Wolbachia endosymbionts of Onchocerca volvulus express a putative periplasmic HtrA-type serine protease

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Microbes and Infection 6 (2004) 141–149 www.elsevier.com/locate/micinf

Original article

Wolbachia endosymbionts of Onchocerca volvulus express a putative periplasmic HtrA-type serine protease> Abbas Jolodar a, Peter Fischer b, Dietrich W. Büttner b, Norbert W. Brattig a,* a

b

Tropical Medicine Section, Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Str. 74, 20359 Hamburg, Germany Department of Helminthology, Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Str. 74, 20359 Hamburg, Germany Received 30 July 2003; accepted 13 October 2003

Abstract Wolbachia are intracellular bacteria of many filarial nematodes. A mutualistic interaction between the endobacteria and the filarial host is likely, because the clearance of Wolbachia by tetracycline leads to the obstruction of embryogenesis and larval development. Databases were searched for exported molecules to identify candidates involved in this mutualism. Fragments of a Wolbachia serine protease from the human filarial parasite Onchocerca volvulus were obtained (Wol-Ov-HtrA) by the use of a PCR technique and primers based on the Rickettsia prowazekii genome. The deduced amino acid sequence exhibited 87% and 81% identity to the homologous Wolbachia proteases identified from Brugia malayi and Drosophila melanogaster, respectively. The full-length cDNA encodes 494 amino acids with a calculated mass of 54 kDa. Three characteristic features, (i) a catalytic triad of serine proteases, (ii) two PDZ domains and (iii) a putative signal peptide, classify the endobacterial protein as a member of the periplasmic HtrA family of proteases known to express chaperone and regulator activity of apoptosis. Using a rabbit antiserum raised against a recombinantly expressed 33-kDa fragment of Wol-Ov-HtrA, strong labelling of the antigen was found associated with endobacteria in hypodermis, oocytes, zygotes, all embryonic stages and microfilariae of O. volvulus. Staining of hypodermal cytoplasm surrounding the endobacteria indicated a possible release of the protein from the Wolbachia. The demonstration of Wol-Ov-HtrA-reactive IgG1 antibodies in sera of O. volvulus-infected persons indicated the exposure to the protein and its recognition by the human immune system. Wol-Ov-HtrA is a candidate for an exported Wolbachia protein that may interact with the filarial host metabolism. © 2003 Elsevier SAS. All rights reserved. Keywords: Filaria; Onchocerca volvulus; Endobacteria; Wolbachia; Serine protease; HtrA protease; Immunolocalisation

1. Introduction The filarial parasite Onchocerca volvulus is the causative agent of onchocerciasis, leading to dermal and lymphatic pathology and to eye disease and blindness (river blindness) [1]. Although the O. volvulus infection can be treated with the microfilaricidal drug ivermectin, there is a need for addi-

Abbreviations: Wol-Ov-HtrA, Wolbachia-O. volvulus HtrA; HtrA, hightemperature requirement A protease; WSP, Wolbachia surface protein; PCR, polymerase chain reaction; ORF, open reading frame; IPTG, isopropyl thio-beta-D-galactoside; APAAP, alkaline phosphatase anti-alkaline phosphatase; ELISA, enzyme-linked immunosorbent assay. > The sequence of Wol-Ov-HtrA has been deposited in the GeneBank database under accession no: AY255127. * Corresponding author. Tel.: +49-40-42818-530; fax: +49-40-42818-309. E-mail address: [email protected] (N.W. Brattig).

© 2003 Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2003.10.013

tional intervention strategies, since ivermectin does not kill the adult filariae. Like many other filariae, O. volvulus harbours abundant intracellular bacteria well-known as residents in a wide range of arthropods [2,3]. These Wolbachia endobacteria are alphaproteobacteria belonging to the order Rickettsiales. Wolbachia were reported to be symbionts in filariae but parasites in most arthropods [2,3]. Although there is increasing knowledge about the phylogeny of Wolbachia, little is known about the interaction between Wolbachia and its filarial host [3,4]. Antibiotic treatment of O. volvulus-infected persons using doxycycline leads to inhibition of embryogenesis [3–5]. The molecular basis for the mutualistic relationship is unkown. Molecules so far identified in Wolbachia include intracellular proteins, i.e., the cell cycle protein ftsZ, heat shock protein 60, aspartate aminotransferase, and a Wolbachia surface protein (WSP) [3,6–8]. However, no endobacterial proteins exported into the filarial host cells have been described yet and

