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Horn, M.

Publications and source records attributed to Horn, M..

At least 19 recordsLinked to original sources
bioRxiv2026

Mimicin, an antimicrobial protein encoded by mimivirus

Antimicrobial peptides (AMPs) are innate defense molecules found in all domains of life. Giant viruses of amoeba are known to thrive among complex microbial relationships within its hosts cells, hinting at the existence of virus-derived antimicrobial strategies. Here we show that viruses belonging to the Mimiviridae and Marseilleviridae families contain a higher density of in silico predicted AMPs per genome size than other viruses of amoeba. The investigation of potential AMPs led to the description of Mimicin, a taxonomically restricted 74 amino acid long protein coded by few mimiviruses. Mimicin contains three smaller predicted AMP sequences within it and has a broad in vitro antimicrobial activity against different bacteria, a yeast and two non-enveloped phages. In contrast, it has no activity against a marseillevirus, a mimivirus or human cell lines. When tested against bacterial endosymbionts co-cultured with Acanthamoeba terricola, Mimicin and its SIM-31 portion were able to control the attenuated Protochlamydia amoebophila but not the highly virulent Parachlamydia acanthamoebae. Based on deposited transcriptomic data, Mimicin is coded by an early gene more active during the beginning of the infection process. No structure could be predicted using Alphafold, while additional structural analysis indicate that Mimicin could be a highly disordered protein. In conclusion, we describe evidence of a biologically relevant antimicrobial activity derived from a giant virus. Mimicin highlights the relevance of AMPs from giant viruses for microbial ecology and opens the way for investigating their biotechnological and clinical potential.

microbiology↗
bioRxiv2026

Identification of MED13 and DDX60 as critical host factors for SARS-CoV-2 infections

The virus responsible for COVID-19, SARS-CoV-2, continues to spread through the world. The ongoing emergence of new variants with increased viral transmission and immune evasion continue to pose a challenge. Although large-scale genetic screens have identified numerous host factors required for SARS-CoV-2 infection, however the function of these hits remain incompletely understood. In this study, we performed a haploid forward genetic screen in chemically mutagenized mouse embryonic stem cells overexpressing human ACE2 and identified MED13 and DDX60 as important host factors involved in the modulation of SARS-CoV-2 infection. In this study, we have identified and characterized the function of these key element factors for SARS-CoV2 infection. Knockdown of CKM subunits--with the exception of CDK8--or the helicase DDX60 was sufficient to reduce SARS-CoV-2 infection across Vero E6, A549, and Calu-3 cells. During SARS-CoV-2 infection, MED13 was found to function downstream of the JAK/STAT interferon pathway, but also showed another function independently of the interferon response pathway. Surprisingly, while DDX60 is traditionally involved in the interferon response pathway, its knockdown reduces SARS-CoV-2 infection, suggesting DDX60 can promote SARS-CoV-2 infection Interestingly, while inactivation of MED13 or DDX60 markedly reduced SARS-CoV-2 and SARS-CoV infection, it did not affect MERS-CoV. Collectively, these results identify MED13 and DDX60 as critical host determinants for SARS-CoV-2 related-coronaviruses with pandemic potential. Impact statementUsing an unbiased haploid genetic screening approach, this study identifies for the first time MED13 and DDX60 as host factors that influence coronavirus replication through transcriptional and interferon-associated pathways. While DDX60 has previously been linked to antiviral signaling, our findings suggest it can promote coronavirus infection. Most interestingly, the effects of these factors extend beyond SARS-CoV-2 to other coronaviruses. These findings broaden the current understanding of coronavirus-host interaction by highlighting transcriptional control and interferon-associated pathways as modulators of infection rather than focusing solely on viral entry. The work provides mechanistic insight into how host regulatory networks influence coronavirus replication and suggests potential targets for host-directed therapies with activity against multiple present and future coronavirus threats. Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. RepositoriesThe data generated in this study are provided in the Supplementary Information/Source Data file. Sequencing data are available on NCBI Sequence Read Archive under the accession number BioProject PRJNA1271794. The next generation sequencing data generated in this study has been deposited in the NCBI Sequence Read Archive (SRA) under accession number SRX29485992, SRX29485993, SRX29485994, SRX29485995, SRX29485996, SRX29485997, SRX29485998, SRX29485999, SRX29486000, SRX29486001, SRX29486002, SRX29486003, SRX29486004, SRX29486005, SRX29486006, SRX29486007, SRX29486010, SRX29486011, SRX29486012, SRX29486013, SRX29486014, SRX29486015, SRX29486016, SRX29486017, SRX29486018, SRX29486019, SRX29486021, SRX29486022, SRX29486023, SRX29486024

