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bioRxiv2021

Mutations in the B.1.1.7 SARS-CoV-2 spike protein reduce receptor-binding affinity and induce a flexible link to the fusion peptide

The B.1.1.7 variant of the SARS-CoV-2 virus shows enhanced infectiousness over the wild type virus, leading to increasing patient numbers in affected areas. A number of single amino acid exchanges and deletions within the trimeric viral spike protein characterize this new SARS-CoV-2 variant. Crucial for viral entry into the host cell is the interaction of the spike protein with the cell surface receptor angiotensin-converting enzyme 2 (ACE2) as well as integration of the viral fusion peptide into the host membrane. Respective amino acid exchanges within the SARS-CoV-2 variant B.1.1.7 affect inter-monomeric contact sites within the spike protein (A570D and D614G) as well as the ACE2-receptor interface region (N501Y), which comprises the receptor-binding domain (RBD) of the viral spike protein. However, the molecular consequences of mutations within B.1.1.7 on spike protein dynamics and stability, the fusion peptide, and ACE2 binding are largely unknown. Here, molecular dynamics simulations comparing SARS-CoV-2 wild type with the B.1.1.7 variant revealed inter-trimeric contact rearrangements, altering the structural flexibility within the spike protein trimer. In addition to reduced flexibility in the N-terminal domain of the spike protein, we found increased flexibility in direct spatial proximity of the fusion peptide. This increase in flexibility is due to salt bridge rearrangements induced by the D614G mutation in B.1.1.7 found in pre- and post-cleavage state at the S2 site. Our results also imply a reduced binding affinity for B.1.1.7 with ACE2, as the N501Y mutation restructures the RBD-ACE2 interface, significantly decreasing the linear interaction energy between the RBD and ACE2. Our results demonstrate how mutations found within B.1.1.7 enlarge the flexibility around the fusion peptide and change the RBD-ACE2 interface, which, in combination, might explain the higher infectivity of B.1.1.7. We anticipate our findings to be starting points for in depth biochemical and cell biological analyses of B.1.1.7, but also other highly contagious SARS-CoV-2 variants, as many of them likewise exhibit a combination of the D614G and N501Y mutation.

biophysics↗

bioRxiv2021

GID E3 ligase supramolecular chelate assembly configures multipronged ubiquitin targeting of an oligomeric metabolic enzyme

To achieve precise cellular regulation, E3 ubiquitin ligases must be configured to match substrate quaternary structures. Here, by studying the yeast GID complex, mutation of which is Glucose-Induced Degradation deficient, we discover supramolecular chelate assembly as an E3 ligase strategy for targeting an oligomeric substrate. Cryo EM structures show that to bind the tetrameric substrate fructose-1,6-bisphosphatase (Fbp1), two otherwise functional GID E3s assemble into a 20-protein Chelator-GIDSR4, which resembles an organometallic supramolecular chelate. The Chelator-GIDSR4 assembly avidly binds multiple Fbp1 degrons and positions Fbp1 so that its protomers are simultaneously ubiquitylated at lysines near its allosteric and substrate binding sites. Significantly, key structural and biochemical features -including capacity for supramolecular assembly - are preserved in the human ortholog, the CTLH E3. Based on our integrative structural, biochemical and cell biological data, we propose that higher-order E3 ligase assembly generally underlies multipronged targeting, capable of simultaneously incapacitating multiple protomers and functionalities of oligomeric substrates.

biochemistry↗

bioRxiv2021

The Centrosomal Swiss Army Knife: A combined in silico and in vivo approach to the structure-function annotation of SPD-2 provides mechanistic insight into its functional diversity.

