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Niche-Specific Requirement for Hyphal Wall protein 1 in Virulence of Candida albicans
Staab, Janet F.
Datta, Kausik
Rhee, Peter
PLoS ONE
Specialized Candida albicans cell surface proteins called adhesins mediate binding of the fungus to host cells. The mammalian transglutaminase (TG) substrate and adhesin, Hyphal wall protein 1 (Hwp1), is expressed on the hyphal form of C. albicans where it mediates fungal adhesion to epithelial cells. Hwp1 is also required for biofilm formation and mating thus the protein functions in both fungal-host and self-interactions. Hwp1 is required for full virulence of C. albicans in murine models of disseminated candidiasis and of esophageal candidiasis. Previous studies correlated TG activity on the surface of oral epithelial cells, produced by epithelial TG (TG1), with tight binding of C. albicans via Hwp1 to the host cell surfaces. However, the contribution of other Tgs, specifically tissue TG (TG2), to disseminated candidiasis mediated by Hwp1 was not known. A newly created hwp1 null strain in the wild type SC5314 background was as virulent as the parental strain in C57BL/6 mice, and virulence was retained in C57BL/6 mice deleted for Tgm2 (TG2). Further, the hwp1 null strains displayed modestly reduced virulence in BALB/c mice as did strain DD27-U1, an independently created hwp1Δ/Δ in CAI4 corrected for its ura3Δ defect at the URA3 locus. Hwp1 was still needed to produce wild type biofilms, and persist on murine tongues in an oral model of oropharyngeal candidiasis consistent with previous studies by us and others. Finally, lack of Hwp1 affected the translocation of C. albicans from the mouse intestine into the bloodstream of mice. Together, Hwp1 appears to have a minor role in disseminated candidiasis, independent of tissue TG, but a key function in host- and self-association to the surface of oral mucosa.
Related Articles
Diagnosis of invasive candidiasis by enzyme-linked immunosorbent assay using the N-terminal fragment of Candida albicans hyphal wall protein 1
Laín, Ana
Elguezabal, Natalia
Brena, Sonia
García-Ruiz, Juan Carlos
del Palacio, Amalia
Moragues, María D
Pontón, José
BMC Microbiology
The diagnosis of invasive candidiasis is difficult because there are no specific clinical manifestations of the disease and colonization and infection are difficult to distinguish. In the last decade, much effort has been made to develop reliable tests for rapid diagnosis of invasive candidiasis, but none of them have found widespread clinical use.
Antibodies against a recombinant N-terminal fragment of the Candida albicans germ tube-specific antigen hyphal wall protein 1 (Hwp1) generated in Escherichia coli were detected by both immunoblotting and ELISA tests in a group of 36 hematological or Intensive Care Unit patients with invasive candidiasis and in a group of 45 control patients at high risk for the mycosis who did not have clinical or microbiological data to document invasive candidiasis. Results were compared with an immunofluorescence test to detect antibodies to C. albicans germ tubes (CAGT). The sensitivity, specificity, positive and negative predictive values of a diagnostic test based on the detection of antibodies against the N-terminal fragment of Hwp1 by immunoblotting were 27.8 %, 95.6 %, 83.3 % and 62.3 %, respectively. Detection of antibodies to the N-terminal fragment of Hwp1 by ELISA increased the sensitivity (88.9 %) and the negative predictive value (90.2 %) but slightly decreased the specificity (82.6 %) and positive predictive values (80 %). The kinetics of antibody response to the N-terminal fragment of Hwp1 by ELISA was very similar to that observed by detecting antibodies to CAGT.
An ELISA test to detect antibodies against a recombinant N-terminal fragment of the C. albicans germ tube cell wall antigen Hwp1 allows the diagnosis of invasive candidiasis with similar results to those obtained by detecting antibodies to CAGT but without the need of treating the sera to adsorb the antibodies against the cell wall surface of the blastospore.
Related Articles
Function of Candida albicans Adhesin Hwp1 in Biofilm Formation
Nobile, Clarissa J.
Nett, Jeniel E.
Andes, David R.
Mitchell, Aaron P.
Eukaryotic Cell
Hwp1 is a well-characterized Candida albicans cell surface protein, expressed only on hyphae, that mediates tight binding to oral epithelial cells. Prior studies indicate that HWP1 expression is dependent upon Bcr1, a key regulator of biofilm formation. Here we test the hypothesis that Hwp1 is required for biofilm formation. In an in vitro model, the hwp1/hwp1 mutant produces a thin biofilm that lacks much of the hyphal mass found in the hwp1/HWP1 reconstituted strain. In a biofilm cell retention assay, we find that the hwp1/hwp1 mutant is defective in retention of nonadherent bcr1/bcr1 mutant cells. In an in vivo rat venous catheter model, the hwp1/hwp1 mutant has a severe biofilm defect, yielding only yeast microcolonies in the catheter lumen. These properties of the hwp1/hwp1 mutant are consistent with its role as a hypha-specific adhesin and indicate that it is required for normal biofilm formation. Overexpression of HWP1 in a bcr1/bcr1 mutant background improves adherence in the in vivo catheter model. This finding provides additional support for the model that Hwp1 is critical for biofilm adhesion. Hwp1 is the first cell surface protein known to be required for C. albicans biofilm formation in vivo and is thus an excellent therapeutic target.
Related Articles
Heterologous Expression of Candida albicans Cell Wall-Associated Adhesins in Saccharomyces cerevisiae Reveals Differential Specificities in Adherence and Biofilm Formation and in Binding Oral Streptococcus gordonii▿
Nobbs, Angela H.
Vickerman, M. Margaret
Jenkinson, Howard F.
Eukaryotic Cell
Colonization and infection of the human host by opportunistic pathogen Candida albicans derive from an ability of this fungus to colonize mucosal tissues and prosthetic devices within the polymicrobial communities present. To determine the functions of C. albicans cell wall proteins in interactions with host or bacterial molecules, Saccharomyces cerevisiae was utilized as a surrogate host to express C. albicans cell wall proteins Als3p, Eap1p, Hwp1p, and Rbt1p. Salivary pellicle and fibrinogen were identified as novel substrata for Als3p and Hwp1p, while only Als3p mediated adherence of S. cerevisiae to basement membrane collagen type IV. Parental S. cerevisiae cells failed to form biofilms on salivary pellicle, polystyrene, or silicone, but cells expressing Als3p or Hwp1p exhibited significant attachment to each surface. Virulence factor Rbt1p also conferred lower-level binding to salivary pellicle and polystyrene. S. cerevisiae cells expressing Eap1p formed robust biofilms upon polystyrene surfaces but not salivary pellicle. Proteins Als3p and Eap1p, and to a lesser degree Hwp1p, conferred upon S. cerevisiae the ability to bind cells of the oral primary colonizing bacterium Streptococcus gordonii. These interactions, which occurred independently of amyloid aggregate formation, provide the first examples of specific C. albicans surface proteins serving as receptors for bacterial adhesins. Streptococcus gordonii did not bind parental S. cerevisiae or cells expressing Rbt1p. Taken collectively, these data suggest that a network of cell wall proteins comprising Als3p, Hwp1p, and Eap1p, with complementary adhesive functions, promotes interactions of C. albicans with host and bacterial molecules, thus leading to effective colonization within polymicrobial communities.
Related Articles
Effect of Piper betle and Brucea javanica on the Differential Expression of Hyphal Wall Protein (HWP1) in Non-Candida albicans Candida (NCAC) Species
Wan Harun, Wan Himratul Aznita
Jamil, Nur Alyaa
Jamaludin, Nor Hazwani
Nordin, Mohd-Al-Faisal
Evidence-based Complementary and Alternative Medicine : eCAM
The study aimed to identify the HWP1 gene in non-Candida albicans Candida species and the differential expression of HWP1 following treatment with Piper betle and Brucea javanica aqueous extracts. All candidal suspensions were standardized to 1 × 106 cells/mL. The suspension was incubated overnight at 37 °C (C. parapsilosis, 35°C). Candidal cells were treated with each respective extract at 1, 3, and 6 mg/mL for 24 h. The total RNA was extracted and reverse transcription-polymerase chain reaction was carried out with a specific primer of HWP1. HWP1 mRNAs were only detected in C. albicans, C. parapsilosis, and C. tropicalis. Exposing the cells to the aqueous extracts has affected the expression of HWP1 transcripts. C. albicans, C. parapsilosis, and C. tropicalis have demonstrated different intensity of mRNA. Compared to P. betle, B. javanica demonstrated a higher suppression on the transcript levels of HWP1 in all samples. HWP1 was not detected in C. albicans following the treatment of B. javanica at 1 mg/mL. In contrast, C. parapsilosis and C. tropicalis were shown to have HWP1 regulation. However, the expression levels were reduced upon the addition of higher concentration of B. javanica extract. P. betle and B. javanica have potential to be developed as oral health product.
Related Articles
Candida albicans adhesin Als3p is dispensable for virulence in the mouse model of disseminated candidiasis
Cleary, Ian A.
Reinhard, Sara M.
