The Cell Wall Integrity (CWI) pathway is the primary signaling cascade that controls the de novo synthesis of the fungal cell wall, and in Saccharomyces cerevisiae this event is highly dependent on the RLM1 transcription factor. Here, we investigated the function of RlmA in the fungal pathogen Aspergillus fumigatus. We show that the ΔrlmA strain exhibits an altered cell wall organization in addition to defects related to vegetative growth and tolerance to cell wall-perturbing agents. A genetic analysis indicated that rlmA is positioned downstream of the pkcA and mpkA genes in the CWI pathway. As a consequence, rlmA loss-of-function leads to the altered expression of genes encoding cell wall-related proteins. RlmA positively regulates the phosphorylation of MpkA and is induced at both protein and transcriptional levels during cell wall stress. The rlmA was also involved in tolerance to oxidative damage and transcriptional regulation of genes related to oxidative stress adaptation. Moreover, the ΔrlmA strain had attenuated virulence in a neutropenic murine model of invasive pulmonary aspergillosis. Our results suggest that RlmA functions as a transcription factor in the A. fumigatus CWI pathway, acting downstream of PkcA-MpkA signaling and contributing to the virulence of this fungus.
Aspergillus fumigatus is an opportunistic human pathogen, which causes the life-threatening disease, invasive pulmonary aspergillosis. In fungi, cell wall homeostasis is controlled by the conserved Cell Wall Integrity (CWI) pathway. In A. fumigatus this signaling cascade is partially characterized, but the mechanisms by which it is activated are not fully elucidated. In this study we investigated the role of protein kinase C (PkcA) in this signaling cascade. Our results suggest that pkcA is an essential gene and is activated in response to cell wall stress. Subsequently, we constructed and analyzed a non-essential A. fumigatus pkcA G579R mutant, carrying a Gly579Arg substitution in the PkcA C1B regulatory domain. The pkcA G579R mutation has a reduced activation of the downstream Mitogen-Activated Protein Kinase, MpkA, resulting in the altered expression of genes encoding cell wall-related proteins, markers of endoplasmic reticulum stress and the unfolded protein response. Furthermore, PkcAG579R is involved in the formation of proper conidial architecture and protection to oxidative damage. The pkcA G579R mutant elicits increased production of TNF-α and phagocytosis but it has no impact on virulence in a murine model of invasive pulmonary aspergillosis. These results highlight the importance of PkcA to the CWI pathway but also indicated that additional regulatory circuits may be involved in the biosynthesis and/or reinforcement of the A. fumigatus cell wall during infection.
Standing among the front defense strategies against pathogens, host phagocytic cells release various oxidants. Therefore, pathogens have to cope with stressful conditions at the site of infection. Peroxiredoxins (Prx) are highly reactive and abundant peroxidases that can support virulence and persistence of pathogens in distinct hosts. Here, we revealed that the opportunistic human pathogen A. fumigatus presents three 1-Cys Prx (Prx6 subfamily), which is unprecedented. We showed that PrxB and PrxC were in mitochondria, while Prx1 was in cytosol. As observed for other Prxs, recombinant Prx1 and PrxC decomposed H2O2 at elevated velocities (rate constants in the 107 M−1s−1 range). Deletion mutants for each Prx displayed higher sensitivity to oxidative challenge in comparison with the wild-type strain. Additionally, cytosolic Prx1 was important for A. fumigatus survival upon electron transport dysfunction. Expression of Prxs was dependent on the SakAHOG1 MAP kinase and the Yap1YAP1 transcription factor, a global regulator of the oxidative stress response in fungi. Finally, cytosolic Prx1 played a major role in pathogenicity, since it is required for full virulence, using a neutropenic mouse infection model. Our data indicate that the three 1-Cys Prxs act together to maintain the redox balance of A. fumigatus.
Purpose Nanoparticles are resources of advanced nanotechnology being present in several products. Titanium dioxide nanoparticles are among the five most widely used NP currently expanding their benefits from the oil industry to the areas of diagnostic medicine due to their properties and small size. However, its impact on human health is still controversial in the literature. We aimed to evaluate the cytotoxicity of a new titanium NP functionalized with sodium carboxylic ligand (COOH − Na + ) in human keratinocytes (HaCaT) and human fibroblasts (HDFn). Methods The physical-chemical characterization was performed by the transmission electron microscopy (TEM), dynamic light scattering (DLS) and zeta potential techniques, respectively. MTT and LDH assays were used to assess cytotoxicity and cell membrane damage respectively, ELISA to identify the inflammatory profile and, reactive oxygen species assay and cytometry to detect reactive oxygen species and their relationship with apoptosis/necrosis mechanisms. Results The results demonstrated a decrease in cell viability at the highest concentrations tested for both cell lines, but no change in LDH release was detected for the HaCaT. The cell membrane damage was found only at 100.0 µg/mL for the HDFn. It was demonstrated that cytotoxicity in the highest concentrations evaluated for both cell lines for the 72 h period. The HDFn showed damage to the cell membrane at a concentration of 100 µg/mL followed by a significant increase in reactive oxygen species production. No inflammatory profile was detected. The HaCaT showed apoptosis when exposed to the highest concentration evaluated and HDFn showed both apoptosis and necrosis for the same concentration. Conclusion Thus, it is possible to conclude that the cytotoxicity mechanism differs according to the cell type evaluated, with HDFn being the most sensitive line in this case, and this mechanism can be defined in a dose and time dependent manner, since the highest concentrations also triggered death cell.
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