Effects on mammalian cell transcriptome reveal a plant extract's medicinal properties: Potential anti-COVID-19 and anti-cancer properties of Sarcopoterium spinosum

Plants with medicinal properties are usually identified based on traditional medicine knowledge or using low-throughput screens for specific pharmacological activities. Here, we suggest a different approach to uncover a range of pharmacological activities of a chosen plant extract without the need for functional screening. This tactic predicts biological activities of a plant extract based on pathway analysis of transcriptome changes caused by the extract in mammalian cell culture. In this work, we identified transcriptome changes after exposure of cultured cells to an extract of the medicinal plant Sarcopoterium spinosum. Gene Set Enrichment Analysis (GSEA) confirmed known anti-inflammatory and anti-cancer activities of the extract and predicted novel biological effects on oxidative phosphorylation and interferon pathways. Experimental validation of these pathways uncovered strong activation of autophagy, including mitophagy, and astounding protection from SARS-CoV-2 infection. Our study shows that gene expression analysis alone is insufficient for predicting biological effects since some of the changes reflect compensatory effects, and additional biochemical tests provide necessary corrections. In conclusion, this study defines the advantages and limitations of an approach towards predicting the biological and medicinal effects of plant extracts based on transcriptome changes caused by these extracts in mammalian cells.

Coxiella burnetii inhibits host immunity by a protein phosphatase adapted from glycolysis

Coxiella burnetii is a bacterial pathogen that replicates within host cells by establishing a membrane-bound niche called the Coxiella-containing vacuole. Biogenesis of this compartment requires effectors of its Dot/Icm type IV secretion system. A large cohort of such effectors has been identified, but the function of most of them remain elusive. Here, by a cell-based functional screening, we identified the effector Cbu0513 (designated as CinF) as an inhibitor of NF-κB signaling. CinF is highly similar to a fructose-1,6-bisphosphate (FBP) aldolase/phosphatase present in diverse bacteria. Further study reveals that unlike its ortholog from Sulfolobus tokodaii, CinF does not exhibit FBP phosphatase activity. Instead, it functions as a protein phosphatase that specifically dephosphorylates and stabilizes IκBα. The IκBα phosphatase activity is essential for the role of CinF in C. burnetii virulence. Our results establish that C. burnetii utilizes a protein adapted from sugar metabolism to subvert host immunity.

Discovery and Biosynthesis of Natural Products from New Zealand Soil Metagenome Libraries

<p>Antibiotic discovery rates dramatically declined following the “golden age” of the 1940’s to the 1960’s. The platforms that underpinned that age of discovery rested upon laboratory cultivation of a small clade of bacteria, the actinomycetes, primarily isolated from soil environments. Fermentation extracts of these isolated bacteria have provided the majority of antibiotics and anticancer small molecules still used today. By applying modern genetic analysis techniques to these same environmental sources that have previously yielded such success, we can uncover new biosynthetic pathways, and bioactive compounds. The work described in this thesis investigated New Zealand soil metagenomes for this purpose. Four large metagenome libraries were constructed from the microbiomes of diverse soil environments. These were then interrogated by a functional screening approach in a knockout Escherichia coli strain, to recover a large collection of the biosynthetic gene clusters responsible for bacterial secondary metabolite production. Using different modes of bioinformatic analysis, these gene clusters were demonstrated to have both phylogenetic divergence, and functional difference from bacterial biosynthesis pathways previously discovered from culture based studies. Two additional biosynthetic pathways were recovered from one of these metagenome libraries, and in each case found to have novel genetic features. These gene clusters were further studied by heterologous expression within Streptomyces albus production hosts. One of these gene clusters produced small aromatic polyketide compounds, the structure of one of which was solved by chemical analytic techniques, and found to be a new chemical entity. The second gene cluster was demonstrated to have similarity to known aureolic acid biosynthesis gene clusters – a class of potent anticancer natural products. Heterologous expression resulted in the production of many metabolites, two of which were characterised and found to be new members of this chemical class. The research in this thesis both validates the use of metagenomic analysis for future natural product discovery efforts, and adds to a growing body of evidence that understudied clades of bacteria have an untapped biosynthetic potential that can be accessed by metagenomic methods.</p>

Discovery and Biosynthesis of Natural Products from New Zealand Soil Metagenome Libraries

