Identification and Characterization of a Novel Aspergillus fumigatus Rhomboid Family Putative Protease, RbdA, Involved in Hypoxia Sensing and Virulence.

Aspergillus fumigatus is the most common pathogenic mold infecting humans and a significant cause of morbidity and mortality in immunocompromised patients. In invasive pulmonary aspergillosis, A. fumigatus spores are inhaled into the lungs, undergoing germination and invasive hyphal growth. The fungus occludes and disrupts the blood vessels, leading to hypoxia and eventual tissue necrosis. The ability of this mold to adapt to hypoxia is regulated in part by the sterol regulatory element binding protein (SREBP) SrbA and the DscA to DscD Golgi E3 ligase complex critical for SREBP activation by proteolytic cleavage. Loss of the genes encoding these proteins results in avirulence. To identify novel regulators of hypoxia sensing, we screened the Neurospora crassa gene deletion library under hypoxia and identified a novel rhomboid family protease essential for hypoxic growth. Deletion of the A. fumigatus rhomboid homolog rbdA resulted in an inability to grow under hypoxia, hypersensitivity to CoCl2, nikkomycin Z, fluconazole, and ferrozine, abnormal swollen tip morphology, and transcriptional dysregulation-accurately phenocopying deletion of srbA. In vivo, rbdA deletion resulted in increased sensitivity to phagocytic killing, a reduced inflammatory Th1 and Th17 response, and strongly attenuated virulence. Phenotypic rescue of the ΔrbdA mutant was achieved by expression and nuclear localization of the N terminus of SrbA, including its HLH domain, further indicating that RbdA and SrbA act in the same signaling pathway. In summary, we have identified RbdA, a novel putative rhomboid family protease in A. fumigatus that mediates hypoxia adaptation and fungal virulence and that is likely linked to SrbA cleavage and activation.

Immunization protocols for use in animal models of candidiasis.

Immunoprotection during most forms of candidiasis (oropharyngeal, invasive) is lacking since most candidiasis patients are immunosuppressed either as a result of their allogeneic transplant, cancer chemotherapy, or HIV infection. Consequently, immunization might be considered as an unlikely way to protect patients from such infection. Nonetheless, there are a number of investigations that indicate active immunization or the passive treatment with hyperimmune, specific antibodies can result in protection in models of experimental candidiasis. The former subject, active immunization, is the subject of this chapter. We focus on recent efforts with the Als family of cell wall proteins to serve as a model, and also offer immunization methods in candidiasis models that can be adapted to any antigen of the organism.

Biomedical coatings based on chitin soluble extract for inhibition of fungal adhesion to polymeric surfaces.

Indwelling medical devices made of polymeric materials, such as intravenous (IV) catheters, are known risk factors for development of fungal infection, particularly systemic candidiasis, that are a significant cause of morbidity and lethality in compromised patients. Candida can form a biofilm on the polymeric surface, serving as a nidus for systemic, difficult to eradicate infection. The current research focuses on development and study of a chitin soluble extract (CSE) coating on polyurethane (PU), in order to reduce the level of adherence of C. albicans to the PU surface. The immobilization of CSE onto the PU surface was performed using both, chemical binding and physical adsorption. Our results indicate that CSE develops a unique tertiary structure in which basic elements in the range of 100 nm build "fingers" and these "fingers" are arranged in a concentric structure around a center. The CSE coated films showed 75% inhibition of C. albicans adhesion for the chemical binding and 83% for the physical adsorption coating and even after 11 weeks the inhibitory effect on adhesion is still significant. Hence, our new coatings may lead to a new generation of medical devices with surfaces that can prevent fungal adhesion.

Infection of HEp2 epithelial cells with Candida albicans: adherence and postadherence events.

We have previously observed that the infection of HEp2 epithelial cells with Candida albicans results in HEp2 cell actin rearrangement, and that a culture filtrate of C. albicans (Candida metabolite) caused the same changes and reduced membrane ruffling and motility. It was found that the Candida metabolite consisted of several proteins and nonproteinaceous components. In this study we report on the identity of three of the main proteins in the Candida metabolite, namely a secretory aspartate protease (Sap), an agglutinin-like adhesion sequence (Als) and a glucan 1,3-beta-glucosidase. The effect on HEp2 cells caused by the Candida metabolite, an inhibitor of the PKC MAP kinase signal pathway - bisindolylmaleimide (BIM), or the actin polymerization inhibitor - cytochalasin D (CyD) were studied alone and in combination. Exposure of HEp2 cells to the Candida metabolite, together with the BIM or CyD, had profound effects on HEp2 cell morphology, as compared to individually treated cells, and also reduced the adherence of the organisms to HEp2 cells. Our results show that the interaction of C. albicans with HEp2 cells is, not unexpectedly, complex, and involves changes in the host cell that may be related to the effect of Candida-secreted biomolecules.

