Aspergillus fumigatus calcium-responsive transcription factors regulate cell wall architecture promoting stress tolerance, virulence and caspofungin resistance.

Aspergillus fumigatus causes invasive aspergillosis, the most common life-threatening fungal disease of immuno-compromised humans. The treatment of disseminated infections with antifungal drugs, including echinocandin cell wall biosynthesis inhibitors, is increasingly challenging due to the rise of drug-resistant pathogens. The fungal calcium responsive calcineurin-CrzA pathway influences cell morphology, cell wall composition, virulence, and echinocandin resistance. A screen of 395 A. fumigatus transcription factor mutants identified nine transcription factors important to calcium stress tolerance, including CrzA and ZipD. Here, comparative transcriptomics revealed CrzA and ZipD regulated the expression of shared and unique gene networks, suggesting they participate in both converged and distinct stress response mechanisms. CrzA and ZipD additively promoted calcium stress tolerance. However, ZipD also regulated cell wall organization, osmotic stress tolerance and echinocandin resistance. The absence of ZipD in A. fumigatus caused a significant virulence reduction in immunodeficient and immunocompetent mice. The ΔzipD mutant displayed altered cell wall organization and composition, while being more susceptible to macrophage killing and eliciting an increased pro-inflammatory cytokine response. A higher number of neutrophils, macrophages and activated macrophages were found in ΔzipD infected mice lungs. Collectively, this shows that ZipD-mediated regulation of the fungal cell wall contributes to the evasion of pro-inflammatory responses and tolerance of echinocandin antifungals, and in turn promoting virulence and complicating treatment options.

Aspergillus fumigatus protein phosphatase PpzA is involved in iron assimilation, secondary metabolite production, and virulence.

Metal restriction imposed by mammalian hosts during an infection is a common mechanism of defence to reduce or avoid the pathogen infection. Metals are essential for organism survival due to its involvement in several biological processes. Aspergillus fumigatus causes invasive aspergillosis, a disease that typically manifests in immunocompromised patients. A. fumigatus PpzA, the catalytic subunit of protein phosphatase Z (PPZ), has been recently identified as associated with iron assimilation. A. fumigatus has 2 high-affinity mechanisms of iron acquisition during infection: reductive iron assimilation and siderophore-mediated iron uptake. It has been shown that siderophore production is important for A. fumigatus virulence, differently to the reductive iron uptake system. Transcriptomic and proteomic comparisons between ∆ppzA and wild-type strains under iron starvation showed that PpzA has a broad influence on genes involved in secondary metabolism. Liquid chromatography-mass spectrometry under standard and iron starvation conditions confirmed that the ΔppzA mutant had reduced production of pyripyropene A, fumagillin, fumiquinazoline A, triacetyl-fusarinine C, and helvolic acid. The ΔppzA was shown to be avirulent in a neutropenic murine model of invasive pulmonary aspergillosis. PpzA plays an important role at the interface between iron starvation, regulation of SM production, and pathogenicity in A. fumigatus.

Xenogeneic mesenchymal stem cell biocurative improves skin wounds healing in diabetic mice by increasing mast cells and the regenerative profile.

Introduction: Diabetes mellitus (DM) is a chronic disease and a major cause of mortality and morbidity worldwide. The hyperglycemia caused by DM induces micro and macrovascular complications that lead, among other consequences, to chronic wounds and amputations. Cell therapy and tissue engineering constitute recent therapeutic alternatives to improve wound healing in diabetic patients. The current study aimed to analyze the effectiveness of biocuratives containing human mesenchymal stem cells (MSCs) associated with a hydrogel matrix in the wound healing process and related inflammatory cell profile in diabetic mice. Methods: Biocuratives containing MSCs were constructed by 3D bioprinting, and applied to skin wounds on the back of streptozotocin (STZ)-induced type 1 diabetic (T1D) mice. The healing process, after the application of biocuratives with or without MSCs was histologically analyzed. In parallel, genes related to growth factors, mast cells (MC), M1 and M2 macrophage profiles were evaluated by RT-PCR. Macrophages were characterized by flow cytometry, and MC by toluidine blue staining and flow cytometry. Results: Mice with T1D exhibited fewer skin MC and delayed wound healing when compared to the non-diabetic group. Treatment with the biocuratives containing MSCs accelerated wound healing and improved skin collagen deposition in diabetic mice. Increased TGF-ß gene expression and M2 macrophage-related markers were also detected in skin of diabetic mice that received MSCs-containing biocuratives. Finally, MSCs upregulated IL-33 gene expression and augmented the number of MC in the skin of diabetic mice. Conclusion: These results reveal the therapeutic potential of biocuratives containing MSCs in the healing of skin wounds in diabetic mice, providing a scientific base for future treatments in diabetic patients.

