dependency and replacing it with a Rab21/Rab22-dependency. Data from cells expressing either a bacterial or a eukaryotic mTOR sensitizing factor uncovered two distinct intraorganellar Arg/Leu pools that fuel mTOR activation in parallel - one regulated by Rab21/22 and the other by Rab5. Consistent with the role of mTOR in expansion of the Legionella-occupied organelle, deletion of Lpg0393 also resulted in premature vacuolar rupture in a mTOR-dependent manner. All together, we have identified a novel bacterial mTOR regulator and consistent with its reported functions we propose Lpg0393 is named as BinA (Bacterial initiator of TORC1 signaling and an activator of Rab5 family GTPases). Author SummaryLegionella pneumophila - a prototypical vacuolar pathogen - manipulates host lipogenesis to sustain membrane biogenesis, which ensures the integrity of the vacuolar compartment is not compromised as demand for housing capacity increases during bacterial replication. Subversion of the host mTOR kinase signaling, which functions as a central regulatory hub coordinating nutrients sufficiency with metabolic output, is one mechanism by which Legionella increases de novo lipogenesis in infected cells. Here, we report a novel mechanism by which the bacteria sustain mTOR signaling through the actions of the type IV secretion system effector BinA. We found that BinA rewires amino acid transport mechanisms by manipulating Rab5 family GTPase to selectively sensitize mTOR to arginine and leucine but not methionine or glutamine stimulation. mTOR sensitization by BinA revealed a switch in upstream regulators from Rab5 to Rab21/Rab22. Our work provides novel insight into how pathogens exert metabolic control over host cells to maximize intracellular replication.

microbiology↗

bioRxiv2024

The T4bSS of Legionella features a two-step secretion pathway with an inner membrane intermediate for secretion of transmembrane effectors

To promote intracellular survival and infection, Legionella spp. translocate hundreds of effector proteins into eukaryotic host cells using a type IV b protein secretion system (T4bSS). T4bSS are well known to translocate soluble as well as transmembrane domain-containing effector proteins (TMD-effectors) but the mechanisms of secretion are still poorly understood. Herein we investigated the secretion of hydrophobic TMD-effectors, of which about 80 were previously reported to be encoded by L. pneumophila. A proteomic analysis of fractionated membranes revealed that TMD-effectors are targeted to and inserted into the bacterial inner membranes of L. pneumophila independent of the presence of a functional T4bSS. While the T4bSS chaperones IcmS and IcmW were critical for secretion of all tested TMD-effectors, they did not influence inner membrane targeting of these proteins. As for soluble effector proteins, translocation of TMD-effectors into host cells depended on a C-terminal secretion signal and this signal needed to be presented towards the cytoplasmic side of the inner membrane. A different secretion behavior of TMD- and soluble effectors and the need for small periplasmic loops within TMD-effectors provided strong evidence that TMD-effectors are secreted in a two-step secretion process: Initially, an inner membrane intermediate is formed, that is extracted towards the cytoplasmic side, possibly by the help of the type IV coupling protein complex and subsequently secreted into eukaryotic host cells by the T4bSS core complex. Overall, our study highlights the amazing versatility of T4bSS to secrete soluble and TMD-effectors from different subcellular locations of the bacterial cell.

microbiology↗

bioRxiv2023

Peribacillus frigoritolerans T7-IITJ, a potential biofertilizer, induces plant growth-promotinggenes of Arabidopsis thaliana

This study aimed to isolate plant growth and drought tolerance-promoting bacteria from the nutrient- poor rhizosphere soil of several plant species from the Thar desert and unravel their molecular mechanisms of plant growth promotion, to develop effective biofertilizers for arid agriculture. Among our isolates of Thar desert rhizobacteria, Enterobacter cloacae C1P-IITJ, Kalamiella piersonii J4-IITJ, and Peribacillus frigoritolerans T7-IITJ, significantly enhanced root and shoot growth in the model plant Arabidopsis thaliana under PEG-induced drought stress in the lab. Whole genome sequencing and biochemical analyses of the non-pathogenic bacterium T7-IITJ revealed its plant growth-promoting traits, viz., solubilization of phosphate, iron, and nitrate and production of exopolysaccharides and auxin. Transcriptome analysis of Arabidopsis thaliana inoculated with T7-IITJ and exposure to drought revealed the induction of plant genes for photosynthesis, auxin and jasmonate signaling, nutrient mining and sequestration, redox homeostasis, and secondary metabolite biosynthesis pathways related to beneficial bacteria-plant interaction, but repression of many stress-responsive genes. Biochemical analyses indicated enhanced proline, chlorophyll, iron, phosphorous, and nitrogen content and reduced reactive oxygen species in plant tissues due to T7-IITJ inoculation. This bacterium could also improve the germination and seedling growth of Tephrosia purpurea, Triticum aestivum, and Setaria italica under drought. Additionally, T7-IITJ inhibited the growth of two plant pathogenic fungi, Rhizoctonia solani, and Fusarium oxysporum. These results suggest P. frigoritolerans T7-IITJ is a potent biofertilizer which can regulate plant genes promoting growth and drought tolerance.

