• View in gallery

    Effects of recombinant Cry5B (rCry5B) on Strongyloides stercoralis first larval stage. Each concentration of rCry5B was used in triplicate wells over three independent experiments. The percentage of deaths is represented as average ± standard error. The asterisk indicates statistical significance compared with the control group (P < 0.001).

  • View in gallery

    Effects of recombinant Cry5B (rCry5B) on Strongyloides stercoralis infective larvae (iL3s). (A) Recombinant Cry5B decreased the viability of S. stercoralis iL3s. The percentage of deaths is represented in average ± standard error. Max % feeding indicates feeding rates of the iL3s in vitro as measured by FITC labeling experiments. The numbers of asterisks indicate levels of statistical significance compared with the control group (*: P < 0.05; ***: P < 0.001). (B) Comparison of Cry5B, ivermectin, and albendazole actions against S. stercoralis iL3s. Results of rCry5B, normalized with max % feeding, are represented by red line.

  • View in gallery

    Effects of Escherichia coli expressing recombinant Cry5B (Cry5B) on Strongyloides stercoralis free-living adults. (A) Time-dependent motility responses of S. stercoralis free-living adult to Cry5B. Data points indicate percentages of motile worms. (B) Virtual blot from the capillary chemiluminescence assay verifying increasing Cry5B concentrations produced by dilutions of suspended E. coli JM103 cells expressing the His-tagged crystal protein. The percentage of adults that were motile is represented as average ± standard error over three independent experiments.

  • View in gallery

    Effects of Escherichia coli expressing Cry5B on ingestion rates of Strongyloides stercoralis. As an index of food ingestion, pulsations of the pharynx were counted for 30 seconds in individual worms. Ingestion rates are represented as average from three experiments ± standard error. The numbers of asterisks indicate levels of statistical significance compared with the control group (*: P < 0.05; ***: P < 0.001).

  • View in gallery

    Effects of recombinant Cry5B (rCry5B) on parasitic females of Strongyloides stercoralis. Percentages of live parasitic adult worms are represented as average from three biological replicates ± standard error. P-value < 0.001 indicates a statistically significant difference compared with the control group.

  • 1.

    Greaves D, Coggle S, Pollard C, Aliyu SH, Moore EM, 2013. Strongyloides stercoralis infection. BMJ 347: f4610.

  • 2.

    Luvira V, Watthanakulpanich D, Pittisuttithum P, 2014. Management of Strongyloides stercoralis: a puzzling parasite. Int Health 6: 273281.

  • 3.

    Schar F, Trostdorf U, Giardina F, Khieu V, Muth S, Marti H, Vounatsou P, Odermatt P, 2013. Strongyloides stercoralis: global distribution and risk factors. PLoS Negl Trop Dis 7: e2288.

    • Search Google Scholar
    • Export Citation
  • 4.

    Prendki V, Fenaux P, Durand R, Thellier M, Bouchaud O, 2011. Strongyloidiasis in man 75 years after initial exposure. Emerg Infect Dis 17: 931932.

  • 5.

    Buonfrate D, Requena-Mendez A, Angheben A, Munoz J, Gobbi F, Van Den Ende J, Bisoffi Z, 2013. Severe strongyloidiasis: a systematic review of case reports. BMC Infect Dis 13: 78.

    • Search Google Scholar
    • Export Citation
  • 6.

    Croker C, Reporter R, Redelings M, Mascola L, 2010. Strongyloidiasis-related deaths in the United States, 1991–2006. Am J Trop Med Hyg 83: 422426.

    • Search Google Scholar
    • Export Citation
  • 7.

    Marcos LA, Terashima A, Canales M, Gotuzzo E, 2011. Update on strongyloidiasis in the immunocompromised host. Curr Infect Dis Rep 13: 3546.

  • 8.

    WHO, 2011. Helminth Control in School-Age Children. A Guide for Managers of Control Programmes. Geneva, Switzerland: World Health Organization. Available at: https://www.who.int/neglected_diseases/resources/9789241548267/en/.

    • Search Google Scholar
    • Export Citation
  • 9.

    WHO, 2012. Research Priorities for Helminth Infections. WHO Technical Report Series. Geneva, Switzerland: World Health Organization. Available at: https://apps.who.int/iris/handle/10665/75922.

    • Search Google Scholar
    • Export Citation
  • 10.

    Gupta S, Jain A, Fanning TV, Couriel DR, Jimenez CA, Eapen GA, 2006. An unusual cause of alveolar hemorrhage post hematopoietic stem cell transplantation: a case report. BMC Cancer 6: 87.

    • Search Google Scholar
    • Export Citation
  • 11.

    Hunter CJ, Petrosyan M, Asch M, 2008. Dissemination of Strongyloides stercoralis in a patient with systemic lupus erythematosus after initiation of albendazole: a case report. J Med Case Rep 2: 156.

    • Search Google Scholar
    • Export Citation
  • 12.

    Kaplan RM, Klei TR, Lyons ET, Lester G, Courtney CH, French DD, Tolliver SC, Vidyashankar AN, Zhao Y, 2004. Prevalence of anthelmintic resistant cyathostomes on horse farms. J Am Vet Med Assoc 225: 903910.

    • Search Google Scholar
    • Export Citation
  • 13.

    Beknazarova M, Whiley H, Ross K, 2016. Advocating for both environmental and clinical approaches to control human strongyloidiasis. Pathogens 5: E59.

    • Search Google Scholar
    • Export Citation
  • 14.

    Bravo A, Likitvivatanavong S, Gill SS, Soberon M, 2011. Bacillus thuringiensis: a story of a successful bioinsecticide. Insect Biochem Mol Biol 41: 423431.

    • Search Google Scholar
    • Export Citation
  • 15.

    Ibrahim MA, Griko N, Junker M, Bulla LA, 2010. Bacillus thuringiensis: a genomics and proteomics perspective. Bioeng Bugs 1: 3150.

  • 16.

    Wei JZ, Hale K, Carta L, Platzer E, Wong C, Fang SC, Aroian RV, 2003. Bacillus thuringiensis crystal proteins that target nematodes. Proc Natl Acad Sci USA 100: 27602765.

