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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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Venn diagram showing stage-specific expression of the 834 filarial (shared betweenD.immitisandB.malayi) genes. Stage-specificity (up to three stages) for each gene was determined for each using differential expression testing by Cuffdiff (Additional file 4: Table S2, q < 0.01). Only life cycle stages expressing the gene (value >0) were considered.
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Fig2: Venn diagram showing stage-specific expression of the 834 filarial (shared betweenD.immitisandB.malayi) genes. Stage-specificity (up to three stages) for each gene was determined for each using differential expression testing by Cuffdiff (Additional file 4: Table S2, q < 0.01). Only life cycle stages expressing the gene (value >0) were considered.

Mentions: Previous cluster analysis of the predicted D. immitis nuclear proteome (version 1.3) with four other nematode proteomes (B. malayi, C. elegans, Trichinella sprialis and Ascaris suum) identified 850 ‘filarial-specific’ proteins in clusters, i.e., conserved proteins uniquely shared by D. immitis and B. malayi, but lacking in the three other nematode species [19]. Comparison of these 850 predicted protein sequences with the improved version 2.2 D. immitis proteome (by BLASTp analysis) removed redundancies and yielded 834 predicted proteins which may represent common filarial nematode targets shared between D. immitis and B. malayi. Assessment of stage-specific transcription of these 834 gene products is listed in Additional file 6: Table S4 and summarized in Figure 2. Notably, only 367 of these genes have functional annotations, twenty of which are listed as hypothetical proteins. Hence, no functional information is available for over half of the 834 genes and many of the existing annotations are relatively vague. Nearly 65% (539) were expressed in all life cycle stages examined while 25 of the 834 D. immitis and B. malayi specific genes were not expressed in any life cycle stage (Additional file 6: Table S4). The majority of genes (711) displayed no stage-associated transcriptional pattern. Conversely, 123 genes (~15%) were significantly upregulated in at least one life cycle stage (Additional file 6: Table S4, Figure 2). Of these 123 D. immitis genes with stage-associated transcriptional patterns, two-thirds were significantly upregulated in the L4 stage of D. immitis (alone or in conjunction with other life cycle stages, Figure 2) and include an apoptotic chromatin condensation inducer protein and a number of hypothetical proteins (Additional file 6: Table S4). Seven genes were mutually upregulated between the L3 and L4 stages. Among these 7 L3/L4 upregulated genes are a 227 kDa spindle- and centromere-associated protein and a subunit of a nicotinic acetylcholine receptor. Fifteen additional genes are upregulated in the L3 stage, which may represent potential L3 targets, and include basement membrane proteoglycans and collagen domain containing proteins. Several genes (15) were upregulated in both AM and AF samples (Figure 2). These potential adulticidal targets include genes for adenylate kinase, a c-terminal binding protein and a bzip transcription factor family protein (Additional file 6: Table S4). An additional 48 genes were upregulated in AF (as well as other stages, Figure 2) including two hypothetical proteins, cell death specification protein 2, an ecdysone induced protein and the ecdysone receptor, transcription of which was previously shown to be upregulated in D. immitis AF [39]. Upregulation of an additional 23 genes was observed for AM (as well as other stages, Figure 2) including a hypothetical protein and an RNA-binding domain containing protein (Additional file 6: Table S4).Figure 2


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)

Venn diagram showing stage-specific expression of the 834 filarial (shared betweenD.immitisandB.malayi) genes. Stage-specificity (up to three stages) for each gene was determined for each using differential expression testing by Cuffdiff (Additional file 4: Table S2, q < 0.01). Only life cycle stages expressing the gene (value >0) were considered.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Venn diagram showing stage-specific expression of the 834 filarial (shared betweenD.immitisandB.malayi) genes. Stage-specificity (up to three stages) for each gene was determined for each using differential expression testing by Cuffdiff (Additional file 4: Table S2, q < 0.01). Only life cycle stages expressing the gene (value >0) were considered.
Mentions: Previous cluster analysis of the predicted D. immitis nuclear proteome (version 1.3) with four other nematode proteomes (B. malayi, C. elegans, Trichinella sprialis and Ascaris suum) identified 850 ‘filarial-specific’ proteins in clusters, i.e., conserved proteins uniquely shared by D. immitis and B. malayi, but lacking in the three other nematode species [19]. Comparison of these 850 predicted protein sequences with the improved version 2.2 D. immitis proteome (by BLASTp analysis) removed redundancies and yielded 834 predicted proteins which may represent common filarial nematode targets shared between D. immitis and B. malayi. Assessment of stage-specific transcription of these 834 gene products is listed in Additional file 6: Table S4 and summarized in Figure 2. Notably, only 367 of these genes have functional annotations, twenty of which are listed as hypothetical proteins. Hence, no functional information is available for over half of the 834 genes and many of the existing annotations are relatively vague. Nearly 65% (539) were expressed in all life cycle stages examined while 25 of the 834 D. immitis and B. malayi specific genes were not expressed in any life cycle stage (Additional file 6: Table S4). The majority of genes (711) displayed no stage-associated transcriptional pattern. Conversely, 123 genes (~15%) were significantly upregulated in at least one life cycle stage (Additional file 6: Table S4, Figure 2). Of these 123 D. immitis genes with stage-associated transcriptional patterns, two-thirds were significantly upregulated in the L4 stage of D. immitis (alone or in conjunction with other life cycle stages, Figure 2) and include an apoptotic chromatin condensation inducer protein and a number of hypothetical proteins (Additional file 6: Table S4). Seven genes were mutually upregulated between the L3 and L4 stages. Among these 7 L3/L4 upregulated genes are a 227 kDa spindle- and centromere-associated protein and a subunit of a nicotinic acetylcholine receptor. Fifteen additional genes are upregulated in the L3 stage, which may represent potential L3 targets, and include basement membrane proteoglycans and collagen domain containing proteins. Several genes (15) were upregulated in both AM and AF samples (Figure 2). These potential adulticidal targets include genes for adenylate kinase, a c-terminal binding protein and a bzip transcription factor family protein (Additional file 6: Table S4). An additional 48 genes were upregulated in AF (as well as other stages, Figure 2) including two hypothetical proteins, cell death specification protein 2, an ecdysone induced protein and the ecdysone receptor, transcription of which was previously shown to be upregulated in D. immitis AF [39]. Upregulation of an additional 23 genes was observed for AM (as well as other stages, Figure 2) including a hypothetical protein and an RNA-binding domain containing protein (Additional file 6: Table S4).Figure 2

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