Characterization and functional analysis of zinc trafficking in the human fungal pathogen Candida parapsilosis.

The zinc restriction and zinc toxicity are part of host defence, called nutritional immunity. The crucial role of zinc homeostasis in microbial survival within a host is established, but little is known about these processes in the opportunistic human fungal pathogen Candida parapsilosis. Our in silico predictions suggested the presence of at least six potential zinc transporters (ZnTs) in C. parapsilosis-orthologues of ZRC1, ZRT3 and ZRT101-but an orthologue of PRA1 zincophore was not found. In addition, we detected a species-specific gene expansion of the novel ZnT ZRT2, as we identified three orthologue genes in the genome of C. parapsilosis. Based on predictions, we created homozygous mutant strains of the potential ZnTs and characterized them. Despite the apparent gene expansion of ZRT2 in C. parapsilosis, only CpZRT21 was essential for growth in a zinc-depleted acidic environment, in addition we found that CpZrc1 is essential for zinc detoxification and also protects the fungi against the elimination of murine macrophages. Significantly, we demonstrated that C. parapsilosis forms zincosomes in a Zrc1-independent manner and zinc detoxification is mediated by the vacuolar importer CpZrc1. Our study defines the functions of C. parapsilosis ZnTs, including a species-specific survival and zinc detoxification system.

Functionalized Mesoporous Silica Nanoparticles for Drug-Delivery to Multidrug-Resistant Cancer Cells.

Background: Multidrug resistance is a common reason behind the failure of chemotherapy. Even if the therapy is effective, serious adverse effects might develop due to the low specificity and selectivity of antineoplastic agents. Mesoporous silica nanoparticles (MSNs) are promising materials for tumor-targeting and drug-delivery due to their small size, relatively inert nature, and extremely large specific surfaces that can be functionalized by therapeutic and targeting entities. We aimed to create a fluorescently labeled MSN-based drug-delivery system and investigate their internalization and drug-releasing capability in drug-sensitive MCF-7 and P-glycoprotein-overexpressing multidrug-resistant MCF-7 KCR cancer cells. Methods and Results: To track the uptake and subcellular distribution of MSNs, particles with covalently coupled red fluorescent Rhodamine B (RhoB) were produced (RhoB@MSNs). Both MCF-7 and MCF-7 KCR cells accumulated a significant amount of RhoB@MSNs. The intracellular RhoB@MSN concentrations did not differ between sensitive and multidrug-resistant cells and were kept at the same level even after cessation of RhoB@MSN exposure. Although most RhoB@MSNs resided in the cytoplasm, significantly more RhoB@MSNs co-localized with lysosomes in multidrug-resistant cells compared to sensitive counterparts. To examine the drug-delivery capability of these particles, RhoB@Rho123@MSNs were established, where RhoB-functionalized nanoparticles carried green fluorescent Rhodamine 123 (Rho123) - a P-glycoprotein substrate - as cargo within mesopores. Significantly higher Rho123 fluorescence intensity was detected in RhoB@Rho123@MSN-treated multidrug-resistant cells than in free Rho123-exposed counterparts. The exceptional drug-delivery potential of MSNs was further verified using Mitomycin C (MMC)-loaded RhoB@MSNs (RhoB@MMC@MSNs). Exposures to RhoB@MMC@MSNs significantly decreased the viability not only of drug-sensitive but of multidrug-resistant cells and the elimination of MDR cells was significantly more robust than upon free MMC treatments. Conclusion: The efficient delivery of Rho123 and MMC to multidrug-resistant cells via MSNs, the amplified and presumably prolonged intracellular drug concentration, and the consequently enhanced cytotoxic effects envision the enormous potential of MSNs to defeat multidrug-resistant cancer.

Lactobacillus acidophilus, L. plantarum, L. rhamnosus, and L. reuteri Cell-Free Supernatants Inhibit Candida parapsilosis Pathogenic Potential upon Infection of Vaginal Epithelial Cells Monolayer and in a Transwell Coculture System In Vitro.

