Project description:The study consists of the pathogens Lichtheimia corymbifera, Lichtheimia ramosa and non-pathogen Lichtheimia hyalospora untreated (control, CTRL) and during HSP90 inhibition (Geldanamycin, GDA), endoplasmic reticulum stress (Dithiothreitol, DTT), thermal stress (42°C, HEAT) and heat stress upon a concentration of 0.5 M NaCl (L. hyalospora only)
Project description:Mucormycosis is a life-threatening disease especially in immunocompromised patients that was caused my mucoralean fungi. The rate of mortality is tremendously increased in the last decades due to the lack of appropriate diagnostic tools, insufficient knowledge about the immune response toward the mucormycosis and unavailability of specific antifungal drugs. Several species of mucoralean fungi cause mucormycosis such as Lichtheimia, Rhizopus, and Mucor. Lichtheimia species ranks the second and third cause of mucormycosis in Europe and the USA, respectively. In this study, we investigated the receptors present on the surface of immune cells that bind to the spores of Lichtheimia. We focus on two strains of L. corymbifera (FSU:9682 and FSU:10164) using resting and heat-killed spores. Additionally, we choose alveolar macrophages (MH-S) to carry out our experiment. MH-S is the first line of defense in the lung and the major component in the innate immune system. MH-S surface proteins were biotinylated and incubated with Lichtheimia spores. The surface proteins and putative binding partners were enriched by streptavidin. LC-MS/MS analysis showed that several proteins are highly expressed in presence of Lichtheimia spores, of which the heat shock protein family A (HSPA8) was one of the most abundant proteins. FACS analysis and immunofluorescence examination confirmed that HSPA8 is highly abundant on the surface of the MH-S, but not on the surface of Lichtheimia spores. Moreover, our study showed that the intensity of HSPA8 on the surface of MH-S depends on the multiplicity of infection (MOI). Additionally, the blocking with anti-HSPA8 antibody reduced the capability of MH-S to engulf the Lichtheimia spores, but not Aspergillus fumigatus spores. This confirms that HSPA8 is specific to Lichtheimia. THis is the first study addressing the determination of surface receptors of alveolar macrophages that in the context of Mucoralean fungi.
Project description:During the infection process, both the host and the pathogen undergo a dynamic cascade of events that result in altered gene expression patterns (Westermann et al. 2012). With dual-RNA analysis, the complex interaction between a pathogen and its host during infection can be revealed simultaneously at their RNA levels (Westermann et al. 2012). Therefore, dual-transcriptomic analysis on JMRC:FSU:09682 and MH-S was performed to understand the pathogenesis at the genetic level to identify what kind of stresses that L. corymbifera encounter as well as the transcriptional response of macrophages during the infection. L. corymbifera (JRMC:FSU:09682) was cultivated for 7 days in KK1 medium (Kraibooj et al. 2014) at 37 °C . Murine alveolar MH-S macrophages (ATCC: CRL-2019) were cultivated in RPMI-1640 supplemented with 10% heat inactivated fetal bovine serum at 37 °C in 5 % CO2. Macrophages were seeded in 6 well plates at 106 cells per well to adhere overnight. Macrophages were infected with spores at a MOI (Multiplicity of Infection) of 5. After 3 hr of co-incubation with MH-S, extracellular spores were removed by at least 3 washing steps with pre-warmed RPMI-1640 (PAA Laboratories) and incubated for 13 hr in prior to the RNA extraction procedure. Controls in these experiments were fungus grown for 16 hours without macrophages and macrophages without fungal spores. Fungal RNA was isolated using RiboPure Yeast Kit (Thermo Fisher) and macrophage RNA was isolated with the RNeasy Mini kit (Qiagen), according to the manufacturers’ instructions. The concentration and purity of RNA samples were assessed using a NanoDrop spectrophotometer and Agilent 2100 bioanalyzer. A total of 681 L. corymbifera genes were differentially expressed during the co-infection with macrophages, with 629 down- (data not shown) and 52 up-regulated (Table 4.1). In order to systematically analyse the function of the differentially expressed genes (DEG), GO enrichment analyses were performed using FungiFun2.