plant biology↗

bioRxiv2023

High diversity of Proteobacteria amid the dominance of a few Aquificae species in the vent-water of a Trans-Himalayan sulfur-borax hot spring

Trans-Himalayan hot spring waters rich in boron, chlorine, sodium and sulfur (but poor in calcium and silicon) are known based on PCR-amplified 16S rRNA gene sequence data to harbor high diversities of infiltrating bacterial mesophiles. Yet, little is known about the community structure and functions, primary productivity, mutual interactions, and thermal adaptations of the microorganisms present in the steaming waters discharged by these geochemically peculiar spring systems. We revealed these aspects of a bacteria-dominated microbiome (microbial cell density [~]8.5 x 104 mL-1; live:dead cell ratio 1.7) thriving in the boiling (85{degrees}C) fluid vented by a sulfur-borax spring called Lotus Pond, situated at 4436 m above the mean sea-level, in the Puga valley of eastern Ladakh, on the Changthang plateau. Assembly, annotation, and population-binning of >15-GB metagenomic sequence illuminated the numeral predominance of Aquificae. While members of this phylum accounted for 80% of all 16S rRNA-encoding reads within the metagenomic dataset, 14% of such reads were attributed to Proteobacteria. Post assembly, only 25% of all protein-coding genes identified were attributable to Aquificae, whereas 41% was ascribed to Proteobacteria. Annotation of metagenomic reads encoding 16S rRNAs, and/or PCR-amplified 16S rRNA genes, identified 163 bacterial genera, out of which 66 had been detected in past investigations of Lotus Ponds vent-water via 16S amplicon sequencing. Among these 66, Fervidobacterium, Halomonas, Hydrogenobacter, Paracoccus, Sulfurihydrogenibium, Tepidimonas, Thermus and Thiofaba (or their close phylogenomic relatives) were presently detected as metagenome-assembled genomes (MAGs). Remarkably, the Hydrogenobacter related MAG alone accounted for [~]56% of the entire metagenome, even though only 15 out of the 66 genera consistently present in Lotus Ponds vent-water have strains growing in the laboratory at >45{degrees}C, reflecting the continued existence of the mesophiles in the ecosystem. Furthermore, the metagenome was replete with genes crucial for thermal adaptation in the context of Lotus Ponds geochemistry and topography. In terms of sequence similarity, a majority of those genes were attributable to phylogenetic relatives of mesophilic bacteria, while functionally they rendered functions such as encoding heat shock proteins, molecular chaperones, and chaperonin complexes; proteins controlling/modulating/inhibiting DNA gyrase; universal stress proteins; methionine sulfoxide reductases; fatty acid desaturases; different toxin-antitoxin systems; enzymes protecting against oxidative damage; proteins conferring flagellar structure/function, chemotaxis, cell adhesion/aggregation, biofilm formation, and quorum sensing. The Lotus Pond Aquificae not only dominated the microbiome numerically but also acted potentially as the main primary producers of the ecosystem, with chemolithotrophic sulfur oxidation (Sox) being the fundamental bioenergetic mechanism, and reductive tricarboxylic acid (rTCA) cycle the predominant carbon fixation pathway. The Lotus Pond metagenome contained several genes directly or indirectly related to virulence functions, biosynthesis of secondary metabolites including antibiotics, antibiotic resistance, and multi-drug efflux pumping. A large proportion of these genes being attributable to Aquificae, and Proteobacteria (very few were ascribed to Archaea), it could be worth exploring in the future whether antibiosis helped the Aquificae overcome niche overlap with other thermophiles (especially those belonging to Archaea), besides exacerbating the bioenergetic costs of thermal endurance for the mesophilic intruders of the ecosystem.

genomics↗

bioRxiv2023

Synergistic activity of IL-2 mutein with tolerogenic ImmTOR nanoparticles leads to massive expansion of antigen-specific Tregs and protection against autoimmune disease

