PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of aacPermissionsJournals.ASM.orgJournalAAC ArticleJournal InfoAuthorsReviewers
 
Antimicrob Agents Chemother. 2010 July; 54(7): 3065–3067.
Published online 2010 May 17. doi:  10.1128/AAC.01825-09
PMCID: PMC2897284

Endocannabinoids Inhibit the Growth of Free-Living Amoebae[down-pointing small open triangle]

Abstract

The cannabinoid Δ9-tetrahydrocannabinol inhibits the growth of some pathogenic amoebae in vitro and exacerbates amoebic encephalitis in animal models. However, the effects of endogenous cannabinoids on amoebae remain unknown. Therefore, we tested several endocannabinoids (N-acyl ethanolamines and 2-O-acyl glycerol) on different genera of amoebae. The results showed that all of the endocannabinoids tested inhibit amoebic growth at subpharmacological doses, with 50% inhibitory concentrations ranging from 15 to 20 μM. A nonhydrolyzable endocannabinoid had similar effects, showing that the inhibition seen results from endocannabinoids per se rather than from a catabolic product.

Some free-living amoebae, such as members of the genera Acanthamoeba and Naegleria, are responsible for severe encephalitis and dermatitis with increasing prevalence, especially in immunocompromised patients (3, 11, 12). In addition to numerous side effects, such as seizures, nausea, and vomiting, granulomatous amebic encephalitis (GAE) is usually fatal to patients who do not have access to efficient therapeutic approaches (11). The plant-derived psychoactive cannabinoid Δ9-tetrahydrocannabinol (Δ9-THC) was shown to inhibit the growth of the pathogen Naegleria fowleri in vitro (14). However, this cannabinoid also shows immunosuppressive activity and exacerbates encephalitis due to opportunistic amoebae in a mouse model of GAE by inhibiting macrophage-like cell activity (2, 3). Unlike Δ9-THC, the endogenous cannabinoid 2-O-arachidonoyl glycerol (2-O-AG; one of the two prototypic endocannabinoids, along with N-arachidonoyl ethanolamine) activates immune responses, including chemotaxis of macrophages and microglial cells (3, 17), but its effects on free-living amoebae remain unknown. Some observations showed that amoebae display an active endocannabinoid system inasmuch as N-acyl phosphatidylethanolamine (the precursor of the N-acyl ethanolamine endocannabinoids) is expressed in a regulated fashion during the development of Dictyostelium discoideum amoeba (8). Moreover, arachidonoyl ethanolamine is metabolized in Tetrahymena pyriformis through fatty acid amide hydrolase activity (7, 9). Endocannabinoids may therefore impact amoebic cell fate. Therefore, we tested the effects of several endocannabinoids (N-arachidonoyl ethanolamine, 2-O-AG, and 2-O-AG ether from Cayman Chemical, Ann Arbor, MI) on different genera of amoebae.

The amoebae used in this study were Acanthamoeba castellanii (By 02.2.4), Hartmannella vermiformis (Ax.5.2e4b), and Willaertia magna c2c Maky (ATCC PTA-7824). Cells were grown at 30°C on a lawn of Escherichia coli on nonnutrient agar and were established in axenic culture at 37°C in serum casein glucose yeast extract medium (SCGYEM) (4).

On day 0 of experiments, amoebae were seeded in culture tubes at a concentration of 2 × 105 cells/ml of SCGYEM which either contained various concentrations of endocannabinoids (see figure legend for details) or did not (controls). Endocannabinoids were added to the medium in an ethanol solution (ethanol at a final concentration of 0.05%). Control tubes also contained ethanol (vehicle). The tubes were placed at 37°C in a slanting position, and the cell concentrations were determined on a daily basis up to day 3 using a hemocytometer. Exposure of different free-living amoebae to N-arachidonoyl ethanolamine at a concentration of 10 μg/ml inhibited the growth of all of the amoebic species tested (Fig. (Fig.11 A). After 3 days, the growth of A. castellanii, W. magna, and H. vermiformis was reduced by 68%, 58%, and 96%, respectively, compared to that of controls (the respective 50% inhibitory concentrations [IC50s] of anandamide were ~17, 20, and 14 μM). Inhibition of amoebic growth was also observed with lower doses (2 and 5 μg/ml) of N-arachidonoyl ethanolamine (data not shown). This growth inhibition is further illustrated in Fig. Fig.1B,1B, showing that anandamide prevented the formation of amoebic cell monolayers. This effect was also obtained using endocannabinoids of the 2-O-acyl glycerol class (Fig. (Fig.1B).1B). The IC50s were in the same range as those of N-arachidonoyl ethanolamine. A toxic effect was observed at a concentration of 20 μg/ml, as shown by the decrease in viable cell concentrations compared to the number of cells seeded at day 0 (Fig. (Fig.1A).1A). These effects may result from a direct action of endocannabinoids on amoebae or from the action of one of their catabolic products. In order to test that possibility, we compared the inhibitory effect of 2-O-AG with that of a nonhydrolyzable analogue (10), i.e., 2-O-AG ether (Table (Table1).1). The two molecules had similar effects on the various amoebae, although H. vermiformis proved to be more sensitive than the other two species at the concentration used (Table (Table11 compares the percentages of inhibition versus those obtained with the control). This observation strongly suggests that the inhibition of cell growth results from endocannabinoids per se rather than from a catabolic product.

