Trichoderma asperelloides Spores Downregulate dectin1/2 and TLR2 Receptors of Mice Macrophages and Decrease Candida parapsilosis Phagocytosis Independent of the M1/M2 Polarization

The intensive use of pesticides to control pests in agriculture has promoted several issues relating to environment. As chemical pesticides remain controversial, biocontrol agents originating from fungi could be an alternative. Among them, we highlight biocontrol agents derived from the fungi genus Trichoderma, which have been documented in limiting the growth of other phytopathogenic fungus in the roots and leaves of several plant species. An important member of this genus is Trichoderma asperelloides, whose biocontrol agents have been used to promote plant growth while also treating soil diseases caused by microorganisms in both greenhouses and outdoor crops. To evaluate the safety of fungal biological agents for human health, tests to detect potentially adverse effects, such as allergenicity, toxicity, infectivity and pathogenicity, are crucial. In addition, identifying possible immunomodulating properties of fungal biocontrol agents merits further investigation. Thus, the aim of this study was to evaluate the effects of T. asperelloides spores in the internalization of Candida parapsilosis yeast by mice phagocytes, in order to elucidate the cellular and molecular mechanism of this interaction, as a model to understand possible in vivo effects of this fungus. For this, mice were exposed to a fungal spore suspension through-intraperitoneal injection, euthanized and cells from the peripheral blood and peritoneal cavity were collected for functional, quantitative and phenotypic analysis, throughout analysis of membrane receptors gene expression, phagocytosis ability and cells immunophenotyping M1 (CCR7 and CD86) and M2 (CCR2 and CD206). Our analyses showed that phagocytes exposed to fungal spores had reduced phagocytic capacity, as well as a decrease in the quantity of neutrophils and monocytes in the peripheral blood and peritoneal cavity. Moreover, macrophages exposed to T. asperelloides spores did not display the phenotypic profile M1/M2, and had reduced expression of pattern recognition receptors, such as TLR2, dectin-1 and dectin-2, all involved in the first line of defense against clinically important yeasts. Our data could infer that T. asperelloides spores may confer susceptibility to infection by C. parapsilosis.

For analysis of the microscopic characteristics (shape and measurements of conidiophores, supporting cells, phialides, conidia, and chlamydospores), slide cultures were performed in PDA and CMA and incubated at 25 °C for 72 hours. All slides were prepared using 3M KOH (Potassium hydroxide) and observed under a microscope.
For molecular analysis, genomic DNA was extracted from the culture incubated at 25 °C in PDA for three days, in darkness (MONTOYA et al., 2016). The partial sequence (ca. 563-615 bp) of the gene encoding for the elongation factor 1 alpha (tef1) was amplified. Amplification conditions and primers used were described according to Atanasova et al. (2013). The obtained amplicons were purified and sequenced by the Sanger method in an ABI 3500 (Life Technologies). The obtained forward and reverse sequences were assembled in BioEdit (HALL, 1999) and the consensus sequence (SAMUELS et al., 2010) was aligned with other phylogenetically related fungal sequences obtained from NCBI-GenBank in MAFFT (KATOH & STANDLEY, 2013). Protodrea pallida (CBS 29978) was used as an out group. The phylogenetic tree was reconstructed using Bayesian Inference in MrBayes v.3.2.2 (RONQUIST et al., 2012). The final tree includes a total of 53 partial tef1 sequences whose final alignment presented a total size of 591 bp. The nucleotide substitution model selected for the data set (HKY + I + G) was calculated in jModelTest 2 (DARRIBA et al., 2012) using the Akaike information criterion with a 95% confidence interval. Two independent runs were performed, each containing three hot chains and one cold chain; each run consisted of the Markov Chain Monte Carlo sampling (MCMC) with 200,000 generations. Finally, the first 25% of the MCMC generations was discarded as burn-in. The final tree was edited using the Adobe Illustrator CS6 (Adobe Systems).

Morphological and molecular identification of the LIBASP02 isolate
In order to identify if the species used in this work was T. asperelloides, after morphological analysis, the isolate showed radial growth with cottony mycelium.
Colonies turned green after 72 hours on PDA medium (Supplementary figure 1A, 1B and 1C). Likewise, the isolate formed conidiophores in the aerial mycelium with green smooth ovoid conidia in ampulliform phialides (Supplementary figure 1D, 1E and 1H). Pustules and chlamydospores were formed in all three analyzed culture media (Supplementary figure 1F and 1G). No soluble pigment was observed on all culture medium. All the morphological characteristics observed are in agreement with the description of T. asperelloides (SAMUELS et al., 2010).
In addition, Blast results showed that the LIBASP02 isolate had 100% of identity with the culture collection strains GJS04-217 and GJS 99-6 (T. asperelloides). Also, phylogenetic analysis based on the partial tef1gene revealed that the isolate LIBASP02 formed a monophyletic group (with a high posterior provability value) with T. asperelloides (supplementary figure 2). This analysis showed T. asperellum as a phylogenetic sister clade of T. asperelloides (supplementary figure 2). Finally, both the morphological and molecular data supported the identification of LIBASP02 isolate as T. asperelloides.     were washed once with PBS and 5x10 5 C. parapsilosis yeasts (ATCC 22019) were added in eachwell for 24 hours. After this period, wells were washed twice with PBS and cells on the slides were fixed with methanol and stained with May-Grünwald, followed by Giemsa. We observed that the yeast make pseudo hyphas what may be difficult to correct evaluate phagocytosis. We believe that an in vivo infection will be better to follow it up.