A case of horizontal gene transfer from Wolbachia to Aedes albopictus C6/36 cell line.

Horizontal gene transfer plays an essential role in evolution and ecological adaptation, yet this phenomenon has remained controversial, particularly where it occurs between prokaryotes and eukaryotes. There are a handful of reported examples of horizontal gene transfer occurring between prokaryotes and eukaryotes in the literature, with most of these documented cases pertaining to invertebrates and endosymbionts. However, the vast majority of these horizontally transferred genes were either eventually excluded or rapidly became nonfunctional in the recipient genome. In this study, we report the discovery of a horizontal gene transfer from the endosymbiont Wolbachia in the C6/36 cell line derived from the mosquito Aedes albopictus. Moreover, we report that this horizontally transferred gene displayed high transcription level. This finding and the results of further experimentation strongly suggest this gene is functional and has been expressed and translated into a protein in the mosquito host cells.

Introduction

Horizontal gene transfer (HGT) is the exchange of genetic elements between phylogenetically distant and reproductively isolated species or organisms. HGT was traditionally thought to be unlikely between prokaryotes and eukaryotes.However, recent studies have revealed that microbial endosymbiotic DNA fragments can integrate into unrelated eukaryotic organisms.- Although, this process is still considered extremely rare, endosymbiotic gene transfer is believed to be possible because of the intimate symbiotic relationship.

Wolbachia is a very important and common bacterial endosymbiont, infecting around 66% of arthropods worldwide including mosquitoes, Drosophila, and beetles.,Wolbachia is able to invade rapidly and spread widely among the host arthropod population, and can manipulate its host’s reproduction and also prevent its host from infecting humans with RNA viruses and other pathogens.- This behavior of Wolbachia, along with the increasing resistance of many arthropods to insecticides, has led to considerable research aimed at using Wolbachia as a biological control for insect-borne diseases., Research efforts have particularly focused on mosquito-borne diseases including malaria, dengue fever, West Nile fever, lymphatic filariasis, which become a significant concern to global human health and cause the mortality and morbidity of hundreds of thousands of people annually.- A large range of insect hosts can be naturally infected with Wolbachia, including four mosquito species, Culex pipiens,Culex quinquefasciatus, Aedes fluviatilis, and Aedes albopictus, such an important vector of dengue, arguable the most important arboviral diseases of humans, leading to tens of thousands of deaths globally each year.

Recent studies have revealed that genetic fragments, ranging in size from single genes to even entire genome, have horizontally transferred from Wolbachia to their insect hosts as illustrated below. Genes originating from Wolbachia were identified and located on the X chromosome of the adzuki bean beetle, Callosobruchus chinensis. Hotopp et al., meanwhile, found nearly the entire Wolbachia genome in Drosophila ananassae Hawaii, while much smaller fragments were found in the other three insects and four nematode species. However, these horizontally transferred genes were all found during tetracycline treatment and their transcription levels were far lower than those of the control gene (act5C) in their recipient, suggesting they could not be expressed or were potentially nonfunctional. Therefore, the mechanism and process of evolution of Wolbachia mediated HGT remains poorly understood. A natural gene-exchange model is required to more fully understand the processes of HGT evolution.

C6/36 cell line was derived from Ae. albopictus which can be naturally infected with both Wolbachia wAlbA and wAlbB strains. However, it is accepted that Ae. albopictus C6/36 cell lines lack Wolbachia endosymbionts (based on professor Scott O’Neill’s personal communication). In this study, we report the discovery of a gene WP0273(C6/36) within Wolbachia-uninfected C6/36 cells of Ae. albopictus and it is highly similar to the gene originating from Wolbachia of Cx. quinquefasciatus Pel wPip strain (the putative transcriptional regulator, WP0273). We further revealed the high transcription level of the horizontally transferred gene WP0273(C6/36) and demonstrated that WP0273(C6/36) encodes a protein in the host cell. Taken together, these findings strongly suggest that this represents a natural HGT event from Wolbachia to Ae. albopictus C6/36 cell line, which is involved in a particular functional role.