Vulvovaginal candidiasis (VVC) is a common clinical condition with symptoms and signs of vaginal inflammation in the presence of Candida species. At least one episode of VVC is experienced in up to 75% of women in the reproductive age group during their lifetime, and 5% to 8% of such women suffer from the chronic form. Most cases of VVC are still caused by C. albicans. However, the incidence of VVC cases by non-albicans Candida (NAC) species, such as C. parapsilosis, is continuously increasing. Despite the prevalence of VVC from NAC, little is known about these species and almost nothing about the mechanisms that trigger the VVC. Lactobacillus spp. are the most widely before represented microorganisms in the vaginal microbiota of healthy women. Here, cell-free supernatants (CFS) obtained from L. acidophilus, L. plantarum, L. rhamnosus, and L. reuteri were assessed for their effect on C. parapsilosis virulence traits. Moreover, we assessed if such an effect persisted even after the removal of the CFS (CFS preincubation effect). Moreover, a transwell coculture system was employed by which the relevant antifungal effect was shown to be attributable to the compounds released by lactobacilli. Our results suggest that lactobacilli can work (i) by reducing C. parapsilosis virulence traits, as indicated by the reduced fungal proliferation, viability, and metabolic activity, and (ii) by improving epithelial resistance to the fungus. Overall, these data suggest that, in the context of the vaginal microbiota, the lactobacilli may play a role in preventing the onset of mucosal C. parapsilosis infection. IMPORTANCE The incidence of VVC by non-albicans Candida (NAC) species, such as C. parapsilosis, is increasing. Treatment failure is common in NAC-VVC because some species are resistant or poorly susceptible to the antifungal agents normally employed. Research on C. parapsilosis's pathogenic mechanisms and alternative treatments are still lacking. C. albicans triggers the VVC by producing hyphae, which favor the loss of epithelial tolerance. Differently, C. parapsilosis only produces pseudohyphae. Hence, different virulence factors may trigger the VVC. Likewise, the therapeutic options could also involve different fungal targets. Substantial in vitro and in vivo studies on the pathogenicity mechanisms of C. parapsilosis are lacking. The data presented here ascribe a novel beneficial role to different Lactobacillus spp., whose CFS provides a postbiotic-like activity against C. parapsilosis. Further studies are needed to unravel the mechanisms involved in the bioactivities of such compounds, to better understand the role of single postbiotics in the CFS.

Signaling through Syk or CARD9 Mediates Species-Specific Anti-Candida Protection in Bone Marrow Chimeric Mice.

The spleen tyrosine kinase (Syk) and the downstream adaptor protein CARD9 are crucial signaling molecules in antimicrobial immunity. Candida parapsilosis is an emerging fungal pathogen with a high incidence in neonates, while Candida albicans is the most common agent of candidiasis. While signaling through Syk/CARD9 promotes protective host mechanisms in response to C. albicans, its function in immunity against C. parapsilosis remains unclear. Here, we generated Syk-/- and CARD9-/- bone marrow chimeric mice to study the role of Syk/CARD9 signaling in immune responses to C. parapsilosis compared to C. albicans. We demonstrate various functions of this pathway (e.g., phagocytosis, phagosome acidification, and killing) in Candida-challenged, bone marrow-derived macrophages with differential involvement of Syk and CARD9 along with species-specific differences in cytokine production. We report that Syk-/- or CARD9-/- chimeras rapidly display high susceptibility to C. albicans, while C. parapsilosis infection exacerbates over a prolonged period in these animals. Thus, our results establish that Syk and CARD9 contribute to systemic resistance to C. parapsilosis and C. albicans differently. Additionally, we confirm prior studies but also detail new insights into the fundamental roles of both proteins in immunity against C. albicans. Our data further suggest that Syk has a more prominent influence on anti-Candida immunity than CARD9. Therefore, this study reinforces the Syk/CARD9 pathway as a potential target for anti-Candida immune therapy. IMPORTANCE While C. albicans remains the most clinically significant Candida species, C. parapsilosis is an emerging pathogen with increased affinity to neonates. Syk/CARD9 signaling is crucial in immunity to C. albicans, but its role in in vivo responses to other pathogenic Candida species is largely unexplored. We used mice with hematopoietic systems deficient in Syk or CARD9 to comparatively study the function of these proteins in anti-Candida immunity. We demonstrate that Syk/CARD9 signaling has a protective role against C. parapsilosis differently than against C. albicans. Thus, this study is the first to reveal that Syk can exert immune responses during systemic Candida infections species specifically. Additionally, Syk-dependent immunity to a nonalbicans Candida species in an in vivo murine model has not been reported previously. We highlight that the contribution of Syk and CARD9 to fungal infections are not identical and underline this pathway as a promising immune-therapeutic target to fight Candida infections.