    • Search Google Scholar
    • Export Citation
  • 17.

    Cappello M, Bungiro RD, Harrison LM, Bischof LJ, Griffitts JS, Barrows BD, Aroian RV, 2006. A purified Bacillus thuringiensis crystal protein with therapeutic activity against the hookworm parasite Ancylostoma ceylanicum .Proc Natl Acad Sci USA 103: 1515415159.

    • Search Google Scholar
    • Export Citation
  • 18.

    Hu Y, Zhan B, Keegan B, Yiu YY, Miller MM, Jones K, Aroian RV, 2012. Mechanistic and single-dose in vivo therapeutic studies of Cry5B anthelmintic action against hookworms. PLoS Negl Trop Dis 6: e1900.

    • Search Google Scholar
    • Export Citation
  • 19.

    Hu Y et al. 2018. Bacillus thuringiensis Cry5B protein as a new pan-hookworm cure. Int J Parasitol Drugs Drug Resist 8: 287294.

  • 20.

    Urban JF Jr., Hu Y, Miller MM, Scheib U, Yiu YY, Aroian RV, 2013. Bacillus thuringiensis-derived Cry5B has potent anthelmintic activity against Ascaris suum .PLoS Negl Trop Dis 7: e2263.

    • Search Google Scholar
    • Export Citation
  • 21.

    Hu Y, Georghiou SB, Kelleher AJ, Aroian RV, 2010. Bacillus thuringiensis Cry5B protein is highly efficacious as a single-dose therapy against an intestinal roundworm infection in mice. PLoS Negl Trop Dis 4: e614.

    • Search Google Scholar
    • Export Citation
  • 22.

    Li X-Q, Tan A, Voegtline M, Bekele S, Chen C-S, Aroian RV, 2008. Expression of Cry5B protein from Bacillus thuringiensis in plant roots confers resistance to root-knot nematode. Biol Control 47: 97102.

    • Search Google Scholar
    • Export Citation
  • 23.

    Hu Y, Platzer EG, Bellier A, Aroian RV, 2010. Discovery of a highly synergistic anthelmintic combination that shows mutual hypersusceptibility. Proc Natl Acad Sci USA 107: 59555960.

    • Search Google Scholar
    • Export Citation
  • 24.

    Hu Y, Miller M, Zhang B, Nguyen TT, Nielsen MK, Aroian RV, 2018. In vivo and in vitro studies of Cry5B and nicotinic acetylcholine receptor agonist anthelmintics reveal a powerful and unique combination therapy against intestinal nematode parasites. PLoS Negl Trop Dis 12: e0006506.

    • Search Google Scholar
    • Export Citation
  • 25.

    Charuchaibovorn S, Sanprasert V, Nuchprayoon S, 2019. The experimental infections of the human isolate of Strongyloides stercoralis in a rodent model (The Mongolian gerbil, Meriones unguiculatus). Pathogens 8: E21.

    • Search Google Scholar
    • Export Citation
  • 26.

    Koga K, Kasuya S, Khamboonruang C, Sukhavat K, Ieda M, Takatsuka N, Kita K, Ohtomo H, 1991. A modified agar plate method for detection of Strongyloides stercoralis. Am J Trop Med Hyg 45: 518521.

    • Search Google Scholar
    • Export Citation
  • 27.

    Nolan TJ, Megyeri Z, Bhopale VM, Schad GA, 1993. Strongyloides stercoralis: the first rodent model for uncomplicated and hyperinfective strongyloidiasis, the Mongolian gerbil (Meriones unguiculatus). J Infect Dis 168: 14791484.

    • Search Google Scholar
    • Export Citation
  • 28.

    Albarqi MM, Stoltzfus JD, Pilgrim AA, Nolan TJ, Wang Z, Kliewer SA, Mangelsdorf DJ, Lok JB, 2016. Regulation of life cycle checkpoints and developmental activation of infective larvae in Strongyloides stercoralis by dafachronic acid. PLoS Pathog 12: e1005358.

    • Search Google Scholar
    • Export Citation
  • 29.

    Ashton FT, Zhu X, Boston R, Lok JB, Schad GA, 2007. Strongyloides stercoralis: amphidial neuron pair ASJ triggers significant resumption of development by infective larvae under host-mimicking in vitro conditions. Exp Parasitol 115: 9297.

    • Search Google Scholar
    • Export Citation
  • 30.

    Stoltzfus JD, Massey HC Jr., Nolan TJ, Griffith SD, Lok JB, 2012. Strongyloides stercoralis age-1: a potential regulator of infective larval development in a parasitic nematode. PLoS One 7: e38587.

    • Search Google Scholar
    • Export Citation
  • 31.

    Bischof LJ, Huffman DL, Aroian RV, 2006. C. elegans: Methods and Applications. Strange K, ed. Methods in Molecular Biology. Totowa, NJ: © Humana Press Inc.

    • Search Google Scholar
    • Export Citation
  • 32.

    Bischof LJ, Huffman DL, Aroian RV, 2006. Assays for toxicity studies in C. elegans with Bt crystal proteins. Methods Mol Biol 351: 139154.

  • 33.

    Los FC, Kao CY, Smitham J, McDonald KL, Ha C, Peixoto CA, Aroian RV, 2011. RAB-5- and RAB-11-dependent vesicle-trafficking pathways are required for plasma membrane repair after attack by bacterial pore-forming toxin. Cell Host Microbe 9: 147157.

    • Search Google Scholar
    • Export Citation
  • 34.

    Griffitts JS, Haslam SM, Yang T, Garczynski SF, Mulloy B, Morris H, Cremer PS, Dell A, Adang MJ, Aroian RV, 2005. Glycolipids as receptors for Bacillus thuringiensis crystal toxin. Science 307: 922925.

    • Search Google Scholar
    • Export Citation
  • 35.

    Griffitts JS, Huffman DL, Whitacre JL, Barrows BD, Marroquin LD, Muller R, Brown JR, Hennet T, Esko JD, Aroian RV, 2003. Resistance to a bacterial toxin is mediated by removal of a conserved glycosylation pathway required for toxin-host interactions. J Biol Chem 278: 4559445602.

