Microbial Warfare on Three Fronts: Mixed Biofilm of Aspergillus fumigatus and Staphylococcus aureus on Primary Cultures of Human Limbo-Corneal Fibroblasts

BackgroundCoinfections with fungi and bacteria in ocular pathologies are increasing at an alarming rate. Two of the main etiologic agents of infections on the corneal surface, such as Aspergillus fumigatus and Staphylococcus aureus, can form a biofilm. However, mixed fungal–bacterial biofilms are rarely reported in ocular infections. The implementation of cell cultures as a study model related to biofilm microbial keratitis will allow understanding the pathogenesis in the cornea. The cornea maintains a pathogen-free ocular surface in which human limbo-corneal fibroblast cells are part of its cell regeneration process. There are no reports of biofilm formation assays on limbo-corneal fibroblasts, as well as their behavior with a polymicrobial infection.ObjectiveTo determine the capacity of biofilm formation during this fungal–bacterial interaction on primary limbo-corneal fibroblast monolayers.ResultsThe biofilm on the limbo-corneal fibroblast culture was analyzed by assessing biomass production and determining metabolic activity. Furthermore, the mixed biofilm effect on this cell culture was observed with several microscopy techniques. The single and mixed biofilm was higher on the limbo-corneal fibroblast monolayer than on abiotic surfaces. The A. fumigatus biofilm on the human limbo-corneal fibroblast culture showed a considerable decrease compared to the S. aureus biofilm on the limbo-corneal fibroblast monolayer. Moreover, the mixed biofilm had a lower density than that of the single biofilm. Antibiosis between A. fumigatus and S. aureus persisted during the challenge to limbo-corneal fibroblasts, but it seems that the fungus was more effectively inhibited.ConclusionThis is the first report of mixed fungal–bacterial biofilm production and morphological characterization on the limbo-corneal fibroblast monolayer. Three antibiosis behaviors were observed between fungi, bacteria, and limbo-corneal fibroblasts. The mycophagy effect over A. fumigatus by S. aureus was exacerbated on the limbo-corneal fibroblast monolayer. During fungal–bacterial interactions, it appears that limbo-corneal fibroblasts showed some phagocytic activity, demonstrating tripartite relationships during coinfection.

The Oxazolidinone Derivative Locostatin Induces Apoptosis in CLL Cells through Inhibition of AKT and MAPK-ERK1/2 Signaling Under Conditions That Mimic the Tumor Microenvironment

BACKGROUND The Raf-1/MEK/MAPK-ERK1/2 pathway plays an important role in the pathogenesis and progression of multiple forms of cancer, including Chronic Lymphocytic Leukemia (CLL). Recent pre-clinical trials of the Raf-1 inhibitor Sorafenib demonstrate that targeting this pathway may represent a therapeutic option for CLL. Raf-1 activity is negatively regulated by the Raf-1 kinase inhibitory protein (RKIP). However, in CLL cells evidence suggests that phosphorylation of RKIP by protein kinase C (PKC) may restrict RKIP binding to Raf-1 and promote its binding and inhibition of G-coupled protein receptor 2 (GRK2). Consequently, GRK2, which would normally bind AKT and MEK1/2, would no longer suppress activity of these proteins. It is conceivable, therefore, that deregulation of RKIP may contribute to the over activity of the Raf-1 and AKT-mediated pathways that has been described in freshly isolated CLL cells. METHODS We sought to determine the effects of inhibiting RKIP on the survival and migratory capacity of CLL cells in vitro by using Locostatin, a specific inhibitor of RKIP binding to Raf-1 and GRK2. The proportion of apoptotic primary CLL cells following Locostatin treatment in either media alone or in the presence of a supportive stroma was assessed by flow cytometry. The mechanisms of action of Locostatin were investigated by western blotting for the phosphorylated forms of AKT and ERK1/2. RESULTS Locostatin induced a dose dependent decrease in the proportion of viable cells from CLL patients (n = 6) and a panel of haematological lines (n = 8). The IC50 for Locostatin was significantly (P < 0.001) lower against primary CLL samples analysed (11.14 +/- 4.86 µM) than in peripheral blood mononuclear cells from healthy volunteers (72.71 +/- 9.85 µM). Locostatin was also effective against CLL cells cultured in the presence of a CD40L-expressing fibroblast layer. Locostatin treatment reduced the expression of the chemokine receptor CXCR4 on the CLL cell surface with a concomitant decrease in their migration along a stroma-derived factor 1α (SDF-1α) gradient and significantly reduced the proportion of cycling CLL cells following induction by CpG oligonucleotide/IL2 (Dsp30/IL-2). We demonstrate by western blotting that RKIP is phosphorylated on S153 in CLL patient samples (Figure 1), supporting our hypothesis that RKIP may not be able to bind Raf-1 in these cells. Furthermore, we show that Locostatin treatment inhibits phosphorylation of AKT (S473) and ERK1/2 in CLL cells cultured in media alone or on CD40L fibroblasts (Figure 2). CONCLUSIONS These data suggest that deregulation of RKIP in CLL cells may contribute to their survival and proliferation by increasing the activities of AKT and ERK1/2. We show that Locostatin, a specific RKIP inhibitor, down-regulates the activity of both AKT and ERK1/2 in CLL cells cultured in media or a model of the tumor microenvironment, decreases cell viability and reduces the migratory and proliferative capacities of the leukemic cells. Our data suggest that RKIP may represent a novel target for therapy in CLL, which can be effectively inhibited by Locostatin. Figure 1 RKIP is phosphorylated on S153 in CLL patient samples. Figure 1. RKIP is phosphorylated on S153 in CLL patient samples. Figure 2 Locostatin inhibits AKT and ERK1/2 phosphorylation in CLL cells cultured in media or on a CD40L-expressing fibroblast monolayer. Figure 2. Locostatin inhibits AKT and ERK1/2 phosphorylation in CLL cells cultured in media or on a CD40L-expressing fibroblast monolayer. Disclosures Mulligan: Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi Aventis: Research Funding; Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria.

Enzymatic profile, adhesive and invasive properties of Candida albicans under the influence of selected plant essential oils.

The influence of essential oils (EOs) used at sublethal level, on the presence and intensity of Candida albicans virulence factors was evaluated. Minimal inhibitory concentrations (MICs) of Lemon balm, Citronella, Geranium and Clove oils were established as 0.097% (v/v). Using the agar plates with substrates for proteases, phospholipases and hemolysins it was shown that C. albicans ATCC 10231 and C. albicans ATCC 90028 strains differed in the type and amount of enzymes produced. No significant difference in their total amount could be detected after pretreatment for 24 h with EOs at ½ MIC. However, the short-term (1 h) acting oils at MIC caused a statistically significant reduction in this activity. In the API ZYM test it was demonstrated that both strains exhibited activity of the same 9 out of 19 enzyme types and that EOs caused a significant decrease in the release of some of them. In the presence of subMIC of EOs, or when the fungus had previously been exposed to the MIC of oil, germ tubes formation was significantly and irreversibly reduced. Such C. albicans spotted on the Spider agar containing EOs at subMICs were unable to penetrate the agar. A significant decrease in the C. albicans adhesion to the fibroblast monolayer with respect to controls was also demonstrated when yeasts had been exposed to EOs at MIC (1 h) in liquid medium. Thus, it has been shown that tested oils, used even at subMIC, exhibit significant activity reducing the presence/quantity of important C. albicans virulence factors.