Genomics of Loa loa, a Wolbachia-free filarial parasite of humans

Introduction

Filarial nematodes dwell within the lymphatics and the subcutaneous tissues of up to 170 million people worldwide and are responsible for notable morbidity, disability and socioeconomic loss¹. Although eight filarial species infect humans, only five cause significant pathology -- Wuchereria bancrofti, Brugia malayi, and Brugia timori, the causative agents of lymphatic filariasis; Onchocerca volvulus, the causative agent of ‘river blindness’ or onchocerciasis; and Loa loa, the African eyeworm; L. loa affects an estimated 13 million people and causes chronic infection most often characterized by localized angioedema (Calabar swelling) and/or subconjunctival migration of adult worms across the eye (“African eyeworm”). Complications of infection include encephalopathy, entrapment neuropathy, glomerulonephritis and endomyocardial fibrosis²; L. loa is restricted geographically to equatorial West and Central Africa, where its deerfly vector (Chrysops spp.) breeds; L. loa microfilariae (L1) are acquired by flies from human blood and subsequently develop into infective larvae (L3) before being reintroduced into a human host during a second blood meal (Supplementary Fig. 1). While L. loa is the least well-studied of the pathogenic filariae, it has been gaining prominence of late because of the severe adverse events (encephalopathy and death) associated with ivermectin treatment³ in mass drug administration campaigns in West and Central Africa.

L. loa was targeted for genomic sequencing for two reasons. First, in contrast to other pathogenic filariae, L. loa lacks the α-proteobacterial endosymbiont Wolbachia. The obligate nature of Wolbachia symbiosis in W. bancrofti, B. malayi, and O. volvulus has been inferred by studies in which antibiotics (e.g. doxycycline) that target Wolbachia (but not the worm itself) have shown efficacy in treating humans with these infections^{4, 5}. Through genomic analysis, Wolbachia have been hypothesized to provide essential metabolic supplementation to their filarial hosts^{6, 7}. The absence of the Wolbachia endosymbiont in L. loa suggests that either there has been lateral transfer of important bacterially-encoded genes or that the obligate relationship between the endosymbiont and its filarial host is dispensable, at least under certain circumstances. Understanding the comparable adaptations of L. loa was considered essential to gain insight into the potential impact of the endosymbiont⁸. Second, as the most neglected of the pathogenic filariae but one gaining increasingly more clinical prominence, understanding the host parasite relationship as it relates to the severe post-treatment reactions typical of both Wolbachia-containing and Wolbachia-free filarial parasites became of paramount importance.

Thus, we generated a draft genome sequence of L. loa generated and produced a refined gene annotation aided by transcriptional data from L. loa microfilariae. We also generated draft genome sequences of two of the most pathogenic (and Wolbachia-containing) filarial species, W. bancrofti and O. volvulus. This approach enabled us to define more comprehensively the genomic differences between L. loa and other filarial parasites.

Genome assemblies and repeat content

The nuclear genome of Loa loa consists of five autosomes plus a sex chromosome. Using 454 whole genome shotgun sequencing, L. loa was sequenced to 20x coverage and assembled into 5774 scaffolds with an N50 of 172 Kb and total size of 91.4 Mb (Table 1). The W. bancrofti and O. volvulus genomes derived from single adult worms (an unsexed juvenile adult worm for W. bancrofti and an adult male worm for O. volvulus) were sequenced to 12x and 5x coverage, respectively (Table 1). Because of the low coverage of the O. volvulus genome, it was not included in further analysis. While the assembly sizes of the L. loa and B. malayi genomes are comparable (91.4 Mb versus 93.7 Mb), the scaffold N50 of the L. loa genome is almost twice that of B. malayi, making the L. loa genome assembly the most contiguous assembly of any filarial nematode to date. The filarial genomes differ widely in repeat content (Table 1, Supplementary Tables 1–14, Supplementary Note) with the L. loa genome being more repetitive than W. bancrofti but less than B. malayi.

