A luciferase reporter for gene expression studies and dynamic imaging of superficial Candida albicans infections.

Real-time imaging of fungal infections is becoming integral to the study of host-pathogen interactions, as it allows monitoring of the spatial and temporal progression of pathogen growth or of the host response in a single animal as well as reducing the number of animals used to obtain significant data. We present different applications of a novel luciferase reporter gene constructed from the coding sequences of the Candida albicans PGA59 gene, encoding a GPI-linked cell wall protein, and the Gaussia princeps luciferase gene. Upon addition of the coelenterazine substrate, light produced by the surface-exposed luciferase can be used to quantify gene expression from a variety of C. albicans promoters as well as monitoring cutaneous, subcutaneous, and vaginal infections.

Th17 cells and IL-17 in protective immunity to vaginal candidiasis.

BACKGROUND: Th17 cells play a major role in coordinating the host defence in oropharyngeal candidiasis. In this study we investigated the involvement of the Th17 response in an animal model of vulvovaginal candidiasis (VVC). METHODS: To monitor the course of infection we exploited a new in vivo imaging technique. RESULTS: i) The progression of VVC leads to a strong influx of neutrophils in the vagina soon after the challenge which persisted despite the resolution of infection; ii) IL-17, produced by vaginal cells, particularly CD4 T cells, was detected in the vaginal wash during the infection, reaching a maximum 14 days after the challenge; iii) The amount and kinetics of IL-23 in vaginal fluids were comparable to those in vaginal cells; iv) The inhibition of Th17 differentiation led to significant inhibition of IL-17 production with consequent exacerbation of infection; v) An increased production of ßdefensin 2 was manifested in cells of infected mice. This production was strongly reduced when Th17 differentiation was inhibited and was increased by rIL-17 treatment. CONCLUSIONS: These results imply that IL-17 and Th17, along with innate antimicrobial factors, have a role in the immune response to vaginal candidiasis.

Beneficial effect of Mentha suaveolens essential oil in the treatment of vaginal candidiasis assessed by real-time monitoring of infection.

BACKGROUND: Vaginal candidiasis is a frequent and common distressing disease affecting up to 75% of the women of fertile age; most of these women have recurrent episodes. Essential oils from aromatic plants have been shown to have antimicrobial and antifungal activities. This study was aimed at assessing the anti-fungal activity of essential oil from Mentha suaveolens (EOMS) in an experimental infection of vaginal candidiasis. METHODS: The in vitro and in vivo activity of EOMS was assessed. The in vitro activity was evaluated under standard CLSI methods, and the in vivo analysis was carried out by exploiting a novel, non-invasive model of vaginal candidiasis in mice based on an in vivo imaging technique. Differences between essential oil treated and saline treated mice were evaluated by the non-parametric Mann-Whitney U-test. Viable count data from a time kill assay and yeast and hyphae survival test were compared using the Student's t-test (two-tailed). RESULTS: Our main findings were: i) EOMS shows potent candidastatic and candidacidal activity in an in vitro experimental system; ii) EOMS gives a degree of protection against vaginal candidiasis in an in vivo experimental system. CONCLUSIONS: This study shows for the first time that the essential oil of a Moroccan plant Mentha suaveolens is candidastatic and candidacidal in vitro, and has a degree of anticandidal activity in a model of vaginal infection, as demonstrated in an in vivo monitoring imaging system. We conclude that our findings lay the ground for further, more extensive investigations to identify the active EOMS component(s), promising in the therapeutically problematic setting of chronic vaginal candidiasis in humans.

The Inflammatory response induced by aspartic proteases of Candida albicans is independent of proteolytic activity.

