Altered phagocytosis and morphogenesis of phenotypic switching-derived strains of the pathogenic Candida tropicalis co-cultured with phagocytic cells.

BACKGROUND AND OBJECTIVE: Candida tropicalis is among the most prevalent human pathogenic yeast species. Switch states of C. tropicalis differ in virulence traits. Here, we evaluate the effect of phenotypic switching on phagocytosis and yeast-hyphae transition in C. tropicalis. METHODS: C. tropicalis morphotypes included a clinical strain and two switch strains (rough variant and rough revertant). In vitro, phagocytosis assay was performed using peritoneal macrophages and hemocytes. The proportion of hyphal cells was ascertained by scoring morphology using optical microscopy. Expression of the WOR1 (White-opaque regulator 1) and EFG1 (Enhanced filamentous growth protein 1) was determined by quantitative PCR. RESULTS: The rough variant was more resistant to in vitro phagocytosis by peritoneal macrophages than that observed for the clinical strain, while hemocytes phagocytosed clinical and rough variant to the same extent. The rough revertant was more phagocytosed than the clinical strain by both phagocytes. During co-incubation with phagocytic cells, the clinical strain of C. tropicalis exists mainly as blastoconidia. The co-culture of the rough variant with macrophages resulted in a higher percentage of hyphae than blastoconidia cells, while in co-culture with hemocytes, no differences were observed between the percentage of hyphae and blastoconidia. The expression levels of WOR1 in the rough variant co-cultured with phagocytes were significantly higher than they were in the clinical strain. CONCLUSIONS: Differences on phagocytosis and hyphal growth between switch states cells of C. tropicalis co-cultured with phagocytic cells were observed. The pronounced hyphal growth may affect the complex host-pathogen relationship and favor the pathogen to escape phagocytosis. The pleiotropic effects of phenotypic switching suggest that this event may contribute to the success of infection associated with C. tropicalis.

Adhesion and biofilm formation by the opportunistic pathogen Candida tropicalis: what do we know?

Candida tropicalis is among the most important Candida species in terms of epidemiology, virulence and resistance. Considering the increase in C. tropicalis incidence and high rates of mortality associated with this species, knowledge of its adhesion and biofilm formation abilities is needed. These traits determine the persistence and survival of yeast on different indwelling medical devices and host sites. C. tropicalis is among the most adherent Candida species, and it has been described as a strong biofilm producer. Environmental factors, phenotypic switching and quorum sensing molecules can affect adhesion and biofilm growth. C. tropicalis can form sexual biofilms, which are promoted by mating pheromones. C. tropicalis biofilms are regulated by a wide and complex network of genes and signaling pathways that are currently poorly understood. Morphological studies showed improved biofilm architecture, which was related to the expression of several hypha-specific genes. Based on recent updates, research is still needed to increase our knowledge on the genetic network of adhesion and biofilm formation by C. tropicalis, as well as the protein diversity that mediates interactions with inert materials and biological surfaces. Here, we have reviewed the main aspects related to adhesion and biofilm formation in C. tropicalis and summarized current knowledge on the significance of these virulence factors in this opportunistic species.

Effect of phenotypic switching on biofilm traits in Candida tropicalis.

Candida tropicalis can undergo multiple forms of phenotypic switching. We have reported a switching system in C. tropicalis that is associated with changes in virulence attributes. We aimed to assess biofilm formation by distinct switch states of C. tropicalis and evaluate whether their sessile cells exhibit altered virulence traits. C. tropicalis strains included the parental phenotype (a clinical isolate) and four switch phenotypes (crepe, rough, revertant of crepe and revertant of rough). Biofilm formation and adhesion capability of sessile cells on polystyrene were assessed through quantification of total biomass. Filamentous forms were characterized by direct counting of sessile cells. A virulence assay was conducted using the Galleria mellonella infection model. Switch variants (crepe and rough) and their revertant counterparts produced higher biofilm biomass (P < 0.05) than the parental strain. Additionally, filamentous forms were enriched among sessile cells of switched strains compared to those observed for sessile cells of the parental strain, with the exception of the revertant of rough. Sessile cells of switched strains showed higher adhesion to polystyrene compared to the parental strain. Sessile cells of the crepe variant and its revertant strain (RC) exhibited higher virulence against G. mellonella larvae than sessile cells of the parental strain. Our findings indicate that switching events in C. tropicalis affect biofilm development and that sessile cells of distinct switch states may exhibit increased adhesion ability and enhanced virulence towards G. mellonella larvae.

