Opportunistic Sampling of Roadkill as an Entry Point to Accessing Natural Products Assembled by Bacteria Associated with Non-anthropoidal Mammalian Microbiomes

Few secondary metabolites have been reported from mammalian microbiome bacteria despite the large numbers of diverse taxa that inhabit warm-blooded higher vertebrates. As a means to investigate natural products from these microorganisms, an opportunistic sampling protocol was developed, which focused on exploring bacteria isolated from roadkill mammals. This initiative was made possible through the establishment of a newly created discovery pipeline, which couples laser ablation electrospray ionization mass spectrometry (LAESIMS) with bioassay testing, to target biologically active metabolites from microbiome-associated bacteria. To illustrate this process, this report focuses on samples obtained from the ear of a roadkill opossum (Dideiphis virginiana) as the source of two bacterial isolates (Pseudomonas sp. and Serratia sp.) that produced several new and known cyclic lipodepsipeptides (viscosin and serrawettins, respectively). These natural products inhibited biofilm formation by the human pathogenic yeast Candida albicans at concentrations well below those required to inhibit yeast viability. Phylogenetic analysis of 16S rRNA gene sequence libraries revealed the presence of diverse microbial communities associated with different sites throughout the opossum carcass. A putative biosynthetic pathway responsible for the production of the new serrawettin analogues was identified by sequencing the genome of the Serratia sp. isolate. This study provides a functional roadmap to carrying out the systematic investigation of the genomic, microbiological, and chemical parameters related to the production of natural products made by bacteria associated with non-anthropoidal mammalian microbiomes. Discoveries emerging from these studies are anticipated to provide a working framework for efforts aimed at augmenting microbiomes to deliver beneficial natural products to a host.Research reported in this publication was supported by the National Institutes of Health, National Institute of Allergy and Infectious Diseases (1R21AI101487) (R.H.C. and B.S.S.). X-ray data were collected by L. Thomas in the OU Macromolecular Crystallography Laboratory, which is supported, in part, by an Institutional Development Award from the National Institutes of Health, General Medical Sciences (P20GM103640). The C. albicans SC5314 culture was kindly provided by A. Dongari-Bagtzoglou, University of Connecticut Health Center. The LC-ESIMS instrument used for this project was funded in part by a Challenge Grant from the Office of the Vice President for Research, University of Oklahoma, Norman Campus, and an award through the Shimadzu Equipment Grant Program (R.H.C.).Ye

Synergy of Human Immunodeficiency Virus Protease Inhibitors with Chloroquine against Plasmodium falciparum In Vitro and Plasmodium chabaudi In Vivo▿

The synergy of the activities between chloroquine and various human immunodeficiency virus protease inhibitors was investigated in chloroquine-resistant and -sensitive malaria parasites. In both in vitro and in vivo assay systems, ritonavir was found to be the most potent in potentiating the antimalarial action of chloroquine

Analysis of m7G-Related signatures in the tumour immune microenvironment and identification of clinical prognostic regulators in breast cancer

Abstract Background Breast cancer is a malignant tumour that seriously threatens women’s life and health and exhibits high inter-individual heterogeneity, emphasising the need for more in-depth research on its pathogenesis. While internal 7-methylguanosine (m7G) modifications affect RNA processing and function and are believed to be involved in human diseases, little is currently known about the role of m7G modification in breast cancer. Methods and Results We elucidated the expression, copy number variation incidence and prognostic value of 24 m7G-related genes (m7GRGs) in breast cancer. Subsequently, based on the expression of these 24 m7GRGs, consensus clustering was used to divide tumour samples from the TCGA-BRCA dataset into four subtypes based on significant differences in their immune cell infiltration and stromal scores. Differentially expressed genes between subtypes were mainly enriched in immune-related pathways such as ‘Ribosome’, ‘TNF signalling pathway’ and ‘Salmonella infection’. Support vector machines and multivariate Cox regression analysis were applied based on these 24 m7GRGs, and four m7GRGs—AGO2, EIF4E3, DPCS and EIF4E—were identified for constructing the prediction model. An ROC curve indicated that a nomogram model based on the risk model and clinical factors had strong ability to predict the prognosis of breast cancer. The prognoses of patients in the high- and low-TMB groups were significantly different (p = 0.03). Moreover, the four-gene signature was able to predict the response to chemotherapy. Conclusions In conclusion, we identified four different subtypes of breast cancer with significant differences in the immune microenvironment and pathways. We elucidated prognostic biomarkers associated with breast cancer and constructed a prognostic model involving four m7GRGs. In addition, we predicted the candidate drugs related to breast cancer based on the prognosis model

Production of Cytotoxic Glidobactins/Luminmycins by <i>Photorhabdus asymbiotica</i> in Liquid Media and Live Crickets

<i>Photorhabdus asymbiotica</i> engages in a two-part life cycle that requires adaptation to both symbiotic and pathogenic phases. The genome of <i>P. asymbiotica</i> contains several gene clusters, which are predicted to be involved in the biosynthesis of unique secondary metabolites that are hypothesized to enhance the bacterium’s pathogenic capabilities. However, recent reports on <i>Photorhabdus</i> secondary metabolite production have indicated that many of its genes are silent under laboratory culture conditions. Using a circumscribed panel of media and alternative fermentation conditions, we have successfully achieved the production of a series of new and known glidobactin/luminmycin derivatives from <i>P. asymbiotica</i> including glidobactin A (<b>1</b>), luminmycin A (<b>2</b>), and luminmycin D (<b>3</b>). These compounds were also obtained upon infection of live crickets with the bacterium. Luminmycin D showed cytotoxicity against human pancreatic cells (IC₅₀ of 0.11 μM), as well as proteasome inhibition (IC₅₀ of 0.38 μM)

