Microbial pathogens often establish infection within particular niches of their host for replication. Determining how infection occurs preferentially in specific host tissues is a key aspect of understanding host-microbe interactions. Here, we describe the discovery of a natural microsporidian parasite of the nematode Caenorhabditis elegans that displays a unique tissue tropism compared to previously described parasites of this host. We characterize the life cycle of this new species, Nematocida displodere, including pathogen entry, intracellular replication, and exit. N. displodere can invade multiple host tissues, including the epidermis, muscle, neurons, and intestine of C. elegans. Despite robust invasion of the intestine very little replication occurs there, with the majority of replication occurring in the muscle and epidermis. This feature distinguishes N. displodere from two closely related microsporidian pathogens, N. parisii and N. sp. 1, which exclusively invade and replicate in the intestine. Comparison of the N. displodere genome with N. parisii and N. sp. 1 reveals that N. displodere is the earliest diverging species of the Nematocida genus. Over 10% of the proteins encoded by the N. displodere genome belong to a single species-specific family of RING-domain containing proteins of unknown function that may be mediating interactions with the host. Altogether, this system provides a powerful whole-animal model to investigate factors responsible for pathogen growth in different tissue niches.
Pathogens infect host organisms and then often establish themselves within a particular niche of the host environment in order to replicate [1–3]. This niche usually resides within a particular cell type or tissue, and is commonly referred to as cellular or tissue tropism. The mechanisms responsible for tissue tropism are broad and potentially multifactorial, and can involve features such as access, specific receptor/ligand interactions, pathogen competence for growth in particular tissue niches, and/or host defense [4–8]. Understanding the mechanistic and evolutionary bases for tissue tropism is key to understanding host/pathogen interactions and discovering therapeutics to prevent pathogens from causing disease. Microsporidia represent a large phylum of obligate intracellular pathogens related to fungi, which can infect a diverse array of hosts from protists to humans [9–12]. They have features consistent with having adapted to proliferate exclusively within the host cellular environment, including greatly reduced genome sizes and the loss of true mitochondria [13]. Different species of microsporidia display a range of different tissue tropisms. For example, the microsporidian species Encephalitozoon cuniculi shows a broad tissue tropism in humans and is able to infect the liver, brain, kidneys, skin, and gastrointestinal tract, while Enterocytozoon bieneusi mainly infects the enterocytes of the small intestine [14]. Studying tissue tropism in higher animals can be confounded by the complexity of the host body plan, making it difficult to comprehensively describe the tissues that are subject to infection in vivo. In some cases, tropism is implied from in vitro studies based on cell types that are infected but may not reflect the true tropism within the live animal [15,16]. The nematode Caenorhabditis elegans is a tractable, whole-animal system to study host/pathogen interactions because of its simple body plan and transparency, which facilitates assessment of tissue tropism in vivo. Sampling of proliferating populations of Caenorhabditis nematodes from rotting plant substrates in wild habitats has demonstrated that they are regularly infected by microsporidia [17–19]. Isolates of two closely related species, Nematocida parisii and Nematocida sp. 1, are thus far the only described microsporidian species found in wild Caenorhabditis nematodes, and both of these species are fecal/oral pathogens that infect and replicate exclusively in C. elegans intestinal cells [9,18]. The intestinal-trophic nature of N. parisii has been well-studied, with all stages of the pathogen being solely observed in the intestine by light, fluorescence, and transmission electron microscopy (TEM) [18]. Additionally, multiple infection-induced changes have been observed in the intestine, including restructuring of the apical cytoskeleton [20] and hijacking of the intestinal recycling endosome pathway for the exit of newly made spores [21]. Here, we report the discovery of a new species of microsporidia found infecting non-intestinal tissues of a wild-caught C. elegans animal. Whole genome sequencing and phylogenomic analysis places this new species in the Nematocida genus, and we have named it Nematocida displodere, based on a vulva bursting mechanism for spore exit. N. displodere displays a distinct tropism from the other described Nematocida species and has the capacity to initially invade a wide array of tissues and cell types, including the intestine, epidermis, muscle, neurons, and specialized phagocytic cells called coelomocytes, with feeding being required for infection. Strikingly, the majority of intestinal infection fails to replicate. Comparison of the N. displodere genome with the other Nematocida species shows that despite N. displodere having a smaller genome, it contains an enormously expanded species-specific gene family encoding for RING-domain containing proteins of unknown function, which may explain its distinct infection life cycle. Altogether, we characterize a new species of microsporidia, Nematocida displodere, with a broader tissue tropism compared to other Nematocida species identified to date, and this system provides a convenient model to study the mechanistic and evolutionary bases of tissue tropism using closely related but distinct pathogens infecting a single tractable host.