As nuclear Wolbachia transfers (nuwts) have been identified in all Wolbachia-colonized and Wolbachia-free filarial nematodes examined⁹, we expected to find similar transfers in the L. loa genome. However, a BLAST-based search of the assembled L. loa genome did not reveal any large transfers of Wolbachia DNA. A more sensitive read-based analysis determined that the L. loa genome does not have any large, recent transfers (> 500 bp, Supplementary Note). It does however have small, presumably “older”, transfers supporting the hypothesis that L. loa was once colonized by Wolbachia but subsequently lost its endosymbiont (Supplementary Table 15 and Supplementary Fig. 2). Of the transfers definitively of Wolbachia ancestry, and not of possible mitochondrial ancestry, there is no evidence that these are functional in L. loa (Supplementary Note).

Gene content and synteny

Initial gene sets were produced for both L. loa and W. bancrofti based on a combination of gene predictors with refinements to the L. loa annotation based on RNA sequence (RNA-Seq) data (see Methods). The final L. loa gene set contains 14,907 genes, 70% of which are supported by RNA-Seq (Table 1, Supplementary Tables 16, 17). The W. bancrofti genome is predicted to encode 19,327 genes (Table 1, Supplementary Note). The filarial genomes show a high degree of synteny (Figure 1), with 40% and 13% of L. loa genes being syntenic relative to B. malayi and W. bancrofti, respectively. Nearly all syntenic breaks between filarial genomes occur at scaffold ends (Supplementary Fig. 3B), suggesting the percentage is limited by assembly contiguity and the true level of synteny is much higher. When the L. loa genome is compared to that of C. elegans, orthologs from a single L. loa scaffold map predominantly to a single C. elegans chromosome (Figure 1). However, only 2% of all L. loa genes were syntenic relative to C. elegans (Supplementary Fig. 3A), supporting the hypothesis that genome rearrangements during filarial evolution were mostly intra-chromosomal⁷. An intermediate level of synteny (12%) is seen between L. loa and related non-filarial parasite Ascaris suum (Supplementary Fig. 3A).

More than half of the genes encoded by the L. loa and W. bancrofti genomes could be assigned functional categories, PFAM domains, Gene Ontology (GO) terms, and/or Enzyme Commission (EC) numbers (Supplementary Fig. 4, Supplementary Tables 16, 18). Relative to other filarial genomes, the L. loa genome is enriched (p < 0.05, Fisher’s exact test) for numerous domains, including pyridoxamine 5’-phosphate oxidases that synthesize vitamin B (Figure 2; see below). The L. loa genome is also enriched for numerous chemoreceptors suggesting that L. loa may be capable of more complex interactions with its host environment than other filarial worms (see below; Supplementary Note). An RNA helicase domain involved in viral DNA replication is enriched in the L. loa genome; this domain was likely horizontally transferred to the L. loa genome from cyclovirus infection (Supplementary Note). While not statistically significant, the L. loa genome encodes many more hyaluronidases (6) than the B. malayi or W. bancrofti genomes (2 each). Hyaluronidases are often involved in tissue penetration and could allow L. loa to move more readily through human host tissue, as L. loa adults are highly mobile whereas B. malayi and W. bancrofti adults are commonly tethered to the lymphatic endothelium.

The genome of W. bancrofti is enriched (p < 0.05, Fisher’s exact test) for genes with domains related to cellular adhesion and the extracellular matrix (e.g., cadherins, laminins, and fibronectins). Whether these are important in mediating the fibrosis associated with lymphatic filarial disease (e.g. elephantiasis, lymphedema) in W. bancrofti infection¹⁰ or with establishing an anatomical niche within the afferent lymphatics where the adults reside awaits clarification.