Miller, C. Lindsay
Murdoch, Craig
Thornhill, Martin H.
Lazzell, Anna L.
Monteagudo, Carlos
Thomas, Derek P.
Saville, Stephen P.
2011;157(Pt 6):1806-1815.
The presence of specific proteins, including Ece1p, Hwp1p and Als3p, distinguishes the Candida albicans hyphal cell wall from that of yeast-form cells. These proteins are thought to be important for the ability of C. albicans cells to adhere to living and non-living surfaces and for the cell-to-cell adhesion necessary for biofilm formation, and also to be pivotal in mediating C. albicans interactions with endothelial cells. Using an in vitro flow adhesion assay, we previously observed that yeast cells bind in greater numbers to human microvascular endothelial cells than do hyphal or pseudohyphal cells. This is consistent with previous observations that, in a murine model of disseminated candidiasis, cells locked in the yeast form can efficiently escape the bloodstream and invade host tissues. To more precisely explore the role of Als3p in adhesion and virulence, we deleted both copies of ALS3 in a wild-type C. albicans strain. In agreement with previous studies, our als3Δ null strain formed hyphae normally but was defective in biofilm formation. Whilst ALS3 was not expressed in our null strain, hypha-specific genes such as ECE1 and HWP1 were still induced appropriately. Both the yeast form and the hyphal form of the als3Δ strain adhered to microvascular endothelial cells to the same extent as a wild-type strain under conditions of flow, indicating that Als3p is not a significant mediator of the initial interaction between fungal cells and the endothelium. Finally, in a murine model of haematogenously disseminated candidiasis the mutant als3Δ remained as virulent as the wild-type parent strain.
Related Articles
Reduced Virulence of HWP1-Deficient Mutants of Candida albicans and Their Interactions with Host Cells
Tsuchimori, Noboru
Sharkey, Laura L.
Fonzi, William A.
French, Samuel W.
Edwards, John E.
Filler, Scott G.
Infection and Immunity
The Candida albicans gene HWP1 encodes a surface protein that is required for normal hyphal development in vitro. We used mutants lacking one or both alleles of HWP1 to investigate the role of this gene in virulence. Mice infected intravenously with the homozygous hwp1 null mutant, CAL3, survived a median of >14 days, whereas mice infected with a control strain containing two functional alleles of HWP1 survived only 3.5 days. After 1 day of infection, all strains produced similar levels of infection in the kidneys, spleen, and blood. However, after 2 and 3 days, there was a significant decrease in the number of organisms in the kidneys of the mice infected with CAL3. This finding suggests that the hwp1 homozygous null mutant is normal in its ability to initiate infection but deficient in its capacity to maintain infection. CAL3 also germinated minimally in the kidneys. The ability of the heterozygous null mutant to germinate and cause mortality in mice was intermediate to CAL3, suggesting a gene dosage effect. To investigate potential mechanisms for the diminished virulence of CAL3, we examined its interactions with endothelial cells and neutrophils in vitro. CAL3 caused less endothelial cell injury than the heterozygous hwp1 mutant. We conclude that the HWP1 gene product is important for both in vivo hyphal development and pathogenicity of C. albicans. Also, the ability to form filaments may be critical for candidal virulence by enabling the fungus to induce cellular injury and maintain a deep-seated infection.
Related Articles
Cigarette smoke condensate increases C. albicans adhesion, growth, biofilm formation, and EAP1, HWP1 and SAP2 gene expression
Semlali, Abdelhabib
Killer, Kerstin
Alanazi, Humidah
Chmielewski, Witold
Rouabhia, Mahmoud
BMC Microbiology
Smokers are more prone to oral infections than are non-smokers. Cigarette smoke reaches the host cells but also microorganisms present in the oral cavity. The contact between cigarette smoke and oral bacteria promotes such oral diseases as periodontitis. Cigarette smoke can also modulate C. albicans activities that promote oral candidiasis. The goal of this study was to investigate the effect of cigarette smoke condensate on C. albicans adhesion, growth, and biofilm formation as well as the activation of EAP1, HWP1 and secreted aspartic protease 2.
Cigarette smoke condensate (CSC) increased C. albicans adhesion and growth, as well as biofilm formation. These features may be supported by the activation of certain important genes. Using quantitative RT-PCR, we demonstrated that CSC-exposed C. albicans expressed high levels of EAP1, HWP1 and SAP2 mRNA and that this gene expression increased with increasing concentrations of CSC.
CSC induction of C. albicans adhesion, growth, and biofilm formation may explain the increased persistence of this pathogen in smokers. These findings may also be relevant to other biofilm-induced oral diseases.
Cigarette smoke; Tobacco; C albicans; Adhesion; Growth; Biofilm; Genes; EAP-1; HWP-1; Sap2
Related Articles
Human Serum Promotes Candida albicans Biofilm Growth and Virulence Gene Expression on Silicone Biomaterial
Samaranayake, Yuthika Hemamala
Cheung, Becky P. K.
Yau, Joyce Y. Y.
Yeung, Shadow K. W.
Samaranayake, Lakshman P.
PLoS ONE
Systemic candidal infections are a common problem in hospitalized patients due to central venous catheters fabricated using silicone biomaterial (SB). We therefore evaluated the effect of human serum on C. albicans biofilm morphology, growth, and the expression of virulence-related genes on SB in vitro.
We cultivated C. albicans SC5314 (wild-type strain, WT) and its derivative HLC54 (hyphal mutant, HM) for 48 h in various conditions, including the presence or absence of SB discs, and human serum. The growth of planktonic and biofilm cells of both strains was monitored at three time points by a tetrazolium salt reduction assay and by scanning electron microscopy. We also analyzed by RT-PCR its expression of the virulence-related genes ALS3, HWP1, EAP1, ECE1, SAP1 - SAP10, PLB1, PLB2, PLC and PLD.
At each time point, planktonic cells of WT strain cultured in yeast nitrogen base displayed a much higher expression of EAP1 and HWP1, and a moderately higher ALS3 expression, than HM cells. In planktonic cells, expression of the ten SAP genes was higher in the WT strain initially, but were highly expressed in the HM strain by 48 h. Biofilm growth of both strains on SB was promoted in the presence of human serum than in its absence. Significant upregulation of ALS3, HWP1, EAP1, ECE1, SAP1, SAP4, SAP6 - SAP10, PLB1, PLB2 and PLC was observed for WT biofilms grown on serum-treated SB discs for at least one time point, compared with biofilms on serum-free SB discs.
Human serum stimulates C. albicans biofilm growth on SB discs and upregulates the expression of virulence genes, particularly adhesion genes ALS3 and HWP1, and hydrolase-encoding genes SAP, PLB1 and PLB2. This response is likely to promote the colonization of this versatile pathogen within the human host.
Related Articles
O-Mannosylation in Candida albicans Enables Development of Interkingdom Biofilm Communities
Dutton, Lindsay C.
Nobbs, Angela H.
Jepson, Katy
Jepson, Mark A.
Vickerman, M. Margaret
Aqeel Alawfi, Sami
Munro, Carol A.
Lamont, Richard J.
Jenkinson, Howard F.
Candida albicans is a fungus that colonizes oral cavity surfaces, the gut, and the genital tract. Streptococcus gordonii is a ubiquitous oral bacterium that has been shown to form biofilm communities with C. albicans. Formation of dual-species S. gordonii-C. albicans biofilm communities involves interaction of the S. gordonii SspB protein with the Als3 protein on the hyphal filament surface of C. albicans. Mannoproteins comprise a major component of the C. albicans cell wall, and in this study we sought to determine if mannosylation in cell wall biogenesis of C. albicans was necessary for hyphal adhesin functions associated with interkingdom biofilm development. A C. albicans mnt1Δ mnt2Δ mutant, with deleted α-1,2-mannosyltransferase genes and thus defective in O-mannosylation, was abrogated in biofilm formation under various growth conditions and produced hyphal filaments that were not recognized by S. gordonii. Cell wall proteomes of hypha-forming mnt1Δ mnt2Δ mutant cells showed growth medium-dependent alterations, compared to findings for the wild type, in a range of protein components, including Als1, Als3, Rbt1, Scw1, and Sap9. Hyphal filaments formed by mnt1Δ mnt2Δ mutant cells, unlike wild-type hyphae, did not interact with C. albicans Als3 or Hwp1 partner cell wall proteins or with S. gordonii SspB partner adhesin, suggesting defective functionality of adhesins on the mnt1Δ mnt2Δ mutant. These observations imply that early stage O-mannosylation is critical for activation of hyphal adhesin functions required for biofilm formation, recognition by bacteria such as S. gordonii, and microbial community development.
In the human mouth, microorganisms form communities known as biofilms that adhere to the surfaces present. Candida albicans is a fungus that is often found within these biofilms. We have focused on the mechanisms by which C. albicans becomes incorporated into communities containing bacteria, such as Streptococcus. We find that impairment of early stage addition of mannose sugars to C. albicans hyphal filament proteins deleteriously affects their subsequent performance in mediating formation of polymicrobial biofilms. Our analyses provide new understanding of the way that microbial communities develop, and of potential means to control C. albicans infections.
