*4.2. Antifungal and Antibacterial Activity*

So far there are not many examples in the literature of IDPs with antifungal activity, despite the two decades elapsed since the definition of IDPs, around the 2000s [98–101].

As mentioned above, Jaburetox presents antifungal activity against filamentous fungi and yeasts [61]. Postal and co-authors observed the toxicity of Jaburetox against the phytopathogenic filamentous fungi *Mucor* sp. (at 10 µM) and *Penicillium herguei* (at 20 µM). *Rhizoctonia solani* was not susceptible to Jaburetox in the tested doses. Regarding yeasts, Jaburetox at 9 µM inhibited the multiplication of *Saccharomyces cerevisiae*, *Candida parapsilosis* and *Pichia membranifaciens* and at 18 µM, the peptide inhibited *Candida tropicalis*, *C. albicans* and *Kluyveromyces marxiannus*. Fluorescence microscopy of *S. cerevisiae* evidenced an increase in membrane permeability in Jaburetox-treated cells, using the SYTOX Green stain. In *C. tropicalis,* exposition to Jaburetox also induced the formation of pseudohyphae. These microscopy experiments were conducted at lower doses of Jaburetox (0.36–0.72 µM). In another work of our group, Broll and co-workers [62] showed that FITC-labeled Jaburetox interacted with *S. cerevisiae* cells, and remained bound to membrane cell debris even after yeast lysis. These results suggested that the target of Jaburetox is present on the yeast external membrane [62].

The antimicrobial activity of Jaburetox against some bacteria such as *Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa* and *Staphylococcus aureus* was observed in preliminary assays in the dose range of 0.25 µM to 13.5 µM [60]. As described earlier, Jaburetox was shown to permeabilize model membranes, as LUVs and PLBs, composed by different phospholipids and net charges; phosphatidylglycerol (PG) and phosphatidic acid (PA) with negative charges and the neutral phosphatidylethanolamine (PE), phosphatidylcholine (PC) and cholesterol (Ch) [66,76]. Many microorganisms contain negatively charged lipids in their membrane compositions, as PG and cardiolipin (CL) [102,103]. The main phospholipids found in the bacterium *S. aureus* membrane are PG, CL, and lysophosphatidylglycerol (LPG) [104,105]. In yeasts, a study using eight *C. albicans* azole-resistant and azole-sensitive strains demonstrated that the major phospholipids compositions in the plasma membrane of all the isolates were PC, PE, phosphatidylinositol (PI) and phosphatidylserine (PS). The percentage of phospholipids varied individually [106]. Interestingly, both microorganisms are susceptible to Jaburetox [60,61].

Soyuretox was also investigated regarding its antifungal activity. It was found to be active against *C. albicans*, *C. parapsilosis* and *S. cerevisiae* at 9 µM and 18 µM concentrations, similar to the fungitoxic doses reported for Jaburetox. For *C. albicans*, at the minimal inhibitory of 5 µM, production of superoxide anions was detected as part of the fungitoxic mode of action of Soyuretox. Binding of Soyuretox to *C. albicans* cells was observed by immunofluorescence [64].

The mechanism of antifungal activity of Jaburetox and Soyuretox remains elusive. It is known that the peptides permeabilize the fungal membrane, and cause change of fungal morphology, inducing formation of pseudohyphae, structures considered a stress and defense response mechanism of yeasts [48]. Moreover, the peptides induced intracellular production of superoxide anions in yeasts, causing oxidative stress. Our data suggest that these peptides probably interact with lipids in the fungal membrane (Figure 4). Although it still lacks experimental demonstration, it is plausible that

the IDP nature of these peptides could be relevant for their antimicrobial activities, as changes in order-disorder states upon ligand binding could possibly modulate their fungitoxic action.

**Figure 3.** Tissue-specific effects of urease-derived peptides in *Rhodnius prolixus*. In the flowchart, the brown boxes are the organs or the cells affected by the urease-derived peptides while the grey boxes indicate no change. The green boxes represent an increase in the assessed effect while the red boxes indicate a decrease. Acronyms stand for: 5-HT, Serotonin; ANTMIDGUT, Anterior midgut; CNS, Central nervous system; ET'S, Extracellular traps; JBTX, Jaburetox; MALP TUBULES, Malpighian tubules; NOS, Nitric oxide synthase; ROS, Reactive oxygen species; SG, Salivary glands; SYTX, Soyuretox; UAP, UDP-*N*-acetylglucosamine pyrophosphorylase; WGA, Wheat germ agglutinin. The numbers between brackets indicate the corresponding references: (1) Fruttero et al., [78]; (2) unpublished results; (3) Moyetta et al., [77]; (4) Fruttero et al., [82]; (5) Coste Grahl et al., [83]; (6) Kappaun et al., [64]; (7) and (8) unpublished results; (9) Staniscuaski et al., [81].

Jaburetox and Soyuretox are peptides prone to aggregation. In the studies aiming to characterize their 3D structures, TCEP, a potent reducing agent, was used to avoid aggregation during NMR experiments. It was demonstrated that Jaburetox and its truncated peptides (jbtx N-ter and jbtx C-ter) tend to form aggregates in solution and that their oligomerization state interfered in biological activities and membrane interactions [58,66,76]. Aggregation is known as an important factor in mode of action of antimicrobial peptides [102].

**Figure 4.** Schematic representation of the antifungal effects of Jaburetox and Soyuretox against filamentous fungi and yeasts.
