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Identification and comparative analysis of subolesin/akirin ortholog from Ornithodoros turicata ticks.

Sultana H, Patel U, Sonenshine DE, Neelakanta G - Parasit Vectors (2015)

Bottom Line: Our study reveals that O. turicata subolesin gene expression is developmentally regulated, where; adult ticks expressed significantly higher levels in comparison to the larvae or nymphal ticks.However, no myristoylation or glycosylation sites were evident in the O. turicata subolesin sequence.Our study provides important insights in recognizing subolesin as a conserved potential candidate for the development of a broad-spectrum anti-vector vaccine to control not only ticks but also several other arthropods that transmit diseases to humans and animals.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Medicine, College of Sciences, Old Dominion University, Norfolk, 23529, VA, USA. hsultana@odu.edu.

ABSTRACT

Background: Subolesin is an evolutionary conserved molecule in diverse arthropod species that play an important role in the regulation of genes involved in immune responses, blood digestion, reproduction and development. In this study, we have identified a subolesin ortholog from soft ticks Ornithodoros turicata, the vector of the relapsing fever spirochete in the United States.

Methods: Uninfected fed or unfed O. turicata ticks were used throughout this study. The subolesin mRNA was amplified by reverse transcription polymerase chain reaction (RT-PCR) and sequenced. Quantitative-real time PCR (QRT-PCR) was performed to evaluate subolesin mRNA levels at different O. turicata developmental stages and from salivary glands and gut tissues. Bioinformatics and comparative analysis was performed to predict potential post-translational modifications in O. turicata subolesin amino-acid sequences.

Results: Our study reveals that O. turicata subolesin gene expression is developmentally regulated, where; adult ticks expressed significantly higher levels in comparison to the larvae or nymphal ticks. Expression of subolesin was evident in both unfed and fed ticks and in the salivary glands and midgut tissues. The expression of subolesin transcripts varied in fed ticks with peak levels at day 14 post-feeding. Phylogenetic analysis revealed that O. turicata subolesin showed a high degree of sequence conservation with subolesin's from other soft and hard ticks. Bioinformatics and comparative analysis predicted that O. turicata subolesin carry three Protein kinase C and one Casein kinase II phosphorylation sites. However, no myristoylation or glycosylation sites were evident in the O. turicata subolesin sequence.

Conclusion: Our study provides important insights in recognizing subolesin as a conserved potential candidate for the development of a broad-spectrum anti-vector vaccine to control not only ticks but also several other arthropods that transmit diseases to humans and animals.

No MeSH data available.


Related in: MedlinePlus

PCR amplification, nucleotide and deduced amino acid sequence ofO. turicatasubolesin gene. A) PCR amplification of subolesin gene is shown. Band of approximately 470 bp was evident in both unfed and fed O. turicata ticks. The 28S rRNA levels were shown as controls. Arrow indicates subolesin PCR fragments. B) Percent identity (horizontally above black boxed diagonal) and divergence (vertically below black boxed diagonal) of O. turicata subolesin nucleotide sequence in comparison to O. erraticus and O. moubata subolesin sequences is shown. C) The nucleotide sequence of O. turicata subolesin gene was obtained by sequencing PCR products cloned in the pGEMT-easy vector. The deduced amino acid sequence is shown as a single letter code below the nucleotide sequence.
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Fig1: PCR amplification, nucleotide and deduced amino acid sequence ofO. turicatasubolesin gene. A) PCR amplification of subolesin gene is shown. Band of approximately 470 bp was evident in both unfed and fed O. turicata ticks. The 28S rRNA levels were shown as controls. Arrow indicates subolesin PCR fragments. B) Percent identity (horizontally above black boxed diagonal) and divergence (vertically below black boxed diagonal) of O. turicata subolesin nucleotide sequence in comparison to O. erraticus and O. moubata subolesin sequences is shown. C) The nucleotide sequence of O. turicata subolesin gene was obtained by sequencing PCR products cloned in the pGEMT-easy vector. The deduced amino acid sequence is shown as a single letter code below the nucleotide sequence.

Mentions: The Subolesin/akirins are evolutionary conserved transcriptional factors in diverse arthropod species [12]. To identify whether O. turicata ticks also encode and express subolesin, cDNA prepared from unfed and fed O. turicata ticks was used as the template for PCR amplification. A band of approximately 470 bp was evident in both unfed and fed samples (Figure 1A). The band from the fed tick sample was excised and processed for sequencing as described in the Methods section. Among the three clones that were sequenced, one of the clones had guanine and the other two clones had adenine nucleobase at position 11 (Additional file 1: Figure S1A). However, these differences in the nucleotide sequences did not affect any change in the annotated amino acid sequences (Additional file 1: Figure S1B). No changes were seen in the rest of the nucleotide sequence of all three clones (Additional file 1: Figure S1A). The subolesin nucleotide sequence from clone one (GenBank accession number: KP708703) was considered for further analysis. Analysis of the nucleotide sequence revealed that the O. turicata sequence showed more than 83% and 86% identity with O. moubata and O. erraticus subolesin sequences respectively (Figure 1B). The subolesin nucleotide sequence was annotated based on the predicted coding region of subolesin orthologs from various hard ticks (Figure 1C).Figure 1


Identification and comparative analysis of subolesin/akirin ortholog from Ornithodoros turicata ticks.