Symbiotic NCR Peptide Fragments Affect the Viability, Morphology and Biofilm Formation of Candida Species.

The increasing rate of fungal infections causes global problems not only in human healthcare but agriculture as well. To combat fungal pathogens limited numbers of antifungal agents are available therefore alternative drugs are needed. Antimicrobial peptides are potent candidates because of their broad activity spectrum and their diverse mode of actions. The model legume Medicago truncatula produces >700 nodule specific cysteine-rich (NCR) peptides in symbiosis and many of them have in vitro antimicrobial activities without considerable toxicity on human cells. In this work we demonstrate the anticandidal activity of the NCR335 and NCR169 peptide derivatives against five Candida species by using the micro-dilution method, measuring inhibition of biofilm formation with the XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) assay, and assessing the morphological change of dimorphic Candida species by microscopy. We show that both the N- and C-terminal regions of NCR335 possess anticandidal activity as well as the C-terminal sequence of NCR169. The active peptides inhibit biofilm formation and the yeast-hypha transformation. Combined treatment of C. auris with peptides and fluconazole revealed synergistic interactions and reduced 2-8-fold the minimal inhibitory concentrations. Our results demonstrate that shortening NCR peptides can even enhance and broaden their anticandidal activity and therapeutic potential.

Candida albicans Enhances the Progression of Oral Squamous Cell Carcinoma In Vitro and In Vivo.

Oral squamous cell carcinoma (OSCC) is associated with oral Candida albicans infection, although it is unclear whether the fungus promotes the genesis and progression of OSCC or whether cancer facilitates fungal growth. In this study, we investigated whether C. albicans can potentiate OSCC tumor development and progression. In vitro, the presence of live C. albicans, but not Candida parapsilosis, enhanced the progression of OSCC by stimulating the production of matrix metalloproteinases, oncometabolites, protumor signaling pathways, and overexpression of prognostic marker genes associated with metastatic events. C. albicans also upregulated oncogenes in nonmalignant cells. Using a newly established xenograft in vivo mouse model to investigate OSCC-C. albicans interactions, oral candidiasis enhanced the progression of OSCC through inflammation and induced the overexpression of metastatic genes and significant changes in markers of the epithelial-mesenchymal transition. Finally, using the 4-nitroquinoline 1-oxide (4NQO) murine model, we directly correlate these in vitro and short-term in vivo findings with the progression of oncogenesis over the long term. Taken together, these data indicate that C. albicans upregulates oncogenes, potentiates a premalignant phenotype, and is involved in early and late stages of malignant promotion and progression of oral cancer. IMPORTANCE Oral squamous cell carcinoma (OSCC) is a serious health issue worldwide that accounts for 2% to 4% of all cancer cases. Previous studies have revealed a higher yeast carriage and diversity in oral cancer patients than in healthy individuals. Furthermore, fungal colonization in the oral cavity bearing OSCC is higher on the neoplastic epithelial surface than on adjacent healthy surfaces, indicating a positive association between oral yeast carriage and epithelial carcinoma. In addition to this, there is strong evidence supporting the idea that Candida contributes to carcinogenesis events in the oral cavity. Here, we show that an increase in Candida albicans burden promotes an oncogenic phenotype in the oral cavity.

Phenotypic Variability in a Coinfection With Three Independent Candida parapsilosis Lineages.

The human pathogenic yeast Candida parapsilosis has gained significant importance over the past decades as one of the principal causes of fungal bloodstream infections. Isolates of C. parapsilosis are known to be able to switch between several different colony morphologies in vitro, which are correlated with different cell shapes, altered cell surface properties, and thus different capacities to form biofilms on indwelling medical devices. In a set of six clinical specimens from a single surgery patient yielding stable smooth- as well as crepe-morphology isolates, we investigated the differences between five of them on a phenotypic and genomic level. In contrast to the initial assumption that they were switched forms of a clonal strain, karyotyping and genome sequencing showed that the patient was colonized by at least three distinct linages. Statistical analysis placed these groups distantly across the population of C. parapsilosis. Interestingly the single blood culture isolate was of smooth morphology and matched with an isolate from the patient's nose of similar morphology. Strong variation between the isolates was seen in adhesin-encoding genes, where repeat regions showed significant variation in length and repeat-numbers, most strikingly in HWP1 of the smooth isolates. Although no differences in drug susceptibility were evident, the high phylogenetic distance separating the individual strains highlights the need for testing of multiple colonies in routine practice. The absence of biofilm formation in the blood stream isolate indicates a lack of respective adhesins in the cell wall, in turn pointing toward lack of adhesion as a positively contributing factor for dissemination.

