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Tissue-specific transcriptomics and proteomics of a filarial nematode and its Wolbachia endosymbiont.

Luck AN, Anderson KG, McClung CM, VerBerkmoes NC, Foster JM, Michalski ML, Slatko BE - BMC Genomics (2015)

Bottom Line: Although many functions are shared between the reproductive tissues, the most significant differences in gene expression were observed between the uterus and testis.Of note, hypothetical proteins were among some of the most abundant Wolbachia proteins identified, which may fulfill some important yet still uncharacterized biological function.The spatial resolution gained from this parallel transcriptomic and proteomic analysis adds to our understanding of filarial biology and serves as a resource with which to develop future therapeutic strategies against filarial nematodes and their Wolbachia endosymbionts.

View Article: PubMed Central - PubMed

Affiliation: Genome Biology Division, New England Biolabs, Inc., 240 County Road, Ipswich, MA, 01938, USA.

ABSTRACT

Background: Filarial nematodes cause debilitating human diseases. While treatable, recent evidence suggests drug resistance is developing, necessitating the development of novel targets and new treatment options. Although transcriptomic and proteomic studies around the nematode life cycle have greatly enhanced our knowledge, whole organism approaches have not provided spatial resolution of gene expression, which can be gained by examining individual tissues. Generally, due to their small size, tissue dissection of human-infecting filarial nematodes remains extremely challenging. However, canine heartworm disease is caused by a closely related and much larger filarial nematode, Dirofilaria immitis. As with many other filarial nematodes, D. immitis contains Wolbachia, an obligate bacterial endosymbiont present in the hypodermis and developing oocytes within the uterus. Here, we describe the first concurrent tissue-specific transcriptomic and proteomic profiling of a filarial nematode (D. immitis) and its Wolbachia (wDi) in order to better understand tissue functions and identify tissue-specific antigens that may be used for the development of new diagnostic and therapeutic tools.

Methods: Adult D. immitis worms were dissected into female body wall (FBW), female uterus (FU), female intestine (FI), female head (FH), male body wall (MBW), male testis (MT), male intestine (MI), male head (MH) and 10.1186/s12864-015-2083-2 male spicule (MS) and used to prepare transcriptomic and proteomic libraries.

Results: Transcriptomic and proteomic analysis of several D. immitis tissues identified many biological functions enriched within certain tissues. Hierarchical clustering of the D. immitis tissue transcriptomes, along with the recently published whole-worm adult male and female D. immitis transcriptomes, revealed that the whole-worm transcriptome is typically dominated by transcripts originating from reproductive tissue. The uterus appeared to have the most variable transcriptome, possibly due to age. Although many functions are shared between the reproductive tissues, the most significant differences in gene expression were observed between the uterus and testis. Interestingly, wDi gene expression in the male and female body wall is fairly similar, yet slightly different to that of Wolbachia gene expression in the uterus. Proteomic methods verified 32 % of the predicted D. immitis proteome, including over 700 hypothetical proteins of D. immitis. Of note, hypothetical proteins were among some of the most abundant Wolbachia proteins identified, which may fulfill some important yet still uncharacterized biological function.

Conclusions: The spatial resolution gained from this parallel transcriptomic and proteomic analysis adds to our understanding of filarial biology and serves as a resource with which to develop future therapeutic strategies against filarial nematodes and their Wolbachia endosymbionts.

No MeSH data available.


Related in: MedlinePlus

Venn diagrams illustrating D. immitis (a) and wDi (b) proteins detected in D. immitis tissues. a Since only 35 D. immitis proteins were identified in the MS tissue sample, the spicule sample was excluded from this figure. However, those 35 proteins were also identified in other tissues and are represented accordingly. b Only FBW, FH, MH and FU samples are shown since no wDi proteins were detected in the FI or MS sample
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Fig4: Venn diagrams illustrating D. immitis (a) and wDi (b) proteins detected in D. immitis tissues. a Since only 35 D. immitis proteins were identified in the MS tissue sample, the spicule sample was excluded from this figure. However, those 35 proteins were also identified in other tissues and are represented accordingly. b Only FBW, FH, MH and FU samples are shown since no wDi proteins were detected in the FI or MS sample

