Anti-Leishmania amazonensis activity of the marine sponge Dercitus (Stoeba)

: The search for new therapeutic strategies for leishmaniasis treatment is essential due to the side effects of available drugs and the increasing incidence of resistance to them. Marine sponges use chemical compounds as a defense mechanism, and several of them present interesting pharmacological properties. The aim of this study was to evaluate the in vitro activity of the aqueous extract of the marine sponge Dercitus (Stoeba) latex against Leishmania amazonensis . MIC and toxicity against mammal cells were evaluated through broth microdilution assays. Transmission electron microscopy analysis was performed to assess possible effects on L. amazonensis ultrastructure. Arginase and proteolytic activities were measured by spectrometric methodologies. The extract of Dercitus (Stoeba) latex displayed antileishmanial activity and moderate toxicity against peritonial macrophages. Ultrastructural changes were observed after the growth of L. amazonensis promastigotes in the presence of the extract at 150 µg.ml -1 (IC 50 ), mainly on acidocalcysomes. The extract was able to inhibit the activity of arginase and serine proteases. This study shows that Dercitus (Stoeba) latex aqueous extract may be a novel potential source of protozoa protease inhibitors and drugs that are less toxic to be used in the treatment of L. amazonensis infections.


INTRODUCTION
Leishmaniasis refers to a group of diseases caused by protozoa Leishmania and is transmitted by the bite of an infected sandfl y.Clinically, the infectioncan progress in several ways, such as a chronic skin ulcer (cutaneous leishmaniasis), an erosive mucosal disease with severe facial disfigurement (mucocutaneous leishmaniasis), or a life-threatening systemic infection with hepato-splenomegaly and bone marrow involvement (visceral leishmaniasis) (Psicopo & Mallia 2009, David & Craft 2009).
Leishmania species express molecules, such as proteases, that play different roles in host tissue invasion and immune evasion mechanisms (Soares et al. 2003, McKerrow 2018).Among them, arginase, a metalloenzyme involved in the hydrolysis of L-arginine and the regulation of nitric oxide synthesis, plays an important role in the subversion of macrophage function during the disease (Balaña-Fouce et al. 2012).
Pentavalent antimonials are the firstchoice therapeutic regimen for the treatment of leishmaniasis.Whenever it does not work or cannot be prescribed, amphotericin B, pentamidine, or paromomycin serve as a second-choice line of treatment.Miltefosine, the only anti-Leishmania drug available for oral treatment, is normally prescribed for visceral leishmaniasis, and azoles, such as fluconazole, have been used for the treatment of the cutaneous form.However, the development of resistant strains has often led to therapeutic failure (Mans et al. 2016, Ponte-Sucre 2017, Rodrigues et al. 2006).
The relevance of non-conventional drugs in the treatment of infectious diseases has increased remarkably in recent years.Marine organismsoffer an unprecedented opportunity for pharmacological exploitation, mainly because their metabolites have already been reported as rich sources of novel chemical compounds which may lead to more effective drugs (Haefner 2003, Mollica et al. 2012).
In the present study, the antileishmanial activity of the endemic brazilian marine sponge Dercitus (Stoeba) latex (Moraes & Muricy 2007) collected from São Pedro and São Paulo Archipelago was evaluated.

Sponge sampling and identification
São Pedro and São Paulo Archipelago is one of the smallest and most isolated set of islands in the world, located 1000 km from the city of Natal, Rio Grande do Norte State, Northeastern Brazil.The sponge was manually collected by scuba diving (0˚55'N 29˚21'W) and preserved in 70% ethanol.The specimen was photographed in situ using a Nikonos V camera with 35 mm Nikkor lens and close-up kit.The positive film was digitalized using a Nikon Coolscan IV ED scanner (Moraes & Muricy 2007).Samples of the sponge were stored at -20˚ C for later evaluation.

Preparation of extracts
The sponge was washed with distilled water and transported to the laboratory under refrigeration.Then, it was kept in milli-Q water for 72 hours at 4 o C for the extraction.Thus, the extract was lyophilized in Speed Vac ® and resuspended in sterile distilled water to a stock solution of 100 mg.ml -1 .

