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Home , Brugia malayi, Onchocerca, Onchocerca volvulus

volvulus-neurotransmitter tyramine is a biomarker for river blindness

Daniel Globischa, Amira Y. Morenoa, Mark S. Hixona,b, Ashlee A. K. Nunesa, Judith R. Denerya, Sabine Spechtc, Achim Hoeraufc, and Kim D. Jandaa,1

aDepartments of Chemistry, Immunology, and Microbial Science, and Worm Institute for Research and Medicine, The Scripps Research Institute, La Jolla, CA 92037; bTakeda California, Inc., San Diego, CA 92121; and cInstitute of Medical Microbiology, Immunology, and , University Hospital Bonn, 53105 Bonn, Germany

Edited* by Peter G. Schultz, The Scripps Research Institute, La Jolla, CA, and approved January 29, 2013 (received for review December 18, 2012) , also known as “river blindness”,isaneglected to , and thus underscores the importance of both infecting millions of people mainly in and treating and monitoring the disease in a cohesive fashion. Simply the Middle East but also in South America and Central America. stated, the elimination of onchocerciasis, regardless of the form of Disease infectivity initiates from the filarial parasitic treatment, will require a method for determining the relationship , which is transmitted by the blackfly vector between infection prevalence, individual infection intensity, and sp. carrying infectious third-stage larvae. transmission intensity (5). has controlled transmission of microfilariae, with an African Pro- Several diagnostics are currently used to monitor the disease gram elimination target date of 2025. However, there is currently state in patients. Among these, skin snips are considered the “gold no point-of-care diagnostic that can distinguish the burden of standard,” but this method is becoming increasingly unpopular infection—including active and/or past infection—and enable the and is being replaced by PCR assays, ELISAs, enzyme immuno- elimination program to be effectively monitored. Here, we describe assays, and surveys (6, 7). A shortcoming of these newer how liquid chromatography-MS–based urine metabolome anal- assays is their inability to monitor disease progression readily. ysis can be exploited for the identification of a unique biomarker, Recently, an -based assay was described, which allows N-acetyltyramine-O,β-glucuronide (NATOG), a neurotransmitter- the parallel detection of four O. volvulus-specific , and CHEMISTRY derived secretion metabolite from O. volvulus. The regulation of thus discrimination among filarial infections (8). Still, humoral this tyramine neurotransmitter was found to be linked to patient response-based diagnostics carry liabilities with the potential of disease infection, including the controversial antibiotic doxycy- antibody persistence even after treatments, hence confounding cline treatment that has been shown to both sterilize and kill adult active/past infections. A potential alternative would be the iden- female worms. Further clues to its regulation have been elucidated tification of specific metabolites in blood or urine samples be- through biosynthetic pathway determination within the nema- cause, in theory, this could provide both infection status and a tode and its human . Our results demonstrate that NATOG quantitative measure of infection based on metabolite concen- tracks O. volvulus metabolism in both worms and humans, and tration (9, 10). Using liquid chromatography (LC)-MS to analyze thus can be considered a host-specific biomarker for onchocerciasis O. volvulus-infected serum samples, we demonstrated the prom- progression. Liquid chromatography-MS–based urine metabolome ise of metabolomics as an onchocerciasis diagnostic (11). How- analysis discovery of NATOG not only has broad implications for a ever, technical constraints, such as a need for multiple mass noninvasive host-specific onchocerciasis diagnostic but provides biomarkers and an inability of compound biomarker assignment, a basis for the metabolome mining of other neglected tropical led us to investigate whether urine analysis might serve as a better diseases for the discovery of distinct biomarkers and monitoring platform for O. volvulus biomarker diagnostics. of disease progression. Results and Discussion nchocerciasis, commonly referred to as “river blindness”, To assess this hypothesis, extraction of nonprotein fractions from Ois a neglected tropical disease that affects more than 37 urine of African onchocerciasis-positive and -negative samples million people worldwide. The disease is endemic in many sub- was undertaken for analysis in global metabolomic profiling. A Saharan countries in Africa, with minor foci in Central and South complete list of all sample origins is detailed in Table S1. Extracts America and in . The infection is caused by the filarial were analyzed by LC-MS, and statistical analysis revealed an MS parasitic nematode Onchocerca volvulus, which is transmitted signal of m/z = 356.1, which was enriched in O. volvulus-positive during a blood meal by a blackfly vector (Simulium sp.) carrying patients compared with African control samples (12). Extraction infectious third-stage larvae (1). These larvae develop into adults of the specific ion chromatogram and the corresponding box-and- in the