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Ethnopharmacological relevance The Vha-Venda people living in rural areas of Limpopo Province of South Africa regularly use traditional plant-based medicines to treat malaria. In our earlier publication, twenty indigenous plant species used to treat malaria or its symptoms by Vha-Venda people were evaluated for antiplasmodial activity. The main objective of the current study was to assess the robustness of NMR-based metabolomics in discriminating classes of secondary compounds that are responsible for the observed antimalarial activity and the isolation of antiplasmodial compounds. Materials and methods Twenty dichloromethane extracts were reconstituted in CDCl3, subjected to 1H NMR-based metabolomic analysis on a Varian 600 MHz spectrometer and the acquired 1H NMR spectra were then evaluated collectively using multivariate data analysis (MDA). Principal Component Analysis (PCA) and Orthogonal Projections to Latent Structures–Discriminant Analysis (OPLS-DA) were used to ‘globally’ discern antiplasmodial profiles. A contribution plot was then generated from the OPLS-DA scoring plot in an attempt to determine the classes of compounds that are responsible for the observed grouping. Further phytochemical analyses were conducted on the lipophilic extracts of Tabernaemontana elegans and Vangueria infausta subsp. infausta. These best candidates were fractionated, purified and their isolated compounds identified based on conventional chromatographic and spectroscopic techniques. Results The PCA did not separate the acquired profiles according to the detected antiplasmodial bioactivity. Application of a supervised OPLS-DA on the 1H NMR profiles resulted in a discrimination pattern that could be correlated to the observed antimalarial bioactivity. A contribution plot generated from the OPLS-DA scoring plot illustrated the classes of compounds responsible for the observed grouping. Prominent peaks were observed in the aromatic, sugar-based/N-containing and aliphatic spectral regions of the contribution plot. Two known indole alkaloids were isolated from T. elegans, and identified as tabernaemontanine (IC50 = 12.0 ± 0.8 µM) and dregamine (IC50 = 62.0 ± 2.4 µM). Friedelin (IC50 = 7.20 ± 0.5 µM) and morindolide (IC50 = 107.1 ± 0.6 µM) were isolated from V. infausta subsp. infausta. This is the first report of the rare iridoid lactone, morindolide's antimalarial activity. While these two compounds have been previously identified, this is the first account of their occurrence in the genus Vangueria. Conclusion The study illustrated the potential of NMR-based metabolomics in discriminating classes of compounds that may be attributed to antiplasmodial activity. Additionally, the study demonstrated the potential of discovering novel antiplasmodial scaffolds from medicinal plants and the rationale for the bioprospecting antimalarial plant species used by Vha-Venda people.

Cryptococcus neoformans is an encapsulated yeast that causes disease mainly in immunosuppressed hosts. It is considered a facultative intracellular pathogen because of its capacity to survive and replicate inside phagocytes, especially macrophages. This ability is heavily dependent on various virulence factors, particularly the glucuronoxylomannan (GXM) component of the polysaccharide capsule. Inflammasome activation in phagocytes is usually protective against fungal infections, including cryptococcosis. Nevertheless, recognition of C. neoformans by inflammasome receptors requires specific changes in morphology or the opsonization of the yeast, impairing proper inflammasome function. In this context, we analyzed the impact of molecules secreted by C. neoformans B3501 strain and its acapsular mutant Δcap67 in inflammasome activation in an in vitro model. Our results showed that conditioned media derived from B3501 was capable of inhibiting inflammasome-dependent events (i.e., IL-1β secretion and LDH release via pyroptosis) more strongly than conditioned media from Δcap67, regardless of GXM presence. We also demonstrated that macrophages treated with conditioned media were less responsive against infection with the virulent strain H99, exhibiting lower rates of phagocytosis, increased fungal burdens, and enhanced vomocytosis. Moreover, we showed that the aromatic metabolite DL-Indole-3-lactic acid (ILA) and DL-p-Hydroxyphenyllactic acid (HPLA) were present in B3501’s conditioned media and that ILA alone or with HPLA is involved in the regulation of inflammasome activation by C. neoformans. These results were confirmed by in vivo experiments, where exposure to conditioned media led to higher fungal burdens in Acanthamoeba castellanii culture as well as in higher fungal loads in the lungs of infected mice. Overall, the results presented show that conditioned media from a wild-type strain can inhibit a vital recognition pathway and subsequent fungicidal functions of macrophages, contributing to fungal survival in vitro and in vivo and suggesting that secretion of aromatic metabolites, such as ILA, during cryptococcal infections fundamentally impacts pathogenesis.

