An oleate-stimulated, phosphatidylinositol 4,5-bisphosphate-independent phospholipase D in Schizosaccharomyces pombe.

Phospholipase D1 (PLD1) is an important enzyme involved in lipid-mediated signal transduction and membrane dynamics in eukaryotes. PLD1 preferentially hydrolyzes phosphatidylcholine to phosphatidic acid. This potent second messenger is involved in cytoskeletal reorganization, secretion, and membrane trafficking in eukaryotic cells. In Saccharomyces cerevisiae, PLD1 is involved in polarized growth and morphogenesis during pheromone response and sporulation. The presence of a PLD activity in Schizosaccharomyces pombe is demonstrated. PLD activity was able to hydrolyze a fluorescently labeled analog of phosphatidylcholine and was capable of performing the transphosphatidylation reaction characteristic of PLDs. Schizosaccharomyces pombe PLD activity was unaffected by phosphatidylinositol 4,5 bisphosphate (PIP(2)), but was slightly stimulated by oleate. PLD activity was shown to increase when the S. pombe cells underwent mating and sporulation. Here, we also report the molecular cloning of the first phospholipase D isoform from an S. pombe genomic DNA library (EMBL accession no. FN547388). Comparisons of three divergent yeasts, S. pombe, S. cerevisiae, and Candida albicans, with respect to the PLD enzymes revealed differences in regulation by oleate and PIP(2). Even with high homology in the protein sequences between the PLD1 enzymes of S. cerevisiae, C. albicans, and S. pombe, there was variation with the effects of the regulators.

An upstream regulator and downstream target of phospholipase D1 activity during pheromone response in Saccharomyces cerevisiae.

Phospholipase D1 (PLD1), which is the product of the SPO14 gene, has been shown to play a role in the process of polarized cell growth (PCG) during the pheromone response in Saccharomyces cerevisiae. PLD1 hydrolyzes phosphatidylcholine to produce phosphatidic acid (PA) and a free choline headgroup. This study investigated the interactions of PLD1 and PA with two proteins known to be involved in the cellular signaling leading to PCG in yeast, the small GTPase Cdc42p and the PAK family kinase Ste20p. Constitutively activated Cdc42p stimulates PLD1 activity. Protein-lipid binding blots confirmed the specific binding of Ste20p to the PLD1 product, PA. Finally, kinase activity assays provided evidence for the stimulation of Ste20p by PA. These findings highlight the important interactions among PLD1, Cdc42p and Ste20p during PCG in S. cerevisiae.

Candida albicans PLD I activity is required for full virulence.

Phospholipase D1 (PLD1) mutants of Candida albicans are defective in important in vivo and in vitro virulence factors. PLD1 mutants colonize the murine alimentary tract as well as PLD1 sufficient strains. In comparison to PLD1 sufficient strains, the PLD1 mutants: (i) are unable to survive in internal organs after intravenous challenge; (ii) do not decrease the body weights of mice after oral challenge; and (iii) are not lethal for immunodeficient mice after oral challenge. In vitro, the PLD1 mutants show a drastically reduced capacity to penetrate epithelial monolayers and they fail to develop hyphae when grown on solid Spider medium. The morphogenic switch from yeast to hyphae is controlled by multiple parallel signaling pathways that couple specific stimuli to the regulation of several transcription factors. Our data suggest that PLD1 functions in at least one of these pathways regulating morphogenesis in vitro and that while the mutants are able to form hyphae in vivo, the hyphae are defective in their ability to cause oroesophageal and gastric candidiasis and to kill the C. albicans-colonized mice.

Use of genetically engineered phage to deliver antimicrobial agents to bacteria: an alternative therapy for treatment of bacterial infections.