Phenotypic switching in Candida tropicalis alters host-pathogen interactions in a Galleria mellonella infection model.

Candida tropicalis is a human pathogen associated with high mortality rates. We have reported a switching system in C. tropicalis consisting of five morphotypes - the parental, switch variant (crepe and rough), and revertant (crepe and rough) strains, which exhibited altered virulence in a Galleria mellonella model. Here, we evaluate whether switching events may alter host-pathogen interactions by comparing the attributes of the innate responses to the various states. All switched strains induced higher melanization in G. mellonella larvae than that induced by the parental strain. The galiomicin expression was higher in the larvae infected with the crepe and rough morphotypes than that in the larvae infected with the parental strain. Hemocytes preferentially phagocytosed crepe variant cells over parental cells in vitro. In contrast, the rough variant cells were less phagocytosed than the parental strain. The hemocyte density was decreased in the larvae infected with the crepe variant compared to that in the larvae infected with the parental strain. Interestingly, larvae infected with the revertant of crepe restored the hemocyte density levels that to those observed for larvae infected with the parental strain. Most of the switched strains were more resistant to hemocyte candidacidal activity than the parental strain. These results indicate that the switch states exhibit similarities as well as important differences during infection in a G. mellonella model.

Assessment of vancomycin resistance transfer among enterococci of clinical importance in milk matrix.

Dissemination of vancomycin resistance in enterococci has been associated with horizontal transfer of mobile genetic elements. Aim of the study was to evaluate if milk matrix is a suitable environment to support transferability of vancomycin resistance (vanA) gene from clinical vancomycin-resistant Enterococcus faecium to vancomycin-sensitive Enterococcus faecalis. Enterococci strains were firstly screened for the presence of cpd (inducible sex pheromone determinant) gene, vanA and tetL genes (vancomycin and tetracycline resistance markers, respectively) and the gelE (extracellular metalloendopeptidase) gene to define the mating pairs. Based on these selection markers, we investigated the transferability of eight plasmid-borne vanA harbored by E. faecium (vanA+, cpd-, tetL- and gelE-) into two E. faecalis (vanA-, cpd+, tetL + and gelE+) recipient strains in milk matrix. The strains were mated in a 1:1 ratio in 7% reconstituted milk and incubated at 37 °C. Transconjugants emerged from all 16 matings within 2 h of incubation and were evidenced by dual antibiotic resistance (vancomycin and tetracycline). The vancomycin-resistance of trasconjugants was maintained even after ten subsequent passages on nonselective medium. Transconjugants were positive for vanA, tetL and gelE genes. This study indicates milk matrix as suitable environment to support gene exchange between Enterococcus species.

How much do we know about hemolytic capability of pathogenic Candida species?

Hemolytic factor production by pathogenic Candida species is considered an important attribute in promoting survival within the mammal host through the ability to assimilate iron from the hemoglobin-heme group. Hemolytic capability has been evaluated for Candida species based on hemolysis zones on plate assay, analysis of hemolytic activity in liquid culture medium, and hemolysis from cell-free culture broth. The production of hemolytic factor is variable among Candida species, where C. parapsilosis is the less hemolytic species. In general, no intraspecies differences in beta-hemolytic activities are found among isolates belonging to C. albicans, C. glabrata, C. krusei, C. tropicalis, and C. parapsilosis. The production of hemolytic factor by Candida species is affected by several factors such as glucose supplementation in the culture medium, blood source, presence of erythrocytes and hemoglobin, and presence of electrolytes. On the basis of existing achievements, more researches are still needed in order to extend our knowledge about the biochemical nature of hemolytic molecules produced by distinct Candida species, the mechanism of hemolysis, and the molecular basis of the hemolytic factor expression.

γ-irradiation from radiotherapy improves the virulence potential of Candida tropicalis.

AIM: To evaluate if radiation used in radiotherapy can cause changes in the virulence potential of Candida tropicalis ATCC 750. MATERIALS & METHODS: C. tropicalis was exposed in vitro to identical dose and scheme of irradiation would be used in patients with head and neck cancer. Some virulence parameters were analyzed before and after irradiation. RESULTS: Colony morphologies were irreversibly affected by irradiation. Increase in growth rate, filamentation, adhesion on cell lines and phagocytosis process were also observed. Overall the irradiated C. tropicalis cells became more efficient at causing systemic infection in mice. CONCLUSION: γ-radiation induced important changes in C. tropicalis increasing its virulence profile, which could directly affect the relationship between yeasts and hosts.

