Aspects of the progesterone response in Hortaea werneckii: Steroid detoxification, protein induction and remodelling of the cell wall.

Progesterone in sublethal concentrations temporarily inhibits growth of Hortaea werneckii. This study investigates some of the compensatory mechanisms which are activated in the presence of progesterone and are most probably contributing to escape from growth inhibition. These mechanisms lead on the one hand to progesterone biotransformation/detoxification but, on the other, are suggested to increase the resistance of H. werneckii to the steroid. Biotransformation can detoxify progesterone efficiently in the early logarithmic phase, with mostly inducible steroid transforming enzymes, while progesterone biotransformation/detoxification in the late logarithmic and stationary phases of growth is not very efficient. The relative contribution of constitutive steroid transforming enzymes to progesterone biotransformation is increased in these latter phases of growth. In the presence of progesterone, activation of the cell wall integrity pathway is suggested by the overexpression of Pck2 which was detected in the stationary as well as the logarithmic phase of growth of the yeast. Progesterone treated H. werneckii cells were found to be more resistant to cell lysis than mock treated cells, indicating for the first time changes in the yeast cell wall as a result of treatment with progesterone.

Molecular characterization of a ribosome-associated Hsp70-homologous gene from Rhizopus nigricans.

A ribosome-associated Hsp70-homologous gene (Rnssb-1) was isolated from the genomic library of the filamentous zygomycete fungus Rhizopus nigricans. The nucleotide sequence of a genomic clone encoded the N-terminal part of a protein with high similarity to the yeast SSB ribosome-associated chaperones. The missing 3' end of the gene was obtained by 3' RACE. The Northern blot analysis showed that the Rnssb-1 gene is constitutively expressed and is not induced upon heat shock at 37 degrees C. The primary structure analyses revealed that the coding region of the Rnssb-1 gene is interrupted by at least four introns. Their splicing was not inhibited by exposure of the organism to heat shock as proven by RT-PCR. A Southern blot analysis of R. nigricans genomic DNA confirmed the presence of two additional gene copies of ribosome-associated Hsp70 genes in the fungal genome.

Isolation, partial length sequence and expression of steroid inducible hps 70 gene from Rhizopus nigricans.

Previous studies have shown that filamentous fungus Rhizopus nigricans responds to addition of different steroids into growth medium with induction of hydroxylation system and that some steroids provoke stress response. The purpose of this study was to investigate whether those steroids provoke induction of Hsp70 gene(s), well studied markers of stress response in different cells and organisms. The expression studies of fungal Hsp70 gene(s) using Northern blot analysis showed that fungal hsp70 mRNA was upregulated after treatment of mycelia with deoxycorticosterone and testosterone, but not after exposure to progesterone. In addition, expression of fungal Hsp70 mRNA was elevated after exposure of mycelia to heat shock (32°C), ethanol, heavy metal (CuSO4), and oxidative stressor (H2O2), whereas treatment of mycelia with osmotic stressor (KCl) didn't have any influence on stress protein expression. The partial nucleotide sequence and deduced amino acid sequence homology search revealed that the cDNA clone (λ hs20/2), isolated from cDNA library prepared from heat shock treated fungal mycelia, contained Hsp70 gene of DnaK subfamily.

Molecular characterization of genes encoding cytosolic Hsp70s in the zygomycete fungus Rhizopus nigricans.

Previous studies have shown that some stressors, including steroid hormones 21-OH progesterone and testosterone, stimulate the accumulation of heat shock protein 70 (hsp70) messenger ribonucleic acid (mRNA) population in the zygomycete filamentous fungus Rhizopus nigricans. In this study we report the cloning of 3 R nigricans hsp70 genes (Rnhsp70-1, Rnhsp70-2, and Rnhsp70-3) encoding cytosolic Hsp70s. With a Southern blot experiment under high stringency conditions we did not detect any additional highly homologous copies of the cytosolic hsp70 genes in the R nigricans genome. Sequence analyses showed that all 3 genes contain introns within the open reading frame. The dynamics of the R nigricans molecular response to progesterone, 21-OH progesterone, and testosterone, as well as to heat shock, copper ions, hydrogen peroxide, and ethanol was studied by temporal analysis of Rnhsp70-1 and Rnhsp70-2 mRNA accumulation. Northern blot experiments revealed that the Rnhsp70-2 transcript level is not affected by testosterone, whereas mRNA levels of both genes are rapidly increased with all the other stressors studied. Moreover, the decrease of transcript levels is notably delayed in ethanol stress, and a difference is observed between the profiles of Rnhsp70-1 and Rnhsp70-2 transcripts during heat stress.

Characterisation and expression of a gene encoding a mutarotase from the fungus Rhizopus nigricans.

A gene coding for a mutarotase was isolated and characterised from the filamentous fungus Rhizopus nigricans. In order to determine the encoded enzyme's activity a recombinant protein was prepared in the baculovirus expression system and the mutarotase activity was determined. Expression studies showed that the gene is repressed by high as well as low concentrations of glucose and derepressed during deficiency of glucose. Besides the regulation at the level of transcription, an accelerative effect of glucose in growth medium on the mutarotase mRNA decay was also demonstrated. Moreover, a Southern hybridisation performed at lower temperatures suggested that the R. nigricans genome harbours a nucleotide sequence, that is homologous to the isolated gene.

Identification and partial characterization of Rhizopus nigricans Gß proteins and their expression in the presence of progesterone.

The mammalian steroid hormone progesterone actuates a signalling pathway in the zygomycete Rhizopus nigricans which includes heterotrimeric G proteins. To investigate the possibility that the Gß subunit of these proteins is involved in the signalling, a cDNA library from R. nigricans exposed to progesterone was prepared and a sequence coding for a Gß subunit was searched for. Using degenerate primers, two sequences, RnGPB1 and RnGPB2, were identified that exhibited a high degree of identity with those for Gß from other filamentous fungi, but not from yeast. The presence of more than one Gß subunit is very rare among the fungi, and it has been to date reported only for Rhizopus oryzae. We have shown that progesterone increases the expression of RnGPB1, but has no influence on the expression of RnGPB2. Therefore, our studies imply the involvement of Gß subunit 1 in the response of R. nigricans to progesterone. Moreover, the Gß subunit is subjected to endogenous ADP-ribosylation in the presence of NAD, which could be important in some, as yet unknown, cell process. Article from a special issue on steroids and microorganisms.

Aspects of the steroid response in fungi.

The number of fungal infections is increasing due to higher numbers of immunocompromised patients. Unfortunately, drug resistance represents a major additional problem in clinical praxis. Therefore factors contributing to infection by opportunistic pathogens, and to their growth and drug resistance are of major importance. It has been known for some time that mammalian steroid hormones are toxic to fungi. In this paper the response of fungi to the presence of steroid hormones will be discussed at different levels. First, the effect of steroid hormones on fungal growth, morphology and virulence will be considered. Processes affecting steroid intracellular concentration will be discussed; steroid uptake and, even more, steroid extrusion are currently of special interest. The role of biotransformation in the detoxification of active steroids will be taken into consideration and phases of steroid metabolism in fungal cells will be compared to phases of classical xenobiotic metabolism. Steroid signaling in fungi is presently not yet clear. It results in a global response of fungi to steroid hormones. Some of the genes differentially expressed in fungi as the result of exposure to steroid hormones may contribute to fungal drug resistance.