Effects of Antifungal Agents in Sap Activity of <Emphasis Type="Italic">Candida albicans</Emphasis> Isolates

Some antifungal agents have shown to exert effects on expression of virulent factors of Candida as the production of secretory aspartyl proteinase (Sap). In this study, we sought to determine and to compare the influence of fluconazole and voriconazole in proteinase activity of this microorganism. Thirty-one isolates obtained from oral mucosa of human immunodeficiency virus positive (HIV⁺) patients were used in this study. The minimal inhibitory concentrations (MIC) of fluconazole and voriconazole were determined using the broth microdilution method with RPMI 1640 medium and with yeast carbon base–bovine serum albumin (YCB–BSA) medium. The Sap activity following by digestion of BSA as substrate was determined for four Candida albicans strains arbitrarily chosen according to susceptibility (susceptible or resistant) to fluconazole or voriconazole. Besides, the SAP1 to SAP7 genes were screened by PCR for the same isolates that were determined by the Sap activity. In vitro susceptibility testing using the two media presented similar MIC values. Increased Sap activity was observed in resistant isolates on presence of drugs, but the Sap activity by susceptible isolates to azoles showed different behavior on the presence of drug. We detected the presence of SAP1 to SAP7 genes from all susceptible or resistant C. albicans isolates. The present study provides important data about the proteinase activity and the presence of genes of SAP family in fluconazole and voriconazole susceptible or resistant C. albicans isolates.     

Virulence and pathogenicity of Candida albicans is enhanced in biofilms containing oral bacteria

This study examined the influence of bacteria on the virulence and pathogenicity of candidal biofilms. Mature biofilms (Candida albicans-only, bacteria-only, C. albicans with bacteria) were generated on acrylic and either analysed directly, or used to infect a reconstituted human oral epithelium (RHOE). Analyses included Candida hyphae enumeration and assessment of Candida virulence gene expression. Lactate dehydrogenase (LDH) activity and Candida tissue invasion following biofilm infection of the RHOE were also measured. Candida hyphae were more prevalent (p < 0.05) in acrylic biofilms also containing bacteria, with genes encoding secreted aspartyl-proteinases (SAP4/SAP6) and hyphal-wall protein (HWP1) up-regulated (p < 0.05). Candida adhesin genes (ALS3/EPA1), SAP6 and HWP1 were up-regulated in mixed-species biofilm infections of RHOE. Multi-species infections exhibited higher hyphal proportions (p < 0.05), up-regulation of IL-18, higher LDH activity and tissue invasion. As the presence of bacteria in acrylic biofilms promoted Candida virulence, consideration should be given to the bacterial component when managing denture biofilm associated candidoses.     

Acid proteinase,phospholipase, and biofilm production of <Emphasis Type="Italic">Candida</Emphasis> species isolated from blood cultures

Three virulence factors comprising proteinase, phospholipase, and biofilm among 68 Candida albicans and 31 non-albicans Candida strains (11 C. tropicalis, 8 C. parapsilosis, 6 C. glabrata, 4 C. guillermondii, 2 C. krusei) isolated from blood cultures were analyzed. In total, 61 (89.7%) C. albicans strains were detected as proteinase positive whereas eight (25.8%) non-albicans Candida strains were proteinase positive (P < 0.05). Phospholipase production was detected in 41 (60.3%) C. albicans strains. All non-albicans Candida strains were phospholipase negative. Biofilm production was determined by both visual and spectrophotometric methods. Eight (11.8%) of C. albicans strains and 13 (41.93%) of 31 non-albicans Candida strains were biofilm positive with two of the methods (P < 0.05). According to our results, we may suggest that detection of hydrolytic enzyme and biofilm production abilities of the Candida isolates in clinical mycology laboratories may warn the clinican for a possible hematogenous infection.     

Phospholipase and Proteinase Activities of Clinical Isolates of Candida from Immunocompromised Patients

Sixty-one isolates of Candida recovered from HIV seropositive and cancer patients were studied for elaboration of putative virulence determinants – phospholipase (PL) and secreted aspartyl proteinase (Sap). Forty two (68.85%) isolates examined were PL producers and 51 (83.6%) were positive for Sap. 57.37% (35/61) isolates produced both enzymes. Enzymatic activity was more pronounced in Candida albicans with 100% PL and 94.1% Sap activity. In contrast, non-C. albicans species demonstrated only 29.6% PL and 70.3% Sap activity, indicating interplay of other virulence determinants in these yeasts in colonization and disease.     