microbiology↗
bioRxiv2025

THC Reverses SIV-Induced Senescence in Astrocytes: Possible Compensatory Mechanism Against HIV Associated Brain Injury?

Despite effective combination antiretroviral therapy (cART), chronic neuroinflammation and glial dysfunction continues to be an important yet understudied issue with people living with HIV (PLWH). The endocannabinoid system is increasingly recognized as a potential therapeutic target for modulating neuroimmune environments, given its role in regulating synaptic plasticity, immune responses, and neuroinflammatory cascades. However, the extent to which cannabinoids influence HIV-associated neuroinflammation remains unclear. This study investigates the impact of {Delta}9-tetrahydrocannabinol (THC) on astrocyte growth characteristics, viability, and senescence-associated cytokine release following exposure to HIV Tat protein using primary mixed glial cultures derived from rhesus macaques. Real-time impedance-based cellular integrity assessments were conducted using the xCELLigence system, while morphological analyses and cytokine quantification were performed using phase-contrast microscopy and multiplex immunoassays. Treatment of macaques with THC protected the astrocytes from virus-induced senescence. Further, THC facilitated a rapid recovery from Tat-induced decline in astrocyte adhesion, suggesting a compensatory effect. THC promoted glial process elongation and morphological complexity, indicative of a shift toward a neuroprotective phenotype. Furthermore, THC significantly reduced inflammatory cytokine secretion, including TNF-, IL-6, and IL-1{beta}, in an apparently dose-dependent manner. These findings suggest that THC may modulate neuroinflammation in PLWH by promoting astrocytic survival, suppressing inflammatory cytokine secretion, and enhancing neurotrophic signaling. However, prolonged exposure to high-dose THC may negatively impact glial survival. The results underscore the complexity of cannabinoid signaling in the CNS and highlight the potential of cannabinoid-based interventions to mitigate HIV-associated neuroinflammation.

neuroscience↗
bioRxiv2025

Giant virus transcription and translation occur at well-defined locations within amoeba host cells

Many giant viruses replicate in the cytoplasm in so-called viral factories. How exactly these viral factories are established and where the different steps of the replication cycle occur remains largely obscure. We have developed a single-molecule messenger RNA fluorescence in situ hybridisation (smFISH) protocol for giant viruses in an Acanthamoeba host. Combined with other labelling techniques (FUNCAT, DiD, rRNA FISH, DAPI), we show the Mimivirus transcription and translation sites during an infection cycle in the amoeba host cell. While viral mRNA localisation changes depending on the infection stage, transcription occurs at well-defined spots within the viral factory. The original viral cores released within the cytoplasm most likely define these spots. When transported outside of the viral factory, the translation of viral mRNA takes place in a well-defined ring surrounding it. With this study, we obtained novel insights into giant virus replication, of which the methods are widely applicable to other viruses for the visualisation and quantification of RNA molecules.