Centrosomes are organelles that function as hubs of microtubule nucleation and organization, with key roles in organelle positioning, asymmetric cell division, and ciliogenesis. Aberrant centrosome structure or function is linked to neurodegenerative diseases, developmental abnormalities, ciliopathies, and tumor development. A major regulator of centrosome biogenesis and function in C. elegans is the highly conserved protein Spindle-defective protein 2 (SPD-2), a homolog of the human CEP-192 protein. CeSPD-2 is required for centrosome maturation, centriole duplication, spindle assembly and cell polarity establishment. Despite its importance, the specific molecular mechanism of CeSPD-2 function is poorly understood. To address this gap in knowledge, we combined computational analysis with cell biology approaches to uncover structure-function relationships of CeSPD-2 that may shed mechanistic light on its function. Domain prediction analysis corroborated and refined previously identified coiled-coils and ASH (Aspm-SPD-2 Hydin) domains and identified new domains and motifs: an additional coiled-coil, a GEF domain, an Ig-like domain, and a PDZ-like domain. Our findings suggest that ASH domain belongs to the same superfold as PapD chaperone domains and Major Sperm Protein (MSP) domains within the larger Immunoglobulin superfamily. We have identified a large novel basic region in the CeSPD-2 ASH domain that harbors most of the predicted protein and nucleic acid contact residues in the ASH domain. In vivo, ASH::GFP localized to centrosomes and centrosome associated microtubules, and forms aggregates in the cytosol when overexpressed. This study lays the groundwork for designing rational hypothesis-based experiments for future analyses to further elaborate the mechanisms of CeSPD-2 function in vivo.

bioinformatics↗

bioRxiv2021

Systematic morphological and morphometric analysis of identified olfactory receptor neurons in Drosophila melanogaster

The biophysical properties of sensory neurons are influenced by their morphometric and morphological features, whose precise measurements require high-quality volume electron microscopy (EM). However, systematic surveys of these nanoscale characteristics for identified neurons are scarce. Here, we characterize the morphology of Drosophila olfactory receptor neurons (ORNs) across the majority of genetically identified sensory hairs. By analyzing serial block-face electron microscopy (SBEM) images of cryofixed antennal tissues, we compile an extensive morphometric dataset based on 122 reconstructed 3D models of 33 identified ORN types. In addition, we observe multiple novel features--including extracellular vacuoles within sensillum lumen, intricate dendritic branching, mitochondria enrichment in select ORNs, novel sensillum types, and empty sensilla containing no neurons--which raise new questions pertinent to cell biology and sensory neurobiology. Our systematic survey is critical for future investigations into how the size and shape of sensory neurons influence their responses, sensitivity and circuit function.

neuroscience↗

bioRxiv2021

Endoplasmic reticulum-targeting but not translation is required for mRNA balancing in trypanosomes

The cell surface of bloodstream form African trypanosomes is covered by a dense coat of immunogenic variant surface glycoproteins (VSGs). By continuously changing the expressed VSG antigen, the parasites can survive the hosts immune response. The VSG is highly expressed in Trypanosoma brucei, accounting for approximately 10 - 20% of total mRNA. Depletion of VSG mRNA is lethal, and a counterbalancing of the mRNA levels occurs when two or more VSGs are simultaneously expressed. How the VSG expression levels are regulated is unknown. Here, by using inducible and constitutive systems for ectopic VSG expression, we have discovered that (i) the endogenous VSG mRNA level is downregulated only when the ectopic VSG is targeted to the ER, (ii) VSG translation is dispensable and in fact, (iii) the regulation of VSG mRNA levels does not depend on a VSG open reading frame. We propose that feedback elicited at the ER regulates the VSG mRNA amounts to avoid overshooting the secretory pathway capacity. In this way, VSG expression is quantitatively and qualitatively fine-tuned. Balancing the overall number of ER-targeted mRNAs could well be a general mechanism in cell biology. The trypanosome system with just one dominant mRNA species provides a versatile model for studying this phenomenon.

microbiology↗

bioRxiv2021

Fusion of single-cell transcriptome and DNA-binding data, for genomic network inference in cortical development

