Differential Expression of Genes in Uninfected and Rickettsia -Infected Dermacentor variabilis Ticks as Assessed by Differential-Display PCR

Abstract

Ticks serve as both the vector and the reservoir for members of the spotted fever group rickettsiae. The molecular interaction(s) that results from this close relationship is largely unknown. To identify genetic factors associated with the tick response to rickettsial infection, we utilized differential-display PCR. The majority of upregulation appeared in the infected tissue. We cloned and sequenced 54 differentially expressed transcripts and compared the sequences to those in the GenBank database. Nine of the 54 clones were assigned putative identities and included a clathrin-coated vesicle ATPase, peroxisomal farnesylated protein, Ena/vasodilatorstimulated phosphoprotein-like protein, ␣-catenin, tubulin ␣-chain, copper-transporting ATPase, salivary gland protein SGS-3 precursor, glycine-rich protein, and Dreg-2 protein. Confirmation of the rickettsial influence on the differential expression in the ovaries for a number of these clones was demonstrated by semiquantitative reverse transcription-PCR and Northern blot analyses, resulting in confirmation of six out of nine and three out of four assessed clones, respectively. Further characterization of the clones identified tissue-dependent expression in the midguts and salivary glands. The potential roles of these molecules in the maintenance and transmission of rickettsiae are discussed.Among tick-borne human pathogens, Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is a serious health concern in the United States. The parameters of transmission make Rocky Mountain spotted fever exceptionally difficult in terms of management and control. Small mammals, and occasionally humans, become infected when infected ticks feed on them during their life cycles. However, spotted fever group (SFG) rickettsiae are primarily maintained in nature through transovarial transmission via the infected germinal tissues of ixodid ticks. Under both laboratory and natural conditions, it has been reported that ticks infected with one species of Rickettsia are unable to pass a second species to their progeny (1,20). While the transovarial transmission-blocking phenomenon has been reported only for SFG rickettsiae within ticks, de la Fuente et al. (3) recently reported that experimental competition results in a selection of only one genotype of Anaplasma marginale in the tick.The mechanism(s) allowing for SFG rickettsial interference remains elusive. We hypothesize that, in response to a primary rickettsial infection, differentially regulated tick-derived molecules prevent a secondary rickettsial infection. The category (i.e., receptor or defense) and mode of action of these tickderived molecules are unknown, and in order to test the hypothesis, differentially expressed candidate molecules must first be identified. We have recently utilized subtractive hybridization as a tool to identify some candidate molecules (21), and characterization of these molecules is the objective of our ongoing project. The objective of this study was to assess the differential expre...

New Tick Defensin Isoform and Antimicrobial Gene Expression in Response to Rickettsia montanensis Challenge

“…Recent reports indicate that genes putatively functioning as receptor/adhesion and immune response effectors are differentially expressed in ovaries from D. variabilis ticks chronically infected with R. montanensis (26,28). Therefore, it was of interest to examine the transcriptional profiles of three important antimicrobial genes in response to rickettsial challenge.…”

“…Of equal interest was the use of a differential display to parse the same experimental design for ovary-specific differentially expressed genes. Macaluso et al (26) found that Ena/ vasodilator-stimulated phosphoprotein-like protein, vacuolar ATPase, and ␣-catenin transcripts were all up-regulated in infected ovaries, reflecting the potential importance of these proteins for rickettsial entry and intra-and intercellular mobility.…”

Comparative population genetics of two invading ticks: Evidence of the ecological mechanisms underlying tick range expansions

“…We used standard PCR to amplify a fragment of the tick mitochondrial 16S rRNA gene using primers 16S+1 (5′-CTGCTCAATGATTTTTTAAATTGCTGT-3′) and 16S-1 (5′-GTCTGAACTCAGATCAAGT-3′) (Macaluso et al 2003, Nadolny et al 2011). PCR reactions were performed in 15 µL reaction volumes, with 0.05 U/µL Taq DNA Polymerase (BioPioneer Inc., San Diego, CA), 1 µM each primer, 1.5 mM MgCl 2 , 1X PCR buffer (Qiagen Inc., Valencia, CA) and 2 µL DNA template.…”

Identification of Differential Gene Expression in Bacteria Associated with Coral Black Band Disease by Using RNA-Arbitrarily Primed PCR

“…To confirm this possibility, semiquantitative RT-PCR was performed with the original samples of RNA, in situ and suspended, from three different experiments. Semiquantitative RT-PCR analysis has previously been used to confirm differences in gene expression (15,24,26). Specific primers were designed to analyze the levels of expression of the genes presented in Table 2.…”