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mediators of the mutualistic relationship between the Wolbachia and the filariae are still unknown. Bacterial HtrA proteases are identified as chaperones and characterised by a catalytic triad of serine proteases, one or more C-terminal PDZ domains and a signal peptide [9,10]. HtrA genes have been implicated in the periplasmic stress response of intracellular bacteria of several genera [10–13]. Mutants lacking HtrA failed to digest misfolded protein in the periplasm [14]. In the present report, a putative periplasmic HtrA-type serine protease of filarial endobacteria that may be involved in protein metabolism or apoptotic processes of the filaria has been identified, cloned and immunolocalised. It may represent a candidate molecule operative in the interaction between the endobacteria and their filarial host.

2. Materials and methods 2.1. Genomic DNA extraction and PCR amplification Purified O. volvulus worm tissue (0.5–1.0 g) isolated from onchocercomas obtained from onchocerciasis patients in Guinea (West Africa) was homogenised in 10 ml of RSB buffer (10 mM Tris–HCl pH 7.4, 10 mM NaCl/25 mM EDTA, SDS 1%) and incubated with DNase-free RNase (20 µg/ml) and in protease K (20 µg/ml) for 1 h at 37 °C. The DNA was extracted twice with an equal volume of phenol/chloroform, and the aqueous phase was then extracted with chloroform. Based on sequence data of the Rickettsia and Drosophila Wolbachia genomes (http://www.tigr.org/tdb/mdb/mdbinprogress.html), primers (WDF: 5′-ATGGCAATAGGTAACCCATTTG and WDR: 5′-CTATTTCTTCAACTTTATCGAAG) were designed to amplify the coding region of the serine protease gene from Wolbachia bacteria of O. volvulus. Based on sequence data of the Brugia Wolbachia genome using the Wolbachia Genome Project database (http://tools.neb.com/wolbachia/ wgspindex.html), two additional primers (WBF: 5′-ATGAAAAGTAAGGTTTTATCTAT and WBR: 5′-TAAACCAAATGGGTTACCTATTGC) were designed to identify the homologous region. PCR amplification was performed using genomic DNA as template with initial denaturation for 5 min at 95 °C, followed by 35 cycles of 45 s at 94 °C, 1 min at 59 °C, and 1 min at 72 °C and, finally, 7 min of incubation at 72 °C in 50 µl PCR SuperMix (Life Technologies, Hamburg, Germany) containing 20 pmol of each primer, deoxynucleotides, 1.5 mM MgCl2, 50 ng genomic DNA, and 1 U Taq polymerase. PCR products were ligated into the TOPO-TA vector (Invitrogen, Hamburg, Germany) and transformed into Escherichia coli TOP10F’. Recombinant colonies were cultured on LB agar supplemented with ampicillin. A commercial kit (QIA mini prep, QIAGEN, Hilden, Germany) was used to prepare plasmid DNA.