BackgroundNetwork models are well-established as very useful computational-statistical tools in cell biology. However, a genomic network model based only on gene expression data can, by definition, only infer gene co-expression networks. Hence, in order to infer gene regulatory patterns, it is necessary to also include data related to binding of regulatory factors to DNA. ResultsWe propose a new dynamic genomic network model, for inferring patterns of genomic regulatory influence in dynamic processes such as development. Our model fuses experiment-specific gene expression data with publicly available DNA-binding data. The method we propose is computationally efficient, and can be applied to genome-wide data with tens of thousands of transcripts. Thus, our method is well suited for use as an exploratory tool for genome-wide data. We apply our method to data from human fetal cortical development, and our findings confirm genomic regulatory patterns which are recognised as being fundamental to neuronal development. ConclusionsOur method provides a mathematical/computational toolbox which, when coupled with targeted experiments, will reveal and confirm important new functional genomic regulatory processes in mammalian development.

bioinformatics↗

bioRxiv2021

Rhizobiales commensal bacteria promote Arabidopsis thaliana root growth via host sulfated peptide pathway

Root-associated commensal bacteria that belong to the order Rhizobiales, which also contains symbiotic and pathogenic bacteria, promote primary root growth of Arabidopsis thaliana. However, the molecular mechanism underlying this root growth promotion (RGP) activity remained unclear. Here, we conducted a transcriptomic analysis of A. thaliana roots inoculated with root-associated commensal bacteria of Rhizobiales and sister lineages and revealed common and strain/lineage-specific transcriptional response, possibly mediated by WRKY and ANAC family of transcription factors. We showed that the observed common response was also partly triggered by a wide range of non-pathogenic bacteria, fungi, and a multikingdom synthetic community (SynCom). This response was characterized by a down-regulation of genes related to intracellular redox regulation, suggesting distinctive redox status between pathogenic and non-pathogenic interactions. By integrating with developmental and cell biological experiments, we identified a crucial role of TYROSYLPROTEIN SULFOTRANSFERASE (TPST) in Rhizobiales RGP. Conversely, none of the known TPST-dependent sulfated peptide pathways appeared to be required for this activity, suggesting an unidentified component in the protein sulfation pathway targeted by Rhizobiales RGP. Finally, we show that TPST is needed for RGP exerted by Rhizobiales but not Pseudomonadales isolates, delineating lineage-specific mechanisms to manipulate host root development.

plant biology↗

bioRxiv2021

Quantifying spinal cord vascular permeability in the mouse using intravital imaging

Sensory perception and motor dexterity is coordinated by in part distinct anatomical centres in the spinal cord. Importantly the spinal cord is the first modulatory relay hub for coordinating sensory and motor inputs to allow control of an organisms response to a sensory experience and to orientate proprioceptive outputs. This is whilst communicating with higher centres within the brain to undertake greater complex neurophysiological function such as pain perception. This begins to outline the complexity of the nervous system communication. To allow this integral system to function efficiently neuronal homeostasis needs to be maintained with energy expenditure matched by proficient delivery of nutrients. This factor introduces the vascular system that extensively interacts in a multifaceted manner with differing aspects of the nervous system. Part of this multi-factoral interaction is through the heterogenic cellular makeup of the vascular network that delivers and modulates the molecular transport of such nutrients to spinal cord tissues, but also controlling penetration and migration of harmful pathogens and agents. Therefore the spinal cord is susceptible to any alterations in the microvessel integrity (e.g. vascular leakage) and/or function (e.g. cessated blood flow) of this vascular network, which principally occurs in times of pathology. Typically investigations into microvessel function have utilised histological and/or tracer based in-vivo assays. Methodologies such as evans blue extravasation have been used inconjunction with in-vitro cell biology assays such as transwell assays to determine microvessel integrity or function that only provides snapshots of developing vasculopathy. Adopting in-vivo imaging approaches, allow for real time functional measurements of the ongoing physiological function within the spinal cord, providing direct measurement of the vascular processes in play, including vascular architecture, blood flow and/or permeability. This technique in mouse allow for direct visualisation of cellular and/or mechanistic influence upon vascular function through utilising disease, transgenic and/or viral approaches. This combination of attributes allows for in depth real time understanding of the function of the vascular network within the spinal cord.

neuroscience↗

bioRxiv2021

MRP5 and MRP9 Play a Concerted Role in Male Reproduction and Mitochondrial Function