The emergence and increasing prevalence of multidrug-resistant bacterial pathogens emphasizes the need for new and innovative antimicrobial strategies. Lytic phages, which kill their host following amplification and release of progeny phage into the environment, may offer an alternative strategy for combating bacterial infections. In this study, however, we describe the use of a nonlytic phage to specifically target and deliver DNA encoding bactericidal proteins to bacteria. To test the concept of using phage as a lethal-agent delivery vehicle, we used the M13 phagemid system and the addiction toxins Gef and ChpBK. Phage delivery of lethal-agent phagemids reduced target bacterial numbers by several orders of magnitude in vitro and in a bacteremic mouse model of infection. Given the powerful genetic engineering tools available and the present knowledge in phage biology, this technology may have potential use in antimicrobial therapies and DNA vaccine development.

Propranolol inhibits hyphal development in Candida albicans.

Propranolol was used to investigate the role of phosphatidic acid (PA) and diacylglycerol in the dimorphic transition in Candida albicans. Propranolol was able to inhibit the appearance of germ tubes without decreasing growth rate. Data suggest that inhibition of morphogenesis may be due to binding by propranolol of PA derived from PLD1 hydrolysis of phosphatidylcholine.

Doc-mediated cell killing in Shigella flexneri using a C1/LacI controlled expression system.

In this report we describe the development of a highly stringent and dually regulated promoter system for Shigella flexneri. Dual regulation was provided by utilizing a promoter susceptible to control by the bacteriophage P1 temperature-sensitive C1 repressor that in turn was under the transcriptional control of LacI. The level of induction/repression ratios observed was up to 3700-fold in S. flexneri. The general utility of this promoter system was evaluated by demonstrating that the bacteriophage P1 post-segregational killer protein Doc mediates a bactericidal effect in S. flexneri. This represents the first report of Doc (death on curing)-mediated killing in this Gram-negative species.

Phosphatidylinositol-4-phosphate 5-kinase activity is stimulated during temperature-induced morphogenesis in Candida albicans.

Phosphoinositides are important lipid signalling molecules in eukaryotic cells. Phosphatidylinositol-4-phosphate 5-kinase (PI4P5K) catalyses the production of phosphatidylinositol 4,5-bisphosphate (PIP2), which stimulates phospholipase D1 (PLD1) activity in mammalian and yeast cells. PLD1 catalyses the formation of phosphatidic acid (PA), which has been shown to activate PI4P5Ks in mammalian and Saccharomyces cerevisiae cells. In the present study, PI4P5K activity in the opportunistic pathogen Candida albicans was identified. A gene with significant sequence homology to the S. cerevisiae PI4P5K was cloned and designated MSS4. This gene was demonstrated to encode a functional PI4P5K by expression in S. cerevisiae. This enzyme was found to be membrane-associated and was stimulated by PA. Within the first 20 min after induction of polarized hyphal growth induced by a shift to elevated temperature, PI4P5K activity increased 2.5-fold. This stimulation was not observed when hyphae were induced by a combination of elevated temperature and serum. A lack of PLD1 activity resulted in the loss of induction of PI4P5K activity during the morphogenetic switch. Furthermore, the addition of propranolol attenuated the stimulation of PI4P5K activity during morphogenesis. These results suggest that PA derived from PLD1 activity stimulates C. albicans PI4P5K during the switch to the hyphal form under some conditions.

Tight regulation and modulation via a C1-regulated promoter in Escherichia coli and Pseudomonas aeruginosa.

We describe the development and analysis of a novel promoter system regulated by the bacteriophage P1 temperature-sensitive C1 repressor. Using transcriptional fusions to the lacZ reporter gene to monitor gene expression, we show that the ratio of induction/repression can be up to 1500-fold in Escherichia coli. The promoters exhibited extremely tight repression and could be modulated over a range of temperatures. The utility of the promoter system was tested in Pseudomonas aeruginosa. C1 effectively repressed transcription; however, only modest induction was achieved. To increase the levels of induction, the amount of c1 was modulated at the mRNA level by using a LacI-regulated promoter. This resulted in a 59-fold induction in gene expression under inducing conditions. As the promoter system was constructed in a broad-host range vector and utilized the C1 repressor from a broad-host range phage, the system will provide the potential for controlled gene expression in Gram-negative bacteria.

