A novel rotifer-derived alkaloid paralyzes schistosome larvae and prevents infection

Schistosomes are parasitic flatworms that infect over 200 million people, causing the neglected tropical disease, schistosomiasis. A single drug, praziquantel, is used to treat schistosome infection. Limitations in mass drug administration programs and the emergence of schistosomiasis in non-tropical areas indicate the need for new strategies to prevent infection. It has been known for several decades that rotifers colonizing the schistosome’s snail intermediate host produce a water-soluble factor that paralyzes cercariae, the life-cycle stage infecting humans. In spite of its potential for preventing infection, the nature of this factor has remained obscure. Here, we report the purification and chemical characterization of Schistosome Paralysis Factor (SPF), a novel tetracyclic alkaloid produced by the rotifer Rotaria rotatoria. We show that this compound paralyzes schistosome cercariae and prevents infection, and does so more effectively than analogous compounds. This molecule provides new directions for understanding cercariae motility and new strategies for preventing schistosome infection.

1 Schistosomiasis, caused by the blood fluke Schistosoma, is a devastating and 19 widespread disease affecting more than 200 million people and threatening over 700 20 million people worldwide 1,2 . Treatment relies exclusively on one drug, praziquantel, for 21 which there are concerns of emerging drug resistance 3,4 and which has shown some 22 limitations in mass drug administration programs 5 . Efforts to develop an effective 23 vaccine to combat schistosomiasis have been unsuccessful; thus, there is an urgent 24 need for alternative drugs and better prevention strategies. Here we report the 25 discovery and purification of a novel tetracyclic alkaloid secreted by the rotifer Rotaria 26 rotatoria, which lives commensally on snails that serve as intermediate hosts for S. 27 mansoni 6 . This compound effectively paralyzes cercariae, the infective larvae of 28 schistosomes, at nanomolar concentrations. Furthermore, we show that worm burden 29 and disease pathology are significantly reduced when cercariae are pre-treated with this 30 Schistosome Paralysis Factor (SPF). Identifying this molecule provides new directions 31 for understanding motility of schistosome cercariae and new strategies for preventing 32

