Is selenoprotein K required for Borrelia burgdorferi infection within the tick vector Ixodes scapularis?
Tick selenoproteins are involved in regulating oxidative and endoplasmic reticulum stress during prolonged tick feeding on mammalian hosts. How selenoproteins are activated upon tick-borne pathogen infection is yet to be defined.
To examine the functional role of selenoprotein K in Borrelia burgdorferi infection within the tick host Ixodes scapularis, RNA interference (RNAi)-based gene silencing was performed.
Selenoprotein K is an endoplasmic reticulum (ER)-resident protein and a component of the ERAD complex involved in ER homeostasis. A qRT-PCR assay revealed the significant upregulation of selenogene K (selenoK) expression in B. burgdorferi-infected tick tissues. Silencing of the selenoK transcript significantly depleted B. burgdorferi copies within the infected tick tissues. Upon selenoK knockdown, another component of the ERAD complex, selenoprotein S (selenoS), was significantly upregulated, suggesting a compensatory mechanism to maintain ER homeostasis within the tick tissues. Knockdown of selenoK also upregulated ER stress-related unfolded protein response (UPR) pathway components, ATF6 and EIF2.
The exact mechanisms that contribute to depletion of B. burgdorferi upon selenoK knockdown is yet to be determined, but this study suggests that selenoK may play a vital role in the survival of B. burgdorferi within the tick host.
KeywordsTick Ixodes scapularis Borrelia burgdorferi Lyme disease Selenoproteins ER stress
endoplasmic reticulum associated degradation
activating transcription factor 6
eukaryotic initiation factor 2
ribosomal protein S4
quantitative real-time polymerase chain reaction
reactive oxygen species
In the USA, reported vector-borne diseases are predominantly tick-borne; for example, there are approximately 329,000 cases of Lyme disease annually in the USA. A recent CDC study based upon vector-borne disease cases reported to the National Notifiable Disease Surveillance System from 2004 to 2016 revealed 491,671 cases of tick-borne infectious diseases (76.5% of all vector-borne diseases) (http://ww.cdc.gov/mmwr) . In the USA, Lyme disease-causing B. burgdorferi spirochetes are primarily harbored by the blacklegged tick I. scapularis .
Tick blood-feeding also generates toxic levels of reactive oxygen species (ROS) that could damage lipids, proteins and DNA, and promote mutation, cellular dysfunction and cell death. To successfully feed and survive, ticks must somehow prevent these detrimental effects and promote the beneficial aspects of ROS, which suggests that there are precise regulatory strategies for maintaining appropriate ROS levels both within the tick and possibly at the tick-host interface. Our previously published studies have shown an adaptive coevolutionary process that has enabled tick-borne pathogen survival by manipulating an antioxidant defense system associated with selenium including a full set of selenoproteins and other antioxidants [3, 4, 5, 6, 7, 8, 9, 10, 11, 12]. Generation of ROS is among the first lines of host defense against invading microbes [13, 14].
Selenoproteins exhibit diverse biological functions such as detoxification of peroxides, regeneration of reduced thioredoxin and reduction of oxidized methionine residues by oxidation of the selenium (Se−) active site [15, 16, 17]. In the last decade, significant progress has been made in clarifying the functions and physiological roles of vertebrate selenoproteins; new selenoprotein families have been identified, and new functions have been assigned to previously characterized selenoproteins. Some of the newer specific functions of selenoproteins involve removal of hydrogen peroxide, repair of oxidatively damaged proteins, control of cytoskeleton/actin assembly, protein folding and mitigating ER stress as part of the endoplasmic reticulum associated degradation (ERAD) complex , among others. Tick selenoproteins have been shown to play an important role in mitigating oxidative stress [3, 4, 5, 6, 7, 8, 9, 10, 11, 12], pathogen colonization [5, 7, 8, 9, 11, 18, 19, 20] and microbiota maintenance [9, 10, 12].
A study using a tick-pathogen model has shown that the initial stage of transmission of a pathogen by an arthropod vector is influenced by gene expression changes in both vectors and pathogens . However, there is a lack of knowledge on how gene expression within the vector is altered by the presence of the vector-borne pathogen. Therefore, the present study was designed to test the hypothesis that B. burgdorferi infection activates upregulation of selenogene K expression in order to survive within the tick host, I. scapularis, before transmission to the mammalian host. To test this hypothesis, a qRT-PCR assay was used to determine the expression of selenoK in B. burgdorferi-infected I. scapularis tissues over the course of a feeding period. An RNA interference approach also was used to silence selenogene K expression in B. burgdorferi-infected ticks to examine pathogen survival within the tick vector.
