Scientists have identified specific compounds from the Brazilian peppertree – a weedy, invasive shrub in Florida – that reduce the virulence of antibiotic-resistant staph bacteria.
Scientific Reports published the research, demonstrating that triterpenoid acids in the red berries of the plant “disarm” dangerous staph bacteria by blocking its ability to produce toxins.
The work was led by the lab of Cassandra Quave, an assistant professor in Emory University’s Center for the Study of Human Health and the Emory School of Medicine’s Department of Dermatology.
The researchers’ laboratory experiments provide the first evidence that triterpenoid acids pack a punch against methicillin-resistant Staphylococcus aureus, known as MRSA.
The Brazilian peppertree (Schinus terebinthifolia), native to South America, is also abundant in Florida, where it forms dense thickets that crowd out native species.
“It is a noxious weed that many people in Florida hate, for good reason,” Quave says.
“But, at the same time, there is this rich lore about the Brazilian Peppertree in the Amazon, where traditional healers have used the plant for centuries to treat skin and soft tissue infections.”
Quave, a leader in the field of medical ethnobotany and a member of the Emory Antibiotic Resistance Center, studies how indigenous people incorporate plants in healing practices to uncover promising candidates for new drugs.
The Centers for Disease Control and Prevention calls antibiotic resistance “one of the biggest public health challenges of our time.”
Each year in the U.S., at least 2.8 million people get antibiotic-resistant infections, leading to more than 35,000 deaths.
“Even in the midst of the current viral pandemic of COVID-19, we can’t forget about the issue of antibiotic resistance,” Quave says.
She notes that many COVID-19 patients are receiving antibiotics to deal with secondary infections brought on by their weakened conditions, raising concerns about a later surge in antibiotic-resistant infections.
In 2017, the Quave lab published the finding that a refined, flavone-rich mix of 27 compounds extracted from the berries of the Brazilian peppertree inhibits formation of skin lesions in mice infected with MRSA.
The extract works not by killing the MRSA bacteria, but by repressing a gene that allows the bacteria cells to communicate with one another.
Blocking that communication prevents the cells from taking collective action, which essentially disarms the bacteria by preventing it from excreting the toxins it uses to damage tissues.
The body’s immune system then stands a better chance of healing a wound.
That approach is different from the typical treatment of blasting deadly bacteria with drugs designed to kill them, which can help fuel the problem of antibiotic resistance. Some of the stronger bacteria may survive these drug onslaughts and proliferate, passing on their genes to offspring and leading to the evolution of deadly “super bugs.”
For the current paper, the researchers wanted to narrow down the scope of 27 major compounds from the berries to isolate the specific chemicals involved in disarming MRSA.
They painstakingly refined the original compounds, testing each new iteration for its potency on the bacteria. They also used a series of analytical chemistry techniques, including mass spectrometry, nuclear magnetic resonance spectroscopy and X-ray crystallography to gain a clear picture of the chemicals involved in the anti-virulence mechanism.
The results showed that three triterpenoid acids worked equally well at inhibiting MRSA from forming toxins in a petri dish, without harming human skin cells. And one of the triterpenoid acids worked particularly well at inhibiting the ability of MRSA to form lesions on the skin of mice.
The researchers also demonstrated that the triterpenoid acids repressed not just one gene that MRSA uses to excrete toxins, but two genes involved in that process.
“Nature is the best chemist, hands down,” Quave says. She adds that weeds, in particular, tend to have interesting chemical arsenals that they may use to protect them from diseases so they can more easily spread in new environments.
The research team plans to do further studies to test the triterpenoid acids as treatments for MRSA infections in animal models. If those studies are promising, the next step would be to work with medicinal chemists to optimize the compounds for efficacy, delivery and safety before testing on humans.
“Plants are so incredibly complex chemically that identifying and isolating particular extracts is like picking needles out of haystacks,” Quave says.
“When you’re able to pluck out molecules with medicinal properties from these complex natural mixtures, that’s a big step forward to understanding how some traditional medicines may work, and for advancing science towards a potential drug development pathway.”
Staphylococcus aureus has long been recognized as a significant cause of both community-acquired (CA) and healthcare associated (HA) infections, such as endocarditis, septic arthritis, osteomyelitis, and necrotizing pneumonia1.
In the past decade, an increasing number of infections by methicillin-resistant S. aureus (MRSA) have been documented and estimates in the United States suggest that MRSA causes between 11,000–18,000 deaths and 80,000 invasive infections annually2,3.
Furthermore, a single clone of CA-MRSA (USA300) has emerged as the most common cause of all skin and soft tissue infections in the United States and continues to pose a serious public health threat in community and healthcare settings4,5.