Antifungal effect and possible mode of activity of a compound from the marine sponge Dysidea herbacea.

OBJECTIVES: Assessment of antifungal activity of a compound isolated from the marine sponge Dysidea herbacea against the fungal pathogens Candida (primarily C. albicans) and Aspergillus (primarily A. fumigatus) species, and investigations of the possible mode of activity of the compound. METHODS: Freeze dried sponges were extracted with EtOAc-MeOH. Bioassay guided separation was used to identify the active compound. Antifungal activity was assessed in vitro by a modified NCCLS technique. For determination of the possible mode of activity of the compound we tested the effect on fungal cellular morphology (light, scanning and transmission electron microscopy) and possible site of activity in the fungal cells, such as cell membrane (ion leakage kinetics) as well as toxicity (cytotoxicity tests). RESULTS AND CONCLUSIONS: The active compound was determined to be 3,5-dibromo-2-(3,5-dibromo-2-methoxyphenoxy) phenol. This compound exhibited in vitro activity against the tested fungal pathogens. The experiments on the mode of activity revealed that there are significant changes in fungal cell morphology, as demonstrated by scanning and transmission electron microscopy. The compound, apparently, affects the fungal cell membrane, expressed primarily in leakage of potassium ions from the fungal cells. Two other bromo diphenyl ethers were also found to be active. Further experiments in in vivo models are planned.

Gene transcription studies of Candida albicans following infection of HEp2 epithelial cells.

Previously we observed that infection of HEp2 epithelial cells with Candida albicans results in HEp2 cell actin rearrangement as well as reduced membrane ruffling and motility and that supernatants of a C. albicans culture (Candida metabolite) caused the same changes. In this study, we used microarray analysis to determine changes in gene transcription of C. albicans following infection of HEp2 cells compared to control cultures grown in the absence of HEp2 cells. We observed 201 genes whose regulation was increased at least 2-fold following a 3 h incubation with HEp2 cells as well as 87 genes that are down-regulated. Among the up-regulated genes were ALS2 and ALS5 both of which encode proteins that provide an adherence function for C. albicans. To confirm the changes in ALS transcription, we measured by RT-PCR ALS1-9 at 1 h intervals for a total of 4 h. After 1 h of infection, several of the ALS genes were up-regulated compared to C. albicans grown alone. At 2-4 h, an increase in most of the ALS genes was observed in both infected and control cultures. ALS7 transcription was observed only at 3-4 h, but transcription was similar in both infected and control cultures. By RT-PCR, ALS2 and 5, similar to the microarray data, were significantly increased in infected cells at 3 h. Our results show that gene transcription following the adherence of C. albicans to HEp2 cells includes the up-regulation of genes encoding members of a family of known host recognition adhesins that may be critical to successful colonization and invasion of the organism.

Effect of a chitin derivative in combination with cleaning and preservation solutions for contact lenses on adherence of fungi.

The objective of the study was to evaluate the effect of supplementation of a chitin soluble extract (CSE) to clinically used commercial contact lenses' cleaning/preservation solutions on adherence of Aspergillus niger and Candida albicans to contact lenses. CSE (25 mg ml-1) was added to Solocare or Oxysept. Adherence of A. niger or C. albicans to contact lenses was assessed by exposing lenses to fungi in test solutions and enumeration of fungal colony forming unit. The study included three parts: (i) the disinfecting and adherence-inhibiting effect of Solocare and Oxysept: (ii) the adherence-inhibiting effect of CSE; (iii) the effect of Solocare + CSE or Oxysept + CSE on the fungal adherence to contact lenses. The experiments showed that: (i) Solocare had a significant disinfecting effect on A. niger and C. albicans while that of Oxysept was only minimal. (ii) Neither Solocare nor Oxysept had an inhibitory effect on the fungal adherence. (iii) CSE inhibited significantly the adherence of A. niger and C. albicans. (iv) Addition of CSE to Solocare or Oxysept resulted in almost complete inhibition of adherence Supplementing CSE to lenses' maintenance solutions increases their antifungal potential by adding anti-adherence activity to the disinfecting effect of these solutions, bearing the potential to serve as a prophylactic means.