| Candida albicans is the composition of oral commensal flora and the most prevalent opportunistic human fungal pathogen, which can cause endogenous infection from superficial to seriously deep-seated mycose under the altered host conditions. G. albicans are also the most common fungal pathogens causing hospital-acquired infections. C. albicans is particularly problematic in immuno-compromised patients such as those with AIDS and cancer, or in organ transplant patients. Although candidemia rates differ between countries, recent epidemiological data from the United States, Europe, Latin America and Asia show an overall increase in the incidence of candidemia in the last decade. Candida infections are often untreatable and sometimes can result in life-threatening diseases with a high rate of mortality approaching 40-70%. Most Candida infections involve biofilm formation and often pose poor prognosis owing to drug tolerance. It is believed that biofilm formation and drug tolerance can lead to the recalcitrant and untreatable nature of some fungal infectious diseases. Biofilm-related drug tolerance may be due to high density and low growth rate within the biofilm, binding of large molecule antibiotics to the exopolymer matrix, and up-regulation of biofilm-associated genes such as MDR1, CDR1, and CDR2. Indeed, increasing evidence indicates that the mechanism of biofilm-related drug tolerance is multifactorial.In 2006, LaFleur and colleagues found that only a small population in the C. albicans biofilm could survive when challenged with lethal antifungal treatment. The sub-population of survivors is known as persisters. Persisters can reconstitute a new biofilm with a similar proportion of persisters and an equal susceptibility to antifungal treatment when they are harvested and re-incubated. Just as for their bacterial counterparts, these C. albicans persisters exhibit a non-hereditary, multi-drug tolerance. Therefore, persisters were implicated as the main determinants of high biofilm tolerance to antifungal. In 2009, LaFleur and colleagues measured the persister levels of 150 Candida albicans isolates from 46 cancer patients undergoing chemotherapy and found that strains isolated from patients with long-term carriage had high levels of persisters. Hip mutants of C. albicans were isolated from each of patients with C. albicans carriage of more than 8 consecutive weeks but from no patients with transient carriage. Similar, K. Lewis and colleagues took advantage of a set of longitudinal P. aeruginosa isolates from a single patient collected over the course of many years and testing persister levels by monitoring survival after challenge with a high dose of ofloxacin showed a dramatic,100-fold increase in surviving cells in the last four isolates. These experiments directly link persisters to the clinical manifestation of the disease and the persisters are believed to be responsible for the therapy failure and the recurring symptoms of of chronic infectious diseases. Moreover, we infected Caenorhabditis elegans with hip or lop C albicans isolates and then treated them with amphotericin B and compared the survival rate. We found that the survival rate of Caenorhabditis elegans infected with lop C albicans strains were much higher than those infected with hip strains. The results confirmed again those persisters within the C. albicans biofilms, which can withstand lethal antifungal treatment, is believed to the main culprit in the therapy failure and the recurring symptoms of fungal infections.C. albicans persister cells constitute a small sub-population of biofilm and are often described as dormant, multidrug-tolerant, non-growing and phenotypic variants of microorganisms. Evidence indicates that persisters may play a major role in recalcitrant chronic candidiasis; however, the mechanism underlying persister formation remains unclear. Although it is known that Candida albicans persisters have so far been observed only in biofilm environment, the biofilm element(s) that trigger(s) persister formation are still unknown. In 2006, LaFleur and colleagues found that some mutants that cannot form mature biofilms, such as ⊿efg1/⊿cph1, could still produce wild-type levels of persister cells. This result indicates that the maturation of biofilms is not the essential condition for Candida persister formation. However, the previous studies have been focused on the persister formation in mature biofilms (48 h) and none of these studies have been conducted to investigate persister formation in the early phases of biofilms. Thus, further studies are still necessary.Moreover, C. albicans persister cells are difficult to isolate and study not only owing to their low levels in C. albicans biofilms, but also owing to their transient, reversible phenotype. Persisters are described as dormant, non-dividing phenotypic variants. The dormant cells are characterized by quiescent metabolisms such as nucleic acid synthesis and protein synthesis. The inability of antimicrobial agents to eradicate persisters is thought to result from the quiescent physiological state because antifungal agents need a physiologically/metabolically active target to function. In 2013, Kwan and colleagues reported recently that the pre-treatment of Escherichia coli with some antibiotics that can halt transcription, translation, or ATP synthesis, dramatically increased persistence. This finding indicated that inhibition of anabolism seems to be the main mechanisms underlying persister formation. Therefore, we speculated that inhibition of nucleic acid synthesis could increase the formation of C. albicans persisters.Objective1. In this study, we tested C. albicans persister formation dynamically at different time points during the process of adhesion and biofilm formation, and tried to further elucidate the possible relationship between C. albicans persisters and the early phases of biofilm formation, especially the surface adhesion phase.2. In this