    • Search Google Scholar
    • Export Citation
  • 36.

    Griffitts JS, Whitacre JL, Stevens DE, Aroian RV, 2001. Bt toxin resistance from loss of a putative carbohydrate-modifying enzyme. Science 293: 860864.

    • Search Google Scholar
    • Export Citation
 
 
 
 

 

 
 
 

 

 

 

 

 

 

Bacillus thuringiensis Cry5B is Active against Strongyloides stercoralis in vitro

View More View Less
  • 1 Lymphatic Filariasis and Tropical Medicine Research Unit, Chulalongkorn Medical Research Center, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand;
  • | 2 Siriraj Center of Excellence for Stem Cell Research (SiSCR), Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand;
  • | 3 Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand;
  • | 4 Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts;
  • | 5 Biology Department, Worcester State University, Worcester, Massachusetts;
  • | 6 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

Strongyloidiasis, caused by Strongyloides stercoralis infection, is an important neglected tropical disease that causes significant public health problems in the tropics and subtropics. The disease can persist in hosts for decades and may be life-threatening because of hyperinfection and dissemination. Ivermectin (mostly) and albendazole are the most common anthelmintics used for treatment. Albendazole is suboptimal for this parasite, and although ivermectin is quite effective in immunocompromised patients, a multiple-course regimen is required. Furthermore, reliance on a single drug class for treating intestinal nematodes is a recipe for future failure. Therefore, it is important to discover new anthelmintics to treat or prevent human strongyloidiasis. One promising candidate is the Bacillus thuringiensis crystal protein Cry5B. Cry5B is highly potent against parasitic nematodes, for example, hookworms and Ascaris suum. Here, we investigated the potential of Cry5B against S. stercoralis. Multiple stages of S. stercoralis, including the first larval stage (L1s), infective stage (iL3s), free-living adult stage, and parasitic female stage, were all susceptible to Cry5B as indicated by impairment of motility and decreased viability in vitro. In summary, Cry5B demonstrated strong potential as an effective anthelmintic for treatment and transmission control of human strongyloidiasis, justifying further experiments to investigate in vivo therapeutic efficacy.

INTRODUCTION

Human strongyloidiasis is a gastrointestinal disease caused by the parasitic threadworms Strongyloides stercoralis and Strongyloides fuelleborni. An estimated 30–100 million people in the tropics and subtropics, including sub-Saharan Africa, South America, and Southeast Asia (including Thailand), are infected with these parasites.13 Strongyloidiasis also causes public health problems among immigrants, refugees, and travelers returning from endemic areas to nonendemic countries, such as the United States and Canada.3 The clinical manifestations of strongyloidiasis can range from asymptomatic to fatal. The parasites are able to complete their life cycles and replicate within hosts as a result of their unique capacity for autoinfection. In immunocompetent individuals, this autoinfection is regulated.4 In immunocompromised patients, however, autoinfection may lead to hyperinfection and dissemination syndrome, with mortality rates potentially reaching 69–87%.57

Only a few anthelmintic drugs are used for strongyloidiasis control. Ivermectin is the major drug of choice against the disease, but it is restricted in several countries because of licensing issues.79 Ivermectin can cause adverse reactions, such as nausea, vomiting, diarrhea, liver dysfunction, and neurological effects.2,10,11 Albendazole can be used as an alternative drug for strongyloidiasis. However, the cure rates of human strongyloidiasis following albendazole therapy are variable, with an average of about 58%.8 Most importantly, for such a potentially lethal parasite, nearly complete reliance on a single drug, that is, ivermectin, represents a significant risk. Resistance to ivermectin is widespread in parasites of veterinary importance.12 Such resistance would severely hamper control of human strongyloidiasis. Data suggest that albendazole and ivermectin resistance could also occur in Strongyloides spp.13 Hence, it is important that we find new drugs to treat these parasitisms.

Bacillus thuringiensis–derived crystal (Cry) proteins are pore-forming proteins that are highly effective against a broad range of insect larvae.14,15 Because of their potency across orders of insects and their safety to humans and other vertebrates, these proteins are extensively used as biopesticides, have been expressed in transgenic crops for insect pest control, and are applied to control vector insects around the world.14 Cry5B, a crystal or “Cry” protein phylogenetically related to those used in insect control, has demonstrated anti-nematode activity.16 Cry5B has anthelmintic activity against a broad range of gastrointestinal parasitic nematodes, including hookworms (Ancylostoma ceylanicum, Ancylostoma caninum, and Necator americanus),1719 large roundworms (Ascaris suum),20 rodent parasitic nematodes (Heligmosomoides bakeri and Nippostrongylus brasiliensis),16,21 and a plant parasitic nematode (Meloidogyne incognita).22 In addition, Cry5B is active against albendazole-resistant and nicotinic acetylcholine receptor agonist–resistant Caenorhabditis elegans and cyathostome parasites.23,24 Here, we provide the first evidence of Cry5B efficacy against isolated life stages of the threadworm S. stercoralis.

MATERIALS AND METHODS

Strongyloides life cycle and parasite preparations.