microbiology↗
bioRxiv2024

The adaptation of chlamydiae to facultative host multicellularity

The phylum Chlamydiota consists of obligate intracellular bacteria comprising the human pathogen Chlamydia trachomatis and a large variety of species infecting animals and protists. Despite their enormous diversity, a feature shared by all known chlamydiae is their biphasic developmental cycle, consisting of intra- and extracellular stages with substantial differences in morphology and physiology. A similarly remarkable shift occurs in the amoeba Dictyostelium discoideum and related dictyostelids in their so-called social life cycle, leading to the formation of spores through aggregation of vegetative trophozoites and the development of multicellular fruiting bodies. Although dictyostelids undergo symbioses with various bacteria, chlamydiae have only recently been found to be associated with this host. Here we report the isolation of a Dictyostelium giganteum strain naturally infected with a chlamydial symbiont, identified as a novel species, Reclusachlamydia socialis. The symbiont is retained in all stages of the hosts social life cycle and notably lacks an extracellular form. Combining fluorescence microscopy and quantitative PCR we showed that transmission is entirely dependent on cell-to-cell contact during the host aggregation stage. The absence of an extracellular stage is further supported by transmission electron microscopy and the lack of genes essential for chlamydial developmental cycle regulation and extracellular survival. This unprecedented variation of a highly conserved developmental feature that evolved more than a billion years ago illustrates the remarkable adaptability of chlamydiae. This study adds to our understanding of endosymbiosis in the face of facultative multicellularity.

microbiology↗
bioRxiv2024

Novel high-quality amoeba genomes reveal widespread codon usage mismatch between giant viruses and their hosts

The need for high-quality protist genomes has prevented in-depth computational and experimental studies of giant virus-host interactions. In addition, our current knowledge of host range is highly biased due to the few hosts used to isolate novel giant viruses. This study presents six high-quality amoeba genomes from known and potential giant virus hosts belonging to two distinct eukaryotic clades: Amoebozoa and Discoba. We employ their genomic data to investigate the predictability of giant virus host range. Using a combination of long and short-read sequencing, we obtained highly contiguous and complete genomes of Acanthamoeba castellanii, Acanthamoeba griffini, Acanthamoeba terricola, Naegleria clarki, Vermamoeba vermiformis, and Willaertia magna, contributing to the collection of sequences for the eukaryotic tree of life. We found that the six amoebae have distinct codon usage patterns and that, contrary to other virus groups, giant viruses often have different and even opposite codon usage with their known hosts. Conversely, giant viruses with matching codon usage are frequently not known to infect or replicate in these hosts. Interestingly, analyses of integrated viral sequences in the amoeba host genomes reveal potential novel virus-host associations. Matching of codon usage preferences is often used to predict virus-host pairs. However, with the broad-scale analyses performed in this study, we demonstrate that codon usage alone appears to be a poor predictor of giant virus host range. We discuss the potential strategies that giant viruses employ to ensure high viral fitness in non-matching hosts. Moreover, this study emphasises the need for more high-quality protist genomes. Finally, the amoeba genomes presented in this study set the stage for future experimental studies to better understand how giant viruses interact with different host species.

evolutionary biology↗
bioRxiv2024

Pythia: Non-random DNA repair allows predictable CRISPR/Cas9 integration and gene editing

CRISPR-based genome engineering holds enormous promise for basic science and therapeutic applications. Integrating and editing DNA sequences is still challenging in many cellular contexts, largely due to insufficient control of the repair process. We find that repair at the genome-cargo interface is predictable by deep-learning models and adheres to sequence context specific rules. Based on in silico predictions, we devised a strategy of triplet base-pair repeat repair arms that correspond to microhomologies at double-strand breaks (trimologies), which facilitated integration of large cargo (>2 kb) and protected the targeted locus and transgene from excessive damage. Successful integrations occurred in >30 loci in human cells and in in vivo models. Germline transmissible transgene integration in Xenopus, and endogenous tagging of tubulin in adult mice brains demonstrated integration during early embryonic cleavage and in non-dividing differentiated cells. Further, optimal repair arms for single- or double nucleotide edits were predictable, and facilitated small edits in vitro and in vivo using oligonucleotide templates. We provide a design-tool (Pythia, pythia-editing.org) to optimize custom integration, tagging or editing strategies. Pythia will facilitate genomic integration and editing for experimental and therapeutic purposes for a wider range of target cell types and applications.

molecular biology↗
bioRxiv2024

Deep learning uncovers sequence-specific amplification bias in multi-template PCR

