Differential adhesion of pathogenic Candida species to epithelial and inert surfaces

Abstract Growth in medium containing 500 mM galactose is known to promote the adhesion of Candida albicans to buccal epithelial cells or to acrylic in vitro. Of 5 other Candida species tested, only C. tropicalis (one strain) showed substantially increased adhesion to buccal cells (but not to acrylic) after growth under these conditions. A second strain of C. tropicalis as well as C. stellatoidea, C. parapsilosis, C. pseudotropicalis, C. guilliermondii and Saccharomyces cerevisiae showed little or no increased adhesion to either surface. However, after growth in medium containing 50 mM glucose, C. tropicalis and C. parapsilosis were significantly more adherent to acrylic than glucose-grown yeasts of the other species, including C. albicans . These results are discussed in relation to the colonization and infection potential of the pathogenic Candida species.     

Factors involved in the adherence of Candida albicans and Candida tropicalis to protein-adsorbed surfaces

The adherence of Candida albicans and C. tropicalis to protein-adsorbed surfaces was investigated with surface-modified glass slides to which serum or salivary proteins were covalently bound. A specific adherence like a ligand-receptor interaction was observed between C. albicans and mucin- or salivary protein-immobilized glass slides. This interaction was eliminated by deglycosylation of the slides, suggesting that the receptor may be an oligosaccharide(s) contained mucin or saliva. A similar specific interaction was also observed between C. tropicalis and fibrinogen-immobilized glass surfaces. When the numbers of adherent cells to deglycosylated protein-immobilized glass glides were plotted against zeta potentials and contact angles of these protein-immobilized glass slides, a significant correaltion was observed between the numbers of adherent cells and zeta potentials in the case of C. albicans (r = –0.87), whereas a significant correlation was observed between cell numbers and contact angles (r = 0.82) in the case of C. tropicalis. These results suggest that the forces governing the adherence of fungi to pellicle in dentures may vary depending upon the surface properties of fungi and substrate.     

Mechanisms involved in the photosensitized inactivation of Acanthamoeba palestinensis trophozoites

Aims:  To advance our understanding of the mechanisms involved in the RLP068 phthalocyanine-photosensitized inactivation of Acanthamoeba palestinensis trophozoites through a precise identification of the targets of the photoprocess in both the cytosolic and mitochondrial compartments.
Methods and Results:  We followed the activities of selected marker enzymes as well as we performed fluorescence and transmission electron microscopy investigations of the alterations induced by the photoprocess in the fine structure of subcellular compartments. RLP068 is preferentially located in the contractile vacuole: the fluorescence in that site is particularly evident in the unirradiated cells and becomes more diffused after irradiation. Electron microscopic analysis of photosensitized A. palestinensis cells clearly shows that the swelling of trophozoites and the appearance of vacuoles spread throughout the cytoplasm after phototreatment. The activity of a typical cytoplasmic enzyme, such as lactate dehydrogenase, underwent a 35% decrease as a consequence of the photoprocess, reflecting the photodamage induced by migrating phthalocyanine molecules in their micro-environment.
Conclusions:  The presence of multiple targets for the phthalocyanine-photosensitized process is of utmost importance because this pattern of cell damage makes it unlikely that photoresistant A. palestinensis strains are gradually selected or mutagenic phenomena are developed as a consequence of the photoinduced damage.
Significance and Impact of the Study:  Photosensitization via phthalocyanines appears to represent an efficient and safe approach for achieving a close control of the population of a potentially pathogenic protozoan such as A. palestinensis , opening new perspectives for the disinfection of microbiologically polluted waters.     

The MgtE Mg2+ transport protein is involved in Aeromonas hydrophila adherence

Aeromonas hydrophila AH-3 strains carrying mutations in mgtE, which encodes a Mg2+ and Co2+ transport system, showed a 50% reduction of in vitro adherence to HEp-2 cells, a reduction in swarming in semisolid swarming agar, and decrease in biofilm formation of over 60% in comparison to the wild-type strain. The cloned A. hydrophila mgtE expressed from a plasmid complements a Salmonella typhimurium strain deleted for all Mg2+ transporters both phenotypically and by measurement of 57Co2+ uptake. Likewise, plasmid-borne mgtE was able to complement the changes observed in A. hydrophila mgtE mutants. We suggest that MgtE and thus Mg2+ and possibly Co2+ have a role in A. hydrophila related to their swarming ability and related consequences such as adherence and biofilm formation.     

