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Localization of Wolbachia-like gene transcripts and peptides in adult Onchocerca flexuosa worms indicates tissue specific expression
© McNulty et al.; licensee BioMed Central Ltd. 2013
Received: 5 November 2012
Accepted: 20 December 2012
Published: 2 January 2013
Most filarial species in the genus Onchocerca depend on Wolbachia endobacteria to successfully carry out their life cycle. O. flexuosa is a Wolbachia-free species, but its genome contains Wolbachia-like sequences presumably obtained from Wolbachia via horizontal gene transfer. Proteogenomic studies have shown that many of these Wolbachia-like sequences are expressed in adult worms.
Six Wolbachia-like sequences in O. flexuosa were chosen for further study based on their sequence conservation with Wolbachia genes, length of predicted open reading frames, and expression at the RNA and/or protein levels. In situ hybridization and immunohistochemical labeling were used to localize Wolbachia-like transcripts and peptides in adult worm tissues.
RNA probes representing three of the six target sequences produced hybridization signals in worm tissues. These probes bound to transcripts in the intestine and lateral chords of both sexes, in the hypodermis, median chords and uteri in females, and in sperm precursor cells in males. Antibodies raised to three peptides corresponding to these transcripts bound to specific bands in a soluble extract of adult O. flexuosa by Western blot that were not labeled by control antibodies in pre-immune serum. Two of the three antibodies produced labeling patterns in adult worm sections that were similar to those of the RNA probes, while the third produced a different pattern.
A subset of the Wolbachia-like sequences present in the genome of the Wolbachia-free filarial species O. flexuosa are transcribed in tissues where Wolbachia reside in infected filarial species. Some of the peptides and/or proteins derived from these transcripts appear to be concentrated in the same tissues while others may be exported to other regions of the worm. These results suggest that horizontally transferred Wolbachia genes and gene products may replicate important Wolbachia functions in uninfected filarial worms.
Filarial nematodes comprise a superfamily of parasitic worms that infect a wide array of hosts, including humans. Three filarial species (Onchocerca volvulus, Wuchereria bancrofti, and Brugia malayi) are important human pathogens in the tropics, and many other filarial species infect wild or domestic animals throughout the world. One feature shared by many filarial pathogens is their association with Wolbachia endobacteria. Prior studies have shown that Wolbachia play important roles in worm growth, molting, reproduction, and pathogenesis [1–4].
Filarial nematodes have been divided into eight subfamilies based on classical parasitological criteria. Two of these subfamilies, the Onchocercinae and Dirofilariinae, appear to be dominated by Wolbachia-dependent species [5–7]. The abundance of Wolbachia-dependent species relative to Wolbachia-free species and the congruencies between the phylogenetic lineages of Wolbachia and their filarial hosts suggest that Wolbachia entered the filarial lineage prior to the differentiation of the Onchocercinae and Dirofilariinae [5–7]. Recent studies have shown that two Wolbachia-free filarial species, Onchocerca flexuosa (subfamily: Onchocercinae) and Acanthocheilonema viteae (subfamily: Dirofilariinae), contain Wolbachia-like DNA sequences in their nuclear genomes, indicating that these species may have been associated with Wolbachia in the ancient past . Since O. flexuosa and A. viteae are relatively distantly related [6, 9], we expect that this trend will prove consistent among other Wolbachia-free onchocercids and dirofilariids.
Our previous surveys of the genome and transcriptome of O. flexuosa identified sequence fragments with similarity to 178 different Wolbachia genes [8, 10]. qRT-PCR reactions and partial transcriptome sequencing indicated that many of these Wolbachia-like sequences are transcribed, and a mass spectrometry study with follow-up immunohistology and Western blot studies indicated that at least two Wolbachia-like sequences were translated into Wolbachia-like peptides . Wolbachia are known to be proficient at transferring genetic material to host cells and many Wolbachia-like sequences are present in the genomes of Wolbachia-dependent filarial worms; however, the Wolbachia-like sequences found in the genomes of dependent species like B. malayi are thought to be degenerate and non-functional . Retention and expression of Wolbachia-like sequences in O. flexuosa suggests that they may have essential roles in the biology of Wolbachia-free filarial worms.