The S. stercoralis life cycle was maintained in 6- to 12-week-old male Mongolian gerbils (Meriones unguiculatus) as previously described.25 Briefly, S. stercoralis–positive stool specimens, obtained from patients of King Chulalongkorn Memorial Hospital, were directly cultured on agar plates at 25°C for 5 days.26 Infective larvae (iL3s) were collected from the surface of the agar plates by washing with buffer saline solution (BU saline) (22 mM KH2PO4, 50 mM Na2HPO4, and 70 mM NaCl) and decontaminated by using low-melting-point agarose. The iL3s were then axenized in BU saline with 1% penicillin–streptomycin, 12.5 μg/mL tetracycline, and 10 μg/mL chloramphenicol for 3 hours. Approximately, 10,000 iL3s were subcutaneously injected into the right shoulders of immunosuppressed gerbils. To immunosuppress the gerbils to induce autoinfection, the infected animals were injected subcutaneously with 2 mg of methylprednisolone acetate (Solu-Medrol; Pfizer Co. Ltd., New York, NY) at days −3, 0, 7, 14, and 21 of infection. All animal experimental protocols were carried out under approval by the Animal Ethical Research Committee, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (IRB no: 022/2560). To obtain the multiple stages of S. stercoralis for in vitro testing with recombinant Cry5B (rCry5B), infected gerbil feces were initially collected after day 14 postinoculation. We used first-stage larval progeny of free-living female S. stercoralis (L1s) for in vitro testing. To isolate these, 1–2 g of feces were collected from infected gerbils and directly cultured on agar plates at 25°C for 2–3 days. Free-living adults were collected from the plates and bleached by using bleaching solutions (20% NaClO and 0.5 M KOH in dH2O) to obtain eggs. The egg pellet was placed on the edge of fresh agar plates and incubated at 25°C overnight to allow larval hatching. The L1s were collected by rinsing the plate surface with BU saline and washed by centrifugation (700 g for 1 minute). To obtain free-living adults, the worms were collected from the surface of the positive agar plates as described previously. For the iL3s, the larvae were collected from 5-day-old agar plate cultures as described previously and then washed with Dulbecco’s modified Eagle’s medium (DMEM) containing 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 5 mM glutathione, 10% canine serum, 1% penicillin–streptomycin, 12.5 μg/mL tetracycline, and 10 μg/mL chloramphenicol.

Parasitic female S. stercoralis were raised in male Mongolian gerbils as described previously.27 All procedures in this phase of the study were conducted in accordance with protocol 804798 and 804893 approved by the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC). Gerbils were euthanized by CO2 asphyxia in accordance with standards established by the American Veterinary Medical Association. All IACUC protocols, as well as routine husbandry care of the animals, were carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals of the U.S. National Institutes of Health. Five-week-old male gerbils were subcutaneously injected with 500 iL3s of S. stercoralis, and 2 mg prednisolone acetate was subcutaneously administered on a weekly basis to induce autoinfection and obtain a larger number of parasitic female worms.27 At 37 days postinfection, gerbils began to show clinical signs of hyperinfection and were euthanized by CO2 asphyxia in an approved chamber. Parasitic female S. stercoralis were harvested from the euthanized gerbils at necropsy on the same day. The small intestines were dissected from the gerbils and their contents removed. Excised intestines were then slit longitudinally and carefully washed with phosphate-buffered saline (PBS). The cleaned intestines were hung from metal clips in 100-mL glass cylinders filled with PBS and incubated at 37°C for 3 hours. The worms were then handpicked from the sediment collected from the bottom of the cylinder, washed three times with PBS, and counted for use in the in vitro study of efficacy against parasitic female worms.

Recombinant Cry5B preparation.

All in vitro studies were conducted with rCry5B. Highly bioactive rCry5B crystals (> 95% rCry5B by protein content) were purified from a novel genetically engineered B. thuringiensis strain that allows for rapid purification of native-like crystals (manuscript in preparation). The protein crystals were stored in dH2O at a final concentration of 2 mg/mL at ‒80°C. For rCry5B experiments, the recombinant protein was dissolved in 20 mM HEPES (pH 8.0) immediately before use.

In vitro efficacy studies.

The L1s of S. stercoralis were obtained as described in Section 2.1. Thirty to fifty L1s were cultured in BU saline containing 1 μg/mL Fungizone and rCry5B. The final concentrations of rCry5B were 0.01, 0.05, 0.1, 0.5, and 5 μg/mL. HEPES buffer (20 mM, pH 8.0) was used as a negative control. Escherichia coli OP50 was used as a food source. After incubation at 25°C for 24 hours, the dead worms were counted using an inverted microscope. Death was confirmed by the lack of worm movement in response to touch with a fine needle probe or to increase in the intensity of light from the microscope illuminator.

As S. stercoralis iL3s are nonfeeding when outside the host, the larvae were induced to feed by culturing in a host-like medium (DMEM with 25 mM HEPES, 5 mM glutathione, 10% canine serum, 1% penicillin–streptomycin, 12.5 μg/mL tetracycline, and 10 μg/mL chloramphenicol) with rCry5B. The in vitro activation of S. stercoralis iL3s was performed as previously described.2830 Briefly, hundreds of iL3s were cultured and isolated from 5-day cultures of infected gerbil’s feces. The iL3s were incubated in 200 μL of the host-like medium at 37°C and 5% CO2 for 21 hours. To determine the percent feeding, 5 μL of fluorescein isothiocyanate (FITC) (20 mg/mL in dimethylformamide) (Sigma-Aldrich, St. Louis, MO) was added to the cultures and then incubated for 3 hours. The larvae were washed five times with M9 buffer (2,000 g) for 5 minutes. The iL3s were then added to an agarose pad with 0.1% sodium azide and observed under a fluorescence microscope. The iL3s with FITC in the pharynx were counted as positive for feeding. The final concentrations of rCry5B varied from 5 μg/mL to 100 μg/mL. Ivermectin (Sigma-Aldrich) and albendazole (Sigma-Aldrich) were prepared in 10% dimethyl sulfoxide and used for comparison. The experimental plates were incubated at 37°C and 5% CO2 for 24 hours. The 50% lethal concentration (LC50) of each drug was calculated using GraphPad Prism software (GraphPad Software, La Jolla, CA).

