The hidden base of the iceberg: gut peptidoglycome dynamics is foundational to its influence on the host.

The intestinal microbiota of humans includes a highly diverse range of bacterial species. All these bacteria possess a cell wall, composed primarily of the macromolecule peptidoglycan. As such, the gut also harbors an abundant and varied peptidoglycome. A remarkable range of host physiological pathways are regulated by peptidoglycan fragments that originate from the gut microbiota and enter the host system. Interactions between the host system and peptidoglycan can influence physiological development and homeostasis, promote health, or contribute to inflammatory disease. Underlying these effects is the interplay between microbiota composition and enzymatic processes that shape the intestinal peptidoglycome, dictating the types of peptidoglycan generated, that subsequently cross the gut barrier. In this review, we highlight and discuss the hidden and emerging functional aspects of the microbiome, i.e. the hidden base of the iceberg, that modulate the composition of gut peptidoglycan, and how these fundamental processes are drivers of physiological outcomes for the host.

Intraflagellar transport speed is sensitive to genetic and mechanical perturbations to flagellar beating.

Two sets of motor proteins underpin motile cilia/flagella function. The axoneme-associated inner and outer dynein arms drive sliding of adjacent axoneme microtubule doublets to periodically bend the flagellum for beating, while intraflagellar transport (IFT) kinesins and dyneins carry IFT trains bidirectionally along the axoneme. Despite assembling motile cilia and flagella, IFT train speeds have only previously been quantified in immobilized flagella-mechanical immobilization or genetic paralysis. This has limited investigation of the interaction between IFT and flagellar beating. Here, in uniflagellate Leishmania parasites, we use high-frequency, dual-color fluorescence microscopy to visualize IFT train movement in beating flagella. We discovered that adhesion of flagella to a microscope slide is detrimental, reducing IFT train speed and increasing train stalling. In flagella free to move, IFT train speed is not strongly dependent on flagella beat type; however, permanent disruption of flagella beating by deletion of genes necessary for formation or regulation of beating showed an inverse correlation of beat frequency and IFT train speed.

Identification of 30 transition fibre proteins in Trypanosoma brucei reveals a complex and dynamic structure.

Transition fibres and distal appendages surround the distal end of mature basal bodies and are essential for ciliogenesis, but only a few of the proteins involved have been identified and functionally characterised. Here, through genome-wide analysis, we have identified 30 transition fibre proteins (TFPs) and mapped their arrangement in the flagellated eukaryote Trypanosoma brucei. We discovered that TFPs are recruited to the mature basal body before and after basal body duplication, with differential expression of five TFPs observed at the assembling new flagellum compared to the existing fixed-length old flagellum. RNAi-mediated depletion of 17 TFPs revealed six TFPs that are necessary for ciliogenesis and a further three TFPs that are necessary for normal flagellum length. We identified nine TFPs that had a detectable orthologue in at least one basal body-forming eukaryotic organism outside of the kinetoplastid parasites. Our work has tripled the number of known transition fibre components, demonstrating that transition fibres are complex and dynamic in their composition throughout the cell cycle, which relates to their essential roles in ciliogenesis and flagellum length regulation.

High-throughput prioritization of target proteins for development of new antileishmanial compounds.

Leishmaniasis, a vector-borne disease, is caused by the infection of Leishmania spp., obligate intracellular protozoan parasites. Presently, human vaccines are unavailable, and the primary treatment relies heavily on systemic drugs, often presenting with suboptimal formulations and substantial toxicity, making new drugs a high priority for LMIC countries burdened by the disease, but a low priority in the agenda of most pharmaceutical companies due to unattractive profit margins. New ways to accelerate the discovery of new, or the repositioning of existing drugs, are needed. To address this challenge, our study aimed to identify potential protein targets shared among clinically-relevant Leishmania species. We employed a subtractive proteomics and comparative genomics approach, integrating high-throughput multi-omics data to classify these targets based on different druggability metrics. This effort resulted in the ranking of 6502 ortholog groups of protein targets across 14 pathogenic Leishmania species. Among the top 20 highly ranked groups, metabolic processes known to be attractive drug targets, including the ubiquitination pathway, aminoacyl-tRNA synthetases, and purine synthesis, were rediscovered. Additionally, we unveiled novel promising targets such as the nicotinate phosphoribosyltransferase enzyme and dihydrolipoamide succinyltransferases. These groups exhibited appealing druggability features, including less than 40% sequence identity to the human host proteome, predicted essentiality, structural classification as highly druggable or druggable, and expression levels above the 50th percentile in the amastigote form. The resources presented in this work also represent a comprehensive collection of integrated data regarding trypanosomatid biology.

