Knockdown of Selenocysteine-Specific Elongation Factor in <i>Amblyomma maculatum</i> Alters the Pathogen Burden of <i>Rickettsia parkeri</i> with Epigenetic Control by the Sin3 Histone Deacetylase Corepressor Complex

<div><p>Selenocysteine is the 21st naturally-occurring amino acid. Selenoproteins have diverse functions and many remain uncharacterized, but they are typically associated with antioxidant activity. The incorporation of selenocysteine into the nascent polypeptide chain recodes the TGA stop codon and this process depends upon a number of essential factors including the selenocysteine elongation factor (SEF). The transcriptional expression of SEF did not change significantly in tick midguts throughout the blood meal, but decreased in salivary glands to 20% at the end of the fast feeding phase. Since selenoprotein translation requires this specialized elongation factor, we targeted this gene for knockdown by RNAi to gain a global view of the role selenoproteins play in tick physiology. We found no significant differences in tick engorgement and embryogenesis but detected no antioxidant capacity in tick saliva. The transcriptional profile of selenoproteins in <i>R. parkeri</i>-infected <i>Amblyomma maculatum</i> revealed declined activity of selenoprotein M and catalase and increased activity of selenoprotein O, selenoprotein S, and selenoprotein T. Furthermore, the pathogen burden was significantly altered in SEF-knockdowns. We then determined the global impact of SEF-knockdown by RNA-seq, and mapped huge shifts in secretory gene expression that could be the result of downregulation of the Sin3 histone deacetylase corepressor complex.</p> </div

Evolutionary relationships of taxa based on the SEF amino acid sequence using the Neighbor-joining method.

<p>Bootstrap values <70 and branch lengths shorter than 0.10 were not displayed.</p

Effects of SEF-knockdown on selenogenes and antioxidant genes.

<p>Normalized fold change in the transcriptional activity of selected selenogenes and associated antioxidant genes in the salivary glands of pathogen-free <i>A. maculatum</i> blood fed seven days and preceded by SEF-dsRNA injection. The transcriptional level of each candidate gene in tissues injected with LacZ-dsRNA was set to 1.0 as a reference point. </p

Evidence of transcriptional knockdown of SDS3 components in control and SEF-knockdowns.

<p>Transcriptional gene expression of two SDS3 components of the Sin3 histone deacetylase corepressor complexes in LacZ- and SEF-dsRNA knockdowns in pathogen-free and <i>R. parkeri-</i>infected <i>A. maculatum</i>.</p

Transcriptional activity of selenogenes in pathogen-free and <i>R. parkeri-</i>infected tick midgut tissues.

<p>Normalized fold change in the transcriptional activity of selected selenoprotein and associated antioxidant genes in pathogen-free or <i>R. parkeri-</i>infected <i>A. maculatum</i> midguts at three and five days post-infestation. Gene expression was normalized to pathogen-free levels in three and five days to highlight differences observed in <i>R. parkeri-</i>infected tick midguts. (*<i>p</i><0.05).</p

Evidence of transcriptional knockdown of SEF in dsRNA-injected ticks.

<p>Normalized fold change in the salivary gland transcriptional activity of SEF in pathogen-free and <i>R. parkeri</i>-infected <i>A. maculatum</i> injected with LacZ-dsRNA or SEF-dsRNA.</p

<i>R. parkeri</i> pathogen burden in RNAi tissues. (n.d.: not detected).

<p><i>R. parkeri</i> pathogen burden in RNAi tissues. (n.d.: not detected).</p

Transcriptional expression of SEF in tick tissues throughout the blood meal.

<p>Normalized fold change in the transcriptional activity of SEF in <i>A. maculatum</i> salivary glands and midgut tissues throughout the blood meal.</p