Related Articles
Complementary adhesin function in C. albicans biofilm formation
Nobile, Clarissa J.
Schneider, Heather A.
Nett, Jeniel E.
Sheppard, Donald C.
Filler, Scott G.
Andes, David R.
Mitchell, Aaron P.
Current biology : CB
Biofilms are surface-associated microbial communities with significant environmental and medical impact. Here we focus on an adherence mechanism that permits biofilm formation by Candida albicans, the major invasive fungal pathogen of humans.
The Als surface protein family has been implicated in biofilm formation, and we show that Als1 and Als3 have critical but redundant roles. Overexpression of several other Als proteins permits biofilm formation in a biofilm-defective als1/als1 als3/als3 strain, thus arguing that Als protein function in this process is governed by their respective expression levels. The surface protein Hwp1 is also required for biofilm formation, and we find that a mixture of biofilm-defective hwp1/hwp1 and als1/als1 als3/als3 strains can form a hybrid biofilm both in vitro and in vivo, in a catheter infection model. Complementary function of Hwp1 and Als1/3 seems to reflect their interaction, because expression of Hwp1 in the heterologous host S. cerevisiae permits adherence to wild-type C. albicans but not to an als1/als1 als3/als3 strain.
The complementary roles of Hwp1 and Als1/3 in biofilm formation are analogous to the roles of sexual agglutinins in mating reactions. This analogy suggests that biofilm adhesin complementarity may promote formation of monospecies biofilms.
Candida albicans; biofilm; adherence; adhesin; cell-surface proteins; complementary adhesins
Related Articles
Real-time PCR expression profiling of genes encoding potential virulence factors in Candida albicans biofilms: identification of model-dependent and -independent gene expression
Nailis, Heleen
Kucharíková, Soňa
Řičicová, Markéta
Van Dijck, Patrick
Deforce, Dieter
Nelis, Hans
Coenye, Tom
BMC Microbiology
Candida albicans infections are often associated with biofilm formation. Previous work demonstrated that the expression of HWP1 (hyphal wall protein) and of genes belonging to the ALS (agglutinin-like sequence), SAP (secreted aspartyl protease), PLB (phospholipase B) and LIP (lipase) gene families is associated with biofilm growth on mucosal surfaces. We investigated using real-time PCR whether genes encoding potential virulence factors are also highly expressed in biofilms associated with abiotic surfaces. For this, C. albicans biofilms were grown on silicone in microtiter plates (MTP) or in the Centres for Disease Control (CDC) reactor, on polyurethane in an in vivo subcutaneous catheter rat (SCR) model, and on mucosal surfaces in the reconstituted human epithelium (RHE) model.
HWP1 and genes belonging to the ALS, SAP, PLB and LIP gene families were constitutively expressed in C. albicans biofilms. ALS1-5 were upregulated in all model systems, while ALS9 was mostly downregulated. ALS6 and HWP1 were overexpressed in all models except in the RHE and MTP, respectively. The expression levels of SAP1 were more pronounced in both in vitro models, while those of SAP2, SAP4 and SAP6 were higher in the in vivo model. Furthermore, SAP5 was highly upregulated in the in vivo and RHE models. For SAP9 and SAP10 similar gene expression levels were observed in all model systems. PLB genes were not considerably upregulated in biofilms, while LIP1-3, LIP5-7 and LIP9-10 were highly overexpressed in both in vitro models. Furthermore, an elevated lipase activity was detected in supernatans of biofilms grown in the MTP and RHE model.
Our findings show that HWP1 and most of the genes belonging to the ALS, SAP and LIP gene families are upregulated in C. albicans biofilms. Comparison of the fold expression between the various model systems revealed similar expression levels for some genes, while for others model-dependent expression levels were observed. This suggests that data obtained in one biofilm model cannot be extrapolated to other model systems. Therefore, the need to use multiple model systems when studying the expression of genes encoding potential virulence factors in C. albicans biofilms is highlighted.
Related Articles
Role of Actin Cytoskeletal Dynamics in Activation of the Cyclic AMP Pathway and HWP1 Gene Expression in Candida albicans▿ †
Wolyniak, Michael J.
Sundstrom, Paula
Eukaryotic Cell
Changes in gene expression during reversible bud-hypha transitions of the opportunistic fungal pathogen Candida albicans permit adaptation to environmental conditions that are critical for proliferation in host tissues. Our previous work has shown that the hypha-specific adhesin gene HWP1 is up-regulated by the cyclic AMP (cAMP) signaling pathway. However, little is known about the potential influences of determinants of cell morphology on HWP1 gene expression. We found that blocking hypha formation with cytochalasin A, which destabilizes actin filaments, and with latrunculin A, which sequesters actin monomers, led to a loss of HWP1 gene expression. In contrast, high levels of HWP1 gene expression were observed when the F-actin stabilizer jasplakinolide was used to block hypha formation, suggesting that HWP1 expression could be regulated by actin structures. Mutants defective in formin-mediated nucleation of F-actin were reduced in HWP1 gene expression, providing genetic support for the importance of actin structures. Kinetic experiments with wild-type and actin-deficient cells revealed two distinct phases of HWP1 gene expression, with a slow, actin-independent phase preceding a fast, actin-dependent phase. Low levels of HWP1 gene expression that appeared to be independent of stabilized actin and cAMP signaling were detected using indirect immunofluorescence. A connection between actin structures and the cAMP signaling pathway was shown using hyper- and hypomorphic cAMP mutants, providing a possible mechanism for up-regulation of HWP1 gene expression by stabilized actin. The results reveal a new role for F-actin as a regulatory agent of hypha-specific gene expression at the bud-hypha transition.
Related Articles
Divergent Targets of Candida albicans Biofilm Regulator Bcr1 In Vitro and In Vivo
Fanning, Saranna
Xu, Wenjie
Solis, Norma
Woolford, Carol A.
Filler, Scott G.
Mitchell, Aaron P.
Eukaryotic Cell
Candida albicans is a causative agent of oropharyngeal candidiasis (OPC), a biofilm-like infection of the oral mucosa. Biofilm formation depends upon the C. albicans transcription factor Bcr1, and previous studies indicate that Bcr1 is required for OPC in a mouse model of infection. Here we have used a nanoString gene expression measurement platform to elucidate the role of Bcr1 in OPC-related gene expression. We chose for assays a panel of 134 genes that represent a range of morphogenetic and cell cycle functions as well as environmental and stress response pathways. We assayed gene expression in whole infected tongue samples. The results sketch a portrait of C. albicans gene expression in which numerous stress response pathways are activated during OPC. This one set of experiments identifies 64 new genes with significantly altered RNA levels during OPC, thus increasing substantially the number of known genes in this expression class. The bcr1Δ/Δ mutant had a much more limited gene expression defect during OPC infection than previously reported for in vitro growth conditions. Among major functional Bcr1 targets, we observed that ALS3 was Bcr1 dependent in vivo while HWP1 was not. We used null mutants and complemented strains to verify that Bcr1 and Hwp1 are required for OPC infection in this model. The role of Als3 is transient and mild, though significant. Our findings suggest that the versatility of C. albicans as a pathogen may reflect its ability to persist in the face of multiple stresses and underscore that transcriptional circuitry during infection may be distinct from that detailed during in vitro growth.
Related Articles
Small-Molecule Inhibitors of the Budded-to-Hyphal-Form Transition in the Pathogenic Yeast Candida albicans
Toenjes, Kurt A.
Munsee, Suzanne M.
Ibrahim, Ashraf S.
Jeffrey, Rachel
Edwards, John E.
Johnson, Douglas I.
Antimicrobial Agents and Chemotherapy
The pathogenic yeast Candida albicans can exist in multiple morphological states, including budded, pseudohyphal, and true hyphal forms. The ability to convert between the budded and hyphal forms, termed the budded-to-hyphal-form transition, is important for virulence and is regulated by multiple environmental and cellular signals. To identify inhibitors of this morphological transition, a microplate-based morphological assay was developed. With this assay, the known actin-inhibiting drugs latrunculin-A and jasplakinolide were shown to inhibit the transition in a dose-dependent and reversible manner. Five novel small molecules that reversibly inhibited the transition and hyphal elongation without affecting budded growth were identified. These molecules inhibited hyphal growth induced by Spider, Lee's, M199 pH 8, and 10% serum-containing media, with two molecules having a synergistic effect. The molecules also differentially affected the hyphal form-specific gene expression of HWP1 and endocytosis without disrupting the actin cytoskeleton or septin organization. Structural derivatives of one of the molecules were more effective inhibiters than the original molecule, while other derivatives had decreased efficacies. Several of the small molecules were able to reduce C. albicans-dependent damage to endothelial cells by inhibiting the budded-to-hyphal-form transition. These studies substantiated the effectiveness of the morphological assay and identified several novel molecules that, by virtue of their ability to inhibit the budded-to-hyphal-form transition, may be exploited as starting points for effective antifungal therapeutics in the future.
