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Published in final edited form as: Invert Neurosci. 2012 May 5;12(1):37–41. doi: 10.1007/s10158-012-0135-8

Bacterial pore-forming proteins as anthelmintics

Yan Hu 1,✉, Raffi V Aroian 2,✉
PMCID: PMC3889471  NIHMSID: NIHMS457796  PMID: 22562659

Abstract

Crystal (Cry) proteins are made by the Gram-positive bacterium Bacillus thuringiensis (Bt). Cry proteins are pore-forming proteins and are the most widely used biological insecticides in the world. Our laboratory found some Cry proteins are highly effective against a broad range of nematodes (roundworms). Here, we discuss our results of Cry protein activity against intestinal roundworms. Both Cry5B and Cry21A have therapeutic activities against infections of the roundworm Heligmosomoides polygyrus bakeri in mice. Cry5B also shows highly therapeutic activity against Ancylostoma ceylanicum infection in hamsters. A. ceylanicum is a minor hookworm parasite of humans, and it is closely related to the more prevalent Ancylostoma duodenale. In addition, Cry proteins show excellent combinatorial therapeutic properties with nicotinic acetylcholine receptor (nAChR) agonists, one of the two classes of compounds approved by the World Health Organization for the treatment for intestinal roundworms in humans. Given their non-toxicity to humans and their broad spectrum of nematicidal action, Cry proteins show great potential as next-generation anthelmintics.

Keywords: Bacillus thuringiensis, Crystal proteins, Anthelmintic, Cry5B, Soil-transmitted helminths, Pore-forming proteins

Hundreds of millions of children and pregnant women who live in impoverished conditions are infected with the soil-transmitted helminths known more commonly as hookworm (Ancylostoma duodenale and Necator americanus), whipworm (Trichuris trichiura), and giant roundworm (Ascaris lumbricoides) (Bethony et al. 2006; Hall et al. 2008; Hotez et al. 2007). In children, these roundworm (nematode) parasites are significant sources of malnutrition, anorexia, growth stunting, cognitive retardation, lethargy, high rates of school absenteeism, and immune defects; children who grow up with these roundworm infections make significantly less money as adults than children who do not have roundworms (Bethony et al. 2006; Hall et al. 2008; Harhay et al. 2010; Hotez et al. 2007; Lim et al. 2009; Tchuem Tchuente 2011). Pregnant women with roundworm infections, especially hookworm infections, are more likely to die, have their baby die, or give birth to low birthweight babies than uninfected women (Haider et al. 2009; Hotez et al. 2004). As such, soil-transmitted helminths are the leading contributors to poverty worldwide.

Because of the paucity of resources in most places where soil-transmitted helminths are endemic, mass drug administration treatment programs require that drugs be cheap, easy to dose, work as a single dose, and affect as many parasites as possible (Keiser and Utzinger 2008). For these reasons, the drugs of choice are the benzimidazoles—mebendazole and albendazole. Pyrantel and levamisole, both L-subtype nicotinic acetylcholine receptor (nAChR) agonists, are less commonly used alternatives. A new drug in a similar class as pyrantel and levamisole, tribendimidine, is now in use in China and may be superior to these two (Xiao et al. 2005). Because the benzimidazoles are variably effective against the parasites and because resistance to these drugs appears to be emerging (Adugna et al. 2007; Albonico et al. 2003; Flohr et al. 2007; Gunawardena et al. 2008; Humphries et al. 2011; Smits 2009; Soukhathammavong et al. 2012; Stepek et al. 2006; Stothard et al. 2009), new and more efficacious anthelmintics (antiroundworm drugs) are urgently needed.

Bacillus thuringiensis (Bt) is a Gram-positive soil bacterium with a long history as the premier biologically produced insect control agent (Sanahuja et al. 2011). Bt was discovered in 1901 and first commercialized as an insecticide in the 1930s. During sporulation, the mother cell produces copious amounts of proteins called Cry proteins (Bulla et al. 1980; Ibrahim et al. 2010; Soberon et al. 2010). Following sporulation, a Bt culture will contain spores, crystals, and mother cell lysate (hence, the end product of Bt sporulation is called spore-crystal lysates). Cry proteins are ingestible pore-forming proteins that bind to the insect midgut, forming pores in these cells and leading to death of the insect (Ibrahim et al. 2010; Soberon et al. 2010). Because Bt and its Cry proteins are non-toxic to vertebrates, Bt spore-crystal lysates are ideal insecticides and in fact are used by organic farmers (Sanahuja et al. 2011; Zehnder et al. 2007). Moreover, transgenic plants that express Cry proteins are now widespread in agriculture around the world in order to improve crop yields and decrease reliance on toxic chemical pesticides (Sanahuja et al. 2011).

