Severe Immune-Related Enteritis after In Utero Exposure to Pembrolizumab.

Immune checkpoint blockade has become standard treatment for many types of cancer. Such therapy is indicated most often in patients with advanced or metastatic disease but has been increasingly used as adjuvant therapy in those with early-stage disease. Adverse events include immune-related organ inflammation resembling autoimmune diseases. We describe a case of severe immune-related gastroenterocolitis in a 4-month-old infant who presented with intractable diarrhea and failure to thrive after in utero exposure to pembrolizumab. Known causes of the symptoms were ruled out, and the diagnosis of pembrolizumab-induced immune-related gastroenterocolitis was supported by the results of histopathological assays, immunophenotyping, and analysis of the level of antibodies against programmed cell death protein 1 (PD-1). The infant's condition was successfully treated with prednisolone and infliximab.

Entropic control of the free-energy landscape of an archetypal biomolecular machine.

Biomolecular machines are complex macromolecular assemblies that utilize thermal and chemical energy to perform essential, multistep, cellular processes. Despite possessing different architectures and functions, an essential feature of the mechanisms of action of all such machines is that they require dynamic rearrangements of structural components. Surprisingly, biomolecular machines generally possess only a limited set of such motions, suggesting that these dynamics must be repurposed to drive different mechanistic steps. Although ligands that interact with these machines are known to drive such repurposing, the physical and structural mechanisms through which ligands achieve this remain unknown. Using temperature-dependent, single-molecule measurements analyzed with a time-resolution-enhancing algorithm, here, we dissect the free-energy landscape of an archetypal biomolecular machine, the bacterial ribosome, to reveal how its dynamics are repurposed to drive distinct steps during ribosome-catalyzed protein synthesis. Specifically, we show that the free-energy landscape of the ribosome encompasses a network of allosterically coupled structural elements that coordinates the motions of these elements. Moreover, we reveal that ribosomal ligands which participate in disparate steps of the protein synthesis pathway repurpose this network by differentially modulating the structural flexibility of the ribosomal complex (i.e., the entropic component of the free-energy landscape). We propose that such ligand-dependent entropic control of free-energy landscapes has evolved as a general strategy through which ligands may regulate the functions of all biomolecular machines. Such entropic control is therefore an important driver in the evolution of naturally occurring biomolecular machines and a critical consideration for the design of synthetic molecular machines.

Structure and dynamics of a processive Brownian motor: the translating ribosome.

There is mounting evidence indicating that protein synthesis is driven and regulated by mechanisms that direct stochastic, large-scale conformational fluctuations of the translational apparatus. This mechanistic paradigm implies that a free-energy landscape governs the conformational states that are accessible to and sampled by the translating ribosome. This scenario presents interdependent opportunities and challenges for structural and dynamic studies of protein synthesis. Indeed, the synergism between cryogenic electron microscopic and X-ray crystallographic structural studies, on the one hand, and single-molecule fluorescence resonance energy transfer (smFRET) dynamic studies, on the other, is emerging as a powerful means for investigating the complex free-energy landscape of the translating ribosome and uncovering the mechanisms that direct the stochastic conformational fluctuations of the translational machinery. In this review, we highlight the principal insights obtained from cryogenic electron microscopic, X-ray crystallographic, and smFRET studies of the elongation stage of protein synthesis and outline the emerging themes, questions, and challenges that lie ahead in mechanistic studies of translation.

Late steps in bacterial translation initiation visualized using time-resolved cryo-EM.

The initiation of bacterial translation involves the tightly regulated joining of the 50S ribosomal subunit to an initiator transfer RNA (fMet-tRNAfMet)-containing 30S ribosomal initiation complex to form a 70S initiation complex, which subsequently matures into a 70S elongation-competent complex. Rapid and accurate formation of the 70S initiation complex is promoted by initiation factors, which must dissociate from the 30S initiation complex before the resulting 70S elongation-competent complex can begin the elongation of translation1. Although comparisons of the structures of the 30S2-5 and 70S4,6-8 initiation complexes have revealed that the ribosome, initiation factors and fMet-tRNAfMet can acquire different conformations in these complexes, the timing of conformational changes during formation of the 70S initiation complex, the structures of any intermediates formed during these rearrangements, and the contributions that these dynamics might make to the mechanism and regulation of initiation remain unknown. Moreover, the absence of a structure of the 70S elongation-competent complex formed via an initiation-factor-catalysed reaction has precluded an understanding of the rearrangements to the ribosome, initiation factors and fMet-tRNAfMet that occur during maturation of a 70S initiation complex into a 70S elongation-competent complex. Here, using time-resolved cryogenic electron microscopy9, we report the near-atomic-resolution view of how a time-ordered series of conformational changes drive and regulate subunit joining, initiation factor dissociation and fMet-tRNAfMet positioning during formation of the 70S elongation-competent complex. Our results demonstrate the power of time-resolved cryogenic electron microscopy to determine how a time-ordered series of conformational changes contribute to the mechanism and regulation of one of the most fundamental processes in biology.

