Factors Affecting The Hemolytic Activity Of Candida Species

BackgroundIn the past 30 years, invasive fungal diseases are an increasingly encountered threat among critically ill patients and are a significant cause of morbidity and mortality. Pathogenic fungi have become an increasing risk factor for people suffering from systemic disease and patients with impaired immune systems. This results from a number of factors, but two main reasons the extensive use of broad-spectrum antibiotics and immunsuppressive therapy are the most important.Although a wide variety of pathogens can be associated with invasive fungal diseases, Candida species have historically been the most common causative organisms. Candida is a common inhabitant of the normal human flora (e.g. skin, gastrointestinal tract, genitourinary tract) and is also found in the environment (especially on surfaces). Numerous Candida have been described, but only a small group, is pathogenic. Candida species can cause superficial mucosal infections and deadly systemic disease. In recent years, the number of Candida infections has increased dramatically and many have resulted in mortality.Therefore, a better understanding of Candida species pathogenicity will undoubtedly be beneficial for the treatment of Candida infections. Although the epidemiology of Candida species has been thoroughly studied, the virulence is not well understood. Virulence factors, such as adherence, extracellular hydrolase production, hemolysis, phenotypic switching, and filamentation may all influence the pathogenesis of Candida species. A variety of virulence factors are expressed by Candida albicans for the adaptation to specific anatomical sites. As one of the most important virulence factors of C. albicans, secreted aspartate proteinases (Saps) have been studied fully, while its hemolytic activity is not well investigated. Hemolytic activity is a potential virulence factor that helps to disseminate candidiasis and facilitate hyphal invasion. The hemolytic activity of Candida is enhanced by growth on glucose-enriched blood agar, as first described by Manns et al.. Candida albicans uses hemolysins to degrade hemoglobin and obtain elemental iron. Therefore, hemolysins are crucial virulence factors that help pathogens to survive and persist, but for Candida isolates, the features of hemolysins are poorly studied.Lachke demonstrated that C. glabrata undergoes reversible, high-frequency phenotypic switching between a white (Wh), light brown (LB), and dark brown (DB) colony phenotype discriminated on an indicator agar containing 1 mM CUSO4. Switching regulates the transcript level of the MT-II metallothionein gene(s) and a newly discovered gene for a hemolysin-like protein (HLP). Luo studied a total of 34 Candida glabrata isolates for their in vitro haemolytic activity by using a previously described plate assay system. The mRNA expression of HLP, a putative haemolysin gene, of these isolates was also evaluated using a semi-quantitative, non-competitive RT-PCR assay. Their data illustrate not only the phenotypic characteristics of haemolysin(s) and HLP expression of a battery of C. glabrata clinical isolates, but also, for the first time, evidence for a role of HLP in haemolysis.A limited number of studies have explored the influence of specific ions on the hemolysins of Candida isolates. N. Yigit and Aktas compared the use of different blood media on the hemolysins of Candida isolates and found that sheep blood Sabouraud dextrose agar is the most suitable for the study of the beta-hemolytic activity of Candida isolates, and Linares et al. observed that CaGl2 affected the hemolytic activity. When 2.5% CaCl2 was added to Sabouraud glucose agar supplemented with sheep blood, the hemolytic activity of C. dubliniensis was reduced and that of the C. albicans strains was stimulated. However, in the absence of 2.5% CaCl2, the hemolytic activities of C. albicans and C. dubliniensis were not different.Despite these previous studies, the effect of specific electrolytes on the hemolysins of Candida strains remains unclear. In particular, the relationship between common electrolytes and the hemolysins of Candida strains has not been explored. Although hemolysis may be effective at promoting the pathogenesis of Candida strains, additional studies of potential hemolytic factors are needed. This study was designed to test the influence of specific electrolytes on the hemolysins of Candida strains.The "white-opaque" transition in Candida albicans was discovered in 1987. For the next fifteen years, a significant body of knowledge accumulated that included differences between the cell types in gene expression, cellular architecture and virulence in cutaneous and systemic mouse models. However, it was not until 2002 that we began to understand the role of switching in the life history of this pathogen, the role of the mating type locus and the molecular pathways that regulated it. Since that year, a number of new observations on the regulation and biology of switching have been made that have significantly increased the perceived complexity of this fascinating phenotypic transition.Phenotypic switching has been suggested to contribute to Candida pathogenesis by providing variability within colonizing populations that allows for the organism to adapt to different challenging environments, including distinct anatomical sites in the human body. Switching might provide cells with a flexibility that results in the adaptation of the organism to the hostile conditions imposed not only by the host but also by the physician treating the infection. A switching event represents an alternative epigenetic state that occurs in a small fraction of the population and is random and reversible. For