Transcriptional response of Candida albicans biofilms following exposure to 2-amino-nonyl-6-methoxyl-tetralin muriate.
ABSTRACT: AIM: To identify changes in the gene expression profile of Candida albicans (C albicans) biofilms following exposed to 2-amino-nonyl-6-methoxyl-tetralin muriate(10b) and clarify the mechanism of 10b against C albicans biofilms. METHODS: Anti-biofilm activity of 10b was assessed by tetrazolium (XTT) reduction assay and the action mechanism against biofilms was investigated by cDNA microarray analysis and real-time RT-PCR assay. RESULTS: Ten differentially expressed genes were directly linked to biofilm formation and filamentous or hyphal growth (eg, NRG1, ECE1 and CSA1). Decreased gene expression was involved in glycolysis (eg, HXK2 and PFK1) and antioxidant defense (eg, SOD5), while increased gene expression was associated with enzymes that specifically hydrolyzed beta-1,3 glucan (XOG1), and with lipid, fatty acid and sterol metabolism (eg, SLD1, ERG6 and ERG2). Functional analysis indicated that addition of anti-oxidant ascorbic acid reduced inhibitory efficiency of 10b on mature biofilm. CONCLUSION: Inhibition of 10b on biofilm formation possibly depends on impairing the ability of C albicans to change its morphology via altering the expression of biofilm formation genes. Mitochondrial aerobic respiration shift and endogenous ROS augmentation might be a major contribution to reduce mature biofilm metabolic activity. The data may be useful for the development of new strategies to reduce the incidence of device-associated infections.
Project description:AIM: To investigate the action mechanism of a novel chemical structural aminotetralin derivate, 2-Amino-Nonyl-6-Methoxyl-Tetralin Muriate (10b), against Candida albicans (C albicans) in the ergosterol biosynthetic pathway. METHODS: Antifungal susceptibility test of 10b was carried out using broth microdilution method, the action mechanism of 10b against C albicans was investigated by GC-MS spectrometry and real-time RT-PCR assay, and cytotoxicity of 10b in vitro was assessed by MTS/PMS reduction assay. RESULTS: 10b reduced the ergosterol content markedly, and the 50% ergosterol content inhibitory concentration (ECIC(50) value) was 0.08 microg/mL. Although the sterol composition of 10b-grown cells was completely identical with that of erg24 strain, the content of ergosta-8,14,22-trienol in 10b-grown cells was much higher than that in erg24 strain. Real-time RT-PCR assay revealed a global upregulation of sterol metabolism genes. In addition, the 50% inhibitory concentration (IC(50) value) of 10b was 11.30 microg/mL for murine embryonic fibroblasts and 35.70 microg/mL for human normal liver cells. CONCLUSION: 10b possessed a mode of action different from that of azoles and morpholines, whose targets were sterol C-14 reductase (encoded by ERG24 gene) and sterol C-5 desaturase (encoded by ERG3) related enzyme. Although 10b seemed to reduce MTS/PMS reduction in a dose dependent manner, IC(50) value for mammalian cells was much higher than 50% minimum inhibitory concentration (MIC(50)) value for C albicans. This indicates that the formulation is preliminarily safe and warrants further study for possible human applications.
Project description:Candida parapsilosis is a pathogenic fungus that is major cause of hospital-acquired infection, predominantly due to growth as biofilms on indwelling medical devices. It is related to Candida albicans, which remains the most common cause of candidiasis disease in humans. The transcription factor Bcr1 is an important regulator of biofilm formation in vitro in both C. parapsilosis and C. albicans. We show here that C. parapsilosis Bcr1 is required for in vivo biofilm development in a rat catheter model, like C. albicans. By comparing the transcription profiles of a bcr1 deletion in both species we found that regulation of expression of the CFEM family is conserved. In C. albicans, three of the five CFEM cell wall proteins (Rbt5, Pga7 and Csa1) are associated with both biofilm formation and acquisition of iron from heme, which is an important virulence characteristic. In C. parapsilosis, the CFEM family has undergone an expansion to 7 members. Expression of three genes (CFEM2, CFEM3, and CFEM6) is dependent on Bcr1, and is induced in low iron conditions. All three are involved in the acquisition of iron from heme. However, deletion of the three CFEM genes has no effect on biofilm formation in C. parapsilosis. Our data suggest that the role of the CFEM family in iron acquisition is conserved between C. albicans and C. parapsilosis, but their role in biofilm formation is not.