human host, ultimately accumulating in subcutaneous or deep whisker plot indicated elevated signal intensities in infected pa- = × −8 nodules called onchocercomas. In onchocerciasis, microfilariae are tient samples (Fig. 1A; P 1.2 10 ). fi born from viviparous females and migrate through nodular tissue To de ne the importance of this biomarker better, we sought to the skin and eyes, resulting in chronic inflammation. Invasion to determine its structure. High-resolution MS provided a mo- = of the cornea by microfilariae leads to blindness, causing the lecular mass of M 355.3398 g/mol, corresponding to the mo- typical pathology associated with this disease. lecular formula C16H21NO8. To decipher the chemical structure, Treatment options for onchocerciasis include the microfilari- cidal drug ivermectin, which has been critical in controlling on- chocerciasis transmission (2), and the antibiotic , which Author contributions: D.G., A.Y.M., A.A.K.N., J.R.D., and K.D.J. designed research; D.G., A.Y.M., M.S.H., and A.A.K.N. performed research; D.G., M.S.H., S.S., and A.H. contributed targets the bacterial , resulting in both new reagents/analytic tools; D.G., A.Y.M., A.A.K.N., J.R.D., and K.D.J. analyzed data; and death and sterilization of adult . Recently, concomitant D.G. and K.D.J. wrote the paper. administration of both drugs resulted in promising and improved The authors declare no conflict of interest. treatment outcomes (3, 4). Despite this success, challenges still re- *This Direct Submission article had a prearranged editor. main, including the development of reliable disease surveillance 1To whom correspondence should be addressed. E-mail: [email protected]. methods to overcome the need for strate- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. gies. Unspecific treatment of whole communities may eventually lead 1073/pnas.1221969110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1221969110 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 Fig. 1. Identification of the onchocerciasis-specific biomarker NATOG. (A) Comparison of the extracted ion currents (EIC) in LC-MS experiments of biomarker NATOG in O. volvulus-positive (red, n = 19) and O. volvulus-negative (black, n = 12) samples, and corresponding box and whisker plots. A (a.u.), area (arbitrary units); pos., positive. (B) Specific MS/MS fragmentation pattern of biomarker NATOG in positive mode at three different collision energies: collision-induced dissociation (CID) = 10 V, 20 V, and 40 V. (C) Assignment of MS/MS fragments of biomarker NATOG.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1221969110 Globisch et al. Downloaded by guest on September 30, 2021 we enriched the biomarker by HPLC purification, and fragmen- After confirmation of the molecular structure of NATOG, we tation of the specific mass signal in LC-tandem MS (MS/MS) sought to quantify it in urine samples, which would reinforce its experiments enabled the detection of three major signals orig- potential as a biomarker for diagnostic use. Toward this goal, we inating from the parent ion (m/z = 356.1340; Fig. 1B). These analyzed a series of O. volvulus-positive and -negative samples signals permitted decoding of the biomarker structure. The first and extended our study with four sample sets to evaluate the fragment signal, m/z = 180.1048 (C10H13NO2), suggested neutral specificity of this biomarker. To permit precise quantification, the = loss of glucuronic acid (m/z 176.0321). At higher fragmentation D3-labeled analog of NATOG (D3-NATOG) was prepared (Fig. = energies, we observed two further fragmentation signals, m/z S3). Importantly, D3-NATOG has the same physical properties as 138.0927 and m/z = 121.0663, which provide evidence for loss of NATOG but can be distinguished due to the molecular mass an aliphatic N-acetyl moiety (13). Confirmation of these two difference that allows parallel analysis (14). Calibration curves fragments allowed us to consider the remaining C8H8O fragment were constructed for precise quantification (Fig. S4), and = (m/z 121.0663). Both additional fragments were assigned to the D3-NATOG was spiked into each analyzed urine sample at a loss of water and ethylene (m/z = 103.0540 and m/z = 93.0701, known concentration before extraction of the nonprotein frac- respectively), indicating a phenolic structure. A systematic com- tions to allow unbiased analysis. bination of all identified fragments allowed assignment to the The obtained quantitative values are summarized in Fig. 2C.As structure of N-acetyltyramine-O,β-glucuronide (NATOG; Fig. anticipated, analysis of a larger sample set confirmed our ob- 1C). Further proof of NATOG came via chemical synthesis (Fig. served qualitative differences of onchocerciasis-positive samples S1). The structure was confirmed by the exact match of the high- compared with African control samples that unequivocally resolution mass of synthetic and natural material, together with revealed elevated values of biomarker NATOG in O. volvulus- examination of both the retention time in coinjection experiments infected individuals, with an average value of 36.9 ± 4.0 μM and LC-MS/MS fragmentation (Fig. 2 A and B and Fig. S2). (±SEM). This represents approximately a sixfold increase compared CHEMISTRY