With the last novel class of antibiotics to be brought to market being discovered in the 1980s, new strategies are necessary to respond to the widespread development of antibiotic resistant infections6.
Some promising future approaches include promoting infection prevention to reduce the need for antibiotics, encouraging investment in antimicrobial agents with new regulatory policies and economic models, slowing the spread of resistance in order to preserve the useful lives of available antibiotics, and developing novel therapeutics that modulate host-microbe interactions without placing direct selective pressures known to drive resistance resistance7.
S. aureus antibiotic resistance can be due to various extrinsic, or acquired, mechanisms such as enzymatic drug modification, mutated drug targets, enhanced efflux pump expression, and altered membrane permeability8.
Additionally, intrinsic mechanisms of resistance such as biofilm formation and production of virulence factors that subvert the host immune response, play a key role in prolonging and increasing the pathogenicity of S. aureus infections.
The production of a suite of superantigens, toxins and exo-enzymes heavily contribute to the invasive nature of staphylococcal infections, and this virulence mechanism is largely controlled by quorum sensing (QS).
S. aureus uses multiple two-component systems to sense and respond to changes in cell density and environmental cues. One of these two-component systems is the Accessory Gene Regulator (agr) system, which has been extensively characterized for its complex regulatory role in global MRSA virulence and its requirement for MRSA skin infection pathogenesis1,2,9.
Agr senses and responds to its cognate auto-inducing peptide (AIP) signal in a cell-density dependent manner. All described AIP signals are between 7 and 12 amino acids long, with the C-terminal amino acids constrained in a thiolactone or lactone ring and an N-terminal “tail” extension10,11. S. aureus has four allelic variants of the agr system (agr-I, II, III, IV), and each recognizes and responds to its cognate AIP signal (AIP-I, II, III, IV). The dominant CA-MRSA USA300 is agr Type-I, while health-care associated MRSA strains are predominantly agr Type-II12,13.
The agrBDCA operon encodes the AIP precursor (AgrD) and integral membrane protease (AgrB) necessary for final AIP processing, as well as the membrane-localized histidine kinase sensor (AgrC) and response regulator (AgrA).
A hypervariable region spanning agrBDC determines the agr type. At sufficient local concentration, cognate AIPs bind AgrC, which dimerizes and phosphorylates the response regulator AgrA.
Downstream, activated AgrA binds chromosomal promoters P2 and P3 to induce the transcription of the agrBDCA operon and the small RNA regulator RNAIII, respectively. As the primary effector of the agr system, RNAIII post-transcriptionally regulates the expression of more than 200 virulence-associated genes including toxins (α, β, δ, and γ), proteases, lipases, superantigens (toxic shock syndrome toxin-1, enterotoxins B, C, and D), and leukocidins10,14,15,16.
Additionally, the RNAIII transcript includes the hld gene for δ-toxin, an amphipathic 26 amino acid peptide with cytolytic activity.
Given such extensive agr-dependent virulence factor production, inhibition of agr signaling (i.e. quorum sensing) has been proposed as an alternative strategy to prevent or treat MRSA skin infections.
Several promising molecules have been reported, including the AgrA inhibitor savarin3, AgrB inhibitor ambuic acid4, and the pan-agr inhibitor apicidin5. Previously, we reported on the quorum sensing inhibitory activity of a refined extract from the fruit of the Brazilian peppertree (Schinus terebinthifolia Raddi, Anacardiaceae) against several MRSA strains6. S. terebinthifolia is an evergreen shrub native to South and Central America and grows as a noxious weed in the southern United States.
It was introduced to the USA just over 100 years ago as an ornamental plant, and has since spread via a process of stratified dispersal around established populations and by long-distance jumps due to human activities17.
In Florida, where we collected it for the present study, S. terebinthifolia is listed as a Category I invasive exotic species throughout the state18. Eradication efforts are underway in Florida, and have included largescale removal in the Everglades, application of herbicides, and most recently, deployment of parasitizing insects as a biocontrol measure19.
However, in Brazil where the plant is valued as a medicinal species and popularly known as “pimenta-rosa”, it is used in folk medicine for the treatment of an array of illnesses, including several associated with infection and inflammation.
Several parts of the plant have been found to contain chemicals with antimicrobial20,21, anti-inflammatory22, antioxidant22,23 and anti-tumor24 bioactivities.
The current study characterizes the anti-virulence activity of three triterpenoid acids isolated from the fruit of S. terebinthifolia against clinically-relevant MRSA strains.