While sampling for nematodes near the Viosne stream in Santeuil, France, we isolated a wild-caught C. elegans infected by a microbe displaying microsporidian-like features in the head of the animal (S1A Fig). For reference, microsporidian species display certain stereotypical hallmarks in their life cycle [22]. Specifically, infection begins when an extracellular, transmissible spore fires an infection apparatus called a polar tube to deliver a single mononucleated parasite cell called a sporoplasm into the host cell. The sporoplasm then develops into a multinucleate, proliferative stage called a meront, and eventually differentiates into new spores that exit the host cell. The wild-caught C. elegans we found had structures that appeared like meronts and spores in an area that is likely the epidermis (S1A Fig). In the lab, we found that this P0 adult was able to transmit infection to its progeny, as observed by the appearance of large, meront-like structures and spores in recipient animals (Fig 1A and 1B). These later stages of infection were seen along the anterior/posterior axis of the animal, including the head (Fig 1A and 1B), the mid-body (S1C Fig, Fig 1C), and the tail (Fig 1C). The majority of animals showed these later stages of infection in the body wall of C. elegans, which is the outer tube of the animal distal to the pseudocoelomic space that includes the epidermis, muscle, and neurons [23]. By contrast, tissues in the intestine (Fig 1A) and gonad remained for the most part symptom-free as observed by light microscopy. Consistent with a lack of infection in the gonad, we did not find that infection was vertically transmitted because the eggs from a bleached population of heavily infected animals developed into a population that remained uninfected for multiple generations (n = 100 animals analyzed by Nomarski and n = 100 animals analyzed by fluorescent in situ hybridization, FISH, over 4 months at 15°C). We confirmed this pathogen as a new species of microsporidia in the Nematocida genus based on whole genome sequencing and phylogenomic comparison (see below), and we named this new species Nematocida displodere. To characterize the infection life cycle of N. displodere we labeled the pathogen with a FISH probe to label the small ribosomal subunit RNA (rRNA) or with a chitin binding dye, Direct Yellow 96 (DY96), to label spore walls. We synchronized wild-type N2 C. elegans animals, infected them with N. displodere spores and then observed them for the main hallmarks of microsporidia infection. These hallmarks include mononucleated sporoplasms observed at 1 day post-infection (dpi), multinucleate meronts from 2–4 dpi, and sporoblasts (pre-spores) and spores at 5 dpi, which eventually fill up a large proportion of the animal by 7 dpi (Fig 1C). We quantified the percent of animals exhibiting the symptoms of each stage of infection using FISH and DY96 staining from 1 to 7 dpi (Fig 1D), and found that 100% of animals in a population exhibited replicative forms of infection by 2 dpi, and 100% exhibited spores by 7 dpi. Thus, similar to the intestinal-trophic N. parisii, N. displodere appears to efficiently infect an entire C. elegans population on a plate in a laboratory setting [18]. To more closely examine the N. displodere life cycle in C. elegans, we conducted transmission electron microscopy (TEM) analysis on infected animals. When compared to uninfected animals (Fig 2A), we observed numerous structures by TEM that looked like distinct stages of microsporidia infection. These structures include large, multinucleate cells that are likely the proliferative meront stage (Fig 2B) and groups of mononucleate cells that likely correspond to the sporont stage of microsporidia, which are thought to be capable of further divisions (Fig 2C) [24]. Additionally, groups of cells were seen with nascent microsporidian spore structures that likely represent sporoblasts, which do not undergo further divisions before becoming spores (Fig 2D and 2E). Finally, darker, more fully differentiated spores were seen (Fig 2F and 2G). From the sporont to the spore stage of N. displodere we observed cross-sections of polar tube coils (the specialized infection apparatus of microsporidia). In spores that appeared fully developed, a maximum of five polar coils were observed per cell (seen in 19 of 70 TEM cross-sections of spores), with two coils on one side and three coils on the other (Fig 2C and 2G). Thus, N. displodere appears to undergo stereotypical developmental features of microsporidia, as assessed by TEM. Our observations of N. displodere infection by light and electron microscopy indicated that meronts and spores were predominantly in non-intestinal tissues, suggesting a different tropism than N. parisii, which exclusively infects the intestine. To simultaneously compare the tissue tropism of these two species, we co-infected N2 animals with N. displodere and N. parisii and found that indeed these two closely related microsporidian species infect distinct areas of the animal (Fig 3A). Next, to determine the range of tissues in which N. displodere can proliferate, we infected a panel of C. elegans strains that express GFP in distinct tissue types, and then looked for multinucleate meronts at 3 dpi by rRNA FISH, and newly differentiated spores at 5 dpi by DY96. Using this approach, we found the epidermis, body-wall muscle, and neurons had N. displodere meronts (Fig 3B) and newly-formed