Fig. 2. Structure confirmation and quantification of biomarker NATOG. (A) Cospiking experiment of the synthetic compound NATOG to a positive urine extract (Top) in comparison to single injections of the positive urine extract (Middle) and the synthetic biomarker NATOG (Bottom). EIC, extracted ion cur- rents. (B) MS/MS fragmentation comparison of the synthetic and natural molecules (collision-induced dissociation = 20 V). (C) Quantitative values of NATOG

using isotopic labeled compound D3-NATOG as an internal standard in onchocerciasis-positive (n = 81) and control urine samples. Error bars represent SEM values. Data were analyzed by ANOVA, followed by Tukey’s multiple comparison method to determine significance (***, P < 0.001). For placebo (n = 14) and doxycycline (n = 24) sample sets, an unpaired two-tailed Student t test was performed (**P = 0.0072). A detailed overview of the quantitative values, number of analyzed samples, and procedure is provided in Tables S2 and S3.(D) Quantitative values of NATOG for placebo- and doxycycline-treated patients. Columns labeled with x represent sample sets, which were treated 6 mo later with ivermectin (Tables S1 and S2).

Globisch et al. PNAS Early Edition | 3of6 Downloaded by guest on September 30, 2021 with African control samples (7.0 ± 2.7 μM; P < 0.001). As establish the value of biomarker NATOG as a means for moni- a second O. volvulus-negative control group, we examined North toring onchocerciasis progression. American samples, which were found to have an average value of Having established the importance of NATOG in West African 1.1 ± 0.2 μM. samples, we then analyzed the presence of NATOG in Central Filarial nematodes contain the endosymbiotic bacteria Wol- American samples. In this case, we observed a low abundance of bachia, and apart from many filarial species, these endo- biomarker NATOG in Guatemalan O. volvulus-positive samples bacteria are present in several human filariae, such as Wuchereria (8.4 ± 1.6 μM; P < 0.001). This finding can be rationalized based bancrofti, malayi, and O. volvulus. In 2000, a landmark on the presence of genetically different O. volvulus species (18). study provided evidence that doxycycline cleared Wolbachia from This result is also in line with previous observations for blood fi the endodermis and uteri of adult female O. volvulus, leading to biomarkers speci c for African O. volvulus infections, including extensive worm sterility (15). Thus, monitoring the influence such a report detailing nonoverlapping polypeptide composition as treatment would have on NATOG could provide an additional well as IgG4 response differences seen between Central American and West African samples (11, 19). Due to the close phylogenetic link between NATOG and worm metabolism. We analyzed urine relationship between O. volvulus and other filarial species, we samples from patients with O. volvulus who received either examined samples of patients with lymphatic filariasis infections. doxycycline or placebo over a 4- to 6-wk period. Sample sets were A remarkably diminished difference in NATOG concentration collected 20 mo after treatment and were shown both to kill the with high statistical significance (4.2 ± 0.7 μM; P < 0.001) was endosymbiont Wolbachia and to sterilize the nematode (16, 17). observed. Although these data suggest a metabolic specificity of fi fi The quanti cation experiments revealed a signi cantly reduced NATOG for O. volvulus and the host, future sampling of other ± concentration of NATOG in doxycycline-treated patients (9.5 human filarial nematode species, such as and 1.7 μM) compared with untreated O. volvulus-positive patients perstans, will help to establish a more complete picture of the (P < 0.001) and placebo-treated patients (33.5 ± 10.7 μM; P = metabolic regulation of NATOG and these other 0.0072). As shown in Fig. 2D, remarkably diminished levels of states (20). NATOG were seen in patients treated with doxycycline. These The metabolic pathway of biomarker NATOG reveals an in- results are consistent with an increasing body of data demon- timate relationship between O. volvulus and its human host (Fig. strating doxycycline’s antifilaricidal capacity, and they further 3A). The “core” moiety of NATOG is tyramine and represents