Waikialoid A Suppresses Hyphal Morphogenesis and Inhibits Biofilm Development in Pathogenic <i>Candida albicans</i>

A chemically prolific strain of <i>Aspergillus</i> was isolated from a soil sample collected near Waikiki Beach, Honolulu, Hawaii. The fungus produced several secondary metabolites, which were purified and placed in our natural products library and were later screened for substances capable of inhibiting biofilm formation by <i>Candida albicans</i>. It was determined that one of the secondary metabolites from the Hawaiian fungal isolate, a new complex prenylated indole alkaloid named waikialoid A (<b>1</b>), inhibited biofilm formation with an IC₅₀ value of 1.4 μM. Another structurally unrelated, presumably polyketide metabolite, waikialide A (<b>15</b>), also inhibited <i>C. albicans</i> biofilm formation, but was much less potent (IC₅₀ value of 32.4 μM). Microscopy studies revealed that compound <b>1</b> also inhibited <i>C. albicans</i> hyphal morphogenesis. While metabolite <b>1</b> appears ineffective at disrupting preformed biofilms, the accumulated data indicate that the new compound may exert its activity against <i>C. albicans</i> during the early stages of surface colonization involving cell adherence, hyphal development, and/or biofilm assembly. Unlike some other stephacidin/notoamide compounds, metabolite <b>1</b> was not cytotoxic to fungi or human cells (up to 200 μM), which makes this an intriguing model compound for studying the adjunctive use of biofilm inhibitors in combination with standard antifungal antibiotics

Waikialoid A Suppresses Hyphal Morphogenesis and Inhibits Biofilm Development in Pathogenic <i>Candida albicans</i>

A chemically prolific strain of <i>Aspergillus</i> was isolated from a soil sample collected near Waikiki Beach, Honolulu, Hawaii. The fungus produced several secondary metabolites, which were purified and placed in our natural products library and were later screened for substances capable of inhibiting biofilm formation by <i>Candida albicans</i>. It was determined that one of the secondary metabolites from the Hawaiian fungal isolate, a new complex prenylated indole alkaloid named waikialoid A (<b>1</b>), inhibited biofilm formation with an IC₅₀ value of 1.4 μM. Another structurally unrelated, presumably polyketide metabolite, waikialide A (<b>15</b>), also inhibited <i>C. albicans</i> biofilm formation, but was much less potent (IC₅₀ value of 32.4 μM). Microscopy studies revealed that compound <b>1</b> also inhibited <i>C. albicans</i> hyphal morphogenesis. While metabolite <b>1</b> appears ineffective at disrupting preformed biofilms, the accumulated data indicate that the new compound may exert its activity against <i>C. albicans</i> during the early stages of surface colonization involving cell adherence, hyphal development, and/or biofilm assembly. Unlike some other stephacidin/notoamide compounds, metabolite <b>1</b> was not cytotoxic to fungi or human cells (up to 200 μM), which makes this an intriguing model compound for studying the adjunctive use of biofilm inhibitors in combination with standard antifungal antibiotics

Spiro Fused Diterpene–Indole Alkaloids from a Creek-Bottom-Derived <i>Aspergillus terreus</i>

Four metabolites, teraspiridoles A–D (<b>2</b>–<b>5</b>), formed from the merger of a diterpene and modified indole scaffold were obtained from an <i>Aspergillus terreus</i> isolate. The structures and absolute configurations of these natural products were established using NMR, mass spectrometry, Marfey’s method, VCD, and ECD data. Teraspiridole B (<b>3</b>) exhibited weak inhibition of planaria regeneration/survival

Polyketide Glycosides from <i>Bionectria ochroleuca</i> Inhibit <i>Candida albicans</i> Biofilm Formation

One of the challenges presented by <i>Candida</i> infections is that many of the isolates encountered in the clinic produce biofilms, which can decrease these pathogens’ susceptibilities to standard-of-care antibiotic therapies. Inhibitors of fungal biofilm formation offer a potential solution to counteracting some of the problems associated with <i>Candida</i> infections. A screening campaign utilizing samples from our fungal extract library revealed that a <i>Bionectria ochroleuca</i> isolate cultured on Cheerios breakfast cereal produced metabolites that blocked the <i>in vitro</i> formation of <i>Candida albicans</i> biofilms. A scale-up culture of the fungus was undertaken using mycobags (also known as mushroom bags or spawn bags), which afforded four known [TMC-151s C–F (<b>1</b>–<b>4</b>)] and three new [bionectriols B–D (<b>5</b>–<b>7</b>)] polyketide glycosides. All seven metabolites exhibited potent biofilm inhibition against <i>C. albicans</i> SC5314, as well as exerted synergistic antifungal activities in combination with amphotericin B. In this report, we describe the structure determination of the new metabolites, as well as compare the secondary metabolome profiles of fungi grown in flasks and mycobags. These studies demonstrate that mycobags offer a useful alternative to flask-based cultures for the preparative production of fungal secondary metabolites