Collinsella aerofaciens Produces a pH-Responsive Lipid Immunogen.

Some members of the human gut microbiota profoundly influence their host's physiology, health, and therapeutic responses, but the responsible molecules and mechanisms are largely unknown. As part of a project to identify immunomodulators produced by gut microbes, we analyzed the metabolome of Collinsella aerofaciens, an actinomycete that figures prominently in numerous association studies. The associations are typically positive correlations of C. aerofaciens with pro-inflammatory responses and undesirable outcomes, but an association with favorable responses to PD-1/PD-L1 cancer immunotherapy is a notable exception. A phenotypic assay-guided screen using dendritic cells (mBMDCs) and cytokine readouts identified the active compound, which was structurally characterized as a lysoglycoglycerolipid with an acetal-bearing ß-galactofuranose head group (CaLGL-1, 1). The structural assignment was confirmed through total synthesis. Assays with tlr2-/-, tlr4-/-, and wt mBMDCs revealed TLR2-dependent signaling. CaLGL-1 is produced by a conversion of a bacterially biosynthesized plasmalogen (CaPlsM, 3) to CaLGL-1 (1) in a low-pH environment.

Cellular Labeling of Phosphatidylserine Using Clickable Serine Probes.

Phosphatidylserine (PS) is a key lipid that plays important roles in disease-related biological processes, and therefore, the means to track PS in live cells are invaluable. Herein, we describe the metabolic labeling of PS in Saccharomyces cerevisiae cells using analogues of serine, a PS precursor, derivatized with azide moieties at either the amino (N-l-SerN3) or carbonyl (C-l-SerN3) groups. The conservative click tag modification enabled these compounds to infiltrate normal lipid biosynthetic pathways, thereby producing tagged PS molecules as supported by mass spectrometry studies, thin-layer chromatography (TLC) analysis, and further derivatization with fluorescent reporters via click chemistry to enable imaging in yeast cells. This approach shows strong prospects for elucidating the complex biosynthetic and trafficking pathways involving PS.

Mitigation and use of biofilms in space for the benefit of human space exploration.

Biofilms are self-organized communities of microorganisms that are encased in an extracellular polymeric matrix and often found attached to surfaces. Biofilms are widely present on Earth, often found in diverse and sometimes extreme environments. These microbial communities have been described as recalcitrant or protective when facing adversity and environmental exposures. On the International Space Station, biofilms were found in human-inhabited environments on a multitude of hardware surfaces. Moreover, studies have identified phenotypic and genetic changes in the microorganisms under microgravity conditions including changes in microbe surface colonization and pathogenicity traits. Lack of consistent research in microgravity-grown biofilms can lead to deficient understanding of altered microbial behavior in space. This could subsequently create problems in engineered systems or negatively impact human health on crewed spaceflights. It is especially relevant to long-term and remote space missions that will lack resupply and service. Conversely, biofilms are also known to benefit plant growth and are essential for human health (i.e., gut microbiome). Eventually, biofilms may be used to supply metabolic pathways that produce organic and inorganic components useful to sustaining life on celestial bodies beyond Earth. This article will explore what is currently known about biofilms in space and will identify gaps in the aerospace industry's knowledge that should be filled in order to mitigate or to leverage biofilms to the advantage of spaceflight.

Akkermansia muciniphila phospholipid induces homeostatic immune responses.

Multiple studies have established associations between human gut bacteria and host physiology, but determining the molecular mechanisms underlying these associations has been challenging1-3. Akkermansia muciniphila has been robustly associated with positive systemic effects on host metabolism, favourable outcomes to checkpoint blockade in cancer immunotherapy and homeostatic immunity4-7. Here we report the identification of a lipid from A. muciniphila's cell membrane that recapitulates the immunomodulatory activity of A. muciniphila in cell-based assays8. The isolated immunogen, a diacyl phosphatidylethanolamine with two branched chains (a15:0-i15:0 PE), was characterized through both spectroscopic analysis and chemical synthesis. The immunogenic activity of a15:0-i15:0 PE has a highly restricted structure-activity relationship, and its immune signalling requires an unexpected toll-like receptor TLR2-TLR1 heterodimer9,10. Certain features of the phospholipid's activity are worth noting: it is significantly less potent than known natural and synthetic TLR2 agonists; it preferentially induces some inflammatory cytokines but not others; and, at low doses (1% of EC50) it resets activation thresholds and responses for immune signalling. Identifying both the molecule and an equipotent synthetic analogue, its non-canonical TLR2-TLR1 signalling pathway, its immunomodulatory selectivity and its low-dose immunoregulatory effects provide a molecular mechanism for a model of A. muciniphila's ability to set immunological tone and its varied roles in health and disease.

Lyme Disease, Borrelia burgdorferi, and Lipid Immunogens.