FIG. 1.
Effects of endocannabinoids on amoebic growth. (A) Effect of N-arachidonoyl ethanolamine on amoebic growth. Cells were cultured for up to 3 days (d) either with (white bars, 10 μg/ml; gray bars, 20 μg/ml) or without (black bars) endocannabinoids. ...
TABLE 1.
Effect of a nonhydrolyzable endocannabinoid on amoebic growtha

These results showed that endocannabinoids displayed similar effects to the one reported for Δ9-tetrahydrocannabinol on amoebic growth (14). Moreover, the inhibitory effects of endocannabinoids were observed at doses lower than or similar to the pharmacological concentrations used in animal studies (16). These observations are particularly interesting since, unlike Δ9-tetrahydrocannabinol, some endocannabinoids, such as 2-O-AG, are known to stimulate the immune response, including macrophage activity (3) (17). This property, taken together with our observations that endocannabinoids strongly inhibit the growth of free-living amoebae, suggests that modulation of the endocannabinoid system may be used in designing therapeutic approaches for pathogenic amoeba infections. Cannabinoids have already been used in the treatment of human glioma tumors with promising results (15). Endocannabinoids administrated in vivo would probably impact signaling through CB1 and CB2 receptors that are mainly expressed in the central nervous system and immune cells, respectively, and neurobehavioral effects (spontaneous activity, hypothermia, antinociception, and catalepsy) would be expected, as shown in animals (6). However, unlike Δ9-tetrahydrocannabinol, these pharmacological effects of endocannabinoids are completely dissipated by 30 min because of their different metabolism and half-life (1, 18). We do not know what mechanisms underlie the effect of endocannabinoids on the growth of free-living amoeba. Phylogenetic studies suggest that protozoans do not express CB1 and CB2 cannabinoid receptors (13). If this is true, it would imply that endocannabinoids act on free-living amoebas through different mechanisms. Indeed, endocannabinoids can modulate intracellular targets (reviewed in reference 17) and they can be metabolized to prostamides upon cyclooxygenase activity (19). A cyclooxygenase-like enzyme has recently been characterized in protozoa (5).

Footnotes

[down-pointing small open triangle]Published ahead of print on 17 May 2010.