Multi-template polymerase chain reaction is a key step in many amplicon sequencing protocols enabling parallel amplification of diverse DNA molecules sharing common adapters in applications, ranging as wide as quantitative molecular biology and DNA data storage. However, this process results in a skewed amplicon abundance, due to sequence-specific amplification biases. In this study, one-dimensional convolutional neural networks (1D-CNNs) were trained on synthetic DNA pools to learn the PCR amplification efficiency of individual templates. These 1D-CNN models can predict poorly amplifying templates based solely on sequence information, achieving an AUROC/AUPRC of up to 0.88/0.44 with very imbalanced prevalence of 2%, thereby greatly outperforming baseline models relying only on GC content and nucleotide frequency as predictors. A new, general-purpose framework for interpreting deep learning models, termed CluMo provides mechanistic insights into the amplification biases. Most strikingly, specific amplification reactions were identified as suffering from adaptor-template self-priming a mechanism previously disregarded in PCR.

molecular biology↗
bioRxiv2023

Chlamydiae as symbionts of photosynthetic dinoflagellates

Chlamydiae are ubiquitous intracellular bacteria and infect a wide diversity of eukaryotes, including mammals. However, chlamydiae have never been reported to infect photosynthetic organisms. Here, we describe a novel chlamydial genus and species, Candidatus Algichlamydia australiensis (A. australiensis thereafter), capable of infecting the photosynthetic dinoflagellate Cladocopium sp. (originally isolated from a scleractinian coral). A. australiensis was confirmed to be intracellular by fluorescence in situ hybridization and confocal laser scanning microscopy, and temporally stable at the population level by monitoring its relative abundance across four weeks of host growth. Using a combination of short- and long-read sequencing, we recovered a high-quality (completeness 91.73% and contamination 0.27%) metagenome-assembled genome of A. australiensis. Phylogenetic analyses show that this chlamydial taxon represents a new genus and species within the Simkaniaceae family. A. australiensis possesses all the hallmark genes for chlamydiae-host interactions, including a complete type III secretion system. In addition, a type IV secretion system is encoded on a plasmid and has previously been observed for only three other chlamydial species. Twenty orthologous groups of genes are unique to A. australiensis, one of which is structurally similar to a protein known from Cyanobacteria and Archaeplastida involved in thylakoid biogenesis and maintenance, hinting at potential chlamydiae interactions with the chloroplasts of Cladocopium cells. Despite Cladocopium being itself a symbiont of cnidarians, a meta-analysis of 12,009 cnidarian 16S rRNA gene metabarcoding samples only returned five samples with A. australiensis sequences, suggesting A. australiensis does not associate with cnidarians. Our study shows that chlamydiae infect dinoflagellate symbionts of cnidarians, the first photosynthetic organism reported to harbor chlamydiae, thereby expanding the breadth of chlamydial hosts and providing a new contribution to the discussion around the role of chlamydiae in the establishment of the primary plastid.

microbiology↗
bioRxiv2023

Chlamydiae in cnidarians: Shared functional potential despite broad taxonomic diversity

Cnidarians, such as corals and sea anemones, associate with a wide range of bacteria that have essential functions, including nutrient cycling and the production of antimicrobial compounds. Within cnidarians, bacteria can colonize all microhabitats including the tissues. Among them are obligate intracellular bacteria of the phylum Chlamydiota (chlamydiae) whose impact on cnidarian hosts and holobionts remain unknown. Here, we conducted a meta-analysis of previously published cnidarian 16S rRNA gene metabarcoding data and eight metagenome-assembled genomes (MAGs) of cnidarian-associated chlamydiae to decipher their diversity and functional potential. While the metabarcoding dataset showed an enormous diversity of cnidarian-associated chlamydiae, five out of eight MAGs were affiliated with the Simkaniaceae family. The other three MAGs were assigned to the Parasimkaniaceae, Rhabdochlamydiaceae, and Anoxychlamydiaceae, respectively. All MAGs were associated with corals and showed a functional potential insufficient for an independent existence, lacking any nucleotide or vitamin and most amino acid biosynthesis pathways. Hallmark chlamydial genes, such as a type III secretion system, nucleotide transporters, and genes for host interaction, were encoded in all MAGs. Together these observations suggest an obligate intracellular lifestyle of cnidarian-associated chlamydiae. Cnidarian-associated chlamydiae lacked unique genes, suggesting the core chlamydial genetic arsenal may be flexible enough to infect many eukaryotic hosts, including cnidarians. Additional studies are needed to understand how chlamydiae interact with their cnidarian host, and other microbes in cnidarian holobionts. This first study of the diversity and functional potential of cnidarian-associated chlamydiae improves our understanding of both the cnidarian microbiome and the chlamydial lifestyle and host range.