Related Articles
Role of Bcr1-Activated Genes Hwp1 and Hyr1 in Candida Albicans Oral Mucosal Biofilms and Neutrophil Evasion
Dwivedi, Prabhat
Thompson, Angela
Xie, Zhihong
Kashleva, Helena
Ganguly, Shantanu
Mitchell, Aaron P.
Dongari-Bagtzoglou, Anna
PLoS ONE
Candida albicans triggers recurrent infections of the oropharyngeal mucosa that result from biofilm growth. Prior studies have indicated that the transcription factor Bcr1 regulates biofilm formation in a catheter model, both in vitro and in vivo. We thus hypothesized that Bcr1 plays similar roles in the formation of oral mucosal biofilms and tested this hypothesis in a mouse model of oral infection. We found that a bcr1/bcr1 mutant did not form significant biofilm on the tongues of immunocompromised mice, in contrast to reference and reconstituted strains that formed pseudomembranes covering most of the tongue dorsal surface. Overexpression of HWP1, which specifies an epithelial adhesin that is under the transcriptional control of Bcr1, partly but significantly rescued the bcr1/bcr1 biofilm phenotype in vivo. Since HWP1 overexpression only partly reversed the biofilm phenotype, we investigated whether additional mechanisms, besides adhesin down-regulation, were responsible for the reduced virulence of this mutant. We discovered that the bcr1/bcr1 mutant was more susceptible to damage by human leukocytes when grown on plastic or on the surface of a human oral mucosa tissue analogue. Overexpression of HYR1, but not HWP1, significantly rescued this phenotype. Furthermore a hyr1/hyr1 mutant had significantly attenuated virulence in the mouse oral biofilm model of infection. These discoveries show that Bcr1 is critical for mucosal biofilm infection via regulation of epithelial cell adhesin and neutrophil function.
Related Articles
The Adhesin Hwp1 and the First Daughter Cell Localize to the a/a Portion of the Conjugation Bridge during Candida albicans Mating
Daniels, Karla J.
Lockhart, Shawn R.
Staab, Janet F.
Sundstrom, Paula
Soll, David R.
Molecular Biology of the Cell
The cell wall protein Hwp1 was originally demonstrated to be expressed exclusively in hyphae of Candida albicans and cross-linked to human epithelium by mammalian transglutaminase. Hwp1 is expressed on the walls of hyphae formed by a/α, a/a, and α/α cells. Hence, it is expressed on hyphae independently of mating type. However, Hwp1 is selectively expressed on the wall of conjugation tubes formed by a/a cells, but not α/α cells, in the mating process. This was demonstrated in all possible crosses between four unrelated natural a/a strains and four unrelated α/α strains. In zygotes, Hwp1 is restricted to that portion of the wall of the conjugation bridge contributed by the a/a parent cell. Hwp1 staining further revealed that the first daughter bud that emerges from the conjugation bridge does so from the a/a-contributed portion. Hwp1 expression and localization during the mating process is, therefore, mating type specific, opaque phase specific, and α-pheromone induced. These results indicate that the mating type-specific contributions to the conjugation bridge during the mating process in C. albicans are qualitatively and functionally distinct and that the a/a portion of the bridge, which selectively contains Hwp1, bears the first daughter cell in the mating process.
Related Articles
C. albicans growth, transition, biofilm formation, and gene expression modulation by antimicrobial decapeptide KSL-W
Theberge, Simon
Semlali, Abdelhabib
Alamri, Abdullah
Leung, Kai P
Rouabhia, Mahmoud
BMC Microbiology
Antimicrobial peptides have been the focus of much research over the last decade because of their effectiveness and broad-spectrum activity against microbial pathogens. These peptides also participate in inflammation and the innate host defense system by modulating the immune function that promotes immune cell adhesion and migration as well as the respiratory burst, which makes them even more attractive as therapeutic agents. This has led to the synthesis of various antimicrobial peptides, including KSL-W (KKVVFWVKFK-NH2), for potential clinical use. Because this peptide displays antimicrobial activity against bacteria, we sought to determine its antifungal effect on C. albicans. Growth, hyphal form, biofilm formation, and degradation were thus examined along with EFG1, NRG1, EAP1, HWP1, and SAP 2-4-5-6 gene expression by quantitative RT-PCR.
This study demonstrates that KSL-W markedly reduced C. albicans growth at both early and late incubation times. The significant effect of KSL-W on C. albicans growth was observed beginning at 10 μg/ml after 5 h of contact by reducing C. albicans transition and at 25 μg/ml by completely inhibiting C. albicans transition. Cultured C. albicans under biofilm-inducing conditions revealed that both KSL-W and amphotericin B significantly decreased biofilm formation at 2, 4, and 6 days of culture. KSL-W also disrupted mature C. albicans biofilms. The effect of KSL-W on C. albicans growth, transition, and biofilm formation/disruption may thus occur through gene modulation, as the expression of various genes involved in C. albicans growth, transition and biofilm formation were all downregulated when C. albicans was treated with KSL-W. The effect was greater when C. albicans was cultured under hyphae-inducing conditions.
These data provide new insight into the efficacy of KSL-W against C. albicans and its potential use as an antifungal therapy.
Antimicrobial peptide; KSL-W; C. albicans; Growth; Hyphae; Gene; EFG1; NRG1; HWP1; SAPs
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Rbt1 Protein Domains Analysis in Candida albicans Brings Insights into Hyphal Surface Modifications and Rbt1 Potential Role during Adhesion and Biofilm Formation
Monniot, Céline
Boisramé, Anita
Da Costa, Grégory
Chauvel, Muriel
Sautour, Marc
Bougnoux, Marie-Elisabeth
Bellon-Fontaine, Marie-Noëlle
Dalle, Frédéric
d'Enfert, Christophe
Richard, Mathias L.
PLoS ONE
Cell wall proteins are central to the virulence of Candida albicans. Hwp1, Hwp2 and Rbt1 form a family of hypha-associated cell surface proteins. Hwp1 and Hwp2 have been involved in adhesion and other virulence traits but Rbt1 is still poorly characterized. To assess the role of Rbt1 in the interaction of C. albicans with biotic and abiotic surfaces independently of its morphological state, heterologous expression and promoter swap strategies were applied. The N-terminal domain with features typical of the Flo11 superfamily was found to be essential for adhesiveness to polystyrene through an increase in cell surface hydrophobicity. A 42 amino acid-long domain localized in the central part of the protein was shown to enhance the aggregation function. We demonstrated that a VTTGVVVVT motif within the 42 amino acid domain displayed a high β-aggregation potential and was responsible for cell-to-cell interactions by promoting the aggregation of hyphae. Finally, we showed through constitutive expression that while Rbt1 was directly accessible to antibodies in hyphae, it was not so in yeast. Similar results were obtained for another cell wall protein, namely Iff8, and suggested that modification of the cell wall structure between yeast and hyphae can regulate the extracellular accessibility of cell wall proteins independently of gene regulation.
Related Articles
A Comprehensive Functional Portrait of Two Heat Shock Factor-Type Transcriptional Regulators Involved in Candida albicans Morphogenesis and Virulence
Znaidi, Sadri
Nesseir, Audrey
Chauvel, Murielle
Rossignol, Tristan
d'Enfert, Christophe
PLoS Pathogens
Sfl1p and Sfl2p are two homologous heat shock factor-type transcriptional regulators that antagonistically control morphogenesis in Candida albicans, while being required for full pathogenesis and virulence. To understand how Sfl1p and Sfl2p exert their function, we combined genome-wide location and expression analyses to reveal their transcriptional targets in vivo together with the associated changes of the C. albicans transcriptome. We show that Sfl1p and Sfl2p bind to the promoter of at least 113 common targets through divergent binding motifs and modulate directly the expression of key transcriptional regulators of C. albicans morphogenesis and/or virulence. Surprisingly, we found that Sfl2p additionally binds to the promoter of 75 specific targets, including a high proportion of hyphal-specific genes (HSGs; HWP1, HYR1, ECE1, others), revealing a direct link between Sfl2p and hyphal development. Data mining pointed to a regulatory network in which Sfl1p and Sfl2p act as both transcriptional activators and repressors. Sfl1p directly represses the expression of positive regulators of hyphal growth (BRG1, UME6, TEC1, SFL2), while upregulating both yeast form-associated genes (RME1, RHD1, YWP1) and repressors of morphogenesis (SSN6, NRG1). On the other hand, Sfl2p directly upregulates HSGs and activators of hyphal growth (UME6, TEC1), while downregulating yeast form-associated genes and repressors of morphogenesis (NRG1, RFG1, SFL1). Using genetic interaction analyses, we provide further evidences that Sfl1p and Sfl2p antagonistically control C. albicans morphogenesis through direct modulation of the expression of important regulators of hyphal growth. Bioinformatic analyses suggest that binding of Sfl1p and Sfl2p to their targets occurs with the co-binding of Efg1p and/or Ndt80p. We show, indeed, that Sfl1p and Sfl2p targets are bound by Efg1p and that both Sfl1p and Sfl2p associate in vivo with Efg1p. Taken together, our data suggest that Sfl1p and Sfl2p act as central "switch on/off" proteins to coordinate the regulation of C. albicans morphogenesis.