The secretion of aspartic proteases (Saps) has long been recognized as a virulence-associated trait of the pathogenic yeast Candida albicans. In this study, we report that different recombinant Saps, including Sap1, Sap2, Sap3, and Sap6, have differing abilities to induce secretion of proinflammatory cytokines by human monocytes. In particular Sap1, Sap2, and Sap6 significantly induced interleukin-1ß (IL-1ß), tumor necrosis factor alpha (TNF-α), and IL-6 production. Sap3 was able to stimulate the secretion of IL-1ß and TNF-α. All Saps tested were able to induce Ca(2+) influx in monocytes. Treatment of these Saps with pepstatin A did not have any effect on cytokine secretion, indicating that their stimulatory potential was independent from their proteolytic activity. The capacity of Saps to induce inflammatory cytokine production was also independent from protease-activated receptor (PAR) activation and from the optimal pH for individual Sap activity. The interaction of Saps with monocytes induced Akt activation and phosphorylation of IκBα, which mediates translocation of NF-κB into the nucleus. Overall, these results suggest that individual Sap proteins can induce an inflammatory response and that this phenomenon is independent from the pH of a specific host niche and from Sap enzymatic activity. The inflammatory response is partially dependent on Sap denaturation and is triggered by the Akt/NF-κB activation pathway. Our data suggest a novel, activity-independent aspect of Saps during interactions of C. albicans with the host.

A beta-glucan-conjugate vaccine and anti-beta-glucan antibodies are effective against murine vaginal candidiasis as assessed by a novel in vivo imaging technique.

The protective capacity of a parenterally administered beta-glucan-conjugate vaccine formulated with the human-compatible MF59 adjuvant was assessed in a murine model of vaginal candidiasis. To monitor infection, an in vivo imaging technique exploiting genetically engineered, luminescent Candida albicans was adopted, and compared with measurements of colony forming units. The vaccine conferred significant protection, and this was associated with production of serum and vaginal anti-beta-glucan IgG antibodies. Vaginal IgG molecules were the likely mediators of protection as inferred by the efficacy of passive transfer of immune vaginal fluid and passive protection by an anti-beta-1,3-glucan mAb. Overall, the in vivo imaging technique was more reliable than vaginal CFU counts in assessing the extent and duration of the vaginal infection, and the consequent protection level.

A multifunctional, synthetic Gaussia princeps luciferase reporter for live imaging of Candida albicans infections.

Real-time monitoring of the spatial and temporal progression of infection/gene expression in animals will contribute greatly to our understanding of host-pathogen interactions while reducing the number of animals required to generate statistically significant data sets. Sensitive in vivo imaging technologies can detect low levels of light emitted from luciferase reporters in vivo, but the existing reporters are not optimal for fungal infections. Therefore, our aim was to develop a novel reporter system for imaging Candida albicans infections that overcomes the limitations of current luciferase reporters for this major fungal pathogen. This luciferase reporter was constructed by fusing a synthetic, codon-optimized version of the Gaussia princeps luciferase gene to C. albicans PGA59, which encodes a glycosylphosphatidylinositol-linked cell wall protein. Luciferase expressed from this PGA59-gLUC fusion (referred to as gLUC59) was localized at the C. albicans cell surface, allowing the detection of luciferase in intact cells. The analysis of fusions to strong (ACT1 and EFT3), oxidative stress-induced (TRX1, TRR1, and IPF9996), and morphogenesis-dependent (HWP1) promoters confirmed that gLUC59 is a convenient and sensitive reporter for studies of gene regulation in yeast or hyphal cells, as well as a flexible screening tool. Moreover, the ACT1-gLUC59 fusion represented a powerful tool for the imaging of disease progression in superficial and subcutaneous C. albicans infections. gLUC59 and related cell surface-exposed luciferase reporters might find wide applications in molecular biology, cell biology, pathobiology, and high-throughput screens.

Dectin-1 is required for human dendritic cells to initiate immune response to Candida albicans through Syk activation.