Differences in proteolytic activity and gene profiles of fungal strains isolated from the total parenteral nutrition patients

Fungal infections constitute a serious clinical problem in the group of patients receiving total parenteral nutrition. The majority of species isolated from infections of the total parenteral nutrition patients belong to Candida genus. The most important factors of Candida spp. virulence are the phenomenon of “phenotypic switching,” adhesins, dimorphism of fungal cells and the secretion of hydrolytic enzymes such as proteinases and lipases, including aspartyl proteinases. We determined the proteolytic activity of yeast-like fungal strains cultured from the clinical materials of patients receiving total parenteral nutrition and detected genes encoding aspartyl proteinases in predominant species Candida glabrata—YPS2, YPS4, and YPS6, and Candida albicans—SAP1–3, SAP4, SAP5, and SAP6. C. albicans released proteinases on the various activity levels. All C. glabrata strains obtained from the clinical materials of examined and control groups exhibited secretion of the proteinases. All 13 isolates of C. albicans possessed genes SAP1–3. Gene SAP4 was detected in genome of 11 C. albicans strains, SAP5 in 6, and SAP6 in 11. Twenty-six among 31 of C. glabrata isolates contained YPS2 gene, 21 the YPS4 gene, and 28 the YPS6 gene. We observed that clinical isolates of C. albicans and C. glabrata differed in SAPs and YPSs gene profiles, respectively, and displayed differentiated proteolytic activity. We suppose that different sets of aspartyl proteinases genes as well as various proteinase-activity levels would have the influence on strains virulence.     

Three distinct secreted aspartyl proteinases in Candida albicans.

The secreted aspartyl proteinases of Candida albicans (products of the SAP genes) are thought to contribute to virulence through their effects on Candida adherence, invasion, and pathogenicity. From a single strain of C. albicans (WO-1) which expresses a phenotypic switching system, three secreted aspartyl proteinases have been identified as determined by molecular weight and N-terminal sequence. Each of the three identified proteins represents the mature form of one of three distinct proteinase isoenzymes, two of which correspond to the recently cloned SAP1 and SAP2 genes (previously referred to as CAP, PEP, or PRA). A genomic library was screened under low-stringency hybridization conditions with a polymerase chain reaction fragment from SAP1. In addition to clones of SAP1 and SAP2, a clone containing SAP3, a novel third secreted proteinase gene, was identified and sequenced. The three aspartyl proteinase isoenzymes differ in primary sequence and pI, suggesting that they may play different roles in virulence and pathogenesis. All three of these proteinases are expressed in the same strain. However, the pattern of proteinase expression is correlated with the switch phenotype of the cell. Opaque cells of strain WO-1 express Sap1 and Sap3, while white cells of the same strain express Sap2. The differential expression of three Sap proteinases may contribute to virulence in C. albicans.     

Impact of glucose levels on expression of hypha-associated secreted aspartyl proteinases in Candida albicans

Background

Ten secreted aspartyl proteinase (Sap) genes were identified in Candida albicans. The products of SAP genes are considered to be virulent factors of C. albicans that participated in causing mucocutaneous and systemic candidiasis in humans. Depending on environmental conditions, C. albicans may stay in yeast-form or convert into invasive hypha-form, and these issues may affect the expression of SAP genes. In this study we explored the component(s) of culture media that may affect the expression of hypha-associated SAP genes.

Results

We demonstrate that glucose levels modulate both the hyphae development and the expression strength of hypha-associated SAP genes (SAP4-6). In contrast to high glucose concentration (2%), lower glucose level (0.1%) is more potent to promote hyphae development and to promptly elicit the expression of hypha-associated Sap proteins during yeast-to-hypha transition of C. albicans. Both Cph1-mediated MAP kinase cascade and Efg1-mediated cAMP/PKA pathway, although the latter seemed dominant, participate in convey the glucose signaling to regulate the expression of hypha-associated SAP genes and this glucose level effect may perform at very early stage of yeast-to-hypha transition. In addition, when C. albicans was co-cultured with THP-1 human monocytes, the engulfed C. albicans was developing hypha efficiently within 1 hr and the expression of hypha-associated Sap proteins could be detected on the distal surface of hyphae.