spores (Fig 3C). Additionally, we occasionally saw meronts of N. displodere in epidermal seam cells and coelomocytes (S2 Fig), but we did not observe new spores in these cells at later time points. In some cases, N. displodere-infected cells appear to become larger than corresponding uninfected cells, as can be seen in the neurons (Fig 3B, bottom) and seam cells (S2A Fig). Additionally, we found that multiple tissues can be infected in the same animal. For example, meronts were found both inside and outside of the GFP-positive muscle in one animal (Fig 3C, middle). We observed far fewer animals infected in the intestine compared to the muscle and epidermis at 3 and 5 dpi, although a small fraction of animals did show meronts and newly made spores in the intestine (S3 Fig). Interestingly, the intestine was frequently invaded with N. displodere sporoplasms at 1 dpi, but was rarely infected with multinucleate meronts at 3 dpi (Fig 3D). This observation suggests that N. displodere can invade this tissue, but often fails to proliferate there. We quantified this observation by counting the total number of meront clusters in an animal at 3 dpi and then calculating the percentage of those clusters that are in a particular GFP-labeled tissue. This analysis was performed separately for each tissue in its respective tissue-specific GFP expression strain, with each cluster of meronts assumed to have formed from a single invasion event. In this manner, we found that only about 5% of multinucleate meronts were found in the intestine, whereas 42% were in the epidermis, 53% in the muscle, 7% in the neurons, and 3% in the coelomocytes (Fig 3E). Although the infections in each tissue were quantified separately using individually marked C. elegans strains, this approach appears to provide a good estimate of the overall tissue distribution of meronts at 3 dpi, because the total percent of pathogen in these five tissues adds up to about 100%. We next looked at other time points to quantify the tissues in which N. displodere could invade to deliver sporoplasms, proliferate into multinucleate meronts, and differentiate into spores. We infected the intestinal, muscle, and epidermal GFP expression strains for 1,3, or 6 days and counted the fraction of animals displaying a given N. displodere stage at that time point in the GFP-labeled tissue. While greater than 90% of animals were initially infected with sporoplasms in either the intestine, epidermis, or muscle at 1 dpi, very few of these invasion events appeared to proliferate and differentiate in the intestine, with less than 10% of animals exhibiting meronts in the intestine at 3 dpi and 6% exhibiting new spores in the intestine at 6 dpi (Fig 3F). By contrast, greater than two-thirds of animals showed meronts and new spores in both the muscle and the epidermis at these later time points. Together, these results suggest that N. displodere can initially invade multiple tissues in C. elegans, including the intestine, but shows preferential proliferation and differentiation in the epidermis and muscle. We next investigated how N. displodere is able to access the host environment to invade host tissues. Invasion via the epidermis or the intestine after feeding are the only two routes described for pathogens infecting C. elegans [25,26], and these two tissues represent the largest surface areas with exposure to the environment. To investigate whether N. displodere infection of C. elegans might occur through feeding or external penetration through the cuticle, we investigated transcriptional responses characteristic of intestinal infection and cuticle damage. First, we analyzed the C. elegans intestinal GFP reporter strains for the genes F26F2. 1 and C17H1. 6, which are highly induced upon infection with both N. parisii and another natural C. elegans intestinal pathogen, the Orsay virus [27]. Here, we found that N. displodere infection caused induction of both the F26F2. 1p: : GFP (Fig 4A, left) and the C17H1. 6p: : GFP reporter strains (S4 Fig). In fact, N. displodere caused a greater degree of induction of these reporter strains than N. parisii. By contrast, an epidermal infection/damage reporter strain for nlp-29, a gene highly induced upon infection by the fungus D. coniospora and epidermal wounding through the cuticle [26,28], did not show induction after infection by N. displodere (Fig 4A, right). N. parisii infection also failed to induce nlp-29, as has been previously described [18]. The lack of nlp-29 induction suggests that despite its capacity to infect the epidermis, N. displodere is unlikely to cause damage through external cuticle disruption, which is known to induce nlp-29 [26]. These results, together with the fact that N. displodere spores were seen in the intestinal lumen soon after infection (S5 Fig), suggest that feeding could be a major route for infection. To further examine if N. displodere infects through C. elegans feeding, we assessed infection in C. elegans strains that have a reduced or no ability to feed. First, we used the temperature sensitive (ts) strain daf-2 (e1368) [29], which constitutively enters the non-feeding dauer stage at the restrictive temperature of 25°C. Dauers of daf-2 (ts) animals grown at 25°C were inoculated with N. displodere and showed no infection in any tissues, while 100% of L3 animals of daf-2 (ts) maintained at a permissive temperature did show infection (Fig 4B). Similar results were