The Effects And Mechanisms Of Polysaccharide Of Candida Albicans On Macrophages

Introduction: Human skin is widely colonized by a variety of fungal species.Some Candida species,especially C.albicans,do not only cause infections by growing into the colonized tissue,but also reside on the skin surface as commensals.However,the resistance mechanism of skin barrier is the most effective,involving residential non-immune and immune cells as well as immune cells specifically recruited to the site of infection.As a result,the skin is an effective barrier against fungal infection.At present,most studies about commensal and pathogenic interaction of Candida species with host epithelial focus on the interaction with mucosal surfaces such as the vaginal and gastrointestinal epithelial,but there is less known research on the mechanisms underlying Candida interaction with the skin.In recent decades,colonization of human skin with bacterial and fungal communities has been widely studied,but the host-fungus interactions are not well understood.At present,different fungi including Malassezia,Cryptococcus,Rhodotorula,and Candida species have been identified as human skin commensals.In patients with primary immunodeficiency,the enrichment of fungal diversity,especially Candida albicans,is significantly increased in the skin.Although some fungi are symbiotic bacteria in the skin microorganism group,some species are still pathogenic.According to the statistics that 20-25% of the world population is affected by fungal skin infections.Fungal skin pathogens can be divided into two classes: dermatophytes and yeast,with Candida speciesbelonging to the latter.Among the 200 known Candida species,only a few,including C.tropicalis,C.parapsilosis,are often found on healthy skin,which can become pathogenic.C.albicans is the Candida species most common cause of symptomatic skin infections.The main symptoms of Candida skin infections include thickening of the skin,hyperkeratosis,and erythema.Candida invasion of the skin is quickly recognized by innate immune receptors,such as pattern recognition receptors(PRRs),which can open an effective immune response.Different fungal structures,such as the cell wall components,?-glucans,mannans and phospholipid mannans,can be recognized by receptors belonging to different kinds of PRRs including Toll-like receptors(TLRs)and C-type lectins.Their contribution to host defense against Candida species is,however,tissue specific and dependent on fungal morphology.Dectin-1 is a C-type lectin expressed on different cell types including monocytes,macrophages,dendritic cells,and neutrophils.It recognizes the fungal cell wall component ?-glucan and is involved in epithelial antifungal defense by Th17 induction in a Syk-and Card9-dependant manner.Langerin is the expression of epidermal cell specific Hans Lange,also has the function of identification of Candida albicans.The interaction between Candida albicans and the skin: In the state of symbiosis,Candida albicans in the noninvasive state are in the yeast phase on the tissue surface.Yeast cells can be identified by Dectin-1 receptor,and Dectin-1 is expressed in the epidermis of Langerhans cells.The activation of Langerhans cells leads to the response of interleukin 6 dependent Th17,and keratinocytes produce antibacterial peptides to resist superficial fungal infection.The symbiotic skin bacteria are also involved in the invasion of Candida albicans through direct and indirect mechanisms.Candida albicans invaded the epidermis can also be detected by competent neurons.It promotes the secretion of interleukin 23 from dermal dendritic cells,and then stimulates the secretion of interleukin 17 and proliferation by T cells.Penetrate the epidermis after Candida albicans by dendritic cell recognition in dermis Th1 responses induced by interleukin 12 on the dermal fibroblasts through activation of TLR2,interleukin 1 beta of antimicrobial resistance directly.Macrophages act as sentinel cells for skin resistance to external microorganisms.Several Toll-like Receptors(TLRs)recognize C.albicans cell wall polysaccharides including TLR-2,which recognizes phospholipomannans,and TLR-4,which can recognize O-linked mannans.The activation of TLRs by their ligands leads to triggering of intracellular signaling pathways,including MAPK(mitogen-activated protein kinase)and NF-?B(nuclear factor kappa-light-chain-enhancer of activated B cells)pathways,that lead to the transcription and secretion of TNF?,IL-6 and/or type I interferons.TLRs are expressed differentially on numerous cell types including keratinocytes,melanocytes,dendritic cells and macrophages and can also cooperate with