Parasitic Worms: An Ally in the War Against the Superbugs

Focus

Parasitic Worms: An Ally in the War Against the Superbugs J.M. Webster, G. Chen and J. Li The entomopathogenic nematodes are now of considerable scientific interest not only because of their function as biological control agents, but because of the potential medical and agricultural importance of the metabolic products of their symbiotic bacteria. Initial exploration in Australia, a decade ago, has blossomed into a significant area of research activity as we discover the nematicidal, anticancer, antimycotic and antibiotic properties of these bacterially derived compounds. Some of the antibiotics are novel compounds, and they might form the basis of the next round of drugs against the superbugs, as discussed here by John Webster, Genhui Chen and Jianxong Li. Nematodes are often viewed as disease agents that are the scourge of tropical rural communities, periodic troublesome irritants in schoolchildren or the bane of the new crop of lambs with scours. However, this evolutionary diverse group of parasites also comprises some common associates of invertebrates, especially insects. The benefits to society of one of these groups of nematodes, the entomopathogenic nematodes, has become very apparent in the worldwide search for effective, environmentally friendly insecticides1–3. The application of entomopathogenic nematodes to control insect pests in agriculture and forestry has met with some commercial success. It seems now that this may be the proverbial ‘tip of the iceberg’, if the potential of the biological and medicinal properties of these nematodes and their bacterial symbionts is fulfilled. Entomopathogenic nematodes Entomopathogenic nematodes, belonging to the genera Steinernema and Heterorhabditis, carry bacterial symbionts, species of Xenorhabdus and Photorhabdus, respectively, in the gut of the infective juvenile stage of the nematode1,2. The infective juvenile penetrates into the haemocoel of the host insect, either directly through the cuticle (for some heterorhabditids) or via the intestinal wall subsequent to ingestion3. Once in the haemocoel, the nematodes release the symbiotic bacteria from their gut and, within 24–48 h, the insect dies from bacterial septicaemia. The bacteria grow to the stationary phase condition in the insect cadaver and the nematodes feed on the growing number of bacteria in order to develop and reproduce. Eventually, the infective juveniles exit into the soil from the degenerating cadaver, and carry in their gut their respective species of bacterial symbiont for transmission to another insect4. This is the general picture of development of these two nematode–bacterial pairs, even though there are significant differences in the biological details of their development. The key point of interest is that the antibacterial and antifungal activities of the John M. Webster, Genhui Chen and Jianxong Li are at the Department of Biological Sciences, Simon Fraser University, Burnaby, Vancouver, BC, Canada V5A 1S6. Tel: +1 604 291 4475, Fax: +1 604 291 3496, e-mail: [email protected] Parasitology Today, vol. 14, no. 4, 1998

metabolites produced by the symbiotic bacteria slow down the putrefaction of the dead insect2,4,5. This enables continued growth and reproduction of the bacterial and nematode symbionts in the relative absence of competition from the soil and insect gut microflora. There are at least 15 species of Steinernema and three of Heterorhabditis, and the number of known species of these ubiquitous nematodes is increasing. Each nematode species is associated with only one species of bacterial symbiont, although each Xenorhabdus or Photorhabdus species may be associated with more than one nematode species. The five species of Xenorhabdus and one species of Photorhabdus are found only in the gut of entomopathogenic nematodes and the body cavities of dying and dead insects; except, that is, for the curious record of X. (Photorhabdus) luminescens isolated from human wounds and blood6. Fighting the superbugs Over 20 years ago, Dutky7 suggested that the bacterial symbiont of the DD136 strain of Neoaplectana (Steinernema) feltiae, namely X. nematophilus, produced antibiotics. Since then, numerous substances and phage have been found associated with species of both genera of entomopathogenic nematodes8 (see Table 1). Not only have bacteriocins been found that mainly attack those species of bacteria closely related to these symbiotic species, but an array of other antimicrobial agents that kill fungi and/or bacteria have also been identified9 (Table 1). Xenorhabdins10 (dithiolopyrrolones), xenocoumacins (benzopyranone derivatives) and a range of indole derivatives11 (Table 1) have been isolated from various strains and species of Xenorhabdus and they all have antibiotic activity. Similarly, Photorhabdus produces hydroxystilbenes that have antibiotic activity (Table 1). In addition to these various antibiotic activities, the metabolites exhibit other bioactive properties, such as nematicidal (K. Hu, pers. commun.) and insecticidal activities. The properties of these compounds not only raise the interesting question as to their role in the insect–nematode–bacterial interaction4, but also lead us to the exciting possibility that they might be effective against drug-resistant human bacterial pathogens, the superbugs. Both the indole and stilbene derivatives are effective against Gram-positive and Gram-negative bacteria: they cause a severe inhibition of RNA synthesis by inducing an accumulation of the regulatory nucleotide, guanosine-3',5'-bis-pyrophosphate in susceptible bacteria12,13. Xenorhabdins, dithiolopyrrolones isolated from X. bovienii, are active against Gram-positive bacteria10. Although the mechanism of action of these dithiolopyrrolones is not fully known, they may be involved in membrane function14,15 or, in yeasts, in the inhibition of RNA and protein synthesis16,17. The hydroxystilbenes and anthraquinones are the only non-protein antibiotic substances that have been isolated from P. luminescens.

