Comparative Analysis of the Secretome from a Model Filarial Nematode (Litomosoides sigmodontis) Reveals Maximal Diversity in Gravid Female Parasites

Filarial nematodes (superfamily Filarioidea) are responsible for an annual global health burden of ∼6.3 million disability-adjusted life-years, which represents the greatest single component of morbidity attributable to helminths affecting humans. No vaccine exists for the major filarial diseases, lymphatic filariasis and onchocerciasis; in part because research on protective immunity against filariae has been constrained by the inability of the human-parasitic species to complete their lifecycles in laboratory mice. However, the rodent filaria Litomosoides sigmodontis has become a popular experimental model, as BALB/c mice are fully permissive for its development and reproduction. Here, we provide a comprehensive analysis of excretory-secretory products from L. sigmodontis across five lifecycle stages and identifications of host proteins associated with first-stage larvae (microfilariae) in the blood. Applying intensity-based quantification, we determined the abundance of 302 unique excretory-secretory proteins, of which 64.6% were present in quantifiable amounts only from gravid adult female nematodes. This lifecycle stage, together with immature microfilariae, released four proteins that have not previously been evaluated as vaccine candidates: a predicted 28.5 kDa filaria-specific protein, a zonadhesin and SCO-spondin-like protein, a vitellogenin, and a protein containing six metridin-like ShK toxin domains. Female nematodes also released two proteins derived from the obligate Wolbachia symbiont. Notably, excretory-secretory products from all parasite stages contained several uncharacterized members of the transthyretin-like protein family. Furthermore, biotin labeling revealed that redox proteins and enzymes involved in purinergic signaling were enriched on the adult nematode cuticle. Comparison of the L. sigmodontis adult secretome with that of the human-infective filarial nematode Brugia malayi (reported previously in three independent published studies) identified differences that suggest a considerable underlying diversity of potential immunomodulators. The molecules identified in L. sigmodontis excretory-secretory products show promise not only for vaccination against filarial infections, but for the amelioration of allergy and autoimmune diseases.Filarial nematodes are the most important helminth parasites of humans in terms of overall impact on public health, with an annual global burden of ∼6.3 million disability-adjusted life-years (1). Lymphatic filariasis (LF)¹ or “elephantiasis,” which affects populations across Africa, South Asia, the Pacific, Latin America, and the Caribbean, accounts for 92% of this toll. The remainder is caused by onchocerciasis or “river blindness,” primarily in sub-Saharan Africa. The major human filarial pathogens are Wuchereria bancrofti (responsible for 90% of LF cases), Brugia malayi and Brugia timori (geographically restricted causes of LF), and Onchocerca volvulus (the sole agent of human onchocerciasis). In addition, Loa loa affects ∼13 million people in West and Central Africa. This parasite usually induces a relatively mild disease, but has been associated with severe and sometimes fatal adverse events following anthelmintic chemotherapy (2). Filarial parasites are primarily drivers of chronic morbidity, which manifests as disabling swelling of the legs, genitals and breasts in LF; or visual impairment and severe dermatitis in onchocerciasis. The filariae are also a major problem in small animal veterinary medicine, with ∼0.5 million dogs in the USA alone infected with Dirofilaria immitis (3), the cause of potentially fatal heartworm disease. However, in domesticated ungulates, filarial infections are generally benign (4).Currently, control of human filarial diseases is almost entirely dependent on three drugs (ivermectin, diethylcarbamazine, and albendazole). Prevention of heartworm also relies on prophylactic treatment of dogs and cats with ivermectin or other macrocyclic lactones. Reports of possible ivermectin resistance in O. volvulus (5) and D. immitis (6) have highlighted the importance of maintaining research efforts in vaccine development against filarial nematodes. However, rational vaccine design has been constrained for several decades (7) by the intrinsic complexity of these metazoan parasites and their multistage lifecycle. Moreover, many filarial species carry obligate bacterial endosymbionts (Wolbachia), which may also stimulate the immune response during infection (8). As part of global efforts to improve prevention and treatment of these diseases, large-scale projects have been undertaken, including sequencing of the nematodes (9–11) and their Wolbachia (10, 12, 13), and proteomic analyses of both whole organisms and excretory-secretory products (ESP) (14, 15). Additionally, two studies (both on B. malayi) have examined lifecycle stage-specific secretomes (16, 17). In the context of vaccine design, the identification of ESP proteins and determination of their expression in each major lifecycle stage can facilitate the prioritization of candidates for efficacy screening in animal models.One barrier to the progression of research in the filarial field is our inability to maintain the full lifecycle of the human parasites in genetically tractable hosts. This lifecycle involves uptake of the first-stage larvae (microfilariae, Mf) by a hematophagous arthropod and two moults in the vector, followed by transmission of third-stage larvae (L3) to a new vertebrate host and two further moults before the nematodes mature as dioecious adults. However, the complete lifecycle of the New World filaria Litomosoides sigmodontis can be maintained in laboratory rodents, including inbred mice (18). This species [incorrectly referred to as L. carinii in the older literature (19)] was first studied in its natural host, the cotton rat (Sigmodon hispidus) (20). Mongolian jirds (Meriones unguiculatus) are also fully permissive for L. sigmodontis infection and are routinely used for maintaining its lifecycle in the laboratory, as they tolerate higher parasite burdens than do laboratory mice. To exploit the full power of murine immunology, including defined knockout strains, L. sigmodontis in mice has been used to address questions regarding the fundamental immunomodulatory mechanisms employed by filarial parasites (21, 22), their ability to mitigate proinflammatory pathology and autoimmune disease (23), and the impact of various vaccine strategies on adult nematode burden and fecundity (24, 25).Using the resource of a newly-determined genome sequence, coupled with a derivative of the intensity-based absolute quantification (iBAQ) proteomic approach, we have examined the stage-specific secretome of L. sigmodontis in vector-derived L3 (vL3), adult males (AM), pre-gravid adult females (pgAF), gravid adult females (gAF), and immature Mf (iMf). In addition to identifying dynamic changes in the ESP profile through the lifecycle, we show important differences in the adult secretomes of L. sigmodontis and B. malayi, especially in the abundance of two novel proteins released by female L. sigmodontis that lack orthologs in B. malayi. As has been observed in other parasitic nematodes, we find transthyretin-like family (TTL) proteins to be particularly dominant in the ESP. Active expulsion of uterine fluid may account for the remarkable diversity of proteins that we detect in gAF ESP, and we highlight several novel proteins that warrant evaluation in vaccine trials and as anti-inflammatory mediators.