More information: Huaqiao Tang et al, Triterpenoid acids isolated from Schinus terebinthifolia fruits reduce Staphylococcus aureus virulence and abate dermonecrosis, Scientific Reports (2020). DOI: 10.1038/s41598-020-65080-3
References
1.Lee, A. S. et al. Methicillin-resistant Staphylococcus aureus. Nat. Rev. Dis. Primers 4, 18033, https://doi.org/10.1038/nrdp.2018.33 (2018).
2.Wilcox, M. et al. Reporting elevated vancomycin minimum inhibitory concentration in methicillin-resistant Staphylococcus aureus: consensus by an International Working Group. Future Microbiol., https://doi.org/10.2217/fmb-2018-0346 (2019).
3.Challagundla, L. et al. Range expansion and the origin of USA300 North American epidemic Methicillin-Resistant Staphylococcus aureus. mBio 9, https://doi.org/10.1128/mBio.02016-17 (2018).
4.King, M. D. et al. Emergence of community-acquired methicillin-resistant Staphylococcus aureus USA 300 clone as the predominant cause of skin and soft-tissue infections. Ann. Intern. Med. 144, 309–317, https://doi.org/10.7326/0003-4819-144-5-200603070-00005 (2006).
5.Stryjewski, M. E. & Chambers, H. F. Skin and soft-tissue infections caused by community-acquired methicillin-resistant Staphylococcus aureus. Clin. Infect. Dis. 46, S368–S377, https://doi.org/10.1086/533593 (2008).
6.Silver, L. L. Challenges of antibacterial discovery. Clin Microbiol Rev 24, 71–109, https://doi.org/10.1128/CMR.00030-10 (2011).
7.Spellberg, B., Bartlett, J. G. & Gilbert, D. N. The future of antibiotics and resistance. N. Engl. J. Med 368, 299–302, https://doi.org/10.1056/NEJMp1215093 (2013).
8.van Duijkeren, E., Schink, A. K., Roberts, M. C., Wang, Y. & Schwarz, S. Mechanisms of bacterial resistance to antimicrobial agents. Microbiol. Spectr. 6, https://doi.org/10.1128/microbiolspec.ARBA-0019-2017 (2018).
9.Cheung, G. Y., Wang, R., Khan, B. A., Sturdevant, D. E. & Otto, M. Role of the accessory gene regulator agr in community-associated methicillin-resistant Staphylococcus aureus pathogenesis. Infect. Immun. 79, 1927–1935, https://doi.org/10.1128/iai.00046-11 (2011).
10.Thoendel, M., Kavanaugh, J. S., Flack, C. E. & Horswill, A. R. Peptide signaling in the staphylococci. Chem. Rev. 111, 117–151, https://doi.org/10.1021/cr100370n (2011).
11.Olson, M. E. et al. Staphylococcus epidermidis agr quorum-sensing system: signal identification, cross talk, and importance in colonization. J. Bacteriol. 196, 3482–3493, https://doi.org/10.1128/JB.01882-14 (2014).
12.Tsuji, B. T., Rybak, M. J., Cheung, C. M., Amjad, M. & Kaatz, G. W. Community- and health care-associated methicillin-resistant Staphylococcus aureus: a comparison of molecular epidemiology and antimicrobial activities of various agents. Diagn. Microbiol. Infect. Dis. 58, 41–47, https://doi.org/10.1016/j.diagmicrobio.2006.10.021 (2007).
13.Grundstad, M. L. et al. Quorum sensing, virulence, and antibiotic resistance of USA100 methicillin-resistant Staphylococcus aureus isolates. mSphere 4, e00553–00519, https://doi.org/10.1128/mSphere.00553-19 (2019).
14.Novick, R. P. & Geisinger, E. Quorum sensing in staphylococci. Annu. Rev. Genet. 42, 541–564, https://doi.org/10.1146/annurev.genet.42.110807.091640 (2008).
15.Parlet, C. P. et al. Apicidin attenuates MRSA virulence through quorum-sensing inhibition and enhanced host defense. Cell Rep 27, 187–198.e186, https://doi.org/10.1016/j.celrep.2019.03.018 (2019).
16.Kobayashi, S. D., Malachowa, N. & DeLeo, F. R. Pathogenesis of Staphylococcus aureus abscesses. Am J Pathol 185, 1518–1527, https://doi.org/10.1016/j.ajpath.2014.11.030 (2015).
17.Williams, D. A., Muchugu, E., Overholt, W. A. & Cuda, J. P. Colonization patterns of the invasive Brazilian peppertree, Schinus terebinthifolius, in Florida. Heredity 98, 284–293, https://doi.org/10.1038/sj.hdy.6800936 (2007).