Multidrug Resistance Proteins (MRPs) are transporters that play critical roles in cancer even though the physiological substrates of these enigmatic transporters are poorly elucidated. In Caenorhabditis elegans, MRP5/ABCC5 is an essential heme exporter as mrp-5 mutants are unviable due to their inability to export heme from the intestine to extra-intestinal tissues. Heme supplementation restores viability of these mutants but fails to restore male reproductive deficits. Correspondingly, cell biological studies show that MRP5 regulates heme levels in the mammalian secretory pathway even though MRP5 knockout (KO) mice do not show reproductive phenotypes. The closest homolog of MRP5 is MRP9/ABCC12, which is absent in C. elegans raising the possibility that MRP9 may genetically compensate for MRP5. Here, we show that MRP5 and MRP9 double KO mice are viable but reveal significant male reproductive deficits. Although MRP9 is highly expressed in sperm, MRP9 KO mice show reproductive phenotypes only when MRP5 is absent. Both ABCC transporters localize to mitochondrial-associated membranes (MAMs), dynamic scaffolds that associate the mitochondria and endoplasmic reticulum. Consequently, DKO mice reveal abnormal sperm mitochondria with reduced mitochondrial membrane potential and fertilization rates. Metabolomics show striking differences in metabolite profiles in the DKO testes and RNA-seq show significant alterations in genes related to mitochondrial function and retinoic acid metabolism. Targeted functional metabolomics reveal lower retinoic acid levels in the DKO testes and higher levels of triglycerides in the mitochondria. These findings establish a model in which MRP5 and MRP9 play a concerted role in regulating male reproductive functions and mitochondrial sufficiency. Significance StatementMRPs are typically implicated in cancer biology. Here, we show that MRP9 and MRP5 localize to mitochondrial-associated membranes and play a concerted role in maintaining mitochondrial homeostasis and male reproductive fitness. Our work fills in significant gaps in our understanding of MRP9 and MRR5 with wider implications in male fertility. It is plausible that variants in these transporters are associated with male reproductive dysfunction.

genetics↗

bioRxiv2021

PlotXpress, a webtool for normalization and visualization of reporter expression data

In molecular cell biology, reporter assays are frequently used to investigate gene expression levels. Reporter assays employ a gene that encodes a light-emitting protein, of which the luminescence is quantified as a proxy of gene expression. Commercial parties provide reporter assay kits that include protocols and specialized detection machinery. However, downstream analysis of the output data and their presentation are not standardized. We have developed plotXpress to fill this gap, providing a free, open-source platform for the semi-automated analysis and standardized visualisation of experimental gene reporter data. Users can upload raw luminescence data acquired from a reporter gene assay with an internal control. In plotXpress, the data is corrected for sample variation with the internal control and the average for each condition is calculated. When a reference condition is selected the fold change is calculated for all other conditions, based on the selected reference. The results are shown as dot plots with a statistical summary, which can be adjusted to create publication-grade plots without requiring coding skills. Altogether, plotXpress is an open-source, low-threshold, web-based tool, that promotes a standardized and reproducible analysis while providing an appealing visualization of reporter data. The webtool can be accessed at: https://huygens.science.uva.nl/PlotXpress/ O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/451595v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1b499aborg.highwire.dtl.DTLVardef@1d4d375org.highwire.dtl.DTLVardef@186bac1org.highwire.dtl.DTLVardef@4ed7bc_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2021