Synthesis and Evaluation of (Bis)benzyltetrahydroisoquinoline Alkaloids as Antiparasitic Agents.

Visceral leishmaniasis and Chagas disease are neglected tropical diseases (NTDs) that severely impact the developing world. With current therapies suffering from poor efficacy and safety profiles as well as emerging resistance, new drug leads are direly needed. In this work, 26 alkaloids (9 natural and 17 synthetic) belonging to the benzyltetrahydroisoquinoline (BI) family were evaluated against both the pro/trypomastigote and amastigote forms of the parasites Leishmania infantum and Trypanosoma cruzi, the causative agents of these diseases. These alkaloids were synthesized via an efficient and modular enantioselective approach based on Bischler-Napieralski cyclization/Noyori asymmetric transfer hydrogenation to build the tetrahydroisoquinoline core. The bis-benzyltetrahydroisoquinoline (BBI) alkaloids were prepared using an Ullmann coupling of two BI units to form the biaryl ether linkage, which enabled a comprehensive survey of the influence of BI stereochemistry on bioactivity. Preliminary studies into the mechanism of action against Leishmania mexicana demonstrate that these compounds interfere with the cell cycle, potentially through inhibition of kinetoplast division, which may offer opportunities to identify a new target/mechanism of action. Three of the synthesized alkaloids showed promising druglike potential, meeting the Drugs for Neglected Disease initiative (DNDi) criteria for a hit against Chagas disease.

Whole cell reconstructions of Leishmania mexicana through the cell cycle.

The unicellular parasite Leishmania has a precisely defined cell architecture that is inherited by each subsequent generation, requiring a highly coordinated pattern of duplication and segregation of organelles and cytoskeletal structures. A framework of nuclear division and morphological changes is known from light microscopy, yet this has limited resolution and the intrinsic organisation of organelles within the cell body and their manner of duplication and inheritance is unknown. Using volume electron microscopy approaches, we have produced three-dimensional reconstructions of different promastigote cell cycle stages to give a spatial and quantitative overview of organelle positioning, division and inheritance. The first morphological indications seen in our dataset that a new cell cycle had begun were the assembly of a new flagellum, the duplication of the contractile vacuole and the increase in volume of the nucleus and kinetoplast. We showed that the progression of the cytokinesis furrow created a specific pattern of membrane indentations, while our analysis of sub-pellicular microtubule organisation indicated that there is likely a preferred site of new microtubule insertion. The daughter cells retained these indentations in their cell body for a period post-abscission. By comparing cultured and sand fly derived promastigotes, we found an increase in the number and overall volume of lipid droplets in the promastigotes from the sand fly, reflecting a change in their metabolism to ensure transmissibility to the mammalian host. Our insights into the cell cycle mechanics of Leishmania will support future molecular cell biology analyses of these parasites.

Comprehensive sub-mitochondrial protein map of the parasitic protist Trypanosoma brucei defines critical features of organellar biology.

We have generated a high-confidence mitochondrial proteome (MitoTag) of the Trypanosoma brucei procyclic stage containing 1,239 proteins. For 337 of these, a mitochondrial localization had not been described before. We use the TrypTag dataset as a foundation and take advantage of the properties of the fluorescent protein tag that causes aberrant but fortuitous accumulation of tagged matrix and inner membrane proteins near the kinetoplast (mitochondrial DNA). Combined with transmembrane domain predictions, this characteristic allowed categorization of 1,053 proteins into mitochondrial sub-compartments, the detection of unique matrix-localized fucose and methionine synthesis, and the identification of new kinetoplast proteins, which showed kinetoplast-linked pyrimidine synthesis. Moreover, disruption of targeting signals by tagging allowed mapping of the mode of protein targeting to these sub-compartments, identifying a set of C-tail anchored outer mitochondrial membrane proteins and mitochondrial carriers likely employing multiple target peptides. This dataset represents a comprehensive, updated mapping of the mitochondrion.