C-type lectins or the inflammasome to drive IL-1?,TNF? and IL12 production.The research on the genetic basis of complex biological phenotype is an important research content of many scholars.The main genetic basis of diversity of biological traits is gene expression diversity,and is regulated by complex and fine genetic diversity.The main aspects of gene regulation are mainly manifested in the following aspects: SNP,CNV,LOH and translocation of genes.In the epigenetic aspect,the methylation,modification on histone and combination of transcripts and mi RNA.Transcriptional studies are all RNA molecules that study a single cell or a population of cells.It is generally used to refer to all RNAs,or just m RNA,depending on the particular experiment.It differs from the exome in that it includes only those RNA molecules found in a specified cell population,and usually includes the amount or concentration of each RNA molecule in addition to the molecular identities.In a sense,it means all m RNA collections.In a given organism or population of cells,the transcripts represented transcriptome.Unlike the genome,genome remained the same,except some gene mutation happens.The environmental conditions can alter the expression of transcriptome.The variation caused by m RNA may lead to the changes happening in transcriptome.The study of transcriptomics,(which includes expression profiling,splice variant analysis etc),evaluates the expression level of RNAs in some kind of cell population,often focusing on m RNA,but sometimes including others such as t RNAs,s RNAs.Transcriptomics techniques include DNA microarrays and next-generation sequencing technologies called RNA-Seq.Transcription can also be studied at the level of individual cells by single-cell transcriptomics.There are two general methods of inferring transcriptome sequences.One approach maps sequence reads onto a reference genome,either of the organism itself(whose transcriptome is being studied)or of a closely related species.The other approach,de novo transcriptome assembly,uses software to infer transcripts directly from short sequence reads.The proteome is the whole unit of proteins presented by a genome,cell,tissue,or organism at a certain time.More specifically,it is the set of expressed proteins in a given type of cell or organism,at a given time,under defined conditions.Proteomics is the study of the proteome.After genomics and transcriptomics,proteomics is the next step in the study of biological systems.It is more complicated than genomics because an organism's genome is more or less constant,whereas the proteome differs from cell to cell and from time to time.Distinct genes are expressed in different cell types,which mean that even the basic set of proteins that are produced in a cell needs to be identified.In the past this phenomenon was done by RNA analysis,but it was found not to correlate with protein content.It is now known that m RNA is not always translated into protein,and the amount of protein produced for a given amount of m RNA depends on the gene it is transcribed from and on the current physiological state of the cell.Proteomics confirms the presence of the protein and provides a direct measure of the quantity present.The integration of transcriptome and proteome is the complementation of both data set.The production of protein and the maintenance of protein level need a complex biological process which includes the transcription of genes,the modification of m RNA and protein.Proteomics gives a different level of understanding than genomics for many reasons: the level of transcription of a gene gives only a rough estimate of its level of translation into a protein.An m RNA produced in abundance may be degraded rapidly or translated inefficiently,resulting in a small amount of protein.as mentioned above many proteins experience post-translational modifications that profoundly affect their activities;for example some proteins are not active until they become phosphorylated.Methods such as phosphoproteomics and glycoproteomics are used to study post-translational modifications.Many transcripts give rise to more than one protein,through alternative splicing or alternative post-translational modifications.Many proteins form complexes with other proteins or RNA molecules,and only function in the presence of these other molecules.Protein degradation rate plays an important role in protein content.The central principle of molecular biology explains the passage of genetic information(gene->m RNA->protein).The PI3K/AKT/m TOR pathway is an intracellular signaling pathway important in regulating the cell cycle.Therefore,it is directly related to