Low dose IL-2 therapy and IL-2 molecules engineered to be selective for the high affinity IL-2 receptor have been shown to expand Tregs in vivo, and, in the case of low dose IL-2 therapy, has demonstrated promising therapeutic benefit in autoimmune diseases. One of the potential limitations of IL-2 therapy is the nonselective expansion of pre-existing Treg populations rather than induction of antigen-specific Tregs, as well as potential activation of effector cells. We have recently developed biodegradable nanoparticles encapsulating rapamycin, called ImmTOR, to induce selective immune tolerance to co-administered antigens, such as immunogenic biologic drugs. Unlike Treg-selective IL-2 therapy, ImmTOR alone does not increase total Treg numbers. However, here we demonstrate that the combination of ImmTOR and an engineered Treg-selective IL-2 variant (termed IL-2 mutein) increases the number and durability of total Tregs, as well as inducing a profound synergistic increase in antigen-specific Treg when combined with a target antigen. We demonstrate that the combination of ImmTOR and an IL-2 mutein leads to durable inhibition of antibody responses to co-administered AAV gene therapy capsid, even at sub-optimal doses of ImmTOR, and provides protection in autoimmune models of type 1 diabetes and primary biliary cholangitis. ImmTOR also showed the potential to increase the therapeutic window of engineered IL-2 molecules by mitigating effector T cell expansion typically observed at higher doses of IL-2 and preventing exacerbation of disease in a model of graft-versus-host-disease. At the same time, engineered IL-2 molecules showed potential for dose-sparing of ImmTOR. Overall, these results establish that the combination of ImmTOR and an IL-2 mutein show synergistic benefit on both safety and efficacy to provide durable antigen-specific immune tolerance to mitigate drug immunogenicity and to treat autoimmune diseases.

immunology↗

bioRxiv2023

Designer grass pea for transgene-free minimal neurotoxin-containing seeds with CRISPR-Cas9

Grass pea seeds are consumed as food in several South Asian and Sub-Saharan African nations. However, the presence of the neurotoxic compound N-oxalyl-L-diamino propionic acid ({beta}-ODAP) has restricted its cultivation. Although various cultivars with low {beta}-ODAP levels have been developed, their cultivation is still limited due to the risk of neurolathyrism from long-term grass pea seed ingestion. In this study, we employed the CRISPR-Cas9-mediated gene editing technique to generate grass pea seeds with zero or minimal {beta}-ODAP levels. We targeted the BAHD-AT3 gene that encodes a key enzyme in the {beta}-ODAP biosynthesis pathway. We developed bahd-at3 knock-out lines using three gRNAs targeting different regions of this gene and characterized them. Cas9-free independent lines from each event carrying the desired on-target mutation were selected and backcrossed twice with the wild-type to eliminate any off-target mutation present therein. Various agronomical parameters were analyzed from the backcrossed mutant lines and they displayed no phenotypic abnormalities. Interestingly, the seed {beta}-ODAP content ranged between 0.001 % - 0.002 % of dry weight which is 99 % lower than the wild-type. Together, our study reports the development of transgene-free, genome-edited grass peas with insignificant levels of {beta}-ODAP in seeds for safer food in the future.

plant biology↗

bioRxiv2023

Maintenance and transformation of representational formats during working memory prioritization

Visual working memory depends on both material-specific brain areas in the ventral visual stream (VVS) that support the maintenance of stimulus representations and on regions in prefrontal cortex (PFC) that control these representations. Recent studies identified stimulus-specific working memory contents via representational similarity analysis (RSA) and analyzed their representational format using deep neural networks (DNNs) as models of the multi-layered hierarchy of information processing. How executive control prioritizes relevant working memory contents and whether this affects their representational formats remains an open question, however. Here, we addressed this issue using a multi-item working memory task involving a retro-cue that prompted participants to maintain one particular item. We exploited the excellent spatiotemporal resolution of intracranial EEG (iEEG) recordings in epilepsy patients and analyzed activity at electrodes in VVS (n=28 patients) and PFC (n=16 patients). During encoding, we identified category-specific information in both VVS and PFC. During maintenance, this information re-occurred in VVS but not in the PFC - suggesting a transformation of PFC representations from encoding to maintenance which putatively reflects the prioritization process. We thus applied RSA in combination with different DNN architectures to investigate the representational format of prioritized working memory contents. Representations during the maintenance period matched representations in deep layers of recurrent but not feedforward DNNs, in both VVS and PFC. While recurrent DNN representations matched PFC representations in the beta band following the retro-cue, they corresponded to VVS representations in a lower theta-alpha frequency range (3-14Hz) towards the end of the maintenance period. Findings could be replicated in recurrent DNNs with two different architectures and using two different training sets. Together, these results demonstrate that VWM relies on representational transformations in VVS and PFC that give rise to distinct coding schemes of prioritized contents.

