CRK1Gene Deletion Influence Interaction Between Candida Albicans And Intestinal Mucosa

Candida albicans is the most frequent human opportunistic fungus. In2010，according to a survey in the United States, Candida already accounts for the fourth hospitalacquired systemic infection, its mortality rate rose to50%,among which C.albicansvirulence is the strongest and the most common in clinical which has aroused greatconcerns of scholars from home and abroad. Most scholars believe that the gastrointestinaltract is the main repository of C albicans，which is also a main source of systemic Candidainfection.Under certain circumstances, such as patients infected with HIV, use ofbroad-spectrum antibiotics, after radiation and chemotherapy, C.albicans can be sostrongly invasive that can penetrate the mucous barrier and result in life-threateningsystemic infections. Therefore, to make certain of pathogenic mechanism about C albicansinteract with intestinal mucosa，which is of great significance for us to better prevent andtreat candidiasis derived from intestinal infection.In1996，using an oligonucleotide probe-based screen，Professor Chen Jiang-ye andher colleagues had isolated CRK1gene, a new member of the Cdc2(CDC2-related proteinkinase1) kinase, They have found, in their research, the protein sequence of Crk1is themost similar to those of Saccharomyces cerevisiae Sgv1and human Pkl1/Cdk9, Underseveral conditions, such as YPD medium or Lee’s medium, deleting both copies of CRK1affect the growth of C.albicans and morphogenesis. Therefore, Based on previous studies,We further research the role of CRK1gene in forming biofilm，adhering and invadingintestinal mucosal barrier and the differences on host immune response. The results willhelp clarify the effect of CRK1genes on interaction between C.albicans and intestinalmucosa and further elucidate the pathogenesis of candidiasis.from intestinal sources.The biofilm formation is one of the most symbols of virulence. To compare the abilityof biofilm formation between ΔCRK1and Standard bacteria SC5314,The morphologyunder scanning electron microscopy (SEM) indicates that ΔCRK1,a mass of elongation ofhyphae, interwoven, closely packed and paved with the bottom of the bottle, but comparedwith SC5314, it is slightly loose and some gaps are still faintly visible, However, Standardbacteria SC5314,a large number of elongation of hyphae and yeast cells are intertwined inthe extracellular matrix， Closely packed, forming multilayer membrane structure.Meanwhile, we quantified by MTT(OD490nm) method and crystal violet (OD620nm) inorder to evaluation cells and extracellular polymeric substances of biofilm. Researches show that the difference of biofilm formation between ΔCRK1and standard bacteriaSC5314have Statistical significance which further come to the conclusion that CRK1genedeletion is against C.albicans biofilm formation.Caco-2cells is a kind of intestinal colon cancer cells. Its structure and function aresimilar to the differentiation of the small intestinal epithelial cells, such as microvilli. Tostudy the CRK1gene deletion influence C.albicans to adhere to intestinal mucosa. Weestablish a model of intestinal mucosa epithelium with Caco2cells in vitro. C.albicansΔCRK1and standard bacteria SC5314were observed with PAS staining after interaction atdifferent time(30、60、90、120min) and the numbers of adherent were counted by colonycounting method. The results show that ΔCRK1and Standard bacteria SC5314reach themaximum number of adhesion at90minutes. Compared with SC5314, ΔCRK1have asignificant reduction in adhesion with human intestinal epithelial cells at60,90minutes,which have statistical significance.To definite the effects of CRK1gene deletion on penetrating intestinal mucousmembrane, We compare the numbers of strains by colony counting method at6h and24hrespectively. At6h, Both ΔCRK1bacteria and standard bacteria SC5314are very few,There is no statistical differences between the two strains. However when at24h, ΔCRK1bacteria is much less than that of standard bacteria SC5314,the difference between Δ CRK1and standard bacteria SC5314have statistical significance.When C.albicans contact with intestinal mucosa.which can cause a series ofinflammation and lead to cytokines released by host cell. To study the CRK1gene deletionaffect the cytokine released by intestinal mucosa epithelial cells. We use ELISA methodsfrom the protein level to compare the difference. The results show that, compared withstandard bacteria SC5314,ΔCRK1induce proinflammatory cytokines such as TNF-α、IL-8、IFN-γ are relatively high, but for Anti-inflammatory cytokine IL-12,TGF-β and IL-10,thestandard bacteria SC5314is higher than ΔCRK1,and the difference between the two havestatistical significance. Proinflammatory cytokines play a key role on antimicrobialinvasion, which make the Pathogenic bacteria to be easier esistance. The results shows thatCRK1genes deletion make it lower pathogenicity toward host cell.Therefore, from the aspects of morphogenesis, adhesion, biofilm and host immuneresponse.We found that CRK1genes deletion influence biofilm formation, and also inhibitC.albicans adhesion, penetrate intestinal mucosa and Produce different cytokines. Thismay be due to CDC2protein kinase family plays a key role in eukaryotes cycle regulation, CRK1genes is related CDC2protein kinase, It may also be involved in cell cycleregulation. Therefore, The result prove that the CRK1genes deletion influence C.albicansinteraction with intestinal mucosa cells from several aspects.