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Concurrent transcriptional profiling of Dirofilaria immitis and its Wolbachia endosymbiont throughout the nematode life cycle reveals coordinated gene expression.

Luck AN, Evans CC, Riggs MD, Foster JM, Moorhead AR, Slatko BE, Michalski ML - BMC Genomics (2014)

Bottom Line: Interestingly, a large proportion of both D. immitis and wDi genes display microfilarial-biased transcriptional patterns.Concurrent transcriptome sequencing identified potential molecular interactions between parasite and endosymbiont that are more prominent during certain life cycle stages.In support of metabolite provisioning between filarial nematodes and Wolbachia, the synthesis of the critical metabolite, heme, by wDi appears to be synchronized in a stage-specific manner (mf-specific) with the production of heme-binding proteins in D. immitis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Microbiology, University of Wisconsin Oshkosh, Oshkosh, WI 54901, USA. michalsk@uwosh.edu.

ABSTRACT

Background: Dirofilaria immitis, or canine heartworm, is a filarial nematode parasite that infects dogs and other mammals worldwide. Current disease control relies on regular administration of anthelmintic preventives, however, relatively poor compliance and evidence of developing drug resistance could warrant alternative measures against D. immitis and related human filarial infections be taken. As with many other filarial nematodes, D. immitis contains Wolbachia, an obligate bacterial endosymbiont thought to be involved in providing certain critical metabolites to the nematode. Correlations between nematode and Wolbachia transcriptomes during development have not been examined. Therefore, we detailed the developmental transcriptome of both D. immitis and its Wolbachia (wDi) in order to gain a better understanding of parasite-endosymbiont interactions throughout the nematode life cycle.

Results: Over 215 million single-end 50 bp reads were generated from total RNA from D. immitis adult males and females, microfilariae (mf) and third and fourth-stage larvae (L3 and L4). We critically evaluated the transcriptomes of the various life cycle stages to reveal sex-biased transcriptional patterns, as well as transcriptional differences between larval stages that may be involved in larval maturation. Hierarchical clustering revealed both D. immitis and wDi transcriptional activity in the L3 stage is clearly distinct from other life cycle stages. Interestingly, a large proportion of both D. immitis and wDi genes display microfilarial-biased transcriptional patterns. Concurrent transcriptome sequencing identified potential molecular interactions between parasite and endosymbiont that are more prominent during certain life cycle stages. In support of metabolite provisioning between filarial nematodes and Wolbachia, the synthesis of the critical metabolite, heme, by wDi appears to be synchronized in a stage-specific manner (mf-specific) with the production of heme-binding proteins in D. immitis.

Conclusions: Our integrated transcriptomic study has highlighted interesting correlations between Wolbachia and D. immitis transcription throughout the life cycle and provided a resource that may be used for the development of novel intervention strategies, not only for the treatment and prevention of D. immitis infections, but of other closely related human parasites as well.

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D. immitischitinase expression. Expression profiles (FPKM values) of the D. immitis chitinase precursor protein (A), cuticular endochitinase (B) and chitinase (C) genes.
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Fig3: D. immitischitinase expression. Expression profiles (FPKM values) of the D. immitis chitinase precursor protein (A), cuticular endochitinase (B) and chitinase (C) genes.

Mentions: Many studies have identified microfilarial-specific chitinase activity in filarial nematodes that release sheathed microfilariae (e.g., B. malayi, Wuchereria bancrofti and Loa loa) purportedly involved in exsheathment and/or penetration of the vector midgut [67, 68]. Therefore, unsurprisingly, chitinase activity was previously identified as upregulated in B. malayi mf [38]. Conversely, filarial species that shed unsheathed mf, including species such as D. immitis, Acanthocheilonema viteae and Onchocerca volvulus, have been shown to utilize a temperature specific (37°C) L3-specific chitinase, homologous to the B. malayi mf-specific chitinase, to possibly degrade the cuticle during the L3 to L4 molt within the mammalian host. Of the three annotated chitinase genes within the D. immitis genome, the chitinase precursor protein (nDi.2.2.2.g01593) was consistently transcribed throughout the life cycle (Figure 3A). Although numerous studies have shown a lack of chitinase activity in D. immitis mf [67, 68] and specific chitinase activity in the L3 stage [69, 70], we observed significant upregulation of the cuticular endochitinase gene (nDi.2.2.2.g09584) in mf as compared to the L3 stage (Additional file 4: Table S2, Figure 3B). When describing the L3-specific chitinase of A. viteae, Wu et al., identified a protein of similar molecular weight as the L3-specific chitinase present in mf that displayed the opposite temperature specificity (expression at 27°C, not 37°C) [69], suggesting the presence of an additional chitinase functional within the insect vector. Hence, there is some evidence to support our findings that expression of this endochitinase gene does occur in unsheathed microfilariae of certain nematode species. The exact role of endochitinase in unsheathed mf that do not require chitinases to infect the vector species (D. immitis mf do not penetrate the midgut of the insect vector but rather enter the Malpighian tubes through the posterior lumen of the vector midgut [71]), remains unclear. However, chitinase may be important to help the microfilariae migrate through the chitinous peritrophic membrane that surrounds the blood meal. Additionally, because L1 to L3 development for many filarial nematodes within the vector is intracellular, chitinase may be required to invade the host cell. As expected, expression levels of the chitinase gene (nDi.2.2.2.g09661) were much higher in the L3 stage (Figure 3C) than any other chitinase-related gene (nDi.2.2.2.g01593 or nDi.2.2.2.g09584) in any other life cycle stage (note difference in scales between Figure 3A/3B and Figure 3C).Figure 3


Concurrent transcriptional profiling of Dirofilaria immitis and its Wolbachia endosymbiont throughout the nematode life cycle reveals coordinated gene expression.