Project description:Biomaterial infections are an increasingly alarming problem, and because of their intrinsic recalcitrance to conventional therapy, a new class of antifungal drugs must be explored. 10b, a 2-aminotetralin derivate, was synthesized as a novel chemical structural antifungal agent and exibited strong anti-biofilm activity. To further investigate the action mechanism, we used microarray analysis to investigate the genes expression profiles of C. albicans biofilms treated or untreated with 10b and found 150 genes were differentially expressed. Of them, 69 showed a decrease in expression and 81 showed an increase in expression -10 differentially expressed genes related to biofilm formation, Filamentous or hypha growth. A gene related to specifically hydrolyzing β-1, 3 glucan was significantly increased. 10 down-regulated genes were involved in glycolysis, fermentation and active oxygen scavenging. 15 overexpressed genes were related to the lipid metabolic process. Of them, 13 genes were directly linked to ergosterol biosynthesis including ERG2, ERG6 and ERG11. 10 genes related to translation were over-expressed. Among them, 2 genes involved in negative regulation of transcription were significantly up-regulated. Overall design: Total RNA from the control SC5314 biofilms and 10b-treated SC5314 biofilms were used to generate target cDNA, and then hybridized to 8k Candida albicans Genome Array Genechips, representing about 7925 characterized Candida albicans genes. Two independent experiments were conducted. Reference strain was control SC5314 biofilms and test strain was SC5314 biofilms treated with 10b.
Project description:Biomaterial infections are an increasingly alarming problem, and because of their intrinsic recalcitrance to conventional therapy, a new class of antifungal drugs must be explored. 10b, a 2-aminotetralin derivate, was synthesized as a novel chemical structural antifungal agent and exibited strong anti-biofilm activity. To further investigate the action mechanism, we used microarray analysis to investigate the genes expression profiles of C. albicans biofilms treated or untreated with 10b and found 150 genes were differentially expressed. Of them, 69 showed a decrease in expression and 81 showed an increase in expression -10 differentially expressed genes related to biofilm formation, Filamentous or hypha growth. A gene related to specifically hydrolyzing β-1, 3 glucan was significantly increased. 10 down-regulated genes were involved in glycolysis, fermentation and active oxygen scavenging. 15 overexpressed genes were related to the lipid metabolic process. Of them, 13 genes were directly linked to ergosterol biosynthesis including ERG2, ERG6 and ERG11. 10 genes related to translation were over-expressed. Among them, 2 genes involved in negative regulation of transcription were significantly up-regulated. Total RNA from the control SC5314 biofilms and 10b-treated SC5314 biofilms were used to generate target cDNA, and then hybridized to 8k Candida albicans Genome Array Genechips, representing about 7925 characterized Candida albicans genes. Two independent experiments were conducted. Reference strain was control SC5314 biofilms and test strain was SC5314 biofilms treated with 10b.
Project description:Azole resistance and varying degrees of cross-resistance to other members of the azole family in clinical isolates have been documented, which has necessitated additional and prolonged use of the antifungal agents available. 2-Amino-Nonyl-6-Methoxyl-Tetralin Muriate (10b), a novel chemical structural aminotetralin derivate, is synthesized as an antifungal agent and exibited strong antifungal activity. To further investigated the action mechanism, we used microarray analysis to investigate the genes expression profiles of C. albicans cells treated or untreated with 10b and found 957 genes were differentially expressed. Of them,457 showed a decrease in expression and 500 showed an increase in expression. 33 down-regulated genes were involved in glycolysis (e.g., PFK1, CDC19 and HXK2), fermentation (e.g., PDC11, ALD5 and ADH1) and respiratory electron transport chain (e.g., CBP3, COR1 and QCR8). 30 differentially expressed genes were found to relate to biofilm formation, filamentous or hyphal growth. It was noticed that striking up-regulation of SFL1 and marked down-regulation of YWP1 directly related to prevent C. albicans from changing its morphology from the yeast form to the hyphal. Two genes related to specifically hydrolyzing beta-1, 3 glucan (e.g., XOG1) and chitin (e.g., CHT1) were significantly increased. 40 overexpressed genes and 15 down-regulated genes were related to the lipid metabolic process. Of them, Eight were directly linked to ergosterol biosynthesis, including ERG2, ERG6 and ERG11. 99 genes related to translation were down-regulated following exposure to 10b, which account for 21.66% in down-regulated genes. This suggested that translation might be lower in SC5314 cells exposed to 10b than in control. Overall design: Total RNA from the control SC5314 cells and 10b-treated SC5314 cells were used to generate target cDNA, and then hybridized to 8k Candida albicans Genome Array Genechips, representing about 7925 characterized Candida albicans genes. Two independent experiments were conducted. Reference strain was control SC5314 cells and test strain was SC5314 cells treated with 10b.