Cytotoxicity assay
Mice were subjected to intraperitoneal stimulation with thioglycolate for 96 hours.
Then, peritoneal macrophages were obtained and incubated (5x10 5 /well) on 96-well plates with various concentrations (promastigotes IC 10 , IC 50 and MIC) of D. (S.) latex extract at 37 o C in 5% CO 2 for 24 h.In vitro cytotoxicity of the extract on peritoneal murine macrophages performed after intraperitoneal stimulation for 96 hours by thioglycolate was assessed by colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) reduction assay (Mosmann 1983).Firstly, macrophages were treated with 5 mg.ml -1 MTT and incubated for 3h at 37° C. Afterwards, dimethyl sulfoxide (DMSO) was added for 1 h to solubilize formazan crystals, and absorbance was measured at 540 nm.Cell viability was expressed as a percentage of average viable control cells.Three independent experiments were carried out in triplicates.Results were expressed as the concentration able to reduce cell viability by 50% (CC 50 ), in comparison to positive control.This protocol was approved by ethical committee of the UFRJ Health Sciences Center under the number IMPG020.

Activity against L. amazonensis amastigotes
Peritoneal macrophages adhered to glass coverslips were washed with PBS pH 7.2 and incubated with promastigotes (5x10 6 parasites.ml -1 ) at stationary growth phase in DMEM medium (Thermo Fisher Scientific, MA, USA) supplemented with 2% FCS at 37° C in 5% CO 2 for one hour.After interaction, cells were washed in PBS and incubated with DMEM supplemented with 10% FCS in the presence or absence of D. (S.) latex extract at promastigotes IC 50 .After 24h and 48h, the material was washed, fixed with methanol for 5 minutes, dehydrated with acetone-xylol, and stained with 36% Giemsa.Infected macrophages were visualized by light microscopy and counted.Three independent experiments were carried out in triplicate.

Transmission electron microscopy
Parasites incubated for 120 h in the presence or absence of D. (S.) latex aqueous extract at promastigotes IC 50 were fixed in 4% formaldehyde, 2.5 % glutaraldehyde, and 0.1 M sodium cacodylate buffer (pH 7.2) for 1h at room temperature.Cells were post-fixed in 1% OsO 4 , dehydrated and embedded as previously described (Bisaggio et al. 2006).Ultrathin sections obtained with a Reichert UltraCut S ultra microtome were stained with uranyl acetate and then analyzed in a FEI Morgagni F 268 transmission electron microscope, operating at 80 kV, equipped with a Megaview G2 Camera.Alternatively, cells were grown in the presence or absence of IC 50 extract for 72h, washed in PBS and placed on Formvar®coated grids (Agar Scientific, United Kingdom) and subsequently observed in the transmission electron microscope.A descriptive analysis of morphometric parameters was performed using ImageJ® (National Institutes of Health, MD, USA).
Morphometric parameters such as number, diameter, circularity and absolute volume were measured.

Determination of arginase activity
Arginase enzyme activity was measured as previously described (Kropf et al. 2005), with slight modifications.Promastigotes were incubated for 72 h in the absence or presence of D. (S.) latex aqueous extract at promastigotes IC 50 concentration.The parasites were lysed with 0.1% Triton X-100, and then 25 mM Tris-HCl pH 8.3 was added.After this, 10 mM MnCl 2 was added, and the enzyme was activated by heating for 10 min at 56° C. Arginine hydrolysis was conducted by incubating the lysate with 0.5 M L-arginine (pH 9.7) at 37° C for 15-20 min.The reaction was stopped with the addition of an acidic solution (H 2 SO 4 (96%)/H 3 PO 4 (85%)/H 2 O (1/3/7, v/v/v).Urea concentration was measured at 540 nm in spectrophotometer (FLUOstar OPTIMA, BMG Labtech, Offenburg, Germany) after addition of α-isonitrosopropiophenone (dissolved in 100% ethanol) followed by heating at 95° C for 30 min.

Statistical analysis
All experiments were repeated at least three times, all systems were performed in triplicate sets, and the results were expressed as means ± standard deviation.Data were analysed by Student´s t-test, and P<0.05 was considered statistically significant.

RESULTS
Antileishmanial activity and cytotoxicity assay D. (S.) latex aqueous extract inhibited the growth of L. amazonensis promastigotes in a dose dependent manner (data not shown), reaching absolute inhibition at 372 μg.ml -1 and the 50% inhibitory concentration (IC 50 ) was 150 µg.ml -1 .The control drugs Amphotericin B and Glucantime exhibited IC 50 of 4 µg.ml - and 60 µg.ml -1 , respectively.The IC 50 of sponge extract against L. amazonensis promastigotes, its CC 50 against mouse peritoneal macrophages, and the selective indexes are presented in Table I.
Treatment with the IC 50 of the sponge extract reduced the macrophage infection by 32.6% within 24 hours, and no significant difference was observed after a 48h-incubation (data not shown).Furthermore, it was observed a 21% reduction in the number of amastigotes per macrophage.