Results

The expression of Wolbachia gene in the Aedes albopictus C6/36 cell line

Protein-protein interactions (PPI) are important for the majority of biological functions. We have found that ribosomal protein L39 (RPL39) of Culex pipiens pallens is a deltamethrin resistance-associated protein,, to explore whether there are some proteins interacting with RPL39 and participate the mechnism of deltamethrin resistance in Cx. Pipiens pallens, we fused the protein RPL39 with tandem affinity peptides, expressed it in Ae. albopictus C6/36 cells and then co-purified the binding proteins through two affinity steps. Surprisingly, our results showed for the first time, a domain of 97 amino acids that is the component of the putative transcriptional regulator of Wolbachia endosymbiont of Culex quinquefasciatus Pel (YP 001975078.1) was 87% identical by mass spectrometry (Fig. 1A). Additional, we demonstrated that WP0273(C6/36) protein binds to RPL39 protein in a His pull-down assay (Fig. 1B).

Figure 1. The interaction between WP0273(C6/36) and RPL39. (A) Partial amino acid sequence of WP0273, the residues identified by mass spectrometry are shown in red. (B) Pull-down with His or His-WP0273(C6/36) proteins with GST-RPL39 protein, followed by western blotting.

Specific PCR detection of Wolbachia WP0273(C6/36) gene

To confirm the absence of Wolbachia in Ae. albopictus C6/36 cell lines, PCR detection of Wolbachia was conducted using the wsp gene, with the Wolbachia infected Ae. albopictus strains as a positive control, we amplified the fragment of expected size in the DNA samples of Ae. albopictus but did not in the C6/36 cells. The results indicate that Wolbachia is not present in the C6/36 cell line, and the presence of WP0237(C6/36) in this cell line probably results from an HGT event.

Specific amplification of the WP0237 gene in C6/36 cells was performed and a DNA segment of 903bp size was detected, which was still amplified in the tetracycline treated C6/36 cells after three generations. DNA sequencing and BLASTN analysis revealed that the target gene WP0273(C6/36) (GenBank accession number KF283997) shared the highest similarity (99% identity) with Wolbachia of Cx. pipiens pallens wPip strain transcriptional regulator WP0273 (GenBank AM999887.1) according to the NCBI database (Fig. 2).

Figure 2. Alignment of nucleic acid sequences WP0273(C6/36) with WP0273. Dashes (−) indicate alignments gaps.

Phylogenetic analysis

To identify the phylogenetic position of WP0273(C6/36), we used the nuclear acid sequence of WP0273, as well as transcriptional regulators of Wolbachia of mosquito and Drosophila hosts, and choose the transcriptional regulator of Wolbachia of Brugia malayi as the outgroup sequence, to construct phylogenetic tree using MEGA5.1. The earliest branching sequences were all from Wolbachia strains infecting in mosquito and Drosophila. The phylogenetic distances in these trees suggested that WP0273(C6/36) was derived from endosymbiont Wolbachia (Fig. 3). This indicates that the WP0273(C6/36) gene found in the mosquito genome cannot be explained by sequence conservation, and was rather inserted into the mosquito genome through HGT.

Figure 3. Phylogenetic relationship of WP0273(C6/36) gene and homologous sequences. A total of 6 genes, including transcriptional regulators of infecting various hosts Wolbachia were analyzed. The same topology was produced by both neighbor-joining (NJ) and minimum-evolution (ME) analyses, NJ bootstrap values were given above branches and ME bootstrap values were given below.

Estimating the transcriptional level of the horizontally transferred gene WP0273(C6/36)

Quantitative PCR analysis was conducted to assess the transcriptional level of WP0273(C6/36) relative to a control gene, act5C, which has been shown to be highly transcribed in the Drosophila genome and thus offers an effective comparison for assess whether horizontally transferred genes are likely to be functional.,, Our results showed that the transcriptional level of WP0273(C6/36) was 1.7 × 103 times lower than that of act5C of C6/36 cells (Fig. 4). This value is significantly higher than the transcriptional level of most horizontally transferred genes from Wolbachia to insect hosts recorded in previous studies (estimated to be 104–107 lower than the control). This result indicates clearly that WP0273(C6/36) was transcribed.