Dectin-1 is a pattern recognition receptor found on monocytes and dendritic cells (DC) able to recognize beta-1,3 and beta-1,6 glucans. It is thought to act via the spleen tyrosine kinase (Syk) to initiate immune response against infectious agents such as Candida albicans, one of the leading causes of invasive fungal disease in immunocompromised individuals. This study addresses the importance of this receptor in the context of human DC response to C. albicans. Upon blockage of Dectin-1, immature DC are less able than untreated cells to bind, phagocytose, and kill C. albicans via oxidative burst. In fact, a consistent decrease in superoxide anion, but not nitric oxide production, was manifested when the Syk pathway was inhibited. C. albicans-induced cytokine production via Dectin-1 recognition is mediated by the Syk activation pathway. Indeed, specific Syk inhibition significantly suppressed the production of IL-12, IL-6, and TNF-alpha. Finally, we observed that Dectin-1 engagement was also involved in DC maturation and subsequent lymphocyte activation. Collectively, these findings identify Dectin-1 as a key receptor influencing critical biological functions of DC in response to C. albicans leading to T cells response alteration. These effects are largely, though not completely, mediated by Syk activation.

A Candida albicans mannoprotein deprived of its mannan moiety is efficiently taken up and processed by human dendritic cells and induces T-cell activation without stimulating proinflammatory cytokine production.

Mannoproteins are cell wall components of pathogenic fungi and play major virulence and immunogenic roles with both their mannan and protein moieties. The 65-kDa mannoprotein (MP65) of Candida albicans is a beta-glucanase adhesin recognized as a major target of the human immune response against this fungus, and its recombinant product (rMP65; devoid of the mannan moiety) is presently under consideration as a vaccine candidate. Here we investigated cellular and molecular aspects of the interaction of rMP65 with human antigen-presenting cells. We also assessed the ability of rMP65 to initiate a T-cell response. Both the native mannosylated MP65 (nMP65) and the recombinant product were efficiently bound and taken up by macrophages and dendritic cells. However, contrarily to nMP65, rMP65 did not induce tumor necrosis factor alpha and interleukin-6 release from these cells. On the other hand, rMP65 was rapidly endocytosed by both macrophages and dendritic cells, in a process involving both clathrin-dependent and clathrin-independent mechanisms. Moreover, the RGD sequence inhibited rMP65 uptake to some extent. After internalization, rMP65 partially colocalized with lysosomal membrane-associated glycoproteins 1 and 2. This possibly resulted in efficient protein degradation and presentation to CD4(+) T cells, which proliferated and produced gamma interferon. Collectively, these results demonstrate that the absence of the mannan moiety does not deprive MP65 of the capacity to initiate the pattern of cellular and molecular events leading to antigen presentation and T-cell activation, which are essential features for further consideration of MP65 as a potential vaccine candidate.

An anti-beta-glucan monoclonal antibody inhibits growth and capsule formation of Cryptococcus neoformans in vitro and exerts therapeutic, anticryptococcal activity in vivo.

In this study we tested the in vitro and in vivo anti-Cryptococcus neoformans activity of an antilaminarin (anti-beta-glucan) monoclonal antibody (MAb 2G8) (immunoglobulin G2b) which was previously shown to inhibit the growth of beta-glucan-exposing Candida albicans cells. Here we show that MAb 2G8 binds to the cell wall of C. neoformans and inhibits its growth to an extent comparable to that observed for C. albicans. Binding and growth inhibition were detected almost equally for encapsulated and acapsular C. neoformans strains. In addition, at subinhibitory concentrations, MAb 2G8 reduced the capsule thickness without affecting protease or phospholipase production. Acapsular fungal cells, but not encapsulated fungal cells, were opsonized by the antibody and more efficiently phagocytosed and killed by human monocytes and by murine peritoneal macrophages. A single administration of MAb 2G8 resulted in a reduction in the fungal burden in the brains and livers of mice systemically infected with a highly virulent, encapsulated C. neoformans strain. This protective effect was also detected in neutropenic mice. Overall, these findings demonstrate that cell wall beta-glucan of encapsulated C. neoformans is accessible to antibodies which can exert remarkable anticryptococcal activities in vitro and in vivo.