schistosomiasis. 33
Schistosomes have a complex life cycle that alternates between an intermediate host 34 (snail) and a definitive host (mammal) via two free-living, water-borne forms called 35 miracidia and cercariae, respectively 7 (Fig. 1a). For decades, inconsistency in cercarial 36 production by snails and infectivity of mammalian hosts has been observed in most 37 schistosome laboratories 8 . Intriguingly, Stirewalt and Lewis reported that rotifer 38 colonization on snail (Biomphalaria glabrata) shells significantly reduced cercariae 39 output, motility, and infectivity 6 . Furthermore, they observed that cercarial motility was 40 affected not only by the presence of rotifers, but also by rotifer-conditioned water, 41 indicating that rotifers released water-soluble molecules with paralytic activity. Almost 42 40 years have passed since this important finding, yet this factor's identity has remained 43 a mystery. 44 Encouraged by this anti-cercarial effect and its potential to prevent schistosome 45 infection, we sought to purify this paralyzing agent. We isolated individual rotifers from 46 snail shells and found two species, Rotaria rotatoria ( Fig. 1b) and Philodina acuticornis 47 ( Fig. 1c), as previously reported 6 . To identify which rotifer was responsible for the 48 paralytic effect, we grew clonal isolates of each species, producing rotifer-conditioned 49 artificial pond water (APW). Adding Rotaria-conditioned APW to freshly collected 50 cercariae resulted in gradual paralysis within five minutes (Fig. 1d). Most cercariae 51 stopped swimming and sank to the bottom of the dish. Tapping the dish could stimulate 52 their movement, but their response was limited to writhing on the dish bottom or short-53 distance swimming before becoming paralyzed again. In contrast, P. acuticornis-54 conditioned water had no effect (Fig. 1e). 55 To purify the paralyzing agent, we performed molecular weight cut-off filtration (MWCO) 56 of rotifer-conditioned water and found that the activity was present in the <650 Da 57 fraction. The <650 Da filtrate was fractionated by reversed-phase high-performance 58 liquid chromatography (RP-HPLC) (Fig 2a) and each fraction was tested on cercariae. 59 Paralysis was only observed following treatment with a peak eluting at 25-27 min (Fig.  60 2b). As expected, this peak was detected only in R. rotatoria-but not P. acuticornis-61 conditioned water (Fig. 2b). A second round of HPLC on this peak revealed one peak 62 (eluting at 24-26 min) with paralytic activity (Fig. 2c). A predominant signal of 273.16 Da 63 (M+H) in this peak was revealed by matrix-assisted laser desorption/ionization mass 64 spectrometry (MALDI-MS) (Fig. 2d). Consistent with the paralysis assay, this signal (m/z 65 273.16) was detected exclusively in the fraction eluting at 24-26 min but not in the 66 fractions before or after (Fig. 2e). These results suggested that the component with m/z 67 273.16 was the paralyzing agent, which we named "Schistosome Paralysis Factor" 68 (SPF). We then determined the monoisotopic mass for protonated SPF using high-69 resolution quadrupole time-of-flight (Q-TOF) MS, 273.1595 Da (Fig. 2f), suggesting 70 C 16 H 20 N 2 O 2 as the best-fitting formula for SPF. 71 To elucidate its structure, we purified SPF from R. rotatoria-conditioned water, obtaining 72 0.126 mg from 25 L. Nuclear magnetic resonance (NMR) spectroscopy revealed a novel 73 tetracyclic structure, which conformed to the best-fitting formula and contained one 74 configuration (110.9 kcal/mol) at the three chiral centers, C12-14. Based on energy-79 minimized models, the proton-proton spatial distance in the (R, S, S) configuration 80 matched the relative intensity of cross-peak signals in NOESY, whereas the distances 81 in the (S, R, R) configuration did not (Extended Data Table 2; Extended Data Fig. 7). 82 To test its dose dependency, we examined the paralytic effect of serially diluted SPF on 83 cercariae by quantifying their movement over time. In the absence of SPF, over 82% of 84 cercariae were free-swimming over three minutes (Fig. 3a). In 2.5 nM SPF, the 85 percentage of free-swimming cercariae dropped to 67% at three-minutes post drug 86 treatment. As the concentration of SPF increased, so did the rate of paralysis, and more 87 cercariae were paralyzed at the end of treatment. We observed maximum effects in 250 88 nM and 2.5 µM SPF, with the majority of cercariae paralyzed within 30 s. 89 Two natural compounds isolated from Streptomyces sp., ht-13-A and ht-13-B 9 , are 90 structurally related to SPF. All three alkaloids share a novel oxepineindole framework 91 fused with a pyrrolidine ring (Fig. 3a-c). Although synthesis of SPF has not been 92 achieved, total syntheses of ht-13-A and ht-13-B have been reported 10-12 . To test 93 whether this shared tetracyclic scaffold is responsible for the paralytic effect, we 94 analyzed structure-activity relationships by using ht-13-A, ht-13-B, three ht-13-A 95 derivatives, and one epimer in cercarial paralysis assays. Importantly, ht-13-A, although 96 not as potent as SPF, also had a paralytic effect on cercariae (Fig. 3b). In contrast, ht-97 13-B did not paralyze cercariae, suggesting that the extra methyl group disrupts 98 interaction with the target (Fig. 3c). Of the three ht-13-A analogs, only ht-13-A-pr 99 effectively paralyzed cercariae; it was more potent than ht-13-A, indicating that the 100 nature of the side chain is important for proper target interaction ( Fig. 3d-e). In contrast 101 to ht-13-A, the epimer was unable to paralyze cercariae, supporting the (R, S) 102 configuration of SPF at C12, 13. 103 Since motility of the cercarial tail is essential for swimming and provides force for skin 104 penetration 13-15 , we examined whether SPF prevented infection. We treated ~200 105 cercariae with different concentrations of SPF for 10 mins, and tested their ability to 106 infect mice after a 30-min exposure to their tails (N=6 for each condition). Six-weeks 107 post infection, we euthanized the mice, counted schistosomes recovered after hepatic 108 portal vein perfusion, and examined liver pathology. From controls, we recovered 83 109 adult worms on average (Fig. 4a), consistent with typical recoveries of ~40% 16 . Livers 110 from these mice appeared dark and contained extensive granulomas (Fig. 4a). In 111 contrast, we did not recover any adult worms from mice after treatment with 250 nM or 112 2.5 µM SPF (Fig. 4b) and no granulomas were observed (Fig. 4a). Histological 113 examination confirmed that these livers were free of schistosome eggs (Fig. 4e), 114 suggesting complete inhibition of infection. These data are consistent with the full 115 paralysis observed after treatment with 250 nM or 2.5 µM SPF (Fig. 3a). Although 25 116 nM SPF paralyzed most cercariae in vitro, the effects on mouse infection were not as 117 severe (Fig. 4a). Mechanical and/or chemical stimuli from mouse tails may overcome 118