Author Summary
Candida albicans can switch from a harmless colonizer of body organs to a life-threatening invasive pathogen. This switch is linked to the ability of C. albicans to undergo a yeast-to-filament shift induced by various cues, including temperature. Sfl1p and Sfl2p are two transcription factors required for C. albicans virulence, but antagonistically regulate morphogenesis: Sfl1p represses it, whereas Sfl2p activates it in response to temperature. We show here that Sfl1p and Sfl2p bind in vivo, via divergent motifs, to the regulatory region of a common set of targets encoding key determinants of morphogenesis and virulence and exert both activating and repressing effects on gene expression. Additionally, Sfl2p binds to specific targets, including genes essential for hyphal development. Bioinformatic analyses suggest that Sfl1p and Sfl2p control C. albicans morphogenesis by cooperating with two important regulators of filamentous growth, Efg1p and Ndt80p, a premise that was confirmed by the observation of concomitant binding of Sfl1p, Sfl2p and Efg1p to the promoter of target genes and the demonstration of direct or indirect physical association of Sfl1p and Sfl2p with Efg1p, in vivo. Our data suggest that Sfl1p and Sfl2p act as central "switch on/off" proteins to coordinate the regulation of C. albicans morphogenesis.
Related Articles
HWP1 Functions in the Morphological Development of Candida albicans Downstream of EFG1, TUP1, and RBF1
Sharkey, Laura L.
McNemar, Mark D.
Saporito-Irwin, Susan M.
Sypherd, Paul S.
Fonzi, William A.
Journal of Bacteriology
The morphological plasticity of Candida albicans is an important determinant of pathogenicity, and nonfilamentous mutants are avirulent. HWP1, a hypha-specific gene, was identified in a genetic screen for developmentally regulated genes and encodes a cell surface protein of unknown function. Heterozygous and homozygous deletions of HWP1 resulted in a medium-conditional defect in hyphal development. HWP1 expression was blocked in a Δefg1 mutant, reduced in an Δrbf1 mutant, and derepressed in a Δtup1 mutant. Therefore, HWP1 functions downstream of the developmental regulators EFG1, TUP1, and RBF1. Mutation of CPH1 had no effect on HWP1 expression, suggesting that the positive regulators of hyphal development, CPH1 and EFG1, are components of separate pathways with different target genes. The expression of a second developmentally regulated gene, ECE1, was similarly regulated by EFG1. Since ECE1 is not required for hyphal development, the regulatory role of EFG1 apparently extends beyond the control of cell shape determinants. However, expression of ECE1 was not influenced by TUP1, suggesting that there may be some specificity in the regulation of morphogenic elements during hyphal development.
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Candida albicans RFX2 Encodes a DNA Binding Protein Involved in DNA Damage Responses, Morphogenesis, and Virulence▿
Hao, Binghua
Clancy, Cornelius J.
Cheng, Shaoji
Raman, Suresh B.
Iczkowski, Kenneth A.
Nguyen, M. Hong
Eukaryotic Cell
We previously showed that Candida albicans orf19.4590, which we have renamed RFX2, expresses a protein that is reactive with antibodies in persons with candidiasis. In this study, we demonstrate that C. albicans RFX2 shares some functional redundancy with Saccharomyces cerevisiae RFX1. Complementation of an S. cerevisiae rfx1 mutant with C. albicans RFX2 partially restored UV susceptibility and the repression of DNA damage response genes. DNA damage- and UV-induced genes RAD6 and DDR48 were derepressed in a C. albicans rfx2 null mutant strain under basal conditions, and the mutant was significantly more resistant to UV irradiation, heat shock, and ethanol than wild-type strain SC5314. The rfx2 mutant was hyperfilamentous on solid media and constitutively expressed hypha-specific genes HWP1, ALS3, HYR1, ECE1, and CEK1. The mutant also demonstrated increased invasion of solid agar and significantly increased adherence to human buccal epithelial cells. During hematogenously disseminated candidiasis, mice infected with the mutant had a significantly delayed time to death compared to the wild type. During oropharyngeal candidiasis, mice infected with the mutant had significantly lower tissue burdens in the oral cavity and esophagus at 7 days and they were less likely to develop disseminated infections because of mucosal translocation. The data demonstrate that C. albicans Rfx2p regulates DNA damage responses, morphogenesis, and virulence.
Related Articles
A Candida albicans Temperature-Sensitive cdc12-6 Mutant Identifies Roles for Septins in Selection of Sites of Germ Tube Formation and Hyphal Morphogenesis
Li, Lifang
Zhang, Chengda
Konopka, James B.
Eukaryotic Cell
Septins were identified for their role in septation in Saccharomyces cerevisiae and were subsequently implicated in other morphogenic processes. To study septins in Candida albicans hyphal morphogenesis, a temperature-sensitive mutation was created that altered the C terminus of the essential Cdc12 septin. The cdc12-6 cells grew well at room temperature, but at 37°C they displayed expected defects in septation, nuclear localization, and bud morphogenesis. Although serum stimulated the cdc12-6 cells at 37°C to form germ tube outgrowths, the mutant could not maintain polarized hyphal growth and instead formed chains of elongated cell compartments. Serum also stimulated the cdc12-6 mutant to induce a hyphal reporter gene (HWP1-GFP) and a characteristic zone of filipin staining at the leading edge of growth. Interestingly, cdc12-6 cells shifted to 37°C in the absence of serum gradually displayed enriched filipin staining at the tip, which may be due to the altered cell cycle regulation. A striking difference from the wild type was that the cdc12-6 cells frequently formed a second germ tube in close proximity to the first. The mutant cells also failed to form the diffuse band of septins at the base of germ tubes and hyphae, indicating that this septin band plays a role in preventing proximal formation of germ tubes in a manner analogous to bud site selection. These studies demonstrate that not only are septins important for cytokinesis, but they also promote polarized morphogenesis and selection of germ tube sites that may help disseminate an infection in host tissues.
Related Articles
Extracellular DNA release confers heterogeneity in Candida albicans biofilm formation
Rajendran, Ranjith
Sherry, Leighann
Lappin, David F
Nile, Chris J
Smith, Karen
Williams, Craig
Munro, Carol A
Ramage, Gordon
BMC Microbiology
Biofilm formation by Candida albicans has shown to be highly variable and is directly associated with pathogenicity and poor clinical outcomes in patients at risk. The aim of this study was to test the hypotheses that the extracellular DNA release by C. albicans is strain dependent and is associated with biofilm heterogeneity.
Initially, biofilm formed by C. albicans high biofilm formers (HBF) or low biofilm formers (LBF) were treated with DNase to find whether eDNA play a role in their biofilm formation. Digestion of biofilm eDNA significantly reduced the HBF biofilm biomass by five fold compared to untreated controls. In addition, quantification of eDNA over the period of biofilm formation by SYBR green assay demonstrate a significantly higher level of 2 to 6 fold in HBF compared to LBF. Biochemical and transcriptional analyses showed that chitinase activity and mRNA levels of chitinase genes, a marker of autolysis, were upregulated in 24 h biofilm formation by HBF compared to LBF, indicating autolysis pathway possibly involved in causing variation. The biofilm biomass and eDNA release by single (∆cht2, ∆cht3) and double knockout (∆cht2/∆cht3) chitinase mutants were significantly less compared to their parental strain CA14, confirming the role of chitinases in eDNA release and biofilm formation. Correlation analysis found a positive correlation between chitinases and HWP1, suggesting eDNA may release during the hyphal growth. Finally, we showed a combinational treatment of biofilms with DNase or chitinase inhibitor (acetazolamide) plus amphotericin B significantly improved antifungal susceptibility by 2 to 8 fold.
Collectively, these data show that eDNA release by C. albicans clinical isolates is variable and is associated with differential biofilm formation. Digestion of biofilm eDNA by DNase may provide a novel therapeutic strategies to destabilise biofilm growth and improves antifungal sensitivity.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-014-0303-6) contains supplementary material, which is available to authorized users.
