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The premise of molecular biology research in China's microbial strain query network is to obtain high quantity and high quality nucleic acids, namely DNA and RNA. Due to the thick capsular membrane outside the cell wall of Cryptococcus, it is very difficult to break the fungal cell wall. The method for reliably extracting Cryptococcus DNA was established in the late 1980s. The researchers have established glass-beading methods, ultrasonic pulverization, liquid nitrogen freezing and grinding techniques. Later, the development of enzymatic methods replaced physical methods, digestion of capsules and cell walls with biological enzymes, preparation of protoplasts, and then the use of detergents to dissolve cell membranes and release DNA. The currently used enzyme is Novozyme 234, which has various activities such as 1,3-a- and 1,3-b-glucanase, chitinase, protease, lichenase and xylanase, and has good wall breaking effect. In addition, Cryptococcus neoformans can produce extracellular DNase, and always maintain a high concentration of EDTA in the reaction system, which can effectively inhibit DNase and prevent DNA degradation.
Second, gene cloning
The genetic map of Cryptococcus neoformans that has been mastered is quite small and quite inaccurate. The whole genome sequence of Cryptococcus neoformans has not been completed yet, and only a few genes have been identified and reported on the chromosome.
1. There are four main pathogenic factors of toxic gene Cryptococcus neoformans: polysaccharide capsule, puerperal pigment, a-joining type and 37 °C can survive. Still and Chang were separated by classical genetic methods and gene complementation techniques, and identified a gene involved in capsular formation, CAP59, which encodes 458 amino acids and contains six introns. The CAP59 gene is localized on chromosome I of Cryptococcus neoformans. The cloning of the two clones, the essential gene, CAP64, has a full-length DNA of 1.9 kb, is expected to encode 522 amino acids, and has eight introns located on chromosome III. The gingivalin gene of Cryptococcus neoformans, CNLAC1, has also been cloned. This gene encodes 642 amino acids and contains 14 introns. The a-ligation type and the a-join type are a pair of alleles, which are related to the sexual reproduction of Cryptococcus neoformans. Genetic analysis shows that there is a correlation between a-type and strain toxicity, and Moore et al. The junction-related gene, MFa, contains a zero-opening 114 bp open reading frame (ORF).
2, structural genes
Edman et al. cloned a gene encoding cryptococcal nucleoside-monophosphate-pyrophosphorylase (OMPase), which consists of 675 nucleotides and 2 introns, using E. coli complementation technology. The son is located on a chromosome of about 1500 kb. Varma and other animal experiments confirmed that the URA5 negative mutant strain has significantly reduced toxicity, showing that the URA5 gene is associated with the pathogenic ability of Cryptococcus neoformans. Edman et al subsequently cloned the gene encoding cryptococcal ribosylamine-imidazole carboxylase---ADE2, phosphoribosamine-imidazole carboxylase in the biosynthesis of alfalfa, and in many microorganisms, purine metabolism and strain pathogenicity There is a strong correlation between them. The ADE2 gene is expected to be a breakthrough in exploring antifungal therapy. Thymidylate synthase catalyzes reductive methylation and plays a key role in DNA biosynthesis. Livi et al. cloned the gene encoding Cryptococcus neoformans thymidylate synthase---CnTS by PCR, and detected the gene sequence. The cloned CnTS gene is 1127kp long, with 3 introns and 1 951bp ORF. A protein with a molecular weight of 35,844 Da was encoded. rRNA is a highly conserved gene of biological evolution. The genes encoding the four rRNA molecules of Cryptococcus neoformans, namely 5.8s, 18s, 25s and 5s rRNA, have been cloned, and their order in the rRNA gene cluster has been determined, which is 5s. -18s-5.8s-25s. Fan et al. believe that the subunits of rRNA are in the same order in both variants, and the size of 5s rRNA is 118 bases. Perfect et al. chromosomal localization of the rRNA gene of Cryptococcus neoformans revealed that the rRNA genes of different Cryptococcus strains were located on chromosomes of different sizes, ranging from greater than 1130 kb to less than 2000 kb.
