The impact of ORF19.36.1 in the pathobiology of Candida albicans.

BACKGROUND: Our previous proteomics data obtained from Candida albicans recovered after serial passage in a murine model of systemic infection revealed that Orf19.36.1 expression correlates with the virulence of the fungus. Therefore, the impact of ORF19.36.1 upon virulence was tested in this study. MATERIALS & METHODS: CRISPR-Cas9 technology was used to construct homozygous C. albicans orf19.36.1 null mutants and the phenotypes of these mutants examined in vitro (filamentation, invasion, adhesion, biofilm formation, hydrolase activities) and in vivo assays. RESULTS: The deletion of ORF19.36.1 did not significantly impact the phenotypes examined or the virulence of C. albicans in two infection models. CONCLUSION: These results suggest that, although Orf19.36.1 expression correlates with virulence, this protein is not essential for C. albicans pathobiology.

Differential role of D cyclins in the regulation of cell cycle by influencing Ki67 expression in HaCaT cells.

D-type cyclins are important regulatory proteins of the G1/S phase of the cell cycle however, their specific functions are only partially understood. We show that silencing of individual D-type cyclins has no effect on the proliferation and morphology of Immortalized non-tumorigenic human epidermal (HaCaT) cells, while double and triple D cyclin silencing results in the failure of the cytokinesis leading to the appearance of large multinucleated cells. Both CDC20 and Ki67 mRNA is downregulated in these cells. Ki67 mRNA silenced cells show similar multinucleated cellular phenotype as double or triple D cyclin silenced cells without affecting D cyclin expression, suggesting that Ki67 is necessary for normal G2/M transition. Our data have revealed that cyclin D1 may have a leading role in G1/S phase regulation and suggest an incomplete functional overlap among D cyclins. Our results indicate that beside their well-known functions during the G0-G1/S phase, D-type cyclins play a pivotal role in the regulation of mitosis via influencing Ki67 expression in a downstream manner probably through E2F1 activation in HaCaT cells.

Elevated catalase expression in a fungal pathogen is a double-edged sword of iron.

Most fungal pathogens of humans display robust protective oxidative stress responses that contribute to their pathogenicity. The induction of enzymes that detoxify reactive oxygen species (ROS) is an essential component of these responses. We showed previously that ectopic expression of the heme-containing catalase enzyme in Candida albicans enhances resistance to oxidative stress, combinatorial oxidative plus cationic stress, and phagocytic killing. Clearly ectopic catalase expression confers fitness advantages in the presence of stress, and therefore in this study we tested whether it enhances fitness in the absence of stress. We addressed this using a set of congenic barcoded C. albicans strains that include doxycycline-conditional tetON-CAT1 expressors. We show that high basal catalase levels, rather than CAT1 induction following stress imposition, reduce ROS accumulation and cell death, thereby promoting resistance to acute peroxide or combinatorial stress. This conclusion is reinforced by our analyses of phenotypically diverse clinical isolates and the impact of stochastic variation in catalase expression upon stress resistance in genetically homogeneous C. albicans populations. Accordingly, cat1Δ cells are more sensitive to neutrophil killing. However, we find that catalase inactivation does not attenuate C. albicans virulence in mouse or invertebrate models of systemic candidiasis. Furthermore, our direct comparisons of fitness in vitro using isogenic barcoded CAT1, cat1Δ and tetON-CAT1 strains show that, while ectopic catalase expression confers a fitness advantage during peroxide stress, it confers a fitness defect in the absence of stress. This fitness defect is suppressed by iron supplementation. Also high basal catalase levels induce key iron assimilatory functions (CFL5, FET3, FRP1, FTR1). We conclude that while high basal catalase levels enhance peroxide stress resistance, they place pressure on iron homeostasis through an elevated cellular demand for iron, thereby reducing the fitness of C. albicans in iron-limiting tissues within the host.

Regulatory networks contributing to psoriasis susceptibility.

The non-involved, healthy-looking skin of psoriatic patients displays inherent characteristics that make it prone to develop typical psoriatic symptoms. Our primary aim was to identify genes and proteins that are differentially regulated in the non-involved psoriatic and the normal epidermis, and to discover regulatory networks responsible for these differences. A cDNA microarray experiment was performed to compare the gene expression profiles of 4 healthy and 4 psoriatic non-involved epidermis samples in response to T-cell lymphokine induction in organotypic cultures. We identified 61 annotated genes and another 11 expressed transcripts that were differentially regulated in the psoriatic tissues. Bioinformatics analysis suggested that the regulation of cell morphology, development and cell death is abnormal, and that the metabolism of small molecules and lipids is differentially regulated in psoriatic epidermis. Our results indicate that one of the early steps of psoriasis pathogenesis may be the abnormal regulation of IL-23A and IL-1B genes in psoriatic keratinocytes.