Candida albicans; Biofilm; eDNA; Chitinases
Related Articles
Therapeutic Potential of Thiazolidinedione-8 as an Antibiofilm Agent against Candida albicans
Feldman, Mark
Al-Quntar, Abed
Polacheck, Itzhak
Friedman, Michael
Steinberg, Doron
PLoS ONE
Candida albicans is known as a commensal microorganism but it is also the most common fungal pathogen in humans, causing both mucosal and systemic infections. Biofilm-associated C. albicans infections present clinically important features due to their high levels of resistance to traditional antifungal agents. Quorum sensing is closely associated with biofilm formation and increasing fungal pathogenicity. We investigated the ability of the novel bacterial quorum sensing quencher thiazolidinedione-8 (S-8) to inhibit the formation of, and eradication of mature C. albicans biofilms. In addition, the capability of S-8 to alter fungal adhesion to mammalian cells was checked. S-8 exhibited specific antibiofilm and antiadhesion activities against C. albicans, at four- to eightfold lower concentrations than the minimum inhibitory concentration (MIC). Using fluorescence microscopy, we observed that S-8 dose-dependently reduces C. albicans–GFP binding to RAW macrophages. S-8 at sub-MICs also interfered with fungal morphogenesis by inhibiting the yeast-to-hyphal form transition. In addition, the tested agent strongly affected fungal cell wall characteristics by modulating its hydrophobicity. We evaluated the molecular mode of S-8 antibiofilm and antiadhesion activities using real-time RT-PCR. The expression levels of genes associated with biofilm formation, adhesion and filamentation, HWP1, ALS3 and EAP1, respectively, were dose-dependently downregulated by S-8. Transcript levels of UME6, responsible for long-term hyphal maintenance, were also significantly decreased by the tested agent. Both signaling pathways of hyphal formation-cAMP-PKA and MAPK-were interrupted by S-8. Their upstream general regulator RAS1 was markedly suppressed by S-8. In addition, the expression levels of MAPK cascade components CST20, HST7 and CPH1 were downregulated by S-8. Finally, transcriptional repressors of filament formation, TUP1 and NRG1, were dramatically upregulated by our compound. Our results indicate that S-8 holds a novel antibiofilm therapeutic mean in the treatment and prevention of biofilm-associated C. albicans infections.
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Hello. Take a look in this funny request please: "You are sitting down, reading a pregnancy/baby magazine, wishing that you were pregnant again. Suddenly, your belly starts growing, and you realize that you are pregnant. You are happy to be pregnant again and you want to get bigger. Your belly gets bigger and bigger, you do not want it to stop. You can feel the kicks of many babies in your belly. The video cuts to later and you now have a massive belly (giant balloon under dress). You are happy with your belly, and you hope you can be pregnant forever. You try and get up, struggling because your belly is very heavy, and walk away to the kitchen."
Hey!!! This is the request:"hey can you do me a pregnancy request were you wear a short dress that you just fit in and have your fake belly grow so much you think your dress is going to rip open and you can carrying as many babies you want. thank you". Of course that i can do ;D
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Hi!!!! This is the request: "Could you make a set with belly full stuffed to maximum, wearing tiny underwear/bikini. Pls set on the bed, dancing, jumping and laying and rolling sexy with a pillow? You then end pls putting oil on your body and taking out bra? show close your belly, thighs and face!"
Miss Pink came on to show you in one video the moust belly requests; like: Belly massaging; Belly pocking; Belly squeeze; Belly button playing If you like belly attention you will fall in love with this video!
Hi people!!! See my request please: "Could you do the same "friend thinks you're pregnant request" that honney recently did? Please :)"
Hello. See this request: "Pls a changing several tiny bikini underwear request with stuffed belly and drinking cachaza or similar. Pending my previous request for september at the beach! Pls continue gaining."
Hello!!! This is the request: "Workout in tight clothes with your belly hanging out". It is going to be funny, lets go!!
Hello. Take a look in this nice request. Its a remake. I am sure that you will enjoy.
I'm doing an outgrown with my old jeans. . .jeans i would dress at the first sets here in the site. . . WATCH AND HAVE FUN!
Carol is getting dressed for work and is runnining ou time. BUT HER PANTS SPLITS OPEN AS SHE BENDS OVER TO PICK UP HER KEYS. EMBARRASSING UNDERWEAR UNDERNEATH! ! ! CAROL GETS EXTREMELY EMBARASED! ! !
THIS IS A TALKING VIDEO! HERE I'M TALKING AND SHOWING YOU MY FAST, SPECIALY THE BELLY AND BOOBS. . .Boobs have been far the part which have grown bigger!
In VERY SMALL BIKINI. . . CLOSE UPS. . . DRINKING BEER. . . TOPLESS. . . Watch the video and have fun ;-)
WALKING, TALKING, MODELING, SIT-UPS, IN MY CORRIDOR! If your fan of walking and moving video, this is a perfect video for you, watch it!
TAKING BEER AND TRYING OUTGROWNED AND TOO TIGHT DRESS. . . I did almoust rip one of my dresses trying to fit on it. . .lol. I though its better drink more and get a bigger belly then try to get the dress buttoned! Watch it! ! !
PUSHING IN AND OUT MY BELLY! TALKING A LOT while pushing the belly in and out the maximun i can! Watch it!
I just have hade a loving time, hmmmm i feel so good! BUT HEY SOMETHING IS HAPPENING IN MY BELLY! Ow my, my belly IS GROWING! IT GOT SO BIG! ! ! LOOKS LIKE I'M 9 MONTHS PREGNANT, WITH 3 BABIES! OW MY! ! ! NOW I FEEL LIKE I'M IN LABOR! If you like fantasy, baloons, this is your video!
Hello, in this video i have a BIG PREGNANCY BELLY and i talk about how does it feels to have this big pregnancy belly! FUN WATCH AND HEARD!
Hello. . . This is a great set to change a little of what we usualy do. TOTAL BALOON SET! BELLY. BOOTY. BOOM. Verry funny video and set, PLEASE WATCH IT!
Hello, today i got a request. . .and right when i did a close up face my PANTS RIPED. . . I FEEL SO EMBARASED! All i have to say is that YOU MUST WATCH THE VIDEO TO SEE ALL ABOUT IT! ! !
Hello. It was so nice to remake this set! In my restroom ONLY IN LINGERY LUBBING LOTION INJ MY BODY! What else can i say? WATCH IT NOW! ;-)
Hello. . .In this request i'm talking about my belly and about gain more weight! Watch the video please!!! :-)

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Candlewick | Mar 26, 2013 | 48 Pages | 6 x 9 | 5-9 years | ISBN 9780763648411
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When two friends who are not exactly alike come down with colds, figuring out what will make each other feel better can be a surprising endeavor!Monkey and Elephant are very good friends who have a lot in common. They both like morning time. They both like shiny rocks. And they both like to hear their own voices: "TAH. TAH. TAH." But Monkey is Monkey and Elephant is Elephant. They are not exactly the same. So when Elephant starts to sneeze — achoo! — Monkey has her own ideas of what will make him feel better. And when Monkey gets the sniffles, Elephant has his own ideas of what will make her feel better. Maybe there's more than one way to enjoy a soft pile of hay, some fresh water, and a bit of entertainment!
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Chemical hair dye over henna
Started by
, Jan 19 2013 10:31 AM
5 replies to this topic
#1Posted 19 January 2013 - 10:31 AM
I (a few months ago) decided to stop using chemical hair dyes and got henna from lush and used that. Result was good but just takes so long to do the whole thing that wondering if should just go back to chemical ... least its quicker and easier! Sure I read somewhere that using chemical hair dye over henna-ed hair can be problematic. Wondering if anyone has done it and their hair has been ok? #2Posted 19 January 2013 - 10:36 AM
I did it and I *think* it might have stuffed up my hair... it just hasn't been the same since.
Love henna on brown hair, it looks amazing! Does nothing for me though. #3Posted 19 January 2013 - 03:00 PM
I wouldn't attempt it until the henna has faded out of your hair.
I use Lush Henna as well and while it's a massive pain in the neck to do it, I like the result so will continue using it. #4Posted 19 January 2013 - 05:17 PM
hmmm i have brown hair and used the brown finish henna, may go for the redder one next time though.
may just stick with the henna, least its natural! & even with chemical dye i only did it once every 3-4 mnths anyway Edited by fefe76, 19 January 2013 - 06:30 PM. #5Posted 21 January 2013 - 10:02 PM
Oh dear I didnt realise you cant put chemical die over the top of henna! Glad I read this post!
I have been using chemical dies for years and its really taking its toll on my thin locks. Love the henna but its a pain to use and I dont live near a Lush store so then there is postage. I'm going to see whether our health food store sells a different henna brand for next time as I really dont want to kill my hair again by using the chemical stuff. It was just starting to look up. #6Posted 22 January 2013 - 08:56 AM
I know Mrs Flannerys and other health places have henna powder for hair
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The School wishes to appoint an experienced ESL teacher to join the Trinity Education Support Services Team, based at the Preparatory School at Strathfield. The ...
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Australian International School Malaysia - Sydney , Nsw
JOB SUMMARY Job Title: EAL Teacher Reporting to: Head of School Direct Reports: (Nil) JOB OUTCOMES • Provide an exemplary student-centered, ICT rich learning ...
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Junior School EAL / ESL Teacher
ESL Teacher Required Immediately for High School in Jiangyan, China - Ref No.16516843
Anz Uk Teaching - Melbourne , Vic
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Randstad - Brisbane , Qld
Teacher - Mathematics
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Diversified Financial Services
Company Overview of EBS Limited
Company Overview
The EBS Building
2 Burlington Road
Dublin, 4
Founded in 1935
613 Employees
Key Executives for EBS Limited
Group Chief Executive, Executive Director, Member of Transactions Committee, Member of Nominations Committee, Member of Credit Approval Committee and Member of Risk Committee
Age: 50
Compensation as of Fiscal Year 2014.