seen with N. parisii infection. A caveat to these results being evidence for a feeding-based mechanism of infection is that the dauer stage not only ceases feeding, but also develops a tougher cuticle surface. Next, we used a C. elegans eat-2 mutant to investigate whether feeding was important for N. displodere infection. The eat-2 mutant shows an approximately 70% reduction in feeding rate compared to wild-type animals, either in the absence or presence of N. displodere spores (S6 Fig). Consistent with this feeding defect, we saw a 66% reduction in sporoplasms in the intestine (Fig 4C). We also saw a 61% reduction in sporoplasms in non-intestinal tissues in eat-2 mutants compared to wild-type animals (Fig 4C). These results, like the dauer results described above, show that feeding is likely a route of entry for both intestinal and non-intestinal tissue infection by N. displodere. Finally, we tested the C. elegans temperature-sensitive endocytosis strain, dyn-1 (ts) which stops feeding and moving at 30°C. We shifted adult N2 and dyn-1 (ts) animals to 30°C for 2. 5 hours and then infected with N. displodere for 30 minutes. Even with this short infection time, wild-type animals showed a substantial level of infection, with an average of 4. 4 sporoplasms inside of the intestine and 1. 4 sporoplasms outside of the intestine (Fig 4D, left). By contrast, the dyn-1 (ts) mutants showed a complete lack of infection in any tissue of the animals. We saw similar results for N. parisii infection (Fig 4D, right), with substantial intestinal infection in wild-type animals, and no infection in dyn-1 (ts) mutants. As a control, we verified that dyn-1 (ts) animals were infected by N. displodere at the permissive temperature (S7 Fig). Altogether, these data strongly suggest that C. elegans feeding is required for the majority, if not all, N. displodere infection in susceptible tissues of C. elegans.
Our study describes a new Nematocida species of microsporidia that infects C. elegans, and has characteristics distinct from the other species in the genus described to date. We found that N. displodere has a broad tissue tropism, with the capacity to infect and replicate in the muscle, neurons, epidermis, intestine, coelomocytes, and seam cells. Compared to N. parisii, which only infects and replicates in the intestine, N. displodere showed preferential tropism for the epidermis and muscle over the intestine. Likely due to this difference in tropism, we also found that N. displodere has an unusual mechanism for newly differentiated spore exit via bursting, while new spores of N. parisii continuously exit through defecation. When we analyzed the genomes of these related Nematocida species for differences, we found that N. displodere has a greatly expanded family of proteins with a RING domain of which only a couple members were found in the intestinal-trophic Nematocida species. Conversely, a separate expanded family was found in N. parisii and N. sp. 1 that was completely absent in N. displodere. To our knowledge, N. displodere has the broadest tissue tropism yet seen for a pathogen infecting C. elegans, and is the first pathogen with the capacity to infect the neurons, muscle, or coelomocytes of C. elegans. N. displodere infection is dependent on C. elegans feeding, which is consistent with other invertebrate-infecting microsporidia which display two major pathways for infection, either through ingestion of spores or transovum/transovarial passage [32]. Once in the intestinal lumen, the polar tube of N. displodere can infect intestinal cells, but is also long enough to theoretically reach other tissues, more so in younger animals than older animals. Because our data shows that N. displodere invasion of non-intestinal tissues can occur very rapidly, and microsporidia are not known to have standard movement apparatuses, like cilia or flagella [22,33], it is likely that the polar tube is used to access other tissues. One model of infection is that N. displodere spores fire their polar tubes in the lumen to invade any tissue, intestinal or non-intestinal, to which the polar tube gains access. This' general access' model could explain why larger tissues like the muscle and epidermis showed a higher percent of pathogen than the neurons or less frequent cells, like seam cell and coelomocytes (see Fig 3D), assuming relatively similar proliferation rates in non-intestinal tissues. By contrast, we found that the polar tube lengths of similarly sized N. parisii spores were three-fold shorter than N. displodere. These measurements are consistent with the number of polar tube coils seen in TEM cross-sections, as N. displodere spores had up to five coils (see Fig 3G) while prior TEM images showed that small N. parisii spores had only one coil [18]. A shorter polar tube likely limits N. parisii infection to shorter distances from the intestinal lumen compared to N. displodere infection. However, given that the distance from the lumen to the basolateral side of the intestine is variable within an animal, with some distances being as short as 2 μm (see Figs 2A and 5A), polar tube lengths are not the only limiting factor for infecting non-intestinal tissues from the intestinal lumen. It is possible that spores must convey enough force on the polar tube upon germination in order to pierce through multiple membranes to reach non-intestinal tissues [31], or that specific proteins are required on the polar