Effect Of Mandelic Acid And Its Metabolism Made On Virulence Of C. Neoformans And Rapidly Identification Of Common Dermatophyte Agents Of Tinea Capitis

BackgroundCryptococcosis is one of the most common clinical invasive fungal disease. On the global scale, It has a high morbidity and mortality. With the deeply research of science technology, cryptococcus has been used as a model fungus,and has widely used to study its pathogenesis, virulence factors, clinical susceptibility and biological and physiological characteristics et al. Through our early research "The physiological function of C. neoformans meiotic genes ISC 10" we found that the virulence of mutant strain ISC10△ of C. neoformans decrease obviously, at the meantime,the tyrosine metabolic pathway particularly in mandelic acid metabolism-related gene expression increased.At present,Mandelic acid is still limited used as anti-parasitic, so in this study, we want to build a mutant strain in mandelic acid and its metabolites to explore its effects on virulence, metabolism and other aspects.Tinea capitis is a common superficial mycoses, and predominantly involving children. In the worldwide.The etiological agent varies from one geographical region to another. In China,especially in the west of Xinjiang, Trichophyton violaceum was the most widespread etiologic agent. However, M.canis was the most common organism isolated from patients with tinea capitis in the east of China, Which is significantly different from the westen regions in China.In recent years,numerous cases of tinea capitis have been reported, but most of the coverage were restricted to a certern local center or individual case research.With the rapid development of our economy, further deeply reform of the health system and the improvement of our everyday’s life, more and more attentions are paid to topic of health. As a consequence,it is of profound significance to have a overall research on the epidemiological character,specially of the pathogens.By the way, the common methods to identify the pathogen still has many drawbacks to solve or some limitations to cross at present, such as conventional identification by standard culture-based identification methods are time-consuming and imprecise, Molecular biology techniques like PCR prefer more professional skills and high-consumption. What’s more, because of the diversity of the phenotypic and the proximity of genetic evolution of dermatophytes, PCR and sequencing analyses hardly identify to species. Furthermore, a early, rapid and accurate diagnosis is rather crucial for the treatment and prognosis of disease.In a conclusion, to explore a novel and convenient method with high sensitivity and specificity is of great practical significance.Objects1. To construct the mutant strains of mandelate racemase, peroxisomal copper amine oxidase and mandelic acid dehydrogenase, and to initially explore its effects on C.neoformans virulence phenotypes.2. To have a analysis and summary of tinea capitis reported in 2005.1-2014.12,and to explore epidemiological satistics about the clinical types and pathogen.3. To compare and analyse two molecular techniques for rapid identification of main dermatophyte agents of tinea capitis, T.violaceum and M.canis.MethodsPart 1. By analyzing the physiological characteristics of mandelate dehydrogenase, peroxisomal copper amine oxidase and mandelate racemase, designed gene-specific primers, and constructed the gene mutation box,and constructed the lmdA, pcao△, mre△ mutant strains.And further analysed their virulence phenotype factors, such as high temperature tolerance, hypertonic stress, melanin secretion, extracellular capsular et al,we hope to lay the foundation for further study of the physiological mechanisms.Part 2. Tinea capitis we searched caused by dermatophytes, with clinical symptoms, and diagnosed by direct microscopy and positive fungus culture results. Through searching cases using Wanfang and Pubmed databases with "tinea capitis" [Mesh] or [Title/Abstract], reported in 2005.1-2014.12, eliminated the cases which had obvious repetition and the information is not complete, finally, made a summary and analysis the epidemiological incidence of tinea capitis.Part 3. In this study, we applied and analysed two molecular techniques (RCA and MLPA) to rapidly detect of T.violaceum and M.canis, causative agents of tinea capitis.Specificity analysis:The strains in this study in total were 77, including 4 American Type Culture Collection (ATCC) strains,13 strains from Centraalbureau voor Schimmelcultures collection (CBS) and 60 clinial isolates, to evaluate the specificity,31 T.violaceum strains and 22 M.canis strains were involved. The probes were designed based on the gene differences in internal transcribed spacer (ITS) of rDNA. Abide by the rule of complementary base pairing, probes and primers can match specifically.Sensitivity analysis:template products of RCA, MLPA were diluted 10-fold concentration in turn, taking the same amount of product in gel electrophoresis to detect the limitation, which turned positive result to negative.Visibility analysis:SYBR Green I can combined with minor groove of DNA duplexe, and detect DNA double-stranded structure, according to connect to a double-stranded DNA during PCR amplification, the fluorescent signal will be greatly enhanced. However, when it does not connect to the double-stranded structure,there is no fluorescence.By this method to detect samples will be more intuitive and simple.Results1. To analyse the effects that mandelic acid and its metabolites made on virulence phenotypes of C.neoformance.By analyzing mandelic acid metabolic pathways and protein bioinformatics studies, found that mandelate racemase consists 438 amino acids in JEC21, and has two domains; while in H99 is composed of three domains, there are differences between the species; mandelate dehydrogenase and peroxisomal copper amine oxidase has a high degree of homology.We Successfully constructed mutant strains (mreA, pcaoA, lmdA). According to further study of the virulence phenotypes changes in C.neoformance found strain mre△ as a racemase, may be more involved in heterogeneous optical chiral mandelic acid,but no involve virulence phenotypes; mutant strain (lmdA and pcaoA) reduce their virulences in high temperature, high permeability, oxidative stress condition, and produces more melanin, and in sexual reproduction part, compared MATlmd△× MAT a JEC20 single mating with JEC21×JEC20,their hyphae and spores generated decrease slightly, but the exact mechanism will need further research.2. To analyse the epidemiological of tinea capitis in recent ten years.Through counted the relevant literatures in the past decade,there were 7828 cases reported in total, including 3519 males,4309 female patients, ratio of them was 0.82:1. Tinea capitis happened more common in children, white ringworm as the most common clinical types, up to a total of 2153 cases, made up 59.48%,followed by the black dot ringworm and kerion; the pathogen of tinea capitis was still M.canis, accounting for 55.58%, T.violacum was up to 14.81%, M.ferrugineum,T. rubrum as followed.3. To detect the common pathogen of tinea capitis, T.violaceum and M.canis,in two molecular techniques (RCA and MLPA).According to this study, RCA and MPLA in detecting the pathogen of tinea capitis, T.violaceum and M.canis, showed a good result of highly sensitivity and specificity. The specificity of these two methods is 100%, sensitivity of T.violaceum in MLPA was 10 copies,102 copies for M.canis. RCA applied to T.violaceum, sensitivity was 10 copies,108 copies for M.canis. Compared with these two methods, MLPA showed more efficient.Conclusion1. Mandelic acid and its metabolism is highly conserved among species of cryptococcus, According to construct mutant strains mre△ (mre::NEO), pcaoA (pcao::NEO)、1md△ (1md::NEO), analyse virulent phenotypes and sexual reproduction, strains mre△, pcao△ showed significantly weakened; on the contrary,strain mreA may mediate optical isomers, but not mediated phenotype changes;2. Tinea capitis developed relative slowly in recent ten years in China, mainly infected children.In clinical, the white ringworm type is the most majority, the main pathogen of it is M.canis and T.violaceum.3. The two molecular techniques, MLPA and RCA, showed highly efficient and specific on dection of M.canis and T. violaceum.4. MLPA method compared to the RCA method showed a higher sensitivity to identify M.canis and T.violaceum.