Noxious weed yields virulence solutions for MRSA

We applied the ethnobotanical approach to drug discovery to identify and test medicinal plants with potential utility in treating virulent staph infections. We offer the first report of triterpenoid acids exhibiting potent anti-virulence effects against MRSA. This is the story behind the paper.

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Growing up in south Florida, the Brazilian peppertree (also known as the Christmas berry) was a familiar piece of my childhood landscape (Fig. 1). Native to Central and South America, it was introduced to the US around 100 years ago as an ornamental plant. Since then, it has escaped the garden landscapes and has become a very successful invasive plant—especially in Florida, where it is classified as a Category I (the most problematic) invasive exotic species. Today, you can find it throughout the Everglades and the deep roadside ditches common throughout the southern part of the state.

Fig. 1: Herbarium voucher specimen of Schinus terebinthifolia collected for this study.

Despite being hated by Florida locals today, this species (scientific name: Schinus terebinthfolia Raddii) in the poison ivy and mango family (Anacardiaceae) actually has a fascinating history as a key medicinal agent in the treatment of wounds and ulcers, as well as for urinary and respiratory infections. My team, the Quave Research Group, found records dating as far back as 1648, when its medical uses were documented by the Dutch naturalist Willem Piso in Historia Naturalis Brasiliae (Fig. 2). As an ethnobotanist, the relationships that people have with plants and especially their medicinal applications are a point of deep fascination for me. Thus, when collecting plants for inclusion in my library of plant natural products for drug discovery, this invasive species was included.

Fig. 2: Historical evidence of Brazilian peppertree used in medicine was found in the 1648 text Historia Naturalis Brasilae by Willem Piso. A digital copy of the text is available from the Biodiversity Heritage Library.

In 2017, my research group together with our longtime collaborators in the Horswill Lab (Fig. 3), first reported on the MRSA anti-virulence effects observed in “430D-F5”, a flavone rich extract from the fruits of this plant.

Fig. 3: Group photo of the Horswill Lab. PI’s Cassandra Quave (Emory University) and Alex Horswill (UC Denver) began their collaboration in 2012 with an R01 project focused on mechanistic studies of agr inhibitors from the European Chestnut (Castanea sativa).

Virulence in Staphylococcus aureus is largely controlled by the accessory gene regulator (agr) system. S. aureus releases autoinducing peptides (AIPs) into the extracellular environment, and this means of quorum sensing is key to the up and down-regulation of a suite of virulence factors, including toxins responsible for hemolysis and tissue damage in a host. We were interested in finding plant natural products that could act as inhibitors of this system as a means of battling infection without necessarily placing the strong selective pressures for resistance observed in classic antibiotic therapies (Fig. 4).

Fig. 4: Brazilian Peppertree fruit are bright red in color, and emit a strong spicy pepper aroma when crushed. My team successfully isolated three bioactive triterpenoid acids from the fruit.

This research project took us to the hot swampy environments of my hometown in DeSoto County, Florida, where I led a group of Emory undergraduate students on a large collection trip to find plant species documented in historic medical texts as remedies for infections (Fig. 5).

Fig. 5: Emory undergraduate students joined our research team in southwest Florida, where we collaborated with local landowners to collect more than 100 species documented in the historic literature for medicinal applications, including the Brazilian peppertree.

Before sunrise each morning, we geared up in field clothes, armed with clippers, plant presses, collection bags and our notebooks, and then set out to different ecosystems—sand dunes, swamplands, pastures, oak hammocks—to collect (Fig. 6). We had no difficulty in finding plenty of peppertree samples for collection due to its abundance throughout the wetland landscapes.

Fig. 6: Our days in the field were long, but fruitful. Here I am pictured on the right with Emory Research Specialist Kate Nelson during a collection expedition in a wetland environment in DeSoto County, Florida.

In the evenings, we worked on carefully arranging our plant specimens in plant presses for herbarium deposit, recording the details of each specimen in our growing database (Fig. 7).

Fig. 7: In addition to bulk plant specimens used in the extractions for this study, we also collected herbarium voucher specimens to serve as a long-term records of the plants found in the region. Plant clippings containing vegetative and reproductive parts were placed in labeled newspaper and pressed between cardboard and slats of wood using the classic botanist’s tool of the plant press. Our active collections resulted in stacks of presses to place in our field drier each day.

Back in the lab, Huaqiao Tang, a visiting scholar from Sichuan Agricultural University in China, worked with the team to isolate the most active constituents found in 430D-F5 (Fig. 8). This involved pulverizing and extracting large batches of the peppertree fruits, and then subjecting them to sequential rounds of partitioning, flash chromatography and preparative HPLC.

Fig. 8: Visiting scholar Huaqiao Tang and Quave Group Research Specialist Marco Caputo used our series of rotary evaporators to remove residual solvents from the liters of fractions produced during the chromatographic isolation of the active triterpenoids.

Each iterative level of fractionation was followed by testing using a suite of agr reporter strains developed by the Horswill Lab. We also examined fractions and compounds for impact of virulence factor outputs, growth inhibition and biofilm inhibition (Fig. 9).  Dr. François Chassagne, a pharmacologist, closely monitored compounds for cytotoxicity against human skin cells (keratinocytes).

Fig. 9: We assessed the impact of the triterpenoid acids on a number of outcomes, including biofilm formation. Here, Quave Research Group Specialist Micah Dettweiler and I examine a biofilm plate stained with crystal violet. 

Once we had some highly active compounds isolated via these screening efforts, natural products chemist, Dr. Gina Porras, and pharmacognosist, Dr. James Lyles, elucidated the structures by mass spectrometry and NMR. Luckily, due to the efficiency of our custom built LEGO® MINDSTORMS robotic open bed fraction collector system (Fig. 10), we were able to isolate sufficient quantities of pure compound to form crystals for single crystal X-ray diffraction with Dr. John Bacsa in the Emory X-ray Crystallography core. These efforts resulted in determination of the full structural conformation of three bioactive triterpenoid acids.

Fig. 10: We leveraged our custom-built open bed fraction collector (run on LEGO MINDSTORM technology) to undertake multiple rounds of preparative chromatographic separation, yielding sufficient quantities of pure compounds for structural elucidation, in vitro and in vivo studies.

In addition to their impact on agr, we wanted to better understand any other relevant targets that these compounds were affecting in S. aureus (Fig. 11). Morgan Brown, Ph.D. candidate in the Horswill Lab, undertook a series of mechanistic assays that revealed an interesting impact on agr-independent nuclease, suggesting potential interactions with the sae system in addition to agr. She also confirmed that the in vitro effects observed in the lab translated to efficacy in a mouse skin infection model.

Fig. 11: We worked with 13 isolates of S. aureus, including the USA300 MRSA Los Angeles Clone isolate (LAC), UAMS-1 MSSA clinical osteomyelitis isolate for biofilm studies, and reporter strains for agr, nuc and mgrA.

The results of this collaborative effort spanning the disciplines of botany, chemistry, pharmacology and microbiology culminated in the first report of triterpenoid acids with potent activity against virulence pathways in S. aureus. The paper reporting these new findings is now available through Open Access in Scientific Reports.

Dr. Cassandra Quave

Assistant Professor and Herbarium Curator, Emory University School of Medicine

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