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Focus Table 1. Antimicrobial agents known from the nematode bacterial symbionts, Xenorhabdus and Photorhabdus spp Species X. nematophilus

Antimicrobial agents Phage Xenocoumacin 1 Xenocoumacin 2 Bacteriocin Xenorhabdicin Nematophin

Propertiesa 1 1, 2 and 3 1, 3 1 1 1, 2

Refs 8, 9, 19 18 18 22 23 20

X. bovienii

Indoles 1–4 Xenorhabdins 1, 2 and/or 4 Bacteriocin Phage Xenorxides

1 1 and 4 1 1 1 and 2

11, 24 10, 24 22 22 19

X. beddingii

Bacteriocin Phage

1

22 22

Xenorhabdus spp

Xenorhabdin 1–4 Xenocoumacin 1

1, 2 and 4 1, 2 and 3

10 18

P. luminescens

Bacteriocin Phage Hydroxystilbene Anthraquinones

1 1 1 and 2 1

9, 22 8 11, 25, 26 15, 26

Photorhabdus spp

Bacteriocin

1

a

1, antibiotic; 2, antimycotic; 3, anti-ulcer; 4, insecticidal.

Xenocoumacins are highly active against Grampositive bacteria, such as certain species of Staphylococcus and Streptococcus18, but most entobacteria and Pseudomonas aeruginosa are resistant to xenocoumacins, as is the drug-resistant strain of Staphylococcus aureus18. Xenocoumacins also exhibit antimycotic activity against species of Cryptococcus, Aspergillus, Trichophyton and Candida18, and both xenocoumacin 1 and 2 show antiulcer activity against stress-induced ulcers when dosed orally in rats18. Xenorhabdins are both antibacterial and antimycotic, and probably function by inhibiting RNA synthesis by microorganisms. Recently, xenorxides19 and nematophin20, produced by X. bovienii and X. nematophilus, respectively, were found to be strong antibiotic agents under certain circumstances. Xenorxides differ from xenorhabdins in that the disulphide in the dithiolopyrrolone ring structure is oxidized. The antibacterial activity of xenorxides appears to be selective for Grampositive bacteria, while the antimycotic activity covers a broad spectrum. Nematophin also is active against Gram-positive bacteria and it has also shown antimycotic activity against the plant pathogen Botrytis cinerea20. The activity of nematophin and xenorxides against drugresistant human pathogens, such as S. aureus, is of particular significance. The apparent specificity of nematophin for S. aureus but not M. luteus under laboratory culture conditions is unusual, especially as it also has antimycotic activity. This in itself may reflect a novel mode of action. The strong activity of both xenorxides and nematophin, with minimum inhibitory concentrations (MICs) of less than 2.0 ␮g ml⫺1 against drug-resistant bacteria, and their relative ease of production by biological or synthetic means could make these two groups of chemicals excellent lead compounds for the development of new drugs19,20. The simple structure of nematophin can be manipulated to generate many bioactive compounds with 162

different properties, and in combination with the newly isolated peptide mimetics, nematophin or its analogues may well result in useful pharmacological products. The well-known antimycotic enzyme chitinase, which was recently identified from Xenorhabdus and Photorhabdus species, inhibits conidial germination and mycelial growth21. However, these bacterial chitinases do not have lysozyme activity and so probably will not confer antibacterial action. The chitinase activity may, of course, have a role in nematode egg hatch in the insect cadaver, as its presence in the complex occurs at that time of nematode development.