Development of a P1 phagemid system for the delivery of DNA into Gram-negative bacteria.

The inability to transform many clinically important Gram-negative bacteria has hampered genetic studies addressing the mechanism of bacterial pathogenesis. This report describes the development and construction of a delivery system utilizing the broad-host-range transducing bacteriophage P1. The phagemids used in this system contain a P1 pac initiation site to package the vector, a P1 lytic replicon to generate concatemeric DNA, a broad-host-range origin of replication and an antibiotic-resistance determinant to select bacterial clones containing the recircularized phagemid. Phagemid DNA was successfully introduced by infection and stably maintained in members of the families Enterobacteriaceae (Escherichia coli, Shigella flexneri, Shigella dysenteriae, Klebsiella pneumoniae and Citrobacter freundii) and Pseudomonadaceae (Pseudomonas aeruginosa). In addition to laboratory strains, these virions were used successfully to deliver phagemids to a number of strains isolated from patients. This ability to deliver genetic information to wild-type strains raises the potential for use in antimicrobial therapies and DNA vaccine development.

The role and relevance of phospholipase D1 during growth and dimorphism of Candida albicans.

The phosphatidylcholine-specific phospholipase D1 (PLD1) in Saccharomyces cerevisiae is involved in vesicle transport and is essential for sporulation. The gene encoding the homologous phospholipase D1 from Candida albicans (PLD1) was used to study the role of PLD1 in this pathogenic fungus. In vitro and in vivo expression studies using Northern blots and reverse transcriptase-PCR showed low PLD1 mRNA levels in defined media supporting yeast growth and during experimental infection, while enhanced levels of PLD1 transcripts were detected during the yeast to hyphal transition. To study the relevance of PLD1 during yeast and hyphal growth, an essential part of the gene was deleted in both alleles of two isogenic strains. In vitro PLD1 activity assays showed that pld1 mutants produced no detectable levels of phosphatidic acid, the hydrolytic product of PLD1 activity, and strongly reduced levels of diacylglycerol, the product of lipid phosphate phosphohydrolase, suggesting no or a negligible background PLD1 activity in the pld1 mutants. The pld1 mutants showed no growth differences compared to the parental wild-type in liquid complex and minimal media, independent of the growth temperature. In addition, growth rates of pld1 mutants in media with protein as the sole source of nitrogen were similar to growth rates of the wild-type, indicating that secretion of proteinases was not reduced. Chlamydospore formation was normal in pld1 mutants. When germ tube formation was induced in liquid media, pld1 mutants showed similar rates of yeast to hyphal transition compared to the wild-type. However, no hyphae formation was observed on solid Spider medium, and cell growth on cornmeal/Tween 80 medium indicated aberrant morphogenesis. In addition, pld1 mutants growing on solid media had an attenuated ability to invade the agar. In a model of oral candidosis, pld1 mutants showed no attenuation of virulence. In contrast, the mutant was less virulent in two different mouse models. These data suggest that PLD1 is not essential for growth and oral infections. However, they also suggest that a prominent part of the phosphatidic acid and diacylglycerol pools is produced by PLD1 and that the level of these components is important for morphological transitions under certain conditions in C. albicans.

Phospholipase D activity is required for dimorphic transition in Candida albicans.

Candida albicans is an opportunistic pathogen that causes significant morbidity and mortality in immunocompromised patients. In this report, the presence of a phospholipase D (PLD) activity in C. albicans, designated CaPLD1, is demonstrated. This is the first description of PLD activity in this organism. CaPLD1 activity was stimulated by inducers of dimorphic transition. Furthermore, transition was stimulated by the addition of exogenous PLD to cells. The addition of 1-propanol to the medium, which resulted in the production of phosphatidylpropanol by CaPLD1 at the expense of the usual product phosphatidic acid, delayed the yeast to hypha transition. These results suggest that CaPLD1 may be an important regulator of dimorphic transition in C. albicans.