2.2. DNA sequence analysis The DNA was sequenced using an Applied Biosystems 373 DNA sequencer. The sequence was determined for both strands by using overlapping fragments. Analyses of nucleotide and deduced amino acid sequences were conducted using the MacMolly Tetra version 3.7. Probable secretory signal peptides were predicted by the Signal P V2.0 program, as described by Henrik et al. [15]. Potential motifs in the protein sequence were predicted using the Pfam collection of hidden Markov models [16]. Prediction of transmembrane helices was done using the TMHMM server V. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). 2.3. Expression and protein purification of a fragment of Wolbachia-O. volvulus-HtrA serine protease SPR (Wol-Ov-HtrA) The coding region of the Wol-Ov-HtrA was amplified by using the described WDF and WDR primers and cloned in the PCRT7/NT-TOPO (Invitrogen) expression vector. This vector enables the N-terminal fusion to a cleavable His-Tag sequence. The target plasmid was established in BL21(DE3) E. coli cells. The authenticity of TOPO recombinants was verified by DNA sequencing. A recombinant colony of BL21 was grown at 37 °C overnight supplemented with ampicillin and chloramphenicol. The overnight culture was diluted with LB medium and incubated until A600 reached an optical density of 0.6. Expression was induced by the addition of isopropyl thio-beta-D-galactoside (IPTG), and incubation for a further 5 h was carried out until the cells were harvested by centrifugation. The recombinant protein was purified by Ni2+ affinity chromatography. The course of the purification was monitored by SDS-polyacrylamide gels and stained with Coomassie brilliant blue. An aliquot of cell lysate or purified protein was diluted with loading buffer (0.05% bromphenol blue–5% SDS–50% glycerol in 225 mM Tris–HCl pH 6.8) and was applied to an SDS-10% polyacrylamide gel. Electrophoresis was carried out in a Protean II cell unit (Bio-Rad Laboratories, München, Germany) at room temperature. 2.4. Production and purification of polyclonal rabbit antibodies against Wol-Ov-HtrA For production of polyclonal antibodies, the purified HisTag fusion protein was cut out from the polyacrylamide gel. A rabbit was then injected subcutaneously on days 0, 14, 28, and 56 with 200 µg of recombinant protein in polyacrylamide gel as adjuvant (Eurogentec, Seraing, Belgium). Blood samples were collected on days 0 (preimmune serum), 38, 66, and 87. 2.5. Immunohistology Onchocercomas embedded in paraffin were available from several studies in Liberia, Uganda, and Ghana

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[5,17,18]. The extirpation of onchocercomas for research had been approved by the Medical Board, Hamburg, Germany, and authorities in the African countries. The nodules had been fixed in 80% ethanol or 4% phosphate-buffered formaldehyde solution. Sixty-two onchocercomas had been extirpated from 18 untreated individuals and 17 nodules from 15 patients treated with ivermectin or 12 or 18 months after administration of doxycycline. The treatment with 100 mg doxycycline per day for six weeks had eliminated the Wolbachia endobacteria from the O. volvulus worms of these patients [5,19]. Specimens with nematodes that did not contain any Wolbachia endobacteria [20] had been processed similarly. Onchocerca flexuosa from deer in northern Germany was supplied by Dr. A. Plenge-Bönig, BNI [21], and Acanthocheilonema viteae worms were from a laboratory strain previously maintained in the BNI. Ascaris suum had been collected at the central abattoir in Hamburg. For immunohistology, the alkaline phosphatase anti-alkaline phosphatase (APAAP) method was applied according to the recommendations given by the manufacturer (Dako Diagnostika, Hamburg, Germany). The polyclonal antiserum raised against Wol-Ov-HtrA in a rabbit was used as primary antibody with dilutions between 1:50 and 1:200. As secondary antibody, anti-rabbit mouse immunoglobulins (clone MR12/53, Dako Diagnostika) were applied. Fast Red TR salt (Sigma) was used as chromogen, and haematoxylin (Merck, Darmstadt, Germany) functioned as the counterstain. Preimmune serum was used as negative control (dilution 1:50). To examine the specificity of the anti-Wol-Ov-HtrA serum, the specific serum against Dirofilaria immitis Wolbachia surface protein (Wol-Di-WSP) was used as primary antibody (diluted 1:4000) [8], kindly supplied by Dr. C. Bandi, Dipartimento di Patologia Animale, Igiene e Sanita Pubblica Veterinaria, Universita di Milano, Italy. 2.6. Antibody analysis Sera from individuals infected with O. volvulus were examined for the presence of IgG1 antibodies reactive with recombinant Wol-Ov-HtrA, using an enzyme-linked immunosorbent assay (ELISA). Sera were obtained from 37 onchocerciasis patients from endemic areas in Ghana and Uganda, whose clinical and parasitological criteria were verified according to WHO recommendations [1]. The examination included a thorough exploration of the skin for dermatological alterations, an inspection for onchocercomas and a search for skin microfilariae. All patients were microfilaria carriers with a range of 1–316 microfilariae per milligram skin. Eleven of the 37 patients had a chronic hyperreactive form of onchocerciasis (sowda). These patients were characterised by low parasite loads and high antibody levels against O. volvulus antigens [22]. For antibody analysis, wells of Maxisorb plates (Nunc, Wiesbaden, Germany) were coated overnight with purified Wol-Ov-HtrA in 0.05 M carbonate buffer, pH 9.6, at 200 ng per well. The ELISA was performed as previously described,