Prior work has shown that Wolbachia endobacteria are restricted to specific tissues in filarial nematodes [7, 12–16]. In early development, the vertically transmitted bacteria that were present in the fertilized oocyte segregate to cells that give rise to the hypodermal lateral chords . Wolbachia from the lateral chords then invade the ovaries and testis prior to sexual maturation [12, 17]. This pattern of localization (i.e., lateral chords and reproductive tissues) may be critical to Wolbachia’s function as a mutualist and to its role in development and reproduction. Therefore, localization studies were performed to gain insight into the possible function(s) of transferred and retained Wolbachia-like sequences in the Wolbachia- free filarial species O. flexuosa.
The present study used in situ hybridization to localize expression of Wolbachia-like transcripts in adult O. flexuosa and immunohistochemical staining to localize peptides corresponding to these sequences. Thus far, all of the RNA probes that produce a signal in adult tissues stain the intestine and lateral chords in both sexes, the hypodermis, median chords, and uteri of females, and developing sperm in males. Two of the three Wolbachia-like peptides were identified in the same tissues and in developing embryos within the females. The third Wolbachia-like peptide was found in similar tissues and in the basal areas of somatic muscles.
O. flexuosa nodules were cut from the skins of freshly-shot European red deer (Cervus elaphus) following community hunts in northern Germany (Schleswig-Holstein) as previously described . Adult B. malayi were obtained from the Filariasis Research Reagent Resource Center . Worms to be used for RNA or DNA isolation were stored in TRIzol (Invitrogen, Carlsbad, CA, USA) or 1x phosphate buffered saline, respectively, at -80°C. O. flexuosa nodules intended for histological examination were fixed in 4% buffered formalin, embedded in paraffin and sectioned at 5μm thickness according to standard histological procedure.
Nucleic acid isolation
DNA was isolated using an E.Z.N.A Tissue DNA Kit (Omega Bio-Tek, Norcross, GA, USA) according to the manufacturer’s suggested protocol. Total RNA was isolated by homogenization in TRIzol (Invitrogen) and organic extraction with 1-bromo-3-chloropropane and purified using an RNeasy Mini Kit with the optional on-column DNase digest (Qiagen, Valencia, CA, USA). A second DNase treatment was performed using the Turbo DNA-free kit (Invitrogen). cDNA was synthesized using qScript cDNA SuperMix (Quanta, Gaithersburg, MD, USA) and tested for DNA contamination by PCR with intron-spanning primer sets as previously described .
The O. flexuosa adult transcriptome was sequenced using Roche/454 Titanium technology and assembled using the Newber v2.5 assembler as described previously . Sequences are available from the Genbank transcriptome shotgun assembly database (BioProject number 62565, accession numbers JI459010-JI484230) and from Nematode.net . Wolbachia-like sequences were identified from O. flexuosa transcripts by BLASTX search against the non-redundant protein database . Measurements of sequence conservation were based on the e-values of hits obtained in this blast search. The relative expression of each O. flexuosa isogroup (unique sequence locus) was estimated based on the number of reads included in each assembled isogroup normalized to the total length of the isogroup (the summed length of all contigs included in the isogroup) (Additional file 1: Table S1). Putative Wolbachia-like peptides were identified by blasting six-frame translations of O. flexuosa transcripts against the non-redundant protein database (>50% sequence identity shared with Wolbachia protein, bit score >35).
RNA probe construction and in situ hybridization
Probes were constructed as previously described . Briefly, 300-600 bp target sequences were PCR amplified from cDNA (see Additional file 2: Table S2 for primers) and cloned using the TOPO TA Cloning Kit Dual Promoter (Invitrogen). Plasmids were purified with the QIAprep Spin Miniprep Kit (Qiagen) and linearized by restriction digest with either EcoRV or BamHI. Following purification of linearized plasmid (QIAquick PCR Purification Kit, Qiagen), biotinlyated antisense RNA probes and sense controls were transcribed from the plasmid template using the MEGAscript SP6 and T7 in vitro transcription kits (Invitrogen) with biotinylated NTPs (Roche Diagnostics, Indianapolis, IN, USA). The final biotinlyated RNA probes were purified and concentrated by ethanol precipitation and stored in 1 × tris-EDTA buffer, pH 8.0.