Activation of Nod2 signaling upon norovirus infection enhances antiviral immunity and susceptibility to colitis.

Over 90% of epidemic non-bacterial gastroenteritis are caused by human noroviruses (NoVs), which persist in a substantial subset of people allowing their spread worldwide. This has led to a significant number of endemic cases and up to 70,000 children deaths in developing countries. NoVs are primarily transmitted through the fecal-oral route. To date, studies have focused on the influence of the gut microbiota on enteric viral clearance by mucosal immunity. In this study, the use of mouse norovirus S99 (MNoV_S99) and CR6 (MNoV_CR6), two persistent strains, allowed us to provide evidence that the norovirus-induced exacerbation of colitis severity relied on bacterial sensing by nucleotide-binding oligomerization domain 2 (Nod2). Consequently, Nod2-deficient mice showed reduced levels of gravity of Dextran sodium sulfate (DSS)-induced colitis with both viral strains. And MNoV_CR6 viremia was heightened in Nod2-/- mice in comparison with animals hypomorphic for Atg16l1, which are prone to aggravated inflammation under DSS. Accordingly, the infection of macrophages derived from WT mice promoted the phosphorylation of Signal Transducer and Activator of Transcription 1 (STAT1) and NOD2's expression levels. Higher secretion of Tumor Necrosis Factor alpha (TNFα) following NOD2 activation and better viral clearance were measured in these cells. By contrast, reduced levels of pSTAT1 and blunted downstream secretion of TNFα were found in Nod2-deficient macrophages infected by MNoV_S99. Hence, our results uncover a previously unidentified virus-host-bacterial interplay that may represent a novel therapeutic target for treating noroviral origin gastroenteritis that may be linked with susceptibility to several common illnesses such as Crohn's disease.

Radial spoke protein 9 is necessary for axoneme assembly in Plasmodium but not in trypanosomatid parasites.

Flagella are important for eukaryote cell motility, including in sperm, and are vital for life cycle progression of many unicellular eukaryotic pathogens. The '9+2' axoneme in most motile flagella comprises nine outer doublet and two central-pair singlet microtubules. T-shaped radial spokes protrude from the outer doublets towards the central pair and are necessary for effective beating. We asked whether there were radial spoke adaptations associated with parasite lineage-specific properties in apicomplexans and trypanosomatids. Following an orthologue search for experimentally uncharacterised radial spoke proteins (RSPs), we identified and analysed RSP9. Trypanosoma brucei and Leishmania mexicana have an extensive RSP complement, including two divergent RSP9 orthologues, necessary for flagellar beating and swimming. Detailed structural analysis showed that neither orthologue is needed for axoneme assembly in Leishmania. In contrast, Plasmodium has a reduced set of RSPs including a single RSP9 orthologue, deletion of which in Plasmodium berghei leads to failure of axoneme formation, failed male gamete release, greatly reduced fertilisation and inefficient life cycle progression in the mosquito. This indicates contrasting selection pressures on axoneme complexity, likely linked to the different mode of assembly of trypanosomatid versus Plasmodium flagella.

Cell envelope growth of Gram-negative bacteria proceeds independently of cell wall synthesis.

All bacterial cells must expand their envelopes during growth. The main load-bearing and shape-determining component of the bacterial envelope is the peptidoglycan cell wall. Bacterial envelope growth and shape changes are often thought to be controlled through enzymatic cell wall insertion. We investigated the role of cell wall insertion for cell shape changes during cell elongation in Gram-negative bacteria. We found that both global and local rates of envelope growth of Escherichia coli remain nearly unperturbed upon arrest of cell wall insertion-up to the point of sudden cell lysis. Specifically, cells continue to expand their surface areas in proportion to biomass growth rate, even if the rate of mass growth changes. Other Gram-negative bacteria behave similarly. Furthermore, cells plastically change cell shape in response to differential mechanical forces. Overall, we conclude that cell wall-cleaving enzymes can control envelope growth independently of synthesis. Accordingly, the strong overexpression of an endopeptidase leads to transiently accelerated bacterial cell elongation. Our study demonstrates that biomass growth and envelope forces can guide cell envelope expansion through mechanisms that are independent of cell wall insertion.