Lipids play a fundamental role in fungal cell biology, being essential cell membrane components and major targets of antifungal drugs. A deeper knowledge of lipid metabolism is key for developing new drugs and a better understanding of fungal pathogenesis. Here we built a comprehensive map of theHistoplasma capsulatumlipid metabolic pathway by incorporating proteomic and lipidomic analyses. We performed genetic complementation and overexpression ofH. capsulatumgenes inSaccharomyces cerevisiaeto validate reactions identified in the map and to determine enzymes responsible for catalyzing orphan reactions. The map led to the identification of both the fatty acid desaturation and the sphingolipid biosynthesis pathways as targets for drug development. We found that the sphingolipid biosynthesis inhibitor myriocin, the fatty acid desaturase inhibitor thiocarlide and the fatty acid analog 10-thiastearic acid inhibitH. capsulatumgrowth in nanomolar to low micromolar concentrations. These compounds also reduced the intracellular infection in an alveolar macrophage cell line. Overall, this lipid metabolic map revealed pathways that can be targeted for drug development.

Histoplasma capsulatum is a dimorphic fungus that most frequently causes pneumonia, but can also disseminate and proliferate in diverse tissues. Histoplasma capsulatum has a complex secretion system that mediates the release of macromolecule-degrading enzymes and virulence factors. The formation and release of extracellular vesicles (EVs) are an important mechanism for non-conventional secretion in both ascomycetes and basidiomycetes. Histoplasma capsulatum EVs contain diverse proteins associated with virulence and are immunologically active. Despite the growing knowledge of EVs from H. capsulatum and other pathogenic fungi, the extent that changes in the environment impact the sorting of organic molecules in EVs has not been investigated. In this study, we cultivated H. capsulatum with distinct culture media to investigate the potential plasticity in EV loading in response to differences in nutrition. Our findings reveal that nutrition plays an important role in EV loading and formation, which may translate into differences in biological activities of these fungi in various fluids and tissues.

Background Damage to enteric neurons and impaired gastrointestinal muscle contractions cause motility disorders in 70% of diabetic patients. It is thought that enteric neuropathy and dysmotility occur before overt diabetes, but triggers of these abnormalities are not fully known. We tested the hypothesis that intestinal contents of mice with and without high-fat diet- (HFD-) induced diabetic conditions contain molecules that impair gastrointestinal movements by damaging neurons and disrupting muscle contractions. Methods Small and large intestinal segments were collected from healthy, standard chow diet (SCD) fed mice. Filtrates of ileocecal contents (ileocecal supernatants; ICS) from HFD or SCD mice were perfused through them. Cultured intact intestinal muscularis externa preparations were used to determine whether ICS and their fractions obtained by solid-phase extraction (SPE) and SPE subfractions collected by high-performance liquid chromatography (HPLC) disrupt muscle contractions by injuring neurons and smooth muscle cells. Key results ICS from HFD mice reduced intestinal motility, but those from SCD mice had no effect. ICS, aqueous SPE fractions and two out of twenty HPLC subfractions of aqueous SPE fractions from HFD mice blocked muscle contractions, caused a loss of nitrergic myenteric neurons through inflammation, and reduced smooth muscle excitability. Lipopolysaccharide and palmitate caused a loss of nitrergic myenteric neurons but did not affect muscle contractions. Conclusions & inferences Unknown molecules in intestinal contents of HFD mice trigger enteric neuropathy and dysmotility. Further studies are required to identify the toxic molecules and their mechanisms of action.

