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. 2020 Jul 15:10:306.
doi: 10.3389/fcimb.2020.00306. eCollection 2020.

Ethanolic Extract of the Fungus Trichoderma asperelloides Induces Ultrastructural Effects and Death on Leishmania amazonensis

Danielle de Sousa Lopes  1 , Uener Ribeiro Dos Santos  1 , Danielle Oliveira Dos Anjos  1 , Lauro José Caires da Silva Júnior  2 , Vanderlúcia Fonseca de Paula  2 , Marcos André Vannier-Santos  3 , Izaltina Silva-Jardim  1 , Thiago Castro-Gomes  4 , Carlos Priminho Pirovani  1 , Jane Lima-Santos  1
Affiliations

Ethanolic Extract of the Fungus Trichoderma asperelloides Induces Ultrastructural Effects and Death on Leishmania amazonensis

Danielle de Sousa Lopes et al. Front Cell Infect Microbiol. .

Abstract

The Trichoderma genus comprises several species of fungi whose diversity of secondary metabolites represents a source of potential molecules with medical application. Because of increased pathogen resistance and demand for lower production costs, the search for new pharmacologically active molecules effective against pathogens has become more intense. This is particularly evident in the case of American cutaneous leishmaniasis due to the high toxicity of current treatments, parenteral administration, and increasing rate of refractory cases. We have previously shown that a fungus from genus Trichoderma can be used for treating cerebral malaria in mouse models and inhibit biofilm formation. Here, we evaluated the effect of the ethanolic extract of Trichoderma asperelloides (Ext-Ta) and its fractions on promastigotes and amastigotes of Leishmania amazonensis, a major causative agent of cutaneous leishmaniasis in the New World. Ext-Ta displayed leishmanicidal action on L. amazonensis parasites, and its pharmacological activity was associated with the low-molecular-weight fraction (LMWF) of Ext-Ta. Ultrastructural analysis demonstrated morphological alterations in the mitochondria and the flagellar pocket of promastigotes, with increased lipid body and acidocalcisome formation, microtubule disorganization of the cytoplasm, and intense vacuolization of the cytoplasm when amastigotes were present. We suggest the antiparasitic activity of Trichoderma fungi as a promising tool for developing chemotherapeutic leishmanicidal agents.

Keywords: Leishmania amazonensis; Trichoderma; chemotherapy; leishmaniasis; leishmanicidal.

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Figures

Figure 1
Figure 1
Ext-Ta treatment decreased L. amazonensis promastigote viability. (A) Promastigotes of L. amazonensis were treated with crescent concentrations of Ext-Ta and pentamidine and (B) different concentrations of HMWF and LMWF for 24 h. (C) J774 macrophages were treated with crescent concentrations of HMWF and LMWF for 24 h. The cell viability was performed by MTT assay. A value of p < 0.05 was considered for statistical significance. One-way ANOVA followed by Tukey post-test was performed to establish the statistical significance between the treatments in relation to the control. Ext-Ta: ethanolic extract of T. asperelloides; HMWF: high-molecular-weight fraction; LMWF: low-molecular-weight fraction; 0: control. *p < 0.05; ***p < 0.001.
Figure 2
Figure 2
LMWF of Ext-Ta induces ultrastructural changes on L. amazonensis promastigotes. Promastigotes were treated with different concentrations of LMWF of Ext-Ta for 24 h, and the ultrastructure was analyzed by transmission electron microscopy. (A) Untreated control protozoan displaying the normal organization of the parasite cells; (B) promastigotes treated with 2.5 ng/μl, (C) 5 ng/μl, (D) 10 ng/μl, and (E,F) 20 ng/μl of LMWF. (B) Flagellar pocket presenting exosomes containing cytoplasmic material (arrowheads) enlarged contractile vacuole (arrow); (C) karyorrhexis (nuclear disruptor) with dense peripheral chromatin in direct contact with the cytoplasm (arrowheads); (D) parasite showing large autophagosome (arrow) and karyorrhectic nucleus displaying nuclear pores in direct contact with the cytoplasm (arrowheads); (E) Ext-Ta-treated parasites usually displayed numerous lipid bodies (L) and acidocalcisomes (arrows); (F) parasites presenting destroyed mitochondria (m). Fp, flagellar pocket; k, kinetoplast; L, lipid bodies; m, mitochondria; n, nucleus.
Figure 3
Figure 3
LMWF of Ext-Ta reduces parasite survival and causes ultrastructural changes on L. amazonensis amastigotes. (A) Phagocytic index of macrophages infected with L. amazonensis promastigotes and treated or not with 2.5 ng/μl of LMWF for 24 h. 0: control (untreated parasites). A value of p < 0.05 was considered for statistical significance; *p < 0.05 by comparison with Student t test. (B) Untreated culture of macrophages infected with L. amazonensis promastigotes (10:1 ratio) presenting intact amastigote inside a parasitophorous vacuole (arrow). (C,D) Macrophages infected and treated with 2.5 ng/μl of LMWF for 24 h showed parasites with numerous lipid bodies (C, L) which eventually coalesced, reaching the parasite cell surface (D, arrowhead). (D) The necrosis of the parasites culminated in the complete destruction of parasites (dp). N, macrophage nucleus.
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