The Polypeptides/Proteins Exuded from Mimosa caesalpiniifolia Seeds Induce Ultrastructural and Biomechanical Modifications of the Caenorhabditis elegans Surface and Inhibit the Nematode Motility.

: Natural products have been studied as potential anthelmintic drugs. This study aimed to evaluate the effects of the polypeptides/proteins exuded from Mimosa caesalpiniifolia mature seeds on the model nematode Caenorhabditis elegans . After seed immersion for 24 h at 25 ºC in sodium phosphate buffer, pH 7.0, containing 0.15 M NaCl and 1.0 M MgSO 4 , the constituent polypeptides/proteins were fractionated using ammonium sulfate precipitation followed by ultrafiltration and their effects on C. elegans motility were assessed. Moreover, we evaluated by Atomic Force Microscopy (AFM) whether exposition of the nematode to the polypeptide fraction Mc SEX10-2 could cause any alteration on the animal’s external surface (cuticle).


INTRODUCTION
Infections caused by parasitic nematodes constitute one of the major public health problems.Although of common occurrence, as they reach more than 24% of the world population, helminth infections are amongst the neglected tropical diseases (NTDs) [1][2][3].To further complicate the issue, NTDs occur mostly in the world's poor populations of low and middle-income countries, which partly explain the limited interests of the pharmaceutical industries on such investments to develop new drugs and diagnoses for these diseases, as they represent low-profit markets [4,5].Less than 1% of global health research is directed to diseases caused by nematodes and to the production and development of new drugs for that purpose [6].Estimates indicate productivity losses of up to US$ 138.9 billion annually due to nematode infection diseases [7].Nematode infection is also a problem in the cattle and small ruminant industry worldwide.For example, Haemonchus contortus is recognized as the most economically important parasitic nematode at endemic zones, chiefly due to its common occurrence and potential to provoke high rates of mortality in small ruminants [8,9].Importantly, the indiscriminate use of anthelmintic drugs has contributed to the selection and establishment of resistant parasites towards the current anthelmintic agents.Therefore, the discovery of novel drugs and the development of alternative therapies are urgently needed [10,11].
Caenorhabditis elegans is a free-living nematode that belongs to the order Rhabditida, naturally found in temperate climate soils [12].It shares many similarities with other parasitic nematodes.As such, it has been used as a model in experimental studies to assess the potential of various substances to act as anthelmintic drugs [13,14], because the animal is readily available, it can be easily handled, and the in vitro assay is inexpensive [15][16][17].
Mature seeds, under specific conditions (germination, for example), exude diverse chemical compounds.Amongst them, some polypeptides and proteins that eventually possess relevant biological activities against

Mimosa caesalpiniifolia seeds
Brazilian Archives of Biology and Technology.Vol.67: e24230057, 2024 www.scielo.br/babtbacteria, fungi, and plant and animal parasitic nematodes [18][19][20][21][22][23][24].For instance, the proteins exuded from Myracrodruon urundeuva mature seeds prevent the larval development of H. contortus [25].Isolation of natural polypeptides/proteins with relevant biological properties is challenging due to their low yield and the presence of contaminants.The use of synthetic bioinspired compounds is becoming more popular [26,27].They can be designed based on the primary structure of previously purified and characterized bioactive polypeptides/proteins, their associated biological and/or toxic activities can be simulated in silico employing publicly available prediction algorithms, and they can be synthesized with a high degree of purity [28].
Mimosa caesalpiniifolia Benth.belongs to the family Leguminosae, subfamily Mimosoideae.It is a native, fast-growing tree of northeastern Brazil, used as forage among other utilities [29][30][31].Encouraged by the need to seek new bioactive compounds against nematodes and considering the attractive and constant growth of the protein and peptide markets due to their lower price, non-toxic nature, and fewer adverse side effects in the treatment of diseases [32], we, recently, studied and reported the anthelminthic activity of the seed exudates of M. caesalpiniifolia against H. contortus [33].
In this work, we fractionated the M. caesalpiniifolia seed exudates into their constituent polypeptides/proteins and evaluated their effects on C. elegans motility.In addition, we assessed by Atomic Force Microscopy (AFM) whether exposition of the nematode to the polypeptide fraction McSEX10-2 could cause any alteration on the animal's external surface (cuticle).

Plant material
Mature seeds of Mimosa caesalpiniifolia, free from chemical or physical treatments, were commercially purchased from Arbocenter (Birigui, São Paulo, Brazil).