study, we tried to induce C. albicans cells into a dormant state to investigate whether inhibition of nucleic acid synthesis could increase C. albicans persister formation.3. To make preliminary screening of the related genes and signaling pathway of the persister formation, we performed a detailed genome-wide transcriptional profiling of the early stages of biofilm development in C. albicans.Methods1. C. albicans persisters were first reported to occur following treatment with a high concentration of amphotericin B or chlorhexidine. Since their identification. Dose-dependent killing has been the only effective and straightforward method of isolating persisters. In the present study, we used a similar protocol with amphotericin B to measure persisters and three C. albicans strains were surveyed for the formation of persisters. The surfaces of 96-wells microtiter plates were pre-coated with C. albicans CAI-4 cells and were checked with microscopy. C. albicans biofilms were induced in 96-wells microtiter plates with or without attachment surfaces. C. albicans persister formation was tested dynamically at different time points during the process of adhesion and biofilm formation. The number of persister cells was determined based on an assessment of cell viability after amphotericin B treatment (100 μg ml-1,24 h) and colony forming unit (CFU) assay. Additionally, the persister levels were also tested in biofilms that were scraped to disrupt surface adhesion prior to amphotericin B treatment2.5-Fluorocytosine (5-FC) is a nucleoside analog with antifungal activities that inhibits nucleic acid and protein synthesis. In this study, we induced C. albicans cells into dormancy with 5- Fluorocytosine and eight C. albicans strains were tested. C. albicans cells were pre-treated with 5-Fluorocytosine (planktonic cells: 0.8 μg ml-1; biofilm cells:1 μg ml-1) for 6 h at 37℃. Nucleic acid synthesis in C. albicans were estimated during the incubation period with 5-Fluorocytosine and the results confirm that 5-Fluorocytosine pre-treatment completely arrest nucleic acid or protein synthesis of the cells. Biofilms and planktonic cultures after 5-Fluorocytosine pre-treatment were surveyed for persisters after amphotericin B treatment and colony forming unit assay.3. Biofilm cells were harvested at different time points in the early stages of biofilm formation. We performed a detailed genome-wide transcriptional analysis. Sequencing data is subjected to quality control (QC) that determines if a resequencing step is needed. If sequencing result passes QC, we will proceed with downstream analysis including gene expression, gene structure refinement, alternative splicing, novel transcript prediction and annotation and SNP detection. Results of gene expression include gene expression levels and differential expression analysis. Expression pattern analysis of differential expression genes (DEGs) was performed. And based on the results of dynamic monitoring of persister formation during the early stages of C. albicans biofilms development, we make preliminary screening of the genes those may be related with the formation of persister. Further, we perform Gene Ontology (GO) enrichment analysis and Pathway enrichment analysis.Results1. Immediately following adhesion of C. albicans cells to the surface, persister cells emerged, and the proportion of persisters reached a peak in approximately 2 h biofilm. As the biofilm matured, the proportion of persisters decreased and was only 0.01-0.02% by 24 h, while the number of persisters remained stable with no significant change. Persisters were not detected in the absence of an attachment surface which was pre-coated. Persisters were also absent in biofilms that were scraped to disrupt surface adhesion prior to amphotericin B treatment.2. Planktonic and biofilm cells of C. albicans appeared to be induced successfully into a dormancy-like state by 5-Fluorocytosine pre-treatment. None of the planktonic cultures, with and without 5-Fluorocytosine pre-treatment, contained persisters. Persister cells were found in biofilms of all tested C. albicans strains, however, the persister levels were not significantly increased in C. albicans biofilms pre-treated with 5-Fluorocytosine.3. Changes in the transcriptome begin within 30 min of contact with the substrate. Based on the formation of persister cells in the early stage of C. albicans biofilm, a totle of 228 genes which may be related with the formation of persister were identified. During the early stage of C. albicans biofilm, the expression trend of 97 genes appeared to be consistent with the formation of persisters, while the expression trend of 131 genes were inconsistent with the formation of persisters.Conclusion1. C. albicans persister cells were only found in biofilms in the presence of surface adhesion and are produced mainly in surface adhesion phase. Furthermore, our findings indicate that that surface adhesion, rather than other steps of biofilm formation and the formation of complex biofilm architecture, is required for the emergence and maintenance of C. albicans persisters. Adhesion of C. albicans to attachment surface triggers cell signal transduction systems prior to biofilm formation. As a result, a subpopulation of cells transfers to persister cells by a stochastic process. This special state protects cells from lethal interactions between antifungal and their targets.2. Inhibition of nucleic acid synthesis did not seem to increase the formation of amphotericin B-tolerant persisters in C. albicans biofilms. These results suggest that dormancy has negligible effect on C. albicans persister formation. Compared with bacterial persisters, the formation of C. albicans persisters is likely to be multifactorial and more complicated.3. Antifungal-tolerant persister cells in C. albicans biofilms are formed through a combination of stochastic and deterministic events. |
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