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1 Department of Biological Sciences, College of Arts and Sciences, Florida International University, Miami, FL 33199, USA
2 Department of Molecular Microbiology and Immunology, College of Medicine, Florida International University, Miami, FL 33199, USA
3 Department of Genetics, Harvard Medical School, and Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA
Correspondence
Kalai Mathee
Kalai.Mathee{at}fiu.edu
Received 7 December 2007
Accepted 19 March 2008
Abbreviations: QS, quorum sensing.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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Conventional anti-pseudomonal treatment includes elevated doses of β-lactam, fluoroquinolone or aminoglycoside antibiotics (Hauser & Sriram, 2005). However, these drugs possess a high degree of toxicity, and mucoid strains of P. aeruginosa are rarely eradicated by these treatments (Hauser & Sriram, 2005; Pedersen, 1992). The failure of existing antibiotics to control infection makes it crucial to find alternatives to currently available drugs. Since pathogenicity in many bacteria is regulated by quorum sensing (QS), or cell-to-cell communication, inhibition of this system can cause attenuation of virulence and protect against infection (Hentzer & Givskov, 2003; Juhas et al., 2005; Smith & Iglewski, 2003a).
Plants have evolved numerous chemical strategies for deterring pathogen attack, including the production of bactericidal and anti-infective compounds, leading to their use as medicines (reviewed by Lewis & Ausubel, 2006). In our previous work, we demonstrated that a number of medicinal plants exhibit anti-QS activity (Adonizio et al., 2006). Extracts of these plants were later shown to have an effect on virulence factor production, biofilm formation, QS gene expression and autoinducer production in P. aeruginosa (Adonizio et al., 2008). In this study, we assessed the ability of three plant extracts to attenuate P. aeruginosa killing of the nematode Caenorhabditis elegans.
Caenorhabditis elegans is well established as a pertinent and practical model for studying bacterial virulence (Darby et al., 1999; Tan & Ausubel, 2000), as a number of P. aeruginosa factors important in the killing of Caenorhabditis elegans are also relevant to mammalian systems (Rahme et al., 1995; Tan et al., 1999a). ‘Fast killing’ of Caenorhabditis elegans by P. aeruginosa strain PA14 (on rich media) is mediated through the production of virulence factors such as phenazines, whereas ‘slow killing’ (on minimal media) occurs via ingestion of the bacteria and subsequent infection (Mahajan-Miklos et al., 1999; Tan et al., 1999a, b). The related strain PAO1 causes death through cyanide poisoning and neuromuscular paralysis (Gallagher & Manoil, 2001). Importantly, P. aeruginosa-mediated killing of Caenorhabditis elegans in all three of these cases is dependent in part on the QS system. Thus addition of plant compounds that have an effect on P. aeruginosa QS should attenuate virulence factor production and the subsequent death of Caenorhabditis elegans.
The advantage of using a live animal model when screening for anti-infective compounds is that both the efficacy and the host toxicity of a plant extract can be tested concurrently. In this report, we show that extracts from three different plant species caused a marked decrease in P. aeruginosa-mediated killing of Caenorhabditis elegans without affecting worm fitness on Escherichia coli. This approach can be expanded to the screening of natural product libraries or native extract sources.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Bacterial strains and growth conditions.
Because the tested extracts have been shown to affect the bacterial QS system (Adonizio et al., 2006, 2008), the QS mutant 
lasR was used as a reference strain. Wild-type P. aeruginosa PAO1 (Holloway & Morgan, 1986) and its isogenic mutant PAO1lasR (Gambello & Iglewski, 1991) were used in the paralytic assay. Wild-type P. aeruginosa PA14 (Rahme et al., 1995) and its PA14lasR (Liberati et al., 2006) were used in the slow-killing and fast-killing assays. E. coli OP50 was used as the control in all assays, as this strain is the standard laboratory food of Caenorhabditis elegans (Brenner, 1974). All bacterial strains were grown overnight in Luria–Bertani broth at 37 °C and transferred to plates, depending on the required conditions.
Nematode culture. The wt Caenorhabditis elegans (Bristol) N2 hermaphrodite strain was used in this study (Brenner, 1974). Worms were synchronized by hypochlorite treatment of gravid adults, hatching of the eggs overnight in M9 minimum buffer (Brenner, 1974) and plating L1-stage worms onto lawns of E. coli on nematode growth medium plates (Brenner, 1974). Synchronized worms were grown to the L4 or young adult stage at 25 °C for use in the killing assays.
Caenorhabditis elegans paralytic assay.
Brain heart infusion agar plates with or without plant extract were seeded with 10 µl of an overnight culture of E. coli OP50 or P. aeruginosa PAO1 or PAO1lasR and incubated at 37 °C for 24 h to form lawns of bacteria (Darby et al., 1999). Nematodes were washed off stock plates and suspended in a minimal volume of M9 buffer (pH 6.5). Droplets containing 20–40 adult nematodes were placed onto the bacterial lawns and the plates were incubated at room temperature (21–23 °C). Worms were evaluated for viability every hour for a total of 4 h. Worms were scored as dead when they no longer responded to physical stimuli.