Perspective The question now arises, will these new antibiotics be effective against the so-called superbugs? One major ad9 vantage that these new antibiotics have over most commercially used antibiotics is that they are not structurally related to current, clinical antibiotics, including penicillin. Consequently, there is less likelihood that the bacteria are already resistant or partially resistant to the antibiotic action of these novel compounds. Tests are progressing to determine the efficacy and pharmacokinetics of these compounds. Meanwhile, other capabilities of this extraordinary array of metabolites, such as their anticancer and nematicidal activity, are being examined. The nematode associates of these bacteria could well be regarded as the ‘good guys’ in the phylum. Acknowledgements The research referred to in this paper is supported largely by the Natural Sciences and Engineering Research Council of Canada and to some degree by several grants and contracts. References 1 Poinar, G.O. (1990) in Entomopathogenic Nematodes in Biological Control (Gaugler, R. and Kaya, H.K., eds), pp 23–62, CRC Press 2 Forst, S. and Nealson, K. (1996) Molecular biology of the symbiotic-pathogenic bacteria Xenorhabdus spp. and Photorhabdus spp. Microbiol. Rev. 60, 21–43 3 Dunphy, G.B. and Thurston, G.S. (1990) in Entomopathogenic Nematodes in Biological Control (Gaugler, R. and Kaya, H.K., eds), pp 301–326, CRC Press 4 Akhurst, R.J. and Dunphy, G.B. (1993) in Parasites and Pathogens of Insects (Vol. 2) (Beckage, N.E., Thompson, S.N. and Federici, B., eds), pp 1–23, Academic Press 5 Chen, G. et al. (1994) Antifungal activity of two Xenorhabdus species and Photorhabdus luminescens, bacteria associated with the nematodes Steinernema species and Heterorhabditis megidis. Biological Control 4, 157–162 6 Farmer, J.J., III et al. (1989) Xenorhabdus luminescens (DNA hybridization group 5) from human clinical specimens. J. Clin. Microbiol. 27, 1594–1600 7 Dutky, S.R. (1974) in Proceedings of the Summer Institute on Biological Control of Plant Insects and Diseases (Maxwell, F.G. and Harris, F.A., eds), pp 576–590, University Press of Mississippi 8 Poinar, G.O., Hess, R.T. and Thomas, G. (1980) Isolation of defective bacteriophages from Xenorhabdus spp. (Enterobacteriaceae). IRCS Med. Sci. 8, 141 9 Baghdiguian, S. et al. (1993) Bacteriocinogenesis in cells of Xenorhabdus nematophilus and Photorhabdus luminescens: enterobacteriaceae associated with entomopathogenic nematodes. Biol. Cell 79, 177–185

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Focus 10 McInerney, B.V. et al. (1991) Biologically active metabolites from Xenorhabdus spp., Part 1. Dithiolopyrrolone derivatives with antibiotic activity. J. Nat. Prod. 54, 774–784 11 Paul, V.J. et al. (1981) Antibiotics in microbial ecology, isolation and structure assignment of several new antibacterial compounds from the insect-symbiotic bacteria Xenorhabdus spp. J. Chem. Ecol. 7, 589–597 12 Sundar, L. and Chang, F.N. (1992) The role of guanosine-3', 5'-bispyrophosphate in mediating antimicrobial activity of the antibiotic 3, 5-dihydroxy-4-ethyl-trans-stilbene. Antimicrob. Agents Chemother. 36, 2645–2651 13 Sundar, L. and Chang, F.N. (1993) Antimicrobial activity and biosynthesis of indole antibiotics produced by Xenorhabdus nematophilus. J. Gen. Microbiol. 139, 3139–3148 14 Ninomiya, Y. et al. (1980) Biochemically active substances from microorganisms. V Pyrrothines, potent platelet aggregation inhibitors of microbial origin. Chem. Pharm. Bull. 28, 3157–3162 15 Szaricskai, F. et al. (1992) Anthraquinones produced by enterobacters and nematodes. Acta Chimica Hungarica 129, 697–707 16 Jiminez, A. et al. (1973) Mode of action of thiolutin, an inhibitor of macromolecular synthesis in Saccharomyces cerevisiae. Antimicrob. Agents Chemother. 3, 729–738 17 Tipper, D.J. (1973) Inhibition of yeast ribonucleic acid polymerases by thiolutin. J. Bacteriol. 116, 245–246 18 McInerney, B.V. et al. (1991) Biologically active metabolites