Luck AN, Evans CC, Riggs MD, Foster JM, Moorhead AR, Slatko BE, Michalski ML - BMC Genomics (2014)

D. immitischitinase expression. Expression profiles (FPKM values) of the D. immitis chitinase precursor protein (A), cuticular endochitinase (B) and chitinase (C) genes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4289336&req=5

Fig3: D. immitischitinase expression. Expression profiles (FPKM values) of the D. immitis chitinase precursor protein (A), cuticular endochitinase (B) and chitinase (C) genes.
Mentions: Many studies have identified microfilarial-specific chitinase activity in filarial nematodes that release sheathed microfilariae (e.g., B. malayi, Wuchereria bancrofti and Loa loa) purportedly involved in exsheathment and/or penetration of the vector midgut [67, 68]. Therefore, unsurprisingly, chitinase activity was previously identified as upregulated in B. malayi mf [38]. Conversely, filarial species that shed unsheathed mf, including species such as D. immitis, Acanthocheilonema viteae and Onchocerca volvulus, have been shown to utilize a temperature specific (37°C) L3-specific chitinase, homologous to the B. malayi mf-specific chitinase, to possibly degrade the cuticle during the L3 to L4 molt within the mammalian host. Of the three annotated chitinase genes within the D. immitis genome, the chitinase precursor protein (nDi.2.2.2.g01593) was consistently transcribed throughout the life cycle (Figure 3A). Although numerous studies have shown a lack of chitinase activity in D. immitis mf [67, 68] and specific chitinase activity in the L3 stage [69, 70], we observed significant upregulation of the cuticular endochitinase gene (nDi.2.2.2.g09584) in mf as compared to the L3 stage (Additional file 4: Table S2, Figure 3B). When describing the L3-specific chitinase of A. viteae, Wu et al., identified a protein of similar molecular weight as the L3-specific chitinase present in mf that displayed the opposite temperature specificity (expression at 27°C, not 37°C) [69], suggesting the presence of an additional chitinase functional within the insect vector. Hence, there is some evidence to support our findings that expression of this endochitinase gene does occur in unsheathed microfilariae of certain nematode species. The exact role of endochitinase in unsheathed mf that do not require chitinases to infect the vector species (D. immitis mf do not penetrate the midgut of the insect vector but rather enter the Malpighian tubes through the posterior lumen of the vector midgut [71]), remains unclear. However, chitinase may be important to help the microfilariae migrate through the chitinous peritrophic membrane that surrounds the blood meal. Additionally, because L1 to L3 development for many filarial nematodes within the vector is intracellular, chitinase may be required to invade the host cell. As expected, expression levels of the chitinase gene (nDi.2.2.2.g09661) were much higher in the L3 stage (Figure 3C) than any other chitinase-related gene (nDi.2.2.2.g01593 or nDi.2.2.2.g09584) in any other life cycle stage (note difference in scales between Figure 3A/3B and Figure 3C).Figure 3

Bottom Line: Interestingly, a large proportion of both D. immitis and wDi genes display microfilarial-biased transcriptional patterns.Concurrent transcriptome sequencing identified potential molecular interactions between parasite and endosymbiont that are more prominent during certain life cycle stages.In support of metabolite provisioning between filarial nematodes and Wolbachia, the synthesis of the critical metabolite, heme, by wDi appears to be synchronized in a stage-specific manner (mf-specific) with the production of heme-binding proteins in D. immitis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Microbiology, University of Wisconsin Oshkosh, Oshkosh, WI 54901, USA. michalsk@uwosh.edu.

ABSTRACT

Background: Dirofilaria immitis, or canine heartworm, is a filarial nematode parasite that infects dogs and other mammals worldwide. Current disease control relies on regular administration of anthelmintic preventives, however, relatively poor compliance and evidence of developing drug resistance could warrant alternative measures against D. immitis and related human filarial infections be taken. As with many other filarial nematodes, D. immitis contains Wolbachia, an obligate bacterial endosymbiont thought to be involved in providing certain critical metabolites to the nematode. Correlations between nematode and Wolbachia transcriptomes during development have not been examined. Therefore, we detailed the developmental transcriptome of both D. immitis and its Wolbachia (wDi) in order to gain a better understanding of parasite-endosymbiont interactions throughout the nematode life cycle.

Results: Over 215 million single-end 50 bp reads were generated from total RNA from D. immitis adult males and females, microfilariae (mf) and third and fourth-stage larvae (L3 and L4). We critically evaluated the transcriptomes of the various life cycle stages to reveal sex-biased transcriptional patterns, as well as transcriptional differences between larval stages that may be involved in larval maturation. Hierarchical clustering revealed both D. immitis and wDi transcriptional activity in the L3 stage is clearly distinct from other life cycle stages. Interestingly, a large proportion of both D. immitis and wDi genes display microfilarial-biased transcriptional patterns. Concurrent transcriptome sequencing identified potential molecular interactions between parasite and endosymbiont that are more prominent during certain life cycle stages. In support of metabolite provisioning between filarial nematodes and Wolbachia, the synthesis of the critical metabolite, heme, by wDi appears to be synchronized in a stage-specific manner (mf-specific) with the production of heme-binding proteins in D. immitis.

Conclusions: Our integrated transcriptomic study has highlighted interesting correlations between Wolbachia and D. immitis transcription throughout the life cycle and provided a resource that may be used for the development of novel intervention strategies, not only for the treatment and prevention of D. immitis infections, but of other closely related human parasites as well.

Show MeSH
Related in: MedlinePlus