Erratum to "Mitogen activated protein kinases (MAPK) and protein phosphatases are involved in Aspergillus fumigatus adhesion and biofilm formation" [Cell Surf. 1 (2018) 43-56].

[This corrects the article DOI: 10.1016/j.tcsw.2018.03.002.].

Activation of the Kinin B1 Receptor by Its Agonist Reduces Melanoma Metastasis by Playing a Dual Effect on Tumor Cells and Host Immune Response.

Metastatic melanoma is an aggressive type of skin cancer leading half of the patients to death within 8-10 months after diagnosis. Kinins are peptides that interact with B1 and B2 receptors playing diverse biological roles. We investigated whether treatment with B1 receptor agonist, des-Arg9-bradykinin (DABK), has effects in lung metastasis establishment after melanoma induction in mice. We found a lower number of metastatic colonies in lungs of DABK-treated mice, reduced expression of vascular cell adhesion molecule 1 (VCAM-1), and increased CD8+T-cell recruitment to the metastatic area compared to animals that did not receive treatment. To understand whether the effects of DABK observed were due to the activation of the B1 receptor in the tumor cells or in the host, we treated wild-type (WT) and kinin B1 receptor knockout (B1-/-) mice with DABK. No significant differences in the number of melanoma colonies established in lungs were seen between WT and B1-/-mice; however, B1-/-mice presented higher VCAM-1 expression and lower CD8+T-cell infiltration. In conclusion, we believe that activation of kinin B1 receptor by its agonist in the host stimulates the immune response more efficiently, promoting CD8+T-cell recruitment to the metastatic lungs and interfering in VCAM-1 expression. Moreover, treatment with DABK reduced establishment of metastatic colonies by mainly acting on tumor cells; hence, this study brings insights to explore novel approaches to treat metastatic melanoma targeting the B1 receptor.

Aspergillus fumigatus High Osmolarity Glycerol Mitogen Activated Protein Kinases SakA and MpkC Physically Interact During Osmotic and Cell Wall Stresses.

Aspergillus fumigatus, a saprophytic filamentous fungus, is a serious opportunistic pathogen of mammals and it is the primary causal agent of invasive aspergillosis (IA). Mitogen activated protein Kinases (MAPKs) are important components involved in diverse cellular processes in eukaryotes. A. fumigatus MpkC and SakA, the homologs of the Saccharomyces cerevisiae Hog1 are important to adaptations to oxidative and osmotic stresses, heat shock, cell wall damage, macrophage recognition, and full virulence. We performed protein pull-down experiments aiming to identify interaction partners of SakA and MpkC by mass spectrometry analysis. In presence of osmotic stress with sorbitol, 118, and 213 proteins were detected as possible protein interactors of SakA and MpkC, respectively. Under cell wall stress caused by congo red, 420 and 299 proteins were detected interacting with SakA and MpkC, respectively. Interestingly, a group of 78 and 256 proteins were common to both interactome analysis. Co-immunoprecipitation (Co-IP) experiments showed that SakA::GFP is physically associated with MpkC:3xHA upon osmotic and cell wall stresses. We also validated the association between SakA:GFP and the cell wall integrity MAPK MpkA:3xHA and the phosphatase PtcB:3xHA, under cell wall stress. We further characterized A. fumigatus PakA, the homolog of the S. cerevisiae sexual developmental serine/threonine kinase Ste20, as a component of the SakA/MpkC MAPK pathway. The ΔpakA strain is more sensitive to cell wall damaging agents as congo red, calcofluor white, and caspofungin. Together, our data supporting the hypothesis that SakA and MpkC are part of an osmotic and general signal pathways involved in regulation of the response to the cell wall damage, oxidative stress, drug resistance, and establishment of infection. This manuscript describes an important biological resource to understand SakA and MpkC protein interactions. Further investigation of the biological roles played by these protein interactors will provide more opportunities to understand and combat IA.

Nutritional Heterogeneity Among Aspergillus fumigatus Strains Has Consequences for Virulence in a Strain- and Host-Dependent Manner.