Knock-in tagging in zebrafish facilitated by insertion into non-coding regions

Zebrafish provide an excellent model for in vivo cell biology studies due to their amenability to live imaging. Protein visualization in zebrafish has traditionally relied on overexpression of fluorescently tagged proteins from heterologous promoters, making it difficult to recapitulate endogenous expression patterns and protein function. One way to circumvent this problem is to tag the proteins by modifying their endogenous genomic loci. Such an approach is not widely available to zebrafish researchers due to inefficient homologous recombination and the error-prone nature of targeted integration in zebrafish. Here, we report a simple approach for tagging proteins in zebrafish on their N- or C termini with fluorescent markers by inserting PCR-generated donor amplicons into non-coding regions of the corresponding genes. Using this approach, we generated endogenously tagged alleles for several genes critical for epithelial biology and organ development including the tight junction components ZO-1 and Cldn15la, the trafficking effector Rab11a, and the ECM receptor {beta}1 integrin. Our approach facilitates the generation of knock-in lines in zebrafish, opening the way for accurate quantitative imaging studies. Summary statementGeneration of endogenously tagged stable zebrafish knock-in lines is simplified by the integration of fluorescent protein cassettes with mRNA splicing elements into non-coding regions of genes.

developmental biology↗

bioRxiv2021

Infinite re-reading of single proteins at single-amino-acid resolution using nanopore sequencing

As identifying proteins is of paramount importance for cell biology and applications, it is of interest to develop a protein sequencer with the ultimate sensitivity of decoding individual proteins. Here, we demonstrate a nanopore-based single-molecule sequencing approach capable of reliably detecting single amino-acid substitutions within individual peptides. A peptide is linked to a DNA molecule that is pulled through the biological nanopore MspA by a DNA helicase in single amino-acid steps. The peptide sequence yields clear stepping ion current signals which allows to discriminate single-amino-acid substitutions in single reads. Molecular dynamics simulations show these signals to result from size exclusion and pore binding. Notably, we demonstrate the capability to rewind peptide reads, obtaining indefinitely many independent reads of the same individual molecule, yielding virtually 100% read accuracy in variant identification, with an error rate less than 10-6. These proof-of-concept experiments constitute a promising basis for developing a single-molecule protein sequencer. One-sentence summaryThis paper presents proof-of-concept experiments and simulations of a nanopore-based approach to sequencing individual proteins.

biophysics↗

bioRxiv2021

Cross-species incompatibility between a DNA satellite and a chromatin protein poisons germline genome integrity

Satellite DNA spans megabases of eukaryotic sequence and evolves rapidly. Paradoxically, satellite-rich genomic regions mediate strictly conserved, essential processes like chromosome segregation and nuclear structure. A leading resolution to this paradox posits that satellite DNA and satellite-associated chromosomal proteins coevolve to preserve these essential functions. We experimentally test this model of intra-genomic coevolution by conducting the first evolution-guided manipulation of both chromosomal protein and DNA satellite. The 359bp satellite spans an 11Mb array in D. melanogaster that is absent from its sister species, D. simulans. This species-specific DNA satellite colocalizes with the adaptively evolving, ovary-enriched protein, Maternal Haploid (MH)-the Drosophila homolog of Spartan. To determine if MH and 359 coevolve, we swapped the D. simulans version of MH ("MH[sim]") into D. melanogaster. MH[sim] triggers ovarian cell death, reduced ovary size, and loss of mature eggs. Surprisingly, the D. melanogaster mh null mutant has no such ovary phenotypes, suggesting that MH[sim] is toxic in a D. melanogaster background. Using both cell biology and genetics, we discovered that MH[sim] poisons oogenesis through a DNA damage pathway. Remarkably, deleting the D. melanogaster-specific 359 satellite array completely restores mh[sim] germline genome integrity and fertility, consistent with a history of coevolution between these two fast-evolving loci. Germline genome integrity and fertility are also restored by overexpressing Topoisomerase II (Top2), suggesting that MH[sim] interferes with Top2-mediated processing of 359. The observed 359-MH[sim] cross-species incompatibility supports a model under which ostensibly inert repetitive DNA and essential chromosomal proteins must coevolve to preserve germline genome integrity.