Conclusion

We propose that the glucose level of bloodstream (approximately 0.1%) may be facilitated for stimulation of C. albicans to develop invasive hypha-form and to elicit promptly production of high-level hypha-associated Sap proteins.     

Species Distribution and Virulence Factors of Candida spp. Isolated from the Oral Cavity of Kidney Transplant Recipients in Brazil

Although yeasts belonging to the genus Candida are frequently seen as commensals in the oral cavity, they possess virulence attributes that contribute for pathogenicity. The aims of the present study were to study the prevalence of Candida spp. isolated from the oral cavity of renal transplant recipients and to analyze strains virulence factors. We isolated a total of 70 Candida strains from 111 transplant recipients, and Candida albicans was the most prevalent species (82.86 %). Oral candidiasis was diagnosed in 14.4 % kidney transplant patients, while 11 isolates (15.7 %) corresponded to non-Candida albicans Candida (NCAC) species. C. albicans adhered to a higher extension than NCAC strains. Some isolates of Candida tropicalis were markedly adherent to human buccal epithelial cells and highly biofilm-forming strains. Regarding proteinase activity, Candida orthopsilosis was more proteolytic than Candida metapsilosis. Candida glabrata and Candida dubliniensis showed very low ability to form biofilm on polystyrene microtiter plates. We have demonstrated here diverse peculiarities of different Candida species regarding the ability to express virulence factors. This study will contribute for the understanding of the natural history and pathogenesis of yeasts belonging to the genus Candida in the oral cavity of patients who were submitted to kidney transplant and are under immunosuppressive therapies.     

Effect of phenotypic switching on expression of virulence factors by Candida albicans causing candidiasis in diabetic patients

A total of 110 strains belonging to seven species of Candida were isolated from various forms of candidiasis in diabetic patients. They were Candida albicans 53 (47%), Candida tropicalis 36 (33%), Candida glabrata 9 (8%), Candida parapsilosis 4 (4%), Candida guilliermondii 2 (2%), Candida krusei 5 (5%) and Candida kefyr 1 (1%). All 53 strains of C. albicans isolated were observed to express virulence factors such as cell surface hydrophobicity (CSH), adherence to human buccal epithelial cell (BEC) and proteinase activity (100%), while phospholipase activity was observed in 52 (98%). Phenotypic switching and its influence on the pathogenicity of C. albicans were studied. Two C. albicans strains isolated from oral and vaginal thrush, respectively, in diabetic individuals, and the control strain C. albicans NCPF 3153A were induced to undergo phenotypic switching by exposure to UV light and the degree of expression of virulence factors by the different morphological forms was determined. Three different morphological forms of C. albicans were obtained, namely Star (S), Wrinkled (W) and Ring (R) types from the original Smooth (O) variety. It was found that proteinase activity was greatest with the W type followed by the R type then the O type. The S type produced the least proteinase. The phospholipase activity was greatest with O type followed by R type. The W and S types produced the least phospholipase. Expression of CSH and adherence was greatest in the O type followed by the R and then the W type and finally the S type. Differential expression of virulence factors occurs with different phenotypic forms of C. albicans and this may provide a particular morphological type with a distinct advantage over other types in causing candidiasis.     

Investigation of extracellular phospholipase and proteinase activities of Candida species isolated from individuals denture wearers and genotypic distribution of Candida albicans strains

In order to determine the relationship between the development of denture related stomatitis (DRS) and the production of phospholipase and proteinase by Candida species, 156 Candida isolates isolated from the individuals in the control group and from the individuals different denture wearers were included in this study. According to the results of the study, C. albicans strains were determined to produce high levels of phospholipase and proteinase. It was also determined that the prevalence of phospholipase and proteinase activities in C. albicans strains isolated from individuals with DRS and from the individuals without DRS was not different. In order to determine genotypic variation of 109 C. albicans strains isolated, CA-INT-L and CA-INT-R primers specific to the site of the transposable group I intron of the 25S rRNA gene (rDNA) region were used. As a result, it was considered that, there were several other virulence factors belonging to the microorganism which played a role in the development mechanisms of the infection caused by C. albicans. In addition, according to the results of microbial genotyping, it was determined that there were no C. albicans strains specifically responsible for the development of DRS.     