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with minor alterations [23]. The sera were diluted at 1:300, 1:600 and 1:1200. As secondary reagent, we applied mouse monoclonal antibodies against IgG1 (clone HP6070; Calbiochem, La Jolla, CA) at 1:1000. As third reagent, horseradish peroxidase-conjugated goat anti-mouse IgG (Dianova, Hamburg, Germany) was used, and as enzyme substrate, tetramethylbenzidine (Sigma) was applied. The titre for each reaction was calculated by generating titration curves [24].

3. Results 3.1. Isolation and characterisation of Wol-Ov-HtrA A product of the Ov-Wolbachia gene of 924 bp was obtained by PCR that revealed an open reading frame (ORF) of 307 amino acids, corresponding to the C-terminal region of the serine protease. Attempts to obtain the entire DNA sequence applying further primers based on the Drosophila Wolbachia sequence data failed. The comparison of the obtained serine protease nucleotide sequence from Drosophila Wolbachia (Wol-Dm-serine protease) with O. volvulusWolbachia (Wol-Ov-HtrA) revealed an insertion of six nucleotides corresponding to position 13 in the Wol-Ov-HtrA. The frame-shift by this insertion causes an extension of the ORF, resulting in the longer sequence in Wol-Dm-serine protease. A DNA fragment of 585 bp, however, yielded when the primers were deduced from a homologous sequence in the B. malayi Wolbachia genome. The nucleotide sequences were assembled, and computer analysis of the ORF with MacMolly Tetra indicated that the contig of the full-length WolOv-HtrA contained a single ORF of 1482 bp, which encoded a polypeptide of 494 amino acids with a theoretical molecular mass of 54 kDa. The sequence of Wol-Ov-HtrA showed 99% and 87% identity with the homologous sequences from Wolbachia of Onchocerca ochengi and B. malayi, respectively, which were also identified and cloned. Approximately 81% identity was found when Wol-Ov-HtrA was compared with the Wolbachia protease from D. melanogaster. In Fig. 1A, the sequences of the three Wolbachia serine proteases are aligned together with other prokaryotic serine proteases from Mesorhizobium loti (NP_103 037), Bartonella henselae (NP P54925) and R. prowazekii (NC_000963), with levels of identity between 38% and 39%. Similarities of 40–43% were also stated for serine proteases of the bacteria Brucella melitensis (NC_003317), Rhodopseudomonas palustris (NZ_AAAF 01000001) and Magnetospirillum magnetotacticum (NZ_AAAP01003865). These results show homology between serine proteases of Wolbachia and those of other intracellular Gram-negative bacteria. While the conserved consensus sequence of the active site of eukaryotic serine proteases is GDSGGP [25], in the bacterial proteases, including Wol-Ov-HtrA, the aspartic acid (D) is replaced by asparagine (N), leading to a GNSGGP motif. The positions of the amino acid residues of the triad

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Fig. 1.

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N SP -20 +1

catalytic domain H107 D137

PDZ 1

S211

PDZ 2

C

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3.3. Production and purification of Wol-Ov-SPR fusion protein

474 aa

Fig. 2. Schematic representation of the primary Wol-Ov-HtrA structure. The signal peptide (SP) is indicated by a black box. The catalytic domain, with the location of the catalytic triad in mature protein (residues 77–254; His107, Asp-137 and Ser-211), is indicated by a white rectangle. PDZ domains (PDZ-1 residues 256–348 and PDZ-2 residues 365–463) are depicted as shaded rectangles.

were identified as His127, Asp157 and Ser 231, numbered from the proposed Met 1 residue (Fig. 1A). Computer analysis of the Wol-Ov-HtrA sequence using the Pfam collection of hidden Markov models (ProfileScan server available at hits.isb-sib.ch) furthermore revealed two PDZ domains at the C-terminal region of the protein (Fig. 2).