Cryptococcus neoformansis an encapsulated yeast that causes disease mainly in immunosuppressed hosts. It is considered a facultative intracellular pathogen because of its capacity to survive and replicate inside phagocytes, especially macrophages. This capacity is heavily dependent on various virulence factors, particularly the glucuronoxylomannan (GXM) component of the polysaccharide capsule, that render the non- or poorly-activated macrophage ineffective against phagocytosed yeast. Strategies utilized by macrophages to prevent this scenario include pyroptosis (a rapid highly inflammatory cell death) and vomocytosis (the expulsion of the pathogen from the intracellular environment without lysis). Inflammasome activation in phagocytes is usually protective against fungal infections, including cryptococcosis. Nevertheless, recognition ofC. neoformansby inflammasome receptors requires specific changes in morphology or the opsonization of the yeast, impairing a proper inflammasome function. In this context, we analyzed the impact of molecules secreted byC. neoformansB3501 strain and its acapsular mutantΔcap67in an inflammasome activationin vitromodel. Our results showed that conditioned media derived from B3501 was capable of inhibiting inflammasome dependent events (i. e. IL-1β secretion and LDH release via pyroptosis) more strongly than conditioned media fromΔcap67, regardless of GXM presence. We also demonstrated that macrophages treated with conditioned media were less responsive against infection with the virulent strain H99, exhibiting lower rates of phagocytosis, increased fungal burdens and enhanced vomocytosis. Moreover, we showed that the aromatic metabolite DL-Indole-3-lactic acid (ILA) was present in B3501’s conditioned media and that this fungal metabolite is involved in the regulation of inflammasome activation byC. neoformans. Overall, the results presented show that conditioned media from a wild-type strain can inhibit an important recognition pathway and subsequent fungicidal functions of macrophages, contributing to fungal survivalin vitroand suggesting that this serves as an important role for secreted molecules during cryptococcal infections.Author’s SummaryCryptococcus neoformansis the agent of cryptococcal meningitis, a disease that can be life-threatening in immunocompromised hosts such as those infected with HIV. The infection thrives in hosts that poorly activate their immune system, mainly because of the yeast’s ability to survive inside macrophages and migrate towards the central nervous system. Emerging data indicate that cryptococci modulate the host immune response, but the underlying mechanisms remain largely uncharacterized. Here we show that secreted molecules from a wild-type strain ofC. neoformansimpair inflammatory responses driven by inflammasome activation, which in turn impact the macrophage antifungal activity. We further show that this inhibition does not involve GXM, the main constituent of the fungal capsule, but rather is partially dependent on DL-Indole-3-lactic acid (ILA), a metabolite not previously implicated in fungal virulence.

Antibodies play a central role in host immunity by directly inactivating or recognizing an invading pathogen to enhance different immune responses to combat the invader. However, the cellular responses of pathogens to the presence of antibodies are not well-characterized. Here, we used different mass spectrometry techniques to study the cellular responses of the pathogenic fungus Histoplasma capsulatum to monoclonal antibodies (mAb) against HSP60, the surface protein involved in infection. A proteomic analysis of H. capsulatum yeast cells revealed that mAb binding regulates a variety of metabolic and signaling pathways, including fatty acid metabolism, sterol metabolism, MAPK signaling and ubiquitin-mediated proteolysis. The regulation of the fatty acid metabolism was accompanied by increases in the level of polyunsaturated fatty acids, which further augmented the degree of unsaturated lipids in H. capsulatum&rsquo, s membranes and energy storage lipids, such as triacylglycerols, phosphatidylcholines, phosphatidylethanolamines and phosphatidylinositols. MAb treatment also regulated sterol metabolism by increasing the levels of cholesterol and ergosterol in the cells. We also showed that global changes in the lipid profiles resulted in an increased susceptibility of H. capsulatum to the ergosterol-targeting drug amphotericin B. Overall, our data showed that mAb induction of global changes in the composition of H. capsulatum membranes can potentially impact antifungal treatment during histoplasmosis.