tube for interaction with tissue-specific host factors on a cell for polar tube entry and invasion [34,35]. Strikingly, although N. displodere can successfully invade the intestine, we found that the majority of intestinal infection fails to thrive in comparison to robust infection of the epidermis and the muscle. One possible reason for this tropism is that competition among other microbes in the intestine has put evolutionary pressure on N. displodere to efficiently proliferate in non-intestinal tissue. Multiple examples of potential competition among microsporidian species have been described. For example, ecological observations have shown that the honey bee-infecting microsporidian Nosema ceranae can almost completely displace other naturally occurring microsporidia species when introduced into a new area [36]. Additionally, laboratory experiments have shown that different microsporidia species of the gypsy moth can exclude or suppress the growth of each other in a particular tissue [37]. For C. elegans in the wild, there may be an increased competition for resources in the intestine compared to other tissues, as several distinct pathogens can naturally infect and proliferate in the intestine [17,18,25]. A potential mechanistic reason for the tropism of N. displodere is that there is distinct induction of or sensitivity to tissue-specific host defense responses. For C. elegans, different tissue-specific transcriptional responses to intracellular pathogens have been identified for the epidermis upon D. coniospora infection and epidermal wounding, and for the intestine upon N. parisii and Orsay virus infection [27,38]. Components of these transcriptional responses have been shown to play a role in tissue-specific host defense against their respective pathogens, including anti-microbial genes induced in the epidermis and Skp1-Cullin-F-box (SCF) ubiquitin ligase components induced in the intestine [26,27]. For the epidermis we found that N. displodere failed to induce a reporter gene representative of the epidermal response, but induced reporter genes representative of the intestinal response to a higher degree than N. parisii. These observations could be representative of differential induction of tissue-specific defense responses upon infection by N. displodere, resulting in better proliferation in the epidermis compared to the intestine. Another possibility is that N. displodere has greater sensitivity to ubiquitin-mediated clearance in the intestine, resulting in the failure of the majority of infection in this tissue [27]. Future studies will investigate the mechanistic basis on the host side for the distinct tissue tropism of N. displodere. To investigate the genetic basis for the distinct features of N. displodere, we sequenced its genome and compared it with the other, intestinal-trophic Nematocida species. N. displodere is the earliest known diverging member of the Nematocida genus, but shares over 70% of its proteins with both N. parisii and N. sp. 1. The most striking difference between the genomes is the presence of species-specific expanded gene families, NemLGF2 and NemLGF1, found in N. displodere and the intestinal-trophic Nematocida, respectively, which are completely absent in any other sequenced genomes. An impressive 10% of the coding sequences in the N. displodere genome, or up to 33% of the proteins not shared with N. parisii and N. sp. 1, belongs to NemLGF2. These gene families likely evolved after the divergence from the last common ancestor and expanded as they adapted to their respective host environments. Considering the fact that microsporidia are obligate intracellular pathogens, it is likely that evolutionary pressure from the host played a role in the expansion, as has been shown for expanded gene families in other microsporidia species [39,40]. In fact, the proteins in these expanded gene families are likely secreted into C. elegans cells at some point during infection, as both NemLGF1 and NemLGF2 have a high percentage of predicted signal peptides among their members. Additionally, almost half of the NemLGF2 proteins from N. displodere contain a C-terminal RING domain, typically found in ubiquitin E3 proteins that function to bind to E2 ligases [41]. The RING domains in NemLGF2 may serve as protein-protein interaction modules in the host cells, allowing these proteins to bind to host proteins and perform some yet unknown function. It is intriguing to speculate that the NemLGF2 proteins may play a role in interacting with the host ubiquitin system, as it has been shown to play a role in response to N. parisii infection [27], and RING domains are found in E3 ubiquitin ligases. Despite having a broader tissue tropism than N. parisii and N. sp. 1, N. displodere has a smaller genome and fewer predicted proteins, which runs contrary to expectations that a broader range might require more genes to adapt to growth in varying niches and avoid different defense responses. However, a related observation was recently made in a study of two microsporidian species that infect mosquitos, as the microsporidia with a broad host range, Vavraia culici, had a smaller genome than the specialist microsporida, Edhazardia aedis, known to infect only one mosquito species [42]. Altogether, the discovery and characterization of N. displodere presents a unique model system in C. elegans to study the mechanistic and evolutionary bases of pathogen tissue tropism.