Transmission electron microscopy
Electron microscopy analysis (Figure 1) showed different degrees of damage in treated parasites, including the discontinuity of the nuclear membrane (Figure 1d, f, black arrows), formation of concentric structures with membranes (Figure 1e, black arrow) and the presence of several axonemes (at least three) in the cytoplasm (Figure 1g, black arrow).These findings indicate the presence of multiple flagella or the internalization of a single one, probably coiledappearing several times in the cut, suggesting effects on the cell cycle.Changes in the electron density of acidocalcisomes were also observed (Figure 1h, i, black arrows).Moreover, descriptive analysis of morphometric parameters revealed an increase in the number of acidocalcisomes and the circularity of these organelles after a 72h-incubation with the IC 50 extract (Figure 2 and Table II).Changes in the electron density of acidocalcisomes were also observed (h, i, black arrows).

Determination of arginase activity
Arginase activity was reduced in 28.66% in the presence of the sponge aqueous extract as demonstrated by the results shown in Figure 3.

Anti-Proteolytic activity
Data obtained revealed that the incubation of the protein cell extract with BSA in phosphate buffer pH 5.5 led to an inhibition of 35% of the proteolytic activity.This result was better than that observed with PMSF, a classic serine protease inhibitor (data not shown).Then, an assay was performed in order to analyze the hydrolysis of a specific fluorogenic substrate for serine protease.When the cysteine proteases were inhibited by E64, it was observed the inhibition of fluorescence emission when PMSF or aqueous extract of D. (S.) latex (IC 50 ) were added to the system (Figure 4).Treatment with E64 led to a significant hydrolysis of the fluorogenic substrate.Moreover, treatment with PMSF and aqueous extract D. (S.) latex significantly decreased substrate cleavage.Table II.Numerical and circular changes in acidocalcisomes of L. amazonensis promastigotes grown in the presence of the extract IC 50 for 72 hours.The (*) represent statistically significant data.

Number of acidocalcisomes
Mean circularity (nm) Mean diameter (nm) Absolute volume (x 10 6 nm 3 ) Control 9 ± 3 0,98 ± 0,03 0,26 ± 0,1 0,016 ± 0,02 Treated 17 ± 9* 0,94 ± 0,06* 0,26 ± 0,1 0,019 ± 0,02 An Acad Bras Cienc (2022) 94(3) e20211090 7 Huang 2015).Despite the changes in the number and shape of acidocalcisomes revealed in this study, protozoan susceptibility to antimicrobial drugs seems to be unrelated, as demonstrated by the use of sitamaquine (López-Martín et al. 2008).However, a recent study using nelfinavir, an HIV protease inhibitor, reported the accumulation of this drug in acidocalcisomes of resistant strains of L. amazonensis, suggesting that an increase in the number of this vesicle might be considered as a resistant profile feature (Kumar et al. 2013).Furthermore, an inositol triphosphate receptor located in acidocalcisomes of Trypanosoma brucei was recently identified and considered essential for the growth and the establishment of an efficient animal infection (Huang et al. 2013).All these characteristics point to acidocalcisome as an important multifunctional organelle associated to crucial biological processes among protozoa.The ability to survive and multiply within macrophages is a feature of several infectious agents, including Trypanosoma cruzi and Leishmania spp.In order to sustain a chronic infection, parasites must subvert macrophageaccessory cell activities and ablate the development of protective immunity (Alexander et al. 2002).Nevertheless, the most important mechanism for the killing of Leishmania and leishmaniasis control is the production of nitric oxide by macrophages (Holzmuller et al. 2006).The inhibition of arginase activity may influence parasite viability in L. major and L. infantum-infected macrophages (Iniesta et al. 2001).It is postulated that among Leishmania species, arginase activity must modulate nitric oxide synthase activity by using L-arginine, which is a common substrate for enzymatic activities (Boucher et al. 1999, Wanasen et al. 2008).In L. amazonensis, the arginase protein is concentrated in glycosomes both in promastigotes and amastigotes, suggesting that its location is important for enzyme activity, which modulates the L-arginine intracellular levels.Knockout parasites were able to infect macrophages but were not able to sustain the infection.It could be explained by a failure in modulating the availability of L-arginine to host cell and leading to an increased production of NO by infected macrophages (Da Silva et al. 2008).Promisingly, besides the effect on acidocalcisomes, the sponge aqueous extract was able to reduce arginase activity of L. amazonensis promastigotes, reinforcing the ability of this extract of modulating distinct mechanisms of L. amazonensis virulence.
Analysis of proteolytic enzymes of pathogenic organisms might lead to the design of powerful chemotherapeutic agents against these microorganisms (Grandgenett et al. 2007, Casgrain et al. 2016).The most studied classes of proteases in Leishmania spp.are cysteine-, metallo-, and serine proteases, since they are directly related to virulence.These enzymes cleave host proteins, neutralize immune response, disrupt fibronectin and extracellular matrix, overall enhancing promastigotes infectivity (Mottram et al. 2004).Then, proteases promote the survival of the parasite and the interaction with host tissues (Silva-Lopez et al. 2005).Results obtained suggest that D. (S.) latex aqueous extract may inhibit L. amazonensis serine proteases activity.Silva-Lopez and colleagues (2007) showed that the serine proteases inhibitors TPCK, benzamidine, and (ShPI-1), isolated from the sea anemone Stichodactyla heliantus affected cell viability and caused ultrastructural changes in the flagellar pocket, changes in membrane parasites, and the formation of intracellular vesicular bodies in L. amazonensis (Silva-Lopez et al. 2007).Nogueira and coworkers (2013) also showed a potent serine protease inhibitor purified from sea anemone which showed significant anti-T.cruzi and moderate anti-L.amazonensis activities (Nogueira et al. 2013).
From the results obtained in our study, it was observed that the aqueous extract of D. (S.) latex has antileishmanial activity, since it was able to disrupt meaningful cellular components and inhibit enzymes associated with virulence in the parasite.The study of the extract of D. (S.) latex is promising and contributes to the advancement in the search for new, more effective, and less toxic drugs that can be used in the treatment of cutaneous leishmaniasis.Furthermore, this study shows that marine animals may be a source of protozoa protease inhibitors.The limitation of a study carried out with extracts in which there is no isolation and identification of compounds is understood; however the contribution to the confirmation of the potential of marine sponges as a source of new drugs is considered.