Mentions: Overall, 4162 of the 12,857 annotated proteins or ~32 % of the predicted D. immitis proteome were verified by at least two unique peptides from the analysis of six D. immitis tissues (FBW: 2408; FU: 3341; FH: 1100; FI: 202; MH: 1183 and MS: 35 proteins) (Table 1, Additional file 11: Dataset S2). Higher protein coverage was obtained with B. malayi [95], however that level of coverage was attained using five life cycle stages whereas our data only covers the proteome of adult D. immitis tissues. A complete list of the peptide spectrum matching counts for every D. immitis protein detected, along with their corresponding gene annotation is provided in Additional file 12: Table S7. Likely due to incomplete lysis of the spicule due to its inherent structural stability, only 35 proteins were detected in this tissue (Additional file 12: Table S7). Because these proteins were also detected in another D. immitis tissue, the spicule sample was omitted from further analysis. Only 3.7 % of identified proteins (n = 152) were common among the remaining five tissue samples (FBW, FU, FH, FI and MH) (Fig. 4a). Not surprisingly, these shared proteins include common structural and motor proteins (actin/collagens/kinesin), galectins, annexins, redox proteins (thioredoxin peroxidases), heat shock proteins, enzymes (enolase, ATP synthase components, lactate dehydrogenase, pyruvate kinase, protein disulfide isomerase, protein phosphatase) and clathrin, among others (Additional file 12: Table S7). Among the 4162 proteins identified, ~49 % (n = 2056) were tissue specific (identified only in one tissue) (Fig. 4a, Additional file 12: Table S7). Perhaps unsurprisingly, the highest number of tissue specific proteins (1458) were identified in the uterus sample (Fig. 4a). Out of the 202 proteins identified in the D. immitis female intestine sample, only 6 proteins were intestine-specific. These potential hidden antigens include: aspartic protease sp-1, transmembrane protein 132a, guanine nucleotide-binding protein α-7 subunit gpa-7, cell division protein kinase 5, α-ulin (catenin vinculin related) family member ctn-1 and a hypothetical protein. Interestingly, both gpa-7 and ctn-1 are expressed in the pharyngeal muscle and intestinal muscle of C. elegans [96, 97].Fig. 4


Tissue-specific transcriptomics and proteomics of a filarial nematode and its Wolbachia endosymbiont.

Luck AN, Anderson KG, McClung CM, VerBerkmoes NC, Foster JM, Michalski ML, Slatko BE - BMC Genomics (2015)

Venn diagrams illustrating D. immitis (a) and wDi (b) proteins detected in D. immitis tissues. a Since only 35 D. immitis proteins were identified in the MS tissue sample, the spicule sample was excluded from this figure. However, those 35 proteins were also identified in other tissues and are represented accordingly. b Only FBW, FH, MH and FU samples are shown since no wDi proteins were detected in the FI or MS sample
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4642636&req=5

Fig4: Venn diagrams illustrating D. immitis (a) and wDi (b) proteins detected in D. immitis tissues. a Since only 35 D. immitis proteins were identified in the MS tissue sample, the spicule sample was excluded from this figure. However, those 35 proteins were also identified in other tissues and are represented accordingly. b Only FBW, FH, MH and FU samples are shown since no wDi proteins were detected in the FI or MS sample
Mentions: Overall, 4162 of the 12,857 annotated proteins or ~32 % of the predicted D. immitis proteome were verified by at least two unique peptides from the analysis of six D. immitis tissues (FBW: 2408; FU: 3341; FH: 1100; FI: 202; MH: 1183 and MS: 35 proteins) (Table 1, Additional file 11: Dataset S2). Higher protein coverage was obtained with B. malayi [95], however that level of coverage was attained using five life cycle stages whereas our data only covers the proteome of adult D. immitis tissues. A complete list of the peptide spectrum matching counts for every D. immitis protein detected, along with their corresponding gene annotation is provided in Additional file 12: Table S7. Likely due to incomplete lysis of the spicule due to its inherent structural stability, only 35 proteins were detected in this tissue (Additional file 12: Table S7). Because these proteins were also detected in another D. immitis tissue, the spicule sample was omitted from further analysis. Only 3.7 % of identified proteins (n = 152) were common among the remaining five tissue samples (FBW, FU, FH, FI and MH) (Fig. 4a). Not surprisingly, these shared proteins include common structural and motor proteins (actin/collagens/kinesin), galectins, annexins, redox proteins (thioredoxin peroxidases), heat shock proteins, enzymes (enolase, ATP synthase components, lactate dehydrogenase, pyruvate kinase, protein disulfide isomerase, protein phosphatase) and clathrin, among others (Additional file 12: Table S7). Among the 4162 proteins identified, ~49 % (n = 2056) were tissue specific (identified only in one tissue) (Fig. 4a, Additional file 12: Table S7). Perhaps unsurprisingly, the highest number of tissue specific proteins (1458) were identified in the uterus sample (Fig. 4a). Out of the 202 proteins identified in the D. immitis female intestine sample, only 6 proteins were intestine-specific. These potential hidden antigens include: aspartic protease sp-1, transmembrane protein 132a, guanine nucleotide-binding protein α-7 subunit gpa-7, cell division protein kinase 5, α-ulin (catenin vinculin related) family member ctn-1 and a hypothetical protein. Interestingly, both gpa-7 and ctn-1 are expressed in the pharyngeal muscle and intestinal muscle of C. elegans [96, 97].Fig. 4