Caenorhabditis elegans fast-killing assay.
Fast-killing plates (peptone/glucose medium with 0.15 M sorbitol; Mahajan-Miklos et al., 1999) with or without plant extract were seeded with 10 µl of an overnight culture of OP50, PA14 or PA14lasR. Plates were incubated for 24 h at 37 °C and then at room temperature (21–23 °C) for another 12 h. Approximately 20 L4-stage Caenorhabditis elegans were transferred with a wire pick onto plates at this time. Worms were evaluated for viability every hour for a total of 4 h. As in the previous assay, worms were considered dead when they no longer responded to physical stimuli.
Caenorhabditis elegans slow-killing assay.
Slow-killing plates (modified nematode growth medium; Tan et al., 1999b) with or without plant extract were seeded with 10 µl of an overnight culture of OP50, PA14 or PA14lasR. Plates were incubated for 24 h at 37 °C and then at room temperature (21–23 °C) for another 24 h. Approximately 20 L4-stage Caenorhabditis elegans were transferred onto plates at this time. Worms were evaluated for viability every 2–4 h for a total of 58 h. As in the previous assays, worms were considered dead when they no longer responded to physical stimuli.
Statistics and reproducibility. All experiments were performed in triplicate. Killing curves represent the mean of three separate experiments. Data were analysed using one-way analysis of variance with a P value of 0.05 using the statistical software package SPSS. As the starting number of worms was different in each case, the percentage of worms still alive rather than the actual number of worms was used for comparison.
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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lasR was used as a reference strain. Although there is some precedence for testing plant extracts in a nematode model system (Rasmussen et al., 2005), this is the first study to evaluate a statistically significant number of worms in both toxin and infection-based assays.
Medicinal plants rescue paralytic killing of Caenorhabditis elegans by P. aeruginosa
Previous studies have shown that QS signalling is required for maximum levels of worm killing (Mahajan-Miklos et al., 1999; Tan et al., 1999b). Thus the success of these plant extracts against P. aeruginosa PAO1 in terms of blocking QS signalling (Adonizio et al., 2008) suggested that they might also reduce death in a PAO1–nematode model. Approximately 50 % of the worms died between 1 and 2 h after transfer to PAO1, with all nematodes dead after 4 h (Fig. 1a
). In contrast, all of the worms on E. coli OP50 remained alive throughout the assay (not shown). As expected, the QS mutant PAO1lasR showed reduced nematode death, with 85 % alive between 1 and 2 h and 74 % still alive at 4 h (Fig. 1a). After 4 h, approximately 85, 84 and 87 % of the worms were alive on PAO1 plates that contained Conocarpus erectus, Callistemon viminalis and B. buceras extracts, respectively (Fig. 1a). In this and all other assays in this study, there was no observable effect of the added plant extracts on worm survival, lifespan or brood size on E. coli OP50 (not shown), indicating a lack of toxicity of the compounds.
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), i.e. there was a significant difference compared with PAO1lasR and the extract-containing plates (P <0.05 in all cases), but not among individual extracts at the end of the assay. All extract plates and the lasR mutant were significantly different from wild-type PAO1 without treatment.
Death via PAO1 is due to cyanide asphyxiation and paralysis of Caenorhabditis elegans (Gallagher & Manoil, 2001). The hcn operon in P. aeruginosa mediates cyanide production and is controlled by the QS regulators LasR and RhlR (Pessi & Haas, 2000). Attenuation of virulence and nematode mortality has been shown with both lasR (Darby et al., 1999) and hcn (Gallagher & Manoil, 2001) strains. Thus the results from the paralytic assay suggested that the addition of these extracts was affecting the production of cyanide either through hcn directly or indirectly via the QS genes. The latter hypothesis agrees with our previous in vitro analysis in PAO1 (Adonizio et al., 2008), which showed a significant reduction in lasR and rhlR gene activity by these extracts.
Medicinal plants prevent fast killing of Caenorhabditis elegans by P. aeruginosa
The results of the fast-killing assay are shown in Fig. 1(b). On PA14, approximately 50 % of the worms were dead within 2 h, with all worms dead by 4 h. At this point, all of the worms on OP50 were still alive (not shown). Again, the QS mutant (PA14lasR) reduced nematode death, with 75 % alive between 1 and 2 h and 47 % alive at 4 h. At the end of the assay, approximately 53, 75 or 90 % of the worms were alive on PAO1 plates with added Conocarpus erectus, Callistemon viminalis and B. buceras extract, respectively (Fig. 1b). As with PAO1, all three of the plant extracts suppressed killing at or above the level of the QS mutant. There was no significant difference between PA14lasR and the Conocarpus erectus extract plates at 4 h (P >0.05); however, the Callistemon viminalis and B. buceras extracts were significantly better at preventing worm death (P <0.01 in both cases). Extract-containing plates were all significantly different from each other, but still showed a large reduction in nematode death when compared with wild-type PA14 without treatment. Overall, there was a pronounced inhibitory effect of the plant extracts on P. aeruginosa PA14 fast killing of Caenorhabditis elegans.