Project description:Streptococcus mutans is a biofilm-forming oral pathogen commonly associated with dental caries. Clinical studies have shown that S. mutans is often detected with Candida albicans in early childhood caries. Although the C. albicans presence has been shown to enhance bacterial accumulation in biofilms, the influence of S. mutans on fungal biology in this mixed-species relationship remains largely uncharacterized. Therefore, we aimed to investigate how the presence of S. mutans influences C. albicans biofilm development and coexistence. Using a newly established haploid biofilm model of C. albicans, we found that S. mutans augmented haploid C. albicans accumulation in mixed-species biofilms. Similarly, diploid C. albicans also showed enhanced biofilm formation in the presence of S. mutans. Surprisingly, the presence of S. mutans restored the biofilm-forming ability of C. albicans bcr1? mutant and bcr1?/? mutant, which is known to be severely defective in biofilm formation when grown as single species. Moreover, C. albicans hyphal growth factor HWP1 as well as ALS1 and ALS3, which are also involved in fungal biofilm formation, were upregulated in the presence of S. mutans. Subsequently, we found that S. mutans-derived glucosyltransferase B (GtfB) itself can promote C. albicans biofilm development. Interestingly, GtfB was able to increase the expression of HWP1, ALS1, and ALS3 genes in the C. albicans diploid wild-type SC5314 and bcr1?/?, leading to enhanced fungal biofilms. Hence, the present study demonstrates that a bacterial exoenzyme (GtfB) augments the C. albicans counterpart in mixed-species biofilms through a BCR1-independent mechanism. This novel finding may explain the mutualistic role of S. mutans and C. albicans in cariogenic biofilms.
Project description:Candida albicans is a fungal species that is part of the normal human microbiota and also an opportunistic pathogen capable of causing mucosal and systemic infections. C. albicans cells proliferate in a planktonic (suspension) state, but they also form biofilms, organized and tightly packed communities of cells attached to a solid surface. Biofilms colonize many niches of the human body and persist on implanted medical devices, where they are a major source of new C. albicans infections. Here, we used an unbiased and global substrate-profiling approach to discover proteolytic activities produced specifically by C. albicans biofilms, compared to planktonic cells, with the goal of identifying potential biofilm-specific diagnostic markers and targets for therapeutic intervention. This activity-based profiling approach, coupled with proteomics, identified Sap5 (Candidapepsin-5) and Sap6 (Candidapepsin-6) as major biofilm-specific proteases secreted by C. albicans Fluorogenic peptide substrates with selectivity for Sap5 or Sap6 confirmed that their activities are highly upregulated in C. albicans biofilms; we also show that these activities are upregulated in other Candida clade pathogens. Deletion of the SAP5 and SAP6 genes in C. albicans compromised biofilm development in vitro in standard biofilm assays and in vivo in a rat central venous catheter biofilm model. This work establishes secreted proteolysis as a promising enzymatic marker and potential therapeutic target for Candida biofilm formation.Biofilm formation by the opportunistic fungal pathogen C. albicans is a major cause of life-threatening infections. This work provides a global characterization of secreted proteolytic activity produced specifically by C. albicans biofilms. We identify activity from the proteases Sap5 and Sap6 as highly upregulated during C. albicans biofilm formation and develop Sap-cleavable fluorogenic substrates that enable the detection of biofilms from C. albicans and also from additional pathogenic Candida species. Furthermore, SAP5 and SAP6 deletions confirm that both proteases are required for proper biofilm development in vitro and in vivo We propose that secreted proteolysis is a promising marker for the diagnosis and potential therapeutic targeting of Candida biofilm-associated infections.