Analysis of Acanthamoeba polyphaga surface carbohydrate exposure by FITC-lectin binding and fluorescence evaluation

AIMS: Characterization of the representative protozoan Acanthamoeba polyphaga surface carbohydrate exposure by a novel combination of flow cytometry and ligand-receptor analysis. METHODS AND RESULTS: Trophozoite and cyst morphological forms were exposed to a panel of FITC-lectins. Population fluorescence associated with FITC-lectin binding to acanthamoebal surface moieties was ascertained by flow cytometry. Increasing concentrations of representative FITC-lectins, saturation binding and determination of K(d) and relative B(max) values were employed to characterize carbohydrate residue exposure. FITC-lectins specific for N-acetylglucosamine, N-acetylgalactosamine and mannose/glucose were readily bound by trophozoite and cyst surfaces. Minor incremental increases in FITC-lectin concentration resulted in significant differences in surface fluorescence intensity and supported the calculation of ligand-binding determinants, K(d) and relative B(max), which gave a trophozoite and cyst rank order of lectin affinity and surface receptor presence. CONCLUSIONS: Trophozoites and cysts expose similar surface carbohydrate residues, foremost amongst which is N-acetylglucosamine, in varying orientation and availability. SIGNIFICANCE AND IMPACT OF THE STUDY: The outlined versatile combination of flow cytometry and ligand-receptor analysis allowed the characterization of surface carbohydrate exposure by protozoan morphological forms and in turn will support a valid comparison of carbohydrate exposure by other single-cell protozoa and eucaryotic microbes analysed in the same manner.     

Phagocytosis in Acanthamoeba: I. A mannose receptor is responsible for the binding and phagocytosis of yeast

We have examined the initial events in phagocytosis by Acanthamoebe castellanii in order to understand this process at the molecular level and have determined that phagocytosis in this organism is mediated by a receptor which recognizes mannose-ricn elements in the particle to be phagocytosed. We demonstrate that the binding and internalization of yeast particles can be inhibited by the sugars D(+)-mannose and D(?)-fructose in a stereospecific, concentration-dependent manner. This inhibition is specific; these sugars did not inhibit the uptake of latex beads by this organism. Using mannosylated neoglycoproteins, which are much more potent inhibitors of particle binding as compared with the free sugar, we demonstrate the presence of a receptor on the amoeba cell surface which is necessary for the binding of yeast as the initial event of phagocytosis. The Acanthamoeba mannose receptor also appears to be able to mediate the delivery of soluble mannose-rich molecules to a degradative compartment such as the lysosome. Knowledge of this receptor will allow a better understanding of the molecular events of phagocytosis.     

QID74 Cell wall protein of Trichoderma harzianum is involved in cell protection and adherence to hydrophobic surfaces

Trichoderma is widely used as biocontrol agent against phytopathogenic fungi, and as biofertilizer because of its ability to establish mycorriza-like association with plants. The key factor to the ecological success of this genus is the combination of very active mycoparasitic mechanisms plus effective defense strategies induced in plants. This work, different from most of the studies carried out that address the attacking mechanisms, focuses on elucidating how Trichoderma is able to tolerate hostile conditions. A gene from Trichoderma harzianum CECT 2413, qid74, was strongly expressed during starvation of carbon or nitrogen sources; it encoded a cell wall protein of 74kDa that plays a significant role in mycelium protection. qid74 was originally isolated and characterized, in a previous work, by a differential hybridization approach under simulated mycoparasitism conditions. Heterologous expression of Qid74 in Saccharomyces cerevisiae indicated that the protein, located in the cell wall, interfered with mating and sporulation but not with cell integrity. The qid74 gene was disrupted by homologous recombination and it was overexpressed by isolating transformants selected for the amdS gene that carried several copies of qid74 gene under the control of the pki promoter. Disruptants and transformants showed similar growth rate and viability when they were cultivated in different media, temperatures and osmolarities, or were subjected to different abiotic stress conditions. However, disruptants produced about 70% mass yield under any condition and were substantially more sensitive than the wild type to cell wall degradation by different lytic preparations. Transformants had similar mass yield and were more resistant to lytic enzymes but more sensitive to copper sulfate than the wild type. When experiments of adherence to hydrophobic surfaces were carried out, the disruptants had a reduced capacity to adhere, whereas that capacity in the overproducer transformants was slightly higher than that of the wild type. Results point to a significant role for Qid74 both in cell wall protection and adhesion to hydrophobic surfaces.     