Bottom Line: Although many functions are shared between the reproductive tissues, the most significant differences in gene expression were observed between the uterus and testis.Of note, hypothetical proteins were among some of the most abundant Wolbachia proteins identified, which may fulfill some important yet still uncharacterized biological function.The spatial resolution gained from this parallel transcriptomic and proteomic analysis adds to our understanding of filarial biology and serves as a resource with which to develop future therapeutic strategies against filarial nematodes and their Wolbachia endosymbionts.

View Article: PubMed Central - PubMed

Affiliation: Genome Biology Division, New England Biolabs, Inc., 240 County Road, Ipswich, MA, 01938, USA.

ABSTRACT

Background: Filarial nematodes cause debilitating human diseases. While treatable, recent evidence suggests drug resistance is developing, necessitating the development of novel targets and new treatment options. Although transcriptomic and proteomic studies around the nematode life cycle have greatly enhanced our knowledge, whole organism approaches have not provided spatial resolution of gene expression, which can be gained by examining individual tissues. Generally, due to their small size, tissue dissection of human-infecting filarial nematodes remains extremely challenging. However, canine heartworm disease is caused by a closely related and much larger filarial nematode, Dirofilaria immitis. As with many other filarial nematodes, D. immitis contains Wolbachia, an obligate bacterial endosymbiont present in the hypodermis and developing oocytes within the uterus. Here, we describe the first concurrent tissue-specific transcriptomic and proteomic profiling of a filarial nematode (D. immitis) and its Wolbachia (wDi) in order to better understand tissue functions and identify tissue-specific antigens that may be used for the development of new diagnostic and therapeutic tools.

Methods: Adult D. immitis worms were dissected into female body wall (FBW), female uterus (FU), female intestine (FI), female head (FH), male body wall (MBW), male testis (MT), male intestine (MI), male head (MH) and 10.1186/s12864-015-2083-2 male spicule (MS) and used to prepare transcriptomic and proteomic libraries.

Results: Transcriptomic and proteomic analysis of several D. immitis tissues identified many biological functions enriched within certain tissues. Hierarchical clustering of the D. immitis tissue transcriptomes, along with the recently published whole-worm adult male and female D. immitis transcriptomes, revealed that the whole-worm transcriptome is typically dominated by transcripts originating from reproductive tissue. The uterus appeared to have the most variable transcriptome, possibly due to age. Although many functions are shared between the reproductive tissues, the most significant differences in gene expression were observed between the uterus and testis. Interestingly, wDi gene expression in the male and female body wall is fairly similar, yet slightly different to that of Wolbachia gene expression in the uterus. Proteomic methods verified 32 % of the predicted D. immitis proteome, including over 700 hypothetical proteins of D. immitis. Of note, hypothetical proteins were among some of the most abundant Wolbachia proteins identified, which may fulfill some important yet still uncharacterized biological function.

Conclusions: The spatial resolution gained from this parallel transcriptomic and proteomic analysis adds to our understanding of filarial biology and serves as a resource with which to develop future therapeutic strategies against filarial nematodes and their Wolbachia endosymbionts.

No MeSH data available.


Related in: MedlinePlus