Phenotypic switching in Candida tropicalis: association with modification of putative virulence attributes and antifungal drug sensitivity.

Although Candida tropicalis has become an increasingly important human pathogen, little is known regarding its potential to cause disease. In this study we evaluated the phenotypic switching ability of C. tropicalis and analyzed the effect of switching on biological properties related to virulence factors. We demonstrated that C. tropicalis switched spontaneously, reversibly and at high frequency (10(-1) to 10(-3)) when grown on yeast extract-peptone-D-glucose (YPD) agar medium. Phenotypic switching in five clinical isolates of C. tropicalis resulted in colonies exhibiting the following morphologies: crepe, rough, crater, irregular center, mycelial and diffuse. The majority of the variant colonies were associated with higher percentages of filamentous growth relative to their parental unswitched isolates. Significant differences (P < 0.05) in the production of hemolytic factor were found between most of the switched variants and their respective parental counterparts. Variant colonies exhibiting the crepe (derived from isolates 49.07 and 100.10) and rough phenotype (derived from isolate 49.07) had higher biofilm formation than their parental counterparts exhibiting a smooth dome surface (P < 0.05). Our data revealed that switching was correlated with changes in the in vitro minimum inhibitory concentrations (MICs) of a subset of the switched variants phenotypes to itraconazole. While the MIC to itraconazole was higher for crepe variant compared with its parental isolate 49.07, the rough variant of 100.10 had a lower MIC to this antifungal agent. The presented data support the role of phenotypic switching in promoting changes in phenotypic expression of putative virulence traits and itraconazole susceptibility of clinical isolates of C. tropicalis.

In vitro antifungal activity of the flavonoid baicalein against Candida species.

The aim of the present study was to evaluate the in vitro activity of baicalein, the flavone constituent of Scutellaria baicalensis, and synergism of the combination of baicalein and fluconazole against Candida albicans, Candida tropicalis and Candida parapsilosis. The MIC(50) (lowest concentration at which there was 50 % inhibition of growth) of baicalein alone against six Candida strains ranged from 13 to 104 µg ml(-1). For the three species tested, exposure to baicalein at the MIC(50) concentrations obtained for each strain resulted in a high loss of viability. The fluconazole plus baicalein combination markedly reduced the MICs of both drugs for all three strains analysed. In addition, a synergistic effect between baicalein and fluconazole was observed for C. parapsilosis in terms of MIC(50) (fractional inhibitory concentration index = 0.207). Scanning electron microscopy analysis revealed that yeast cells exposed to baicalein at MIC(50) produced a profusely flocculent extracellular material, resembling a biofilm-like structure. In conclusion, these results showed the antifungal capability of baicalein against Candida species and highlight a promising role of baicalein when used in combination with fluconazole against Candida infections.

Ultrastructural architecture of colonies of different morphologies produced by phenotypic switching of a clinical strain of Candida tropicalis and biofilm formation by variant phenotypes.

Candida tropicalis has been identified as one of the most prevalent pathogenic yeast species of the Candida-non-albicans (CNA) group. Study of switching in C. tropicalis has not been the subject of extensive research. Therefore, we investigated switching event and characterized the ultrastructural architecture of different phenotypes and biofilm produced in a C. tropicalis clinical strain. Cells switched heritably, reversibly, and at a high frequency between four phenotypes readily distinguishable by the shape of colonies formed on agar at 25°C. SEM analysis was used to verify the architecture of whole Candida colonies at ultrastructural level. The smooth phenotype (parental phenotype) colony showed a hemispherical shape character, while the semi-smooth was characterized by the presence of shallow marginal depressions. The ring and rough phenotypes exhibited more complex architecture and were characterized by the presence of deep central and peripheral depressions areas. The biofilm-forming ability varied among the switch phenotypes. After 12h incubation, the smooth phenotype formed less biofilm compared to the other phenotypes (P<0.05). The electron microscopy analysis revealed that filamentation (pseudohyphae) was associated with ring and rough colonies. The ultrastructural analysis allowed the observation of the arrangement of individual cells within the colonies. At the deep central and peripheral depressions areas of the ring and rough colonies extracellular material was seen in different arrangements. The data presented here open new avenues to study a possible role for extracellular material in the formation and maintenance of the architecture of switch phenotypes in C. tropicalis. It is therefore essential that more strains be investigated to determine the biological significance of extracellular material in C. tropicalis phenotypic switching phenomenon.