All common laboratory supplies and chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), Fisher Scientific (Grand Island, NY, USA) or Bio-Rad (Hercules, CA, USA) unless otherwise specified.
Bioinformatics analysis of selenoprotein K
The full-length gene sequence of I. scapularis selenoK (GenBank: XM_002403043.1) was obtained from the NCBI database. Corresponding protein sequences for I. scapularis selenoK (NCBI Accession number: XP_002403087.1) were aligned with selenoK protein sequences from other organisms such as Amblyomma maculatum, A. americanum, Gallus gallus, Rattus norvegicus, Mus musculus, Equus caballus, Homo sapiens, Macaca mulatta, Pan troglodytes and Pongo abelii by using Clustal Omega  for multiple sequence alignment. selenoK protein sequences aligned by Clustal Omega were refined by eye and presented by Jalview v.2.7 . Additionally, the freely available online tool SECISsearch3 (http://seblastian.crg.es/) was used for the prediction of the SECIS (Selenocysteine Insertion Sequence) element in the tick selenoK nucleotide sequence, required for amino acid selenocysteine (Sec) insertion during translation . Subcellular localization of tick selenoK was also predicted by using the freely available online algorithm DeepLoc-1.0 (http://www.cbs.dtu.dk/services/DeepLoc/index.php) which predicts the subcellular localization of eukaryotic proteins .
Ticks and tissue dissections
Ticks were purchased from the Oklahoma State University Tick Rearing Facility. Adult male and female I. scapularis were kept according to standard practices  and maintained in the laboratory as described in our previously published work . Unfed female adult I. scapularis were infected with laboratory grown B. burgdorferi strain B31.5A19 using the capillary feeding method at Tulane National Primate Research Center . Naturally B. burgdorferi-infected adult I. scapularis were collected from field (Kingston, Rhode Island) and maintained in the laboratory using standard procedures . Rabbit was used as host for tick blood-feeding. The blood-fed adult female I. scapularis were dissected within 60 min of removal and collection from the rabbit. Tick tissues were dissected and washed in M-199 buffer as described previously . Salivary glands and midguts from individual I. scapularis were stored in RNAlater (Life Technologies, Carlsbad, CA, USA) at − 80 °C until used.
Total RNA isolation, cDNA synthesis, dsRNA preparation and transcriptional expression
The methods to extract total RNA, cDNA synthesis, double-stranded RNA for selenoK, irrelevant gene LacZ, and qRT-PCR assays were performed as described previously [9, 12]. RNA was extracted from individually-dissected tick tissues (salivary gland, midgut), and cDNA was synthesized from each of them to check whether they were infected or not. Only tissues which were found infected were pursued for further work. The concentration of dsRNA used was 1000 ng/µl. One microliter of dsRNA was injected into the tick hemocoel. To investigate the role of selenoK in tick feeding success and pathogen survival, 45 unfed adult female capillary-fed (B. burgdorferi culture) ticks were injected with 1000 ng of selenoK-dsRNA into the hemocoel using a Hamilton syringe fitted with a 33-gauge needle . As a control, a total of 45 unfed adult capillary-fed (B. burgdorferi culture) ticks were injected with 1000 ng of lacZ-dsRNA (an irrelevant control dsRNA). After the injection of dsRNA, ticks were kept at 37 °C overnight under high humidity to confirm tick survival. Ixodes scapularis ticks were then placed on rabbit ears . Ixodes scapularis naturally infected with B. burgdorferi were collected from Rhode Island to determine the role of selenoK silencing on pathogen infection. A total of 90 female adult ticks were divided into two groups: one group of 45 female unfed ticks was injected with irrelevant dsRNA-lacZ; the second with selenoK dsRNA followed by tick infestation on rabbit ears as described earlier.
PCR-based detection of B. burgdorferi
Borrelia burgdorferi was detected in the tick tissues using the flaB gene in a PCR assay . The flaB gene and other primers used in the experiments are provided in Additional file 1: Table S1. The PCR conditions were followed from a previous study with slight modification : 1 cycle of 94 °C for 5 min; 50 cycles of 94 °C for 30 s, 50 °C for 30 s and 68 °C for 1 min; and 1 cycle of 72 °C for 8 min.