Acquisition and subsequent metabolism of different carbon and nitrogen sources have been shown to play an important role in virulence attributes of the fungal pathogen Aspergillus fumigatus, such as the secretion of host tissue-damaging proteases and fungal cell wall integrity. We examined the relationship between the metabolic processes of carbon catabolite repression (CCR), nitrogen catabolite repression (NCR) and virulence in a variety of A. fumigatus clinical isolates. A considerable amount of heterogeneity with respect to the degree of CCR and NCR was observed and a positive correlation between NCR and virulence in a neutropenic mouse model of pulmonary aspergillosis (PA) was found. Isolate Afs35 was selected for further analysis and compared to the reference strain A1163, with both strains presenting the same degree of virulence in a neutropenic mouse model of PA. Afs35 metabolome analysis in physiological-relevant carbon sources indicated an accumulation of intracellular sugars that also serve as cell wall polysaccharide precursors. Genome analysis showed an accumulation of missense substitutions in the regulator of protease secretion and in genes encoding enzymes required for cell wall sugar metabolism. Based on these results, the virulence of strains Afs35 and A1163 was assessed in a triamcinolone murine model of PA and found to be significantly different, confirming the known importance of using different mouse models to assess strain-specific pathogenicity. These results highlight the importance of nitrogen metabolism for virulence and provide a detailed example of the heterogeneity that exists between A. fumigatus isolates with consequences for virulence in a strain-specific and host-dependent manner.

Mitogen-Activated Protein Kinase Cross-Talk Interaction Modulates the Production of Melanins in Aspergillus fumigatus.

The pathogenic fungus Aspergillus fumigatus is able to adapt to extremely variable environmental conditions. The A. fumigatus genome contains four genes coding for mitogen-activated protein kinases (MAPKs), which are important regulatory knots involved in diverse cellular responses. From a clinical perspective, MAPK activity has been connected to salvage pathways, which can determine the failure of effective treatment of invasive mycoses using antifungal drugs. Here, we report the characterization of the Saccharomyces cerevisiae Fus3 ortholog in A. fumigatus, designated MpkB. We demonstrate that MpkB is important for conidiation and that its deletion induces a copious increase of dihydroxynaphthalene (DHN)-melanin production. Simultaneous deletion of mpkB and mpkA, the latter related to maintenance of the cell wall integrity, normalized DHN-melanin production. Localization studies revealed that MpkB translocates into the nuclei when A. fumigatus germlings are exposed to caspofungin stress, and this is dependent on the cross-talk interaction with MpkA. Additionally, DHN-melanin formation was also increased after deletion of genes coding for the Gα protein GpaA and for the G protein-coupled receptor GprM. Yeast two-hybrid and coimmunoprecipitation assays confirmed that GpaA and GprM interact, suggesting their role in the MpkB signaling cascade.IMPORTANCEAspergillus fumigatus is the most important airborne human pathogenic fungus, causing thousands of deaths per year. Its lethality is due to late and often inaccurate diagnosis and the lack of efficient therapeutics. The failure of efficient prophylaxis and therapy is based on the ability of this pathogen to activate numerous salvage pathways that are capable of overcoming the different drug-derived stresses. A major role in the protection of A. fumigatus is played by melanins. Melanins are cell wall-associated macromolecules classified as virulence determinants. The understanding of the various signaling pathways acting in this organism can be used to elucidate the mechanism beyond melanin production and help to identify ideal drug targets.

Mitogen activated protein kinases (MAPK) and protein phosphatases are involved in Aspergillus fumigatus adhesion and biofilm formation.

The main characteristic of biofilm formation is extracellular matrix (ECM) production. The cells within the biofilm are surrounded by ECM which provides structural integrity and protection. During an infection, this protection is mainly against cells of the immune system and antifungal drugs. A. fumigatus forms biofilms during static growth on a solid substratum and in chronic aspergillosis infections. It is important to understand how, and which, A. fumigatus signal transduction pathways are important for the adhesion and biofilm formation in a host during infection. Here we investigated the role of MAP kinases and protein phosphatases in biofilm formation. The loss of the MAP kinases MpkA, MpkC and SakA had an impact on the cell surface and the ECM during biofilm formation and reduced the adherence of A. fumigatus to polystyrene and fibronectin-coated plates. The phosphatase null mutants ΔsitA and ΔptcB, involved in regulation of MpkA and SakA phosphorylation, influenced cell wall carbohydrate exposure. Moreover, we characterized the A. fumigatus protein phosphatase PphA. The ΔpphA strain was more sensitive to cell wall-damaging agents, had increased ß-(1,3)-glucan and reduced chitin, decreased conidia phagocytosis by Dictyostelium discoideum and reduced adhesion and biofilm formation. Finally, ΔpphA strain was avirulent in a murine model of invasive pulmonary aspergillosis and increased the released of tumor necrosis factor alpha (TNF-α) from bone marrow derived macrophages (BMDMs). These results show that MAP kinases and phosphatases play an important role in signaling pathways that regulate the composition of the cell wall, extracellular matrix production as well as adhesion and biofilm formation in A. fumigatus.