Figure 4. Transcript levels of WP0273(C6/36) genes in Ae. albopictus C6/36 cell line, relative to act5C gene transcription. WP0273(C6/36) and act5C genes expression in Aedes albopictus C6/36 cell lines were conducted by quantitative PCR and normalized with β-actin. Results are expressed as the mean ± SEM. The values shown above are the average of four different assays on independent experiments. Error bar indicate standard error.

Demonstrating the protein expression of WP0273(C6/36) in the host cells

We constructed the prokaryotic expression vector via recombination of a pET-32a plasmid and the open reading frame of WP0273(C6/36), and expressed the protein for polyclonal antibody preparation. Western immunoblotting was conducted to demonstrate the expression of protein WP0273(C6/36) with non-blood-sucking female and Wolbachia-free Anopheles sinensis mosquitoes as a negative control. The WP0273(C6/36) gene translated into the protein in the environment of mosquito host cells. Conversely, the WP0273 gene did not express in the Anopheles sinensis (Fig. 5).

Figure 5. Identification of WP0273(C6/36) protein expression. The expression of WP0273(C6/36) protein was demonstrated using western blot. A, Anopheles sinensis; B, Aedes albopictus C6/36 cells. β-actin was chosen as the internal control immunoblotted by anti-β-actin and polyclonal rabbit anti-WP0273(C6/36) (Abgent) was used to detect WP0273(C6/36) protein.

Analysis of protein sequence and prediction of protein function

We constructed the secondary structure and a 3D model of the WP0273(C6/36) protein. The detailed template information of all alignment coverage suggested that the WP0273(C6/36) protein was very likely a transcriptional regulator or a DNA-binding protein. Subsequently, motif scan results revealed that the whole sequence contained two helix-turn-helix motifs, which were composed of two α-helix motifs joined by a short strand of amino acids and a major structure capable of binding DNA and which can regulate gene expression. We also found a structure highly similar to the Ankyrin repeat domain (ANK), which could mediate protein–protein interaction and has previously been found in some arthropod-infecting Wolbachia.

Discussion

Our results strongely support the conclusion that WP0273(C6/36), which is highly similar to the gene WP0273 originating from Wolbachia, has horizontally transferred from the symbiotic bacteria Wolbachia to the Ae. albopictus C6/36 cell line. C6/36 cell line was isolated from Ae. albopictus (Singh), we speculate this case of HGT event has occurred in the original Ae. albopictus (Singh) before the C6/36 cell line constructed, so we couldn’t detect the WP0273(C6/36) in Wolbachia-free Ae. albopictus strains (China). Most of these genes horizontally transferred between Wolbachia and their hosts (including mosquitoes, Drosophila, filarial nematodes, and beetles) appear to be nonfunctional in the recipient genome. These transferred genes are commonly reported to be transcriptionally inactive, or to contain stop codons or other structural disruptions such as frameshifts and retroelements insertions. In contrast, this study demonstrated that the horizontally transferred gene, WP0273(C6/36), is transcriptionally active in the C6/36 cell line (as measured by comparison of transcriptional levels with a control gene, act5C). Our results indicate that WP0273(C6/36) is translated into a functional protein which participates in protein-protein interaction. In addition, we confirmed (by western blotting analysis) that the endosymbiontic gene WP0273(C6/36) can be expressed into a protein in eukaryotic cells.

In general, horizontally transferred genes must meet three requirements to attain a novel functionality. First, the donor DNA has to be delivered into the recipient cell. Second, the target sequences must integrate into the host’s genome. Third, these genes could be expressed in host organisms or cells. Obviously, the horizontally transferred gene WP0273(C6/36) has met all the needs described above, suggesting WP0273(C6/36) has obtained a new function. However, what the new capacity of WP0273(C6/36) is and which role it plays in the host cell remains unknown. Further studies are now required to determine whether WP0273(C6/36) acts to regulate transcription or engenders other effects.

