Although microbes influence the health and disease of all animals, we still understand little about mechanisms underlying microbial specificity for hosts and tissues or microbial contributions to host biology. To address these questions, my lab investigates a relatively simple tripartite association in which nematodes of the genus Steinernema parasitize insects by virtue of their mutualistic association with Xenorhabdus bacteria. The Steinernema-Xenorhabdus-insect symbiosis offers numerous advantages as a model to understand the specificity and processes of beneficial and pathogenic microbial colonization of host tissues. These include its experimental tractability: Each member of the tripartite association can be reared separately or together in varied combinations in the laboratory, at least one representative of each has a sequenced genome and transcriptome, and numerous genetic techniques have been developed for Xenorhabdus bacteria. Furthermore, investigations are simplified by the fact that Steinernema are colonized by a monoxenic culture of a specific species of Xenorhabdus bacteria, rather than a complex microbial consortium. Thus, the system can be probed to understand the molecular basis for specificity and the impacts of bacteria on animal host behavior, reproduction, immunity, and physiology. Finally, the Steinernema-Xenorhabdus mutualism is a conserved characteristic of both genera, facilitating evolutionary and comparative analyses of symbiosis mechanisms. The potential of the Steinernema-Xenorhabdus symbiosis to reveal fundamental molecular and cellular processes mediating symbiosis is inhibited by a lack of genetic and molecular tools to manipulate the nematode. Furthermore, although several C. elegans researchers have made advances in understanding several aspects of Steinernema molecular biology, much remains to be learned regarding Steinernema physiology, behavior, development, and immunity with respect to their symbiosis with Xenorhabdus.

Research in my lab focuses on understanding the molecular basis of bacterial symbiosis with animal hosts, and what distinguishes beneficial and pathogenic interactions. I will focus my talk on the molecular basis of species specificity between nematodes and bacteria. We have determined that X. nematophila bacterial association with its S. carpocapsae nematode host is tissue- and species-specific and requires the integral outer membrane protein NilB; Bacteria adhere to the anterior intestinal epithelial surface of adult and juvenile nematodes only when expressing nilB. The consequences of this surface colonization are as yet unknown, but possibilities include bacteria-host cell signaling that promotes pheromone production or immunity. The molecular function of NilB also is unknown. It is a beta-barrel protein with surface exposed loops both necessary for nematode interactions and a predicted small channel with an electronegative strip. These regions may be important for NilB binding to host cell surfaces or for transport of host-derived molecules. NilB is the only characterized member of a family of OMPs found exclusively in symbionts of animals (e.g., Neisseria, Haemophilus, Moraxella, Actinobacillus). Therefore, understanding NilB function in Xenorhabdus–Steinernema mutualism will provide insights into NilB homolog function in human associated microbes.

There are ~23,000 described species of nematodes, and an estimated 2-10 million species exist. Genomics research on nematodes (particularly Caenorhabditis elegans) has delivered important information on core biological processes. Adding additional nematode genomes to that of C. elegans will allow the specific instance of C. elegans to be contextualised, and will we hope feed research on comparative genomics of nematodes, the evolutionary biology of genome change, and the biology of the (many) parasitic nematodes, amongst other fields.

Why (only) 959 nematode genomes?

All adult female hermaphrodite C. elegans have 959 somatic cells. The definition of the embryonic lineage of C. elegans from fertilised zygote to fertile adult was a milestone in C. elegans developmental biology. Just as the tree of the C. elegans embryonic lineage was a key underpinning of later work on this model nematode, we hope that a Nematoda phylogeny with 959 genome-sequenced taxa will underpin the investigation of nematode biology in general. Next generation sequencing technologies already can deliver 100-fold coverage of a new genome in under a week: our challenge is to recruit the next generation of (bioinformatics-capable, genome-savvy) nematologists to drive this aspiration.

Schistosomiasis is a parasitic disease caused by blood flukes (Phylum Platyhelminthes) of the genus Schistosoma. As many as 800 million people are affected in parts of South America, the Western Pacific and China with the majority (>95%) living in sub-Saharan Africa.  Children as young as six months are infected with what can be a life-long affliction.  In the severest of cases, damage to internal organs can kill. However, the disease is better known for its insidious ability to rob those who already live at or below subsistence of their physical and cognitive vitality.  This translates to missed school days, decreased ability to work and lost potential regarding both personal and societal productivity. Often, schistosomiasis is just one of a number of overlapping parasitic diseases, including other worm infections, which when combined degrade the well-being of whole communities.

Although parasitic flatworms can display bewildering life-cycle complexity, schistosomes are relatively easy to keep in the lab requiring as hosts mice (or hamsters) and a robust fast-breeding snail vector – a fortunate situation given the importance of the global disease.  This ease has not only facilitated a wealth of ‘classical’ life-history, biochemical, host-pathogen, and drug discovery studies, but also advances in ‘-omic’ applications over the last ten years with continually-improving annotations of genome, transcriptome and proteome data supported by interrogable databases. In turn, there has been a real effort in and measured success with a range of ‘post-genomic’ reverse genetic tools, typically involving both transient RNAi and transgenesis, but also viral-mediated germ line transgenesis. These developments are timely given the growing concern in the schistosome community at the precarious reliance on just one drug, praziquantel, to treat ‘continents of people’ and the consequent need to find new drug and vaccine targets, and diagnostic candidates.  Nonetheless, a wish-list for a standardized schistosome ‘molecular tool kit’ would include such items as complete in vitro cultivation of the parasite without recourse to mammalian (or molluscan) hosts, targeted germ line integration and the generation of cell lines. On the flip side, an example of employing C. elegans as a functional screening tool for schistosome drug discovery is presented and this approach bodes well for future drug discovery efforts.  Finally, a list of resources available to those potentially interested in working with schistosomes, even ‘on the side,’ is provided.