Gene products associated with immunologic responses

Each of the filarial parasites interacts with both its definitive mammalian and intermediate arthropod hosts (Chrysops spp. in the case of L. loa) during its life cycle (Supplementary Fig. 1). The parasite is thought not only to have its own innate immune system to protect itself from microbial pathogens, but also to have evolved mechanisms to exploit and/or subvert host and vector defense mechanisms. Although adaptive immune molecules such as immunoglobulins or Toll-like receptors (TLRs) are absent in L. loa and other filarial nematodes, L. loa, like other filariae, appears to possess a primordial Toll-related pathway (Supplementary Table 19, Supplementary Note). The innate immune system encoded by the L. loa genome also includes C-type lectins, galectins, jacalins, and scavenger receptors; L. loa contains a number of lipopolysaccharide binding proteins, proteins implicated in modulating the effects of host bacteria or microbial translocation products. Like B. malayi, the L. loa (and W. bancrofti) genomes do not encode antibacterial peptides described in C. elegans and Ascaris suum⁷, suggesting that these molecules are either dispensable in filariae or too divergent to detect.

Analysis of L. loa genes identified a number of human cytokine and chemokine mimics/antagonists including: genes encoding macrophage migration inhibition (MIF) family signaling molecules, transforming growth factor-β and their receptors, members of the interleukin (IL)-16 family, an IL-5 receptor antagonist, an interferon regulatory factor, a homolog of suppressor of cytokine signaling (SOCS)-7, and two members of the chemokine-like family (Supplementary Table 19). In addition, the L. loa genome encodes 17 serpins and 7 cystatins shown to interfere with antigen processing and presentation to T cells¹¹, 2 indoleamine 2,3 dioxygenase (IDO) genes that encode immunomodulatory proteins implicated in strategies of immune subversion, and a number of the Wnt family of developmental regulators that typically modulate immune activation. The L. loa genome encodes proteins that have sequences similar to human autoantigens (Supplementary Note). Although some of these can also be found in the other filariae, the slight expansion of these in L. loa suggests that antibodies induced by L. loa infection may be more autoreactive than those induced by other parasites.

Protein kinases

In addition to elucidating host-pathogen interactions, pathogen genomes can be evaluated for potential drug targets such as protein kinases. We therefore annotated protein kinases in the L. loa genome and compared them to those in other nematode genomes (Supplementary Tables 20–23, Supplementary Fig. 5). Numerous differences were observed between filarial and non-parasitic nematode kinases, particularly regarding meiosis. The widely-conserved TTK kinase (Mps1), which plays a key role in eukaryotic meiosis¹², is present in L. loa and absent in C. elegans. By contrast, filarial nematodes lack the nearly universally conserved RAD53-family kinase chk-2, which is present in C. elegans. In most eukaryotes RAD53 is involved in initiating cell-cycle arrest when DNA damage is detected but in C. elegans is essential for chromosome synapsis and nuclear rearrangement during meiosis¹³. This reciprocal difference suggests that meiosis in filarial parasites may be regulated in a manner more like typical eukaryotes than C. elegans (Supplementary Note). Six L. loa protein kinases are orthologous to targets of drugs currently approved for humans (Supplementary Table 23), including the tyrosine kinase inhibitor imatinib that has been shown to kill schistosomes ¹⁴ and Brugia parasites of all stages at concentrations ranging from 5 to 50 uM (unpublished data). Therefore repurposing already approved drugs that target these kinases may be promising in treating filarial (and other helminth) infections¹⁵.

Nematode phylogenomics

To examine the evolution of filarial parasites in the context of other nematodes, we estimated a phylogeny from 921 single copy core orthologs across nine nematode genomes using maximum likelihood, parsimony, and Bayesian methods. All methods converged on a single topology with 100% support (either bootstrap values or posterior probabilities) at all nodes (Figure 3). This phylogeny indicates that Meloidogyne hapla occupies a position basal to a clade of Rhabdtina (i.e; C. elegans, C. briggsae, and Pristionchus pacificus) and to the Spirurina (i.e. filarial worms and Ascaris suum). While these results contrast with previous studies based on ribosomal subunits that placed M. hapla closer to Rhabdtina than the filarial worms^{16, 17}, our analysis utilized a larger gene set and has higher nodal support values.

