Osmotic behavior and permeability of osmotically lysed mitochondria.

Experiments were carried out with water-treated isolated rat liver mitochondria (mitochondria ghosts) previously studied by Caplan and Greenawalt (Caplan, A.I., and J.W. Greenawalt. 1966. J. Cell Biol. 31:455-472) and Vasington and Greenawalt (Vasington, F., and J. Greenawalt. 1968. J. Cell Biol. 39:661-675). The ghosts have permeability properties and osmotic behavior comparable to those of isolated mitochondria. Although they have lost most of their internal contents, they must have resealed. Four properties were found which have not been previously described in systems derived from biological membranes: (a) an osmotic behavior in the virtual absence of internal components. (b) a self-arranging property in the formation of invaginations corresponding in morphology to the cristae. The results suggest that the assembly of the molecular components of the inner membrane is sufficient to specify the morphology. Hence the surface area to volume ratio of the vesicles may specify the presence or absence of cristae-like folds. (c) an increase in the permeability of the membranes to sucrose in the presence of iso-osmotic concentrations of sucrose. (d) an independence of the light transmitted by suspensions of the vesicles from the refractive index of the external medium. This observations run counter to the general previous experience with either mitochondria or liposomes.

Cloning and characterization of KNR4, a yeast gene involved in (1,3)-beta-glucan synthesis.

k9 killer toxin from Hansenula mrakii was used to select a number of resistant mutants from Saccharomyces cerevisiae. Preliminary biochemical and genetic studies showed that some of them acquired structural defects in the cell wall. One of these mutants, the knr4-1 mutant, displays a number of cell wall defects, including osmotic sensitivity; sensitivity to cercosporamide, a known antifungal agent; and resistance to Zymolyase, a (1,3)-beta-glucanase. We report here the isolation and analysis of the KNR4 gene. DNA sequence analysis revealed an uninterrupted open reading frame which contains five potential start codons. The longest coding template encodes a protein of 505 amino acids with a calculated molecular mass of 57,044 Da. A data base search revealed 100% identity with a nuclear protein, SMI1p. Disruption of the KNR4 locus does not result in cell death; however, it leads to reduced levels of both (1,3)-beta-glucan synthase activity and (1,3)-beta-glucan content in the cell wall. The gene was mapped to the right arm of chromosome VII.

Novel antifungal drugs.

There have been many new developments in antifungal therapy in the past few years. Some antifungal drugs have been reformulated to reduce toxicity (e.g. new lipid formulations of polyenes), and new derivatives of drugs have been developed to enhance potencies. The search for unique drug targets will be enhanced by the availability of sequencing data from whole genome sequencing projects.

Antifungals: what's in the pipeline.

The therapeutic landscape for mycotic infections is shifting. New generation azoles that are active against clinically relevant, drug-resistant fungal pathogens have improved bioavailability, half-lives and safety profiles. Acylated cyclic peptide inhibitors of beta(1,3)glucan synthesis with origins as fungal metabolites provide an alternative and highly-selective mode of action, targeting cell-wall biogenesis in important pathogens such as Candida and Aspergillus species. The development, in each structural class, of compounds that have advanced to late-stage clinical trials is summarized in this review.

Isolation and characterization of the structural gene encoding elongation factor 3.

The unique yeast translational factor EF-3 participates in the elongation cycle by stimulating the function of EF-1 alpha in binding aminoacyl-tRNA to the ribosome. We have isolated the structural gene encoding EF-3 from the yeast Saccharomyces cerevisiae. The YEF3 gene is found in one copy per haploid genome and is essential for vegetative growth. DNA sequence analysis reveals that the YEF3 gene contains an open reading frame of 1044 codons. The deduced amino acid sequence has two repeats of a nucleotide-binding motif. Each of these repeats shows similarity to the nucleotide-binding motif of hydrophilic, membrane-associated ATPases including human multidrug resistant protein MDR. Factor 3 manifests ribosome-dependent ATP hydrolysis. Introduction of the YEF3 gene on a high copy number plasmid into yeast strains increases the ribosome-dependent ATPase activity and EF-3 protein levels by 3-5-fold. Yeast strains containing elevated EF-3 protein levels also exhibit increased sensitivity to the aminoglycoside antibiotics hygromycin and paromomycin. These drugs are known to increase translational errors. These observations suggest that EF-3 may affect translational accuracy.