Outer membrane vesicles produced by Gram-negative bacteria have been studied for half a century but the possibility that Gram-positive bacteria secrete extracellular vesicles (EVs) was not pursued until recently due to the assumption that the thick peptidoglycan cell wall would prevent their release to the environment. However, following their discovery in fungi, which also have cell walls, EVs have now been described for a variety of Gram-positive bacteria. EVs purified from Gram-positive bacteria are implicated in virulence, toxin release, and transference to host cells, eliciting immune responses, and spread of antibiotic resistance. Listeria monocytogenes is a Gram-positive bacterium that causes listeriosis. Here we report that L. monocytogenes produces EVs with diameters ranging from 20 to 200 nm, containing the pore-forming toxin listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC). Cell-free EV preparations were toxic to mammalian cells, the murine macrophage cell line J774.16, in a LLO-dependent manner, evidencing EV biological activity. The deletion of plcA increased EV toxicity, suggesting PI-PLC reduced LLO activity. Using simultaneous metabolite, protein, and lipid extraction (MPLEx) multiomics we characterized protein, lipid, and metabolite composition of bacterial cells and secreted EVs and found that EVs carry the majority of listerial virulence proteins. Using immunogold EM we detected LLO at several organelles within infected human epithelial cells and with high-resolution fluorescence imaging we show that dynamic lipid structures are released from L. monocytogenes during infection. Our findings demonstrate that L. monocytogenes uses EVs for toxin release and implicate these structures in mammalian cytotoxicity.

Bile acids are metabolic links between hosts and their gut microbiomes, yet little is known about the roles they play in microbe-to-microbe interactions. Here we present a study designed to investigate the effect that a common probiotic,Lactobacillus acidophilus, has on microbial interactions that lead to formation of secondary bile acids. A model microbial consortium was built from three human gut isolates,Clostridium scindens, Collinsella aerofaciens,andBlautia obeum, and cultured under different bile acid and probiotic treatments. A multi-omics platform that included mass spectrometry-based metabolomics and activity-based proteomic probes was used to produce two major results. The first, was that an uncommon secondary bile acid – ursocholate – was produced by a multi-species chemical synthesis pathway. This result highlights a new microbe-to-microbe interaction mediated by bile acids. The second finding was that the probiotic strain,L. acidophilus,quenched the observed interactions and effectively halted consortial synthesis of ursocholate. Little is known about the role that ursocholate plays in human health and development. However, we did discover that a decrease in ursocholate abundance corresponded with successful weight loss in patients after gastric bypass surgery versus those who did not lose weight after surgery. Hence, this study uncovered basic knowledge that may aid future designs of custom probiotic therapies to combat obesity.

Outer membrane vesicles produced by Gram-negative bacteria have been studied for half a century but the possibility that Gram-positive bacteria secreted extracellular vesicles (EVs) was not pursued due to the assumption that the thick peptidoglycan cell wall would prevent their release to the environment. However, following discovery in fungi, which also have cell walls, EVs have now been described for a variety of Gram-positive bacteria. EVs purified from Gram-positive bacteriaare implicated in virulence, toxin release and transference to host cells, eliciting immune responses, and spread of antibiotic resistance.Listeria monocytogenesis a Gram-positive bacterium that is the etiological agent of listeriosis. Here we report thatL. monocytogenesproduces EVs with diameter ranging from 20-200 nm, containing the pore-forming toxin listeriolysin O(LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC). Using simultaneousmetabolite,protein, andlipidextraction (MPLEx) multi-omics we characterized protein, lipid and metabolite composition of bacterial cells and secreted EVs and found that EVs carry the majority of listerial virulence proteins. Cell-free EV preparations were toxic to the murine macrophage cell line J774.16, in a LLO-dependent manner, evidencing EV biological activity. The deletion ofplcAincreased EV toxicity, suggesting PI-PLC can restrain LLO activity. Using immunogold electron microscopy we detect LLO localization at several organelles within infected human epithelial cells and with high-resolution fluorescence imaging we show that dynamic lipid structures are released fromL. monocytogenesthat colocalize with LLO during infection. Our findings demonstrate thatL. monocytogenesutilize EVs for toxin release and implicate these structures in mammalian cytotoxicity.