Subcellular protein localisation of Trypanosoma brucei bloodstream form-upregulated proteins maps stage-specific adaptations.

Background: Genome-wide subcellular protein localisation in Trypanosoma brucei, through our TrypTag project, has comprehensively dissected the molecular organisation of this important pathogen. Powerful as this resource is , T. brucei has multiple developmental forms and we previously only analysed the procyclic form. This is an insect life cycle stage, leaving the mammalian bloodstream form unanalysed. The expectation is that between life stages protein localisation would not change dramatically (completely unchanged or shifting to analogous stage-specific structures). However, this has not been specifically tested. Similarly, which organelles tend to contain proteins with stage-specific expression can be predicted from known stage specific adaptations but has not been comprehensively tested. Methods: We used endogenous tagging with mNG to determine the sub-cellular localisation of the majority of proteins encoded by transcripts significantly upregulated in the bloodstream form, and performed comparison to the existing localisation data in procyclic forms. Results: We have confirmed the localisation of known stage-specific proteins and identified the localisation of novel stage-specific proteins. This gave a map of which organelles tend to contain stage specific proteins: the mitochondrion for the procyclic form, and the endoplasmic reticulum, endocytic system and cell surface in the bloodstream form. Conclusions: This represents the first genome-wide map of life cycle stage-specific adaptation of organelle molecular machinery in T. brucei.

TrypTag.org: from images to discoveries using genome-wide protein localisation in Trypanosoma brucei.

TrypTag was a 4-year project to tag the N- and C-termini of almost all Trypanosoma brucei proteins with a fluorescent protein and record the subcellular localisation through images and manual annotation. We highlight the new routes to cell biological discovery this transformative resource is enabling for parasitologists and cell biologists.

Genome-wide subcellular protein map for the flagellate parasite Trypanosoma brucei.

Trypanosoma brucei is a model trypanosomatid, an important group of human, animal and plant unicellular parasites. Understanding their complex cell architecture and life cycle is challenging because, as with most eukaryotic microbes, ~50% of genome-encoded proteins have completely unknown functions. Here, using fluorescence microscopy and cell lines expressing endogenously tagged proteins, we mapped the subcellular localization of 89% of the T. brucei proteome, a resource we call TrypTag. We provide clues to function and define lineage-specific organelle adaptations for parasitism, mapping the ultraconserved cellular architecture of eukaryotes, including the first comprehensive 'cartographic' analysis of the eukaryotic flagellum, which is vital for morphogenesis and pathology. To demonstrate the power of this resource, we identify novel organelle subdomains and changes in molecular composition through the cell cycle. TrypTag is a transformative resource, important for hypothesis generation for both eukaryotic evolutionary molecular cell biology and fundamental parasite cell biology.

Microbiota-induced active translocation of peptidoglycan across the intestinal barrier dictates its within-host dissemination.

Peptidoglycan, the major structural polymer forming the cell wall of bacteria, is an important mediator of physiological and behavioral effects in mammalian hosts. These effects are frequently linked to its translocation from the intestinal lumen to host tissues. However, the modality and regulation of this translocation across the gut barrier has not been precisely addressed. In this study, we characterized the absorption of peptidoglycan across the intestine and its systemic dissemination. We report that peptidoglycan has a distinct tropism for host organs when absorbed via the gut, most notably by favoring access to the brain. We demonstrate that intestinal translocation of peptidoglycan occurs through a microbiota-induced active process. This process is regulated by the parasympathetic pathway via the muscarinic acetylcholine receptors. Together, this study reveals fundamental parameters concerning the uptake of a major microbiota molecular signal from the steady-state gut.

Nucleolar targeting in an early-branching eukaryote suggests a general mechanism for ribosome protein sorting.