A plate assay31 was used for testing the effects of rCry5B on the free-living stages of S. stercoralis. Briefly, overnight cultures of E. coli JM103 pQE9-Cry5B were induced with 50 μM isopropyl β-D-1-thiogalactopyranoside for 1 hour. A dose–response series for E. coli expressing rCry5B (0–100%) was made by mixing different ratios of E. coli JM103 pQE9-Cry5B with the E. coli JM103 pQE9-empty vector (E. coli JM103 pQE9-Cry5B: E. coli JM103 pQE9-empty vector; 100%—100 μL: 0; 75%—75 μL: 25 μL; 50%—50 μL: 50 μL; 25%—25 μL: 75 μL; and 0%—0: 100 μL). Then, 30 μL of the induced cell suspension was spread on enriched nematode growth (ENG) plates and cultured at room temperature overnight. Expression of rCry5B in the E. coli was verified using the ProteinSimple capillary immunoassay (Wes; ProteinSimple, San Jose, CA). Briefly, proteins were size-separated in capillaries, where they were incubated with 1: 50 of mouse anti-6× His tag (1st antibody), 1: 300 of horseradish peroxidase-conjugated goat anti-mouse IgG (2nd antibody), and finally luminol/peroxidase. The resulting chemiluminescence was detected and automatically quantified using Compass software (ProteinSimple), which displays the chemiluminescent signal as a virtual blot-like image. For testing, 10–20 female free-living adults were added to the rCry5B-expressing E. coli lawn and incubated at 25°C. Motility was observed and scored at 4-hour intervals. Motility scores were categorized by convention, with a score of 3 representing vigorous movement similar to control worms, 2 representing whole-body movements significantly slower than the control, 1 representing sluggish worms (worms that moved only when stimulated by touching with a probe or by increasing the intensity of light from the microscope illuminator), and 0 representing dead worms (worms that did not move even when stimulated by touching with a probe or increasing light intensity). To assess the effects of Cry5B on pharyngeal pumping, pulsations of the pharynx were counted for 30 seconds in individual worms.

To investigate the effect of rCry5B on parasitic adult S. stercoralis, cohorts of 60 parasitic females recovered at necropsy from the small intestines of infected gerbils were incubated at 37°C and 5% CO2 for 48 hours in 100 μL of DMEM containing 50 μg/mL gentamicin and 0 (control), 1, and/or 250 μg/mL of rCry5B. Experiments were carried out in 96-well plates and checked twice daily during the 48-hour incubation. Toxicity of rCry5B to parasitic females was measured by counting the number of dead worms at 48 hours in culture. Worm death was confirmed by the absence of movement on touching with a worm pick. Survivorship of parasitic females in the presence of rCry5B, as indicated by the percentage of worms alive at 48 hours in culture, was compared with survivorship of controls cultured in the absence of the protein.

All experiments were performed in three independent trials. Each independent trial was performed in triplicate wells. All rCry5B assays were modified from standard methods.32

Statistical analysis.

All data are represented in the article and figures as average ± standard error. Statistical analysis was performed using GraphPad Prism software version 5.0. ANOVA with the Bonferroni post hoc test was used for multiple group comparisons. Statistical probability P < 0.05 was the criterion for significance.

RESULTS

Recombinant Cry5B decreased the viability of post–free-living S. stercoralis L1s.

To ascertain whether S. stercoralis is susceptible to Cry5B, we initially evaluated the susceptibility of Cry5B to S. stercoralis in the first larval stage. After 24 hours, > 95% of the L1s fed with rCry5B were moribund or dead at 5 μg/mL (Figure 1), whereas 90% L1s developed into L4s or young free-living adults in the control group. The L1s showed a dose-dependent response to rCry5B. At a dose of only 10 ng/mL rCry5B, 44% of S. stercoralis L1s were unable to develop normally.

Figure 1.
Figure 1.

Effects of recombinant Cry5B (rCry5B) on Strongyloides stercoralis first larval stage. Each concentration of rCry5B was used in triplicate wells over three independent experiments. The percentage of deaths is represented as average ± standard error. The asterisk indicates statistical significance compared with the control group (P < 0.001).

Citation: The American Journal of Tropical Medicine and Hygiene 101, 5; 10.4269/ajtmh.19-0083

Recombinant Cry5B was highly potent against S. stercoralis iL3s.

Significant mortality occurred among S. stercoralis iL3s fed with 5 μg/mL (35.7 nM) of rCry5B. Frequency of mortality among S. stercoralis iL3s was proportional to the dose of rCry5B and approached the mean frequency of feeding (56%) as a limit (Figure 2A). Mortality among iL3s in the control group was less than 5%. The effects of ivermectin and albendazole were also tested. More than 80% of the iL3s exposed to these drugs were moribund or dead at > 100 μM (Figure 2B). The LC50s for Cry5B, ivermectin, and albendazole were 39 nM, 9.76 μM, and 32.75 μM, respectively.

Figure 2.
Figure 2.

Effects of recombinant Cry5B (rCry5B) on Strongyloides stercoralis infective larvae (iL3s). (A) Recombinant Cry5B decreased the viability of S. stercoralis iL3s. The percentage of deaths is represented in average ± standard error. Max % feeding indicates feeding rates of the iL3s in vitro as measured by FITC labeling experiments. The numbers of asterisks indicate levels of statistical significance compared with the control group (*: P < 0.05; ***: P < 0.001). (B) Comparison of Cry5B, ivermectin, and albendazole actions against S. stercoralis iL3s. Results of rCry5B, normalized with max % feeding, are represented by red line.

Citation: The American Journal of Tropical Medicine and Hygiene 101, 5; 10.4269/ajtmh.19-0083

Recombinant Cry5B decreased motility of S. stercoralis free-living adults.

Our preliminary results indicated that S. stercoralis free-living adults could not be cultured in liquid medium (data not shown). Therefore, the susceptibility of rCry5B against S. stercoralis free-living adults was evaluated by incubating the worms with the E. coli expressing rCry5B on ENG plates. The free-living adults were subjected to various doses of rCry5B, and percentages of motile adults were measured over time. The percentage of motile S. stercoralis free-living adults fed with E. coli expressing rCry5B decreased at 8 hours in culture, and no motile larvae were observed in the rCry5B group at 36 hours. By contrast, 95% of the adults remained motile in the control group fed vector-only bacteria (Figure 3A). Internal hatching was also found in the treated worms, but internally hatched larvae were not included in analyses. The free-living adults showed a dose-dependent response to the rCry5B from transformed E. coli (Figure 3B). Feeding of C. elegans hermaphrodites on rCry5B inhibits pharyngeal pumping.33 As shown (Figure 4), rCry5B inhibits pharyngeal pumping in free-living S. stercoralis adults as it does with C. elegans, although the inhibition is not as penetrant.

Figure 3.
Figure 3.