Quantification of B. burgdorferi in I. scapularis tissues
The principle of quantification of B. burgdorferi load was followed from our studies established to quantify the load of the spotted fever group rickettsia, Rickettsia parkeri, in tick tissues . Borrelia burgdorferi load in I. scapularis tissues were estimated by quantifying the number of copies of B. burgdorferi flagellin gene, flaB  present per copy of housekeeping gene Rps4 . A list of all primers used is provided in Additional file 1: Table S1. The gene flaB from B. burgdorferi and Rps4 from I. scapularis were amplified, purified, sequenced and verified prior to further assays. Their standard curves were determined by qRT-PCR based on serially-diluted PCR products. qRT-PCR conditions were as follows: 50 °C for 3 min, 95 °C for 10 min; followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s.
All data were expressed as mean values ± standard error of the mean (SEM) unless otherwise indicated. Statistical significance between two experimental groups or their respective controls was determined by a t-test (P-value, < 0.05). P < 0.05 was considered statistically significant. Comparative differences among multiple experimental groups were determined by analysis of variance with P-values < 0.05 considered statistically significant (GraphPad Prism 6.05; GraphPad Software, La Jolla, CA, USA). Transcriptional expression levels were determined using Bio-Rad CFX MANAGER v.3.1, and the gene expression values obtained were considered statistically significant if a P-value of 0.05 was obtained when compared with the control.
Results and discussion
In the present work, subcellular localization of tick selenoK was predicted by DeepLoc-v.1.0 server which uses neural network algorithms based on the experimental localization of Uniprot proteins . To include the protein sorting pathways into the algorithm, a hierarchical tree with multiple nodes was also designed. Each node of the tree represents a binary attempt to assign the protein right pathway from high to low in a hierarchical classification. As shown in Additional file 3: Figure S2, the probability score of tick selenoK is much higher at the ER/Golgi node than other organelles. Thus, our data suggest that the localization of tick selenoK is in the endoplasmic reticulum ER/Golgi membrane (Additional file 3: Figure S2). A previous study on Drosophila has also reported selenoK integration into both of these cell organelles . Attention plot (Alpha) in Additional file 3: Figure S2 indicated that the specific interspersed region of the selenoprotein K (residue number ~ 20–40 from the N-terminal) contributes in binding to ER/Golgi membrane.
It has been suggested that all of the characterized selenoproteins can reduce ROS levels in the cell. However, at the physiological level, their specific roles are yet to be discovered in arthropod vectors like ticks. selenoK lacks the defined redox motif C-X-X-Sec (X = any amino acid) found in other known selenoproteins. Thus, at this time, it cannot be firmly stated that selenoK has an antioxidant role in vivo. It could be speculated that selenoK is part of the protein complex, which demonstrates its antioxidant property (Additional file 3: Figure S2). The exact cellular localization and function of tick selenoprotein in hematophagy and pathogen infection has yet to be determined.
Temporal expression during ingestion of the blood meal
SelenoK knockdown and B. burgdorferi infection
This proof-of-concept study suggests a role for the ER stress machinery in B. burgdorferi colonization and survival inside ticks. Our data provide a deeper insight into the possible role of selenoK in the pathogen colonization of tick vectors. The nymphal ticks play an epidemiologically important role in the infection of humans with B. burgdorferi and a detailed mechanistic study to investigate the role of ER-resident selenoproteins in pathogen infection and transmission is still needed.
We thank Latoyia Downs for her technical assistance in pathogen quantification and critical comments.
Conceived and designed the experiments: DK and SK. Performed the experiments: DK and SK. Analyzed the data: DK and SK. Contributed reagents/materials/analysis tools: DK, ME, TNM and SK. Wrote the paper: DK and SK. All authors read and approved the final manuscript.
This work was supported by grants from the National Institutes of General Medical Sciences #P20 and GM103476, USDA NIFA award #2017-67017-26171 & 2016-09395, and US Department of State award # 2017-67016-26864. The funders had no role in study design, data collection, analysis, decision to publish or manuscript preparation.
Ethics approval and consent to participate
All animal experiments were conducted in strict accordance with recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, USA. The protocol for tick blood-feeding on sheep was approved by the Institutional Animal Care and Use Committee of Southern Mississippi University (protocol #18022801and #15101501.1).
Consent for publication
The authors declare that they have no competing interests.
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