Interleukin-1 receptors are differentially expressed in normal and psoriatic T cells.

This study was carried out to examine the possible role of interleukin-1 (IL-1) in the functional insufficiency of regulatory T cells in psoriasis, by comparing the expression of IL-1 receptors on healthy control and psoriatic T cells. Patients with moderate-to-severe chronic plaque psoriasis and healthy volunteers, matched in age and sex, were selected for all experiments. CD4(+)CD25(-) effector and CD4(+)CD25(+)CD127(low) regulatory T cells were separated and used for the experiments. Expression of the mRNA of IL-1 receptors (IL-1R1, IL-1R2, and sIL-1R2) was determined by quantitative real-time RT-PCR. Cell surface IL-1 receptor expression was assessed by flow cytometry. Relative expression of the signal transmitting IL-1 receptor type 1 (IL-1R1) mRNA is higher in resting psoriatic effector and regulatory T cells, and activation induces higher IL-1R1 protein expression in psoriatic T cells than in healthy cells. Psoriatic regulatory and effector T cells express increased mRNA levels of the decoy IL-1 receptors (IL-1R2 and sIL-1R2) upon activation compared to healthy counterparts. Psoriatic T cells release slightly more sIL-1R2 into their surrounding than healthy T cells. In conclusion, changes in the expression of IL-1 receptors in psoriatic regulatory and effector T cells could contribute to the pathogenesis of psoriasis.

Characterising phagocytes and measuring phagocytosis from live Galleria mellonella larvae.

Over the last 20 years, the larva of the greater waxmoth, Galleria mellonella, has rapidly increased in popularity as an in vivo mammalian replacement model organism for the study of human pathogens. Experimental readouts of response to infection are most often limited to observing the melanization cascade and quantifying larval death and, whilst transcriptomic and proteomic approaches, and methods to determine microbial load are also used, a more comprehensive toolkit of profiling infection over time could transform the applicability of this model. As an invertebrate, Galleria harbour an innate immune system comprised of both humoral components and a repertoire of innate immune cells - termed haemocytes. Although information on subtypes of haemocytes exists, there are conflicting reports on their exact number and function. Flow cytometry has previously been used to assay Galleria haemocytes, but protocols include both centrifugation and fixation - physical methods which have the potential to affect haemocyte morphology prior to analysis. Here, we present a method for live haemocyte analysis by flow cytometry, revealing that Galleria haemocytes constitute only a single resolvable population, based on relative size or internal complexity. Using fluorescent zymosan particles, we extend our method to show that up to 80% of the Galleria haemocyte population display phagocytic capability. Finally, we demonstrate that the developed assay reliably replicates in vitro data, showing that cell wall ß-1,3-glucan masking by Candida albicans subverts phagocytic responses. As such, our method provides a new tool with which to rapidly assess phagocytosis and understand live infection dynamics in Galleria.

Nature of ß-1,3-Glucan-Exposing Features on Candida albicans Cell Wall and Their Modulation.