Ocimum sanctum essential oil inhibits virulence attributes in Candida albicans

Candida albicans is an opportunistic human fungal pathogen which causes disease mainly in immunocompromised patients. Activity of hydrolytic enzymes is essential for virulence of C. albicans and so is the capacity of these cells to undergo transition from yeast to mycelial form of growth. Ocimum sanctum is cultivated worldwide for its essential oil which exhibits medicinal properties. This work evaluates the anti-virulence activity of O. sanctum essential oil (OSEO) on 22 strains of C. albicans (including a standard strain ATCC 90028) isolated from both HIV positive and HIV negative patients. Candida isolates were exposed to sub-MICs of OSEO. In vitro secretion of proteinases and phospholipases was evaluated by plate assay containing BSA and egg yolk respectively. Morphological transition from yeast to filamentous form was monitored microscopically in LSM. For genetic analysis, respective genes associated with morphological transition (HWP1), proteinase (SAP1) and phospholipase (PLB2) were also investigated by Real Time PCR (qRT-PCR). Results were analyzed using Student's t-test. OSEO inhibits morphological transition in C. albicans and had a significant inhibitory effect on extracellular secretion of proteinases and phospholipases. Expression profile of respective selected genes associated with C. albicans virulence by qRT-PCR showed a reduced expression of HWP1, SAP1 and PLB2 genes in cells treated with sub-inhibitory concentrations of OSEO. This work suggests that OSEO inhibits morphological transition in C. albicans and decreases the secretion of hydrolytic enzymes involved in the early stage of infection as well as down regulates the associated genes. Further studies will assess the clinical application of OSEO and its constituents in the treatment of fungal infections.     

A Rat Model of Neonatal Candidiasis Demonstrates the Importance of Lipases as Virulence Factors for <Emphasis Type="Italic">Candida albicans</Emphasis> and <Emphasis Type="Italic">Candida parapsilosis</Emphasis>

The host factors that contribute to the increased susceptibility of preterm neonates to invasive candidiasis have not been fully identified. In addition, there has been a lack of suitable models to study this problem. We show that rat pups, similar to premature neonates, display increased susceptibility to experimental Candida albicans infection. Further, we find that both C. albicans and Candida parapsilosis lipase disruptant mutants exhibit decreased virulence in rat pups, demonstrating the utility of the model to evaluate the impact of specific genes in disease pathogenesis. Our findings highlight the contribution of lipases to the virulence of C. albicans and C. parapsilosis and provide a new system to study the increased susceptibility of neonates to Candida infections.     

Molecular Identification of <Emphasis Type="Italic">Candida orthopsilosis</Emphasis> Isolated from Blood Culture

The incidence of candidemia and invasive candidiasis have increased markedly due to the increasing number of immunocompromised patients. There are five major medically important species of Candida with their frequency of isolation in the diminishing order namely Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata and Candida krusei. In addition, there are numerous other species of Candida which differ in their genetic makeup, virulence properties, drug susceptibilities and sugar assimilation capabilities. In this report, an unusual Candida species was isolated from the blood of two leukaemic patients. Conventional culture and biochemical tests identified the Candida species as C. parapsilosis. Using fungal-specific oligonucleotide primers ITS1 and ITS4, we managed to amplify the ribosomal RNA gene and its internal transcribed spacer region from the genomic DNA of these isolates. The PCR products were then purified and subjected to automated DNA sequencing using BLAST and CLUSTAL sequence analysis identified these isolates to be Candida orthopsilosis. Candida orthopsilosis is a new species recently identified in 2005, being morphologically indistinguishable from C. parapsilosis and was previously classified as a subspecies of C. parapsilosis. This report highlights the importance of complementing traditional culture and biochemical-based identification methods with DNA-based molecular assays such as PCR as the latter is more superior in terms of its discriminatory power and speed.     

CYP52 (cytochrome P450alk) multigene family in Candida maltosa: molecular cloning and nucleotide sequence of the two tandemly arranged genes

Southern blot analysis under low-stringency conditions using a previously isolated n-alkane-inducible cytochrome P450 (P450alk) gene as a probe revealed the presence of multiple P450alk-related genes in the genome of Candida maltosa. Nine P450alk-related genes (one reported previously and eight in the present report) were isolated from a genomic library constructed from this strain, and these were classified on the basis of sequence similarities into three pairs of putative allelic genes and three nonallelic genes. Two pairs of these alleles were tandemly arranged in the genome. The complete nucleotide sequences of one of these pairs were determined and compared to other members of this P450 family (CYP52) in C. maltosa and C. tropicalis. Northern blot analysis further showed that these genes were regulated by carbon sources. These results provide evidence for a P450alk (CYP52) multigene family in C. maltosa.     

