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Biomedical subjects

Caroline Westwater

Publications and source records attributed to Caroline Westwater.

17 recordsLinked to original sources

Differential Candida albicans lipase gene expression during alimentary tract colonization and infection.

The human pathogenic fungus Candida albicans, which can reside as a benign commensal of the gut, possesses a large family of lipase encoding genes whose extracellular activity may be important for colonization and subsequent infection. The expression of the C. albicans lipase gene family (LIP1-10) was investigated using a mouse model of mucosal candidiasis during alimentary tract colonization (cecum contents) and orogastric infection. LIPs4-8 were expressed in nearly every sample prepared from the cecum contents and infected mucosal tissues (stomach, hard palate, esophagus and tongue) suggesting a maintenance function for these gene products. In contrast, LIPs1, 3, and 9, which were detected consistently in infected gastric tissues, were essentially undetectable in infected oral tissues. In addition, LIP2 was expressed consistently in cecum contents but was undetectable in infected oral tissues suggesting LIP2 may be important for alimentary tract colonization, but not oral infection. The host responded to a C. albicans infection by significantly increasing expression of the chemokines MIP-2 and KC at the site of infection. Therefore, differential LIP gene expression was observed during colonization, infection and at different infected mucosal sites.

Animals↗

Divergent chemokine, cytokine and beta-defensin responses to gastric candidiasis in immunocompetent C57BL/6 and BALB/c mice.

Previous studies of animal models of candidiasis have produced conflicting results concerning the cytokines and host defence mechanisms that are most relevant for protection against Candida infections. In this study, the host defence mechanisms evoked by two different immunocompetent murine strains following oral colonization with Candida albicans were assessed. beta-Defensin (mBD1, mBD3 and mBD4), chemokine (MIP-2 and KC) and cytokine (TNF-alpha, IFN-gamma, IL-4, IL-10, IL-12 and IL-15) gene expression in germ-free (gf) and C. albicans-infected (gastric) C57BL/6 and BALB/c mice was contrasted. Gf C57BL/6 and BALB/c mice expressed significantly different basal levels of mBD3, mBD4, TNF-alpha and IL-12 in gastric tissues; however, gf C57BL/6 and BALB/c mice were equally susceptible to intestinal colonization with C. albicans and had similar fungal burdens in gastric tissues 4 weeks after oral challenge. C57BL/6 mice responded to colonization and gastric candidiasis with increased expression of mBD1, mBD3, mBD4, TNF-alpha, MIP-2, KC and IL-12. Conversely, a much more specific and attenuated response was observed in Candida-infected gastric tissues from BALB/c mice. Therefore, different strains of mice that were equally susceptible to gastric candidiasis after oral challenge had divergent cytokine, chemokine and beta-defensin responses. This suggests that conflicting data as to the relevance of cytokines and other host factors in murine resistance to candidiasis may be explained, at least in part, by the strain of mouse studied.

Animals↗

Candida albicans-conditioned medium protects yeast cells from oxidative stress: a possible link between quorum sensing and oxidative stress resistance.

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.

Anticoagulants↗

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.

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 Peyer's 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.

Administration, Oral↗

Mnt1p and Mnt2p of Candida albicans are partially redundant alpha-1,2-mannosyltransferases that participate in O-linked mannosylation and are required for adhesion and virulence.

The MNT1 gene of the human fungal pathogen Candida albicans is involved in O-glycosylation of cell wall and secreted proteins and is important for adherence of C. albicans to host surfaces and for virulence. Here we describe the molecular analysis of CaMNT2, a second member of the MNT1-like gene family in C. albicans. Mnt2p also functions in O-glycosylation. Mnt1p and Mnt2p encode partially redundant alpha-1,2-mannosyltransferases that catalyze the addition of the second and third mannose residues in an O-linked mannose pentamer. Deletion of both copies of MNT1 and MNT2 resulted in reduction in the level of in vitro mannosyltransferase activity and truncation of O-mannan. Both the mnt2Delta and mnt1Delta single mutants were significantly reduced in adherence to human buccal epithelial cells and Matrigel-coated surfaces, indicating a role for O-glycosylated cell wall proteins or O-mannan itself in adhesion to host surfaces. The double mnt1Deltamnt2Delta mutant formed aggregates of cells that appeared to be the result of abnormal cell separation. The double mutant was attenuated in virulence, underlining the importance of O-glycosylation in pathogenesis of C. albicans infections.

Candida albicans↗

beta-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.

Animals↗

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.

Animals↗

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.

Animals↗

Development of a thermally regulated broad-spectrum promoter system for use in pathogenic gram-positive species.

