Current Research Directions

I. Anthelmintics (curing roundworm diseases in humans):

Goal: Harnessing crystal proteins made by the common soil bacterium Bacillus thuringiensis as a new, natural, and effective cure for intestinal helminth parasites.

Background: Soil-transmitted helminths (STHs), including hookworm, whipworm, and Ascaris, are diseases of the world's most poor and are likely the most common human parasite. These parasites infect the gastrointestinal (GI) tracts of 1 in 3 people in the world and may cause as much morbidity as malaria. STH infections in children result in growth and cognitive stunting and severely impact learning, school attendance, and future income potential. The World Health Assembly (WHA) in 2001 has urged the deworming of 75% at-risk school-aged children (nearly 400 million children). Over 44 million hookworm-infected pregnant women are at increased risk for premature delivery, low birth weight, maternal ill-health, and maternal death. Recent data suggest STH infections worsen the effects of malaria, HIV, and TB. STHs are one of the greatest neglected diseases of our time.

Only four anti-nematode drugs (anthelmintics) that fall into two classes are approved by the WHA for STH therapy in humans: the benzimidazoles (mebendezole, albendazole) and nicotinic acetylcholine receptor (nAChR) agonists (levamisole, pyrantel). The problem with having so few anthelmintic classes is the emergence of parasite resistance. In veterinary medicine, every parasitic nematode has been able to develop resistance to every class of anthelmintic.  In human therapy studies, resistance to anthelmintics is suspected in Australia, Zanzibar, Vietnam, and Mali. Experiences from veterinary helminth programs paint a grim picture of the future if we take no action to prevent the emergence of resistance.  This realization has evoked urgent and repeated cries for the development of new anthelmintics.

Our approach: Our laboratory is pioneering work on a new class of anthelmintics, crystal (Cry) proteins made by the soil bacterium Bacillus thuringiensis (Bt). Over five decades of intensive use of these proteins as insecticides, including in major aerial spraying and mosquito control programs and in transgenic food crops, have proven that Bt and its crystal proteins are non-toxic to vertebrates. However, Cry proteins have been overlooked for their potential as anthelmintics. A few years ago, we demonstrated that the Bt Cry protein, Cry5B, can kill free-living soil nematodes (Wei et al., 2003). Cry5B, like other Cry proteins, is predicted to have a high safety profile in vertebrates since its receptor is found only in invertebrates (Griffitts and Aroian, 2005). Cry5B has a mechanism of action unique from all currently used anthelmintics.

We were the first laboratory to show that a crystal protein, namely Cry5B,can indeed be used to cure a nematode infection in vivo. In collaboration with the Cappello laboratory, Cry5B protein was delivered orally (gavaged) into hamsters infected with the hookworm Ancylostoma ceylanicum. Analyses of these hamsters showed that the crystal protein was able to effect a near complete cure of the infection (Cappello et al., 2006).

We are currently researching to improve the efficacy of Cry5B, approaching this issue from many different angles, including optimization of its toxicity against the free-living nematode, Caenorhabditis elegans (e.g., by mutagensis of the protein) and testing these improvements against other intestinal parasitic worms. We are also testing other crystal proteins for their efficacy in models of human parasitic roundworms. Our goal is to bring Cry5B into human clinical trials as a new and natural remedy for parasitic roundworms.

We gratefully acknowledge the National Institute of Allergy and Infectious Diseases at the NIH and the American taxpayer for supporting this research.

--Team Wormfree

II. Pore-forming toxins

Goal: Study how animal cells guard against the largest and single more important class of toxins made by bacteria-- pore-forming toxins and use this knowledge to cure bacterial diseases.

Background: Pore-forming toxins (PFTs) are the single most abundant protein virulence factor made by disease-causing bacteria and are important for the virulence of many important human pathogens including Staphylococcus aureus, Streptococcus pyogenes, Clostridium perfringens, and Aeromonas hydrophilia. They are thus arguably the single-most important class of toxins made by the bacteria that cause sickness in humans.

Despite their importance, these toxins as a class have been greatly understudied. Part of the problem is that in the past there has not been a genetic model organism available to study these protein toxins. The crystal proteins made by Bacillus thuringiensis (Bt) are PFTs that target the intestinal cells of nematodes and insects. Although used as natural pesticides, Bt Cry PFTs have similar effects on target cells as PFTs made by human pathogenic bacteria do on human cells. Thus, by studying the effects of Cry proteins on the genetic model organism, Caenorhabditis elegans, we can provide a genetic in vivo model for the effects of PFTs that attack human cells as part of the attack of pathogenic bacteria.