<p>Antibiotic discovery rates dramatically declined following the “golden age” of the 1940’s to the 1960’s. The platforms that underpinned that age of discovery rested upon laboratory cultivation of a small clade of bacteria, the actinomycetes, primarily isolated from soil environments. Fermentation extracts of these isolated bacteria have provided the majority of antibiotics and anticancer small molecules still used today. By applying modern genetic analysis techniques to these same environmental sources that have previously yielded such success, we can uncover new biosynthetic pathways, and bioactive compounds. The work described in this thesis investigated New Zealand soil metagenomes for this purpose. Four large metagenome libraries were constructed from the microbiomes of diverse soil environments. These were then interrogated by a functional screening approach in a knockout Escherichia coli strain, to recover a large collection of the biosynthetic gene clusters responsible for bacterial secondary metabolite production. Using different modes of bioinformatic analysis, these gene clusters were demonstrated to have both phylogenetic divergence, and functional difference from bacterial biosynthesis pathways previously discovered from culture based studies. Two additional biosynthetic pathways were recovered from one of these metagenome libraries, and in each case found to have novel genetic features. These gene clusters were further studied by heterologous expression within Streptomyces albus production hosts. One of these gene clusters produced small aromatic polyketide compounds, the structure of one of which was solved by chemical analytic techniques, and found to be a new chemical entity. The second gene cluster was demonstrated to have similarity to known aureolic acid biosynthesis gene clusters – a class of potent anticancer natural products. Heterologous expression resulted in the production of many metabolites, two of which were characterised and found to be new members of this chemical class. The research in this thesis both validates the use of metagenomic analysis for future natural product discovery efforts, and adds to a growing body of evidence that understudied clades of bacteria have an untapped biosynthetic potential that can be accessed by metagenomic methods.</p>

Functional screening of a human saliva metagenomic DNA reveal novel resistance genes against sodium hypochlorite and chlorhexidine

Objective Many sections of the health care system are facing a major challenge making infectious disease problematic to treat; antimicrobial resistance (AMR). Identification and surveillance of the resistome have been highlighted as one of the strategies to overcome the problem. This study aimed to screen for AMR genes in an oral microbiota, a complex microbial system continuously exposed to antimicrobial agents commonly used in dental practice. Materials and methods As a significant part of the oral microbiome cannot be conventionally cultured, a functional metagenomic approach was chosen. The human oral metagenomic DNA was extracted from saliva samples collected from 50 healthy volunteers in Norway. The oral metagenomic library was then constructed by ligating partially digested oral metagenome into pSMART BAC vector and introducing into Escherichia coli. The library was screened against antimicrobials in dental practices. All resistant clones were selected and analyzed. Results Screening of the oral metagenomic library against different antimicrobials detected multiple clones with resistance against chlorhexidine, triclosan, erythromycin, tetracycline, and sodium hypochlorite. Bioinformatic analysis revealed both already known resistance genes, including msr, mef(A), tetAB(46), and fabK, and genes that were not previously described to confer resistance, including recA and accB conferring resistance to sodium hypochlorite and chlorhexidine, respectively. Conclusion Multiple clones conferring resistance to antimicrobials commonly used in dental practices were detected, containing known and novel resistant genes by functional-based metagenomics. There is a need for more studies to increase our knowledge in the field.

The science of “smell” and the noble for “hugs:” making “sense?”

David Julius and Ardem Patapoutian were jointly awarded the 2021 Nobel Prize in Physiology and Medicine for their discoveries of receptors for temperature and touch. It was a phenomenal moment for the scientific community, more for the discovery that forms the basic fabric of our everyday life—ever imagined how life would have been without feeling the aroma around? Or even the dangers of accidentally touching a heated object. Dr Julius and Dr Patapoutian, independently discovered key mechanisms of how living organisms sense heat, cold, and touch. The journey started when Dr Julius, at the University of California, San Francisco, used a key ingredient in hot chili peppers to identify a protein in nerve cells that respond to these stimuli. Using capsaicin, the pungent component of chili peppers, he provided fundamental insights into mechanisms of pain. Then using a meticulous cDNA library-based functional screening from sensory neurons to search for the gene(s) that could confer capsaicin sensitivity, Dr Julius identified for the first time a novel ion channel (now called transient receptor potential [TRP] vanilloid 1) belonging to the family of TRP ion channels associated with the pain sensitivity. A painful exercise indeed! While Prof Julius was exploring the oceans and skies to hunt for sensory pain pathways, quite independently, Dr Patapoutian of Scripps Research Institute La Jolla, California, was searching for a similar thing that seemed to bother him equally. How do we sense touches? After all, there are so many emotions packaged in this small five-letter word, touch. The mother’s touch is the first sensation that every single of us always cherishes. Dr Patapoutian research is centered around finding candidate genes in a mechanosensitive cell line that could respond to mechanical stimuli. After a thorough search, the team identified two mechanically-activated ion channels, PIEZO1 and PIEZO2, representing an entirely novel class of mechanical sensors-based ion channels. What is fascinating is the idea that discovery of the smell and touch receptor stretches far beyond just touch and temperature sensations only. Mutations in other TRP channels are involved in neurodegenerative disorders and skeletal dysplasia, while mutations in PIEZO channels help control critical functions such as respiration and blood pressure regulation. So now, the mysterious world surrounding us looks more transparent and clearer. The molecular landscape is defined with precision. We now have a chemical entity behind all these emotions and intuition. The warm hug that makes our day is now millions of ions crisscrossing the ion channels. There is a different side to this too. How will the world look if everything is defined as a chemical entity? Won’t we lose the charm? After all, so much within the subtleness remains charmful when wrapped within the veil of ignorance. Knowing too much about something steals the show.