Isolation and identification of Acanthamoeba species related to amoebic encephalitis and nonpathogenic free‐living amoeba species from the rice field

Aims: Isolation and characterization of the clinically relevant amphizoic amoebas in vegetated farmlands, which may present a risk to farmers’ health. Methods and Results: Acanthamoeba species was isolated and characterized via morphological and molecular means in the rice field where the patient was exposed to rice paddy water which most probably was the point of infection. An Acanthamoeba sp. abundant in the rice field was identified. Genotyping showed the strain to be genotype T4, which was identical to the amoebic parasite found in patient’s cerebrospinal fluid. During the course of the study, three nonpathogenic free‐living amoeba species were also isolated and characterized for the first time in Taiwan. Conclusions: This study successfully located a possible source of granulomatous amoebic encephalitis in a patient and provided the first evidence that Acanthamoeba genotype T4 may be a potential pathogen in Taiwan. Significance and Impact of the Study: The integration of field survey, clinical data and morphological and genetic examination represents a sound strategy for investigation of the possible role of free‐living amoebae in causing human diseases. Future work should include investigating the potential contributory role of other nonpathogenic free‐living protozoa in disease of livestock or even human.     

Specific Detection of Acanthamoeba species using Polyclonal Peptide Antibody Targeting the Periplasmic Binding Protein of A. castellanii

Acanthamoeba keratitis (AK) is a rare ocular disease, but it is a painful and sight-threatening infectious disease. Early diagnosis and adequate treatment are necessary to prevent serious complications. While AK is frequently diagnosis via several PCR assays or Acanthamoeba-specific antibodies, a more specific and effective diagnostic method is required. This study described the production of a polyclonal peptide antibody against the periplasmic binding protein (PBP) of A. castellanii and investigated its diagnostic potential. Western blot analysis showed that the PBP antibody specifically reacted with the cell lysates of A. castellanii. However, the PBP antibody did not interact with human corneal epithelial (HCE) cells and the other 3 major causative agents of keratitis. Immunocytochemistry (ICC) results revealed the specific detection of A. castellanii trophozoites and cysts by PBP antibodies when A. castellanii were co-cultured with HCE cells. PBP antibody specificity was further confirmed by co-culture of A. castellanii trophozoites with F. solani, S. aureus, and P. aeruginosa via ICC. The PBP antibody specifically reacted with the trophozoites and cysts of A. polyphaga, A. hatchetti, A. culbertsoni, A. royreba, and A. healyi, thus demonstrated its genus-specific nature. These results showed that the PBP polyclonal peptide antibody of A. castellanii could specifically detect several species of Acanthamoeba, contributing to the development of an effective antibody-based AK diagnostics.     

The role of proteases in the differentiation of Acanthamoeba castellanii

Proteases are significant determinants of protozoan pathogenicity and cytolysis of host cells. However, there is now growing evidence of their involvement in cellular differentiation. Acanthamoeba castellanii of the T4 genotype elaborates a number of proteases, which are inhibited by the serine protease inhibitor phenylmethylsulphonyl fluoride. Using this and other selective protease inhibitors, in tandem with siRNA primers, specific to the catalytic site of Acanthamoeba serine proteases, we demonstrate that serine protease activity is crucial for the differentiation of A. castellanii . Furthermore, both encystment and excystment of A. castellanii was found to be dependent on serine protease function.     