Cultivation of Caenorhabditis elegans
Cultivation of the Caenorhabditis elegans wild-type N2 was done according to the methodology described by Katiki [12].Cultures were maintained in the Nematode Growth Medium (NGM) at the Laboratory of Parasite Control of the Center for Biological and Health Sciences, UFMA.Briefly, C. elegans was kept in Petri dishes that contained NGM seeded with 50 µL of 1% (w/w) Escherichia coli (NA22 strain) suspension in Luria Bertani-LB medium (10 g Bacto-tryptone, 5 g Bactoyeast, 5 g NaCl, H2O to 1 liter, and pH adjusted to 7.0 with 1 M NaOH) [34,35].Nematodes were identified as been at the L1-L3 juvenile larval stages, and adults (L4), based on their size and the presence and extent of vulvar development [36].
To select L4 animals, four 4-7-day old cultures of C. elegans were transferred to a tube and the desired volume completed with the M9 buffer, pH 7.0, prepared with 3.0 g KH2PO4, 6.0 g Na2HPO4, 5.0 g NaCl, and 1.0 mL 1 M MgSO4 dissolved to 1.0 L distilled H2O.The suspension obtained, which contained animals at all stages of the nematode life cycle, was laid in a rack or 3 min to allow settling of the largest nematodes.The nematodes were separated according to their size using a 32 μm sieve.This procedure permitted to retain the largest nematodes, whereas the smaller ones passed through the lattice.The retained animals, which represented young adults, adults, and large dead nematodes, were recovered in the M9 buffer and the suspension obtained passed through a 53 μm sieve.The living, active young adult and adult nematodes were not retained, whereas the inactive, dead ones were trapped by the sieve.

Preparation and collection of exudates
The visually healthy M. caesalpiniifolia mature seeds were surface sterilized with 70% (v/v) ethanol for 5 min, rinsed three times with distilled water, and air-dried at room temperature [33].To collect the exudates, selected seeds with no visible cracks were distributed in 32 groups, each weighting on average 3 g, and transferred to 50 mL Erlenmeyer flasks.Then, the seeds were immersed in 12.0 mL of the M9 buffer and maintained at 25 °C for 24 h.The choice of the M9 buffer was because it was used in the assays with the nematode.The M. caesalpiniifolia seed exudates (McSEX) samples were collected, and the volumes recorded.
Brazilian Archives of Biology and Technology.Vol.67: e24230057, 2024 www.scielo.br/babtPreparation of the constituent polypeptide/protein fractions and protein determination.
The polypeptides/proteins present in the McSEX fraction were precipitated by addition of an amount of crystalline ammonium sulfate required to reach 90% saturation [37], while kept in an ice bath under continuous agitation.Next, the suspension was left standing for 4 h at 10 °C.The precipitated polypeptides/proteins were recovered by centrifugation at 15,000 x g, 4 °C, 30 min (Thermo scientific Heraeus Megafuge 16R centrifuge, DJB Labcare Limited, UK) and resuspended with and dialyzed (2 kDa cut off) against the M9 buffer for 24 h (with three buffer changes), at 10 °C.Then, the suspension obtained was heated to 80ºC in a water bath for 15 min and centrifuged at 15,000 x g for 10 min, at 4 ºC [38].The supernatant obtained, denominated McSEX90%/80°C, was recovered.The McSEX90%/80°C fraction was further fractionated using tangential flow filtration (TFF) coupled with a 30 kDa cut-off ultrafiltration membrane and centrifugation at 5,5 g for 2 h at 4 o C. The material that passed through the 30 kDa cut-off ultrafiltration membrane was then forced to pass, by centrifugation, through a 10 kDa cut-off ultrafiltration membrane.The retentate, which did not pass through the 10 kDa cut-off ultrafiltration membrane, was denominated McSEX30-10 fraction and comprised the proteins with molecular mass between 30 and 10 kDa.The permeate, which passed through the 10 kDa cut-off ultrafiltration membrane, was denominated McSEX10-2 fraction and encompassed the polypeptides with the molecular mass varying from 10 kDa to 2 kDa.The McSEX10-2 fraction was dialyzed (2 kDa cut-off membrane) for 24 h against the M9 buffer (with three buffer changes) at 10 °C.C. elegans motility was evaluated in 96-well culture plates, in which 100 μL adult nematodes, suspended in 10 mL M9 buffer, were dropped in each well.Next, 100 μL of the samples, at concentrations varying from 163.75 to 1.25 μg mL -1 , were added.In the controls, 100 μL of the M9 buffer was added instead of fractions.The 96-well culture plates were incubated, for 24 h, in a BOD-type greenhouse regulated to operate at 24 ± 1 ºC, RH ≥ 80 % [12].Next, motile and non-motile nematodes were observed for 5 s using an inverted microscope (Zeiss Lab A1, AXIO).The nematodes were considered alive when they showed some type of motility.Animals were considered dead when they did not present tail, head, or pharyngeal movements.