Iron Metabolism, Pseudohypha Production, and Biofilm Formation through a Multicopper Oxidase in the Human-Pathogenic Fungus Candida parapsilosis.

Among all the essential micronutrients, iron plays an important role in mammalian biology. It is also essential for pathogens infecting mammalian hosts, including bacteria, fungi, and protozoans. As the availability of accessible iron is limited within the mammalian host, several human-pathogenic fungal pathogens, such as Candida albicans, Cryptococcus neoformans, Candida glabrata, and Aspergillus fumigatus, have developed various iron uptake mechanisms. Although Candida parapsilosis is the second or third most common non-albicans Candida species associated with systemic and superficial Candida infections in immunocompromised patients, the mechanisms of iron uptake and homoeostasis remain unknown in this fungus. In the current report, we show that a homologue of the multicopper oxidase gene FET3 is present in the genome of C. parapsilosis (CPAR2_603600) and plays a significant role in iron acquisition. We found that homozygous deletion mutants of CPAR2_603600 showed defects under low-iron conditions and were also sensitive to various stressors. Our results also revealed that the levels of pseudohypha formation and biofilm formation were reduced in the null mutants compared to the wild type. This phenotypic defect could be partially rescued by supplementation with excess iron in the growth medium. The expression levels of the orthologues of various iron metabolism-related genes were also altered in the mutants compared to the parental strain. In conclusion, our report describes the role of CPAR2_603600 in iron homoeostasis maintenance as well as morphology and biofilm formation regulation in this pathogenic fungus.IMPORTANCEC. parapsilosis is the second or third most common opportunistic human-pathogenic Candida species, being responsible for severe fungal infections among immunocompromised patients, especially low-birth-weight infants (0 to 2 years of age). Among the major virulence factors that pathogenic fungi possess is the ability to compete with the host for essential micronutrients, including iron. Accessible iron is required for the maintenance of several metabolic processes. In order to obtain accessible iron from the host, pathogenic fungi have developed several iron acquisition and metabolic mechanisms. Although C. parapsilosis is a frequent cause of invasive candidiasis, little is known about what iron metabolic processes this fungus possesses that could contribute to the species' virulent behavior. In this study, we identified the multicopper oxidase FET3 gene that regulates iron homeostasis maintenance and also plays important roles in the morphology of the fungus as well as in biofilm formation, two additional factors in fungal virulence.

Functional Characterization of Secreted Aspartyl Proteases in Candida parapsilosis.

Candida parapsilosis is an emerging non-albicans Candida species that largely affects low-birth-weight infants and immunocompromised patients. Fungal pathogenesis is promoted by the dynamic expression of diverse virulence factors, with secreted proteolytic enzymes being linked to the establishment and progression of disease. Although secreted aspartyl proteases (Sap) are critical for Candida albicans pathogenicity, their role in C. parapsilosis is poorly elucidated. In the present study, we aimed to examine the contribution of C. parapsilosisSAPP genes SAPP1, SAPP2, and SAPP3 to the virulence of the species. Our results indicate that SAPP1 and SAPP2, but not SAPP3, influence adhesion, host cell damage, phagosome-lysosome maturation, phagocytosis, killing capacity, and cytokine secretion by human peripheral blood-derived macrophages. Purified Sapp1p and Sapp2p were also shown to efficiently cleave host complement component 3b (C3b) and C4b proteins and complement regulator factor H. Additionally, Sapp2p was able to cleave factor H-related protein 5 (FHR-5). Altogether, these data demonstrate the diverse, significant contributions that SAPP1 and SAPP2 make to the establishment and progression of disease by C. parapsilosis through enabling the attachment of the yeast cells to mammalian cells and modulating macrophage biology and disruption of the complement cascade.IMPORTANCE Aspartyl proteases are present in various organisms and, among virulent species, are considered major virulence factors. Host tissue and cell damage, hijacking of immune responses, and hiding from innate immune cells are the most common behaviors of fungal secreted proteases enabling pathogen survival and invasion. C. parapsilosis, an opportunistic human-pathogenic fungus mainly threatening low-birth weight neonates and children, possesses three SAPP protein-encoding genes that could contribute to the invasiveness of the species. Our results suggest that SAPP1 and SAPP2, but not SAPP3, influence host evasion by regulating cell damage, phagocytosis, phagosome-lysosome maturation, killing, and cytokine secretion. Furthermore, SAPP1 and SAPP2 also effectively contribute to complement evasion.