In December 2012, the results of the much awaited Global Burden of Disease (GBD) Study 2010 were released.  Aside from visceral leishmaniasis, the most important neglected tropical diseases (NTDs) when measured in disability adjusted life years (DALYs) were helminth infections led by the intestinal nematode infections (with hookworm infection accounting for two-thirds of the DALYs lost) followed by schistosomiasis, lymphatic filariasis, and food-borne trematode infections.  Together these NTDs in addition to onchocerciasis and trachoma are being targeted for either “elimination” (LF, onchocerciasis, and trachoma) or “control” (the intestinal nematode infections and schistosomiasis) through a scaled up program of mass drug administration (MDA) with support from USAID, UKAID, and a private END (Ending Neglected Disease Fund), and under the auspices of a 2012 London Declaration and a 2013 World Health Assembly Resolution.  However, it is unclear whether these goals can be achieved without the development of additional or improved control tools, i.e., drugs, diagnostics, vaccines, and monitoring for resistance or other operational parameters.  For instance, failure with benzimidazole anthelminthics is widespread for hookworm and trichuriasis, especially when they are used in a single dose, although the basis of this observation is not well understood.  It is uncertain whether drug resistance has emerged and is an ongoing concern.  Moreover, there are fundamental aspects of the basic biology of intestinal nematodes that we do not understand such as tissue tropisms and ecological niches, and the basis of long-term survival both within and outside of the human host.   Similar questions remain also for schistosomes and food-borne trematodes (and for praziquantel Rx) and we are just beginning to understand the basis for helminth-induced carcinogenesis and schistosome-induced female urogenital schistosomiasis and HIV/AIDS transmission.  Early stage vaccines for hookwrom and schistosomiasis are also in clinical development.  There remain a number of outstanding questions on the fundamental biology of filarial worms that cause lymphatic filariasis, onchocerciasis, and loiasis, including the basis of parasite tropisms, longevity, and immune evasion, the basis of microfilarial periodicitiy, and how parasite death occurs and its link to host immunopathogenesis.  There is a need to develop a macrofilaricide but few leads on how to achieve drug or vaccine development for this purpose.  Finally there are several important human helminth infections, such as toxocariasis and strongyloidiasis, for which we know little about in terms of disease burden and yet which may be as important as the other helminthiases mentioned.  Human Strongyloides infection has a unique interface between pathogenesis and nematode developmental biology, while toxocariasis has emerged as an important helminth infection the United States and it many be linked to asthma, developmental delays, and epilepsy, especially among under represented minority populations.

Over 140 million people are afflicted with filarial nematode-based diseases with over a billion at risk in over 70 countries. Of the estimated 120 million people afflicted with Lymphatic Filariasis, caused by Wuchereria bancrofti, Brugia malayi, and B. timori, 15 million present with swelling of the soft tissue, with the most extreme case known as Elephantiasis, while 25 million men present with scrotal hydrocele, causing great pain and disability. Another 18 million are afflicted with Onchocerciasis, caused by O. volvulus, which results in the deterioration of skin and eye tissue, with River Blindness being the most severe form. Much of the tissue damage is a result of the patient’s immune reaction against Wolbachia released from the nematodes. In addition to human suffering, these conditions also result in large economic losses to already poor communities.

In each of these diseases, the afflicted carry the long-lived (5-15 years) adult nematodes (macrofilariae), which in turn produce new generations of microfilaria that are released into the blood stream. Microfilariae are then taken up by blood-sucking insects, where they develop into infective larvae. During the next insect feeding on human blood, the larvae are transmitted to new hosts, where they develop into the next adult generation. Importantly, all current anti-filarial medications are only effective against microfilariae and do not destroy the long-lived adults.

Fortunately, it was recently discovered that all human parasitic filarial nematodes but one require the symbiotic bacteria Wolbachia for fertility and adult viability and this has opened up entirely new avenues for drug development. In addition to Wolbachia‘s role in the nematode’s livelihood, these bacteria have also been found to directly contribute to disease symptoms by triggering the victim’s inflammatory immune response. In fact, disease symptoms are frequently aggravated upon medication-induced worm death, due to additional release of Wolbachia from the nematode into the host. Recently, treatment with antibiotics has been shown to increase the efficacy of traditional anthelmintics and to reduce the side effects that are attributed to the release of the symbiotic bacteria.

Despite the biomedical relevance of Wolbachia in filariasis, little is known about the mechanisms of this mutualistic symbiosis. In addition to efforts focused on the development of anti-Wolbachia based therapies, many outstanding questions remain regarding the role of Wolbachia in the filarial immune escape, survival, and fertility. In other words, the interplay between Wolbachia, the filarial nematode, and the mammalian host forming a tripartite system, remains to be better defined.

We will introduce Brugia malayi, a causative agent of human elephantiasis as a model organism, and the technical possibilities and limitations associated with research on filarial nematodes. Using combinations of RNAi and immunofluorescence experiments, our lab is interested in understanding the cell and molecular mechanisms of Wolbachia transmission during the life of Brugia. During embryogenesis, Wolbachia rely on polarity signals to asymetrically segregate into hypodermal precursors. In late larval stages, hypodermal Wolbachia show an ovarian tropism and colonize the female germcells. To better define the basis of the symbiosis, we examined cell defects in Wolbachia-depleted worms. We will report these defects, which include centrosomal and embryonic polarity defects.