Sultana H, Patel U, Sonenshine DE, Neelakanta G - Parasit Vectors (2015)

PCR amplification, nucleotide and deduced amino acid sequence ofO. turicatasubolesin gene. A) PCR amplification of subolesin gene is shown. Band of approximately 470 bp was evident in both unfed and fed O. turicata ticks. The 28S rRNA levels were shown as controls. Arrow indicates subolesin PCR fragments. B) Percent identity (horizontally above black boxed diagonal) and divergence (vertically below black boxed diagonal) of O. turicata subolesin nucleotide sequence in comparison to O. erraticus and O. moubata subolesin sequences is shown. C) The nucleotide sequence of O. turicata subolesin gene was obtained by sequencing PCR products cloned in the pGEMT-easy vector. The deduced amino acid sequence is shown as a single letter code below the nucleotide sequence.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4359563&req=5

Fig1: PCR amplification, nucleotide and deduced amino acid sequence ofO. turicatasubolesin gene. A) PCR amplification of subolesin gene is shown. Band of approximately 470 bp was evident in both unfed and fed O. turicata ticks. The 28S rRNA levels were shown as controls. Arrow indicates subolesin PCR fragments. B) Percent identity (horizontally above black boxed diagonal) and divergence (vertically below black boxed diagonal) of O. turicata subolesin nucleotide sequence in comparison to O. erraticus and O. moubata subolesin sequences is shown. C) The nucleotide sequence of O. turicata subolesin gene was obtained by sequencing PCR products cloned in the pGEMT-easy vector. The deduced amino acid sequence is shown as a single letter code below the nucleotide sequence.
Mentions: The Subolesin/akirins are evolutionary conserved transcriptional factors in diverse arthropod species [12]. To identify whether O. turicata ticks also encode and express subolesin, cDNA prepared from unfed and fed O. turicata ticks was used as the template for PCR amplification. A band of approximately 470 bp was evident in both unfed and fed samples (Figure 1A). The band from the fed tick sample was excised and processed for sequencing as described in the Methods section. Among the three clones that were sequenced, one of the clones had guanine and the other two clones had adenine nucleobase at position 11 (Additional file 1: Figure S1A). However, these differences in the nucleotide sequences did not affect any change in the annotated amino acid sequences (Additional file 1: Figure S1B). No changes were seen in the rest of the nucleotide sequence of all three clones (Additional file 1: Figure S1A). The subolesin nucleotide sequence from clone one (GenBank accession number: KP708703) was considered for further analysis. Analysis of the nucleotide sequence revealed that the O. turicata sequence showed more than 83% and 86% identity with O. moubata and O. erraticus subolesin sequences respectively (Figure 1B). The subolesin nucleotide sequence was annotated based on the predicted coding region of subolesin orthologs from various hard ticks (Figure 1C).Figure 1

Bottom Line: Our study reveals that O. turicata subolesin gene expression is developmentally regulated, where; adult ticks expressed significantly higher levels in comparison to the larvae or nymphal ticks.However, no myristoylation or glycosylation sites were evident in the O. turicata subolesin sequence.Our study provides important insights in recognizing subolesin as a conserved potential candidate for the development of a broad-spectrum anti-vector vaccine to control not only ticks but also several other arthropods that transmit diseases to humans and animals.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Medicine, College of Sciences, Old Dominion University, Norfolk, 23529, VA, USA. hsultana@odu.edu.

ABSTRACT

Background: Subolesin is an evolutionary conserved molecule in diverse arthropod species that play an important role in the regulation of genes involved in immune responses, blood digestion, reproduction and development. In this study, we have identified a subolesin ortholog from soft ticks Ornithodoros turicata, the vector of the relapsing fever spirochete in the United States.

Methods: Uninfected fed or unfed O. turicata ticks were used throughout this study. The subolesin mRNA was amplified by reverse transcription polymerase chain reaction (RT-PCR) and sequenced. Quantitative-real time PCR (QRT-PCR) was performed to evaluate subolesin mRNA levels at different O. turicata developmental stages and from salivary glands and gut tissues. Bioinformatics and comparative analysis was performed to predict potential post-translational modifications in O. turicata subolesin amino-acid sequences.

Results: Our study reveals that O. turicata subolesin gene expression is developmentally regulated, where; adult ticks expressed significantly higher levels in comparison to the larvae or nymphal ticks. Expression of subolesin was evident in both unfed and fed ticks and in the salivary glands and midgut tissues. The expression of subolesin transcripts varied in fed ticks with peak levels at day 14 post-feeding. Phylogenetic analysis revealed that O. turicata subolesin showed a high degree of sequence conservation with subolesin's from other soft and hard ticks. Bioinformatics and comparative analysis predicted that O. turicata subolesin carry three Protein kinase C and one Casein kinase II phosphorylation sites. However, no myristoylation or glycosylation sites were evident in the O. turicata subolesin sequence.

Conclusion: Our study provides important insights in recognizing subolesin as a conserved potential candidate for the development of a broad-spectrum anti-vector vaccine to control not only ticks but also several other arthropods that transmit diseases to humans and animals.

No MeSH data available.


Related in: MedlinePlus