Novel fungal metabolites as cell wall active antifungals: fermentation, isolation, physico-chemical properties, structure and biological activity.

Two novel antifungal compounds, 1 (SCH 466457), and 2 (SCH 466456), active in a "cell wall" assay, were isolated from the fermentation broth of an unidentified fungus. The active compounds were separated from the broth filtrate by adsorption on a macroreticular resin and were purified on reverse phase HPLC. Detailed mass spectrometric and NMR experiments and degradative studies helped in elucidating the structures of these compounds. The compounds were identified to be peptides containing amino acids such as alanine, aminoisobutyric acid, proline, leucine, valine, glycine and a previously identified beta-keto acid, 2-methyl 3-oxotetradecanoic acid. (5) Both compounds were active against Candida, dermatophytes and Aspergillus (Geometric Mean MIC's, 8.9, 20 and 16 microg/ml, and 64, 128 and 23 microg/ml, respectively for 1 and 2).

Analysis of beta-glucans and chitin in a Saccharomyces cerevisiae cell wall mutant using high-performance liquid chromatography.

We have previously shown that mutations in the yeast KNR4 gene resulted in pleiotropic cell wall defects, including resistance to killer 9 toxin, elevated osmotic sensitivity to SDS and increased resistance to zymolyase, a (1-->3)-beta-glucanase. In this report, we further demonstrated that knr4 mutant cells were more permeable to a chromogenic substrate, X-GAL, suggesting that the mutant cell walls were leakier to certain non-permeable molecules. To determine if these defects resulted from structural changes in the cell walls, we analysed the alkali-insoluble cell wall components using HPLC assays developed for this purpose. Comparative analysis using four isogenic strains from a 'knr4 disrupted' tetrad demonstrated that mutant cell walls contained much less (1-->3)-beta-glucan and (1-->6)-beta-glucan; however, the level of chitin, a minor cell wall component, was found to be five times higher in the mutant strains compared to the wild-type strains. The data suggested that the knr4 mutant cell walls were dramatically weakened, which may explain the pleiotropic cell wall defects.

The heat shock response is self-regulated at both the transcriptional and posttranscriptional levels.

When Drosophila cells are shifted from 25 degrees C to 37 degrees C, the synthesis of a small group of proteins (the heat shock proteins or HSPs) is rapidly induced, while most preexisting synthesis is repressed. On return to normal growing temperatures, synthesis of HSPs is gradually repressed and normal synthesis is restored. We show that production of HSP 70 (the major heat-induced protein in these cells) is quantitatively correlated with the degree of stress. The level of synthesis is controlled both transcriptionally and posttranscriptionally through repression of HSP 70 mRNA synthesis and destabilization of HSP 70 transcripts. These regulatory mechanisms depend upon the accumulation of the HSPs themselves; when the production of functional HSPs is blocked, HS transcription continues and HS mRNAs are stable, accumulating in vast quantities; if the block is released, a specific quantity of functional HSP must accumulate before HS transcription is repressed and preexisting HS mRNAs are destabilized. Evidence is also presented that indicates that the same quantity of HSP 70 is required to release the block in normal protein synthesis.

Intracellular localization of heat shock proteins in Drosophila.

