Joseph W. Dolan

Use of Genetically Engineered Phage To Deliver Antimicrobial Agents to Bacteria: an Alternative Therapy for Treatment of Bacterial Infections

ABSTRACT 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.

Overcoming the Phage Replication Threshold: a Mathematical Model with Implications for Phage Therapy

ABSTRACT Prior observations of phage-host systems in vitro have led to the conclusion that susceptible host cell populations must reach a critical density before phage replication can occur. Such a replication threshold density would have broad implications for the therapeutic use of phage. In this report, we demonstrate experimentally that no such replication threshold exists and explain the previous data used to support the existence of the threshold in terms of a classical model of the kinetics of colloidal particle interactions in solution. This result leads us to conclude that the frequently used measure of multiplicity of infection (MOI), computed as the ratio of the number of phage to the number of cells, is generally inappropriate for situations in which cell concentrations are less than 107/ml. In its place, we propose an alternative measure, MOIactual, that takes into account the cell concentration and adsorption time. Properties of this function are elucidated that explain the demonstrated usefulness of MOI at high cell densities, as well as some unexpected consequences at low concentrations. In addition, the concept of MOIactual allows us to write simple formulas for computing practical quantities, such as the number of phage sufficient to infect 99.99% of host cells at arbitrary concentrations.

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.

Undecylenic acid inhibits morphogenesis of Candida albicans.

ABSTRACT Resilient liners are frequently used to treat denture stomatitis, a condition often associated with Candida albicansinfections. Of 10 liners tested, 2 were found to inhibit the switch from the yeast form to hyphae and a third was found to stimulate this switch. The inhibitor was determined to be undecylenic acid.

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.

Candida albicans PLD1 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.

Endogenous mutations in human uncoupling protein 3 alter its functional properties

Human uncoupling protein 3 (UCP3) is a mitochondrial transmembrane carrier that uncouples oxidative phosphorylation and is a candidate gene for obesity. Expression of native human UCP3 mutations in yeast showed complete loss (R70W), significant reduction (R143X), or no effect (V102I and IVS6+1G>A) on the uncoupling activity of UCP3. It is concluded that certain mutations in UCP3 alter its functional impact on membrane potential (ΔΨ), possibly conferring susceptibility to develop metabolic diseases.

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.

Propranolol Inhibits Hyphal Development in Candida albicans

ABSTRACT 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.

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.