neuroscience↗

bioRxiv2022

Two Muscle-Specific and Direct Transcriptional Targets of DAF-16/FOXO Activated by Reduced Insulin/IGF-1 Signaling

C. elegans insulin/insulin-like growth factor 1 signaling, IIS, affects diverse physiological processes through the DAF-16/FOXO transcription factor. Despite its ubiquitous presence in somatic cells, DAF-16s effects exhibit prevalent tissue specificity as well as tissue crosstalk. This implies that tissue-specific DAF-16 transcriptional programs contribute to functional diversity of IIS. To further investigate this possibility, we sought muscle-cell-specific DAF-16 transcriptional targets. Using fluorescence-activated cell sorting to enrich for body wall muscle cells from young hermaphroditic adults, we compared the muscle cell mRNA transcriptomes under conditions of high and low IIS activity, with and without DAF-16. We further analyzed DAF-16as binding sites in muscle and intestine cells by chromatin-immunoprecipitation sequencing. Combined output of these analyses is 12 candidate DAF-16 targets enriched for muscle cells. Transcriptional and translational reporters for three out of the four top candidates - a secreted protein C54F6.5, a calcium-binding protein CEX-1/calexcitin, and a fatty acid metabolic enzyme MLCD-1/MCD - showed DAF-16-dependent activation specifically in body wall muscle cells. Notably, reporters for C54F6.5 and cex-1 exhibit DAF-16-independent, constitutive expression in non-muscle cells, explaining their low rank or absence from the DAF-16 target lists generated by whole-animal microarray or mRNA-sequencing analyses. These results highlight the need to examine FOXO targets in a cell-type-specific manner. Article SummaryThis study is relevant to those interested in functional specificity of signaling pathways. It describes a rigorous workflow to identify tissue-specific transcriptional changes activated by DAF-16/FOXO, a key effector of insulin signaling, its tissue-specific chromatin binding sites, and experimental validation of three previously unknown DAF-16 targets in body wall muscle cells. These findings highlight the intricacy of tissue-specific regulation exerted by a signaling pathway that is present and operates across tissues.

genomics↗

bioRxiv2022

Cytokine profiles in adults with imported malaria: insights from the PALUREA cohort study

The increase in worldwide travel is making imported malaria a growing health concern in nonendemic countries. Most data on the pathophysiology of malaria come from endemic areas. Little is known about cytokine profiles during imported malaria. We report cytokine profiles in adults with Plasmodium falciparum malaria included in PALUREA, a prospective cohort study conducted in France between 2006 and 2010. The patients were classified as having uncomplicated malaria (UM) or severe malaria (SM), with this last further categorized as very severe malaria (VSM) or less severe malaria (LSM). At hospital admission, eight blood cytokines were assayed in duplicate using Luminex technology: interleukin (IL)-1, IL-1{beta}, IL-2, IL-4, IL-10, tumor necrosis factor (TNF), interferon (IFN){gamma}, and macrophage migration inhibitory factor (MIF). These assays were repeated on days 1 and 2 in the SM group. Of the 278 patients, 134 had UM and 144 SM. At hospital admission, over half the patients had undetectable levels of IL-1, IL-1 {beta}, IL-2, IL-4, IFN {gamma}, and TNF, while IL-10 and MIF were significantly higher in the SM vs. the UM group. Higher IL-10 was significantly associated with higher parasitemia (R=0.32 [0.16-0.46]; P=0.0001). In the SM group, IL-10 elevation persisting from admission to day 2 was significantly associated with subsequent nosocomial infection. Of eight tested cytokines, only MIF and IL-10 were associated with disease severity in adults with imported P. falciparum malaria. At admission, many patients had undetectable cytokine levels, suggesting that circulating cytokine assays may not be helpful as part of the routine evaluation of adults with imported malaria. Author SummaryPlasmodium falciparum malaria is increasingly common in nonendemic areas. Improved understanding of its pathophysiology might help to decrease mortality. We therefore routinely assayed eight cytokines in 278 adults with imported P. falciparum malaria at hospital admission; in the group with severe malaria (n=144), we repeated the assays on the next two days. The cytokine levels were often undetectable, suggesting that cytokine storm might not be a driving mechanism at the time of clinical presentation. IL-10 and macrophage migration inhibitory factor (MIF) were significantly higher in the group with severe vs. uncomplicated disease. Thus, the roles for these two cytokines in severe malaria, may deserve further investigation. A complicating factor is that greater IL-10 elevation may be a response to a heavy parasite burden and/or may promote parasite replication. IL-10 elevation that persisted over the first 2 days after admission was significantly associated with subsequent nosocomial infections in the group with severe malaria suggesting its possible role in acquired immune suppression syndrome.