Candida albicans exists as a commensal of mucosal surfaces and the gastrointestinal tract without causing pathology. However, this fungus is also a common cause of mucosal and systemic infections when antifungal immune defenses become compromised. The activation of antifungal host defenses depends on the recognition of fungal pathogen-associated molecular patterns (PAMPs), such as ß-1,3-glucan. In C. albicans, most ß-1,3-glucan is present in the inner cell wall, concealed by the outer mannan layer, but some ß-1,3-glucan becomes exposed at the cell surface. In response to host signals, such as lactate, C. albicans induces the Xog1 exoglucanase, which shaves exposed ß-1,3-glucan from the cell surface, thereby reducing phagocytic recognition. We show here that ß-1,3-glucan is exposed at bud scars and punctate foci on the lateral wall of yeast cells, that this exposed ß-1,3-glucan is targeted during phagocytic attack, and that lactate-induced masking reduces ß-1,3-glucan exposure at bud scars and at punctate foci. ß-1,3-Glucan masking depends upon protein kinase A (PKA) signaling. We reveal that inactivating PKA, or its conserved downstream effectors, Sin3 and Mig1/Mig2, affects the amounts of the Xog1 and Eng1 glucanases in the C. albicans secretome and modulates ß-1,3-glucan exposure. Furthermore, perturbing PKA, Sin3, or Mig1/Mig2 attenuates the virulence of lactate-exposed C. albicans cells in Galleria. Taken together, the data are consistent with the idea that ß-1,3-glucan masking contributes to Candida pathogenicity. IMPORTANCE Microbes that coexist with humans have evolved ways of avoiding or evading our immunological defenses. These include the masking by these microbes of their "pathogen-associated molecular patterns" (PAMPs), which are recognized as "foreign" and used to activate protective immunity. The commensal fungus Candida albicans masks the proinflammatory PAMP ß-1,3-glucan, which is an essential component of its cell wall. Most of this ß-1,3-glucan is hidden beneath an outer layer of the cell wall on these microbes, but some can become exposed at the fungal cell surface. Using high-resolution confocal microscopy, we examine the nature of the exposed ß-1,3-glucan at C. albicans bud scars and at punctate foci on the lateral cell wall, and we show that these features are targeted by innate immune cells. We also reveal that downstream effectors of protein kinase A (Mig1/Mig2, Sin3) regulate the secretion of major glucanases, modulate the levels of ß-1,3-glucan exposure, and influence the virulence of C. albicans in an invertebrate model of systemic infection. Our data support the view that ß-1,3-glucan masking contributes to immune evasion and the virulence of a major fungal pathogen of humans.

Transcriptional differences in gene families of the ascorbate-glutathione cycle in wheat during mild water deficit.

When comparing the responses of two wheat (Triticum aestivum L.) genotypes, the drought-tolerant Plainsman V and the drought-sensitive Cappelle Desprez, to reduced amounts of irrigation water, we found differences in ascorbate metabolism: both ascorbate oxidation and transcription levels of enzymes processing ascorbate were changed. Relative transcript levels of ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) isoenzymes, predicted to localize in distinct subcellular organelles, showed different transcriptional changes in the two genotypes. Among APX coding mRNAs, expression levels of two cytosolic (cAPX I, II) and a thylakoid-bound (tAPX) variants increased significantly in Plainsman V while a cytosolic (cAPX I) and a stromal (sAPX II) APX coding transcripts were found to be higher in Cappelle Desprez after a 4-week-long water-deficit stress. Examining the MDARs, two cytosolic isoforms (cMDAR I, II) displayed significant up-regulation of mRNA levels in the sensitive genotype, whereas only one of them (cMDAR II) did in the tolerant cultivar. We found an up-regulated chloroplastic DHAR (chlDHAR) mRNA only in the sensitive Cappelle Desprez. However, increased expression levels of a cytosolic GR (cGR) and a chloroplastic GR (chlGR) were detected only in the tolerant Plainsman V. After 4 weeks of reduced irrigation, a significantly lower ascorbate/dehydroascorbate ratio was detected in leaves of the sensitive Cappelle Desprez than in the tolerant Plainsman V. Our results indicate that more robust transcription of ascorbate-based detoxification machinery may prevent an adverse shift of the cellular redox balance.

The expressions of ABCC4 and ABCG2 xenobiotic transporters in human keratinocytes are proliferation-related.

Xenobiotic transporters of the ATP-binding cassette (ABC) protein superfamily play important roles in maintaining the biochemical barrier of various tissues, but their precise functions in the skin are not yet known. Screening of the expressions of the known xenobiotic transporter genes in two in vitro keratinocyte differentiation models revealed that the ABCC4 and ABCG2 transporters are highly expressed in proliferating keratinocytes, their expressions decreasing along with differentiation. Abrogation of the ABCC4 and ABCG2 protein functions by siRNA-mediated silencing and chemical inhibition did not affect the proliferation of HaCaT cells. In contrast, disruption of the ABCG2 function had no effect on normal human epidermal keratinocyte proliferation, while the inhibition of ABCC-type transporters by probenecid resulted in a striking decrease in the proliferation of the cells. These results indicate that, besides their possible therapy-modulating effects, xenobiotic transporters may contribute significantly to other keratinocyte functions, such as cell proliferation.

Specific inhibition of the ABCG2 transporter could improve the efficacy of photodynamic therapy.