Ratchet, swivel, tilt and roll: a complete description of subunit rotation in the ribosome.

Protein synthesis by the ribosome requires large-scale rearrangements of the 'small' subunit (SSU; ∼1 MDa), including inter- and intra-subunit rotational motions. However, with nearly 2000 structures of ribosomes and ribosomal subunits now publicly available, it is exceedingly difficult to design experiments based on analysis of all known rotation states. To overcome this, we developed an approach where the orientation of each SSU head and body is described in terms of three angular coordinates (rotation, tilt and tilt direction) and a single translation. By considering the entire RCSB PDB database, we describe 1208 fully-assembled ribosome complexes and 334 isolated small subunits, which span >50 species. This reveals aspects of subunit rearrangements that are universal, and others that are organism/domain-specific. For example, we show that tilt-like rearrangements of the SSU body (i.e. 'rolling') are pervasive in both prokaryotic and eukaryotic (cytosolic and mitochondrial) ribosomes. As another example, domain orientations associated with frameshifting in bacteria are similar to those found in eukaryotic ribosomes. Together, this study establishes a common foundation with which structural, simulation, single-molecule and biochemical efforts can more precisely interrogate the dynamics of this prototypical molecular machine.

Increasing the accuracy of single-molecule data analysis using tMAVEN.

Time-dependent single-molecule experiments contain rich kinetic information about the functional dynamics of biomolecules. A key step in extracting this information is the application of kinetic models, such as hidden Markov models (HMMs), which characterize the molecular mechanism governing the experimental system. Unfortunately, researchers rarely know the physicochemical details of this molecular mechanism a priori, which raises questions about how to select the most appropriate kinetic model for a given single-molecule data set and what consequences arise if the wrong model is chosen. To address these questions, we have developed and used time-series modeling, analysis, and visualization environment (tMAVEN), a comprehensive, open-source, and extensible software platform. tMAVEN can perform each step of the single-molecule analysis pipeline, from preprocessing to kinetic modeling to plotting, and has been designed to enable the analysis of a single-molecule data set with multiple types of kinetic models. Using tMAVEN, we have systematically investigated mismatches between kinetic models and molecular mechanisms by analyzing simulated examples of prototypical single-molecule data sets exhibiting common experimental complications, such as molecular heterogeneity, with a series of different types of HMMs. Our results show that no single kinetic modeling strategy is mathematically appropriate for all experimental contexts. Indeed, HMMs only correctly capture the underlying molecular mechanism in the simplest of cases. As such, researchers must modify HMMs using physicochemical principles to avoid the risk of missing the significant biological and biophysical insights into molecular heterogeneity that their experiments provide. By enabling the facile, side-by-side application of multiple types of kinetic models to individual single-molecule data sets, tMAVEN allows researchers to carefully tailor their modeling approach to match the complexity of the underlying biomolecular dynamics and increase the accuracy of their single-molecule data analyses.

Amoebicidal effect of synthetic indoles against Acanthamoeba spp.: a study of cell death.

Organic and synthetic chemistry plays a crucial role in drug discovery fields. Moreover, chemical modifications of available molecules to enhance their efficacy, selectivity and safety have been considered as an attractive approach for the development of new bioactive agents. Indoles, a versatile group of natural heterocyclic compounds, have been widely used in pharmaceutical industry due to their broad spectrum of activities including antimicrobial, antitumoral and anti-inflammatory among others. Herein, we report the amoebicidal activity of different indole analogs on Acanthamoeba castellanii Neff. Among the 40 tested derivatives, eight molecules were able to inhibit this protistan parasite. The structure-activity relationship (SAR) analysis of their anti-Acanthamoeba activity would suggest that a carboxylation of C-3 position and the incorporation of halogen as chlorine/fluorine would enhance their biological profile, presumably by increasing their lipophilicity and therefore their ability to cross the cell membrane. Fluorescence image base system was used to investigate the effect of indole 6o c-6 on the cytoskeleton network and various programmed cell death features. We were able to highlight that the methyl 6-chloro-1H-indole-3-carboxylate could induce program cell death by the mitochondrial dysfunction.