immunology↗

bioRxiv2022

Developmental transitions coordinate assembly of the Coxiella burnetii Dot/Icm type IV secretion system

Coxiella burnetii is an obligate intracellular bacterial pathogen that has evolved a unique bi-phasic developmental cycle. The infectious form of C. burnetii is the dormant small cell variant (SCV) that transitions to a metabolically active large cell variant (LCV) that replicates inside the lysosome-derived host vacuole. A Dot/Icm type IV secretion system (T4SS), which can deliver over 100 effector proteins to host cells, is essential for the biogenesis of the vacuole and for intracellular replication. How the distinct C. burnetii life cycle impacts the assembly and function of the Dot/Icm T4SS has remained unknown. Here, we combine advanced cryo-focused ion beam (cryo-FIB) milling and cryo-electron tomography (cryo-ET) imaging to visualize all developmental transitions and assembly of the Dot/Icm T4SS. Importantly, assembled Dot/Icm T4SSs were not present in the infectious SCV. The appearance of an assembled Dot/Icm machine correlated with the transition of the SCV to the LCV intracellularly. Furthermore, temporal characterization of C. burnetii morphological changes revealed regions of the inner membrane that invaginated to form tightly packed stacks during the LCV to SCV transition at late stages of infection, which could enable the SCV to LCV transition that occurs upon infection of a new host cell. Overall, these data establish how C. burnetii developmental transitions control critical bacterial processes to promote intracellular replication and transmission.

microbiology↗

bioRxiv2022

Thermal endurance by a hot-spring-dwelling phylogenetic relative of the mesophilic Paracoccus

High temperature growth/survival was revealed in a phylogenetic relative (strain SMMA_5) of the mesophilic Paracoccus isolated from the 78-85{degrees}C water of a Trans- Himalayan sulfur-borax spring. After 12 h at 50{degrees}C, or 45 minutes at 70{degrees}C, in mineral salts thiosulfate (MST) medium, SMMA_5 retained [~]2% colony-forming units (CFUs), whereas comparator Paracoccus had 1.5% and 0% CFU left at 50{degrees}C and 70{degrees}C respectively. After 12 h at 50{degrees}C, the thermally-conditioned sibling SMMA_5_TC exhibited [~]1.5 time increase in CFU-count; after 45 minutes at 70{degrees}C, SMMA_5_TC had 7% of the initial CFU-count intact. 1000-times diluted Reasoners 2A medium, and MST supplemented with lithium, boron or glycine-betaine (solutes typical of the SMMA_5 habitat), supported higher CFU-retention/CFU-growth than MST. With or without lithium/boron/glycine-betaine in MST, a higher percentage of cells always remained viable (cytometry data), compared with what percentage remained capable of forming single colonies (CFU data). SMMA_5, compared with other Paracoccus, contained 335 unique genes, mostly for DNA replication/recombination/repair, transcription, secondary metabolites biosynthesis/transport/catabolism, and inorganic ion transport/metabolism. Its also exclusively enriched in cell wall/membrane/envelope biogenesis, and amino acid metabolism, genes. SMMA_5 and SMMA_5_TC mutually possessed 43 nucleotide polymorphisms, of which 18 were in protein-coding genes with 13 nonsynonymous and seven radical amino acid replacements. Such biochemical and biophysical mechanisms could be involved in thermal stress mitigation which streamline the cells energy and resources towards system-maintenance and macromolecule-stabilization, thereby relinquishing cell-division for cell-viability. Thermal conditioning apparently helped memorize those potential metabolic states which are crucial for cell-system maintenance, while environmental solutes augmented the indigenous stability-conferring mechanisms. IMPORTANCEFor a holistic understanding of microbial lifes high-temperature adaptation it is imperative to explore the biology of the phylogenetic relatives of mesophilic bacteria which get stochastically introduced to geographically and geologically diverse hot spring systems by local geodynamic forces. Here, in vitro endurance of high heat up to the extent of growth under special (habitat-inspired) conditions was discovered in a hot- spring-dwelling phylogenetic relative of the mesophilic Paracoccus species. Thermal conditioning, extreme oligotrophy, metabolic deceleration, presence of certain habitat- specific inorganic/organic solutes, and typical genomic specializations were found to be the major enablers of this conditional (acquired) thermophilicity. Feasibility of such phenomena across the taxonomic spectrum can well be paradigm-changing for the established scopes of microbial adaptation to the physicochemical extremes. Applications of conditional thermophilicity in microbial process biotechnology may be far reaching and multi-faceted.

microbiology↗