Over 200 Cry proteins are known, some of which kill Lepidoptera (caterpillars), some of which kill Coleoptera (beetles), and some of which kill Diptera (mosquitoes) (Sanahuja et al. 2011; Soberon et al. 2010). A small number are also demonstrated to kill nematodes or roundworms (Wei et al. 2003).

Feeding of Cry proteins, most notably Cry5B, to free-living roundworms like Caenorhabditis elegans, results in lethargy, anorexia, pale coloration, brood size reduction, developmental arrest, and/or death of the roundworm (Bischof et al. 2008; Griffitts et al. 2001; Marroquin et al. 2000; Wei et al. 2003). Cry5B is predicted to have significant structural similarity to insecticidal Cry proteins, like Cry1 family proteins used in transgenic crops (Xia et al. 2008). Cry5B also has pore-forming activity similar to insecticidal Cry proteins (Kao et al. 2011). Genetic, molecular, cell biological, and biochemical analyses reveal that the receptors for Cry5B in C. elegans are carbohydrate structures present on lipids on the intestinal surface of the roundworm (Barrows et al. 2006, 2007; Griffitts and Aroian 2005; Griffitts et al. 2001, 2003, 2005). Similar lipids appear to bind Cry5B in the hookworm Ancylostoma ceylanicum (Cappello et al. 2006). These glycolipids that bind Cry5B are known as arthroseries glycolipids and are specific to insects and roundworms (nematodes); they are lacking in mammals and vertebrates (Griffitts and Aroian 2005; Griffitts et al. 2005). Thus, at least part of the reason that Cry proteins like Cry5B are non-toxic to vertebrates is the lack of the Cry5B arthroseries glycolipid receptors in vertebrates.

Because they are safe to vertebrates and have significant intoxicating effects against free-living nematodes, it was investigated whether nematicidal Cry proteins can cure parasitic roundworm infections in vivo. To date, two different rodent–parasite systems have been tested in this regard, with promising results (Cappello et al. 2006; Hu et al. 2010a, b; Table 1). Hamsters infected with the human hookworm A. ceylanicum (Fig. 1a) are cleared of 89 % of their parasites when treated with a triple dose (~14 mg/kg; 100 nM/kg) of Cry5B (Cappello et al. 2006). Production of parasite eggs is also profoundly affected with this treatment, showing about 80 % reduction (Cappello et al. 2006). Mice infected with the mouse parasite Heligmosomoides polygyrus bakeri (aka H. polygyrus, aka H. bakeri Fig. 1b) are cleared of about 70 % of their parasites when treated with single-dose Cry5B spore-crystal lysates (90–100 mg/kg; ~715 nM/kg; Hu et al. 2010a). Parasite egg production is also profoundly impacted by this treatment, showing a 98 % reduction (Hu et al. 2010a). Relative to the treatments with other anthelmintics against the same parasite, H. polygyrus bakeri, Cry5B efficacy is excellent when compared on a molar basis (Table 2). Furthermore, greater than 99 % of the protein is digested in simulated gastric fluids (Hu et al. 2010a), conditions analogous to those in the mammalian stomach. Based on the fact that Cry5B compares very favorably to current anthelmintics without even taking into account this degradation, then these results suggest that Cry proteins have the potential to be anthelmintics superior to those currently in use. Although less studied, Cry21A also has therapeutic activity in vivo against H. polygyrus bakeri (Hu et al. 2010b).

Table 1.

Summary of the efficacy of Cry proteins against roundworms infection in vivo

Cry protein Dose Roundworm % Intestinal worm reduction References
Cry5B ~100 nM/kg (14 mg/kg), triple dose A. ceylanicum 89 Cappello et al. (2006)
Cry5B ~715 nM/kg (100 mg/kg), single dose H. polygyrus bakeri 70 Hu et al. (2010a)
Cry21A 99 nM/kg (13 mg/kg), triple dose H. polygyrus bakeri 40 Hu et al. (2010b)
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Fig. 1.

Fig. 1

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Parasitic roundworms covered in these studies. a A. ceylanicum, b Heligmosomoides polygyrus bakeri. M male; F female

Table 2.