Assessment of the effects of the McSEX/10-2 fraction on the cuticle structure of C. elegans
To assess the possible alterations on the cuticle structure of C. elegans after treatment with McSEX/10-2, optical microscopy and atomic force microscopy (AFM) were employed.Adult nematodes, incubated for 24 h in the M9 buffer (control) or in the McSEX/10-2 fraction prepared in M9, at 1.34 µg mL -1 final concentration, were fixed in 5% formalin for 24 h before being transferred to glass slides, for AFM observations.

Atomic force microscopy analysis
AFM measurements were performed using a Multimode 8 microscope (Bruker, Santa Barbara, CA) in PeakForce Tapping Quantitative Nanomechanics mode, in air (23 o C temperature and 44% air humidity), for the acquisition of topography, adhesion, roughness, and stiffness data.Silicon-made probes, with a nominal spring constant of 0.4 N/m and tip radius of 2 nm, were used.The images were obtained approximately in the central region of the adult C. elegans, with a scan size of 5 x 5 µm and of 256 x 256 force curves.The mean square roughness (Rq) values were obtained from the height maps, according to the methodology used in [40].The adhesion and stiffness data were obtained from the 65,536 force curves produced on the nematode cuticle.The control and McSEX/10-2-treated nematodes were compared.For the adhesion calculation, the maximum negative deflection values for each force curve were evaluated.For stiffness data, the values were obtained from the slope of the force curve in the sample contact portion.

The hemolysis assay of McSEX/10-2 against bovine erythrocytes
The in vitro hemolysis assay was carried out by measuring the lysis of bovine erythrocytes as previously described [41], with minor modifications.Bovine whole blood was collected from the jugular vein of calves in Brazilian Archives of Biology and Technology.Vol.67: e24230057, 2024 www.scielo.br/babt a glass bottle containing heparin (5 IU mL -1 ) as anticoagulant.The red blood cells (RBC) were recovered and washed three times with 0.15 M NaCl.They were recovered by centrifugation at 300 x g (centrifuge Mikro 200R, Hettich, Germany), 5 min, 4 °C, followed by resuspension in 0.15 M NaCl.To carry out the hemolysis assay, the McSEX/10-2 fraction was dialyzed (2 kDa cut off) for 24 h, at 10 °C, against 0.05 M Na-phosphate buffer, pH 7.2, containing 0.15 M NaCl (PBS).Then, the protein content of the dialysate was quantified [39].
Aliquots (100 μL) of a 2.5% RBC suspension were transferred to microtubes (1.5 mL) and incubated for 30 min at 37 °C with 100 μL of the McSEX/10-2 fraction prepared in PBS at 45.00, 22.50, 11.25, 5.63, 2.81, and 1.41 μg mL -1 final concentrations followed by centrifugation at 300 x g, 5 min, 4 °C.The supernatant was collected and transferred to flat bottom wells of 96-well culture plates and hemolysis determined by measuring the absorbance at 450 nm using an automated microplate reader (Epoch, BioTek Instruments Inc., USA).Negative (0%) and positive (100%) hemolysis was achieved by treatment of RBC with PBS and 0.1% (v/v) Triton X-100, respectively, in the place of McSEX/10-2.The percentage of hemolysis was calculated using the following equation: [(Abs450 nm of RBC treated with McSEX/10-2 -Abs450 nm of RBC treated with PBS) / [(Abs450 nm of RBC treated with 0.1 % Triton X-100 -Abs450 nm of RBC treated with PBS)] x 100.

Statistical Analysis
Data from protein content were compared using ANOVA followed by the Tukey test (P<0.05).Inhibitory Concentration (IC50) of samples against nematodes was calculated using GraphPad Prism 8.0 (San Diego, CA, USA).The data of adult C. elegans observed in AFM and FIJI/IMAGEJ were analyzed using D'Agostino & Pearson for normality [42], and the means were compared using the Kolmogorov-Smirnov test (P<0.05).