CD7 activation regulates cytotoxicity-driven pathology in systemic sclerosis, yielding a target for selective cell depletion.

OBJECTIVES: Cytotoxic T cells and natural killer (NK) cells are central effector cells in cancer and infections. Their effector response is regulated by activating and inhibitory receptors. The regulation of these cells in systemic autoimmune diseases such as systemic sclerosis (SSc) is less defined. METHODS: We conducted ex vivo analysis of affected skin and blood samples from 4 SSc patient cohorts (a total of 165 SSc vs 80 healthy individuals) using single-cell transcriptomics, flow cytometry and multiplex immunofluorescence staining. We further analysed the effects of costimulatory modulation in functional assays, and in a severely affected SSc patient who was treated on compassionate use with a novel anti-CD3/CD7 immunotoxin treatment. RESULTS: Here, we show that SSc-affected skin contains elevated numbers of proliferating T cells, cytotoxic T cells and NK cells. These cells selectively express the costimulatory molecule CD7 in association with cytotoxic, proinflammatory and profibrotic genes, especially in recent-onset and severe disease. We demonstrate that CD7 regulates the cytolytic activity of T cells and NK cells and that selective depletion of CD7+ cells prevents cytotoxic cell-induced fibroblast contraction and inhibits their profibrotic phenotype. Finally, anti-CD3/CD7 directed depletive treatment eliminated CD7+ skin cells and stabilised disease manifestations in a severely affected SSc patient. CONCLUSION: Together, the findings imply costimulatory molecules as key regulators of cytotoxicity-driven pathology in systemic autoimmune disease, yielding CD7 as a novel target for selective depletion of pathogenic cells.

Characterizing the Conformational Free-Energy Landscape of RNA Stem-Loops Using Single-Molecule Field-Effect Transistors.

We have developed and used single-molecule field-effect transistors (smFETs) to characterize the conformational free-energy landscape of RNA stem-loops. Stem-loops are one of the most common RNA structural motifs and serve as building blocks for the formation of complex RNA structures. Given their prevalence and integral role in RNA folding, the kinetics of stem-loop (un)folding has been extensively characterized using both experimental and computational approaches. Interestingly, these studies have reported vastly disparate timescales of (un)folding, which has been interpreted as evidence that (un)folding of even simple stem-loops occurs on a highly rugged conformational energy landscape. Because smFETs do not rely on fluorophore reporters of conformation or mechanical (un)folding forces, they provide a unique approach that has allowed us to directly monitor tens of thousands of (un)folding events of individual stem-loops at a 200 µs time resolution. Our results show that under our experimental conditions, stem-loops (un)fold over a 1-200 ms timescale during which they transition between ensembles of unfolded and folded conformations, the latter of which is composed of at least two sub-populations. The 1-200 ms timescale of (un)folding we observe here indicates that smFETs report on complete (un)folding trajectories in which unfolded conformations of the RNA spend long periods of time wandering the free-energy landscape before sampling one of several misfolded conformations or the natively folded conformation. Our findings highlight the extremely rugged landscape on which even the simplest RNA structural elements fold and demonstrate that smFETs are a unique and powerful approach for characterizing the conformational free-energy of RNA.

Molecular mechanisms and treatment responses of pulmonary fibrosis in severe COVID-19.