Effects of Escherichia coli expressing recombinant Cry5B (Cry5B) on Strongyloides stercoralis free-living adults. (A) Time-dependent motility responses of S. stercoralis free-living adult to Cry5B. Data points indicate percentages of motile worms. (B) Virtual blot from the capillary chemiluminescence assay verifying increasing Cry5B concentrations produced by dilutions of suspended E. coli JM103 cells expressing the His-tagged crystal protein. The percentage of adults that were motile is represented as average ± standard error over three independent experiments.

Citation: The American Journal of Tropical Medicine and Hygiene 101, 5; 10.4269/ajtmh.19-0083

Figure 4.
Figure 4.

Effects of Escherichia coli expressing Cry5B on ingestion rates of Strongyloides stercoralis. As an index of food ingestion, pulsations of the pharynx were counted for 30 seconds in individual worms. Ingestion rates are represented as average from three experiments ± standard error. The numbers of asterisks indicate levels of statistical significance compared with the control group (*: P < 0.05; ***: P < 0.001).

Citation: The American Journal of Tropical Medicine and Hygiene 101, 5; 10.4269/ajtmh.19-0083

Recombinant Cry5B was effective against S. stercoralis parasitic females.

Survivorship among parasitic females was significantly lower (P < 0.001) in groups treated with rCry5B than in controls. This effect was dose-dependent, and more than 95% of parasitic females that fed on rCry5B at a concentration of 250 μg/mL died after 48 hours (Figure 5). In addition to confirming activity against the parasitic stage, these results also demonstrate that Cry5B is active against a canine isolate of S. stercoralis, increasing confidence that it will be broadly effective against this parasite.

Figure 5.
Figure 5.

Effects of recombinant Cry5B (rCry5B) on parasitic females of Strongyloides stercoralis. Percentages of live parasitic adult worms are represented as average from three biological replicates ± standard error. P-value < 0.001 indicates a statistically significant difference compared with the control group.

Citation: The American Journal of Tropical Medicine and Hygiene 101, 5; 10.4269/ajtmh.19-0083

DISCUSSION

Here, we demonstrate for the first time that the B. thuringiensis pore-forming protein Cry5B is highly active against multiple life cycle stages of the major pathogenic species of the human threadworm parasite S. stercoralis. Feeding rCry5B to the parasite results in impaired pharyngeal pumping, motility, and viability of the parasites, consistent with the protein’s toxicity in C. elegans.

Recombinant Cry5B is active against S. stercoralis L1s (Figure 1). At a lower dose of rCry5B, treated larvae were unable to develop normally. Such larvae were recognized by impaired growth compared with controls and loss of cuticular integrity. Most of the larvae were dead at a higher dose of the protein. The data demonstrated that early larval stages of S. stercoralis are more sensitive to rCry5B than the infective stage, free-living adults, and parasitic adult worms, consistent with a previous study in A. ceylanicum.17 Consistent with previous findings that Cry5B is an ingested anthelmintic protein, our findings suggest that S. stercoralis iL3s are susceptible to Cry5B only to the extent that they are induced to feed by culturing under host-like conditions. iL3s fed with rCry5B exhibited a 55% mortality rate. This is strikingly similar to the 56% feeding rate observed in iL3s from the population under study. Recombinant Cry5B was more potent than ivermectin (respective LC50s of 0.039 μM versus 9.76 μM) and albendazole (respective LC50s of 0.039 μM versus 32.75 μM) on a molar basis. Although the first larval and infective stages of parasitic nematodes are not therapeutic targets, these stages of the S. stercoralis life cycle are important. The most unique characteristic of S. stercoralis’life cycle is autoinfection in which the L1s in the host’s intestines become the iL3s and complete their parasitic life cycle in the host. In immunosuppressed hosts, autoinfection rates are dramatically increased, resulting in hyperinfection and dissemination, which can be fatal. Our results showing rCry5B activity against both L1s and iL3s suggest that rCry5B may be able to block hyperinfection and dissemination. Recombinant Cry5B was active against S. stercoralis free-living adults (Figure 3). The effects of rCry5B on free-living adults were consistent with the previous studies in several free-living nematodes, including C. elegans, Pristionchus pacificus, Panagrellus redivivus, and Distolabrellus veechi.16 Our results demonstrate a broad target range of Cry5B against free-living nematodes.

Most importantly, rCry5B was highly active against parasitic females of S. stercoralis, which represent a therapeutic target in vivo. Parasitic male S. stercoralis do not exist; parasitic females produce eggs via parthenogenesis, and their progeny are able to complete their life cycle within the host, allowing infections to persist for up to 75 years. Our data demonstrate that Cry5B is a promising new candidate for treating strongyloidiasis. Because the mode of action of Cry5B has a novel mechanism, binding to specific glycolipid receptors,3436 which overcomes and differs from all known anthelmintics, Cry5B could be used to break resistance to these compounds or combined with them to improve the efficacy and delay the onset of resistance.

Cry5B protein is rapidly digested in simulated gastric juices.21 Nevertheless, oral dosing of Cry5B still has high therapeutic effects against hookworms and ascarids in rodents, dogs, and pigs.1721,24 Furthermore, Cry5B efficacy can be improved by neutralization of stomach acid with stomach pump inhibitors, showing a near-complete elimination of hookworms in hamsters at 6 mg/kg, a dose comparable with those used with known anthelmintics against these same infections.19 In fact, on a molar basis, Cry5B is far (> 100×) more potent than current anthelmintics.19,21 In addition, protecting proteins from stomach digestion using enteric coatings is common. Taken together, these data indicate that Cry5B can be successfully delivered orally for small intestinal nematodes and holds great promise in treatment of the diseases that these parasites cause. Further work on formulation for optimal delivery is ongoing.

In summary, we demonstrated for the first time that Cry5B is highly effective against multiple stages of S. stercoralis. Further studies should be performed to assess the therapeutic effects of Cry5B in vivo.

Acknowledgments:

We appreciate the staff members of the Lymphatic Filariasis and Tropical Medicine Research Unit, Department of Parasitology, Faculty of Medicine, Chulalongkorn University for their technical assistance in the laboratory work. We also thank David Gazzola for rCry5B expression and Xinshe Li and Hongguang Shao for their help in maintaining S. stercoralis at the University of Pennsylvania.

REFERENCES

  • 1.