SPF-induced paralytic effects at low SPF concentrations. Notably, neither Ht-13-A nor 119
Ht-13-A-pr blocked infection as completely as 250 nM SPF, even at 25 µM (Fig. 4a, c, d, 120 f, g). Under more realistic infection conditions, in which mouse tails were lifted 1-2 cm 121 from the bottom of the test tube containing cercariae, so they had to swim actively 122 towards the tail to infect the mouse, Ht-13-A and Ht-13-A-pr were still not as effective as 123 SPF, which completely blocked infection (Extended Data Fig. 8). 124 This work has identified a novel tetracyclic alkaloid, produced by the rotifer R. rotatoria, 125 that paralyzes the infective larvae of schistosomes. Although its mechanism of action 126 remains unknown, its chemical structure provides important clues. SPF contains a 127 serotonin backbone, suggesting that SPF might antagonize serotonin signaling, perhaps 128 via G protein-coupled receptors (GPCRs) or serotonin-gated channels. Consistent with 129 this idea, the structurally related compounds, ht-13-A and ht-13-B, bind several human 130 serotonin receptors 9 . In schistosomes, serotonin has been implicated in neuromuscular 131 functions in multiple life stages 17-20 ; knocking down a serotonergic GPCR (Sm5HTR) in 132 schistosomulae and adult worms led to decreased movement 21 . Interestingly, 133 praziquantel partially activates human HT2BR, suggesting that it may also target 134 schistosome serotonergic GPCRs 22 . 135 The chemical ecology underlying R. rotatoria's production of SPF is also unclear. 136 Whether SPF is used naturally to combat other aquatic creatures (e.g., to prevent other 137 rotifers from colonizing areas where R. rotaria live) and, thus, the effect on schistosome 138 cercariae is indirect, or whether SPF benefits the rotifer's commensal host will require 139 further study. In the past few decades, the discovery and development of natural   NanoMate robot (Advion, NY) 27 . Full spectra were acquired with resolution set at 100k. 207

Nuclear magnetic resonance (NMR) analysis 208
Purified bioactive materials were dissolved in 250 µL of CD 3 OD and transferred into a 5 209 mm Shigemi NMR tube with a glass magnetic plug with susceptibility matched to 210 CD 3 OD on the bottom. All NMR data were collected at 40°C on an Agilent VNMRS 750 211 MHz spectrometer equipped with a 5 mm Varian indirect detection probe with z gradient 212 capability. Collected NMR data included 1H spectrum, gradient selected correlation 213 spectroscopy (gCOSY), total correlation spectroscopy (TOSCY), nuclear overhauser 214 enhancement spectroscopy (NOESY) with a mixing time of 500 ms, heteronuclear 215 single quantum coherence spectroscopy ( 1 H-13 C HSQC) and heteronuclear multiple-216 bond correlation spectroscopy ( 1 H-13 C HMBC). The NMR spectra were analyzed using 217 Mnova NMR software (Mestrelab Research, Spain). 218

Determination of SPF concentration 219
The

Cercarial paralysis assay 231
To capture the whole field while avoiding excess reflected light in a well, we used the lid 232 of 96-well plate (Costar). 40 µL of artificial pond water containing ~50 cercariae were 233 added to each shallow well on the lid. 10 µL of SPF (dissolved in APW) was then added 234 to reach the final concentration indicated. Using a high-speed camera (Olympus i-235 SPEED TR), attached to a stereomicroscope (Leica MZ125), we recorded cercariae 236 movement at 20-60 fps at 1.25X magnification just prior to addition of test compounds 237 until 3-4 min after treatment started. Raw movies were converted to .avi files using i-238 SPEED Viewer and compressed into JPEG format using ImageJ (addition of compound 239 is considered time 0). We then counted the numbers of free swimming/paralyzed 240 cercariae every 5 s for 1 min and every 30 s thereafter for 3 min. The number of dead 241 cercariae (those that never swim before and after SPF treatment) were subtracted from 242 data. Experiments were performed in biological triplicate. 243

Mouse infectivity assay 244
Mouse infections were performed by exposing mouse tails to S. mansoni cercariae 245 according to standard protocol from BRI 16 with slight modifications. Briefly, we secured 246 mice in rodent restrainers (Thomas Scientific, Cat #551-BSRR) and put them vertically 247 on top of a rack with grids. We pipetted 100 µL of each drug at proper concentration into 248 a skinny glass tube (Fisher Scientific, Cat #14-958A) inside a 12 X 75 mm holding glass 249 tube (VWR, Cat # 47729-570). 300 µL of APW containing ~200 cercariae were pipetted 250 into each skinny tube and incubated for 10 mins before we inserted the mouse tail. 251 Mouse tails were wiped with APW-moistened Kimwipes, inserted into the skinny tube, 252 and exposed to cercariae for 30 mins. The mouse tail was touching the bottom of the 253 test tube unless otherwise specified. We euthanized and perfused these mice six week-254 post infection according to standard protocols 16 . For each drug, we initially used three 255 mice for controls (APW only) and three mice for each concentration tested except for 25 256 nM Ht-13-A and Ht-13-A-pr. We then repeated the experiments again with three mice 257 for each condition. In addition to that, we included six mice for 25 nM Ht-13-A and Ht-258 13-A-pr. 259

Statistical analysis 276
GraphPad Prism (Version 7) was used for all statistical analyses.