A 924-bp fragment was amplified, and the PCR product was directly cloned into a TOPO-TA expression vector. The fragment includes approximately three-fourths of the WolOv-HtrA coding sequence, corresponding to nucleotide positions 561-1482 and to the amino acids Met 187-Lys 494, respectively (Fig. 1A). Lysates of cells induced with IPTG displayed a new protein band with a molecular mass of approximately 33 kDa (plus 3 kDa for the His-Tag) not present in uninduced cells. The expression levels of Wol-OvHtrA and Wol-Bm-HtrA were similar, as judged by electrophoresis of lysates from IPTG-induced cultures on SDSPAGE and staining with Coomassie blue. After purification of the Wol-Ov-HtrA fusion protein, we obtained 0.2 mg of protein per 100 ml.

3.2. Leader peptide sequence analysis 3.4. Immunolocalisation of Wol-Ov-HtrA Computer analysis for the hydropathy profile of the deduced amino acid sequence at the N-terminus region, using networks trained on sequences from Gram-negative bacteria, indicated that one hydrophobic region directly follows the initiation codon. Based on the “(-3, -1)-rule” [26], a putative 20-amino-acid signal peptide sequence was identified, and the Arg at position 21 was assumed to represent the start of the mature protein (Fig. 1A,B). In contrast, the homologous sequence from Wolbachia in D. melanogaster contained six additional inserted nucleotides, leading to a prolongation of the hydrophobic h-region of two amino acids, alanine and phenylalanine, which results in transmembrane localisation, according to the TMHMM Server V. 2 (Fig. 1B). This would result in a secreted protein of 474 residues with a predicted molecular mass of 52 kDa. When the resulting predicted amino acid sequence of Wolbachia protein was compared with the database, it was found that the leader of the deduced zymogen bears less similarity to that of other serine proteases.

When preimmune serum was applied, none of the worm sections demonstrated any staining. In contrast, immune serum showed strong staining of all sections of O. volvulus (Fig. 3B–G). Granular structures were stained in hypodermis, oocytes, and embryos. Based on the size and localisation of the Wol-Ov-HtrA-positive granules, they were assumed to be Wolbachia endobacteria. Hence, their distribution pattern in the filarial tissues was compared with the pattern of endobacteria in consecutive paraffin sections labelled specifically by anti-Wol-Di-WSP serum shown to distinctly stain the surface of Wolbachia (Fig. 3A,B) [27]. The localisation in consecutive sections was identical with both antisera. The labelled granules were observed in the median layer of the hypodermis (Fig. 3C), whereas the outermost and innermost layers with the basal labyrinths of cell membrane folds were mostly free. In many oocytes in ovary, oviduct and uterus (Fig. 3D) and in zygotes, the granules showed the typical bipolar distribution of endobacteria. In contrast, only a few cells containing endobacteria were positively stained in em-

Fig. 1. (A) Alignments of the amino acid sequences of Wol-Ov-HtrA (Wolbachia-Ov) with homologous serine proteases. The Wol-Ov-HtrA protein sequence is shown aligned with homologues from Wolbachia of B. malayi (Wolbachia-Bm), D. melanogaster (Wolbachia-Dm), M. loti (NP_103037), B. henselae (P54925), and R. prowazekii (NC_000963). Active sites are boxed and catalytic triad residues are marked with bold stars. An arrow denotes the putative signal peptide cleavage site. Shading indicates identity (black) or conservative substitutions (grey) relative to O. volvulus. The aligment was generated using Clustal W1.8, together with the boxshade server (http://www.ch.embnet.org/software/BOX_form.html). (B) Comparison of the signal peptide alignments of the three Wolbachia sequences revealing the insertion of two hydrophobic amino acids in the h-region of the D. melanogaster Wolbachia protease.