Based on our results examining protein structure and motif analysis, we hypothesize that WP0273(C6/36) probably regulates gene expression in C6/36 cells via DNA-binding sites. We will aim to test this hypothesis in future work using Chromatin Immunoprecipitation (Chip) and EMSA-electrophoretic mobility shift assays. Ankyrin repeats consist of 33 amino-acid sequences and the first ANK-containing proteins to be characterized were the yeast cell cycle regulator Swi6/Cdc10 and the Drosophila cell signaling Notch protein.- ANK abounds in arthropod-infecting Wolbachia (for example, 54 in wPip, 23 in wMel, and 34 in wAna). Ank may play an interesting role in the relationship between host and Wolbachia because ANK is one of the most common protein-protein interaction motifs in nature, involved in many physiological processes including cell signaling, apoptosis, and cell cycle control. Previous results have showed that the protein WP0273(C6/36) could interact with the protein RPL39, indicating it is worthy of further study as there are any other features of WP0273(C6/36).

HGT is a crucial driving force in bacterial evolution, with a remarkable impact on pathogenicity and antibiotic resistance of human associated microbes., Transfer of the vanA gene from an E. faecium isolate of animal origin to an E. faecium isolate of human origin can occur in the intestines of humans. “Pathogenicity islands” horizontally acquired were major contributors to the virulence of many pathogenic bacteria., Successful adaptive HGT is traditionally considered beneficial either to hosts or to the transferred genes. However, the mechanisms by which Wolbachia is able to manipulate its insect host (to affect reproductive characters, disrupt pathogen infection, or shorten hosts lifespan) is still poorly understood. One hypothesis posits that some Wolbachia strains interfere with a range of human pathogens in a manner correlated with the innate immune response in the insect., Here, we propose that interaction of Wolbachia-host and the regulation of insect behavior by Wolbachia are mediated by the effect of horizontally transferred genes that can obtain a new functionin the recipient.

It is unknown whether there are other cases of HGT between Wolbachia and Ae. albopictus C6/36 cell line. Elucidating this will require a large-scale exhaustive search using PCR and specific quantitative PCR detection in the C6/36 cell line. Should additional transcriptionally active genes be detected, then the Ae. albopictus C6/36 cell line will offer an ideal model to study HGT mechanisms and evolutionary processes. It will nevertheless be necessary to identify the biological function of any further transferred genes identified, including WP0273(C6/36).

The results of this study provide strong evidences that WP0273(C6/36) discovered in Ae. albopictus C6/36 cells is an event of HGT and it appears to be functional. Importantly, our results highlight a novel mechanism of Wolbachia-host interaction and establish a basis for the further study of HGT between endosymbionts and eukaryotes.

Materials and Methods

Cell culture and mosquito strains

Aedes albopictus C6/36 cells were obtained from the China Center for Type Culture Collection (Wuhan, China). Cells were maintained in DMEM/High Glucose media (Hyclone) in a 5% CO2-humidified incubator at 28 °C. Aedes albopictus were provided by Disease Prevention and Control Center of Nanjing Military Region. Non-blood-sucking female Anopheles sinensis were supplied by Jiangsu Institute of Parasitic Diseases (Wuxi, China).

Tandem affinity purification and mass spectrometry

C6/36 cells were stably transfected with the pIB/V5-GS vector (pIB/V5-GS vector was introduced with ORF of RPL39 and GS tag sequence in-frame fusion which was inserted between EcoRV and Not I sites). Cells were then lysed and the purified eluted proteins separated on a SDS-PAGE gel and stained with silver. The differentially expressed protein was then analyzed by both MALDI-TOF mass spectrometry and tandem TOF/TOF mass spectrometry performed on a time-of-flight Ultraflex II mass spectrometer (Biflex).

Plasmid Construction

Rosetta (DE3) strain of Escherichia coli was used for the protein expression. pGEX-6P-1 GST expression vector was used to express GST-RPL39 fusion protein, the ORF of RPL39 was inserted between BamHI and EcoRI sites. pET32a vector was used to express His-WP0273(C6/36) fusion protein, the ORF of WP0273(C6/36) was inserted between EcoRI and NotI sites.

In vitro His pull-down assay

A total of 1ug purified His or His-WP0273(C6/36) proteins was applied to His resins and incubated for 1 h at 4 °C. The resins were washed and mixed with purified GST-RPL39 protein and were incubated for 4 h at 4 °C. After washing, bound proteins were eluted and subjected to SDS-PAGE, followed by western blotting.

