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. 2017 Sep:88:37-47.
doi: 10.1016/j.ibmb.2017.07.006. Epub 2017 Jul 21.

Amblyomma maculatum SECIS binding protein 2 and putative selenoprotein P are indispensable for pathogen replication and tick fecundity

Khemraj Budachetri  1 , Gary Crispell  1 , Shahid Karim  2
Affiliations

Amblyomma maculatum SECIS binding protein 2 and putative selenoprotein P are indispensable for pathogen replication and tick fecundity

Khemraj Budachetri et al. Insect Biochem Mol Biol. 2017 Sep.

Abstract

Selenium, a vital trace element, is incorporated into selenoproteins to produce selenocysteine. Our previous studies have revealed an adaptive co-evolutionary process that has enabled the spotted fever-causing tick-borne pathogen Rickettsia parkeri to survive by manipulating an antioxidant defense system associated with selenium, which includes a full set of selenoproteins and other antioxidants in ticks. Here, we conducted a systemic investigation of SECIS binding protein 2 (SBP2) and putative selenoprotein P (SELENOP) by transcript silencing in adult female Gulf-coast ticks (Amblyomma maculatum). Knockdown of the SBP2 and SELENOP genes depleted the respective transcript levels of these tick selenogenes, and caused differential regulation of other antioxidants. Importantly, the selenium level in the immature and mature tick stages increased significantly after a blood meal, but the selenium level decreased in ticks after the SBP2 and SELENOP knockdowns. Moreover, the SBP2 knockdown significantly impaired both transovarial transmission of R. parkeri to tick eggs and egg hatching. Overall, our data offer new insight into the relationship between the SBP2 selenoprotein synthesis gene and the putative tick SELENOP gene. It also augments our understanding of selenoprotein synthesis, selenium maintenance and utilization, and bacterial colonization of a tick vector.

Keywords: Amblyomma maculatum; RNA interference; Rickettsia parkeri; Selenium; Selenoproteins.

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Conflict of interest statement

Competing interests: The authors have declared that no competing interests exist.

Figures

Fig. 1
Fig. 1. Transcriptional expression of SECIS binding protein 2 (SBP2), selenoprotein P (SELENOP) and selenophosphate synthetase (SEPHS2) genes post-blood meal during Rickettsia parkeri infection in Amblyomma maculatum
Transcriptional gene expression for (A) SBP2, (B) SELENOP, and (C) SEPHS2 was estimated in unfed ticks, slow feeding (4-day) ticks, and fast feeding (8-day) ticks in adult female A. maculatum. The expression value for the unfed ticks was set to 1 for reference. (D) Tick SBP2, SELENOP and SEPHS2 genes were up-regulated in R. parkeri-infected tick midgut and salivary glands tissues. The transcriptional activities were normalized against tick glyceraldehyde 3-phosphate dehydrogenase.
Fig. 2
Fig. 2. Selenium levels in immature and mature ticks
Selenium levels across (A) embryonic and immature stages of blood fed and unfed (UF) ticks, (B) adult fed and UF male and female Amblyomma maculatum ticks with (+) or without (–) Rickettsia parkeri infection before and after a blood meal. (C) Selenium levels in UF and 8-day partially fed (pF) female Ixodes scapularis (IS) and Amblyomma americanum (AA) ticks. (D) Selenium concentrations in the control, and in the SBP2- and SELENOP-silenced Amblyomma maculatum (AM) ticks. At least three biological replicates were used to estimate the selenium levels via the inductively coupled plasma mass spectrometry (ICP-MS) technique.
Fig. 3
Fig. 3. SECIS binding protein 2 (SBP2) gene (A) and selenoprotein P (SELENOP) gene (B) knockdowns in Amblyomma maculatum
Transcriptional expression of the selenogenes was assessed in SBP2 and SELENOP knockdown tick tissues. Quantitative reverse transcriptase PCR was used to determine the transcriptional expression levels of the tick selenoproteins using tick GAPDH as the reference gene. The expression levels of the target genes in the control samples were set to 1.
Fig. 4
Fig. 4. Immune-blot analysis of SECIS binding protein 2 (SBP2) and selenoprotein P (SELENOP) proteins in Amblyomma maculatum
(A) SDS-PAGE gel (4–20%) stained with GelCode Blue; (B) the corresponding immunoblots showing cross reactivity to an anti-human SELENOP antibody; (C) immunoblot incubated with SBP2 antibodies and (D) immunoblot incubated with anti-glyceraldehyde phosphate dehydrogenase monoclonal antibodies. M: Broad range molecular weight protein standard. Lanes 1 and 2 show the no-treatment ticks and the dsLacZ-injected A. maculatum female ticks, respectively. Lanes 3 and 4 show dsSBP2-and dsSELENOP-injected A. maculatum female ticks, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5
Fig. 5. Rickettsia parkeri and symbiont loads in tick SECIS binding protein 2 (SBP2) gene and selenoprotein P (SELENOP) gene knockdown tissues
(A) R. parkeri load in tick midguts (MG) and salivary glands (SG). (B) Francisella-like endosymbiont (FLE) load in dsRNA-injected tick tissues 5 days after the injections. (C) Candidatus Midichloria mitochondrii (CMM) load in dsRNA-injected tick tissues. (D) Bacterial load in midguts and salivary glands in the dsRNA-injected ticks, as determined by 16S rRNA analysis.
Fig. 6
Fig. 6. Impact of the SECIS binding protein 2 (SBP2) on tick fecundity and transovarial transmission of Rickettsia parkeri from SBP2 and SELENOP gene knockdowns
Eggs from the control (dsLacZ-injected) (a, b) and dsSBP2 injected (c, d) ticks. (e, f) R. parkeri load in tick eggs after SBP2 and SELENOP silencing.
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