Trk1-mediated potassium uptake contributes to cell-surface properties and virulence of Candida glabrata.

The absence of high-affinity potassium uptake in Candida glabrata, the consequence of the deletion of the TRK1 gene encoding the sole potassium-specific transporter, has a pleiotropic effect. Here, we show that in addition to changes in basic physiological parameters (e.g., membrane potential and intracellular pH) and decreased tolerance to various cell stresses, the loss of high affinity potassium uptake also alters cell-surface properties, such as an increased hydrophobicity and adherence capacity. The loss of an efficient potassium uptake system results in diminished virulence as assessed by two insect host models, Drosophila melanogaster and Galleria mellonella, and experiments with macrophages. Macrophages kill trk1Δ cells more effectively than wild type cells. Consistently, macrophages accrue less damage when co-cultured with trk1Δ mutant cells compared to wild-type cells. We further show that low levels of potassium in the environment increase the adherence of C. glabrata cells to polystyrene and the propensity of C. glabrata cells to form biofilms.

Myeloid-Specific Deletion of Mcl-1 Yields Severely Neutropenic Mice That Survive and Breed in Homozygous Form.

Mouse strains with specific deficiency of given hematopoietic lineages provide invaluable tools for understanding blood cell function in health and disease. Whereas neutrophils are dominant leukocytes in humans and mice, there are no widely useful genetic models of neutrophil deficiency in mice. In this study, we show that myeloid-specific deletion of the Mcl-1 antiapoptotic protein in Lyz2 Cre/Cre Mcl1 flox/flox (Mcl1 ΔMyelo) mice leads to dramatic reduction of circulating and tissue neutrophil counts without affecting circulating lymphocyte, monocyte, or eosinophil numbers. Surprisingly, Mcl1 ΔMyelo mice appeared normally, and their survival was mostly normal both under specific pathogen-free and conventional housing conditions. Mcl1 ΔMyelo mice were also able to breed in homozygous form, making them highly useful for in vivo experimental studies. The functional relevance of neutropenia was confirmed by the complete protection of Mcl1 ΔMyelo mice from arthritis development in the K/B×N serum-transfer model and from skin inflammation in an autoantibody-induced mouse model of epidermolysis bullosa acquisita. Mcl1 ΔMyelo mice were also highly susceptible to systemic Staphylococcus aureus or Candida albicans infection, due to defective clearance of the invading pathogens. Although neutrophil-specific deletion of Mcl-1 in MRP8-CreMcl1 flox/flox (Mcl1 ΔPMN) mice also led to severe neutropenia, those mice showed an overt wasting phenotype and strongly reduced survival and breeding, limiting their use as an experimental model of neutrophil deficiency. Taken together, our results with the Mcl1 ΔMyelo mice indicate that severe neutropenia does not abrogate the viability and fertility of mice, and they provide a useful genetic mouse model for the analysis of the role of neutrophils in health and disease.

Specific pathways mediating inflammasome activation by Candida parapsilosis.