evolutionary biology↗

bioRxiv2021

2020 BioImage Analysis Survey: Community experiences and needs for the future

In this paper, we summarize a global survey of 484 participants of the imaging community, conducted in 2020 through the NIH Center for Open BioImage Analysis (COBA). This 23-question survey covered experience with image analysis, scientific background and demographics, and views and requests from different members of the imaging community. Through open-ended questions we asked the community to provide feedback for the open-source tool developers and tool user groups. The communitys requests for tool developers include general improvement of tool documentation and easy-to-follow tutorials. Respondents encourage tool users to follow the best practices guidelines for imaging and ask their image analysis questions on the Scientific Community Image forum (forum.image.sc). We analyzed the communitys preferred method of learning, based on level of computational proficiency and work description. In general, written step-by-step and video tutorials are preferred methods of learning by the community, followed by interactive webinars and office hours with an expert. There is also enthusiasm for a centralized location online for existing educational resources. The survey results will help the community, especially developers, trainers, and organizations like COBA, decide how to structure and prioritize their efforts. Impact statementThe Bioimage analysis community consists of software developers, imaging experts, and users, all with different expertise, scientific background, and computational skill levels. The NIH funded Center for Open Bioimage Analysis (COBA) was launched in 2020 to serve the cell biology communitys growing need for sophisticated open-source software and workflows for light microscopy image analysis. This paper shares the result of a COBA survey to assess the most urgent ongoing needs for software and training in the community and provide a helpful resource for software developers working in this domain. Here, we describe the state of open-source bioimage analysis, developers and users requests from the community, and our resulting view of common goals that would serve and strengthen the community to advance imaging science.

bioinformatics↗

bioRxiv2021

Development of a universal nanobody-binding Fab module for fiducial-assisted cryo- EM studies of membrane proteins

With conformation-specific nanobodies being used for a wide range of structural, biochemical, and cell biological applications, there is a demand for antigen-binding fragments (Fabs) that specifically and tightly bind these nanobodies without disturbing the nanobody-target protein interaction. Here we describe the development of a synthetic Fab (termed NabFab) that binds the scaffold of an alpaca-derived nanobody with picomolar affinity. We demonstrate that upon CDR grafting onto this parent nanobody scaffold, nanobodies recognizing diverse target proteins and derived from llama or camel can cross-react with NabFab without loss of affinity. Using NabFab as a fiducial and size enhancer (50 kDa), we determined the high-resolution cryo-EM structures of nanobody-bound VcNorM and ScaDMT, both small membrane proteins of ~50 kDa. Using an additional anti-Fab nanobody further facillitated reliable initial 3D structure determination from small cryo-EM test datasets. Given that NabFab is of synthetic origin, humanized, and can be conveniently expressed in E. coli in large amounts, it may not only be useful for structural biology, but also for biomedical applications.

molecular biology↗

bioRxiv2021

Characterization of USH1C/harmonin in the human retina provides insights into pathophysiology and therapy options for Usher syndrome

Usher syndrome (USH) is the most common form of hereditary deafness-blindness in humans. USH is a complex genetic disorder, assigned to three clinical subtypes differing in onset, course, and severity, with USH1 being the most severe. Rodent USH1 models do not reflect the ocular phenotype observed in human patients to date; hence, little is known about the pathophysiology of USH1 in the human eye. One of the USH1 genes, USH1C, exhibits extensive alternative splicing and encodes numerous harmonin protein isoforms that function as scaffolds for organizing the USH interactome. RNA-seq analysis of human retinas uncovered harmonin_a1 as the most abundant transcript of USH1C. Bulk RNA-seq analysis and immunoblotting showed abundant expression of harmonin in Muller glia cells (MGCs) and retinal neurons. Furthermore, harmonin was localized in the terminal endfeet and apical microvilli of MGCs, presynaptic region (pedicle) of cones, and outer segments of rods as well as at adhesive junctions of MGCs and photoreceptors in the outer limiting membrane (OLM). Our data provide evidence for the interactions of harmonin with OLM molecules in photoreceptors (PRCs) and MGCs and rhodopsin in PRCs. Subcellular expression and colocalization of harmonin correlate with the clinical phenotype observed in USH1C patients. In addition, primary cilia defects in USH1C patient-derived fibroblasts could be reverted by the delivery of harmonin_a1 transcript isoform. Our data provide novel insights into PRC cell biology, USH1C pathophysiology, and for developing gene therapy treatment.