cellular death,proliferation,autophagy,and cancer.The phosphorylation of PI3 K and the activation of Akt isoforms happened in the plasma membrane.The Akt pathway is the one most frequently altered as related to cell cycles,cell apoptosis,metabolism and other processes.Moreover,as it is mostly involved with malignant transformation of tumors,along with their growth,proliferation,and metastasis,it seemed that directing our efforts at investigating the effects of mannoprotein on the Akt pathway may provide some new perspectives on the mechanisms involved in these responses.This pathway is necessary,however,to promote growth and proliferation over differentiation of adult stem cells,neural stem cells specifically.It is the difficulty in finding an appropriate amount of proliferation versus differentiation that researchers are trying to determine in order to utilize this balance in the development of various therapies.Additionally,this pathway has been found to be a necessary component in neural long term potentiation.Based on the above researches,this experiment brings forward several questions:How to acquire the purified polysaccharide of Candida albicans? How to analyze the structure of these molecules? Which effects do the polysaccharides on mouse macrophages? To solve the above questions,polysaccharides of Candida albicans were analysed and evaluated by High performance gel filtration chromatography,using high-output sequencing transcriptome and proteome to focus on several differentiated genes and proteins,and summarize the signaling pathways,evaluating the PI3K-AKT and MAPK-ERK signaling pathway.?-(1,6)(1,2)-mannoprotein of Candida albicans cell wall had immune-enhancing effect by activating Akt pathway.Understanding the biological characteristics of polysaccharide on macrophages will provide a broad vision in prevention and treatment of fungal infectious diseases.Material and Methods:1.Strains and Cell lineCandida albicans strain(ATCC11006,Manassas,VA,USA)was cultured in sabouraud's liquid medium at 25 degree and collected in centrifuge tube after rotation of3000 rpm 5 minutes.Mouse macrophage line Ana-1 cells were purchased from Beina Culture Collection(BNCC338182)and maintained in a cell incubator at 37°C,5% CO2,in RRMI 1640 medium supplemented with 10% fetal bovine serum and penicillin(100units/m L)and streptomycin(100 mg/m L).After incubation,they were seeded at the density of 1*10 5/m L each well.The cultured cells were used for experiment when they adhered to the culture plat and the confluence reached 70%~80%.2.The extraction,purification and identification of plysaccaride of Candida albicansWe used thin alkali method to extract mannan,mannoprotein and b-glucan of C.albicans.Briefly,the fresh C.albicans cells were freeze-dried and extracted with 3%Na OH at 100 C for 3 h.The homogeneity and molecular weight were determined using high performance gel permeation chromatography(HPGPC).The results showed that the MW of CABI-A,CABI-B1 and CABI-B2 were 33.9 k Da,145 k Da and 10.2 k Da,respectively.CABI-A,CABI-B1,CABI-B2,CABII-A were each hydrolyzed with 2mol/L TFA and the monosaccharide composition was analyzed by high performance liquid chromatography(HPLC)on a C18 reverse-phase column after derivatization with PMP(shown in supplementary figure).For NMR analysis,25 mg of each fraction(CABI-A,CABI-B1,CABI-B2)was dissolved in 0.5 m L D2 O and CABII-A was dissolved in d6-Me2 SO.The preliminary structural characterization was performed by using sugar composition analysis and NMR method.The results were as follows.The major fraction,CABI-A was mainly an a-(1,6)(1,2)-mannan.CABI-B1 was an a-(1,6)(1,2)-mannan with minor protein content.CABI-B2 was an a-(1,6)(1,2)-mannan with a higher protein content,which was used in this study as mannoprotein.The protein in CABI-B1 and CABI-B2 was due to the incomplete b-elimination during alkaline extraction.CABII-A was a b-(1,3)-glucan in soluble water.3.Differential genes expression with transcriptome methodIllumina Nextseq was used to obtain the whole transcriptional data,and it was found that the genes expressed differently from the date in Genebank were found.The analysis of the gene abundance of the KEGG pathway can be divided into key signaling pathways.4.Differential proteins expression with proteome methodi TRAQ reagent was used to label the peptide segments,and then separated by ion exchange column.And then the differential proteins were identified by MALDI-TOF/TOF mass spectrometry.Finally,Uniprot and Cytoskpe software were used to analyze the relationship between different proteins in biological networks.5.The integration of transcriptome and proteome data