Fig. 1. (continued)

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Fig. 3. Demonstration of serine proteases localised in endobacteria and spermatozoa of O. volvulus. (A) Cross-section of a female worm stained by Wolbachia-specific anti-Wol-Di-WSP serum shows the endobacteria in the hypodermis (arrow). (B) A consecutive section to (A) stained by anti-Wol-Ov-HtrA serum shows the same pattern of strong labelling of the bacteria in the hypodermis (arrow). The uterus contains positive spermatozoa. (C–F) Wolbachia endobacteria (arrows) are distinctly labelled by anti-Wol-Ov-HtrA serum in the hypodermal cord (C, E), in oocytes (D), in morulae (E), and in stretched microfilariae (F). The epithelia of intestine and uterus and the muscles of the body wall are negative. (G, H, J) Mature spermatozoa in the testis (G, J) and in the uterus (H) are labelled by the anti-Wol-Ov-HtrA serum due to a cross-reaction of the antiserum with a filarial serine protease. (G) Specific staining of the endobacteria in the hypodermis. (H) Light staining of the uterus epithelium due to the presence of strongly labelled spermatozoa. (J) shows a male worm without endobacteria 18 months after beginning anti-wolbachial treatment with l00 mg doxycycline per day for 6 weeks. hy, hypodermis; i, intestine; mu, muscles; sp, spermatozoa, ut, uterus. Scale bar = 20 µm.

bryos (Fig. 3E) and microfilariae in the uterus (Fig. 3F) and in the nodule tissues. No granular structures were stained in the muscles of the body wall (Fig. 3C,E) and the uterus or in the epithelia of the intestine or the male or female genital tracts—tissues that do not contain Wolbachia. It was concluded that the anti-Wol-Ov-HtrA serum labelled, as assumed, specifically the Wolbachia endobacteria in the filarial worms and no other granular structures of the worms. This conclusion was confirmed by the examination of adult O. volvulus worms in onchocercomas from doxycyclinetreated patients, which did not contain endobacteria. No staining of granular structures was observed in any of these worms (Fig. 3J). Whereas anti-Wol-Di-WSP serum does not stain any stages of sperms, the anti-Wol-Ov-HtrA serum stained mature spermatozoa in male and female worms (Fig. 3G,H) but not immature sperms. In portions of the uterus containing many labelled spermatozoa, sometimes the inner layer of the

uterus epithelium was stained (Fig. 3H), possibly due to Wol-Ov-HtrA from the spermatozoa. The spermatozoa were also labelled in doxycycline-treated, Wolbachia-free O. volvulus worms (Fig. 3J). They were further stained in filaria species that principally do not contain Wolbachia, such as O. flexuosa from red deer and A. viteae from rodents. Even the spermatozoa of the roundworm A. suum were stained by the antiserum. Since spermatozoa of filariae and roundworms do not contain Wolbachia endobacteria, it was concluded that the anti-Wol-Ov-HtrA serum cross-reacts with a serine protease of the nematodes expressed in their sperms. The antiserum also strongly reacted with the serine protease in the granules of mast cells [28] observed in the human tissues of the onchocercomas. Applying antibodies directed against the Wolbachia surface protein (Wol-Di-WSP), the endobacteria were distinctly labelled, while the filarial cytoplasm of the hypodermis was not [27]. In contrast, the cytoplasm around the endobacteria

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Fig. 4. Recognition of a recombinant Wol-Ov-HtrA fragment by IgG1 antibodies in O. volvulus-infected persons. ELISA IgG1 titres of sera from a group of 26 patients with a generalised form of onchocerciasis, of 11 patients with a hyperreactive form (sowda) in comparison with sera from 12 healthy European controls. The figure shows box plots and percentiles (10th, 25th, 50th, 75th, and 90th). Significant differences exist between IgG1 titres for the controls and the total group of patients as well as between both patient groups, when analysed by the Mann–Whitney U–test (P < 0.001 and P < 0.02, respectively).