Slides from various nodules were tested with control probes to determine the quality of RNA preservation. This step is necessary due to the short lifespan of O. flexuosa, as studies have indicated that 20% of nodules collected (even from very young deer) contain dead worms . In situ hybridizations showed inconsistent results in older, mf producing worms. This may have been due to reduced penetration of the fixative through larger, tougher nodules or due to reduced transcription rates in the older worms. Slides prepared from small, soft nodules containing young adult worms showed better RNA integrity. Therefore, we chose to focus on young worms in our in situ hybridization studies.
In situ hybridizations using formalin-fixed, paraffin-embedded specimens were carried out as previously described . Briefly, slides were hybridized with 1μg/mL of RNA probe in hybridization buffer (50% formamide, 5x SSC, 0.3mg/mL yeast tRNA, 100μg/mL heparin, 1x Denhardt’s reagent, 0.1% CHAPS and 5mM EDTA) overnight at 60°C. Stringency washes (60°C for 30m) were carried out using reagents from the “In situ Hybridization Detection System” (Dako, Carpinteria, CA, USA). The same kit was used for colorimetric detection according to the manufacturer’s suggested protocol. Slides were viewed using an Olympus-BX40 microscope (Olympus, Tokyo, Japan) and photographed with an Infinity2 digital microscope camera using Infinity Capture software (Lumenera, Ottowa, Ontario, Canada). For fluorescent detection, washed sections were incubated with 5μg/mL streptavidin Alexa Fluor 488 (Invitrogen) in 1x phosphate buffered saline with 0.1% bovine serum albumin in the dark, at room temperature. After 20 minutes, 5μg/mL wheat germ aggutinin Alexa Fluor 633 was applied to the slide to highlight cell membranes, and the incubation was allowed to proceed for 10 more minutes. Finally, sections were rinsed in 1x tris buffered saline and cover slips were mounted with ProLong Gold Antifade Reagent with DAPI (Invitrogen). Fluorescent labeling was viewed with a Zeiss Axioskop 2 Mot Plus fluorescence microscope equipped with an Axiocam MRm monochrome camera, and images were captured using Axiovision 4.6 software (Carl Zeiss Inc., Thornwood, NY, USA).
Anti-peptide antibodies were produced and purified by LifeTein LLC (South Plainfield, NJ, USA). Targeted portions of Wolbachia-like peptides were selected based on predicted chemical and immunogenic properties (Additional file 3: Table S3). Target peptides were synthesized and coupled to a keyhole limpit hemocyanin (KLH) carrier protein. Rabbits were bled to collect pre-immune serum and then immunized with the peptide/KLH conjugates. Antibody production was allowed to proceed for 12-15 weeks with 3 booster immunizations. Following the terminal bleed, polyclonal antibodies were affinity purified from serum using the target peptide (without the KLH carrier) and tested by ELISA prior to use. Polyclonal antibodies against the KLH carrier protein were produced and purified in the same manner. Total IgG was purified from rabbit pre-immune sera using the Protein A Agarose Kit (KPL, Gaithersburg, MD, USA).
Binding of antibodies to Wolbachia-like peptides present in O. flexuosa antigen extract by Western blot
Western blots were performed as previously described . Briefly, O. flexuosa total worm homogenate separated by SDS-PAGE gel electrophoresis and transferred to a nitrocellulose membrane. Blot strips were probed with peptide antibodies (2.5μg/mL for antibodies against the peptides from isotig12596 and isotig21532, 5μg/mL for antibodies against the HlyD peptide) and purified IgG from the corresponding rabbits’ pre-immune sera (5μg/mL from rabbits used to produce antibodies against the peptides from isotig12596 and isotig21532, 10μg/mL from the rabbit used to produce antibodies against the HlyD peptide) in 1x phosphate buffered saline with 0.5% Tween (PBS/T) overnight at 4°C and washed with PBS/T at room temperature. Anti-rabbit IgG(Fc) AP conjugate (Promega, Sunnyvale, CA, USA) was diluted 1:3,500 in PBS/T and applied to the strips for 1h at 37°C. Strips were again washed with PBS/T and developed using NBT/BCIP substrate (Promega).