The protein content of the seed exudates and fractions
The

Ultrastructural and nanomechanical characteristics of C. elegans treated with McSEX/10-2
Analyses by Atomic force microscopy analysis revealed (Figures 1A and 1B) the ultrastructural morphology and biomechanical properties of the nematode were altered, as revealed by AFM data.Figures 1A (control) and 1B (treated), in combination with the corresponding three-dimensional maps (Figures 1C  and 1D), revealed a significant (p<0.05)decrease in the cuticle height in adult C. elegans (571.0 ± 185.9 nm) incubated with McSEX/10-2 (1.34 µg mL -1 ), in comparison with that of control animals (734.5 ± 244.7 nm).Moreover, the AFM images showed Rq values of 39.7 nm for the McSEX/10-2-treated nematodes and 68.9 nm for the control animals.Such a significant (p<0.05)difference denotes a decrease in the surface roughness of the McSEX/10-2-treated animals.The AFM results also revealed differences in the biomechanical properties of the animals treated with McSEX/10-2 in comparison with controls (Figure 2).

Assessment of the cytotoxic activity of McSEX/10-2 against mammalian cells
The McSEX/10-2 fraction exhibited low hemolytic activity on animal red blood cells, resulting in only 1.8% hemolysis at a protein concentration of 45.00 μg mL -1 (Table 2).

DISCUSSION
During evolution, plants were exposed to a variety of abiotic and/or biotic factors, which, under certain circumstances, may have been detrimental to their growth, development and productivity [43,44].Seed exudates represent a useful source of defense molecules, which are mainly polypeptides/proteins and secondary metabolites, some of which have the capacity to specifically inhibit the development of certain pathogens [20,22,[45][46][47][48].
The use of C. elegans as an experimental model for anthelmintic testing of plant substances is of great importance for the development of new products [17,49].The experimental inhibition of the motility of adult C elegans observed herein, promoted by the McSEX, McSEX90%/80˚C, McSEX/30-10, and McSEX/10-2 fractions, may be a consequence of some deleterious effects that these products exerted on the animal, which could, eventually, influence survival [50].Several proteins exuded from seeds, such as proteases, chitinases, and lectins may interfere with the general physiology of nematodes, for example [22,25,[51][52][53].Nevertheless, our work shows that the M. caesalpiniifolia mature seed exudates and their constituent polypeptide/protein fractions presented anthelmintic potential (Table 1), as their concentrations to inhibit the motility of the exposed adult C. elegans by 50% were lower than those reported by other studies [25,54,55].
There is a plethora of published works that report the anthelminthic activity of plant peptides, highlighting the importance of these molecules as potential agents to combat nematodes.For instance, the peptides extracted from the leaves of Oldenlandia affinis display activity against Schistosoma [56].The nematicidal activity of cyclotids, namely kalata B1, cycloviolacin O2, and hyen D, which were isolated from plants, was assessed on C. elegans larvae [17].In addition, the bioactive proteins exuded by M. caesalpiniifolia mature seeds showed stage-specific inhibition of H. contortus [25].These anthelmintic molecules may have access to the nematode target tissues after oral ingestion or diffusion across the animal cuticle, which although a highly impermeable barrier between the animal and its environment, it is a permeable exoskeleton for several drugs that can diffuse across this extracellular matrix and reach the plasma membrane [57,58], to which they can exert local deleterios effects or internalized.Indeed, a strong example is that regarless the nematodes behave as a free-living, animal or plant parasites, they are sensitive to the crystal (Cry) protein Cry5B.It belongs to a family of membrane pore-forming proteins synthetized by the Gram-positive bacterium, Bacillus thuringiensis.The constituent monomers of the Cry protein monomers interact with the intestinal cell receptors, oligomerize, get inserted into the plasma membrane and form pores that disrupt the cell integrity, ionic balance, and membrane potentials, causing death or severe disruption of the cell activity [58].Thus, besides to be essential for maintenance of the body morphology and integrity, this exoskeleton has a critical role in locomotion via attachments to body-wall muscles, confers environmental protection, and allows growth by molting [57].
Brazilian Archives of Biology and Technology.Vol.67: e24230057, 2024 www.scielo.br/babtTo verify whether the McSEX/10-2 fraction would alter the nematode's motility by inducing modifications of its cuticle, we used AFM.This constituted a valuable approach since McSEX/10-2 promoted remarkable ultrastructural and nanomechanical alterations on the C. elegans cuticle (Figures 1 and 2).The cuticle surface is especially important for the host-parasite interaction.For instance, when the parasitic nematode H. contortus physically interacts with the host tissue, changes in the lipids of its surface might establish the onset of the infection process [59,60].
To the best of our knowledge, for the first time, this study shows that the constituent polypeptides/proteins from M. caesalpiniifolia mature seed exudates promote the reduction of the height and roughness of the C. elegans cuticle (Figures 1B and 1D).The C. elegans cuticle consists mainly of cross-linked collagens associated with the insoluble proteins named cuticlins and with glycoproteins and lipids [57].Previously, it was shown that residues of cyclotides, a class of disulfide-rich cyclic peptides from plants [61], bind to the constituent lipids of the cuticle surface by hydrophobic interactions [62].Nevertheless, further studies are needed to define the responsible agent(s) and elucidate the interaction (mechanisms) of the constituent polypeptides/proteins of the M. caesalpiniifolia exudates with the nematode cuticle and the importance of the cuticle height and roughness reduction on the mechanism of action of these molecules.As depicted in Figure 2, the AFM approach used allowed us to detect nanoscale alterations on the C. elegans cuticle stiffness and adhesion, after treatment with McSEX/10-2.As the control and the McSEX/10-2-treated nematodes were analyzed at the same life cycle stage, the alterations observed may be related to the interaction of the yet unknown polypeptide(s)/protein(s) of the M. caesalpiniifolia exudates with the nematode cuticle.We also observed increased adhesion forces on the C. elegans cuticle after incubation of the nematode with McSEX/10-2.It is important to notice that adhesion forces are a combination of electrostatic, van der Waals and capillary forces promoted by chemical bonds [63].Especially in the cases of non-functionalized probes (as used in this research), the adhesion forces are taken as nonspecific interactions and it is not possible to separate individually the contribution of each one of these forces.However, as the probes used in the AFM analyses were made of the same material (Si) and the experiments and observations were performed under the same conditions (temperature and air humidity), differences in the adhesion forces between the controls and the McSEX/10-2-treated animals are plausible.The scatter plots (Figure 2B) show values that suggest a trend of increasing adhesion force after the McSEX/10-2 treatment of C. elegans.As reported by Essmann and coworkers [64], in their Young's modulus results of the C. elegans cuticle over 18 days, the scattering direction suggests the trend of the kinetic events.
To treat nematode infection orally with any drug, one should be aware of the possible side effects it can cause in the host.The McSEX/10-2 fraction tested caused very low hemolysis of bovine blood cells (Table 2), even at concentrations higher than the IC50 values needed to arrest C. elegans motility.Nevertheless, as a perspective, aiming at achieving higher anthelmintic efficacy of the synergistic effect of McSEX/10-2, when used in combination or together with other anthelmintic drugs, and at different nematode life-stage, must be addressed, as they furnish directions for the bio-inspired design of novel medicines.