19 20 21 22 23

24 25 26

from Xenorhabdus spp., Part 2. Benzopyran-1-one derivatives with gastroprotective activity. J. Nat. Prod. 54, 785–795 Li, J. et al. Novel heterocyclic antibiotics from Xenorhabdus bovienii. J. Nat. Prod. (in press) Li, J. et al. (1997) Nematophin, a novel antimicrobial substance produced by Xenorhabdus nematophilus (Enterobactereaceae). Can. J. Microbiol. 43, 770–773 Chen, G. et al. (1996) Chitinase activity of Xenorhabdus and Photorhabdus species, bacterial associates of entomopathogenic insects. J. Invertebr. Pathol. 68, 101–108 Boemare, N.E. et al. (1992) Lysogeny and bacteriocinogeny in Xenorhabdus nematophilus and other Xenorhabdus spp. Appl. Environ. Microbiol. 58, 3032–3037 Thaler, J., Baghdiguian, S. and Boemare, N. (1995) Purification and characterization of xenorhabdicin, a phage tail-like bacteriocin, from lysogenic strain F1 of Xenorhabus nematophilus. Appl. Environ. Microbiol. 61, 2049–2052 Li, J. et al. (1995) Antimicrobial metabolites from a bacterial symbiont. J. Nat. Prod. 58, 1081–1085 Richardson, W.H. et al. (1988) Identification of an anthraquinone pigment and a hydroxystilbene antibiotic from Xenorhabdus luminescens. Appl. Environ. Microbiol. 54, 1602–1605 Li, J. et al. (1995) Identification of two pigments and a hydroxystilbene antibiotic from Photorhabdus luminescens. Appl. Environ. Microbiol. 61, 4329–4333

Techniques

Representation of Differential Expression: A New Approach to Study Differential Gene Expression in Trypanosomatids M.A. Krieger and S. Goldenberg During the past five years, several methods have been described that allow the isolation and cloning of stagespecific or cell-specific genes. The characterization of genes expressed at different stages of parasite development is of the utmost importance for the understanding of the mechanisms involved in the regulation of gene expression. Here, Samuel Goldenberg and Marco Aurelio Krieger describe a method for the amplification and cloning of Trypanosoma cruzi genes expressed specifically at different times of the metacyclogenesis process. This method, representation of differential expression (RDE), should be useful for the isolation and cloning of any trypanosomatid gene transcribing differentially expressed messenger RNA. Trypanosomatids are members of the order Kinetoplastida and can be pathogenic to animals and plants1. During their life cycle, these parasites can alternate between different morphological and functional types according to the intermediary host. Despite many differences, all trypanosomatids have some characteristic features in common, such as the presence of a kinetoplast2 and a sequence at the 5'-end of their mRNAs3. This mini-exon (ME) or spliced-leader (SL) RNA is encoded Marco Aurelio Krieger and Samuel Goldenberg are at the Departamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz, FIOCRUZ, Avenida Brasil 4365, Rio de Janeiro, RJ, 21045-900 Brazil. Tel: +5521 290 7549, Fax: +5521 590 3495, e-mail: [email protected] Parasitology Today, vol. 14, no. 4, 1998

by a separate gene and is added post-transcriptionally to the 5'-ends of mRNA primary transcripts by a transsplicing mechanism4. The study of trypanosomatid stage-specific genes, in addition to its relevance in elucidating the biological behavior of these parasites and their interaction with their respective hosts, might provide important tools for unravelling the mechanisms involved in the regulation of gene expression in these parasites. An adaptation of the method of Lisitsyn et al. (representational difference analysis method)5, for the isolation and cloning of trypanosomatid stage-specific genes, is described below. The method is based on the polymerase chain reaction (PCR)-amplification of DNA sequences unique to a cell population (tester) after subtractive hybridization with DNA sequences of a related cell population (driver). Tester DNA sequences are annealed to a large molar excess of driver DNA following denaturation; removal of both hybrids and self-reassembled driver molecules should result in a large increase in the relative concentration of testerspecific sequences after several cycles of hybridization. Previous work in our laboratory6,7 resulted in the development of chemically defined conditions that mimic the metacyclogenesis process of Trypanosoma cruzi (transformation of epimastigotes into metacyclic trypomastigotes). Epimastigotes growing in liver infusion tryptose (LIT) medium are harvested by centrifugation and incubated in triatomine artificial urine

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