A facile regio- and stereoselective synthesis of mannose octasaccharide of the N-glycan in human CD2 and mannose hexasaccharide antigenic factor 13b

A highly concise and effective synthesis of the mannose octasaccharide of the N-linked glycan in the adhesion domain of human CD2 was achieved via TMSOTf-promoted selective 6-glycosylation of a trisaccharide 4,6-diol acceptor with a pentasaccharide donor, followed by deprotection. The pentasaccharide was constructed by selective 3,6-diglycosylation of 1,2-O-ethylidene-beta-D-mannopyranose with 2-O-acetyl-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate, while the trisaccharide was obtained by selective 3-O-glycosylation of allyl 4,6-O-benzylidene-alpha-D-mannopyranoside with the same disaccharide trichloroacetimidate, followed by debenzylidenation. The mannose hexasaccharide antigenic factor 13b was synthesized by condensation of a trisaccharide donor, 2-O-acetyl-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->3)-4,6-di-O-acetyl-2-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate, with a trisaccharide acceptor, methyl 3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranoside, followed by deprotection.     

Synthesis of a mannose nonasaccharide existing in the exopolysaccharide of Cryphonectria parasitica

alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->6)[alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)]-alpha-D-Manp-(1-->6)-[alpha-D-Manp-(1-->2)]-alpha-D-Manp, existing in the exopolysaccharide of Cryphonectria parasitica was synthesized as its allyl glycoside in a regio- and stereoselective manner.     

Characterization of a Peptide Antibody Specific to the Adenylyl Cyclase-Associated Protein of Acanthamoeba castellanii

Acanthamoeba keratitis (AK) is a rare infectious disease and accurate diagnosis has remained arduous as clinical manifestations of AK were similar to keratitis of viral, bacterial, or fungal origins. In this study, we described the production of a polyclonal peptide antibody against the adenylyl cyclase-associated protein (ACAP) of A. castellanii, and evaluated its differential diagnostic potential. Enzyme-linked immunosorbent assay revealed high titers of A. castellanii-specific IgG and IgA antibodies being present in low dilutions of immunized rabbit serum. Western blot analysis revealed that the ACAP antibody specifically interacted with A. castellanii, while not interacting with human corneal epithelial (HCE) cells and other causes of keratitis such as Fusarium solani, Pseudomonas aeruginosa, and Staphylococcus aureus. Immunocytochemistry (ICC) results confirmed the specific detection of trophozoites and cysts of A. castellanii co-cultured with HCE cells. The ACAP antibody also specifically interacted with the trophozoites and cysts of 5 other Acanthamoeba species. These results indicate that the ACAP antibody of A. castellanii can specifically detect multiple AK-causing members belonging to the genus Acanthamoeba and may be useful for differentially diagnosing Acanthamoeba infections.     

Isolates from ancient permafrost help to elucidate species boundaries in Acanthamoeba castellanii complex (Amoebozoa: Discosea)

Acanthamoeba castellanii species complex (genotype T4) comprises of more than ten species with unclear synonymy. Its molecular phylogeny has several conflicts with published morphological data. In this paper, we analyze morphometric traits and temperature preferences in six new strains belonging to A. castellanii complex isolated from Arctic permafrost in the framework of molecular phylogeny. This integrative approach allows us to cross-link genotypic and phenotypic variability and identify species-level boundaries inside the complex. We also analyze previously known and newly found discrepancies between the nuclear and mitochondrial gene-based phylogenies. We hypothesize that one reason for these discrepancies may be the intragenomic polymorphism of ribosomal RNA genes.     

Photosensitized inactivation of Acanthamoeba palestinensis in the cystic stage

AIMS: To develop alternative approaches for medical and environmental control of pathogenic Acanthamoeba spp. by means of photodynamic treatment with a tetracationic Zn(II)-phthalocyanine (RLP068). METHODS AND RESULTS: Incubation of cyst cultures with RLP068 for 1 h caused an accumulation of readily detectable concentrations of the phthalocyanine, even at doses as low as 0.5 micromol l(-1). RLP068 exhibited no dark toxicity towards cysts up to 5 micromol l(-1) concentration. A decrease of c. 50% in cyst survival in comparison with controls was measured upon incubation of the cysts with 0.5 micromol l(-1) RLP068, followed by exposure to light (600-700 nm) for 20 min at a fluence rate of 50 mW cm(-2) (60 J cm(-2)). After incubation with 3 and 5 micromol l(-1) RLP068 and irradiation, the cysts lost their excystment ability as early as day 5 and up to day 10, and were clearly damaged when observed under an interference contrast microscope. CONCLUSIONS: These data indicate the promising use of RLP068 in phototreatment of diseases caused by pathogenic amoebae and in initial disinfection of wastewaters. SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid and extensive photodamage may be induced in the highly resistant cystic stages by means of 600- to 700-nm light sources.     