microbiology↗
bioRxiv2023

Loss of Nkd1 is dominant over loss of Axin2 in regulating Wnt signaling

Wnt signaling is a crucial developmental pathway involved in early development as well as stem cell maintenance in adults and its misregulation leads to numerous diseases. Thus, understanding the regulation of this pathway becomes vitally important. Axin2 and Nkd1 are widely utilized negative feedback regulators in Wnt signaling where Axin2 functions to destabilize cytoplasmic {beta}-catenin, and Nkd1 functions to inhibit the nuclear localization of {beta}-catenin. Here, we set out to further understand how Axin2 and Nkd1 regulate Wnt signaling by creating axin2-/-, nkd1-/- single mutants and axin2-/-;nkd1-/- double mutant zebrafish using sgRNA/Cas9. All three Wnt regulator mutants were viable and had impaired heart looping, neuromast migration defects, and behavior abnormalities in common, but there were no signs of synergy in the axin2-/-;nkd1-/- double mutants. Further, Wnt target gene expression by qRT-PCR, and RNA-seq analysis and protein expression by mass spectrometry demonstrated that the double axin2-/-;nkd1-/- mutant resembled the nkd1-/- phenotype demonstrating that Axin2 functions upstream of Nkd1 and that loss of Nkd1 is epistatic to the loss of Axin2. In support of this, the data further demonstrates that Axin2 uniquely alters the properties of {beta}-catenin-dependent transcription having novel readouts of Wnt activity compared to nkd1-/- or the axin2-/-;nkd1-/- double mutant. We also tested the sensitivity of the Wnt regulator mutants to exacerbated Wnt signaling, where the single mutants displayed characteristic heightened Wnt sensitivity, resulting in an eyeless phenotype. Surprisingly, this phenotype was rescued in the double mutant, where we speculate that cross-talk between Wnt/{beta}-catenin and Wnt/Planar Cell Polarity pathways could lead to altered Wnt signaling in some scenarios. Collectively, the data emphasizes both the commonality and the complexity in the feedback regulation of Wnt signaling.

developmental biology↗
bioRxiv2023

A microfluidic model of colonocyte-microbiota interaction mimicking the colorectal cancer microenvironment

Changes in the abundance of certain bacterial species within the colorectal microbiota correlate with colorectal cancer development. While carcinogenic mechanisms of single pathogenic bacteria have been characterized in vitro, limited tools are available to investigate interactions between pathogenic bacteria and both commensal microbiota and colonocytes in a physiologically relevant tumor microenvironment. To address this, we developed a microfluidic device that can be used to co-culture colonocytes and colorectal microbiota. The device was used to explore the effect of Fusobacterium nucleatum, an opportunistic pathogen associated with colorectal cancer development in humans, on colonocyte gene expression and microbiota composition. F. nucleatum altered the transcription of genes involved in cytokine production, epithelial-to-mesenchymal transition, and proliferation in colonocytes in a contact-independent manner; however, most of these effects were diminished by the presence of fecal microbiota. Interestingly, F. nucleatum significantly altered the abundance of multiple bacterial clades associated with mucosal immune responses and cancer development in the colon. Our results highlight the importance of evaluating the potential carcinogenic activity of pathogens in the context of a commensal microbiota, and the potential to discover novel inter-species microbial interactions in the colorectal cancer microenvironment.

bioengineering↗
bioRxiv2022

Endozoicomonas-chlamydiae interactions in cell-associated microbial aggregates of the coral Pocillopora acuta

