David A. Schofield

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.

Candida albicans-Conditioned Medium Protects Yeast Cells from Oxidative Stress: a Possible Link between Quorum Sensing and Oxidative Stress Resistance

ABSTRACT Candida albicans, the most frequent fungal pathogen of humans, encounters high levels of oxidants following ingestion by professional phagocytes and through contact with hydrogen peroxide-producing bacteria. In this study, we provide evidence that C. albicans is able to coordinately regulate the oxidative stress response at the global cell population level by releasing protective molecules into the surrounding medium. We demonstrate that conditioned medium, which is defined as a filter-sterilized supernatant from a C. albicans stationary-phase culture, is able to protect yeast cells from both hydrogen peroxide and superoxide anion-generating agents. Exponential-phase yeast cells preexposed to conditioned medium were able to survive levels of oxidative stress that would normally kill actively growing yeast cells. Heat treatment, digestion with proteinase K, pH adjustment, or the addition of the oxidant scavenger alpha-tocopherol did not alter the ability of conditioned medium to induce a protective response. Farnesol, a heat-stable quorum-sensing molecule (QSM) that is insensitive to proteolytic enzymes and is unaffected by pH extremes, is partly responsible for this protective response. In contrast, the QSM tyrosol did not alter the sensitivity of C. albicans cells to oxidants. Relative reverse transcription-PCR analysis indicates that Candida-conditioned growth medium induces the expression of CAT1, SOD1, SOD2, and SOD4, suggesting that protection may be mediated through the transcriptional regulation of antioxidant-encoding genes. Together, these data suggest a link between the quorum-sensing molecule farnesol and the oxidative stress response in C. albicans.

Diagnostic Bioluminescent Phage for Detection of Yersinia pestis

ABSTRACT Yersinia pestis is the etiological agent of the plague. Because of the diseases inherent communicability, rapid clinical course, and high mortality, it is critical that an outbreak, whether it is natural or deliberate, be detected and diagnosed quickly. The objective of this research was to generate a recombinant luxAB (“light”)-tagged reporter phage that can detect Y. pestis by rapidly and specifically conferring a bioluminescent signal response to these cells. The bacterial luxAB reporter genes were integrated into a noncoding region of the CDC plague-diagnostic phage φA1122 by homologous recombination. The identity and fitness of the recombinant phage were assessed through PCR analysis and lysis assays and functionally verified by the ability to transduce a bioluminescent signal to recipient cells. The reporter phage conferred a bioluminescent phenotype to Y. pestis within 12 min of infection at 28°C. The signal response time and signal strength were dependent on the number of cells present. A positive signal was obtained from 102 cells within 60 min. A signal response was not detectable with Escherichia coli, although a weak signal (100-fold lower than that with Y. pestis) was obtained with 1 (of 10) Yersinia enterocolitica strains and 2 (of 10) Yersiniapseudotuberculosis strains at the restrictive temperature. Importantly, serum did not prevent the ability of the reporter phage to infect Y. pestis, nor did it significantly quench the resulting bioluminescent signal. Collectively, the results indicate that the reporter phage displays promise for the rapid and specific diagnostic detection of cultivated Y. pestis isolates or infected clinical specimens.

Phage-based platforms for the clinical detection of human bacterial pathogens

Bacteriophages (phages) have been utilized for decades as a means for uniquely identifying their target bacteria. Due to their inherent natural specificity, ease of use, and straightforward production, phage possess a number of desirable attributes which makes them particularly suited as bacterial detectors. As a result, extensive research has been conducted into the development of phage, or phage-derived products to expedite the detection of human pathogens. However, very few phage-based diagnostics have transitioned from the research lab into a clinical diagnostic tool. Herein we review the phage-based platforms that are currently used for the detection of Mycobacterium tuberculosis, Yersinia pestis, Bacillus anthracis and Staphylococcus aureus in the clinical field. We briefly describe the disease, the current diagnostic options, and the role phage diagnostics play in identifying the cause of infection, and determining antibiotic susceptibility.