Figure 1 .
Figure 1.Analysis by TEM of L. amazonensis promastigotes in absence (a-c) or presence (d-i) of IC 50 D. (S.) latex aqueous extract.Different degrees of damage were observed in treated cells, including the discontinuity of the nuclear membrane (d and f arrowhead), formation of concentric structures with membranes (e, black arrow), and the presence of several axonemes (at least three) in the cytoplasm (g, black arrow).Changes in the electron density of acidocalcisomes were also observed (h, i, black arrows).

Figure 2 .
Figure 2. Numeric analysis of the acidocalcisomes in L. amazonensis.The number of the organelles was analysed and compared between control and treated cells.The cells treated with the sponge aqueous extract were subdivided into two groups representing cells between 11 and 20, and more than 20 acidocalcisomes per cell.Results are expressed in percentage of cell populations.

| 11 DISCUSSION
Natural products have been successfully used in the search for compounds with anti-Leishmania activity (daSilva et al. 2018).Although most of natural compounds with antimicrobial activity are derived from plant, the use of marine substances seems to be very promising(Donia & Hamman 2003, Laport et al. 2009).In this study, D. (S.) latex aqueous extract presented antileishmanial activity against promastigote forms, and microscopy analysis revealed damage on several structures, including acidocalcisomes.These structures are dense acidic organelles with a high concentration of phosphorus, presented mostly in the form of polyphosphates complexed with calcium and other elements.Several functions have been attributed to the acidocalcisomes, such as the storage of high energy compounds, calcium and other cations, and the regulation of the intracellular pH and osmolarity(DoCampo &

Figure 3 .
Figure 3.Effect of the aqueous extract of Dercitus (Stoeba) latex on arginase activity of L. amazonensis promastigotes.Parasites were grown on Schneider medium at 26 o C for 72 h in the presence of 150 µg.ml -1 Dercitus (Stoeba) latex aqueous extract.Then, parasites were washed, lysed, and arginase activity was measured as described at Materials and Methods section.Values represent means +/-standard deviation of three different experiments.(*): P < 0.05.

Figure 4 .
Figure 4. Effect of Dercitus (Stoeba) latex aqueous extract on the proteolytic activity of L. amazonensis.Hydrolysis of the fluorogenic substrate Z-Phe-Arg 4-methoxy-β-naphthylamide by L. amazonensis (LA) promastigotes cell extract in the presence or absence of proteolytic inhibitors PMSF and/or E64 and/or D. (S.) latex aqueous extract.Trypsin was used as control.The results represent the average of three experiments in triplicate ± standard deviation.(*): P < 0.05.

Table I .
Inhibition of promastigote growth and peritoneal murine macrophages citotoxicity by D. (S.) latex aqueous extract.