A method for taxonomic determination ofCandida albicans with DNA probes

Determination ofCandida species represents an important problem derived from the clinical implications of the species belonging to this genus. DNA probes have already been used for the epidemiology ofCandida albicans, as well as for taxonomic analysis ofCandida and other genera, although these probes are based on non-species-specific DNA sequences. In this work we carried out a 48-h assay, allowing the identification ofC. albicans from clinical isolates, using DNA probes based onC. albicans LEU2 andURA3 genes. Another probe related toC. albicans SEC18 gene was shown not to beC. albicans specific.     

Usefulness of the Non-conventional <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> Model to Assess <Emphasis Type="Italic">Candida</Emphasis> Virulence

Invasive candidiasis is caused mainly by Candida albicans, but other Candida species have increasing etiologies. These species show different virulence and susceptibility levels to antifungal drugs. The aims of this study were to evaluate the usefulness of the non-conventional model Caenorhabditis elegans to assess the in vivo virulence of seven different Candida species and to compare the virulence in vivo with the in vitro production of proteinases and phospholipases, hemolytic activity and biofilm development capacity. One culture collection strain of each of seven Candida species (C. albicans, Candida dubliniensis, Candida glabrata, Candida krusei, Candida metapsilosis, Candida orthopsilosis and Candida parapsilosis) was studied. A double mutant C. elegans AU37 strain (glp-4;sek-1) was infected with Candida by ingestion, and the analysis of nematode survival was performed in liquid medium every 24 h until 120 h. Candida establishes a persistent lethal infection in the C. elegans intestinal tract. C. albicans and C. krusei were the most pathogenic species, whereas C. dubliniensis infection showed the lowest mortality. C. albicans was the only species with phospholipase activity, was the greatest producer of aspartyl proteinase and had a higher hemolytic activity. C. albicans and C. krusei caused higher mortality than the rest of the Candida species studied in the C. elegans model of candidiasis.     

Impact of oxidative and osmotic stresses on Candida albicans biofilm formation

Candida albicans possesses an ability to grow under different host-driven stress conditions by developing robust protective mechanisms. In this investigation the focus was on the impact of osmotic (2M NaCl) and oxidative (5 mM H₂O₂) stress conditions during C. albicans biofilm formation. Oxidative stress enhanced extracellular DNA secretion into the biofilm matrix, increased the chitin level, and reduced virulence factors, namely phospholipase and proteinase activity, while osmotic stress mainly increased extracellular proteinase and decreased phospholipase activity. Fourier transform infrared and nuclear magnetic resonance spectroscopy analysis of mannan isolated from the C. albicans biofilm cell wall revealed a decrease in mannan content and reduced β-linked mannose moieties under stress conditions. The results demonstrate that C. albicans adapts to oxidative and osmotic stress conditions by inducing biofilm formation with a rich exopolymeric matrix, modulating virulence factors as well as the cell wall composition for its survival in different host niches.     

Modulation of Candida albicans virulence by bacterial biofilms on titanium surfaces

Whilst Candida albicans occurs in peri-implant biofilms, its role in peri-implantitis remains unclear. This study therefore examined the virulence of C. albicans in mixed-species biofilms on titanium surfaces. Biofilms of C. albicans (Ca), C. albicans with streptococci (Streptococcus sanguinis, S. mutans) (Ca-Ss-Sm) and those incorporating Porphyromonas gingivalis (Ca-Pg and Ca-Ss-Sm-Pg) were developed. Expression of C. albicans genes associated with adhesion (ALS1, ALS3, HWP1) and hydrolytic enzymes (SAP2, SAP4, SAP6, PLD1) was measured and hyphal production by C. albicans quantified. Compared with Ca biofilms, significant (p<0.05) up-regulation of ALS3, HWP1, SAP2 and SAP6, and hyphal production occurred in biofilms containing streptococci (Ca-Ss-Sm). In Ca-Pg biofilms, down-regulation of HWP1 and SAP4 expression, with reduced hyphal production occurred. Ca-Ss-Sm-Pg biofilms had increased hyphal proportions and up-regulation of ALS3, SAP2 and SAP6. In conclusion, C. albicans expressed virulence factors in biofilms that could contribute to peri-implantitis, but this was dependent on associated bacterial species.