Corals are associated with a variety of bacteria, which occur in the surface mucus layer, gastrovascular cavity, skeleton, and tissues. Some tissue-associated bacteria form clusters, termed cell-associated microbial aggregates (CAMAs), which are poorly studied. Here, we provide a comprehensive characterization of CAMAs in the coral Pocillopora acuta. Combining imaging techniques, laser capture microdissection, and amplicon and metagenome sequencing we show that CAMAs: (i) are located in the tentacle tips and may be intracellular; (ii) contain Endozoicomonas, Kistimonas (both Gammaproteobacteria), and Simkania (Chlamydiota) bacteria; (iii) Endozoicomonas may provide vitamins to its host and use secretion systems and/or pili for colonization and aggregation; (iv) Endozoicomonas and Simkania occur in distinct, but adjacent, CAMAs; (v) Simkania may rely on acetate and heme provided by neighboring Endozoicomonas. Our study provides detailed insight into coral endosymbionts, which will guide the assessment of their suitability for probiotic approaches to mitigate coral bleaching.

microbiology↗
bioRxiv2022

A novel widespread MITE element in the repeat-rich genome of the Cardinium endosymbiont of the spider Oedothorax gibbosus

Free-living bacteria have evolved multiple times to become host-restricted endosymbionts. The transition from a free-living to a host-restricted lifestyle comes with a number of different genomic changes, including a massive loss of genes. In host-restricted endosymbionts, gene inactivation and genome reduction is facilitated by mobile genetic elements, mainly insertion sequences (ISs). ISs are small autonomous mobile elements, and one of, if not the most, abundant transposable elements in bacteria. Proliferation of ISs is common in some facultative endosymbionts, and is likely driven by the transmission bottlenecks, which increase the level of genetic drift. In the current study we present a manually curated genome annotation for a Cardinium endosymbiont of the dwarf spider Oedothorax gibbosus. Cardinium species are host-restricted endosymbionts that, similarly to Wolbachia spp., include strains capable of manipulating host reproduction. Through the focus on mobile elements, the annotation revealed a rampant spread of ISs, extending earlier observations in other Cardinium genomes. We found that a large proportion of IS elements are actually pseudogenised, with many displaying evidence of recent inactivation. Most notably, we describe the lineage-specific emergence and spread of a novel IS-derived Miniature Inverted repeat Transposable Element (MITE), likely being actively maintained by intact copies of its parental IS982-family element. This work highlights the relevance of manual curation of these repeat-rich endosymbiont genomes for the discovery of novel MITEs, as well as the possible role these understudied elements might play in genome streamlining.

genomics↗
bioRxiv2022

One to host them all: genomics of the diverse bacterial endosymbionts of the spider Oedothorax gibbosus

Bacterial endosymbionts of the groups Wolbachia, Cardinium and Rickettsiaceae are well-known for their diverse effects on their arthropod hosts, ranging from mutualistic relationships to reproductive phenotypes. Here, we analyzed a unique system in which the dwarf spider Oedothorax gibbosus is co-infected with up to five different endosymbionts affiliated with Wolbachia, Candidatus Tisiphia (formerly Torix group Rickettsia), Cardinium, and Rhabdochlamydia. Using short-read genome sequencing data, we show that the endosymbionts are heterogeneously distributed among O. gibbosus populations and are frequently found co-infecting spider individuals. To study this intricate host-endosymbiont system on a genome resolved level, we used long-read sequencing to reconstruct closed genomes of the Wolbachia, Ca. Tisiphia and Cardinium endosymbionts. We provide insights in the ecology and evolution of the endosymbionts and shed light on the interactions with their spider host. We detected high quantities of transposable elements in all endosymbiont genomes and provide evidence that ancestors of the Cardinium, Ca. Tisiphia and Wolbachia endosymbionts have co-infected the same hosts in the past. Our findings contribute to broadening our knowledge about endosymbionts infecting one of the largest animal phyla on earth and show the usefulness of transposable elements as an evolutionary "contact-tracing" tool. Data summaryAll supporting data, code and protocols have been provided within the article or through supplementary data files. Seven supplementary figures and seven supplementary tables are available with the online version of this article. Sequencing data used in this study was generated and previously published by Hendrickx et al., 2021. Genome assemblies generated in this study have been deposited under the project PRJEB52003 at DDBJ/ENA/GenBank. The MAG of R. oedothoracis OV001 was deposited at DDBJ/ENA/GenBank under the sample SAMN28026840. The genome of Candidatus Rhabdochlamydia oedothoracis W744x776 was previously published by Halter et al., 2022 and is available at DDBJ/ENA/GenBank (accession: CP075587-CP075588). The collection of genomes and proteomes, all files for phylogenetic analyses including gene alignments, concatenated alignments, and tree files, and original output files of the HGT and SNP predictions used in this study are available at zenodo (https://doi.org/10.5281/zenodo.6362846).