The human immune system detects potentially pathogenic microbes with receptors that respond to microbial metabolites. While the overall immune signaling pathway is known in considerable detail, the initial molecular signals, the microbially produced immunogens, for important diseases like Lyme disease (LD) are often not well-defined. The immunogens for LD are produced by the spirochete Borrelia burgdorferi, and a galactoglycerolipid (1) has been identified as a key trigger for the inflammatory immune response that characterizes LD. This report corrects the original structural assignment of 1 to 3, a change of an α-galactopyranose to an α-galactofuranose headgroup. The seemingly small change has important implications for the diagnosis, prevention, and treatment of LD.

Capsular polysaccharide correlates with immune response to the human gut microbe Ruminococcus gnavus.

Active inflammatory bowel disease (IBD) often coincides with increases of Ruminococcus gnavus, a gut microbe found in nearly everyone. It was not known how, or if, this correlation contributed to disease. We investigated clinical isolates of R. gnavus to identify molecular mechanisms that would link R. gnavus to inflammation. Here, we show that only some isolates of R. gnavus produce a capsular polysaccharide that promotes a tolerogenic immune response, whereas isolates lacking functional capsule biosynthetic genes elicit robust proinflammatory responses in vitro. Germ-free mice colonized with an isolate of R. gnavus lacking a capsule show increased measures of gut inflammation compared to those colonized with an encapsulated isolate in vivo. These observations in the context of our earlier identification of an inflammatory cell-wall polysaccharide reveal how some strains of R. gnavus could drive the inflammatory responses that characterize IBD.

Mapping the Substrate-Binding Sites in the Phosphatidylserine Synthase in Candida albicans.

The fungal phosphatidylserine (PS) synthase, a membrane protein encoded by the CHO1 gene, is a potential drug target for pathogenic fungi, such as Candida albicans. However, both substrate-binding sites of C. albicans Cho1 have not been characterized. Cho1 has two substrates: cytidyldiphosphate-diacylglycerol (CDP-DAG) and serine. Previous studies identified a conserved CDP-alcohol phosphotransferase (CAPT) binding motif, which is present within Cho1. We tested the CAPT motif for its role in PS synthesis by mutating conserved residues using alanine substitution mutagenesis. PS synthase assays revealed that mutations in all but one conserved amino acid within the CAPT motif resulted in decreased Cho1 function. In contrast, there were no clear motifs in Cho1 for binding serine. Therefore, to identify the serine binding site, PS synthase sequences from three fungi were aligned with sequences of a similar enzyme, phosphatidylinositol (PI) synthase, from the same fungi. This revealed a motif that was unique to PS synthases. Using alanine substitution mutagenesis, we found that some of the residues in this motif are required for Cho1 function. Two alanine substitution mutants, L184A and R189A, exhibited contrasting impacts on PS synthase activity, and were characterized for their Michaelis-Menten kinetics. The L184A mutant displayed enhanced PS synthase activity and showed an increased Vmax. In contrast, R189A showed decreased PS synthase activity and increased Km for serine, suggesting that residue R189 is involved in serine binding. These results help to characterize PS synthase substrate binding, and should direct rational approaches for finding Cho1 inhibitors that may lead to better antifungals.

Enteroendocrine cells sense bacterial tryptophan catabolites to activate enteric and vagal neuronal pathways.

The intestinal epithelium senses nutritional and microbial stimuli using epithelial sensory enteroendocrine cells (EEC). EECs communicate nutritional information to the nervous system, but whether they also relay signals from intestinal microbes remains unknown. Using in vivo real-time measurements of EEC and nervous system activity in zebrafish, we discovered that the bacteria Edwardsiella tarda activate EECs through the receptor transient receptor potential ankyrin A1 (Trpa1) and increase intestinal motility. Microbial, pharmacological, or optogenetic activation of Trpa1+EECs directly stimulates vagal sensory ganglia and activates cholinergic enteric neurons by secreting the neurotransmitter 5-hydroxytryptamine (5-HT). A subset of indole derivatives of tryptophan catabolism produced by E. tarda and other gut microbes activates zebrafish EEC Trpa1 signaling. These catabolites also directly stimulate human and mouse Trpa1 and intestinal 5-HT secretion. These results establish a molecular pathway by which EECs regulate enteric and vagal neuronal pathways in response to microbial signals.

Plasmalogen Biosynthesis by Anaerobic Bacteria: Identification of a Two-Gene Operon Responsible for Plasmalogen Production in Clostridium perfringens.