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The Oxford English Dictionary's overarching definition of the transitive verb "publish" is "to make public." An early use, dating to 1382 is "to prepare and issue copies of (a book, newspaper, piece of music, etc.)." This is probably how most publishers think of the term: public distribution of a text. In usage dating from 1573, … Continue reading
Revisiting Kent Anderson's post based on his FOIA request documents show that PubMed Central spends most of its money tagging author manuscripts, and that its stricter rules for NIH authors may double its costs. Continue reading
As we explore the new world of data-driven discovery tools, we must also examine their utility, their trustworthiness and what impact they may have on the creative process. Continue reading
First released in 1935 as a game to teach children the evils of unchecked market capitalism, MONOPOLY-The Publishers' edition keeps the tradition going. Continue reading
As we consider the future of scholarly publishing generally and of open access in particular, we need to keep in mind the deep differences between the humanities and the applied sciences when it comes to both the production and the consumption of scholarship–and the implications of those differences for new dissemination models. Continue reading
Lack of information about how books are actually used has resulted in a set of actions that don't make solid economic sense. Now that more end-user information is becoming available, the book business is likely to adjust its practices. Continue reading
As we drift into a scholarly economy with centralized payment mechanisms and greater dependence on government funding, are we truly setting ourselves up for long-term independence and success? Continue reading
There's no such thing as "too big to fail," and this applies to libraries as much as it does to car makers, steel companies, and search engines. My guess is that Ozymandias never saw it coming. Continue reading
The emerging spectre of cyberwar and cyberterror has real implications for academic and scientific publishers, who already deal with the side effects and may become targets in the future. Continue reading
A recent study finds that academic press offices exaggerate claims in their press releases about published research. Worse, the vast majority of these find their way into subsequent reporting. Continue reading


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Relevant offers
OPINION: It turns out Milford Dart tunnel promoters were just banging their heads on a rock wall.
However, Conservation Minister Nick Smith's decision to decline the $180 million project that would have cut through 11km of mountain and halved the travel time between Queenstown and Milford Sound is interestingly worded.
Dr Smith cites three major reasons - the half-million tonnes of tunnel spoil, the impact of new roads and portals, particularly at the entrance to the Routeburn track, and that the engineering works and tunnel are deemed inconsistent with the Fiordland and Mt Aspiring National Park management plans.
All of which he sums up as nature getting the benefit of any doubt. But then he is very quick to add, in effect, that he had concerns that Milford Dart Ltd would do the job properly. To make such a long, narrow tunnel safe would require huge investment in ventilation and emergency services and Dr Smith identifies a risk that corners would be cut, or the project left half-completed with the public left with a clean-up liability. Though it didn't make his top three reasons, the suspicion arises that it loomed rather large in his thinking for all that.
By contrast, it is worth noting for the record that the often-rumbled warnings that Unesco world heritage status would be seriously imperilled by the tunnel were a fat red herring. Such an impact was unlikely, the minister acknowledged, given that Unesco had no concerns about the similarly scaled Manapouri tailrace tunnel, nor visitor facility developments in world heritage areas elsewhere in the world.
Milford Dart managing director Tony Elworthy sees Dr Smith's decision was essentially political. There was certainly a great deal of political heat based on fears not only for the environment, but the economic consequences for a bypassed Te Anau. So this could be called a political decision. Or, given the greater scale of demonstrated opposition, a democratic one.
The tunnel idea wasn't inherently a terrible one at all. The broader notion of getting tourists more easily to their destination is a good one. As things stand the bus trip from Queenstown to Milford and back, for all that it offers memorable rewards, is also a grinding endurance test and needlessly so.
The Greens argue that the agenda should be to slow visitors down so they stay longer and spend more, rather than pushing them 11km underground "to save a few hours' travel". Apart from the question of how much better off we, or our visitors, really are keeping all those buses thundering along the existing, longer road route, there's the small matter of how wilful we should be about denying them a choice. It's one thing to tempt them to spend time; another to prevent them having much of an alternative.
Te Anau has problems. In contrast to Wanaka and Queenstown it hasn't benefited half as much as it wants from tourist growth. Nor will it, until we find a way to make it easier or more attractive for tourists to spend time there.
The separate proposal from Riverstone Holdings for the Fiordland Link Experience, taking passengers from Queenstown to Te Anau Downs via catamaran, all-terrain vehicle and monorail through the Snowdon Forest area, has passed the public submission stage, with the minister yet to make his decision. Even though the monorail passes over DOC estate but not national park land, it will also face ferocious opposition.
We'll see what happens. But roll on the day that the Haast-Hollyford road proposal gets the go-ahead. The case for connecting the West Coast to Southland is just compelling.
- The Southland Times
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No regrets from coach Jamie Joseph as Highlanders' Super Rugby charge stalls
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Lack of foresight and vision hampering Invercargill - retailer


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Showing based on selected map area. Don't limit my results by the map.
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3 bedroom with All UTILITIES INCLUDED 3 Bedroom row home 1 Bathroom (Ceramic tile) Brand new... More -
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2850 sqftBeautiful Brownstone Rehabbed w/ custom features throughout. This amazing 2850 sq ft property... More -
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$750 2BR 1.5BA 215 S. Calhoun St. Cute row home, partially finished basement, W/D hookups. NO... More -
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You''ll have plenty of room in this Private, 2nd Floor Apartment. Fenced In Backyard,... More -
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Totally Renovated Rowhouse in Baltimore. Close to downtown, the beltway, 295, Ravens Stadium and... More -
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Wonderful 3 bedroom Row home. Freshly painted, close to transportation (Public and MARC) and... More -
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Immaculate 3 Story house that offer easy access to the expressway and downtown. This house is a... More -
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OPEN 3/24/2015 1:00-1:30 GORGEOUS KITCHEN W/DISHWASHER DRAWERS, GRANITE AND GLASS TILES,... More -
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1367 sqftHardwood floors, Central Air, New kitchen, new cabinets, Newly painted, New flooring, Clean open... More -
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1000 sqftNewly Renovated 3 bedroom and 1 bath walking distance to the harbor Nice 3 bedroom and 1 bathroom... More
Summary for Baltimore
Average price per square foot for Baltimore MD was $132, a decrease of 2.2% compared to the same period last year. The median sales price for homes in Baltimore MD for Jan 15 to Apr 15 was $159,700 based on 1,162 home sales. Compared to the same period one year ago, the median home sales price decreased 6%, or $10,200, and the...


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Community Classifieds(Deadline for submission is the Friday before publication. Most items are published for two weeks.) MWS continues to recycle all toner cartridges and cell phones, just bring them to either office. Storage is easier if toner boxes are recycled at home. Thank you! Full-Size Violin For Sale: Has your violin player sprouted up this fall and finally need a full-sized instrument? For less than the cost of renting, you could own your own violin! Gently used for several years, asking $400. For information, call Aly Fullagar at 274-0057. (12/18) Swell Construction has a few openings for indoor carpentry/renovation work this winter. Please inquire with Adrian Bossi (MWS parent) at 841-1355 or firstname.lastname@example.com. (12/18) Quality Toys for Sale: Wooden kitchen heirloom quality "Made by Real Elves in Maine". Was $200 new, now $60.00. Includes some small wooden kitchen accouterments. BIKE BIKE BIKE, Has your child out grown his bike? We're looking for a gently used mountain bike for Finn. He's 12, he's 5'2" or so and would fit a 17" bike. Please call Rosita or Will at 688-4715 or email firstname.lastname@example.com.(12/11) Friday Afternoon Family Skating for the Winter Months: This posting is to announce the possibility of MWS family skating at Falmouth Family Ice on Friday afternoons from 1:40 to 2:40 beginning January 11th and running every Friday, except for a few, until April 12th. There are several ways to participate. You can pay a discounted lump sum for the season for you and your family to skate every Friday; you can pay a ' pay as you go' sum for you and your family to skate whenever you can make it; or you can pay a single child rate either as a lump sum or as he/she can make it if your child will be joining another family to skate. If you and your family are interested, or if you have questions, please email or call me as soon as you can so that I can get all the details ironed out. Julia Dodge at email@example.com or 712-523. Thanks so much. Think winter and skating! (12/11) Property next to the Merriconeag Early Childhood Center for sale: 2.5 acre lot of cleared, open field for sale. This land is adjacent to the school property by the Early Childhood Center. Soil tested, southern exposure. Price: $80,000.00. Please contact Ruby or TJ McDermott, 66 Desert Rd., 207-233-2051 or 207-865-1300 email: firstname.lastname@example.com. (Note: TJ did the beautiful carvings on the classroom doors in the ECC!) (12/11)
Board Meeting:Tues, April 14, 6:15 pm
Community Hall.
"You Can't Take It With You":Grade 8 Play
Wed, April 15, 1:00 pm.
Thurs, April 16, 7:00 pm.
Community Hall, 57 Desert Rd, Freeport.
The 8th Graders will present their play, "You Can't Take It With You." Please join us. Bring A Friend Day:Fri, April 17, 8:00 am - 3:00 pm.