Candida albicans and C. parapsilosis are human pathogens causing severe infections. The NLRP3 inflammasome plays a crucial role in host defence against C. albicans, but it has been previously unknown whether C. parapsilosis activates this complex. Here we show that C. parapsilosis induces caspase-1 activation and interleukin-1ß (IL-1ß) secretion in THP-1, as well as primary, human macrophages. IL-1ß secretion was dependent on NLRP3, K+-efflux, TLR4, IRAK, Syk, caspase-1, caspase-8 and NADPH-oxidase. Importantly, while C. albicans induced robust IL-1ß release after 4 h, C. parapsilosis was not able to stimulate the production of IL-1ß after this short incubation period. We also found that C. parapsilosis was phagocytosed to a lesser extent, and induced significantly lower ROS production and lysosomal cathepsin B release compared to C. albicans, suggesting that the low extent of inflammasome activation by C. parapsilosis may result from a delay in the so-called "signal 2". In conclusion, this is the first study to examine the molecular pathways responsible for the IL-1ß production in response to a non-albicans Candida species, and these results enhance our understanding about the immune response against C. parapsilosis.

Candida parapsilosis Secreted Lipase as an Important Virulence Factor.

The prevalence of Candida parapsilosis, an opportunistic human pathogenic fungal species, is increasing at an alarming rate in the hospital environment. Patients at risk for C. parapsilosis infection include those with immunosuppression, such as individuals with cancer, AIDS, and low birth weight premature neonates as well as patients that had undergone abdominal surgery. Neonatal candidiasis caused by C. parapsilosis has been widely reported across the globe. Various reports have shown that, compared to other Candida species, certain C. parapsilosis clinical isolates were less susceptible to antifungals such as amphotericin B, fluconazole, and caspofungin. In addition, some studies have even reported multi-echinocandin or multi-azole resistant strains of C. parapsilosis. C. parapsilosis has several virulence factors that contribute to its capacity for host invasion and among these factors extracellular lipases have a major role in pathogenesis. In this review we have collected all the recent relevant studies that confirm the involvement of secreted lipases in C. parapsilosis pathogenesis, using both in vitro and in vivo models of infection. Of particular note, an available lipase deficient C. parapsilosis strain has been utilized to demonstrate that the lack of secreted lipases decreased virulence, reduced tissue damage, and was less able to survive within phagocytes or mice compared to the wild type. Since fungal secreted lipases have different characteristics than lipolytic enzymes present in humans, C. parapsilosis extracellular lipases may be potential targets for the development of novel antifungal drugs.

Analysis of oral yeast microflora in patients with oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is the most common form of oral cancer, in this study, the association between OSCC and oral yeast carriage was investigated. FINDINGS: 20 patients having OSCC as well as 40 healthy controls were tested for the presence of yeasts in the oral cavity. Fungal burdens were examined by colony forming unit determinations, while the different yeast genera in patient samples were identified by matrix-associated laser desorption/ionization-time of flight-mass spectrometry. We found that the level of oral yeast carriage was significantly higher in patients with OSCC that was accompanied by a higher diversity of yeasts in the oral cavity of these patients. We also examined the extracellular enzyme production of isolated Candida spp.; however, we found that there was no association between the lipase/protease producing capacity of Candida strains and the higher colonisation rate of neoplastic epithelium. CONCLUSIONS: In conclusion, our results corroborate the findings of previous studies regarding the association between oral yeast carriage and epithelial carcinoma.

The cytoprotective effect of biglycan core protein involves Toll-like receptor 4 signaling in cardiomyocytes.

AIMS: Exogenously administered biglycan (core protein with high-molecular weight glycosaminoglycan chains) has been shown to protect neonatal cardiomyocytes against simulated ischemia/reperfusion injury (SI/R), however, the mechanism of action is not clear. In this study we aimed to investigate, which structural component of biglycan is responsible for its cardiocytoprotective effect and to further explore the molecular mechanisms involved in the cytoprotection. METHODS AND RESULTS: A pilot study was conducted to demonstrate that both native (glycanated) and deglycanated biglycan can attenuate cell death induced by SI/R in a dose-dependent manner in primary neonatal cardiomyocytes isolated from Wistar rats. In separate experiments, we have shown that similarly to glycanated biglycan, recombinant human biglycan core protein (rhBGNc) protects cardiomyocytes against SI/R injury. In contrast, the glycosaminoglycan component dermatan sulfate had no significant effect on cell viability, while chondroitin sulfate further enhanced cell death induced by SI/R. Treatment of cardiomyocytes with rhBGNc reverses the effect of SI/R upon markers of necrosis, apoptosis, mitochondrial membrane potential, and autophagy. We have also shown that pharmacological blockade of Toll-like receptor 4 (TLR4) signaling or its downstream mediators (IRAK1/4, ERK, JNK and p38 MAP kinases) abolished the cytoprotective effect of rhBGNc against SI/R injury. Pretreatment of cardiomyocytes with rhBGNc for 20h resulted in increased Akt phosphorylation and NO production without having significant effect on phosphorylation of ERK1/2, STAT3, and on the production of superoxide. Treatment over 10min and 1h with rhBGNc increased ERK1 phosphorylation, while the SI/R-induced increase in superoxide production was attenuated by rhBGNc. Blockade of NO synthesis also prevented the cardiocytoprotective effect of rhBGNc. CONCLUSIONS: The core protein of exogenous biglycan protects myocardial cells from SI/R injury via TLR4-mediated mechanisms involving activation of ERK, JNK and p38 MAP kinases and increased NO production. The cytoprotective effect of rhBGNc is due to modulation of SI/R-induced changes in necrosis, apoptosis and autophagy.