BACKGROUND: Coronavirus disease 2019 (COVID-19) patients can develop pulmonary fibrosis (PF), which is associated with impaired outcome. We assessed specific leukocytic transcriptome profiles associated with PF and the influence of early dexamethasone (DEXA) treatment on the clinical course of PF in critically ill COVID-19 patients. METHODS: We performed a pre-post design study in 191 COVID-19 patients admitted to the Intensive Care Unit (ICU) spanning two treatment cohorts: the pre-DEXA- (n = 67) and the DEXA-cohort (n = 124). PF was identified based on radiological findings, worsening of ventilatory parameters and elevated circulating PIIINP levels. Longitudinal transcriptome profiles of 52 pre-DEXA patients were determined using RNA sequencing. Effects of prednisone treatment on clinical fibrosis parameters and outcomes were analyzed between PF- and no-PF-patients within both cohorts. RESULTS: Transcriptome analyses revealed upregulation of inflammatory, coagulation and neutrophil extracellular trap-related pathways in PF-patients compared to no-PF patients. Key genes involved included PADI4, PDE4D, MMP8, CRISP3, and BCL2L15. Enrichment of several identified pathways was associated with impaired survival in a external cohort of patients with idiopathic pulmonary fibrosis. Following prednisone treatment, PF-related profiles reverted towards those observed in the no-PF-group. Likewise, PIIINP levels decreased significantly following prednisone treatment. PF incidence was 28% and 25% in the pre-DEXA- and DEXA-cohort, respectively (p = 0.61). ICU length-of-stay (pre-DEXA: 42 [29-49] vs. 18 [13-27] days, p < 0.001; DEXA: 42 [28-57] vs. 13 [7-24] days, p < 0.001) and mortality (pre-DEXA: 47% vs. 15%, p = 0.009; DEXA: 61% vs. 19%, p < 0.001) were higher in the PF-groups compared to the no-PF-groups within both cohorts. Early dexamethasone therapy did not influence these outcomes. CONCLUSIONS: ICU patients with COVID-19 who develop PF exhibit upregulated coagulation, inflammation, and neutrophil extracellular trap-related pathways as well as prolonged ICU length-of-stay and mortality. This study indicates that early dexamethasone treatment neither influences the incidence or clinical course of PF, nor clinical outcomes.

Validation of non-invasive point of care blood content analysis using the TensorTip™ MTX device: a method comparison study.

OBJECTIVES: The TensorTip™ MTX is a non-invasive device designed to determine several physiological parameters with additional analysis of haemoglobin, haematocrit and blood gas analysis by interpreting blood diffusion colour of the finger skin based on spectral analysis. The aim of our study was to investigate the accuracy and precision of the TensorTip MTX in a clinical setting in comparison with routine analysis of blood samples. METHODS: Forty-six patients, scheduled for elective surgery, were enrolled in this study. Placement of an arterial catheter had to be part of the standard of care. Measurements were performed during the perioperative period. The measurements obtained with the TensorTip MTX were compared with the results of routine analysis of the blood samples as a reference using correlation, Bland-Altman analysis and mountain plots. RESULTS: No significant correlation was present in the measurements. Measurement of haemoglobin with the TensorTip MTX had a mean bias of 0.4 mmol/L, haematocrit's bias was 3.0 %. Bias of partial pressure of carbon dioxide and oxygen was 3.6 and 66.6 mmHg, respectively. Calculated percentage errors were 48.2 , 48.9, 39.9 and 109.0 %. Proportional bias was present in all Bland-Altman analyses. Less than 95 % of the differences fell within the pre-set limits of allowable error. CONCLUSIONS: Non-invasive blood content analysis with the TensorTip MTX device is not equivalent to and did not correlate sufficiently with conventional laboratory analysis. None of the parameters measured showed results within the limits of allowable error. Therefore, the use of the TensorTip MTX is not recommended for perioperative care.

The alarmones (p)ppGpp directly regulate translation initiation during entry into quiescence.

Many bacteria exist in a state of metabolic quiescence where energy consumption must be minimized so as to maximize available resources over a potentially extended period of time. As protein synthesis is the most energy intensive metabolic process in a bacterial cell, it would be an appropriate target for down-regulation during the transition from growth to quiescence. We observe that when Bacillus subtilis exits rapid growth, a subpopulation of cells emerges with very low protein synthetic activity. This phenotypic heterogeneity requires the production of the nucleotides (p)ppGpp, which we show are sufficient to inhibit protein synthesis in vivo. We then show that one of these molecules, ppGpp, inhibits protein synthesis by preventing the allosteric activation of the essential GTPase Initiation Factor 2 (IF2) during translation initiation. Finally, we demonstrate that the observed attenuation of protein synthesis during the entry into quiescence is a consequence of the direct interaction of (p)ppGpp and IF2.

A promiscuous archaeal cardiolipin synthase enables construction of diverse natural and unnatural phospholipids.

