A Candida parapsilosis Overexpression Collection Reveals Genes Required for Pathogenesis.

Relative to the vast data regarding the virulence mechanisms of Candida albicans, there is limited knowledge on the emerging opportunistic human pathogen Candida parapsilosis. The aim of this study was to generate and characterize an overexpression mutant collection to identify and explore virulence factors in C. parapsilosis. With the obtained mutants, we investigated stress tolerance, morphology switch, biofilm formation, phagocytosis, and in vivo virulence in Galleria mellonella larvae and mouse models. In order to evaluate the results, we compared the data from the C. parapsilosis overexpression collection analysis to the results derived from previous deletion mutant library characterizations. Of the 37 overexpression C. parapsilosis mutants, we identified eight with altered phenotypes compared to the controls. This work is the first report to identify CPAR2_107240, CPAR2_108840, CPAR2_302400, CPAR2_406400, and CPAR2_602820 as contributors to C. parapsilosis virulence by regulating functions associated with host-pathogen interactions and biofilm formation. Our findings also confirmed the role of CPAR2_109520, CPAR2_200040, and CPAR2_500180 in pathogenesis. This study was the first attempt to use an overexpression strategy to systematically assess gene function in C. parapsilosis, and our results demonstrate that this approach is effective for such investigations.

Radioactive environment adapted bacterial communities constituting the biofilms of hydrothermal spring caves (Budapest, Hungary).

The thermal waters of Gellért Hill discharge area of the Buda Thermal Karst System (Hungary) are characterized by high (up to 1000 Bq/L) 222Rn-activity due to the radium-accumulating biogeochemical layers. Samples were taken from these ferruginous and calcareous layers developed on spring cave walls and water surface. Accumulation of potentially toxic metals (e.g. As, Hg, Pb, Sn, Sr, Zn) in the dense extracellular polymeric substance containing bacterial cells and remains was detected by inductively coupled plasma mass spectrometry. The comparison of bacterial phylogenetic diversity of the biofilm samples was performed by high throughput next generation sequencing (NGS). The analysis showed similar sets of mainly unidentified taxa of phyla Chloroflexi, Nitrospirae, Proteobacteria, Planctomycetes; however, large differences were found in their abundance. Cultivation-based method complemented with irradiation assay was performed using 5, 10 and 15 kGy doses of gamma-rays from a 60Co-source to reveal the extreme radiation-resistant bacteria. The phyla Actinobacteria, Firmicutes, Proteobacteria (classes Alpha- Beta- and Gammaproteobacteria), Bacteriodetes and Deinococcus-Thermus were represented among the 452 bacterial strains. The applied irradiation treatments promoted the isolation of 100 different species, involving candidate novel species, as well. The vast majority of the isolates belonged to bacterial taxa previously unknown as radiation-resistant microorganisms. Members of the genera Paracoccus, Marmoricola, Dermacoccus and Kytococcus were identified from the 15 kGy dose irradiated samples. The close relatives of several known radiation-tolerant bacteria were also detected from the biofilm samples, alongside with bacteria capable of detoxification by metal accumulation, adsorption and precipitation in the form of calcium-carbonate which possibly maintain the viability of the habitat. The results suggest the establishment of a unique, extremophilic microbiota in the studied hydrothermal spring caves.

Deinococcus budaensis sp. nov., a mesophilic species isolated from a biofilm sample of a hydrothermal spring cave.

Following the exposure of a biofilm sample from a hydrothermal spring cave (Gellért Hill, Budapest, Hungary) to gamma radiation, a strain designated FeSTC15-38T was isolated and studied by polyphasic taxonomic methods. The spherical-shaped cells stained Gram-negative, and were aerobic and non-motile. The pH range for growth was pH 6.0-9.0, with an optimum at pH 7.0. The temperature range for growth was 20-37 °C, with an optimum at 28 °C. Phylogenetic analysis based on the 16S rRNA gene sequence of the isolate indicated that the organism belongs to the genus Deinococcus. The highest sequence similarities appeared with Deinococcus hopiensis KR-140T (94.1 %), Deinococcus aquaticus PB314T (93.3 %) and Deinococcus aerophilus 5516T-11T (92.7 %). The DNA G+C content of the novel strain was 68.2 mol%. The predominant fatty acids (>10 %) were iso-C16 : 0 and C16 : 1ω7c, and the cell-wall peptidoglycan type was A3ß l-Orn-Gly2-3, corroborating the assignment of the strain to the genus Deinococcus. Strain FeSTC15-38T contained MK-8 as the major menaquinone and several unidentified phospholipids, glycolipids and phosphoglycolipids. Resistance to gamma radiation (D10) of strain FeSTC15-38T was <3.0 kGy. According to phenotypic and genotypic data, strain FeSTC15-38T represents a novel species for which the name Deinococcus budaensis sp. nov. is proposed. The type strain is FeSTC15-38T (=NCAIM B.02630T=DSM 101791T).