Further characterization of the role of the mitochondrial high-mobility group box protein in the intracellular redox environment of Aspergillus nidulans.

HmbB, a predominantly mitochondrial high-mobility group box (HMGB) protein, of Aspergillus nidulans affects diverse biological activities, such as sterigmatocystin production, the maintenance of mitochondrial DNA copy number, germination of asexual and sexual spores, and protection against oxidative stress agents. We hypothesized that the latter correlates with an unbalanced intracellular redox state, in which case, a not yet fully characterized physiological function could be attributed to this mitochondrial HMGB protein. Here, we studied the intracellular redox environment and oxidative stress tolerance in hmbB+ and hmbBΔ strains under normal and oxidative stress conditions by measuring glutathione redox couple, intracellular reactive oxygen species (ROS) content and ROS-protecting enzyme activities. Our results revealed that the intracellular redox environment is different in hmbBΔ conidia and mycelia from that of hmbB+, and shed light on the seemingly contradictory difference in the tolerance of hmbBΔ mycelia to diamide and menadione oxidative stressors.

Candida parapsilosis produces prostaglandins from exogenous arachidonic acid and OLE2 is not required for their synthesis.

Prostaglandins are C20 fatty acid metabolites with diverse biological functions. In mammalian cells, prostaglandins are produced from arachidonic acid (AA) via cyclooxygenases (COX1 and COX2). Although fungi do not possess cyclooxygenase homologues, several pathogenic species are able to produce prostaglandins from host-derived arachidonic acid. In this study, we characterized the prostaglandin profile of the emerging human pathogen Candida parapsilosis with HPLC-MS and compared it to that of C. albicans. We found that both species synthesized prostaglandins (mainly PGD2 and PGE2) from exogenous AA. Furthermore, as OLE2 has been associated with prostaglandin synthesis in C. albicans, we generated homozygous OLE2 deletion mutants in C. parapsilosis and examined their PGE2 production. However, the PGE2 production of the OLE2 KO strain was similar to that of wild type (WT), indicating that OLE2 is not required for prostaglandin synthesis in C. parapsilosis. Interestingly, analyses of the fatty acid composition of WT and OLE2 KO cells by gas chromatography (GC) highlighted the accumulation of palmitoleic and oleic acid in the OLE2 deletion mutant. The OLE2 KO cells were killed more efficiently by human monocytes-derived macrophages (MDMs) as well as induced higher interleukin-10 (IL-10) secretion, indicating that OLE2 affects the virulence of C. parapsilosis. Taken together, these results contribute to the better understanding of fatty acid biosynthesis pathways in C. parapsilosis.

Secreted Candida parapsilosis lipase modulates the immune response of primary human macrophages.