stained by anti-Wol-Ov-HtrA serum was often found more or less stained. Weak staining was also observed in the hypodermis of female worms after doxycycline treatment, when a dilution of 1:50 was used. No labelling was seen in treated, bacteria-free worms using a dilution of 1:200. 3.5. Demonstration of IgG1 antibodies reactive with a recombinant Wol-Ov-HtrA fragment in O. volvulus-infected persons To examine a possible recognition of Wol-Ov-HtrA by O. volvulus-infected and therefore Wolbachia-exposed persons, sera from patients with onchocerciasis and noninfected control persons were tested for reactivity with the recombinantly expressed fragment of Wol-Ov-HtrA using ELISA (Fig. 4). Significant IgG1 reactivities with Wol-OvHtrA could be shown in the total group of 37 patients with onchocerciasis when compared with control sera of 12 healthy Europeans (P < 0.001; Mann–Whitney U test). The highest IgG titres were observed in sera from patients with a hyperreactive onchocerciasis, when comparing the subgroups of 26 persons with a generalised form of onchocerciasis vs. 11 patients with a hyperreactive form (P < 0.02).

4. Discussion Wolbachia are obligatory symbiotic intracellular Gramnegative alpha-proteobacteria. Several experimental and clinical studies revealed that their filarial hosts, including O. volvulus, do not develop and grow, but are sterilised and may die when the endobacteria are eliminated by antibiotic treatment [3,5,20,29]. So far unknown, however, is the physiological basis for the mutualistic relationship between bacteria and filariae and the transfer of metabolites between the

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cohabitating species. Of major interest are molecules exported from the bacterium into the filarial host cells, i.e., into embryonic cells or the hypodermal syncytium. In the present report, the first candidate for an endobacterial molecule that may interact with the filarial host metabolism, thereby contributing to the mutualistic relationship, has been identified and cloned. Wol-Ov-HtrA was identified as serine protease belonging to the SA clan of the chymotrypsin family, according to the order of the three putative catalytic domains containing a triad of His, Asp and Ser residues and the respective distances between them. This clan comprises a diverse spectrum of proteases, including several families of microbial proteases [30]. The bacterial origin of Wol-Ov-HtrA is confirmed by alignments found with other bacterial serine proteases exhibiting, in contrast to eukaryotic serine proteases, aspartate (N) instead of aspartic acid (D) within the central serine motif (Fig. 1A). The corresponding staining of Wolbachia in endobacteria-harbouring filarial tissues by antibodies against Wol-Ov-HtrA as well as against WSP on the one hand, and the absence of bacteria-associated staining in Wolbachiadepleted worms on the other hand, clearly indicate that WolOv-HtrA represents a bacterial protein. Furthermore, the serine protease genes from the Wolbachia of O. volvulus, B. malayi and of D. melanogaster showed no introns and were highly identical. Analysis of the N-terminus of Wol-Ov-HtrA includes a differentiation between a secretory or transmembrane protein [26]. Three domains (residues 1–4, 5–14, and 15–20) of the Wol-Ov-HtrA sequence completely satisfy the criteria proposed for a cleavable signal, strongly indicating a secretory/periplasmic protein. Of interest, although the identity of the amino acid sequences of Wol-Ov-HtrA and Drosophila (Wol-Dm-serine protease) is over 80%, differences in the N-terminus between the protease of the symbiotic filarial Wolbachia and of the parasitic Wolbachia in the arthropod predict that the Wol-Dm-serine protease, in contrast to WolOv-HtrA, is a type II transmembrane protein. The h-region of the N-terminus of Wol-Dm-serine protease is two amino acids longer than that of Wol-Ov-HtrA and represents an uncleaved signal peptide, leading to membrane anchoring of the protein. It has to be clarified whether this discriminating feature may relate to the mutualistic respective parasitic relationship. In contrast to the distinct histological localisation of the surface protein WSP, strictly found associated with the bacteria [5,27], the staining of Wol-Ov-HtrA was not found to be exclusively bacteria associated but could also be detected externally and in the vicinity of the endobacteria. An argument in favour of the secretion of Wol-Ov-HtrA, furthermore, is the absence of staining in the vicinity of the few remaining bacteria in worms, when the majority of the bacteria had been depleted by doxycycline treatment. It cannot be completely excluded that the Wol-Ov-HtrA reacted differently from the Wol-Di-WSP during the fixation and processing of the sections. In addition to the bacteria-associated labelling, the

