Transcriptional profiling of the early stages of germination in Candida albicans by real-time RT-PCR

By using real-time RT-PCR, we profiled the expression of CGR1, CaMSI3, EFG1, NRG1, and TUP1 in Candida albicans strains JCM9061 and CAI4 under several conditions, including induction of morphological transition, heat shock, and treatment with calcium inhibitors. Expression of CaMSI3 changed under these growth conditions except during heat shock. CGR1 expression increased during the early stages of hyphal growth in JCM9061, while expression was strain-dependent during heat shock. Both EFG1 and NRG1 were similarly expressed under hypha-inducing conditions and heat shock. Expression of TUP1 was slightly different from the expression of EFG1 or NRG1.     

HWP1 Functions in the Morphological Development of Candida albicans Downstream of EFG1, TUP1, and RBF1

The morphological plasticity of Candida albicans is an important determinant of pathogenicity, and nonfilamentous mutants are avirulent. HWP1, a hypha-specific gene, was identified in a genetic screen for developmentally regulated genes and encodes a cell surface protein of unknown function. Heterozygous and homozygous deletions of HWP1 resulted in a medium-conditional defect in hyphal development. HWP1 expression was blocked in a Deltaefg1 mutant, reduced in an Deltarbf1 mutant, and derepressed in a Deltatup1 mutant. Therefore, HWP1 functions downstream of the developmental regulators EFG1, TUP1, and RBF1. Mutation of CPH1 had no effect on HWP1 expression, suggesting that the positive regulators of hyphal development, CPH1 and EFG1, are components of separate pathways with different target genes. The expression of a second developmentally regulated gene, ECE1, was similarly regulated by EFG1. Since ECE1 is not required for hyphal development, the regulatory role of EFG1 apparently extends beyond the control of cell shape determinants. However, expression of ECE1 was not influenced by TUP1, suggesting that there may be some specificity in the regulation of morphogenic elements during hyphal development.     

Expression of mdr49 and mdr65 multidrug resistance genes in larval tissues of Drosophila melanogaster under normal and stress conditions

In situ expression of 2 multidrug resistance genes, mdr49 and mdr65, of Drosophila melanogaster was examined in wild-type third instar larval tissues under physiological conditions and after heat shock or colchicine feeding. Expression of these 2 genes was also examined in tumorous tissues of lethal (2) giant larvae I(2)gl4 mutant larvae. These 2 mdr genes show similar constitutive expression in different larval tissues under physiological conditions. However, they are induced differentially by endogenous (tumorous growth) and exogenous stresses (colchcine feeding or heat shock): whereas heat shock and colchicine feeding induce mdr49, tumorous condition is accompanied by enhanced expression of mdr49 and mdr65 genes.     

Regulation and oxidation of two large monofunctional catalases

The two Neurospora crassa catalase genes cat-1 and cat-3 were shown to encode Cat-1 and Cat-3 large monofunctional catalases. cat-1 and cat-3 genes are regulated differentially during the asexual life cycle and under stress conditions. A stepwise increase in catalase activity occurs during conidiation. Conidia have 60 times more catalase activity than exponentially growing hyphae. Cat-1 activity was predominant in conidia, during germination and early exponential growth. It was induced during prestationary growth and by ethanol or heat shock. Cat-3 activity was predominant during late exponential growth and at the start of the conidiation process. It was induced under stress conditions, such as H(2)O(2), paraquat, cadmium, heat shock, uric acid, and nitrate treatment. In general, Cat-1 activity was associated with nongrowing cells and Cat-3 activity with growing cells. The Cat-3 N-terminus sequence indicates that this catalase is processed and presumably secreted. Paraquat caused modification and degradation of Cat-1. Under heat shock both Cat-1 and Cat-3 were modified and degraded and Cat-1 was resynthesized. Paraquat and heat shock effects were observed only in the presence of air and are probably related to in vivo generation of singlet oxygen. Purified Cat-3 was modified with a photosensitizing reaction in which singlet oxygen is produced.     