Antifungal drugs effect adherence of Candida albicans to acrylic surfaces by changing the zeta-potential of fungal cells

The effect of sub-inhibitory concentrations of antifungal drugs on the adherence of Candida albicans to acrylic surfaces was investigated. Among five antifungals tested, azalomycin F and aculeacin A significantly enhanced the adherence. The zeta-potential of fungal cells was affected by antifungal drugs, whereas no significant change in cell surface hydrophobicity was observed. The relationship obtained between the change in the adherence and that in zeta-potential suggests that the enhanced adherence was caused by decreased electric repulsive forces.     

Oral ingestion of mannose alters the expression level of deaminoneuraminic acid (KDN) in mouse organs

Deaminoneuraminic acid (KDN) is a unique member of the sialic acid family. We previously demonstrated that free KDN is synthesized de novo from mannose as its precursor sugar in trout testis, and that the amount of intracellular KDN increases in mouse B16 melanoma cells cultured in mannose-rich media [Angata et al. (1999) J. Biol. Chem. 274, 22949–56; Angata et al. (1999) Biochem. Biophys. Res. Commun. 261, 326–31]. In the present study, we first demonstrated a mannose-induced increase in intracellular KDN in various cultured mouse and human cell lines. These results led us to examine whether KDN expression in mouse organs is altered by exogenously administered mannose. Under normal feeding conditions, intracellular free KDN was present at very low levels (19–48 pmol/mg protein) in liver, spleen, and lung, and was not detected in kidney or brain. Oral ingestion of mannose, both short-term (90 min) and long-term (2 wk), resulted in an increase of intracellular KDN up to 60–81 pmol/mg protein in spleen and lung and 6.9–18 pmol/mg protein in kidney and brain; however, no change was observed in liver. The level of KDN in organs appears not to be determined only by the KDN 9-phosphate synthase activity, but might also be affected by other enzymes that utilize mannose 6-phosphate as a substrate as well as the enzymes that breakdown KDN, like KDN-pyruvate lyase. In blood, the detectable amount of free KDN did not change on oral ingestion of mannose. These findings indicate that mannose in the diet affects KDN metabolism in various organs, and provide clues to the mechanism of altered KDN expression in some tumor cells and aged organs.     

A zinc finger-like domain of the molecular chaperone DnaJ is involved in binding to denatured protein substrates.

The Escherichia coli heat-shock protein DnaJ cooperates with the Hsp70 homolog DnaK in protein folding in vitro and in vivo. Little is known about the structural features of DnaJ that mediate its interaction with DnaK and unfolded polypeptide. DnaJ contains at least four blocks of sequence representing potential functional domains which have been conserved throughout evolution. In order to understand the role of each of these regions, we have analyzed DnaJ fragments in reactions corresponding to known functions of the intact protein. Both the N-terminal 70 amino acid 'J-domain' and a 35 amino acid glycine-phenylalanine region following it are required for interactions with DnaK. However, only complete DnaJ can cooperate with DnaK and a third protein, GrpE, in refolding denatured firefly luciferase. As demonstrated by atomic absorption and extended X-ray absorption fine structure spectroscopy (EXAFS), the 90 amino acid cysteine-rich region of DnaJ contains two Zn atoms tetrahedrally coordinated to four cysteine residues, resembling their arrangement in the C4 Zn binding domains of certain DNA binding proteins. Interestingly, binding experiments and cross-linking studies indicate that this Zn finger-like domain is required for the DnaJ molecular chaperone to specifically recognize and bind to proteins in their denatured state.