Cardiolipins (CL) are a class of lipids involved in the structural organization of membranes, enzyme functioning, and osmoregulation. Biosynthesis of CLs has been studied in eukaryotes and bacteria, but has been barely explored in archaea. Unlike the common fatty acyl chain-based ester phospholipids, archaeal membranes are made up of the structurally different isoprenoid-based ether phospholipids, possibly involving a different cardiolipin biosynthesis mechanism. Here, we identified a phospholipase D motif-containing cardiolipin synthase (MhCls) from the methanogen Methanospirillum hungatei. The enzyme was overexpressed in Escherichia coli, purified, and its activity was characterized by LC-MS analysis of substrates/products. MhCls utilizes two archaetidylglycerol (AG) molecules in a transesterification reaction to synthesize glycerol-di-archaetidyl-cardiolipin (Gro-DACL) and glycerol. The enzyme is nonselective to the stereochemistry of the glycerol backbone and the nature of the lipid tail, as it also accepts phosphatidylglycerol (PG) to generate glycerol-di-phosphatidyl-cardiolipin (Gro-DPCL). Remarkably, in the presence of AG and PG, MhCls formed glycerol-archaetidyl-phosphatidyl-cardiolipin (Gro-APCL), an archaeal-bacterial hybrid cardiolipin species that so far has not been observed in nature. Due to the reversibility of the transesterification, in the presence of glycerol, Gro-DPCL can be converted back into two PG molecules. In the presence of other compounds that contain primary hydroxyl groups (e.g., alcohols, water, sugars), various natural and unique unnatural phospholipid species could be synthesized, including multiple di-phosphatidyl-cardiolipin species. Moreover, MhCls can utilize a glycolipid in the presence of phosphatidylglycerol to form a glycosyl-mono-phosphatidyl-cardiolipin species, emphasizing the promiscuity of this cardiolipin synthase that could be of interest for bio-catalytic purposes.

Multiplexed genomic encoding of non-canonical amino acids for labeling large complexes.

Stunning advances in the structural biology of multicomponent biomolecular complexes (MBCs) have ushered in an era of intense, structure-guided mechanistic and functional studies of these complexes. Nonetheless, existing methods to site-specifically conjugate MBCs with biochemical and biophysical labels are notoriously impracticable and/or significantly perturb MBC assembly and function. To overcome these limitations, we have developed a general, multiplexed method in which we genomically encode non-canonical amino acids (ncAAs) into multiple, structure-informed, individual sites within a target MBC; select for ncAA-containing MBC variants that assemble and function like the wildtype MBC; and site-specifically conjugate biochemical or biophysical labels to these ncAAs. As a proof-of-principle, we have used this method to generate unique single-molecule fluorescence resonance energy transfer (smFRET) signals reporting on ribosome structural dynamics that have thus far remained inaccessible to smFRET studies of translation.

Synthesis of phosphatidic acids via cobalt(salen) catalyzed epoxide ring-opening with dibenzyl phosphate.

With a CoIII(salen)OTs catalyst, dibenzyl phosphate ring-opens a variety of terminal epoxides with excellent regio-selectively and yields up to 85%. The reaction is used in a highly efficient synthesis of enantiopure mixed-diacyl phosphatidic acids, including a photoswitchable phosphatidic acid mimic.

Pathogenic free-living amoebae from water sources in Cape Verde.

Free-living amoebae (FLA) are protozoa which have been reported in different countries worldwide from diverse sources (water, soil, dust, air), contributing to the environmental microbiological contamination. Most of the FLA species present a life cycle with two different phases: an active vegetative and physiologically form named trophozoite, and an extremely resistant phase called cyst. Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris, Sapinia pedata, Vahlkampfia spp., Paravahlkampfia spp. and Vermamoeba vermiformis have been reported not only as causal agents of several opportunistic diseases including fatal encephalitis or epithelial disorders, but also as capable to favour the intracellular survival of common pathogenic bacteria, which could avoid the typical water disinfection systems, non-effective against FLAs cysts. Even though Santiago Island possesses high levels of humidity compared to the rest of the archipelago of Cape Verde, the water resources are scarce. Therefore, it is important to carry out proper microbiological quality controls, which currently do not contemplate the FLA presence in most of the countries. In the present work, we have reported the presence of Acanthamoeba spp. (69.2%); Vannella spp. (15.4%); Vermamoeba vermiformis (7.7%) and the recently discovered Stenamoeba dejonckheerei (7.7%) in different water sources of Santiago Island.

Dysregulated Innate and Adaptive Immune Responses Discriminate Disease Severity in COVID-19.