molecular biology↗

bioRxiv2021

LectinOracle - A Generalizable Deep Learning Model for Lectin-Glycan Binding Prediction

Ranging from bacterial cell adhesion over viral cell entry to human innate immunity, glycan-binding proteins or lectins abound in nature. Widely used as staining and characterization reagents in cell biology, and crucial for understanding the interactions in biological systems, lectins are a focal point of study in glycobiology. Yet the sheer breadth and depth of specificity for diverse oligosaccharide motifs has made studying lectins a largely piecemeal approach, with few options to generalize. Here, we present LectinOracle, a model combining transformer-based representations for proteins and graph convolutional neural networks for glycans to predict their interaction. Using a curated dataset of 564,647 unique protein-glycan interactions, we show that LectinOracle predictions agree with literature-annotated specificities for a wide range of lectins. We further identify clusters of lectins with related binding specificity that are not clustered based on sequence similarity. Using a range of specialized glycan arrays, we show that LectinOracle predictions generalize to new glycans and lectins, with qualitative and quantitative agreement with experimental data. We further demonstrate that LectinOracle can analyze whole lectomes and their role in host-microbe interactions. We envision that the herein presented platform will advance both the study of lectins and their role in (glyco)biology.

bioinformatics↗

bioRxiv2021

Pathogenic, but not non-pathogenic, Rickettsia manipulate inflammasome-dependent IL-1 responses to facilitate their replication and host dissemination.

Rickettsia species (spp.) are strict obligate intracellular bacteria, with some being pathogenic in their mammalian host, including humans. One critical feature of these stealthy group of pathogens is their ability to manipulate hostile cytosolic environments to their benefits. Although our understanding of Rickettsia cell biology and pathogenesis are evolving, the mechanisms by which pathogenic Rickettsia spp. evade host innate immune detection remains elusive. Here, we showed that disease severity in wild-type (WT) C57BL/6J mice infected with R. typhi (etiologic agent of murine typhus) and R. rickettsii (etiologic agent of Rocky Mountain Spotted Fever), but not with non-pathogenic R. montanensis, correlated with levels of bacterial burden as detected in the spleens, as well as the serum concentrations of pro-inflammatory cytokine IL-1 and to a lesser extent IL- 1{beta}. Antibody-mediated neutralization of IL-1 confirmed a key role in controlling mortality rates and bacterial burdens of rickettsiae-infected WT mice. As macrophages are a primary source of both IL-1 and IL-1{beta} cytokines, we determined the mechanism of the anti-rickettsial activities using bone-marrow-derived macrophages. We found that pathogenic R. typhi and R. rickettsii, but not non-pathogenic R. montanensis, eluded pro- IL-1 induction and benefited pre-dominantly from the reduced IL-1 secretion, via a Caspase-11-Gsdmd-dependent pathway, to facilitate intracytosolic replication. Adoptative transfer experiments identified that IL-1 secretion by macrophages was critical for controlling rickettsiosis in WT mice. In sum, we identified a previously unappreciated pathway by which pathogenic, unlike non-pathogenic, rickettsiae preferentially target the Caspase-11-Gsdmd-IL-1 signaling axis in macrophages thus supporting their replication within the host. IMPORTANCECurrently, no vaccines are available to prevent rickettsioses, while vector-borne rickettsial infections in humans are on the rise globally. In fact, the insufficient understanding of how pathogenic Rickettsia species circumvent host immune defense mechanisms has significantly hindered the development of more effective therapeutics. Here, we identified a previously unappreciated role for the Caspase-11-Gsdmd-IL-1 signaling axis, to limiting the replication of pathogenic R. rickettsia and R. typhi species in murine macrophages and wild-type (WT) C57BL/6J mice. Adoptative transfer studies further identified IL-1-secreting macrophages as critical mediators in controlling rickettsial infection in WT mice. Collectively, these findings provide insight into the potential mechanism of how pathogenic, but not non-pathogenic Rickettsia spp., benefit from a reduction in the Caspase-11-Gsdmd-mediated release of IL-1 to support host colonization.

microbiology↗