*2.3. Hemagglutinating and Antimicrobial Activities of T. maculosa Nattectin-Like Toxin*

To confirm the lectin activity of the nattectin-like protein, hemagglutinating activity was evaluated (Figure 4). Nattectin-like protein agglutinated A type-tested human erythrocytes at a dose of 10 μg. Moreover, when this dose of nattectin-like toxin was previously incubated with D-galactosamine or D-Mannose, the agglutination capacity of erythrocytes by nattectin-like was preserved.

**Figure 4.** Assessment of the hemagglutinating activity of *Thalassophryne maculosa* toxin-TmC on human erythrocytes (**A**). TmC pre-incubation with D-galactosamine or D-Mannose was tested to confirm the permanence of the hemagglutinating pattern (**B**). Negative and positive controls were made by the respective addition of phosphate-buffered saline—PBS and distilled water—H2O.

Antimicrobial activity of nattectin-like toxin was performed against *Micrococcus luteus* A270, *Escherichia coli* SBS 363, and *Candida albicans* strains and the Nattectin from *T. nattereri* was tested in parallel as an intern control. We found that 10 μg of nattectin-like toxin did not inhibit the growth of the Gram-negative bacteria (*E. coli*) tested. Furthermore, corroborating the Nattectin effect (that inhibited the growth of all three strains evaluated at 10 μg), this dose of nattectin-like lectin showed an inhibitory effect on *M. luteus* and *C. albicans*.

#### *2.4. TmC4-47.2 Toxin-Induced Alterations in the Microcirculation*

The ability of nattectin-like toxin to induce changes in the microcirculation was evaluated using intravital microscopy assay in cremaster muscle of mice using the intra-scrotal application of 10 μg of the toxin and evaluation after 3 h of the injection. We observed in Figure 5 an intense leukocyte recruitment and rolling in the post-capillary venules immediately after the 3 h rest period (0 min.) that increased with time or stayed intense up to 30 min, as it can see from Figure 5B–E. Additionally, we registered a decrease in vessel flow after 10 min, followed by a complete stop of flow in venules and arterioles, possibly

due to fibrin thrombus formation after 20 min. Nattectin-like lectin did not induce changes in the caliber of arterioles or damage to muscle fibers.

**Figure 5.** Evaluation of changes in the cremaster muscle microcirculation by intravital microscopy after 3 h of the application of 10 μg of TmC4-47.2 toxin, according to the summarized protocol illustrated in the top-left corner. The tissue microvasculature was evaluated by an optical microscope coupled to a photographic camera in the control group treated with PBS (**A**) and in the set times of 0, 10, 20, and 30 min after the 3 h of exposure (**B**–**E**). An intense migration and rolling of leukocytes have been observed. In the 6E inset, arrows evidence the leukocytes in venules. Ar: arterioles; Vn: venules.

Our results presented in Figure 6A demonstrate that the intense and prolonged rolling of leukocytes induced by the nattectin-like toxin was followed by adhesion in the postcapillary venules, indicating the toxin's ability to promote extravasation of leukocytes into the surrounded interstitial tissue [22].

**Figure 6.** The number of rolling and adherent leukocytes controlled by 10 μg of the nattectin-like toxin TmC4-47.2 (Tmc) were counted in the post-capillary venules of mice at 0 to 30 min after 3 h of exposure using bright field intravital microscopy (**A**). The process of leukocyte recruitment, rolling (**B**), and adherence (**C**) was visualized in the cremaster vasculature of mice with inhibited alpha and beta integrins by neutralizing antibodies before intrascrotal toxin injection. The "\*" represents a statistically significant difference with negative control (non-treated) represented by the dashed basal line (**B**,**C**), and the "#" represents statistically significant difference of integrins-treated groups with the *Thalassophryne maculosa* toxins (Tmc), *p* < 0.05.

Integrins are a family of ubiquitous αβ heterodimeric receptors which combine with various ligands in physiological processes and disease, playing a crucial role in cell proliferation, tissue repair, inflammation, infection, and angiogenesis [23]. Next, using bright field intravital microscopy, we visualized the process of leukocyte recruitment in the cremaster vasculature of mice with alpha and beta integrins inhibited by neutralizing antibodies before intra-scrotal toxin injection. We found that treatment of mice with anti-CD29 (beta 1 integrin), anti-CD49e (alpha 5 integrin), and anti-CD49b (alpha 2 integrin) blocked 83%, 57%, and 69%, respectively, of the rolling leukocytes compared to untreated mice (Figure 6B). No inhibition was induced in mice pre-treated with anti-CD49a (alpha 1 integrin) or anti-CD106 (VCAM-1) neutralizing Abs (Figure 6B). Furthermore, the adherent leukocytes induced by nattectin-like lectin were entirely inhibited by anti-CD29, anti-CD49e, and anti-CD49a neutralizing Abs (Figure 6C). In contrast, anti-CD49b or anti-CD106 did not inhibit the adherence of leukocytes to venules.

#### *2.5. Induction of Acute Inflammation by Nattectin-Like Protein*

We used a mouse model of peritonitis to evaluate the inflammatory response profile induced by the nattectin-like toxin TmC4-47.2. Balb/c mice received 10 μg of the toxin intraperitoneally diluted in 500 μL of sterile phosphate-buffered saline (PBS), and control mice received only sterile PBS. Six, 16, and 24 h after the exposure, the animals were sacrificed, and the peritoneal cavity was washed to obtain the cell suspension. We analyzed the leukocyte influx recruited to the peritoneum by labeling surface molecules typical for each cell population and the dosage of cytokines (IL-1β, IL-6, and TNF-α) and chemokines (MCP-1, KC, and eotaxin) involved in the inflammatory process.

Our results in Figure 7A show that mice injected with the nattectin-like toxin exhibited intense leukocyte extravasation into the peritoneal cavity after 6 h of injection that was persistent for 24 h. The acute phase (6 h) of inflammation was characterized by the influx of eosinophils (Figure 7B) and mainly neutrophils (Figure 7C). After 16 h, macrophages (Figure 7D) entered the inflamed peritoneal cavity and remained for 24 h in the presence of a large number of both granulocytes.

**Figure 7.** The inflammatory response profile induced by the *Thalassophryne maculosa* toxin TmC4-47.2 (Tmc) was evaluated in a Balb/c mice model of peritonitis. The toxin was applied intraperitoneally at 10 μg in 500 μL of sterile phosphate-buffered saline (PBS). Control mice received sterile PBS. At six, 16, and 24 h after the exposure, the cell suspension from the peritoneal cavity was collected. The total cell number (**A**), the leukocyte influx to the peritoneum (**B**–**D**), and the dosage of IL-6 (**E**) and eotaxin (**F**) involved in the inflammatory process was analyzed.The "\*" represents a statistically significant difference with negative control (non-treated), *p* < 0.05.

Finally, we observed the production of IL-6, an important cytokine involved in the inflammatory process, only within 6 h after the 10 μg toxin injection (Figure 7E). Eotaxin, a chemotactic for eosinophils, was produced 6 h after injection, and increasing levels were observed up to 24 h (Figure 7F). In contrast, the injection did not promote the secretion of the cytokines IL-1β and TNF-α, as well as the neutrophil chemotactic factor, KC (data not shown).