When cells and tissues of Drosophila are subjected to elevated temperatures, the pattern of protein synthesis shifts from the production of a broad spectrum of different proteins to the vigorous production of a small number of new, heat shock proteins. The intracellular distribution of these proteins has been investigated through autoradiographic analysis of cells labeled with 3H-leucine at 23 degrees and 37 degrees C. After examining sections of cultured cells from D. melanogaster and polytene cells of D. virilis by electron and light microscopy, we conclude that little (if any) heat shock protein becomes associated with mitochondria, despite the many lines of evidence linking the response to respiratory stress. Confirming earlier reports on the presence of heat shock proteins in nuclei, we find the proteins are very highly concentrated there and that their transport to the nucleus occurs very rapidly. Interestingly, their free concentration in the nuclear sap is extremely low; they are, in fact, quantitatively associated with chromosomes. This association occurs in a nonrandom manner, their concentration in highly condensed chromatin being very low relative to that of other chromosomal loci.

Heat shock and recovery are mediated by different translational mechanisms.

When Drosophila cells are shifted from 25 degrees C to 37 degrees C, protein synthesis is rapidly redirected from the complex pattern characteristic of normal growth to the simple pattern of heat shock proteins (HSPs). On return to 25 degrees C, synthesis of normal proteins is gradually reactivated and that of HSPs is repressed. In quantifying many different recovery experiments, we found that preexisting mRNAs always behaved as a cohort, with messages for different proteins returning to translation at the same rate. Heat shock mRNAs (HS mRNAs), on the other hand, never behaved as a cohort. Their repression was asynchronous, with translation of hsp70 always the first and translation of hsp82 always the last to be repressed. Although recovery times varied enormously (depending on the severity of the heat treatment), repression of hsp70 was always correlated with restoration of normal synthesis, suggesting a link between the two events, hsp70 repression was not simply due to competition with reactivated 25 degrees C mRNAs. A general decline in the translation efficiency of hsp70 mRNA was not observed. Instead, an increasing number of messages were translationally inactivated, while those remaining in the translational pool retained full ribosome loading. Unlike inactive 25 degrees C mRNAs, which are stable during heat shock, inactive HSP mRNAs are degraded during recovery.

The GEF1 gene of Saccharomyces cerevisiae encodes an integral membrane protein; mutations in which have effects on respiration and iron-limited growth.

We have isolated a new class of respiration-defective, i.e petite, mutants of the yeast Saccharomyces cerevisiae. Mutations in the GEF1 gene cause cells to grow slowly on rich media containing carbon sources utilized by respiration. This phenotype is suppressed by adding high concentrations of iron to the growth medium. Gef1- mutants also fail to grow on a fermentable carbon source, glucose, when iron is reduced to low concentrations in the medium, suggesting that the GEF1 gene is required for efficient metabolism of iron during growth on fermentable as well as respired carbon sources. However, activity of the iron uptake system appears to be unaffected in gef1- mutants. Fe(II) transporter activity and regulation is normal in gef1- mutants. Fe(III) reductase induction during iron-limited growth is disrupted, but this appears to be a secondary effect of growth rate alterations. The wild-type GEF1 gene was cloned and sequenced; it encodes a protein of 779 amino acids, 13 possible transmembrane domains, and significant similarity to chloride channel proteins from fish and mammals, suggesting that GEF1 encodes an integral membrane protein. A gef1- deletion mutation generated in vitro and introduced into wild-type haploid strains by gene transplacement was not lethal. Oxygen consumption by intact gef1- cells and by mitochondrial fractions isolated from gef1- mutants was reduced 25-50% relative to wild type, indicating that mitochondrial function is defective in these mutants. We suggest that GEF1 encodes a transport protein that is involved in intracellular iron metabolism.

Isolation, DNA sequence, and regulation of a meiosis-specific eukaryotic recombination gene.

The SPO11 gene, required for meiotic recombination in Saccharomyces cerevisiae, has been cloned by direct selection for complementation of the spo11-1 phenotype: lack of meiotic recombination and low spore viability. DNA sequencing indicates that the gene encodes a 398-amino acid protein having a predicted molecular mass of 45.3 kDa. There is no significant similarity between the SPO11 protein and other protein sequences, including those from genes known to be involved in DNA recombination or repair. Strains bearing a disruption allele are viable, indicating that SPO11 is dispensable for mitotic growth. RNA analyses demonstrate that SPO11 produces a 1.5-kilobase transcript that is developmentally regulated and expressed early in the sporulation process.