Wild nematodes were sampled from the woods near the Viosne stream in Santeuil, France on September 30,2014 using methods previously described [17]. Wild Caenorhabditis animals that looked ‘sick’ were individually picked to nematode growth media (NGM) plates seeded with E. coli strain OP50-1, as described [43], and incubated at 20°C. P0 adults were allowed to self-fertilize to produce F1 progeny and these P0 adults were analyzed by light microscopy for infection. N. displodere (designated isolate JUm2807, ZooBank ID, urn: lsid: zoobank. org: act: 35CF055F-C311-4D9B-BFF0-B7B09FC441E4) was found in the head of the P0 of wild C. elegans strain (designated JU2807), isolated from the rotting stem of an Asteraceae plant (GPS coordinates: 49. 12165,1. 95101) containing a proliferating population of approximately 500 C. elegans of various stages. C. elegans strain JU2807 containing N. displodere isolate JUm2807 was cleared of all bacterial and fungal contamination by thoroughly washing a starved population of infected animals with sterile H2O and incubating 1 h in 15 ml H2O. Animals were incubated 2 h in S-basal (50 mM potassium phosphate, pH 6. 0,100 mM NaCl, 5 μg/ml cholesterol) containing 100 μg/ml gentamycin, 50 μg/ml carbenicillin, 50 μg/ml kanamycin, 20 μg/ml tetracyclin, and 50 μg/ml streptomycin. Sodium dodecyl sulfate (SDS) was added to a final concentration of 1% and incubated for 15 m. Finally, animals were washed with H2O and plated on NGM plates containing 50 μg/ml carbenicillin, 25 μg/ml kanamycin, 12. 5 μg/ml tetracyclin, and 37. 5 μg/ml chloramphenicol seeded with concentrated OP50-1 bacteria, and incubated at 15°C for 5 d. N. displodere spores were prepared as previously described for N. parisii [20]. Briefly, N. displodere JUm2807 was cultured by expanding large-scale cultures of antibiotic-treated C. elegans JU2807, followed by mechanical disruption of the nematodes, and then filtering to isolate spores away from animal debris. Similar methods were used to make N. parisii spore preparations using the isolate ERTm1 infected in C. elegans N2. All C. elegans strains were maintained as previously described [43]. The intestinal GFP strain ERT413 jySi21[spp-5p: : GFP; cb-unc-119 (+) ] II was made in this study using Mos1-mediated single-copy insertion (MosSCI) [44]. Additional strains used in this study include: FISH was performed as described using FISH probes to the small subunit rRNA conjugated to CAL Fluor Red 610 (CF610) or 5-Carboxyfluorescein (FAM), with slight modification [27]. For single-species infections, a mixture of Nematocida-specific probes MicroA-CF610 (CTCTGTCCATCCTCGGCAA), MicroC-CF610 (CAGAATCAACCTGGTGCCTT), MicroD-CF610 (CGAAGGTTTCCTCGGATGTC), and MicroE-CF610 (GTACTGGAAATTCCGTGTTC) were used at 2. 5 μg/ml each, with hybridization at 46°C and washes at 48°C. As indicated, the chitin-staining dye direct yellow 96 (DY96) was added at 10 μg/ml to the hybridization buffer to stain microsporidia spores in the animals [21]. For co-infection, a N. displodere-binding probe Microsp1A-FAM (CAGGTCACCCCACGTGCT) and a N. parisii-specific probe MicroF-CF610 (AGACAAATCAGTCCACGAATT) were used at 5 μg/ml each, with hybridization at 52°C and washes at 54°C. C. elegans strains were infected on NGM plates with purified N. displodere spores (isolate JUm2807) or N. parisii spores (isolate ERTm1) as described previously [9]. All infections in this study were conducted using a standardized dose of N. displodere or N. parisii, defined here as 3. 5 x 104 spores per cm2 (calculated from infecting a 6 cm NGM plate with 1. 