The compartmentalised eukaryotic cell demands accurate targeting of proteins to the organelles in which they function, whether membrane-bound (like the nucleus) or non-membrane-bound (like the nucleolus). Nucleolar targeting relies on positively charged localisation signals and has received rejuvenated interest since the widespread recognition of liquid-liquid phase separation (LLPS) as a mechanism contributing to nucleolus formation. Here, we exploit a new genome-wide analysis of protein localisation in the early-branching eukaryote Trypanosoma brucei to analyse general nucleolar protein properties. T. brucei nucleolar proteins have similar properties to those in common model eukaryotes, specifically basic amino acids. Using protein truncations and addition of candidate targeting sequences to proteins, we show both homopolymer runs and distributed basic amino acids give nucleolar partition, further aided by a nuclear localisation signal (NLS). These findings are consistent with phase separation models of nucleolar formation and physical protein properties being a major contributing mechanism for eukaryotic nucleolar targeting, conserved from the last eukaryotic common ancestor. Importantly, cytoplasmic ribosome proteins, unlike mitochondrial ribosome proteins, have more basic residues - pointing to adaptation of physicochemical properties to assist segregation.

Cellular electron tomography of the apical complex in the apicomplexan parasite Eimeria tenella shows a highly organised gateway for regulated secretion.

The apical complex of apicomplexan parasites is essential for host cell invasion and intracellular survival and as the site of regulated exocytosis from specialised secretory organelles called rhoptries and micronemes. Despite its importance, there are few data on the three-dimensional organisation and quantification of these organelles within the apical complex or how they are trafficked to this specialised region of plasma membrane for exocytosis. In coccidian apicomplexans there is an additional tubulin-containing hollow barrel structure, the conoid, which provides a structural gateway for this specialised apical secretion. Using a combination of cellular electron tomography and serial block face-scanning electron microscopy (SBF-SEM) we have reconstructed the entire apical end of Eimeria tenella sporozoites; we report a detailed dissection of the three- dimensional organisation of the conoid and show there is high curvature of the tubulin-containing fibres that might be linked to the unusual comma-shaped arrangement of protofilaments. We quantified the number and location of rhoptries and micronemes within cells and show a highly organised gateway for trafficking and docking of rhoptries, micronemes and microtubule-associated vesicles within the conoid around a set of intra-conoidal microtubules. Finally, we provide ultrastructural evidence for fusion of rhoptries directly through the parasite plasma membrane early in infection and the presence of a pore in the parasitophorous vacuole membrane, providing a structural explanation for how rhoptry proteins may be trafficked between the parasite and the host cytoplasm.

Stage-specific transcription activator ESB1 regulates monoallelic antigen expression in Trypanosoma brucei.

Variant surface glycoprotein (VSG) coats bloodstream form Trypanosoma brucei parasites, and monoallelic VSG expression underpins the antigenic variation necessary for pathogenicity. One of thousands of VSG genes is transcribed by RNA polymerase I in a singular nuclear structure called the expression site body (ESB), but how monoallelic VSG transcription is achieved remains unclear. Using a localization screen of 153 proteins we found one, ESB-specific protein 1 (ESB1), that localized only to the ESB and is expressed only in VSG-expressing life cycle stages. ESB1 associates with DNA near the active VSG promoter and is necessary for VSG expression, with overexpression activating inactive VSG promoters. Mechanistically, ESB1 is necessary for recruitment of a subset of ESB components, including RNA polymerase I, revealing that the ESB has separately assembled subdomains. Because many trypanosomatid parasites have divergent ESB1 orthologues yet do not undergo antigenic variation, ESB1 probably represents an important class of transcription regulators.

Bacterial sensing via neuronal Nod2 regulates appetite and body temperature.

Gut bacteria influence brain functions and metabolism. We investigated whether this influence can be mediated by direct sensing of bacterial cell wall components by brain neurons. In mice, we found that bacterial peptidoglycan plays a major role in mediating gut-brain communication via the Nod2 receptor. Peptidoglycan-derived muropeptides reach the brain and alter the activity of a subset of brain neurons that express Nod2. Activation of Nod2 in hypothalamic inhibitory neurons is essential for proper appetite and body temperature control, primarily in females. This study identifies a microbe-sensing mechanism that regulates feeding behavior and host metabolism.

Genome sequence of Leishmania mexicana MNYC/BZ/62/M379 expressing Cas9 and T7 RNA polymerase.

We present the genome sequence of Leishmania mexicana MNYC/BZ/62/M379 modified to express Cas9 and T7 RNA-polymerase, revealing high similarity to the reference genome (MHOM/GT2001/U1103). Through RNAseq-based annotation of coding sequences and untranslated regions, we provide primer sequences for construct and sgRNA template generation for CRISPR-assisted gene deletion and endogenous tagging.