Protein synthesis in yeast. Structural and functional analysis of the gene encoding elongation factor 3.

The yeast translational elongation factor 3 (EF-3) stimulates EF-1 alpha-dependent binding of aminoacyl-tRNA by the ribosome. The requirement for EF-3 is unique to fungi; a functional analog has not been found in prokaryotes or other eukaryotes. We have isolated and characterized the structural gene, YEF3, that encodes EF-3. The YEF3 gene is present in one copy/haploid genome and is essential for vegetative growth. DNA sequence analysis revealed that the YEF3 gene contains an open reading frame of 1044 codons. The deduced amino acid sequence contains two repeats of a nucleotide-binding motif, which is similar to the nucleotide-binding consensus sequences of hydrophilic, membrane-associated ATPases. EF-3 catalyzes ATP hydrolysis in a ribosome-dependent manner. A modified assay procedure has been developed that allows measurement of the ATP hydrolytic activity of EF-3 in cell-free extracts without interference by other nucleotide hydrolyase activities. Using this modified assay, we have shown that the wild-type YEF3 gene restores heat stable EF-3 activity in a yeast strain containing a temperature-sensitive EF-3. Introduction of the YEF3 gene on a high copy number plasmid into yeast strains increases the ribosome-dependent ATPase activity. The level of EF-3 protein is also increased 3-5-fold. Elevated EF-3 protein levels did not cause a significant increase in EF-1 alpha and EF-2 protein. Yeast strains containing elevated EF-3 protein levels are more sensitive to the aminoglycoside antibiotics hygromycin and paromomycin. These drugs are known to increase translational errors. This observation suggests that EF-3 may indirectly affect translational accuracy.

A genetic system for studying the activity of a proteolytic enzyme.

We describe a genetic system for monitoring the activity of a specific proteolytic enzyme by taking advantage of the properties of the yeast transcriptional activator GAL4. The GAL4 protein contains two separable and functionally essential domains: the amino-terminal DNA binding domain and the carboxyl-terminal transcriptional activating domain. We constructed two hybrid proteins by inserting between the DNA binding domain and the activation domain of GAL4 either (i) a self-cleaving protease (3C protease of a picornavirus, coxsackievirus B3) or (ii) a mutant form of the protease that is unable to cleave. We show that, although the hybrid protein containing the mutant protease activates transcription of GAL1-lacZ reporter gene, the hybrid protein bearing the wild-type protease is proteolytically cleaved and fails to activate transcription. Our approach to monitor the proteolytic activity could be used to develop simple genetic systems to study other proteases.

Comparison of pathogenesis and host immune responses to Candida glabrata and Candida albicans in systemically infected immunocompetent mice.

Cytokine-mediated host defense against Candida glabrata infection was compared to that against C. albicans, using immunocompetent murine models of systemic candidiasis. The pathogenesis of infection was evaluated morphologically and by culture of target organs, while the kinetics of induction of cytokine mRNAs and corresponding proteins were determined in kidneys by real-time reverse transcription-PCR and cytokine-specific murine enzyme-linked immunosorbent assays, respectively. Systemic infection with C. glabrata resulted in a chronic, nonfatal infection with recovery of organisms from kidneys, while intravenous inoculation with C. albicans resulted in rapid mortality with logarithmic growth of organisms in kidneys and recovery of C. albicans from the spleen, liver, and lungs. Survival of C. glabrata-infected mice was associated with rapid induction of mRNAs and corresponding immunoreactive proteins for the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-12 (IL-12), and gamma interferon (IFN-gamma) and the lack of induction of protein for the anti-inflammatory cytokine IL-10. In contrast, mortality in C. albicans-infected mice was associated with induction of mRNA and corresponding protein for IL-10 but delayed (i.e., TNF-alpha) or absent (i.e., IL-12 and IFN-gamma) induction of immunoreactive proinflammatory cytokines. Mice were subsequently treated with cytokine-specific neutralizing monoclonal antibodies (MAbs) to TNF-alpha, IL-12, or IFN-gamma, and the effect on growth of C. glabrata in kidneys was assessed. Neutralization of endogenous TNF-alpha resulted in a significant increase in C. glabrata organisms compared to similarly infected mice administered an isotype-matched control MAb, while neutralization of endogenous IL-12 or IFN-gamma had no significant effect on C. glabrata replication. These results demonstrate that in response to intravenous inoculation of C. glabrata, immunocompetent mice develop chronic nonfatal renal infections which are associated with rapid induction of the proinflammatory cytokines TNF-alpha, IL-12, and IFN-gamma. Furthermore, TNF-alpha plays a key role in host defense against systemic candidiasis caused by either C. glabrata or C. albicans, as the absence of endogenous TNF-alpha activity was associated with enhanced tissue burden in both infection models.