Photodynamic therapy is based on the selective accumulation of a photosensitizer in tumors, followed by destruction of the target tissue by a light source. Protoporphyrin IX, a well-known photosensitizer, was recently reported as an endogenous substrate for the multidrug transporter ABCG2. We investigated the role of ABCG2 protein in the porphyrin extrusion ability of keratinocytes, with regard to the impact of the specific inhibition of ABCG2 by a non-toxic fumitremorgin C analog, Ko-134, on photodynamic therapy efficacy. We studied the level of porphyrin accumulation in response to delta-aminolevulinic acid pretreatment in proliferating and highly differentiated HaCaT keratinocytes. An in vitro model of photodynamic therapy on HaCaT cells was established with a therapeutically approved narrow-bandwidth red-light source. The porphyrin extrusion ability of HaCaT cells proved to correlate with their ABCG2 expression which was higher in proliferating cells than in differentiated cells. Moreover, the specific inhibition of ABCG2 by Ko-134 enhanced the sensitivity of keratinocytes to photodynamic therapy in vitro. These results suggest that ABCG2 may serve as a target molecule via which to improve the photodynamic therapy of skin lesions: its inhibition by the non-toxic Ko-134 is a promising therapeutic modality.

Protective effects of a phosphatidylcholine-enriched diet in lipopolysaccharide-induced experimental neuroinflammation in the rat.

Our goal was to characterize the neuroprotective properties of orally administered phosphatidylcholine (PC) in a rodent model of systemic inflammation. Sprague-Dawley rats were killed at 3 h, 1 day, 3 days, or 7 days after i.p. administration of lipopolysaccharide (LPS) to determine the plasma levels of tumor necrosis factor α (TNF-α) and interleukin 6 cytokines. The control group and one group of LPS-treated animals were nourished with standard laboratory chow, whereas another LPS-treated group received a special diet enriched with 1% PC for 5 days before the administration of LPS and thereafter during the 7-day observation period. Immunohistochemistry was performed to visualize the bromodeoxyuridine and doublecortin-positive neuroprogenitor cells and Iba1-positive microglia in the hippocampus, whereas the degree of mucosal damage was evaluated on ileal and colon biopsy samples after hematoxylin-eosin staining. The activities of proinflammatory myeloperoxidase and xanthine-oxidoreductase and the tissue nitrite/nitrate (NOx) level were additionally determined, and the cognitive functions were monitored via Morris water maze testing. The inflammatory challenge transiently increased the hippocampal NOx level and led to microglia accumulation and decreased neurogenesis. The intestinal damage, mucosal myeloperoxidase, xanthine-oxidoreductase, and NOx changes were less pronounced, and long-lasting behavioral alterations were not observed. Phosphatidylcholine pretreatment reduced the plasma TNF-α and hippocampal NOx changes and prevented the decreased neurogenesis. These data demonstrated the relative susceptibility of the brain to the consequences of transient peripheral inflammatory stimuli. Phosphatidylcholine supplementation did not reduce the overall extent of peripheral inflammatory activation, but efficiently counteracted the disturbed hippocampal neurogenesis by lowering circulating TNF-α concentrations.

In vitro dedifferentiation of melanocytes from adult epidermis.

In previous work we described a novel culture technique using a cholera toxin and PMA-free medium (Mel-mix) for obtaining pure melanocyte cultures from human adult epidermis. In Mel-mix medium the cultured melanocytes are bipolar, unpigmented and highly proliferative. Further characterization of the cultured melanocytes revealed the disappearance of c-Kit and TRP-1 and induction of nestin expression, indicating that melanocytes dedifferentiated in this in vitro culture. Cholera toxin and PMA were able to induce c-Kit and TRP-1 protein expressions in the cells, reversing dedifferentiation. TRP-1 mRNA expression was induced in dedifferentiated melanocytes by UV-B irradiated keratinocyte supernatants, however direct UV-B irradiation of the cells resulted in further decrease of TRP-1 mRNA expression. These dedifferentiated, easily accessible cultured melanocytes provide a good model for studying melanocyte differentiation and possibly transdifferentiation. Because melanocytes in Mel-mix medium can be cultured with human serum as the only supplement, this culture system is also suitable for autologous cell transplantation.

COP1 contributes to UVB-induced signaling in human keratinocytes.

UVB irradiation has been shown to trigger a broad range of changes in gene expression in human skin; however, factors governing these events are still not well understood. In this study, we show that human constitutive photomorphogenic protein-1 (huCOP1), an E3 ligase, contributes to the orchestration of UVB response of keratinocytes. Accordingly, our data show that (i) huCOP1 protein is expressed both in the nucleus and in the cytoplasm of cultured keratinocytes, (ii) UVB reduces the levels of the huCOP1 mRNA and protein, and (iii) induces changes in the subcellular localization of huCOP1. Finally, we show that gene-specific silencing of huCOP1 induces the accumulation of the tumor suppressor p53 protein, which is further increased after UVB irradiation.