Selectively regulating gene expression is an essential molecular tool that is lacking for many pathogenic gram-positive bacteria. In this report, we describe the evaluation of a series of promoters regulated by the bacteriophage P1 temperature-sensitive C1 repressor in Enterococcus faecium, Enterococcus faecalis, and Staphylococcus aureus. Using the lacZ gene to monitor gene expression, we examined the strength, basal expression, and induced expression of synthetic promoters carrying C1 operator sites. The promoters exhibited extremely low basal expression and, under inducing conditions, gave high levels of expression (100- to 1,000-fold induction). We demonstrate that the promoter system could be modulated by temperature and showed rapid induction and that the mechanism of regulation occurred at the level of transcription. Controlled expression with the same constructs was also demonstrated in the gram-negative bacterium Escherichia coli. However, low basal expression and the ability to achieve derepression were dependent on both the number of mismatches in the C1 operator sites and the promoter driving c1 expression. Since the promoters were designed to contain conserved promoter elements from gram-positive species and were constructed in a broad-host-range plasmid, this system will provide a new opportunity for controlled gene expression in a variety of gram-positive bacteria.

Bacteriophage P1↗

Detection of Candida albicans mRNA from formalin-fixed, paraffin-embedded mouse tissues by nested reverse transcription-PCR.

Histopathology archives represent a vast source of infectious disease specimens that can be used to elucidate important disease processes. In this report, we describe a method to detect Candida albicans gene expression from infected, formalin-fixed, paraffin-embedded mouse tissue. By use of glass beads to break open the fungal cells and proteinase K treatment, RNA was extracted routinely from tissue sections that had been fixed for up to 72 h. Upon reverse transcription of the RNA and nested PCR, the procedure detected C. albicans "housekeeping" and putative virulence genes.

Animals↗

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.

Bacterial Proteins↗

Glycosylation and epitope mapping of the 5T4 glycoprotein oncofoetal antigen.

The human 5T4 oncofoetal antigen is a focus for development of several antibody-directed therapies on the basis of the murine monoclonal antibody against 5T4 (mAb5T4), which recognizes a conformational epitope. 5T4 molecules are highly N-glycosylated transmembrane glycoproteins whose extracellular domain contains two regions of leucine-rich repeats (LRRs) and associated flanking regions, separated by an intervening hydrophilic sequence. Using a series of deletion and mutated cDNA constructs as well as chimaeras with the murine homologue, we have mapped the mAb5T4 epitope to the more membrane-proximal LRR2 or its flanking region. Analysis of the glycosylation of the seven consensus Asp-Xaa-Ser/Thr sites was consistent with all of the sites being glycosylated. A combination of two high-mannose chains (predominantly octasaccharide) and five mostly sialylated bi-, tri- and tetra-antennary complex chains with minor quantities of core fucose were detected. The two glycosylation sites, which are the most likely to have predominantly high-mannose chains, are in the only two regions that show significant differences between the human and the 81% identical mouse sequence. A site-directed mutation, which abolished glycosylation at one of these sites (position 192), did not alter antigenicity. The other, which is nearest to the N-terminus in the human, has an Asn-Leu-Thr to Asn-Leu-Leu conversion in the mouse, so cannot be glycosylated in the latter species. The large complex glycosylation at the other sites is likely to influence the antigenicity and tertiary structure generating the 5T4 epitope.

Animals↗

5T4 interacts with TIP-2/GIPC, a PDZ protein, with implications for metastasis.

Overexpression of the 5T4 transmembrane glycoprotein can have marked effects on both the actin cytoskeleton and cell migration. Using a yeast two-hybrid approach, we describe a novel interaction between 5T4 and TIP-2/GIPC, a cytoplasmic interacting protein containing a PDZ domain. The cytoplasmic tail of 5T4 contains a class I PDZ-binding motif (Ser-Asp-Val) and we demonstrate that this region, in particular the terminal valine, is required for 5T4 interaction with TIP-2/GIPC. HeLa cells expressing hemagglutinin-tagged TIP-2/GIPC (HA-TIP-2/GIPC) have an altered distribution of endogenous 5T4, which colocalizes with HA-TIP-2/GIPC, thus supporting an interaction. Furthermore, TIP-2/GIPC can be coimmunoprecipitated with 5T4 from HeLa cell lysates. Identification of the 5T4 and TIP-2/GIPC interaction provides the first link between 5T4 and the actin cytoskeleton. Since other proteins, like 5T4, associate with TIP-2/GIPC and are linked with cancer, we explore the possibility that TIP-2/GIPC may be a common factor involved in the cancer process.

Adaptor Proteins, Signal Transducing↗

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.

Bacteriophage P1↗

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.

Bacteriophages↗

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.

Amino Acid Sequence↗

Overcoming the phage replication threshold: a mathematical model with implications for phage therapy.

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 10(7)/ml. In its place, we propose an alternative measure, MOI(actual), 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 MOI(actual) 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.

Bacteriophage M13↗