Register your grade school friend through firstname.lastname@example.com April Break - No School:Mon, April 20 through Fri, April 24.
School is closed for April Break. HS Parent Evening:Wed, April 29, 7:00 p.m. Merriconeag Poetry Festival:Sun, May 3, 3:00 - 4:30 pm, Community Hall, 57 Desert Rd, Freeport. Free admission. Featuring noted poet, Jeffrey Harrison as Festival Judge and student poet finalists from the following high schools: Cape Elizabeth, Catherine McAuley, Deering, Falmouth, Gorham, Hyde, Merriconeag Waldorf, Morse, Waynflete, and Yarmouth. G 2, 4, 5 & 6 Parent Evenings:Tues, May 5, 7:00 pm. Senior Project Presentations:Thurs, May 7, 6:30 pm. Community Hall, 57 Desert Rd, Freeport. Tea & Play Session:Fri, May 8, 9 - 10 am.
Early Childhood Center, 60 Desert Rd, Freeport.
Prospective parents: Bring your child, share
a cup of tea with us and learn more about our
programs for young children. Storytime at 9:30.
For more info & to register: 207.865.3900,
Ext 163 or
May Celebration and Medieval Faire:Saturday, May, 10 am - 1 pm.
57 Desert Rd, Freeport


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Find records of Ruth Warner
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La Crosse area school officials are double checking payroll security after hackers made off with $150,000 from a western Wisconsin school district.
The money was designated as pay for teachers and staff working for Stanley-Boyd public schools in Chippewa County, but the funds disappeared last week somewhere in the course of digital transfers between the school district, a Madison-based bank and employee bank accounts.
La Crosse area school administrators say they work closely with banks
to make sure payroll is handled securely. However, safety measures vary, depending on the financial institution charged with receiving and distributing direct deposits.
"We do try and protect to the best of our ability," said Larry Dalton, finance director for the Onalaska School District.
Dalton called Merchants Bank on Wednesday to ask about the situation at Stanley-Boyd schools. Merchants handles the Onalaska district's payroll.
Dalton wants to know more about incident to determine if Onalaska schools need stronger security.
"Hopefully, we'll all learn from this and see if there's a way of tightening things up," Dalton said.
Janet Rosseter, finance director for the La Crosse School District, said officials take preventative measures to keep payroll information from falling into the wrong hands.
Security is discussed during regular meetings between La Crosse school officials and Wells Fargo, the district's bank, Rosseter said.
"That's not a conversation that happens in reaction to something," Rosseter said. "It's a proactive conversation that we have at an ongoing basis."
In a digital world where nearly all payroll information is handled online, school districts already use special encryptions and passwords to protect against hackers.
Some take extra safety measures when it comes to the transmission of private financial data like direct deposit information. Holmen public schools use a double verification system that involves additional codes and communication between the district and the bank, said Jason Austin, the district's finance director.
Austin is confident the added buffer is enough to confound a would-be hacker.
"I'd like to see him try," Austin said.
Still, no security measure is foolproof. Even the latest technology is only part of an endless battle of wills between organizations and the people they want to protect against, Dalton said.
"For them, it's a game of figuring out how to get in," Dalton said. "For us, it's a game of heightening security."


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£120,000 Offers in Excess of
Offers in Excess of £120,000
3 bedroom semi-detached house for sale Millmount Road, Hoyland, Barnsley
NO UPPER VENDOR CHAIN** This three bedroom semi detached house occupies a wonderful position with views over the surrounding area. Ideally suited to the family purchaser, there are gardens to three sides as well as a detached garage and a separate driveway. With plenty of scope for extension subj... More details › -
- 9
- 0
£119,950 Offers in Region ofOffers in Region of £119,950
3 bedroom semi-detached house for sale 163 Cherry Tree Street, Hoyland, Barnsley, S74 9RW
A very well presented 3 bedroom semi detached house with good sized gardens, drive, ample off-street parking and garage, ideally situated within a popular small South Yorkshire town centre, convenient for junction 36 of the M1 motorway and the Dearne Valley Parkway. More details › -
- 11
- 0
3 bedroom semi-detached house for sale Longfields Crescent, Hoyland, Barnsley
Having recently undergone a course of modernisation this three bedroom semi detached property enjoys contemporary fixtures and fittings, in addition to spacious rooms. Being set on a generous plot, this home enjoys gardens to front and rear with ample off street parking. Having three double bedro... More details › -
3 bedroom semi-detached house for sale Eaden Crescent, Hoyland, S74 9RH
Of particular interest to first time buyers or a growing family alike is this bedroom semi detached house situated within this very popular and convenient residential area. In brief the property consists of an entrance hallway, a large dual aspect lounge/diner, modern kitchen with fitted applianc... More details › -
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2 bedroom semi-detached house for sale Willow Close, Hoyland, BARNSLEY, South Yorkshire
Two bedroom semi detached property in a sought after area in Hoyland. This well proportioned property is a credit to the current owners and would make a fantastic buy for someone looking to downsize or a first time purchase. The accommodation briefly comprises a kitchen, living/dining room, dow... More details › -
£105,000 Guide Price
Guide Price £105,000
3 bedroom semi-detached house for sale Springfield Road, Hoyland
GUIDE PRICE OF £105,000 TO £115,000 Lovely three bedroom semi-detached property located in Hoyland Common and ready to view today. With two reception rooms and spacious garden, this is a must see. More details › -
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- 0
£105,000 Guide PriceGuide Price £105,000
3 bedroom town house for sale Kirk Balk Hoyland Barnsley
*** FOR SALE BY THE FAST MOVE METHOD, TERMS & CONDITIONS APPLY, SPEAK TO THE AGENT FOR FURTHER DETAILS *** A well presented brick built double fronted three bedroomed inner town house having been recently re roofed, with a modern fixture and finish throughout and currently benefiting from ful... More details ›Reduced on 21/03/2015 by Lancasters Property Services, Stocksbridge. Call: 0114 467 1623 Local call rate
£100,000 Offers Over
Offers Over £100,000
3 bedroom town house for sale Longfields Crescent, Hoyland, Barnsley
A MUST SEE FAMILY HOME! Offered for sale is this immaculatley presented, 3 bedroomed end town house, the property give access to local amenities and Hoyland town centre and has gas central heating and double glazing. There is an extensive driveway to the front and may suit a range of purchasers. More details ›


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How do I find the end position of the Caret in a WPF textbox so I can`t move right anymore with the caret?
Take the 2-minute tour
Stack Overflow is a question and answer site for professional and enthusiast programmers. It's 100% free, no registration required.
If you need to find the CaretIndex look at the following question. However, if you are looking to jump to the next TextBox under certain conditions check out the following sample. Here I use the TextBox property MaxLength and the KeyUp event to jump to the next TextBox when one is completed. Here is the XAML:
Here is the KeyUp event from the code-behind:


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Mark Berenbaum
Tue Dec 31
written by Bryan Mears on Jan 1st, 2014
Follow them at @bryan_mears
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NBA Playoffs Preview: Clippers vs. Spurs
Two of the league's best teams square off in the first round. Who will advance?
Daniel Lindsey
Fri Apr 17


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I'd really really like to trade with someone for these I'm Cass1968 on pin pics and am pretty up to date.Anyone able to help me in my quest?Post here or private e-mail me at firstname.lastname@example.com. cass
Hi! Just thought I'd move this over to the pin trading forum for you and give the post a bump. Any luck making a trade??
Thanks for the bump. No not even a nibble Wouldn't anyone be willing to trade with me pretty please with sugar or even cheddar cheese soup on top Again I'm Cass1968 on pin pics emaik is email@example.com. Please please pleases come play pin trading with meinkbounc inkbounc Cass


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This information is intended for educational purposes only. These statements have not been evaluated by the Food and Drug Administration. These products are not intended to diagnose, treat, cure or prevent disease.
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Tap Here to view the Mobile Optimized TFLN
You love popeyes more than me
does delicious chicken come out of your vagina?
it always starts out as a suggestion then three hours later I have cum in my eye.
im at a loss of words.... a stripper is dancing to a Justin Bieber song.
Cop gave me a ticket for public drunkedness, and then I convinced him to drive me back to the party
Nobody is wearing shirts anymore. What is happening.
I want you to tape your fingers together and give me a lobster claw hand job.
I am nonfunctional stoned. I had to ask ben to put me somewhere away from all the people I'm sitting on someones bed watching a wall. Not alright. Should not have come.
An hour ago, you were stranded out of state, and now you're getting laid? You are a god. Whatever you do, don't ask her name.
She is just sitting by the bathroom like a little puppy waiting for a knight in shining armor to take her in there to fuck her. New low?
Good news! I don't have Hep C! Better news! I still hate you!
Yeah. I was about to call 911 but I ended up breaking the door frame off then ran and puked all the way home.
I actually have to watch Breaking Bad to make me feel better about my choices last night.
I want to put in my resignation as an adult. From now on I will be spending my time drinking beer and skiing.
This is the Taco Bell dump we've all been waiting for.
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