Susceptibility of Germfree Phagocyte Oxidase- and Nitric Oxide Synthase 2-Deficient Mice, Defective in the Production of Reactive Metabolites of Both Oxygen and Nitrogen, to Mucosal and Systemic Candidiasis of Endogenous Origin

ABSTRACT Mice deficient for phagocyte oxidase (Phox) and nitric oxide synthase 2 (NOS2) (gp91phox−/−/NOS2−/−), defective in the production of both reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI), were used to investigate the role of phagocytic cells during mucosal and systemic candidiasis of endogenous origin. The alimentary tracts of germfree mice were colonized with Candida albicans wild type or each of two hyphal signaling-defective mutants (efg1/efg1 and efg1/efg1 cph1/cph1). All Candida-colonized gp91phox−/−/NOS2−/− mice were moribund within 12 to 15 days after oral inoculation. C. albicans wild-type and mutant strains colonized the alimentary tracts equally well and were able to translocate, most likely via Peyers patches and mesenteric lymph nodes, to the internal organs and trigger the formation of abscesses; however, the wild-type and mutant strains did not survive in the abscessed murine tissues. Surprisingly, there was no significant difference in the ability of peritoneal exudate cells from gp91phox−/−/NOS2−/−, NOS2−/−, gp91phox−/−, or immunocompetent C57BL/6 mice to kill C. albicans in vitro. This suggests that anti-Candida factors other than ROI and RNI can control the growth of C. albicans and that gp91phox−/−/NOS2−/− mice die due to the inability of the host to control its inflammatory response to Candida. Correspondingly, reverse transcription-PCR analysis showed increased expression of the cytokines gamma interferon, tumor necrosis factor alpha, and the chemokines MIP-2 and KC at the site of infection, while interleukin-15 expression remained relatively unchanged between germfree and infected tissues. These studies indicate that defects in ROI and RNI enabled C. albicans to translocate and disseminate to the internal organs, resulting in an uncontrolled immune response, severe pathology, and death; however, ROI and RNI were not required for the killing of phagocytized C. albicans, indicating that other anti-Candida factors either compensate or are sufficient for the killing of phagocytized Candida.

Hydrolytic Gene Expression during Oroesophageal and Gastric Candidiasis in Immunocompetent and Immunodeficient Gnotobiotic Mice

To investigate whether host immunocompetence influences hydrolytic gene expression, we compared secretory aspartyl proteinase gene (SAP) and phospholipase B gene (PLB) expression during gastric candidiasis in immunocompetent and defined immunodeficient gnotobiotic mice, by reverse-transcription polymerase chain reaction. The use of immunodeficient gnotobiotic mice with combined defects in T cells and natural killer cells enabled a comprehensive study of virulence gene expression in various mucosal sites during lethal oroesophageal (tongue, palate, and esophagus) and gastric candidiasis. All 10 SAP and both PLB genes were expressed in both immunocompetent and specific immunodeficient mice, which suggests that the absence of important components of the host defense did not alter gene expression during gastric candidiasis. Although similar patterns of gene expression were evident in different oral tissues, we detected specific differences between Candida albicans-infected oroesophageal and gastric tissues and differences at various time points during the progression of gastric candidiasis.

β-Defensin Expression in Immunocompetent and Immunodeficient Germ-Free and Candida albicans-Monoassociated Mice