    Greaves D, Coggle S, Pollard C, Aliyu SH, Moore EM, 2013. Strongyloides stercoralis infection. BMJ 347: f4610.

  • 2.

    Luvira V, Watthanakulpanich D, Pittisuttithum P, 2014. Management of Strongyloides stercoralis: a puzzling parasite. Int Health 6: 273281.

  • 3.

    Schar F, Trostdorf U, Giardina F, Khieu V, Muth S, Marti H, Vounatsou P, Odermatt P, 2013. Strongyloides stercoralis: global distribution and risk factors. PLoS Negl Trop Dis 7: e2288.

    • Search Google Scholar
    • Export Citation
  • 4.

    Prendki V, Fenaux P, Durand R, Thellier M, Bouchaud O, 2011. Strongyloidiasis in man 75 years after initial exposure. Emerg Infect Dis 17: 931932.

  • 5.

    Buonfrate D, Requena-Mendez A, Angheben A, Munoz J, Gobbi F, Van Den Ende J, Bisoffi Z, 2013. Severe strongyloidiasis: a systematic review of case reports. BMC Infect Dis 13: 78.

    • Search Google Scholar
    • Export Citation
  • 6.

    Croker C, Reporter R, Redelings M, Mascola L, 2010. Strongyloidiasis-related deaths in the United States, 1991–2006. Am J Trop Med Hyg 83: 422426.

    • Search Google Scholar
    • Export Citation
  • 7.

    Marcos LA, Terashima A, Canales M, Gotuzzo E, 2011. Update on strongyloidiasis in the immunocompromised host. Curr Infect Dis Rep 13: 3546.

  • 8.

    WHO, 2011. Helminth Control in School-Age Children. A Guide for Managers of Control Programmes. Geneva, Switzerland: World Health Organization. Available at: https://www.who.int/neglected_diseases/resources/9789241548267/en/.

    • Search Google Scholar
    • Export Citation
  • 9.

    WHO, 2012. Research Priorities for Helminth Infections. WHO Technical Report Series. Geneva, Switzerland: World Health Organization. Available at: https://apps.who.int/iris/handle/10665/75922.

    • Search Google Scholar
    • Export Citation
  • 10.

    Gupta S, Jain A, Fanning TV, Couriel DR, Jimenez CA, Eapen GA, 2006. An unusual cause of alveolar hemorrhage post hematopoietic stem cell transplantation: a case report. BMC Cancer 6: 87.

    • Search Google Scholar
    • Export Citation
  • 11.

    Hunter CJ, Petrosyan M, Asch M, 2008. Dissemination of Strongyloides stercoralis in a patient with systemic lupus erythematosus after initiation of albendazole: a case report. J Med Case Rep 2: 156.

    • Search Google Scholar
    • Export Citation
  • 12.

    Kaplan RM, Klei TR, Lyons ET, Lester G, Courtney CH, French DD, Tolliver SC, Vidyashankar AN, Zhao Y, 2004. Prevalence of anthelmintic resistant cyathostomes on horse farms. J Am Vet Med Assoc 225: 903910.

    • Search Google Scholar
    • Export Citation
  • 13.

    Beknazarova M, Whiley H, Ross K, 2016. Advocating for both environmental and clinical approaches to control human strongyloidiasis. Pathogens 5: E59.

    • Search Google Scholar
    • Export Citation
  • 14.

    Bravo A, Likitvivatanavong S, Gill SS, Soberon M, 2011. Bacillus thuringiensis: a story of a successful bioinsecticide. Insect Biochem Mol Biol 41: 423431.

    • Search Google Scholar
    • Export Citation
  • 15.

    Ibrahim MA, Griko N, Junker M, Bulla LA, 2010. Bacillus thuringiensis: a genomics and proteomics perspective. Bioeng Bugs 1: 3150.

  • 16.

    Wei JZ, Hale K, Carta L, Platzer E, Wong C, Fang SC, Aroian RV, 2003. Bacillus thuringiensis crystal proteins that target nematodes. Proc Natl Acad Sci USA 100: 27602765.

    • Search Google Scholar
    • Export Citation
  • 17.

    Cappello M, Bungiro RD, Harrison LM, Bischof LJ, Griffitts JS, Barrows BD, Aroian RV, 2006. A purified Bacillus thuringiensis crystal protein with therapeutic activity against the hookworm parasite Ancylostoma ceylanicum .Proc Natl Acad Sci USA 103: 1515415159.

    • Search Google Scholar
    • Export Citation
  • 18.

    Hu Y, Zhan B, Keegan B, Yiu YY, Miller MM, Jones K, Aroian RV, 2012. Mechanistic and single-dose in vivo therapeutic studies of Cry5B anthelmintic action against hookworms. PLoS Negl Trop Dis 6: e1900.

    • Search Google Scholar
    • Export Citation
  • 19.

    Hu Y et al. 2018. Bacillus thuringiensis Cry5B protein as a new pan-hookworm cure. Int J Parasitol Drugs Drug Resist 8: 287294.

  • 20.

    Urban JF Jr., Hu Y, Miller MM, Scheib U, Yiu YY, Aroian RV, 2013. Bacillus thuringiensis-derived Cry5B has potent anthelmintic activity against Ascaris suum .PLoS Negl Trop Dis 7: e2263.

    • Search Google Scholar
    • Export Citation
  • 21.

    Hu Y, Georghiou SB, Kelleher AJ, Aroian RV, 2010. Bacillus thuringiensis Cry5B protein is highly efficacious as a single-dose therapy against an intestinal roundworm infection in mice. PLoS Negl Trop Dis 4: e614.

    • Search Google Scholar
    • Export Citation
  • 22.

    Li X-Q, Tan A, Voegtline M, Bekele S, Chen C-S, Aroian RV, 2008. Expression of Cry5B protein from Bacillus thuringiensis in plant roots confers resistance to root-knot nematode. Biol Control 47: 97102.

    • Search Google Scholar
    • Export Citation
  • 23.

    Hu Y, Platzer EG, Bellier A, Aroian RV, 2010. Discovery of a highly synergistic anthelmintic combination that shows mutual hypersusceptibility. Proc Natl Acad Sci USA 107: 59555960.