Relative to the genomes of non-parasitic nematodes, numerous orthologs were identified as unique to the filarial parasites (Figure 3). Proteins encoded by the filarial genomes show enrichment of immunogenic domains such as extracellular and cell adhesion domains, and in a metabolic context are enriched for trehalase domains involved in trehalose degradation (q < 0.05, Fisher’s exact test; Supplementary Fig. 6). Trehalose is known to be involved in the protection of nematodes from environmental stress¹⁸ and could potentially play a key role in filarial survival. Trehalose levels have also been associated with increased lifespan in C. elegans¹⁹ and might support the idea that an increased use of trehalose by filarial nematodes could be related to their relatively long lifespan.

The filarial genomes lack a wide array of seven transmembrane G-protein-coupled chemoreceptors (7 TM GPCRs; Supplementary Fig. 6). Profiling of 7 TM GPCRs revealed a pattern of progressive loss of many families in the transition from non-parasitic to parasitic lifestyles (Figure 4). For example, filarial nematodes and T. spiralis completely lack the Str superfamily, including odr-10, known to be involved in detection of volatiles²⁰, and Kin-29, a protein kinase that regulates Str expression in C. elegans²¹. If the Str superfamily is more broadly involved in odorant detection, this could explain why these molecules are lacking in filarial nematodes and T. spiralis parasites that only live in aqueous environments, while they are retained in A. suum and M. hapla, which are exposed to volatiles in part of their life cycle. Only the Srab, Srx, Srsx, and Srw families were conserved across all nematodes, suggesting that these GPCRs mediate vital nematode functions.

Filarial genomes are also depleted in both soluble and receptor guanylate cyclases – these genes are involved in the regulation of environmental sensing and complex sensory integration functions (Figure 4). However, gcy-35 and gcy-36, which are involved in the detection of molecular oxygen in solution²², are encoded in the filarial genomes. Protein kinase profiling revealed 18 receptor guanylate cyclases which are present in C. elegans but not in filarial worms, including environmental sensors gcy-14 and gcy-22 (Supplementary Table 23). Depletion of these and other kinases involved in olfactory and gustatory sensing, including kin-29, suggests that the environments of filarial nematodes are less complex in terms of chemosensory inputs than are those inhabited by non-parasitic nematodes (Supplementary Note). The L. loa genome does, however, encode significantly more chemoreceptors than do other filarial nematodes (p < 0.05, Fisher’s exact test), which may be related to the increased mobility of L. loa adult worms.

Phylogenetic profiling of metabolism

Previous genomic analysis identified five biosynthetic pathways (heme, riboflavin, FAD, glutathione and nucleotides) present in Wolbachia but missing from its relatives, e.g; Rickettsia. These Wolbachia-encoded pathways were hypothesized to provide metabolites needed by their filarial hosts⁶. As L. loa lacks Wolbachia, it was theorized that the L. loa genome must encode genes to replace these pathways, potentially laterally transferred from Wolbachia to an ancestor of L. loa. However, no transfers relating to these metabolic functions were apparent (see above). Thus, we generated complete metabolic pathway reconstructions for nine nematode and four Wolbachia genomes (Table 2, Supplementary Tables 24, 25) to determine how L. loa acquires these metabolites and placed the results in an evolutionary context. Unexpectedly, none of the five “complementary” pathways differ between L. loa and the other filarial nematodes, calling into question the role of these pathways in filarial-Wolbachia symbiosis.

Furthermore, in only two pathways (heme and nucleotide synthesis) did the filarial genomes differ from the other nematodes. The FAD and glutathione pathways are complete in all nematode genomes, while the riboflavin pathway is missing from all nematode genomes. The heme biosynthesis pathway, previously reported to be absent in B. malayi⁷, is missing from not only the filarial worms but from all nematode genomes characterized to date. Experimental work on C. elegans (also Wolbachia-free) has shown that it cannot synthesize heme de novo²³; B. malayi has been previously noted as having a single member of the heme synthesis pathway, ferrochelatase (an enzyme that catalyzes the last step in heme synthesis⁷ [Supplementary Note]). The gene encoding ferrochelatase is also present in the L. loa and W. bancrofti genomes, but absent in all other nematode genomes, including A. suum. It is possible that this gene in filarial nematodes is not involved in heme synthesis, but rather in an alternate, unknown pathway.