Candida parapsilosis is an important opportunistic pathogen with increasing prevalence. Extracellular lipases have been shown to play an important role in the virulence of pathogenic Candida species. However, studying the role of secreted lipase in C. albicans is challenging due to the lack of a mutant strain deficient in all 10 lipase genes. In contrast, we have previously constructed a lipase mutant C. parapsilosis strain lacking both CpLIP1 and CpLIP2, and shown that it has significantly decreased virulence in various infection models, and is killed more efficiently by mouse macrophages. In the present study, we compared the response of human peripheral blood monocyte-derived macrophages to a wild type (wt) as well as a lipase-deficient (lip(-/-)) C. parapsilosis strain that has been previously established in our lab. Although macrophages phagocytosed both strains with similar efficiency, lipase mutants were killed more efficiently according to fluorescent microscopic analysis. The more efficient killing of lip(-/-) cells was confirmed by CFU-determinations. Phagocytosis of wt and lip(-/-)C. parapsilosis was also examined by flow cytometry, revealing that both strains were internelized to the similar extent by macrophages. Additionally, quantitative imaging analysis revealed that the rate of phagolysosome fusion was higher in case of lip(-/-)C. parapsilosis. Interestingly, macrophages stimulated with lip(-/-)C. parapsilosis showed at least 1.5-fold higher expression of TNFα, IL-1ß, IL-6, IL-8, and PTGS-2 after 12 h compared with those infected with wt C. parapsilosis, as determined by qRT-PCR. Furthermore, the lip(-/-)C. parapsilosis strain induced significantly higher TNFα, IL-1ß, IL-6, and IL-10 protein production in macrophages after 24 h compared with the wt strain. These findings confirm the role of fungal lipases as important virulence factors during C. parapsilosis infection.

Transcriptome profile of the murine macrophage cell response to Candida parapsilosis.

Candida parapsilosis is a human fungal pathogen with increasing global significance. Understanding how macrophages respond to C. parapsilosis at the molecular level will facilitate the development of novel therapeutic paradigms. The complex response of murine macrophages to infection with C. parapsilosis was investigated at the level of gene expression using an Agilent mouse microarray. We identified 155 and 511 differentially regulated genes at 3 and 8h post-infection, respectively. Most of the upregulated genes encoded molecules involved in immune response and inflammation, transcription, signaling, apoptosis, cell cycle, electron transport and cell adhesion. Typical of the classically activated macrophages, there was significant upregulation of genes coordinating the production of inflammatory cytokines such as TNF, IL-1 and IL-15. Further, we used both primary murine macrophages and macrophages differentiated from human peripheral mononuclear cells to confirm the upregulation of the TNF-receptor family member TNFRSF9 that is associated with Th1 T-helper cell responses. Additionally, the microarray data indicate significant differences between the response to C. parapsilosis infection and that of C. albicans.

The role of pancreatic ductal secretion in protection against acute pancreatitis in mice*.

OBJECTIVES: A common potentially fatal disease of the pancreas is acute pancreatitis, for which there is no treatment. Most studies of this disorder focus on the damage to acinar cells since they are assumed to be the primary target of multiple stressors affecting the pancreas. However, increasing evidence suggests that the ducts may also have a crucial role in induction of the disease. To test this hypothesis, we sought to determine the specific role of the duct in the induction of acute pancreatitis using well-established disease models and mice with deletion of the Na/H exchanger regulatory factor-1 that have selectively impaired ductal function. DESIGN: Randomized animal study. SETTING: Animal research laboratory. SUBJECTS: Wild-type and Na/H exchanger regulatory factor-1 knockout mice. INTERVENTIONS: Acute necrotizing pancreatitis was induced by i.p. administration of cerulein or by intraductal administration of sodium taurocholate. The pancreatic expression of Na/H exchanger regulatory factor-1 and cystic fibrosis transmembrane conductance regulator (a key player in the control of ductal secretion) was analyzed by immunohistochemistry. In vivo pancreatic ductal secretion was studied in anesthetized mice. Functions of pancreatic acinar and ductal cells as well as inflammatory cells were analyzed in vitro. MEASUREMENTS AND MAIN RESULTS: Deletion of Na/H exchanger regulatory factor-1 resulted in gross mislocalization of cystic fibrosis transmembrane conductance regulator, causing marked reduction in pancreatic ductal fluid and bicarbonate secretion. Importantly, deletion of Na/H exchanger regulatory factor-1 had no deleterious effect on functions of acinar and inflammatory cells. Deletion of Na/H exchanger regulatory factor-1, which specifically impaired ductal function, increased the severity of acute pancreatitis in the two mouse models tested. CONCLUSIONS: Our findings provide the first direct evidence for the crucial role of ductal secretion in protecting the pancreas from acute pancreatitis and strongly suggest that improved ductal function should be an important modality in prevention and treatment of the disease.