The clinical spectrum of COVID-19 varies and the differences in host response characterizing this variation have not been fully elucidated. COVID-19 disease severity correlates with an excessive proinflammatory immune response and profound lymphopenia. Inflammatory responses according to disease severity were explored by plasma cytokine measurements and proteomics analysis in 147 COVID-19 patients. Furthermore, peripheral blood mononuclear cell cytokine production assays and whole blood flow cytometry were performed. Results confirm a hyperinflammatory innate immune state, while highlighting hepatocyte growth factor and stem cell factor as potential biomarkers for disease severity. Clustering analysis revealed no specific inflammatory endotypes in COVID-19 patients. Functional assays revealed abrogated adaptive cytokine production (interferon-γ, interleukin-17, and interleukin-22) and prominent T-cell exhaustion in critically ill patients, whereas innate immune responses were intact or hyperresponsive. Collectively, this extensive analysis provides a comprehensive insight into the pathobiology of severe to critical COVID-19 and highlights potential biomarkers of disease severity.

5'-UTR recruitment of the translation initiation factor eIF4GI or DAP5 drives cap-independent translation of a subset of human mRNAs.

During unfavorable conditions (e.g. tumor hypoxia or viral infection), canonical, cap-dependent mRNA translation is suppressed in human cells. Nonetheless, a subset of physiologically important mRNAs (e.g. hypoxia-inducible factor 1α [HIF-1α], fibroblast growth factor 9 [FGF-9], and p53) is still translated by an unknown, cap-independent mechanism. Additionally, expression levels of eukaryotic translation initiation factor 4GI (eIF4GI) and of its homolog, death-associated protein 5 (DAP5), are elevated. By examining the 5' UTRs of HIF-1α, FGF-9, and p53 mRNAs and using fluorescence anisotropy binding studies, luciferase reporter-based in vitro translation assays, and mutational analyses, we demonstrate here that eIF4GI and DAP5 specifically bind to the 5' UTRs of these cap-independently translated mRNAs. Surprisingly, we found that the eIF4E-binding domain of eIF4GI increases not only the binding affinity but also the selectivity among these mRNAs. We further demonstrate that the affinities of eIF4GI and DAP5 binding to these 5' UTRs correlate with the efficiency with which these factors drive cap-independent translation of these mRNAs. Integrating the results of our binding and translation assays, we conclude that eIF4GI or DAP5 is critical for recruitment of a specific subset of mRNAs to the ribosome, providing mechanistic insight into their cap-independent translation.

Antiamoebic effects of sesquiterpene lactones isolated from the zoanthid Palythoa aff. clavata.

Opportunistic parasitic protozoa of genus Acanthamoeba are responsible to cause severe infections in humans such as Acanthamoeba Keratitis or Amoebic Granulomatous Encephalitis. Current treatments are usually toxic and inefficient and there is a need to access new therapeutic agents. The antiamoebic effects of nephthediol (1) and fourteen germacranolide and eudesmanolide sesquiterpene lactones (2-5, 7-12) isolated from the indigenous zoanthid Palythoa aff. clavata collected at the coast of Lanzarote, Canary Islands were studied against Acanthamoeba castellanii Neff, and the clinical strains A. polyphaga and A. griffini. 4-epi-arbusculin A (11) presented the lowest IC50 value (26,47 ± 1,69 µM) against A. castellanii Neff and low cytotoxicity against murine macrophages, followed by isobadgerin (2), which also showed to be active against A. castellanii Neff cysts. The studies on the mode of action of compounds 2 and 11 revealed these sesquiterpene lactones induce mechanisms of PDC on A. castellanii Neff.

Converting Escherichia coli into an archaebacterium with a hybrid heterochiral membrane.

One of the main differences between bacteria and archaea concerns their membrane composition. Whereas bacterial membranes are made up of glycerol-3-phosphate ester lipids, archaeal membranes are composed of glycerol-1-phosphate ether lipids. Here, we report the construction of a stable hybrid heterochiral membrane through lipid engineering of the bacterium Escherichia coli By boosting isoprenoid biosynthesis and heterologous expression of archaeal ether lipid biosynthesis genes, we obtained a viable E. coli strain of which the membranes contain archaeal lipids with the expected stereochemistry. It has been found that the archaeal lipid biosynthesis enzymes are relatively promiscuous with respect to their glycerol phosphate backbone and that E. coli has the unexpected potential to generate glycerol-1-phosphate. The unprecedented level of 20-30% archaeal lipids in a bacterial cell has allowed for analyzing the effect on the mixed-membrane cell's phenotype. Interestingly, growth rates are unchanged, whereas the robustness of cells with a hybrid heterochiral membrane appeared slightly increased. The implications of these findings for evolutionary scenarios are discussed.