BACKGROUND Defensins are important components of innate immunity and play a key role in the fight against infectious diseases; however, little is known about their role in resistance to fungal infection. METHODS We examined gene expression of mouse beta -defensins (mBDs) 1-4 in orogastric tissues from germ-free (gf) and Candida albicans-monoassociated immunocompetent (C57BL/6) and immunodeficient (Tg epsilon 26 or gp91(phox-/-)/NOS2(-/-)) mice, using competitive reverse-transcriptase polymerase chain reaction. RESULTS The basal levels of beta -defensin gene expression in orogastric tissues from gf mice varied significantly between immunodeficient and immunocompetent mice and by the particular tissue analyzed. During gastric and lethal oroesophageal candidiasis, expression of mBD1, -3, and -4 was induced at the infection sites (stomach and tongue) and was concomitant with an induction of tumor necrosis factor- alpha expression in Tg epsilon 26 mice, compared with that in tissues from gf mice. Induction of mBD4 expression in response to gastric candidiasis, however, was dependent on the immune competency of the host. A C. albicans mutant that lacked key genes important for filamentation and virulence also significantly induced expression of mBD1, -3, and -4 in Tg epsilon 26 mice. CONCLUSIONS These data not only demonstrate quantitative and qualitative differences in beta -defensin expression in gf and gnotobiotic mice, they also suggest a role for these peptides in resistance to gastric and lethal oroesophageal candidiasis.

Development Of An Engineered Bioluminescent Reporter Phage For Detection Of Bacterial Blight Of Crucifers

ABSTRACT Bacterial blight, caused by the phytopathogen Pseudomonas cannabina pv. alisalensis, is an emerging disease afflicting important members of the Brassicaceae family. The disease is often misdiagnosed as pepper spot, a much less severe disease caused by the related pathogen Pseudomonas syringae pv. maculicola. We have developed a phage-based diagnostic that can both identify and detect the causative agent of bacterial blight and differentiate the two pathogens. A recombinant “light”-tagged reporter phage was generated by integrating bacterial luxAB genes encoding luciferase into the genome of P. cannabina pv. alisalensis phage PBSPCA1. The PBSPCA1::luxAB reporter phage is viable and stable and retains properties similar to those of the wild-type phage. PBSPCA1::luxAB rapidly and sensitively detects P. cannabina pv. alisalensis by conferring a bioluminescent signal response to cultured cells. Detection is dependent on cell viability. Other bacterial pathogens of Brassica species such as P. syringae pv. maculicola, Pseudomonas marginalis, Pectobacterium carotovorum, Xanthomonas campestris pv. campestris, and X. campestris pv. raphani either do not produce a response or produce significantly attenuated signals with the reporter phage. Importantly, the reporter phage detects P. cannabina pv. alisalensis on diseased plant specimens, indicating its potential for disease diagnosis.

GLUTATHIONE S-TRANSFERASE IN HORMONAL CARCINOGENESIS

Treatment with testosterone propionate (TP) and diethylstilbestrol (DES) or TP and estradiol (E2) for 8-9 months causes development of leiomyosarcomas in the vas deferens or uterus of Golden Syrian hamsters at a frequency of 100%. In males, treatment with estrogens alone results in renal tumors, fatal within 6 months. No leiomyosarcomas have been detected after treatment with estrogens alone, perhaps due to this high mortality rate. In tissue culture, treatment with the glucocorticoid (GC) triamcinolone acetonide (TA) results in an increased expression of a mu-class glutathione S-transferase (GST) (hGSTYBX). We have characterized this induction as a secondary response, i.e. requiring new protein synthesis. Here we describe homologies to known transcription factor-binding sites in the hGSTYBX promoter which may be involved in the induction event. hGSTYBX is a member of a superfamily of detoxification enzymes, induced by genotoxic compounds and reactive oxygen species (ROS). We also describe the effects of several known inducers of other GST family members on hGSTYBX. While implicated in many chemotherapeutic-resistant tumors, GST enzymes have not yet been characterized as a functional agent in hormonal carcinogenesis. This latter possibility is the focus of our investigations. To study the effects of hormone treatment on GST levels in vivo, we have developed a polyclonal antibody to hGSTYBX, and conducted immunohistochemistry on tissues from control and treated animals. Treatment with TP and E2 causes a loss of hGSTYBX expression in the epithelium of the vas deferens. We hypothesize that the loss of this protective enzyme leaves the cells vulnerable to the genotoxic effects of estrogen or estrogenic metabolites.

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.