18.Pernas, T. et al. Florida Exotic Pest Plant Council’s 2019 List of Invasive Plant Species. (Florida Exotic Pest Plant Council, 2019).
19.Gioeli, K. T., Enloe, S. F., Minteer, C. R. & Langeland, K. A. Brazilian peppertree control. 1-5 (University of Florida Agronomy Department, UF/IFAS Extension. SS-AGR-17, 2018).
20.da Silva Dannenberg, G., Funck, G. D., Mattei, F. J., da Silva, W. P. & Fiorentini, Â. M. Antimicrobial and antioxidant activity of essential oil from pink pepper tree (Schinus terebinthifolius Raddi) in vitro and in cheese experimentally contaminated with Listeria monocytogenes. Innov. Food Sci. Emerg. Technol. 36, 120–127 (2016).
21.Silva, A. et al. Antibacterial activity, chemical composition, and cytotoxicity of leaf’s essential oil from Brazilian pepper tree (Schinus terebinthifolius, Raddi). Braz. J. Microbiol. 41, 158–163 (2010).
22.da Silva, M. M. et al. Schinus terebinthifolius: phenolic constituents and in vitro antioxidant, antiproliferative and in vivo anti-inflammatory activities. Rev Bras Farmacogn 27, 445–452 (2017).
23.Jeribi, C., Karoui, I. J., Hassine, D. B. & Abderrabba, M. Comparative study of bioactive compounds and antioxidant activity of Schinus terebinthifolius Raddi fruits and leaves essential oils. Int. J. Sci. Res. 3, 453–458 (2014).
24.Ramos, D. M. B. et al. Evaluation of antitumor activity and toxicity of Schinus terebinthifolia leaf extract and lectin (SteLL) in sarcoma 180-bearing mice. J. Ethnopharmacol. 233, 148–157 (2019).
25.Muhs, A. et al. Virulence inhibitors from Brazilian Peppertree block quorum sensing and abate dermonecrosis in skin infection models. Sci. Rep. 7, 42275, https://doi.org/10.1038/srep42275, https://www.nature.com/articles/srep42275#supplementary-information (2017).
26.Shirane, N., Hashimoto, Y., Ueda, K., Takenaka, H. & Katoh, K. Ring-A cleavage of 3-oxo-olean-12-en-28-oic acid by the fungus Chaetomium longirostre. Phytochemistry 43, 99–104, https://doi.org/10.1016/0031-9422(96)00266-X (1996).
27.Mulholland, D. A. & Nair, J. J. Triterpenoids from Dysoxylum pettigrewianum. Phytochemistry 37, 1409–1411, https://doi.org/10.1016/S0031-9422(00)90421-7 (1994).
28.Morais, T. R. et al. Antiparasitic activity of natural and semi-synthetic tirucallane triterpenoids from Schinus terebinthifolius (Anacardiaceae): structure/activity relationships. Molecules 19, 5761–5776, https://doi.org/10.3390/molecules19055761 (2014).
29.Paharik, A. E. et al. Coagulase-negative staphylococcal strain prevents Staphylococcus aureus colonization and skin infection by blocking quorum sensing. Cell Host Microbe 22, 746–756.e745, https://doi.org/10.1016/j.chom.2017.11.001 (2017).
30.Vandeputte, O. M. et al. Identification of catechin as one of the flavonoids from Combretum albiflorum bark extract that reduces the production of quorum-sensing-controlled virulence factors in Pseudomonas aeruginosa PAO1. Appl. Environ. Microbiol. 76, 243, https://doi.org/10.1128/AEM.01059-09 (2010).
31.Vattem, D. A., Mihalik, K., Crixell, S. H. & McLean, R. J. C. Dietary phytochemicals as quorum sensing inhibitors. Fitoterapia 78, 302–310, https://doi.org/10.1016/j.fitote.2007.03.009 (2007).
32.Rasmussen, T. B. & Givskov, M. Quorum sensing inhibitors: a bargain of effects. Microbiology 152, 895–904, https://doi.org/10.1099/mic.0.28601-0 (2006).
33.Xie, P. et al. Enhanced extraction of hydroxytyrosol, maslinic acid and oleanolic acid from olive pomace: Process parameters, kinetics and thermodynamics, and greenness assessment. Food Chem. 276, 662–674, https://doi.org/10.1016/j.foodchem.2018.10.079 (2019).