Various dilutions of primary antibodies were tested in order to optimize signal/background. Antibodies against peptides from isotig12596, isotig21532, HlyD and KLH (negative control) were used at 9.7μg/mL, 5.7μg/mL, 7.4μg/mL, and 1.4 μg/mL, respectively. Visualization was mostly performed with alkaline phosphatase anti-alkaline phosphatase reagents (Dako) as previously described [12, 22], and slides were viewed using an Olympus-BX40 microscope (Olympus) and photographed with an Infinity2 digital microscope camera using Infinity Capture software (Lumenera). FITC labeled goat anti-mouse IgG (1:300; Sigma, St. Louis, MO, USA) was used as secondary antibodies for immunofluorescent labeling, and DAPI and wheat germ agglutinin Alexa Fluor 633 conjugate were used to visualize DNA and cell membranes, respectively (Invitrogen). Fluorescent labeling was viewed with a Zeiss Axioskop 2 Mot Plus fluorescence microscope equipped with an Axiocam MRm monochrome camera, and images were captured using Axiovision 4.6 software (Carl Zeiss Inc.).
Results and discussion
Ranked lists of Onchocerca flexuosa Wolbachia -like transcripts with highest expression levels, highest sequence conservation with Wolbachia proteins, and longest open reading frames
Most abundantly expressed
Greatest sequence conservation withWolbachiaproteins
Longest open reading frames
Wolbachia -like sequences from the Onchocerca flexuosa transcriptome chosen for localization studies
Accession of best BLAST match
Wolbachiastrain of best BLAST match
Annotation of best BLAST match
hypothetical protein OW4-D
Foster et al.  reported that the Wolbachia endosymbionts of B. malayi are capable of de novo nucleotide synthesis, and they suggested that the bacteria may play a role in metabolic provisioning. A recent analysis of the genome of the Wolbachia endosymbiont of Onchocerca ochengi further supported this hypothesis, suggesting that Wolbachia may supplement the host nucleotide pool . It is interesting that enzymes related to energy metabolism and purine synthesis were identified using our selection algorithm, which did not include potential enzymatic function as a selection criterion. Future work will be required to determine whether these sequences encode enzymes that are functional in O. flexuosa.
In situ hybridization
RNA probes corresponding to the five sequences of interest selected from the O. flexuosa transcriptome and Wolbachia HlyD were constructed and used in in situ hybridization studies. Because no genomic or transcriptomic sequence related to Wolbachia HlyD has been found in O. flexuosa, the HlyD probe was amplified from the Wolbachia endosymbiont of B. malayi. Probes against isotig12596 and Wolbachia HlyD produced strong signals in O. flexuosa tissues (Figure 2 and Additional file 4: Figure S1, respectively). The probe against O. flexuosa isotig21532 produced a much weaker signal (Additional file 5: Figure S2), and the probes against isotig14485, isotig12363 and contig10287 produced no signal at all. This was surprising, because expression levels for these transcripts were not estimated to be significantly lower than that of isotig12596 (Additional file 1: Table S1). It could be that these three sequences are expressed under conditions that are not represented in the particular worm specimens we examined or that some Wolbachia-like sequences are expressed at a low level throughout the body rather than concentrating in specific tissues, thus making them difficult to detect using this method. It is also possible that technical issues with the RNA probes led to this result.
A shared expression pattern can be taken as evidence that a group of genes is under the control of either the same or functionally similar promoters or other regulatory elements. Approximately 15% of the genes encoded by B. malayi are organized into operons [31, 32]. Since the complete genome of O. flexuosa has not been sequenced, we cannot rule out the possibility that some of the Wolbachia-like sequences are organized in this way, giving rise to a characteristic pattern of expression. However, clusters of Wolbachia-like sequences were not identified in genomic surveys of O. flexuosa, so it is unlikely that they are all organized into operons.