Interaction of Candida parapsilosis isolates with human hair and nail surfaces revealed by scanning electron microscopy analysis.

Candida parapsilosis is found frequently as commensal organism on epithelial tissues, and is also an increasing cause of nosocomial infection. Scanning electron microscope (SEM) observations were used to analyse the capability of C. parapsilosis cells to adhere and grow as biofilm on human natural substrates and to compare the adherence pattern of isolates exhibiting distinct phenotypes. Cells from the crepe phenotype are predominantly elongated and form pseudohyphae whereas cells from the smooth phenotype are yeast-shaped, either in liquid cultures or on human nail and hair surfaces. The electron micrographs revealed that C. parapsilosis cells from the smooth phenotype adhered in higher number to both surfaces compared to the observed for the crepe phenotype. SEM analysis of human hair surface revealed that cells from the smooth phenotype appear as clumped blastoconidia of uniform morphology embedded in a flocculent extracellular material forming biofilm. The extracellular material and biofilm were seeing in a less extension in the crepe phenotype. A distinct adherence pattern was observed when human nail was used as substrate. Here C. parapsilosis cells seem to be linked to surface structures of human nail plate. Fibrillar extracellular material was observed connecting neighbouring cells as well as nail surface.

Production and regulation of cuticle-degrading proteases from Beauveria bassiana in the presence of Rhammatocerus schistocercoides cuticle.

Extracellular proteases have been shown to be virulence factors in fungal pathogenicity toward insects. We examined the production of extracellular proteases, subtilisin-like activity (Pr1) and trypsin-like activity (Pr2), by Beauveria bassiana CG425, which is a fungus of interest for control of the grasshopper Rhammatocerus schistocercoides. To access the role of these proteases during infection of R. schistocercoides, we analyzed their secretion during fungus growth either in nitrate-medium or in cuticle-containing medium supplemented with different amino acids. The enhancing effect of cuticle on Pr1 and Pr2 production suggests that these protease types may be specifically induced by components of the grasshopper cuticle. In medium supplemented with methionine a high level of Pr1 was observed. The remaining amino acids tested did not induce the protease to the levels seen with cuticle. The amino acid methionine seems to play a regulatory role in Pr1 secretion by B. bassiana, since both induction and repression seem to be dependent on the concentration of the amino acid present in the culture medium.

Agrobacterium-mediated insertional mutagenesis of the ochratoxigenic fungus Aspergillus westerdijkiae.

Aspergillus westerdijkiae is a potent ochratoxin A (OTA) producer that has been found in coffee beans. OTA is known to have nephrotoxic effects and carcinogenic potential in animal species. Here we report for the first time the Agrobacterium-mediated transformation for Aspergillus westerdijkiae and the generation of ochratoxin-defective mutants. Conidia were transformed to hygromycin B resistance using strain AGL-1 of Agrobacterium tumefaciens. The obtained transformation frequency was up to 47 transformants per 10(6) target conidia. Among 600 transformants, approximately 5% showed morphological variations. Eight transformants with consistently reduced OTA production were obtained. Two of these transformants did not produce OTA (detection limit: 0.1 microg/kg); the other six mutants produced lower amounts of OTA (1%-32%) compared with the wild-type strain. By using thermal asymmetric interlaced polymerase chain reaction, we successfully identified a putative flavin adenine dinucleotide monooxygenase gene.

Efficient genetic transformation system for the ochratoxigenic fungus Aspergillus carbonarius.

Aspergillus carbonarius is a potent ochratoxin A producer that has been found in products such as grapes, coffee, spices, and cocoa. Ochratoxin A has nephrotoxic effect, and it has been classified as a possible carcinogenic substance for humans. Here we describe for the first time a transformation system for A. carbonarius, providing an important step towards its genetic manipulation. Conidia were transformed to acquire hygromycin B resistance using the AGL-1 strain of Agrobacterium tumefaciens. Genetic transformation was evaluated growing A. tumefaciens cells in induction medium containing or not acetosyringone prior to co-cultivation. The mean transforming efficiencies in IM+AS and IM-AS conditions were 62.2 and 44.5 transformants per 10(5) conidia, respectively. The hph gene was random integrated into the genome of A. carbonarius. Fungal sequences flanking the insertion site could be amplified by TAIL-PCR.