Our approach: Our primary focus has been on understanding how cells targeted by PFTs mount defenses against these toxins. Our laboratory was the first to show that C. elegans can use a signal transduction pathway, namely the p38 MAPK pathway, to defend against a PFT (Huffman et al., 2004). In collaboration with the Van der Goot laboratory, this observation was extended to mammalian cells. More recently, we screened through the entire Ahringer collection of 16757 RNAi clones to identify genes that, when eliminated from the worm, make the worm hypersensitive to PFT. We identified ~250 genes that fall into this class. We are now using an array of analytical techniques, both molecular and cell biological, to connect these genes into pathways and these pathways with other pathways to understand at a global level how cells respond to and protect against pore-forming toxins.

By underdanding how cells defend against pore-forming toxins, our ultimate goal is to design therapies based upon this knowledge to improve our ability to cure bacterial infections. An attractive feature of focusing on this approach is that it aims to complement the use of antibiotics-- that is, an approach of attacking bacteria back by attacking their pore-forming toxins could work in cases where antibiotics no longer do, due to bacterial antibiotic resistance.

We gratefully acknowledge the National Institute of General Medical Sciences at the NIH and the American taxpayer for supporting this research.

-- Team INCED

III. Crystal protein resistance and use in transgenic plants to control plant-parasitic nematodes

Goal: Study how invertebrate pests develop resistance to the largest natural pesticide in use around the world today-- Bt crystal proteins.

Background: The bacterium Bacillus thuringiensis (Bt) is by far the most widely used natural, biologically produced pesticide in the world today. It is used as a topical spray by farmers to kill insect pests and used by governments and NGOs around the world in the control of insects (mosquitoes, black flies) that carry diseases. It is the favorite pesticide of organic farmers. Bt is very safe-- over 50 years of use and laboratory testing have proven that even at high levels, Bt is non-toxic to vertebrates. The active ingredient in Bt is its crystal (Cry) proteins. Our laboratory has shown at the molecular level why Bt may be so safe to use-- one of the receptors that Cry proteins bind to in order to kill insects and nematodes is missing from vertebrates (Griffitts et al., 2005).

More recently, Cry proteins have been put into transgenic crops. Although controversial for some, these "genetically modified organisms (GMOs)" have been shown to have a positive effect on agriculture. The use of safe, natural, organic Cry proteins in corn, cotton, and rice have allowed farmers to significantly reduce their chemical pesticide use because they no longer need to spray so often with hazardous compounds to kill insects (Huang et al., 2005; Qaim and Zilberman, 2003). That has helped the environment as well as improved farmer health. At the same time, expression of Cry proteins has allowed crop yields to go up. Given increasing demands for better yields from our crops to feed an ever-growing world, Bt Cry-protein expressing crops can provide a positive boost to our environment and yields.

There is, nonetheless, a serious concern that the extensive use of Bt in transgenic crops and as sprays will give rise to resistance. That is to say, the insects will no longer be intoxicated by the proteins. If this were to happen,this tremendous resource of nature would be lost to us.

Our approach: To try to help circumvent the resistance problem and to better understand how Cry proteins intoxicate their targets, our laboratory is actively screening for genes that, when mutated, give rise to Cry protein resistance in C. elegans. We have screened for resistance to the crystal protein, Cry21A, and have found an entirely new resistance pathway not previously identified in resistance screens in insects and nematodes. In addition, we have used RNAi-based screening with protease genes in the C. elegans genome, which identified a signal transduction pathway that also can mutate to resistance. These studies are greatly expanding our knowledge of how resistance to Cry proteins can develop and of the pathways invoked in cells that aid in the intoxication process.

We have also worked to test whether or not transgenic crops expressing nematicidal Cry proteins that protect crops against plant-parasitic nematodes, much the way that transgenic crops expressing inseciticidal Cry proteins protect against insect pests. Currently, nematode crop pests are controlled with very toxic compounds like methyl bromide. Finding alternatives to these chemicals is very desirable and organic Cry proteins might be one such alternative.

We expressed several nematicidal Cry proteins in plants and found that expression of two of these proteins (e.g., Cry6A) can inhibit the ability of a plant-parasitic nematode to produce progeny (Li et al., 2007; Li et al., 2008). Our data demonstrate that Cry proteins have excellent potential to control plant-parasitic nematodes. Although we are not currently working to develop this idea further, we are willing to help those that are (see contact page if you are interested).

We gratefully acknowledge the National Science Foundation and the American taxpayer for currently supporting this research.

-- Team MechIntox