0 x 106 spores). N. displodere was capable of being continually transmitted within C. elegans for multiple generations at 15°C, so all experiments were conducted at this temperature unless otherwise indicated. For kinetics of infection, synchronized N2 L1 larvae were infected with a standard dose and sampled at 1–7 dpi for small subunit rRNA FISH in 24 h increments. Fifty animals per replicate were analyzed for sporoplasms, meronts, sporoblasts, and spores at each time point. For microscopy with the tissue-specific GFP expression lines, strains ERT413, AU189, HC46, and OH441 were infected with the standard dose of N. displodere as synchronized L1 larvae. Animals were fixed at 3 dpi and stained by small subunit rRNA FISH, or at 5 dpi and stained by rRNA FISH plus DY96. For analysis of the tissue distribution of N. displodere infection, synchronized L1 larvae of strains ERT413, AU189, HC46, and OH441 were infected at 15°C with half a standard dose of N. displodere spores in duplicate and fixed at 3 dpi for small subunit rRNA FISH. A total of 50 infected animals for each replicate were analyzed by confocal microscopy for meronts or meront clusters in GFP-positive tissues or GFP-negative tissues. Meront clusters were counted once if they were in distinct areas of the animal and/or separated from another cluster by at least 10 μm. The percent of meront clusters in the GFP-positive tissue was calculated based on the total number of meront clusters calculated for each replicate. For analyzing the percent of animals with tissue-localized symptoms at different time points, synchronized L1 larvae of strains ERT413, AU189, and HC46 were infected in duplicate at 15°C with half a standard dose of N. displodere spores in duplicate. Note that for 1 dpi animals, the L1 larvae were infected at the end of the L1 stage, by first growing for 22 h at 15°C before infecting with N. displodere so that the animals were in the L3 stage by the end of the experiment and express enough GFP for analysis. Animals were fixed at 24 hpi and 144 hpi for 1 dpi and 6 dpi, respectively, and stained by small subunit rRNA FISH for 1 dpi and FISH plus DY96 (10 μg/ml) for 6 dpi. The 3 dpi animals were calculated from the tissue distribution experiment (see above). Animals were analyzed for sporoplasms at 1 dpi, meronts at 3 dpi, and DY96-stained spores at 6 dpi and then the percent of animals with the symptom in the GFP-positive tissue were calculated for fifty animals per replicate. TEM was performed at the Electron Microscopy Facility, Department of Cellular and Molecular Medicine, UCSD. Synchronized N2 L1 larvae were infected with a standard dose of N. displodere at 15°C and harvested at 6 dpi and 8 dpi, and an uninfected sample was collected at 6 dpi. Animals were fixed with 2% of paraformaldehyde, 2. 5% of glutaraldehyde in 150 mM sodium cacodylate buffer (SC), and washed with 150 mM SC buffer. Samples were post-fixed 3 h in 2% osmium tetroxide in 150 mM SC on ice, washed in 150 mM SC followed by ddH2O, pelleted in 2% agarose, and incubated in 2% uranyl acetate overnight at 4°C. Samples were dehydrated on ice with a graded series of ethanol from 50% to 100%, followed with 50% ethanol/50% acetone for 20 m, and twice in 100% acetone for 10 m. Samples were incubated in a graded series of Durcupan from 25% to 100% at RT. Finally, samples were incubated in 100% Durcupan ON at 60°C. Blocks were cut on Leica microtome with a diamond knife to 60 nm sections and collected on 300 mesh grids. Digital images were collected on a Tecnai TEM (Field Emission Inc.) at 80 kv by using an Eagle 4K digital camera. Synchronized ERT54, ERT71, and AU189 L1 larvae were grown on OP50-1 for 20°C for 24 h and 15°C for 24 h to the L3 larval stage. Animals were split and infected in triplicate with a standard dose of either N. displodere or N. parisii for 20 h at 15°C with approximately 800 animals per replicate. Animals were harvested and washed with M9 buffer and loaded onto a COPAS Biosort (Union Biometrica) to measure GFP, dsRed fluorescence, and time-of-flight (TOF) of each animal. Data was analyzed using the R package COPASutils [47], with GFP expression of ERT54 and ERT71 normalized to TOF and AU189 normalized to pcol-12: : dsRed expression. For the eat-2 mutant, synchronized N2 and eat-2 L1 larvae were grown at 15°C to the gravid adult stage for 3 and 4 d, respectively, and then infected for 24 h with N. displodere at 1/10 the standard dose (3. 5 x 103 spores per cm2) to reduce the number of infection events per animal. For the temperature-sensitive feeding mutant dyn-1 (ts), synchronized N2 and ERT125 L1 larvae were grown to the adult stage at 20°C for 3 d. Animals were then shifted for 2. 