Some effects of growth conditions on steady state and heat shock induced htpG gene expression in continuous cultures of Escherichia coli

Most of the data concerning heat shock gene expression reported in the literature are derived from batch culture experiments under substrate and nutrient sufficient conditions. Here, the effects of dilution rate and medium composition on the steady state and heat shock induced htpG gene expression have been investigated in continuous cultures of Escherichia coli, using a chromosomal htpG-lacZ gene fusion. During steady state growth temperature dependent patterns of the relative htpG expression were found to be largely similar, irrespective of the growth condition. However, nitrogen-limited growth resulted in a markedly reduced specific steady state htpG expression as compared to growth under carbon limitation or in complex medium, correlating qualitatively with the total cellular protein content. During heat shock, tight temperature controlled expression was evident. While the relative heat shock induced expression was largely identical at various dilution rates in a given growth medium, significantly different response patterns were observed in the three growth media at any give dilution rate. From these results a clearly temperature regulated htpG expression during both, steady and transient state growth in continuous culture is evident, which is further significantly affected by the growth condition used.     

An analysis of the Impact of <Emphasis Type="Italic">NRG1</Emphasis> Overexpression on the <Emphasis Type="Italic">Candida albicans</Emphasis> Response to Specific Environmental Stimuli

The ability of the opportunistic fungal pathogen Candida albicans to form filaments has been strongly linked to its capacity to cause disease in humans. We previously described the construction of a strain in which filamentation can be modulated both in vitro and in vivo by placing one copy of the NRG1 gene under the control of a tetracycline-regulatable promoter. To further characterize the role of NRG1 in controlling filamentous growth, and in an attempt to determine whether NRG1 downregulation is a requirement for filamentation per se, or is only necessary under certain environmental conditions, we have conducted an analysis of the growth of the tet-NRG1 strain under a variety of in vitro conditions. Through overexpression of NRG1, we were able to block filamentation of C. albicans in both liquid media and on solid media. Filamentation in response to the low-oxygen environment of embedded growth was also inhibited. In all of these conditions, normal filamentation could be restored by down regulating expression from the tet-NRG1 allele. Interestingly, although elevated NRG1 levels were able to inhibit the formation of true hyphae in response to a wide range of environmental stimuli, elevated NRG1 expression did not affect the formation of pseudohyphae on nitrogen-limiting synthetic low ammonia dextrose (SLAD) medium. This work further illustrates the key role played by NRG1 in the control of filamentation and suggests that, although NRG1 repression plays a key role in regulating true hyphal growth, it apparently does not regulate pseudohyphal growth in the same fashion.     

Heat stress regulates the expression of TPK1 gene at transcriptional and post-transcriptional levels in Saccharomyces cerevisiae

In Saccharomyces cerevisiae cAMP regulates different cellular processes through PKA. The specificity of the response of the cAMP-PKA pathway is highly regulated. Here we address the mechanism through which the cAMP-PKA pathway mediates its response to heat shock and thermal adaptation in yeast. PKA holoenzyme is composed of a regulatory subunit dimer (Bcy1) and two catalytic subunits (Tpk1, Tpk2, or Tpk3). PKA subunits are differentially expressed under certain growth conditions. Here we demonstrate the increased abundance and half-life of TPK1 mRNA and the assembly of this mRNA in cytoplasmic foci during heat shock at 37 °C. The resistance of the foci to cycloheximide-induced disassembly along with the polysome profiling analysis suggest that TPK1 mRNA is impaired for entry into translation. TPK1 expression was also evaluated during a recurrent heat shock and thermal adaptation. Tpk1 protein level is significantly increased during the recovery periods. The crosstalk of cAMP-PKA pathway and CWI signalling was also studied. Wsc3 sensor and some components of the CWI pathway are necessary for the TPK1 expression upon heat shock. The assembly in foci upon thermal stress depends on Wsc3. Tpk1 expression is lower in a wsc3? mutant than in WT strain during thermal adaptation and thus the PKA levels are also lower. An increase in Tpk1 abundance in the PKA holoenzyme in response to heat shock is presented, suggesting that a recurrent stress enhanced the fitness for the coming favourable conditions. Therefore, the regulation of TPK1 expression by thermal stress contributes to the specificity of cAMP-PKA signalling.     