fungi, this event manifests as an altered colony morphology, which occurs at a rate that is higher than the somaticmutation rate. Phenotypic switching was originally described in C. albicans strains but is also known to exist in several other Candida species, including C. tropicalis, C. krusei and C. parapsilosis, etc.Various studies have evaluated C. parapsilosis in hospitalized patient. Although only 16% of yeast isolates were C. parapsilosis, this species accounted for the main cause of critical infectons in newly born patients. Whether phenotypic switching will effect the hemolysis of Candida species, especially for C. parapsilosis, is currently unknown. So we designed to study the influence of C. parapsilosis phenotypic switching on the hemolysins.Objective1 To assess the influence of different electrolytes on the hemolytic activity of Candida strains and the hemolysins difference of the same electrolytes on different concentrations.2 To investigate the effects of C. parapsilosis phenotypic switching on the hemolytic activity and the hemolysins difference of the same colony phenotype on different concentrations of CaCl2.3 To confirm the role of HLP on the hemolytic activity of Candida strains and screen any other hemolytic genes which may still exist.Methods1. Preparation of Candida speciesSixteen strains of C. glabrata, four C. albicans and one C. tropicalis recovered from clinical specimens of different patients at the Guangzhou Eighth People’s Hospital and preserved in the laboratory were included in the study. Additionally, a single strain each of C. albicans (ATCC 90028) (American Type Culture Collection, Manassas, VA, USA), C. glabrata (ATCC 90030), C. krusei (ATCC 6258), C. dubliniensis (ATCC MYA 646), C. parapsilosis (ATCC 22019) and C. tropicalis (ATCC 13803) were included for data comparison purposes. Sabouraud dextrose agar, Gram Stain, CHROMagar Candida plates and the API 20C Aux Identification Kit (Bio Merieux SA, Lyon, France) were used to identity all the isolates.All the clinical isolates used in this study, which were not traceable to the donors, have been used previously and details of their isolation have been described. The hemolytic activity of every strain used in this study were determined twice.2. Changes in the hemolytic activity of Candida species by common electrolytesThe control medium was prepared by adding 7 ml of fresh sheep blood (Hemostat, Dixon, CA, USA) to 100 ml of Sabouraud dextrose agar containing 3% glucose (final concentration, wt/vol; Guangzhou Detgerm Microbiology Technology Co., Guangzhou, China). The pH was 5.6±0.2. Sodium chloride or potassium chloride was added to the above medium at concentrations of 1%,2.5% or 5% (wt/vol), while calcium chloride was added at concentrations of 0.5%,1% or 2.5% (wt/vol).Isolates were cultured in Sabouraud dextrose agar at 37℃ for 24 h. Then, the cultures were collected and washed with sterile saline and a yeast suspension was prepared from an inoculum of 1×10 cells/mL by hemocytometric counts. Ten microliters was spotted on each plate with diffenent electrolytes. Plates were cultured at 37℃ in a 5% CO2 atmosphere for 48 h.The method of Luo et al. was used to assess hemolytic activity. A translucent halo around the inoculum indicated the presence of hemolytic activity. The ratio calculated by dividing the total diameter of the colony plus the translucent halo by the diameter of the colony was defined as the hemolytic index (Hi), which represented the intensity of hemolysin production. Moreover, a dark ring was observed at the periphery of the distinctive translucent halo on every plate. The ratio calculated by dividing the diameter of the peripheral dark ring, the translucent halo and the colony by the diameter of the colony was defined as the peripheral hemolytic index (Hp), which represented the intensity of the peripheral hemolysin production.3. The effect of phenotypic switching of C. parapsilosis on the hemolytic activityThe hemolytic activities were conducted respectively for different colony phenotype of C. parapsilosis. And the hemolytic activitiy of certain colony phenotype on difference concentrations of the same electrolyte were analyzed and compared.4. Detection of HLP on Candida speciesBy using PCR amplification and sequencing methods, all Candida species were analyzed to confirm the role of HLP on the hemolytic activity and to screen any other hemolytic gene which might exist.Statistical analysisAll experiments were performed in duplicate. All data were assessed for homogeneity of variance using the Levene index and expressed as the mean± standard deviation. Hemolytic activities in the presence of a specific electrolyte at different concentrations were analyzed by the repeated measures test. A one-way analysis of variance (ANOVA) was used to assess the degree of change in hemolytic activity for each electrolyte at different concentrations. If there was heterogeneity of variance, the rank-sum test was used instead of the one-way ANOVA. Bivariate correlation analysis was applied to calculate the relationship between Hi and Hp. All statistical analyses were carried out using SPSS 13.0 (SPSS Inc., Chicago, IL, USA) and p< 0.05 was considered to be statistically significant, while p< 0.01 was considered highly statistically significant.Result1 After 24 to 48 hour of culturing on Sabouraud dextrose agar medium, Candida strains formed a middle-size, cheese-like, moist colonies, and with a yeast-like odor. Gram stains of Candida strains generally showed positive, but different species had different performance under the microscope. Thick walled spores and pseudohyphae could be observed for C. albicans. The cells of C. glabrata were relatively small, located at the top of the spores and gathered