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staining at a distance from the endobacteria and in bacteriafree worm tissues like spermatozoa indicates that, as a second staining reaction, the antibody directed against the Wolbachia protein cross-reacts with a thus far unknown homologous filarial serine protease. This similarity is not surprising, since it is assumed that HtrA serine proteases of eukaryotes had evolved from the prokaryotic homologues during endosymbiosis of alpha-proteobacteria [31]. Furthermore, antisera against other Wolbachia proteins also show cross-reactions with homologous filarial proteins, such as Wol-Ov-HSP60, and Wol-Ov-aspartate aminotransferase [6,20]. The three characteristic features of the Wol-Ov-HtrA including (i) the signal peptide, (ii) the serine protease-active triad, and in addition, (iii) two C-terminal PDZ domains classify the protease as a member of the HtrA/DegP family expressed in several obligatory and facultative intracellular bacteria of the genera Rickettsia, Legionella, Brucella, Bartonella, Yersinia, and Mycobacterium [9–13,32]; some of those have been included in the alignment (Fig. 1A). HtrA proteins have been reported to act as protease, chaperone, periplasmic stress sensor, and regulator of apoptosis [10,11,33,34]. Crystal structure analysis revealed that two HtrA trimers form a cavity where the C-terminal, highly flexible PDZ domains—known to be involved in protein– protein interaction—may function as “tentacular arms” capturing protein substrates and transferring them into the inner cavity [33]. In a chaperone conformation, the catalytic site is blocked by the PDZ domain acting as “gatekeepers”, while it is active in a proteolytic conformation. These domains are key elements in building protein complexes that play a central role in transport, localisation of receptors and signalling assemblies [35]. As part of a molecular scaffold, PDZ domains bind C-terminal tags, especially of membrane proteins such as ion channels. HtrA proteins can monitor the folded state of other proteins that can be refolded or degraded [10,33,34]. Furthermore, some HtrA proteins have been shown to have proapoptotic activity involved in regulatory processes in apoptosis by interacting with inhibitors of apoptosis (IAP). Apoptosis has been shown to play a major role in tissue development and homeostasis of nematodes, well-known from Caenorhabditis elegans [31,36]. So far, only one protein of O. volvulus has been related with apoptosis [37]. A serine protease exported from the endobacteria could interact or be controlled by inhibitors of serine proteases (serpins) of the filarial host, several of which have been identified [38]. Thus far, the filarial serine protease inhibitors have been reported to neutralise serine proteases degranulated from human effector cells and discussed as an evasion factor of the filaria [39]. Recently, however, the inhibition of the host’s serine proteases by filarial serpins could not be confirmed [40], which may favour the view that filarial serpins may interact or regulate both filarial and endobacterial serine proteases. Exported Wolbachia molecules are supposed to be exposed to the human host when the filaria degenerate naturally

or in consequence of host defence mechanisms. Such an exposure should lead to immune recognition and antibody production against an immunogenic protein. Indeed, IgG1 type antibodies were detected in sera from O. volvulusinfected persons reactive with recombinant Wolbachia serine protease in ELISA. The antibody reactions were found highest in patients with a hyperreactive form of onchocerciasis as known from earlier studies [22,24]. This finding indicated that Wol-Ov-HtrA is recognised by the human immune system, as demonstrated before for endobacterial WSP and aspartate aminotransferase [6]. The present report presents the first characterisation of a candidate exported Wolbachia molecule that may interfere with the filarial host metabolism by inducing apoptosis, protein folding or cleavage during filarial development.

Acknowledgements This work was supported by a fellowship (A.J.) from the Alexander von Humboldt Foundation, Bonn, and a scholarship (P.F.) from the “Vereinigung der Freunde des Tropeninstituts”, Hamburg, Germany. The authors acknowledge the excellent assistance of Ingeborg Albrecht and Insa Bonow.

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