Fast killing of Caenorhabditis elegans is mediated by the production of virulence factors such as phenazines (Mahajan-Miklos et al., 1999). A phnAphnB deletion mutant has been shown to abolish nematode death completely, whilst a TnphoA mutation of the related gene phzB has been shown to greatly reduce mortality in mice and Arabidopsis (Mahajan-Miklos et al., 1999). Like many virulence factors, phenazines are partially under the control of the QS gene rhlR (Brint & Ohman, 1995; Latifi et al., 1995). The results from the PA14 fast-killing assay suggested that the addition of extracts affected phenazine production, either directly through the phz and phn genes or indirectly through the QS system via rhlR. All three extracts were shown previously to significantly affect the rhlI/R system; however, Conocarpus erectus has less of an effect on N-acylhomoserine lactone production and biofilm formation (strain PAO1; Adonizio et al., 2008) than either B. buceras or Callistemon viminalis. Although still successful, Conocarpus erectus was less efficient in preventing nematode death in the fast-killing assay than the other extracts.
Plant extracts reduce the mortality of Caenorhabditis elegans due to slow killing by P. aeruginosa
The slow-killing assay left 50 % of nematodes dead on PA14 between 48 and 50 h, with all worms dead by 58 h (Fig. 1c). The control worms on E. coli OP50 remained alive throughout the assay (not shown). The QS mutant (PA14lasR) reduced nematode death, with 75 % alive between 48 and 50 h and 53 % alive at 58 h. At this time, approximately 60, 59 and 57 % of worms were alive on PA14 plates with added Conocarpus erectus, Callistemon viminalis and B. buceras extract, respectively (Fig. 1c). All three of the plant extracts, when added to plates containing wild-type PA14, suppressed killing to the level of the QS mutant. There was no significant difference between PA14lasR and the extract plates or between individual extracts at 58 h (P >0.05 in all cases); however, all extracts were significantly different from PA14 without treatment, suggesting a marked effect of the plant extracts on P. aeruginosa infection of Caenorhabditis elegans.
Slow killing of Caenorhabditis elegans occurs over approximately 60 h due to ingestion of and subsequent infection by P. aeruginosa (Tan et al., 1999a). Nematode mortality is attenuated by TnphoA mutations of lasR and gacA (Tan et al., 1999b), suggesting that QS is required for the infection process. The addition of plant extracts in this assay drastically reduced nematode death, suggesting an effect on lasR or gacA. Previous work on these extracts corroborated the inhibitory effect on lasR; however, the effect on gacA was not tested directly (Adonizio et al., 2008). An effect on either of these factors remains a plausible hypothesis.
Conclusions
The three plant extracts from Conocarpus erectus (Combretaceae), Callistemon viminalis (Myrtaceae) and B. buceras (Combretaceae), in all three assays, showed a highly significant reduction in virulence when compared with wild-type PAO1 and PA14 without treatment. Overall, the tested plant extracts reduced nematode death by approximately 60–90 % on wild-type P. aeruginosa. In each case, this reduction was equal to or greater than that of the corresponding QS mutant strain. The fact that the plant extracts reduced virulence across the board suggests that they are possibly affecting an upstream QS gene such as las or rhl, or perhaps a global regulator such as GacA. This further corroborates our previous data on the anti-QS effect of these plant extracts (Adonizio et al., 2008). All extracts inhibited nematode death without any significant bactericidal effect, leaving QS inhibition as a plausible hypothesis. In addition, none of the tested plants showed any toxicity in the nematode model, making them reasonable candidates for purification and drug development.
Conocarpus erectus, B. buceras and Callistemon viminalis (and other closely related species) have been used medicinally to treat bacterial infections either as teas or as poultices (Burkhill, 1985; Irvine, 1961; Melendez, 1982; Morton, 1981; Stewart & Percival, 1997). Thus the plants were extracted with hot water to provide greater congruity with traditional preparation methods. Although teas and poultices are many steps removed from modern formulae, traditional use suggests the potential success of topical or enteral routes of administration.
With the increase in bacterial resistance to antibiotics, we should look to the past in the hope of finding solutions for the future. Plants have been used medicinally for thousands of years and, even without marked antibiotic activity, these three plants are still efficacious in ameliorating disease. We have previously shown the activity of these plants on P. aeruginosa alone and, although the exact mechanism of action is not yet known, the nematode experiments described in this paper are consistent with their previous and potential further use as anti-infectives.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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