Wolbachia are commonly detected in the lateral chords, embryos, developing (but not mature) sperm, and sometimes in the intestine of infected filarial nematodes [7, 12]. Interestingly, the expression pattern of Wolbachia-like sequences in O. flexuosa mimics the distribution of Wolbachia in infected species. This may indicate that some of the same tissues (e.g., lateral chords, reproductive organs, intestine etc.) that harbor Wolbachia in infected species produce Wolbachia-related products in Wolbachia-independent species.
Western blot results
It is not possible to estimate the masses of the O. flexuosa proteins based on our transcriptome data due to heavy fragmentation. The masses of the top blastx matches to isotigs12596 (aminopeptidase P from the Wolbachia endosymbiont of Muscidifurax uniraptor) and 21532 (a hypothetical protein from the Wolbachia endosymbiont of Onchocerca volvulus) and the HlyD protein are estimated to be approximately 58 kDa, 83 kDa, and 56 kDa, respectively, based on their reported amino acid sequences. The band detected by the antibodies to the predicted peptide from isotig12596 was similar in size to the corresponding Wolbachia protein (50 kDa vs. 58 kDa), as was one of the bands detected by the antibodies to the HlyD peptide (48 kDa vs. 56 kDa). The bands detected by the antibodies to the predicted peptide from isotig21532 were much larger than the putative Wolbachia homolog (120 and 260 kDa vs. 83 kDa), perhaps due to the incorporation of extra domains from the O. flexuosa genome. Of course, the full amino acid sequences of the O. flexuosa proteins detected by these antibodies are not known at this time.
It is not surprising that Wolbachia-like proteins were detected in tissues or sub-cellular compartments where the corresponding mRNA was not found (e.g., somatic muscles, uterus of mature females, nucleus vs. cytoplasm, etc). Many proteins are produced in a specific tissue (or subset of tissues) but have important functional roles in other regions of the body. In fact, Wolbachia gene products are believed to be exported from the endobacteria to interact with host cells, and some of them have been detected among the excretory/secretory products of Wolbachia-dependent worms .
Different patterns of localization observed for different putative Wolbachia-like peptides could indicate that the proteins containing these peptides are responsible for different functions in the worms. Darby et al.  proposed a dual role for Wolbachia in filarial biology based on their recent genomic analysis of the Wolbachia endosymbiont of the cattle parasite O. ochengi; they speculated that Wolbachia may engage in metabolic provisioning and also serve as a diversion for the vertebrate host immune system. If we hypothesize that Wolbachia-like sequences in O. flexuosa function as a substitute for the endosymbiont, the localization patterns of proteins with Wolbachia-like sequences may provide clues as to which of these two tasks they are most likely to perform. For example, proteins present in somatic muscles and reproductive tissues may be involved in worm metabolism, perhaps granting O. flexuosa novel biochemical capabilities compared to other, Wolbachia-dependent filarial species (e.g., de novo heme, nucleotide or riboflavin synthesis [29, 30]). Likewise, proteins with transmembrane domains or secretion peptides that are present in the hypodermis or lateral chords may be released from the worm to distract the host’s immune system by promoting an ineffective Th1 type immune response . Further work will be needed to define the roles of these proteins in the biology of Wolbachia-free filarial parasites like O. flexuosa.
The results of this study further support the notion that Wolbachia- like sequences are expressed in a Wolbachia-free filarial parasite at both the RNA and protein levels; this expression is highly regulated with regard to tissue and parasite stage. Thus far, all of the Wolbachia-like RNA probes that have successfully labeled O. flexuosa tissues have produced the same labeling pattern: intestine and lateral chords of both sexes, the hypodermis, median chords and uteri of females, and sperm precursors in the male testis. Polyclonal antibodies against predicted Wolbachia-like peptides bound to specific bands in Western blots performed with O. flexuosa adult worm lysate and to specific tissues in fixed and sectioned parasite specimens. Wolbachia-like peptides were sometimes found in the same tissues where the corresponding RNAs were produced and sometimes in other locations. The localization of these proteins within the worm may provide clues regarding their functions by suggesting a role in worm metabolism or in host immune modulation.
The authors would like to thank Katherine Mann for technical assistance and C. Dohr and E. Bach for organizing the deer hunt and granting us access to the hunted deer. This work was supported by a grant from the Barnes Jewish Hospital Foundation.
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