Project description:Candida albicans causes superficial and life-threatening systemic infections. These are difficult to treat often due to drug resistance, particularly because C. albicans biofilms are inherently resistant to most antifungals. Sophorolipid (SL), a glycolipid biosurfactant, has been shown to have antimicrobial and anticancer properties. In this study, we investigated the effect of SL on C. albicans biofilm formation and preformed biofilms. SL was found to inhibit C. albicans biofilm formation as well as reduce the viability of preformed biofilms. Moreover, SL, when used along with amphotericin B (AmB) or fluconazole (FLZ), was found to act synergistically against biofilm formation and preformed biofilms. Effect of SL on C. albicans biofilm formation was further visualized by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM), which revealed absence of hyphae, typical biofilm architecture and alteration in the morphology of biofilm cells. We also found that SL downregulates the expression of hypha specific genes HWP1, ALS1, ALS3, ECE1 and SAP4, which possibly explains the inhibitory effect of SL on hyphae and biofilm formation.
Project description:In the past, biofilm-related research has focused mainly on axenic biofilms. However, in nature, biofilms are often composed of multiple species, and the resulting polymicrobial interactions influence industrially and clinically relevant outcomes such as performance and drug resistance. In this study, we show that Escherichia coli does not affect Candida albicans tolerance to amphotericin or caspofungin in an E. coli/C. albicans biofilm. In contrast, ofloxacin tolerance of E. coli is significantly increased in a polymicrobial E. coli/C. albicans biofilm compared to its tolerance in an axenic E. coli biofilm. The increased ofloxacin tolerance of E. coli is mainly biofilm specific, as ofloxacin tolerance of E. coli is less pronounced in polymicrobial E. coli/C. albicans planktonic cultures. Moreover, we found that ofloxacin tolerance of E. coli decreased significantly when E. coli/C. albicans biofilms were treated with matrix-degrading enzymes such as the ?-1,3-glucan-degrading enzyme lyticase. In line with a role for ?-1,3-glucan in mediating ofloxacin tolerance of E. coli in a biofilm, we found that ofloxacin tolerance of E. coli increased even more in E. coli/C. albicans biofilms consisting of a high-?-1,3-glucan-producing C. albicans mutant. In addition, exogenous addition of laminarin, a polysaccharide composed mainly of poly-?-1,3-glucan, to an E. coli biofilm also resulted in increased ofloxacin tolerance. All these data indicate that ?-1,3-glucan from C. albicans increases ofloxacin tolerance of E. coli in an E. coli/C. albicans biofilm.
Project description:The oral cavity is a complex environment harboring diverse microbial species that often co-exist within biofilms formed on oral surfaces. Within a biofilm, inter-species interactions can be synergistic in that the presence of one organism generates a niche for another enhancing colonization. Among these species are the opportunistic fungal pathogen Candida albicans and the bacterial species Streptococcus mutans, the etiologic agents of oral candidiasis and dental caries, respectively. Recent studies have reported enhanced prevalence of C. albicans in children with caries indicating potential clinical implications for this fungal-bacterial interaction. In this study, we aimed to specifically elucidate the role of C. albicans-derived polysaccharide biofilm matrix components in augmenting S. mutans colonization and mixed biofilm formation. Comparative evaluations of single and mixed species biofilms demonstrated significantly enhanced S. mutans retention in mixed biofilms with C. albicans. Further, S. mutans single species biofilms were enhanced upon exogenous supplementation with purified matrix material derived from C. albicans biofilms. Similarly, growth in C. albicans cell-free spent biofilm culture media enhanced S. mutans single species biofilm formation, however, the observed increase in S. mutans biofilms was significantly affected upon enzymatic digestion of polysaccharides in spent media, identifying C. albicans secreted polysaccharides as a key factor in mediating mixed biofilm formation. The enhanced S. mutans biofilms mediated by the various C. albicans effectors was also demonstrated using confocal laser scanning microscopy. Importantly, a clinically relevant mouse model of oral co-infection was adapted to demonstrate the C. albicans-mediated enhanced S. mutans colonization in a host. Analyses of harvested tissue and scanning electron microscopy demonstrated significantly higher S. mutans retention on teeth and tongues of co-infected mice compared to mice infected only with S. mutans. Collectively, the findings from this study strongly indicate that the secretion of polysacharides from C. albicans in the oral environment may impact the development of S. mutans biofilms, ultimately increasing dental caries and, therefore, Candida oral colonization should be considered as a factor in evaluating the risk of caries.