REFERENCES

1. Barnett, G., V. Licko, and T. Thompson. 1985. Behavioral pharmacokinetics of marijuana. Psychopharmacology (Berl.) 85:51-56. [PubMed]
2. Cabral, G. A., and F. Marciano-Cabral. 2004. Cannabinoid-mediated exacerbation of brain infection by opportunistic amebae. J. Neuroimmunol. 147:127-130. [PubMed]
3. Cabral, G. A., E. S. Raborn, L. Griffin, J. Dennis, and F. Marciano-Cabral. 2008. CB2 receptors in the brain: role in central immune function. Br. J. Pharmacol. 153:240-251. [PMC free article] [PubMed]
4. De Jonckheere, J. 1977. Use of an axenic medium for differentiation between pathogenic and nonpathogenic Naegleria fowleri isolates. Appl. Environ. Microbiol. 33:751-757. [PMC free article] [PubMed]
5. Dey, I., K. Keller, A. Belley, and K. Chadee. 2003. Identification and characterization of a cyclooxygenase-like enzyme from Entamoeba histolytica. Proc. Natl. Acad. Sci. U. S. A. 100:13561-13566. [PubMed]
6. Di Marzo, V., C. S. Breivogel, Q. Tao, D. T. Bridgen, R. K. Razdan, A. M. Zimmer, A. Zimmer, and B. R. Martin. 2000. Levels, metabolism, and pharmacological activity of anandamide in CB(1) cannabinoid receptor knockout mice: evidence for non-CB(1), non-CB(2) receptor-mediated actions of anandamide in mouse brain. J. Neurochem. 75:2434-2444. [PubMed]
7. Ellingson, J. S. 1980. Identification of N-acylethanolamine phosphoglycerides and acylphosphatidylglycerol as the phospholipids which disappear as Dictyostelium discoideum cells aggregate. Biochemistry 19:6176-6182. [PubMed]
8. Ellingson, J. S., and H. C. Dischinger. 1985. Concurrent disappearance of N-acylethanolamine glycerophospholipids and phagolysosomes enriched in N-acylethanolamine glycerophospholipids as Dictyostelium discoideum cells aggregate. Biochim. Biophys. Acta 812:255-260. [PubMed]
9. Karava, V., P. M. Zafiriou, L. Fasia, D. Anagnostopoulos, E. Boutou, C. E. Vorgias, M. Maccarrone, and A. Siafaka-Kapadai. 2005. Anandamide metabolism by Tetrahymena pyriformis in vitro. Characterization and identification of a 66 kDa fatty acid amidohydrolase. Biochimie 87:967-974. [PubMed]
10. Kishimoto, S., S. Oka, M. Gokoh, and T. Sugiura. 2006. Chemotaxis of human peripheral blood eosinophils to 2-arachidonoylglycerol: comparison with other eosinophil chemoattractants. Int. Arch. Allergy Immunol. 140(Suppl. 1):3-7. [PubMed]
11. Marciano-Cabral, F., and G. Cabral. 2003. Acanthamoeba spp. as agents of disease in humans. Clin. Microbiol. Rev. 16:273-307. [PMC free article] [PubMed]
12. Marciano-Cabral, F., R. Puffenbarger, and G. A. Cabral. 2000. The increasing importance of Acanthamoeba infections. J. Eukaryot. Microbiol. 47:29-36. [PubMed]
13. McPartland, J. M., I. Matias, V. Di Marzo, and M. Glass. 2006. Evolutionary origins of the endocannabinoid system. Gene 370:64-74. [PubMed]
14. Pringle, H. L., S. G. Bradley, and L. S. Harris. 1979. Susceptibility of Naegleria fowleri to delta 9-tetrahydrocannabinol. Antimicrob. Agents Chemother. 16:674-679. [PMC free article] [PubMed]
15. Salazar, M., A. Carracedo, I. J. Salanueva, S. Hernandez-Tiedra, M. Lorente, A. Egia, P. Vazquez, C. Blazquez, S. Torres, S. Garcia, J. Nowak, G. M. Fimia, M. Piacentini, F. Cecconi, P. P. Pandolfi, L. Gonzalez-Feria, J. L. Iovanna, M. Guzman, P. Boya, and G. Velasco. 2009. Cannabinoid action induces autophagy-mediated cell death through stimulation of ER stress in human glioma cells. J. Clin. Invest. 119:1359-1372. [PMC free article] [PubMed]
16. Smith, P. B., D. R. Compton, S. P. Welch, R. K. Razdan, R. Mechoulam, and B. R. Martin. 1994. The pharmacological activity of anandamide, a putative endogenous cannabinoid, in mice. J. Pharmacol. Exp. Ther. 270:219-227. [PubMed]
17. Sugiura, T., S. Kishimoto, S. Oka, and M. Gokoh. 2006. Biochemistry, pharmacology and physiology of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand. Prog. Lipid Res. 45:405-446. [PubMed]
18. Willoughby, K. A., S. F. Moore, B. R. Martin, and E. F. Ellis. 1997. The biodisposition and metabolism of anandamide in mice. J. Pharmacol. Exp. Ther. 282:243-247. [PubMed]
19. Woodward, D. F., R. W. Carling, C. L. Cornell, H. G. Fliri, J. L. Martos, S. N. Pettit, Y. Liang, and J. W. Wang. 2008. The pharmacology and therapeutic relevance of endocannabinoid derived cyclo-oxygenase (COX)-2 products. Pharmacol. Ther. 120:71-80. [PubMed]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)