Helichrysum umbraculigerum Less. has been reported to be a prolific producer of phytocannabinoids from the alkyl-, aralkyl-, normal-, and abnormal types. Investigation of an acetone extract from the aerial parts of the plant afforded two novel amorfrutin-type phytocannabinoids (3b, 4) and the new geranylated phloroglucinol 5a. The presence of cannabigerol (CBG, 1a) and its acidic precursor (pre-CBG, CBGA, 1b), previously reported from this plant, could not be confirmed, but the phenethyl analogue of CBG (Heli-CBG, 2a) and the methyl ester of its carboxylated version (2b) were isolated. Heli-CBG (2a) was assayed against a series of metabotropic (CB1 and CB2)- and ionotropic (thermo-TRPs) targets of phytocannabinoids, comparing its profile with the one of cannabigerol (CBG). A decreased affinity for cannabinoid receptor was observed, along with substantial retention of the thermo-TRP profile. The biogenetic relationships between the isoprenylated phenolics from H. umbraculigerum are discussed, highlighting the relevance of this species for biogenetic investigations on phytocannabinoids

Helichrysum umbraculigerum Less. has been reported to be a prolific producer of phytocannabinoids from the alkyl-, aralkyl-, normal-, and abnormal types. Investigation of an acetone extract from the aerial parts of the plant afforded two novel amorfrutin-type phytocannabinoids ( 3b , 4 ) and the new geranylated phloroglucinol 5a . The presence of cannabigerol (CBG, 1a ) and its acidic precursor (pre-CBG, CBGA, 1b ), previously reported from this plant, could not be confirmed, but the phenethyl analogue of CBG (Heli-CBG, 2a) and the methyl ester of its carboxylated version ( 2b ) were isolated. Heli-CBG ( 2a ) was assayed against a series of metabotropic (CB 1 and CB 2 )- and ionotropic (thermo-TRPs) targets of phytocannabinoids, comparing its profile with the one of cannabigerol (CBG). A decreased affinity for cannabinoid receptor was observed, along with substantial retention of the thermo-TRP profile. The biogenetic relationships between the isoprenylated phenolics from H. umbraculigerum are discussed, highlighting the relevance of this species for biogenetic investigations on phytocannabinoids

The rise of infection caused by ‘superbugs’ is alarming and one of the most problematic resistant bacteria is methicillin-resistant Staphylococcus aureus (MRSA). This bacterium can cause a range of ailments like pneumonia, mastitis, meningitis, urinary tract infection, and post operational infection. Ten medicinal plants were investigated for their efficacy against drug-sensitive and drug-resistant strains of S. aureus. Ethanol extracts of Melianthus comosus Vahl (Melianthaceae), Melianthus major L (Melianthaceae), Dodonaea viscosa Jacq. var. angustifolia (L.f.) Benth (Sapindaceae) and Withania somnifera L. Dunal (Zygophyllaceae) were found to have good inhibitory activity against both drug-sensitive and drug-resistant strains of S. aureus. Minimum inhibitory concentrations of these plants ranged from 0.391 to 1.56 mg/ml. Ethanol extracts of all these plants were further tested for cytotoxicity on Vero cells using the XTT method. M. major exhibited a 50% inhibitory concentration (IC50) of 52.76 μg/ml and ...