Fig. 3. Proposed biosynthesis of NATOG. (A) Biosynthetic pathways of NATOG in O. volvulus and the human host. (B) Metabolic and degradation pathway of L-tyrosine in humans.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1221969110 Globisch et al. Downloaded by guest on September 30, 2021 a structural counterpart of vertebrate neurotransmitters (21). NATOG, and this outcome argues that endosymbiotic targeting The path to tyramine requires L-tyrosine decarboxylation by appears to be therapeutically efficacious. The discovery of NATOG L-tyrosine decarboxylase and represents the major conversion of as a biomarker for O. volvulus should prove valuable in the de- L-tyrosine in invertebrates. The discovery of a specific tyramine velopment of a diagnostic, which will assist in facilitating disease receptor in infers an importance of this elimination (31). neurotransmitter in nematodes (22). Accordingly, a high level of activity of this decarboxylase was identified in the third-stage larvae Materials and Methods of O. volvulus, suggesting a critical role for this neurotransmitter in Sample Preparation and Metabolite Extraction. Solvents used were of HPLC the worm’s development (23), a point in fact that readily justifies grade. A methanol precipitation of proteins was conducted by adding 400 mL the reduced concentration of biomarker NATOG in doxycycline- of ice-cold methanol to 100-mL aliquots of urine samples spiked with a known treated patients, which leads to the sterilization of adult worms concentration of isotopic-labeled internal standard D3-NATOG. The samples were vigorously shaken for 30 s and allowed to rest on ice for 30 min. After (16, 17). Based on literature precedents, we concluded that × N-acetylation of tyramine by arylalkylamine N-acetyltransferase centrifugation at 13,780 g for 5 min, the metabolite-containing superna- tant was removed from the precipitated protein pellet and transferred to (AANAT) is the second metabolic step (24, 25) in the bio- fresh tubes. The supernatant samples were dried in a GeneVac EX-2 Evap- synthesis of NATOG. Importantly, N-acetylation and mono- oration System (GeneVac, Inc.) at ambient temperature and then resus- amine oxidation by monoamine oxidases (MAOs) are the two pended to a volume of 50 mL in water/acetonitrile (95:5 ratio), vigorously deactivation mechanisms of excess neurotransmitters that shaken for 30 s, and centrifuged again at 13,780 × g for 5 min. After being nematodes use before excretion. To support this notion further, transferred to prelabeled LC vials, samples were stored at 4 °C and trans- an AANAT has been isolated from O. volvulus, which has a high ferred to the LC-MS thermostated autosampler cooled to 8 °C. degree of specificity for arylalkylamines like tyramine, tryptamine, or octopamine over arylamines or polyamines (25). LC-Electrospray Ionization-TOF-MS Analysis. The samples (3-μL injection vol- On excretion of the deactivated nematode neurotransmitter ume) were analyzed with electrospray ionization (ESI) TOF-MS (Agilent TOF N-acetyltyramine into the human host, it is undoubtedly trans- 6210; Agilent Technologies) and chromatographic separation by HPLC (Agilent 1200 LC; Agilent Technologies) with a flow rate of 70 μL/min using a ported into different tissues, including the liver, kidney, or spleen. × In these tissues, glucuronidation of the phenol moiety by a glu- T3 Atlantis column (3 mm, 1.0 mm 150 mm; Waters Corporation). The column temperature was maintained at 30 °C. Eluting buffers were buffer A curonosyltransferase yields NATOG before excretion (Fig. 3A). (0.1% HCOOH in H2O) and buffer B (0.1% HCOOH in MeOH). Gradients were Mammals use this metabolic pathway to convert exogenous phe- → → → → as follows: 0 4 min, 2% 2% buffer B; 4 25 min, 2% 95% buffer B; CHEMISTRY nols into more hydrophilic compounds that possess better excre- 25 → 30 min, 95% → 2% buffer B; 30 → 33 min, 2% buffer B; and 33 → 58 tion properties (26). min, and 2% → 2% buffer B. The chromatographic eluent was directly