into a group. They had no capsule and hyphae. C. tropicalis were generally distributed in groups, sometimes pseudohyphae could be seen. The colony morphology, color, gram stains of C. dubliniensis were similar to C. albicans, and they were easily confused. Soft circular blastospores and many pseudohyphae could be seen for C. parapsilosis. The spores of C. krusei were relatively large.2 Different colors can be seen for different species of Candida culturing on CHROMagar Candida plates. Green was for C. albicans or C. dubliniensis. The color of C. tropicalis colony was blue, while pink was for C. krusei. C. glabrata colony showed purple and other Candida species were white. All the strains showed hemolytic activity.3 Effect of calcium chloride on the hemolytic activity of Candida isolatesTo determine whether calcium chloride will affect the hemolytic activity of Candida isolates, we used plates supplemented with calcium chloride at 0.5% 1%, or 2.5%(wt/vol). Any decrease in the observed hemolytic activity was compared with the control plates. Comparison of the hemolytic indices among the groups treated with 0.5% CaCl2 (Hi=2.247±0.079),1% CaCl2 (Hi=2.013±0.092),2.5% CaCl2 (Hi=2.150±0.066) and the control (Hi=2.749±0.103) reached statistical significance (p< 0.001).4 Effect of sodium chloride on the hemolytic activity of Candida isolatesTo determine whether sodium chloride will affect the hemolytic activity of Candida isolates, plates including sodium chloride at 1%,2.5% or 5%(wt/vol) were used. Any decrease in the observed hemolytic activity was compared with the control plates. Comparison of the hemolytic indices among the groups treated with 1% NaCl (Hi=2.158±0.078),2.5% NaCl (Hi=1.724±0.077),5% NaCl (Hi=1.746±0.102) and the control (Hi= 2.839±0.156) reached statistical significance (p< 0.001).5 Effect of potassium chloride on the hemolytic activity of Candida isolatesTo determine whether potassium chloride will affect the hemolytic activity of Candida isolates, plates containing potassium chloride at 1%,2.5% or 5%(wt/vol) were used. Any decrease in the observed hemolytic activity observed was compared with the control plates. Comparison of the hemolytic indices among the groups treated with 1% KCl(Hi=1.867±0.077),2.5% KCl (Hi=1.642±0.079),5% KCl (Hi=1.707±0.089) and the control (Hi=2.839±0.156) reached statistical significance (p< 0.001).6 Relative change of hemolytic activity in response to different concentrations of specific electrolytesThe relative changes in hemolytic activity for all three electrolytes at different concentrations were calculated according to the method described above. Significant differences existed between different concentrations for the NaCl group (F=13.967, p<0.001), while no significant differences existed between the different concentrations for the CaCl2 group (F=2.727, p=0.074). The Levene index of the KC1 group was 0.001; therefore, the rank-sum test was used. The result of the KC1 group was χ2= 3.734, p-0.155. For multiple comparisons among different concentrations, no significant difference existed in the KC1 group.7 Relationship between the peripheral hemolytic index and the hemolytic indexAfter integrating all the data from the CaCl2 group, the NaCl group and the KC1 group, the coefficient for the correlation between the peripheral hemolytic index and the hemolytic index was 0.656 (p< 0.001).8 C. parapsilosis culturing on Sabouraud dextrose agar medium had two different colony morphology. One was smooth phenotype. It was hemispherical, smooth and shiny. Another was irregular wrinkle phenotype. It was wrinkled and irregular. Gram stains of these two different colony morphology showed that smooth phenotype had more yeast-like structure and many soft circular blastospores, while more hyphae-like structures and many pseudohyphae could be seen for the irregular wrinkle phenotype. The API 20C Aux Identification Kit (Bio Merieux SA, Lyon, France) were used to identity these two different colony morphology and results showed that both of them were C. parapsilosis.9 The hemolytic activities were conducted for the two different colony morphology of C. parapsilosis, and results showed that smooth phenotype could produce hemolysis while irregular wrinkle phenotype could not. The hemolytic activity of irregular wrinkle phenotype were all negative under any concentrations of CaCl2.10 Agarose gel electrophoresis of 27 samples revealed that:all C. glabrata can be amplified the specific band, including C. glabrata (ATCC 90030) and all C. glabrata clinical isolates. Other Candida strains can not be amplified the band, only one exception. A clinical isolate of C. albicans named 16a amplified the band. All the PCR products were sent to Invitrogen Corporation (Shanghai) for sequencing. Specialized software were used for analysis and comparisons.Conclusion1. Our findings indicate that different electrolytes can modulate the hemolytic activity of Candida species. All the electrolytes tested in this study CaCl2, NaCl, KC1 produced a decrease in the hemolysis of Candida species.2. To our knowledge, this is the first time that a correlation between the peripheral hemolytic index and the translucent hemolytic index has been identified.3. Our study has revealed that phenotype switching can influence the hemolytic activity of C. parapsilosis. The hemolytic activity of two colony phenotypes were completely different. The hemolytic activity of white phenotype was positive while irregular wrinkle phenotype showed negative. And all the hemolytic activity of irregular wrinkle phenotype were negative on different concentrations of CaCl2.4. The present study has confirmed that the role of HLP on the hemolytic activity of Candida species and implied that other hemolytic genes might exist.