setBased on the central principle of molecular biology,the integration of two data sets can facilitate the dynamic expression of genes.The level of m RNA and protein expression is detected under certain circumstance.In correlation analysis,certain protein has expression in transcriptional level.6.The screening of key genes and proteins in PI3K-AKT-mTOR and MAPK-ERK signaling pathwaysUsing realtime PCR to find out the differentiation direction of macrophages,using western blotting to test key proteins in several signaling pathways,using flow cytometry to test the cell cycle and apoptosis,and adding into inhibitors of pathways to see the changes in phenotype.7.Statistical analysisThe statistical analysis was carried out by GraphPad Prism 7.0.The quantitative results were expressed as mean ± SD.For more than two groups of experiments,the differences between each group were analysed by one-way analysis of variance(ANOVA).The unpaired t-test or Mann-Whitney tests were performed for comparisons of group means.When p < 0.05,it will be considered statistically significant.Results:1.The extraction,purification and identification of plysaccaride of Candida albicansThe precipitant of Candida albicans was fractionated and purification by ultrafiltration,anion-exchange chromatography and gel filtration chromatiography.Five homogeneous polysaccharides(CABI,CABI-A,CABI-B1,CAB-B2 and CABII-A)were obtained and identified by HPLC.Analyzed by High performance gel filtration chromatography,we found out that CABI-A was?-(1,6)(1,2)-mannoprotein with a small amount of ?-glucan.CABI-B1 was?-(1,6)(1,2)-mannoprotein.CABI-B2 was?-(1,6)(1,2)-mannoprotein with higher protein level.CABII-A was water-insoluble?-(1,3)-glucan.2.Differential genes expression with transcriptome methodAdding mannoprotein into macrophages and analyzed through transcriptome,we get66 differential expressed genes,classified them into signaling pathways,we found the genes accumulate in tuberculosis,TNF,PI3K-AKT,and Toll-like receptor signaling pathway and so on.3.Differential proteins expression with proteome methodAdding mannoprotein into macrophages and analyzed through proteome,we get 63 differential expressed proteins,51 up-expressed proteins and 12 down-expressed proteins,classified them into signaling pathways,we found the genes accumulate in immune related pathways including m Tor,PI3K-AKT and NF-k B signaling pathways and so on.4.The integration of transcriptome and proteomeAfter comparing and correlation of two data sets,we locate TNFa-IP 2 as the co-upregulated point.It can be the production of TNF and played as the mediator of inflammation reaction.5.The screening of key genes and proteins in PI3K-AKT-m TOR and MAPK-ERK signaling pathwaysMacrophages differentiate into M1 under the influence of mannoprotein.Several key signal pathways are identified by Western blot to identify Akt and ERK signaling pathways.The immuno-enhancing effect is processed through these pathways.In order to further investigate in activation of Akt signaling pathway,we applied Akt signaling pathway inhibitor LY294002 into experiments and discovered it could reverse the activation of Akt signaling pathway,pro-proliferation effect and anti-apoptosis effect which were caused by ?-(1,6)(1,2)-mannoprotein.6.Virulence Activator ToxR Regulates ROS Resistance by Mn transportation in Vibrio choleraThe EI Tor C6706 of Vibrio Cholerae can produce cholera toxin to induce watery diarrhea.During infection,Vibrio Cholerae can resist extracellular stimuli including hydrogen peroxide.In this study,the oxidative resistance mechanism of Vibrio Cholerae was investigated.Manganese was the main cation to protect cells from ROS accumulation.Tox R induced the expression of omp U to import manganese,mne A export manganese to keep the balance.The regulation of manganese by tox R,omp U and mne A could be a beneficial to develop target pharmaceuticals focused on the oxidative procedure during infections.Conclusion: 1.We firstly extracted polysaccharide from Candida albicans cell wall in alkaline method,and identify their structure and molecular component.2.We screen out immune related differential genes and proteins through transcriptome and proteome,and locate several signaling pathways.3.The co-upregulated point in the integration of transcriptome and proteome is TNFa-IP 2 which is related to cellular inflammation reaction.4.The mannoprotein of C.albicans promote the differentiation of macrophages to M1,and promote cell proliferation and inhibit cell apoptosis by activation of AKT and ERK signaling pathway.5.Virulence Activator Tox R Regulates ROS Resistance by Mn transportation in Vibrio cholera.