DNA extraction, RNA extraction, and cDNA synthesis

Total mosquito C6/36 cells DNA was extracted using TAKARA MiniBEST Universal Genomic DNA Extraction Kit Ver4.0 (TAKARA) according to the manufacturer’s protocol. Total RNA from mosquitoes and cells was extracted using TriZol Reagent (Invitrogen) according to the manufacturer’s protocol. The cDNA was reverse transcribed from the RNA using the SuperScript® VILO™ cDNA Synthesis kit (Invitrogen) according to the manufacturer's instructions.

PCR detection of Wolbachia genes

PCR detection of Wolbachia genes was conducted using the primers listed in Table 1, PCR reactions were performed using TAKARA’s Ex TaqVersion 2.0 DNA Polymerase (TAKARA). PCR reactions were composed as follows: Total DNA 200 ng, 25 ul Premix Ex Taq, 1 ul of each 20 uM primer, and ddH2O was added to bring up the total volume to 50 ul. The amplification of wsp gene was performed as follows: 94 °C for 5 min, 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 60 s, followed by a final extension at 72 °C for 7 min. The amplification of WP0273(C6/36) gene was performed as follows: 94 °C for 5 min, 35 cycles of 94 °C for 30 s, 53 °C for 30 s, and 72 °C for 90 s, followed by a final extension at 72 °C for 7 min. The products were separated by 1% agarose gel electrophoresis and purified using TAKARA MiniBEST Agarose Gel DNA Extraction Kit Ver3.0. The purified PCR products were sequenced by the Beijing Genomics Institute (Shanghai, China).

Table 1. Primers used for detection of Wolbachia and WP0273(C6/36)

	Primer sequence (5′-3′)	Product size (bp)	Reference
wsp	F-TGGTCCAATAAGTGAAGAAACR-AAAAATTAAACGCTACTCCA	590–632	23, 24
WP0273(C6/36)	F-ATGTTTGTTTCTGTAAGAGATATTR-TCAATTTTTACACTCATCAGC

Quantitative PCR analysis

Quantitative PCR was performed using the ABI PRISM 7300 (Applied Biosystems) with LightCycler FastStart DNA Master SYBR Green I (Roche) as the detection dye, according to the manufacturer’s instructions. The primers used were listed in Table 2. β-actin was used as the internal control. The relative gene expression level was calculated from the threshold cycle (Ct) value of each reaction.

Table 2. Primers used for quantitative PCR

	Primer sequence (5′-3′)
WP0273(C6/36)	F-GGAGGCAAGATTAGAAGTAGCAAAAR-GAATCAGTGCAGACTTCTTTCTCTGT
act5C	F-ATCGTACGAACTTCCCGATGR-ACAGATCCTTTCGGATGTCG
β-actin	F-CCACCATGTACCCAGGAATCR-CACCGATCCAGACGGAGTAT

Protein extraction from cells and mosquitoes

The culture media was removed from the cell culture plate, washed twice with PBS, then 300 µL RIPA buffer, and 3 µL PMSF was added. Cells were harvested and then lysed. Collected cells were placed in a centrifuge tube on ice for 30 min and then in a water bath at 100 °C with loading buffer for 5 min. This solution was centrifuged at 12,000 rpm for 5 min and the supernatant then transferred into a new tube and stored at −20 °C.

Five non-blood-sucking female Anopheles sinensis mosquitoes were placed in a centrifuge tube to which was added 300 µL RIPA buffer and 3 µL PMSF. The mosquitoes were ground completely on ice and left to stand for 30 min. The supernatant was transferred into a new tube, placed in a water bath at 100 °C with loading buffer for 5 min, then centrifuged at 12,000 rpm for 5 min, and stored at −20 °C. These protein extracts were used to perform Western immunoblotting.

Phylogenetic Analysis

Phylogenetic analyses were conducted by the maximum-likelihood (ML), neighbor-joining (NJ), and minimum-evolution (ME) method by using MEGA 5.1.

Analysis and prediction of protein sequence and structure

The 3D structure of the protein was constructed using Phyre2 server (protein homology/analogy recognition engine). Protein function was predicted using the Phyre2 server SMART software (http://smart.embl-heidelberg.de/) and Myhits Motif Scan (http://myhits.isb-sib.ch/cgi-bin/motif_scan).,