Molecular characterization of the Escherichia coli htrD gene: cloning, sequence, regulation, and involvement with cytochrome d oxidase.

The Escherichia coli htrD gene was originally isolated during a search for new genes required for growth at high temperature. Insertional inactivation of htrD leads to a pleiotropic phenotype characterized by temperature-sensitive growth in rich medium, H2O2 sensitivity, and sensitivity to cysteine. The htrD gene was cloned and sequenced, and an htrD::mini-Tn10 insertion mutation was mapped within this gene. The htrD gene was shown to encode a protein of approximately 17.5 kDa. Expression of the htrD gene was examined by using an phi (htrD-lacZ) operon fusion. It was found that htrD is not temperature regulated and therefore is not a heat shock gene. Further study revealed that htrD expression is increased under aerobic growth conditions. Conversely, under anaerobic growth conditions, htrD expression is decreased. In addition, a mutation within the nearby cydD gene was found to drastically reduce htrD expression under all conditions tested. These results indicate that htrD is somehow involved in aerobic respiration and that the cydD gene product is necessary for htrD gene expression. In agreement with this conclusion, htrD mutant bacteria are unable to oxidize the cytochrome d-specific electron donor N,N,N',N'-tetramethyl-p-phenylenediamine.     

Phosphatidylinositol 3-kinase VPS34 of Candida albicans is involved in filamentous growth, secretion of aspartic proteases, and intracellular detoxification

The phosphatidylinositol (PI) 3-kinase Vps34p of Candida albicans influences vesicular intracellular transport, filamentous growth and virulence. To get a clearer understanding how these phenomena are connected, we analysed hyphal growth in a matrix under microaerophilic conditions at low temperature, the detoxification of metal ions and antifungal drugs, the secretion of aspartic proteinases (Saps), as well as expression of adhesion-associated proteins of the C. albicans vps34 null mutant strain. The hyphal growth in a matrix, which is repressed in the wild-type strain by Efg1p, was derepressed in the mutant. CZF1, which encodes an activator of hyphal growth in a matrix, was up-regulated in the mutant. In addition, CZF1 expression was pH-dependent in the wild-type. Expression of EFG1 was not changed. Examination of Saps secretion showed a reduction in the vps34 null mutant. Determination of sensitivity against metal ions and antimycotic drugs revealed defects in detoxification. Expression studies indicated that the vps34 mutant reacts to the phenotypical defects with an up-regulation of genes involved in these processes, including the aspartyl proteinases SAP2 and SAP9, adhesion proteins ALS1 and HWP1, and the ABC transporters CDR1 and HST6. We also found an increased expression of the PI 4-kinase LSB6 indicating a complex feed-back mechanism for the compensation of the multiple defects arising from the lack of the PI3-kinase VPS34.     

Expression of heat shock protein70 in pig oocytes: heat shock response during oocyte growth

The heat shock response of growing and fully-grown pig oocytes was analyzed in vitro by determining heat shock protein70 (HSP70) synthesis under both normal conditions (39 degrees C; 0 and 6h) and after heat shock (43 degrees C; 1, 4 and 6h). The expression of HSP70 in oocytes was detected by immunoblotting analysis. Growing oocytes measuring 80-99 microm synthesized a high number of HSP70 without heat shock effect, and these were capable of increasing the synthesis of HSP70 after heat shock to a maximum after 1h. Growing oocytes measuring 100-115 microm also synthesized HSP70 without heat shock and after it, but the HSP70 synthesis was not statistically changed by increasing duration of heat shock. In fully-grown oocytes, great amounts of HSP70 were found without heat shock treatment, and the contents of HSP70 significantly decreased after heat shock. These results indicate that growing oocytes are able to synthesize HSP70 after heat shock. This ability declines at the end of the growth period, and fully-grown oocytes are unable to induce HSP70 synthesis after heat shock. HSP70 is synthesized and stored during oocyte growth. The high HSP70 synthesis in non-heat-treated growing oocytes and a great amount of HSP70 in fully-grown oocytes support the hypothesis that HSP70 is important for oocyte growth and maturation.