Understanding the lack of NATOG in healthy donors is also injected into the ion source without prior splitting. Data were collected critical. The simplest interpretation is that tyramine is not a using positive ESI mode scanning in the centroid mode from 100 to 500 m/z major metabolite of L-tyrosine conversion in humans, and is thus with a scan rate of 1.0 spectrum per second in a 2-GHz extended dynamic considered a trace amine. Diverse metabolic pathways are known range. The capillary voltage was 3,500 V; the nebulizer pressure, drying gas flow, and gas temperature were set to 20 psig, 7 L/min, and 350 °C, re- for L-tyrosine with hydroxylation to form L-3,4-dihydroxy- spectively. Parameters of the mass spectrometer were tuned prior to each phenylalanine (L-DOPA) by the L-tyrosine hydroxylase as the sample set with Agilent ESI tune mix for TOF systems (Agilent Technologies). major pathway (Fig. 3B), wherein L-DOPA is decarboxylated to LC-MS/MS fragmentation patterns were obtained on an Accurate Mass dopamine, followed by hydroxylation and methylation to epi- quadrupole TOF LC/MS 6520 (Agilent Technologies) at the indicated colli- nephrine. Furthermore, tyramine is not a product in the degra- sion-induced dissociation energy. dation process of L-tyrosine because the initial metabolic step is the conversion of the amine to 4-hydroxyphenylpyruvate by L- Data Preprocessing, Pattern Determination, and Statistical Analysis. The initial tyrosine transaminase (Fig. 3B). Indeed, the major pathway of screening for the identification of potent biomarker mass signals was ana- tyramine metabolism in humans is the oxidative deamination by lyzed using the statistical analysis program XCMS online (11). Different MAO to yield p-hydroxyphenylacetic acid, while N-acetyltyramine sample sets of onchocerciasis-positive and African-negative control samples is formed as a minor metabolite (27). AANATs in humans reg- were analyzed using the standard parameters for an ESI-TOF system. An ulate acetylation of arylalkylamines also using tyramine as a sub- example output for this initial screening analysis is shown in Fig. 1A. Sta- strate for this enzyme class (28). In a side context, it has been tistical comparison of the intensity data was conducted using the statistical ’ shown in rats that inhibition of MAO leads to increased forma- analysis program XCMS online built in Welch s t test. Quantitative data were tion of N-acetyltyramine as an alternative metabolic pathway obtained with manual mass peak integration using Mass Hunter Qualitative Analysis software, version B.03.01 (Agilent Technologies). ANOVA and an (27). This anomaly of L-tyrosine metabolism has also been de- unpaired two-tailed Student t test were performed using GraphPad Prism, scribed for CNS dysfunctions (29). It is interesting to note that the version 5.0b for Mac OS X (GraphPad Software). “ ” so-called has been linked to onchocerciasis and A complete description of materials and methods, including synthetic whether N-acetyltyramine has an impact on the pathology seen procedures, an ethics statement, and detailed quantification procedures, can may merit additional research (30). be found in SI Materials and Methods. In summary, we have identified an exogenous human urine metabolite NATOG that can be traced to an O. volvulus bio- ACKNOWLEDGMENTS. We thank Sara Lustigman, Peter Enyong, Nidia Rizzo, synthetic pathway. The unique structure of NATOG, coupled with Nancy Cruz-Ortiz, Mauricio Sauerbrey, Eduardo Catú, and Frank O. Richards for their assistance with sample collection. This study was supported by the its origin as a worm metabolite, demonstrates an intimate link with Worm Institute for Research and Medicine at The Scripps Research Institute the O. volvulus life cycle. It is noteworthy that doxycycline treat- and by a postdoctoral fellowship from the German Academic Exchange Ser- ment for O. volvulus infections greatly reduces the concentration of vice (to D.G.).

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