genomics↗
bioRxiv2022

Ecology and evolution of chlamydial symbionts of arthropods

The phylum Chlamydiae consists of obligate intracellular bacteria including major human pathogens and diverse environmental representatives. Here we investigated the Rhabdochlamydiaceae, which is predicted to be the largest and most diverse chlamydial family, with the few described members known to infect arthropod hosts. Using published 16S rRNA gene sequence data we identified at least 388 genus-level lineages containing about 14 051 putative species within this family. We show that rhabdochlamydiae are mainly found in freshwater and soil environments, suggesting the existence of diverse, yet unknown hosts. Next, we used a comprehensive genome dataset including metagenome assembled genomes classified as members of the family Rhabdochlamydiaceae, and we added novel complete genome sequences of Rhabdochlamydia porcellionis infecting the woodlouse Porcellio scaber, and of Candidatus R. oedothoracis associated with the linyphiid dwarf spider Oedothorax gibbosus. Comparative analysis of basic genome features and gene content with reference genomes of well-studied chlamydial families with known host ranges, namely Parachlamydiaceae (protist hosts) and Chlamydiaceae (human and other vertebrate hosts) suggested distinct niches for members of the Rhabdochlamydiaceae. We propose that members of the family represent intermediate stages of adaptation of chlamydiae from protists to vertebrate hosts. Within the genus Rhabdochlamydia, pronounced genome size reduction could be observed (1.49-1.93 Mb). The abundance and genomic distribution of transposases suggests transposable element expansion and subsequent gene inactivation as a mechanism of genome streamlining during adaptation to new hosts. This type of genome reduction has never been described before for any member of the phylum Chlamydiae. This study provides new insights into the molecular ecology, genomic diversity, and evolution of representatives of one of the most divergent chlamydial families.

genomics↗
bioRxiv2021

GFAT2 and AMDHD2 act in tandem to control the hexosamine biosynthetic pathway

The hexosamine biosynthetic pathway (HBP) produces the essential metabolite UDP-GlcNAc and plays a key role in metabolism, cancer, and aging. The HBP is controlled by its rate-limiting enzyme glutamine fructose-6-phosphate amidotransferase (GFAT) that is directly inhibited by UDP-GlcNAc in a feedback loop. HBP regulation by GFAT is well studied but other HBP regulators have remained obscure. Elevated UDP-GlcNAc levels counteract the glycosylation toxin tunicamycin (TM) and thus we screened for TM resistance in haploid mouse embryonic stem cells (mESCs) using random chemical mutagenesis to pinpoint new HBP regulators. We identified the N-acetylglucosamine deacetylase AMDHD2 that catalyzes a reverse reaction in the HBP and its loss strongly elevated UDP-GlcNAc. To better understand AMDHD2, we solved the crystal structure and found that loss-of-function is caused by protein destabilization or interference with its catalytic activity. Finally, we show that mESCs express AMDHD2 together with GFAT2 instead of the more common paralog GFAT1. Compared with GFAT1, GFAT2 had a much lower sensitivity to UDP-GlcNAc inhibition, explaining how AMDHD2 loss-of-function resulted in HBP activation. This HBP configuration in which AMDHD2 serves to balance GFAT2 activity was also observed in other mESCs and, consistently, the GFAT2/GFAT1 ratio decreased with differentiation of mouse and human embryonic stem cells. Together, our data reveal a critical function of AMDHD2 in limiting UDP-GlcNAc production in cells that use GFAT2 for metabolite entry into the HBP.

biochemistry↗