5 h to either 30°C to stop ERT125 pharyngeal pumping (as monitored on a dissecting scope) or 20°C as a control, and infected with five times the standard dose (1. 75 x 105 spores per cm2) for 30 m at the respective temperatures. FISH was conducted as above and sporoplasms were counted and localized using a Zeiss LSM700 confocal microscope with a 40x objective. To test dauer infection, synchronized daf-2 (e1368) L1 larvae were grown for 2 d at 25°C to initiate dauer formation. As a control, daf-2 (e1368) and N2 L1 larvae were grown for 2 d at 15°C to the L3 stage. All animals were infected with the standard dose of N. displodere at 25°C for 18 h. FISH was conducted as described above, except all animals were fixed in 100% acetone for 10 m to permeate the dauer cuticle. Synchronized N2 L1 larvae were infected or mock-infected in triplicate with the standard dose of N. displodere and N. parisii at 100–200 animals per 6 cm plate, and grown at 15°C for 11 d. Infected adults were transferred to new plates at 5–8 dpi to remove the F1 generation. At 5–11 dpi all animals from each condition were analyzed by a dissecting microscope for a bursting phenotype and removed. Synchronized N2 L1 larvae were infected in duplicate with the standard dose of N. displodere and N. parisii and grown at 15°C for 10 d. For quantifying spores produced in the animals, 20 animals were picked into 1 ml PBS + 0. 1% Tween-20 (PBS-T) at 4–10 dpi for N. displodere and 3–8 dpi for N. parisii, and washed three times with 1 ml PBS-T. Animals were lysed and the number of spores produced per animal was counted as described [48]. For quantifying spores shed by the animals, 20 animals were picked into 500 μl of a 1: 10 OP50-1 and M9 mixture at 5–10 dpi for N. displodere and 4–8 dpi for N. parisii. Animals were incubated for 4 h at RT with rotation, and secreted spores were separated from the animals and counted as previously described [21].
Pathogens evolve under selective pressure from host organisms to successfully invade and proliferate in different cells and tissues of the host for their own benefit. Microsporidia represent one of the most successful phyla of pathogens, with severely reduced genomes and loss of core cellular and metabolic pathways making them dependent on host cells for their own proliferation. We sampled around Paris, France, for wild nematodes infected with natural pathogens, and discovered a wild Caenorhabditis elegans that was infected with a new species of microsporidia. We characterize the life cycle of this new species, showing the pathogen enters via host feeding, replicates in multiple host tissues, and exits as new spores via a novel vulva bursting mechanism, leading us to name this species Nematocida displodere. Despite the capacity of N. displodere to invade multiple host tissues during infection, we found that the parasite showed very little replication in the intestine. This unique tissue specificity of N. displodere stands in stark contrast to its two closest-related species, Nematocida parisii and Nematocida sp. 1, which exclusively infect and proliferate in the intestine of C. elegans. We compared the genomes of these related species and found that N. displodere devotes over 10% of its genome to a single large gene family not found in any other species, and propose that their encoded proteins may be interacting with host factors during infection.
skin
invertebrates
medicine and health sciences
integumentary system
pathology and laboratory medicine
caenorhabditis
pathogens
microbiology
parasitic diseases
animals
nematode infections
animal models
developmental biology
caenorhabditis elegans
model organisms
gastroenterology and hepatology
microsporidia
epidermis
fungal pathogens
digestive system
research and analysis methods
mycology
medical microbiology
microbial pathogens
gastrointestinal infections
pathogenesis
gastrointestinal tract
anatomy
host-pathogen interactions
nematoda
biology and life sciences
metamorphosis
larvae
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Discovery of a Natural Microsporidian Pathogen with a Broad Tissue Tropism in Caenorhabditis elegans