34.Silva-Júnior, E. et al. Phytochemical compounds and pharmacological properties from Schinus molle Linnaeus and Schinus terebinthifolius Raddi (Anacardiaceae). J. Chem. Pharm. Res. 7, 389–393 (2015).
35.Morais, T. R. et al. Application of an ionic liquid in the microwave assisted extraction of cytotoxic metabolites from fruits of Schinus terebinthifolius Raddi (Anacardiaceae). J. Braz. Chem. Soc. 28, 492–497 (2017).
36.Jain, M. K. et al. Specific competitive inhibitor of secreted phospholipase A2 from berries of Schinus terebinthifolius. Phytochemistry 39, 537–547, https://doi.org/10.1016/0031-9422(94)00960-2 (1995).
37.Da Silva, G. F. et al. G. Lanost-7-en triterpenes from stem bark of Santiria trimera. Phytochemistry 29, 1629–1632, https://doi.org/10.1016/0031-9422(90)80135-4 (1990).
38.Boles, B. R. & Horswill, A. R. Agr-mediated dispersal of Staphylococcus aureus biofilms. PLoS Pathog. 4, e1000052–e1000052, https://doi.org/10.1371/journal.ppat.1000052 (2008).
39.Lister, J. L. & Horswill, A. R. Staphylococcus aureus biofilms: recent developments in biofilm dispersal. Front Cell Infect Microbiol 4, https://doi.org/10.3389/fcimb.2014.00178 (2014).
40.Gilabert, M. et al. Sesqui- and triterpenoids from the liverwort Lepidozia chordulifera inhibitors of bacterial biofilm and elastase activity of human pathogenic bacteria. Phytomedicine 22, 77–85, https://doi.org/10.1016/j.phymed.2014.10.006 (2015).
41.Garo, E. et al. Asiatic acid and corosolic acid enhance the susceptibility of Pseudomonas aeruginosa biofilms to tobramycin. Antimicrob. Agents Chemother. 51, 1813–1817, https://doi.org/10.1128/aac.01037-06 (2007).
42.Hu, J. F. et al. Bacterial biofilm inhibitors from Diospyros dendo. J. Nat. Prod. 69, 118–120, https://doi.org/10.1021/np049600s (2006).
43.Ren, D. et al. Differential gene expression for investigation of Escherichia coli biofilm inhibition by plant extract ursolic acid. Appl. Environ. Microbiol. 71, 4022–4034, https://doi.org/10.1128/aem.71.7.4022-4034.2005 (2005).
44.Li, H. E. et al. Glycyrrhetinic acid protects mice from Staphylococcus aureus pneumonia. Fitoterapia 83, 241–248, https://doi.org/10.1016/j.fitote.2011.10.018 (2012).
45.Tugume, P. et al. Ethnobotanical survey of medicinal plant species used by communities around Mabira Central Forest Reserve, Uganda. J. Ethnobiol. Ethnomed. 12, 5, https://doi.org/10.1186/s13002-015-0077-4 (2016).
46.Crosby, H. A. et al. The Staphylococcus aureus ArlRS two-component system regulates virulence factor expression through MgrA. Mol. Microbiol. 113, 103–122, https://doi.org/10.1111/mmi.14404 (2020).
47.Crosby, H. A. et al. The Staphylococcus aureus global regulator MgrA modulates clumping and virulence by controlling surface protein expression. PLoS Pathog. 12, e1005604–e1005604, https://doi.org/10.1371/journal.ppat.1005604 (2016).
48.Yan, H., Wang, Q., Teng, M. & Li, X. The DNA-binding mechanism of the TCS response regulator ArlR from Staphylococcus aureus. J. Struct. Biol. 208, 107388, https://doi.org/10.1016/j.jsb.2019.09.005 (2019).
49.Quave, C. L. et al. Castanea sativa (European Chestnut) leaf extracts rich in ursene and oleanene derivatives block Staphylococcus aureus virulence and pathogenesis without detectable resistance. PLoS One 10, e0136486, https://doi.org/10.1371/journal.pone.0136486 (2015).
50.Quave, C. & Horswill, A. in Methods in Molecular Biology 1673, 363–370 (2018)
51.Quave, C. L. et al. Ellagic acid derivatives from Rubus ulmifolius inhibit Staphylococcus aureus biofilm formation and improve response to antibiotics. PLoS One 7, e28737, https://doi.org/10.1371/journal.pone.0028737 (2012).
52.Beenken, K. E., Blevins, J. S. & Smeltzer, M. S. Mutation of sarA in Staphylococcus aureus limits biofilm formation. Infect. Immun. 71, 4206–4211, https://doi.org/10.1128/iai.71.7.4206-4211.2003 (2003).