Efficacy of different anthelmintics against H. bakeri as compared to Cry5B in vivo (Modified from Hu and Aroian 2012)

Anthelmintic Dose given (μM/kg) Dose relative to Cry5B Day(s) treatment was given (PI = post infection) % Reduction in intestinal worm burden Publication references
Cry5B 0.7 1x 15 days PI 70 Hu et al. (2010a)
Levamisole 49 70x 12 days PI 90 Fonseca-Salamanca et al. (2003)
Ivermectin 5.7 8x 18 days PI 87 Githiori et al. (2003a)
Pyrantel 84 120x 18 days PI 99 Githiori et al. (2003b)
Mebendazole 75 107x 9–15 days PI 84 Wabo Pone et al. (2009)
Tribendimidine 2.2 3x 15 days PI 70 Hu et al. (2010a)
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An important consideration in the establishment of any new anthelmintic is its mechanism of action relative to known anthelmintics such that roundworms/nematodes resistant to one anthelmintic are still susceptible to the other class. The benzimidazoles are thought to act via their effects on tubulin, whereas the nAChR agonists act at the neuromuscular junction (Holden-Dye and Walker 2007). Both mechanisms are different than plasma membrane-acting pore-forming Cry proteins. The independence of resistance pathways was formally tested by (1) taking C. elegans mutants resistant to nAChR agonists and benzimidazoles and treating them with Cry5B; and (2) taking C. elegans mutants resistant to Cry5B and treating them with nAChR agonists (Hu et al. 2010b). The results of these experiments confirmed independence of resistance pathways between Cry5B and nAChR agonists and benzimidazoles. Moreover, a very striking result was seen. C. elegans resistant to nAChR agonists were hypersusceptible to Cry5B, and C. elegans resistant to Cry5B were hypersusceptible to nAChR agonists (Hu et al. 2010b; Table 3). Hypersusceptibility is a phenomenon whereby as organisms targeted by Drug A become resistant to Drug A and they conversely become more susceptible than wild-type organisms to a Drug B. This phenomenon has been well characterized in human immunodeficiency virus (HIV) chemotherapy, and HIV drug treatment combinations that take advantage of hypersusceptibility have good clinical efficacy (Demeter et al. 2008; Katzenstein et al. 2003; Zaccarelli et al. 2004). The source of mutual hypersusceptibility between a pore-forming protein that attacks the roundworm intestine and acetylcholine receptor agonists that target the neuromuscular junction is as yet unknown and an interesting question for future research.

Table 3.

Sensitivity of various C. elegans mutants to anthelmintics

Genotype Drug LC50 (μg/mL) LC50 (μM) 95 % Confidence limits Fold changec Resultd
N2 (wild type) Cry5B 7.16 0.05 6.56–7.82
lev-8(ye493)a 5.02 0.04 4.62–5.46 0.70 H
unc-50(ye494)a 5.61 0.04 5.01–6.26 0.78 H
N2 (wild type) Tribendimidine 51.76 114.4 45.70–58.97
bre-5(ye17)b 9.39 20.7 8.28–10.64 0.18 H
N2 (wild type) Levamisole 18.07 75.1 15.10–21.32
bre-5(ye17)b 9.19 38.2 7.57–10.81 0.50 H
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The LC50 values along with 95 % confidence limits are shown (modified from Hu et al. 2010b)

a

These mutants are resistant to nAChR agonists like tribendimidine and levamisole

b

This mutant is resistant to Cry5B

c

The ratio of the LC50 of the mutant to the LC50 of wild-type. Fold change values <1 indicate the mutant is more sensitive to the drug than wild type (i.e., hypersusceptible)

d

H hypersusceptible

These results suggested that Cry5B and nAChR agonists might be usefully combined. In the follow-up, Cry5B and nAChR agonists were then tested for synergistic interactions (Hu et al. 2010b). Synergy is defined as a combination of drugs that shows efficacy greater than predicted from the additive effects of each drug individually. Using the experimental design and algorithm for synergy of Chou and Talalay (Chou 2006), we found that Cry5B and nAChR agonists show strong synergy (Table 4). Taken together with the hypersusceptibility experiments, these results indicate that Cry5B and nAChR agonists represent a potentially powerful combination therapy against roundworm parasites.

Table 4.

Combination index values of Cry5B and nAChR agonists at different effect dose (ED) levels (e.g., at the combination dose at which 50 % of the C. elegans roundworms are killed, Cry5B and levamisole show a combination index value of 0.38)

Combinations Combination index valuea
ED50 ED75 ED95
Cry5B + tribendimidine 0.52 0.30 0.12
Cry5B + levamisole 0.38 0.26 0.15
Cry5B + pyrantel 0.64 0.54 0.40
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a

Combination index values <1 are indicative of synergy. The combination index values shown here are generally indicative of strong synergy

Acknowledgments

This work was supported by National Institutes of Health grant to RVA (NIAID R01 AI056189).

Footnotes

Conflict of interest None.

Contributor Information

Yan Hu, Email: yahu@ucsd.edu, Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, Mail Code 0322, La Jolla, CA 92093-0322, USA.

Raffi V. Aroian, Email: raroian@ucsd.edu, Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, Mail Code 0322, La Jolla, CA 92093-0322, USA

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