CONCLUSION
The results presented herein indicate that the polypeptides/proteins present in the exudates from M. caesalpiniifolia mature seeds caused harmful effects against the nematode C. elegans.Accordingly, they have the potential to bioinspire the design of new synthetic peptides that could control, alone or in combination with other drugs, parasitic nematodes.

McSEX/ 10 - 2
promoted a decrease in the nematode cuticle stiffness to 8.586 ± 0.558 N/m in relation to 11.11 ± 0.863 N/m observed in the control animals (p<0.05).Contrarily, the cuticle surface adhesion increased to 6.693 ± 3.537 nN in the McSEX/10-2-treated C. elegans in comparison to 5.029 ± 1.499 nN verified for the control animals (Figures 2A and 2B).

Figure 1 .
Figure 1.Structure of Caenorhabditis elegans cuticle.(A) Atomic force microscopy images of C. elegans treated with the M9 buffer (control) and with (B) the McSEX/10-2 fraction (1.34 μg mL -1 final concentration), showing its respective three-dimensional maps (C and D).

Table 1 .
).Effect of McSEX, McSEX90%/80˚C, McSEX/30-10, and McSEX/10-2 on Caenorhabditis elegans motility IC50 denotes the protein concentration that inhibited the motility of 50% of the nematode population tested.The values represent the mean ± standard deviation of three independent experiments.** 95% CL means the average of inferior and superior confidence limits at 95 % probability. *