Plasmalogens are vinyl ether-containing lipids produced by mammals and bacteria. The aerobic biosynthetic pathway in eukaryotes and bacteria is known, but the anaerobic pathway has remained a mystery. Here, we describe a two-gene operon (plasmalogen synthase, pls) responsible for plasmalogen production in the anaerobic bacterium Clostridium perfringens. While aerobic plasmalogen biosynthesis involves an oxidative conversion of an ether to a vinyl ether, anaerobic plasmalogen biosynthesis uses the reductive conversion of an ester to an aldehyde equivalent. Heterologous expression of the C. perfringens pls operon in E. coli conferred the ability to produce plasmalogens. The pls operon is predicted to encode a multidomain complex similar to benzoyl-CoA reductase/hydroxylacyl-CoA dehydratase (BCR/HAD) enzymes. Versions of this operon can be found in a wide range of obligate and facultative anaerobic bacteria, including many human gut microbes.

Research Tales from the Clardy Laboratory: Function-Driven Natural Product Discovery.

Over the past 70 years, the search for small molecules from nature has transformed biomedical research: natural products are the basis for half of all pharmaceuticals; the quest for total synthesis of natural products fueled development of methodologies for organic synthesis; and their biosynthesis presented unprecedented biochemical transformations, expanding our chemo-enzymatic toolkit. Initially, the discovery of small molecules was driven by bioactivity-guided fractionation. However, this approach yielded the frequent rediscovery of already known metabolites. As a result, focus shifted to identifying novel scaffolds through either structure-first methods or genome mining, relegating function as a secondary concern. Over the past two decades, the laboratory of Jon Clardy has taken an alternative route and focused on an ecology-driven, function-first approach in pursuit of uncovering bacterial small molecules with biological activity. In this review, we highlight several examples that showcase this ecology-first approach. Though the highlighted systems are diverse, unifying themes are (1) to understand how microbes interact with their host or environment, (2) to gain insights into the environmental roles of microbial metabolites, and (3) to explore pharmaceutical potential from these ecologically relevant metabolites.

Ruminococcus gnavus, a member of the human gut microbiome associated with Crohn's disease, produces an inflammatory polysaccharide.

A substantial and increasing number of human diseases are associated with changes in the gut microbiota, and discovering the molecules and mechanisms underlying these associations represents a major research goal. Multiple studies associate Ruminococcus gnavus, a prevalent gut microbe, with Crohn's disease, a major type of inflammatory bowel disease. We have found that R. gnavus synthesizes and secretes a complex glucorhamnan polysaccharide with a rhamnose backbone and glucose sidechains. Chemical and spectroscopic studies indicated that the glucorhamnan was largely a repeating unit of five sugars with a linear backbone formed from three rhamnose units and a short sidechain composed of two glucose units. The rhamnose backbone is made from 1,2- and 1,3-linked rhamnose units, and the sidechain has a terminal glucose linked to a 1,6-glucose. This glucorhamnan potently induces inflammatory cytokine (TNFα) secretion by dendritic cells, and TNFα secretion is dependent on toll-like receptor 4 (TLR4). We also identify a putative biosynthetic gene cluster for this molecule, which has the four biosynthetic genes needed to convert glucose to rhamnose and the five glycosyl transferases needed to build the repeating pentasaccharide unit of the inflammatory glucorhamnan.

Overproduction of Phospholipids by the Kennedy Pathway Leads to Hypervirulence in Candida albicans.

Candida albicans is an opportunistic human fungal pathogen that causes life-threatening systemic infections, as well as oral mucosal infections. Phospholipids are crucial for pathogenesis in C. albicans, as disruption of phosphatidylserine (PS) and phosphatidylethanolamine (PE) biosynthesis within the cytidine diphosphate diacylglycerol (CDP-DAG) pathway causes avirulence in a mouse model of systemic infection. The synthesis of PE by this pathway plays a crucial role in virulence, but it was unknown if downstream conversion of PE to phosphatidylcholine (PC) is required for pathogenicity. Therefore, the enzymes responsible for methylating PE to PC, Pem1 and Pem2, were disrupted. The resulting pem1Δ/Δ pem2Δ/Δ mutant was not less virulent in mice, but rather hypervirulent. Since the pem1Δ/Δ pem2Δ/Δ mutant accumulated PE, this led to the hypothesis that increased PE synthesis increases virulence. To test this, the alternative Kennedy pathway for PE/PC synthesis was exploited. This pathway makes PE and PC from exogenous ethanolamine and choline, respectively, using three enzymatic steps. In contrast to Saccharomyces cerevisiae, C. albicans was found to use one enzyme, Ept1, for the final enzymatic step (ethanolamine/cholinephosphotransferase) that generates both PE and PC. EPT1 was overexpressed, which resulted in increases in both PE and PC synthesis. Moreover, the EPT1 overexpression strain is hypervirulent in mice and causes them to succumb to system infection more rapidly than wild-type. In contrast, disruption of EPT1 causes loss of PE and PC synthesis by the Kennedy pathway, and decreased kidney fungal burden during the mouse systemic infection model, indicating a mild loss of virulence. In addition, the ept1Δ/Δ mutant exhibits decreased cytotoxicity against oral epithelial cells in vitro, whereas the EPT1 overexpression strain exhibits increased cytotoxicity. Taken altogether, our data indicate that mutations that result in increased PE synthesis cause greater virulence and mutations that decrease PE synthesis attenuate virulence.