Like B. malayi, both L. loa and W. bancrofti lack the ability to synthesize nucleotides de novo. All three filarial genomes lack the majority of the proteins involved in the purine synthesis pathway as well as the first enzyme involved in the pyrimidine synthesis pathway (Table 2, Supplementary Table 24). Other nematodes have also lost portions of these pathways; the purine synthesis pathway has been largely lost in P. pacificus and M. hapla, while the first two enzymes in the pyrimidine synthesis pathway have been lost in T. spiralis. These multiple and likely independent losses could underscore a general flexibility in the need for de novo nucleotide synthesis in nematodes. All nematodes, including the filariae, have complete sets of purine and pyrimidine interconversion pathways (Supplementary Table 24), implying that they could generate all necessary nucleotides from a single purine and pyrimidine source, a concept supported by experimental data in B. malayi²⁴. Filarial genomes do encode two purine-specific 5’ nucleotidases for salvage whereas all other nematodes encode only one; the extra copy in the filariae appears to arise from a single gene duplication event and diverged significantly from the ancestral gene (Supplementary Fig. 7). Additionally, we profiled known nematode and Wolbachia transporters linked to to these pathways, and found no evidence of differences between filarial and non-filarial nematodes nor among Wolbachia endosymbionts (Supplementary Note and Supplementary Fig. 8). Given the uniformity of these pathways across nematodes and the apparent lack of any related transfers of Wolbachia DNA to the L. loa genome, it is likely that the symbiotic role played by Wolbachia in filarial nematodes either lies outside these pathways or involves more subtle metabolic supplementation rather than the wholesale provision of unproduced metabolites.

The only metabolic pathway found to differ in gene content between L. loa and other nematodes with sequenced genomes is vitamin B6 synthesis and salvage. Most nematode genomes encode single copies of the two enzymes involved in vitamin B6 salvage, while the L. loa genome encodes five copies of the second enzyme, pyridoxal 5’-phosphate synthase (Supplementary Note). This pathway also differed among Wolbachia genomes. While both of the insect Wolbachia genomes also encoded two genes involved in the synthesis of vitamin B6 (pdxJ and pdxK), neither of the filarial Wolbachia genomes did (the difference between wBm and wMel having been noted previously⁶). If the filarial Wolbachia endosymbionts need to acquire vitamin B6 exogenously, this could explain a metabolic need of Wolbachia fulfilled by the nematode. However, with that hypothesis in mind, it is unclear why L. loa, the one pathogenic filarial nematode without Wolbachia, would encode a greater number of vitamin B6 salvage genes than either B. malayi or W. bancrofti. We could not exclude differences in pyroxidine transporters as no orthologs of known transporters could be identified in either nematode or Wolbachia genomes (Supplementary Note).

Conclusion

The study of some of the nematode genomes has already provided great insight into the genomic structure, biology and evolution of this major division of nematode parasites. With the release of the genome of L. loa, a human pathogen and parasitic nematode that does not contain Wolbachia, we have been able provide surprising insights into the dispensability of this endosymbiont that deepen the mystery surrounding the “essential nature” of Wolbachia for many filarial worms.

Through large-scale genomic comparisons within the phylum Nematoda, we have not only been able to define molecules and pathways that are either Loa loa- or filaria-specific but also, by comparison with the non-parasitic nematodes (e.g; C. elegans), we have gained a first glimpse into the nature of parasitism itself. Moreover, this effort has identified new targets for intervention that should aid programs aimed at control and elimination of these important but neglected parasites.

Online Methods

Supplementary Material