Homologs of the yeast neck filament associated genes: isolation and sequence analysis of Candida albicans CDC3 and CDC10.

Morphogenesis in the yeast Saccharomyces cerevisiae consists primarily of bud formation. Certain cell division cycle (CDC) genes, CDC3, CDC10, CDC11, CDC12, are known to be involved in events critical to the pattern of bud growth and the completion of cytokinesis. Their products are associated with the formation of a ring of neck filaments that forms at the region of the mother cell-bud junction during mitosis. Morphogenesis in Candida albicans, a major fungal pathogen of humans, consists of both budding and the formation of hyphae. The latter is thought to be related to the pathogenesis and invasiveness of C. albicans. We have isolated and characterized C. albicans homologs of the S. cerevisiae CDC3 and CDC10 genes. Both C. albicans genes are capable of complementing defects in the respective S. cerevisiae genes. RNA analysis of one of the genes suggests that it is a regulated gene, with higher overall expression levels during the hyphal phase than in the yeast phase. Not surprisingly, DNA sequence analysis reveals that the proteins share extensive homology at the amino acid level with their respective S. cerevisiae counterparts. Related genes are also found in other species of Candida and, more importantly, in filamentous fungi such as Aspergillus nidulans and Neurospora crassa. A database search revealed significant sequence similarity with two peptides, one from Drosophila and one from mouse, suggesting strong evolutionary conservation of function.

Nonfilamentous C. albicans mutants are avirulent.

Candida albicans and Saccharomyces cerevisiae switch from a yeast to a filamentous form. In Saccharomyces, this switch is controlled by two regulatory proteins, Ste12p and Phd1p. Single-mutant strains, ste12/ste12 or phd1/phd1, are partially defective, whereas the ste12/ste12 phd1/phd1 double mutant is completely defective in filamentous growth and is noninvasive. The equivalent cph1/cph1 efg1/efg1 double mutant in Candida (Cph1p is the Ste12p homolog and Efg1p is the Phd1p homolog) is also defective in filamentous growth, unable to form hyphae or pseudohyphae in response to many stimuli, including serum or macrophages. This Candida cph1/cph1 efg1/efg1 double mutant, locked in the yeast form, is avirulent in a mouse model.

Antibiotic susceptibility profiles of Escherichia coli strains lacking multidrug efflux pump genes.

The contribution of seven known and nine predicted genes or operons associated with multidrug resistance to the susceptibility of Escherichia coli W3110 was assessed for 20 different classes of antimicrobial compounds that include antibiotics, antiseptics, detergents, and dyes. Strains were constructed with deletions for genes in the major facilitator superfamily, the resistance nodulation-cell division family, the small multidrug resistance family, the ATP-binding cassette family, and outer membrane factors. The agar dilution MICs of 35 compounds were determined for strains with deletions for multidrug resistance (MDR) pumps. Deletions in acrAB or tolC resulted in increased susceptibilities to the majority of compounds tested. The remaining MDR pump gene deletions resulted in increased susceptibilities to far fewer compounds. The results identify which MDR pumps contribute to intrinsic resistance under the conditions tested and supply practical information useful for designing sensitive assay strains for cell-based screening of antibacterial compounds.