Labeling of Phosphatidylinositol Lipid Products in Cells through Metabolic Engineering by Using a Clickable myo-Inositol Probe.

Phosphatidylinositol (PI) lipids control critical biological processes, so aberrant biosynthesis often leads to disease. As a result, the capability to track the production and localization of these compounds in cells is vital for elucidating their complex roles. Herein, we report the design, synthesis, and application of clickable myo-inositol probe 1 a for bioorthogonal labeling of PI products. To validate this platform, we initially conducted PI synthase assays to show that 1 a inhibits PI production in vitro. Fluorescence microscopy experiments next showed probe-dependent imaging in T-24 human bladder cancer and Candida albicans cells. Growth studies in the latter showed that replacement of myo-inositol with probe 1 a led to an enhancement in cell growth. Finally, fluorescence-based TLC analysis and mass spectrometry experiments support the labeling of PI lipids. This approach provides a promising means for tracking the complex biosynthesis and trafficking of these lipids in cells.

PS, It's Complicated: The Roles of Phosphatidylserine and Phosphatidylethanolamine in the Pathogenesis of Candida albicans and Other Microbial Pathogens.

The phospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) play important roles in the virulence of Candida albicans and loss of PS synthesis or synthesis of PE from PS (PS decarboxylase) severely compromises virulence in C. albicans in a mouse model of systemic candidiasis. This review discusses synthesis of PE and PS in C. albicans and mechanisms by which these lipids impact virulence in this fungus. This is further compared to how PS and PE synthesis impact virulence in other fungi, parasites and bacteria. Furthermore, the impact of PS asymmetry on virulence and extracellular vesicle formation in several microbes is reviewed. Finally, the potential for PS and PE synthases as drug targets in these various kingdoms is also examined.

Role of phosphatidylserine synthase in shaping the phospholipidome of Candida albicans.

Phosphatidylserine (PS) synthase (Cho1p) and the PS decarboxylase enzymes (Psd1p and Psd2p), which synthesize PS and phosphatidylethanolamine (PE), respectively, are crucial for Candida albicans virulence. Mutations that disrupt these enzymes compromise virulence. These enzymes are part of the cytidine diphosphate-diacylglycerol pathway (i.e. de novo pathway) for phospholipid synthesis. Understanding how losses of PS and/or PE synthesis pathways affect the phospholipidome of Candida is important for fully understanding how these enzymes impact virulence. The cho1Δ/Δ and psd1Δ/Δ psd2Δ/Δ mutations cause similar changes in levels of phosphatidic acid, phosphatidylglycerol, phosphatidylinositol and PS. However, only slight changes were seen in PE and phosphatidylcholine (PC). This finding suggests that the alternative mechanism for making PE and PC, the Kennedy pathway, can compensate for loss of the de novo synthesis pathway. Candida albicans Cho1p, the lipid biosynthetic enzyme with the most potential as a drug target, has been biochemically characterized, and analysis of its substrate specificity and kinetics reveal that these are similar to those previously published for Saccharomyces cerevisiae Cho1p.

SB-224289 Antagonizes the Antifungal Mechanism of the Marine Depsipeptide Papuamide A.

In order to expand the repertoire of antifungal compounds a novel, high-throughput phenotypic drug screen targeting fungal phosphatidylserine (PS) synthase (Cho1p) was developed based on antagonism of the toxin papuamide A (Pap-A). Pap-A is a cyclic depsipeptide that binds to PS in the membrane of wild-type Candida albicans, and permeabilizes its plasma membrane, ultimately causing cell death. Organisms with a homozygous deletion of the CHO1 gene (cho1ΔΔ) do not produce PS and are able to survive in the presence of Pap-A. Using this phenotype (i.e. resistance to Pap-A) as an indicator of Cho1p inhibition, we screened over 5,600 small molecules for Pap-A resistance and identified SB-224289 as a positive hit. SB-224289, previously reported as a selective human 5-HT1B receptor antagonist, also confers resistance to the similar toxin theopapuamide (TPap-A), but not to other cytotoxic depsipeptides tested. Structurally similar molecules and truncated variants of SB-224289 do not confer resistance to Pap-A, suggesting that the toxin-blocking ability of SB-224289 is very specific. Further biochemical characterization revealed that SB-224289 does not inhibit Cho1p, indicating that Pap-A resistance is conferred by another undetermined mechanism. Although the mode of resistance is unclear, interaction between SB-224289 and Pap-A or TPap-A suggests this screening assay could be adapted for discovering other compounds which could antagonize the effects of other environmentally- or medically-relevant depsipeptide toxins.