    • Search Google Scholar
    • Export Citation
  • 24.

    Hu Y, Miller M, Zhang B, Nguyen TT, Nielsen MK, Aroian RV, 2018. In vivo and in vitro studies of Cry5B and nicotinic acetylcholine receptor agonist anthelmintics reveal a powerful and unique combination therapy against intestinal nematode parasites. PLoS Negl Trop Dis 12: e0006506.

    • Search Google Scholar
    • Export Citation
  • 25.

    Charuchaibovorn S, Sanprasert V, Nuchprayoon S, 2019. The experimental infections of the human isolate of Strongyloides stercoralis in a rodent model (The Mongolian gerbil, Meriones unguiculatus). Pathogens 8: E21.

    • Search Google Scholar
    • Export Citation
  • 26.

    Koga K, Kasuya S, Khamboonruang C, Sukhavat K, Ieda M, Takatsuka N, Kita K, Ohtomo H, 1991. A modified agar plate method for detection of Strongyloides stercoralis. Am J Trop Med Hyg 45: 518521.

    • Search Google Scholar
    • Export Citation
  • 27.

    Nolan TJ, Megyeri Z, Bhopale VM, Schad GA, 1993. Strongyloides stercoralis: the first rodent model for uncomplicated and hyperinfective strongyloidiasis, the Mongolian gerbil (Meriones unguiculatus). J Infect Dis 168: 14791484.

    • Search Google Scholar
    • Export Citation
  • 28.

    Albarqi MM, Stoltzfus JD, Pilgrim AA, Nolan TJ, Wang Z, Kliewer SA, Mangelsdorf DJ, Lok JB, 2016. Regulation of life cycle checkpoints and developmental activation of infective larvae in Strongyloides stercoralis by dafachronic acid. PLoS Pathog 12: e1005358.

    • Search Google Scholar
    • Export Citation
  • 29.

    Ashton FT, Zhu X, Boston R, Lok JB, Schad GA, 2007. Strongyloides stercoralis: amphidial neuron pair ASJ triggers significant resumption of development by infective larvae under host-mimicking in vitro conditions. Exp Parasitol 115: 9297.

    • Search Google Scholar
    • Export Citation
  • 30.

    Stoltzfus JD, Massey HC Jr., Nolan TJ, Griffith SD, Lok JB, 2012. Strongyloides stercoralis age-1: a potential regulator of infective larval development in a parasitic nematode. PLoS One 7: e38587.

    • Search Google Scholar
    • Export Citation
  • 31.

    Bischof LJ, Huffman DL, Aroian RV, 2006. C. elegans: Methods and Applications. Strange K, ed. Methods in Molecular Biology. Totowa, NJ: © Humana Press Inc.

    • Search Google Scholar
    • Export Citation
  • 32.

    Bischof LJ, Huffman DL, Aroian RV, 2006. Assays for toxicity studies in C. elegans with Bt crystal proteins. Methods Mol Biol 351: 139154.

  • 33.

    Los FC, Kao CY, Smitham J, McDonald KL, Ha C, Peixoto CA, Aroian RV, 2011. RAB-5- and RAB-11-dependent vesicle-trafficking pathways are required for plasma membrane repair after attack by bacterial pore-forming toxin. Cell Host Microbe 9: 147157.

    • Search Google Scholar
    • Export Citation
  • 34.

    Griffitts JS, Haslam SM, Yang T, Garczynski SF, Mulloy B, Morris H, Cremer PS, Dell A, Adang MJ, Aroian RV, 2005. Glycolipids as receptors for Bacillus thuringiensis crystal toxin. Science 307: 922925.

    • Search Google Scholar
    • Export Citation
  • 35.

    Griffitts JS, Huffman DL, Whitacre JL, Barrows BD, Marroquin LD, Muller R, Brown JR, Hennet T, Esko JD, Aroian RV, 2003. Resistance to a bacterial toxin is mediated by removal of a conserved glycosylation pathway required for toxin-host interactions. J Biol Chem 278: 4559445602.

    • Search Google Scholar
    • Export Citation
  • 36.

    Griffitts JS, Whitacre JL, Stevens DE, Aroian RV, 2001. Bt toxin resistance from loss of a putative carbohydrate-modifying enzyme. Science 293: 860864.

    • Search Google Scholar
    • Export Citation

Author Notes

Address correspondence to Surang Nuchprayoon, Department of Parasitology, Faculty of Medicine, Chulalongkorn University, 1873 Rama IV Rd., Bangkok 10330, Thailand. E-mail: fmedstt@gmail.com

Financial support: This research was financially supported by the 100th Anniversary Chulalongkorn University for Doctoral Scholarship and by National Institutes of Health National Institute of Allergy and Infectious Diseases (R. V. A., grant number R01 AI056189; J. B. L., grant number R01 AI022662) and Agriculture and Food Research Initiative competitive grant (no. 2015-11323) to R. V. A. from the USDA National Institute of Food and Agriculture.

Authors’ addresses: Sarit Charuchaibovorn, Siriraj Center of Excellence for Stem Cell Research (SiSCR), Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand, E-mail: sarit.src@gmail.com. Vivornpun Sanprasert and Surang Nuchprayoon, Department of Parasitology, Lymphatic Filariasis and Tropical Medicine Research Unit, Chulalongkorn Medical Research Center (Chula MRC), Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand, E-mails: vivornpun@chula.md and fmedstt@gmail.com. Nataya Sutthanont, Lymphatic Filariasis and Tropical Medicine Research Unit, Chulalongkorn Medical Research Center (Chula MRC), Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand, E-mail: nanzzy0704@gmail.com. Yan Hu, Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, and Biology Department, Worcester State University, Worcester, MA, E-mail: yhu@worcester.edu. Ambily Abraham, Gary R. Ostroff, and Raffi V. Aroian, Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, E-mails: ambily.abraham@umassmed.edu, gary.ostroff@umassmed.edu, and raffi.aroian@umassmed.edu. Tegegn G. Jaleta and James B. Lok, Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, E-mails: tjaleta@vet.upenn.edu and jlok@vet.upenn.edu.

These authors contributed equally to this work.

Save