3. Regulatory genes In order to carefully analyze the functional region of the CAP59 gene, Chang et al. placed the CAP59 gene under the control of the cryptococcal GAL7 promoter (pGAL7), and only one of the several different GAL7:CAP59 fusion genes contained complete CAP. The open reading frame of the gene transforms the non-capsulated mutant under galactose induction to form a capsule. This suggests that the entire open reading frame is required for CAP59 encoding proteins. To study the regulation mechanism of Cryptococcus neoformans actin gene, Toffalettit et al. used the promoter of Cryptococcus neoformans actin gene (ACT) and the reporter gene of E. coli---b-galactosidase The gene (LACZ) was fused to construct an expression plasmid, ACTp:LACZ, to determine the function of the actin promoter by detecting the activity of b-galactosidase in vitro. As a result, the activity of the recombinant expression of b-galactosidase was different in the logarithmic growth phase and in the latent growth phase, and both showed temperature reliability, that is, culture at 37 ° C than at 30 ° C. Higher expression activity under conditions. It is suggested that the cryptococcal actin gene is regulated by temperature, and the authors speculate that other cryptococcal genes are also regulated by temperature, which explains why "37 °C can survive" is one of the pathogenic factors of cryptococcus. It has been found that the transcriptional regulation system of fungal genes is different from mammals or plants, but Zhang et al., while studying the cryptococcal toxic gene CNLAC1, found that its regulation mechanism is very similar to mammals or plants, ie The purpose of regulating gene transcription is achieved by blocking multiple DNA binding sites in the upstream region of the transcriptional origin and by using glutamate-rich enhancers such as Sp1.
Third, molecular epidemiology
Epidemiological data is very important for infectious diseases. It is an important basis for determining the source of infection and the route of transmission, and is also an important basis for the development of group prevention measures. As early as the late 1980s and early 1990s, molecular techniques were used to classify Cryptococcus neoformans. These molecular epidemiological experimental methods include: multi-site enzyme electrophoresis typing, pulsed electric field gel electrophoresis karyotype analysis, restriction fragment length polymorphism analysis (RFLPs) DNA fingerprinting, mitochondrial DNA probes and random amplified polymorphisms. DNA analysis (RFPD) and the like.
Although the banding pattern of clinical isolates and environmental isolates is relatively invisible when cultured in vitro, Cryptococcus neoformans exhibits genetic polymorphism under certain environmental stress. The genotypes of some strains are inherently inferior to other strains. This rapidly changing genetic polymorphism is manifested at both microsatellite levels and large fragment DNA levels (chromosomes). Therefore, when using very sensitive genotyping methods, it is necessary to consider the phenomenon of Cryptococcus neoformans micro-evolution under certain environmental and host pressures.
RAPD analysis is the amplification of target cell DNA by PCR reaction using a single oligonucleotide primer that is randomly synthesized. A technique in which amplification products are subjected to gel electrophoresis to analyze DNA fragment size and number polymorphisms to compare target gene differences. Since its inception in 1990, RAPD has been used for the typing, classification and epidemiological studies of yeasts and filamentous fungi. This method is applicable to the taxonomic study of various ranks of fungi. Because the fungal genome is large, RAPD can perform a general analysis of the entire genome sequence, especially for fungi with little research on molecular biology and genomic DNA sequences. The basic method it borrows is PCR technology, which is easy and fast to operate and easy to use on a large scale.
Meyer et al. used (GTG) 5, (GACA) 4 and phage M13 coding sequence (GAGGTGCGGCTCT) as a random single primer for RAPD to study 42 strains of Cryptococcus neoformans. RAPD markers (also known as PCR fingerprints) were found to be highly reproducible and differ in species, variety and individual strain levels. The fingerprints of the A and D serotypes of the new Cryptococcus neoformans are completely different, while the fingerprints of the B and C serotypes of the Ghent variant are not easy to distinguish. The two serotypes of the Ghent variant are genetically more homologous than the two serotypes of the nascent variety. Using warm-sea (GTG)5, (GACA)4 and (GATA)4 as primers, PCR amplified 30 clinical isolates and wild strains of Cryptococcus neoformans from Milan, Italy, and 12 clinical isolates from Shanghai, China. . The results clearly distinguish between different species of Cryptococcus, different serotypes, and different strains of the same serotype, similar to Meyer's report. Gu Julin et al. (GACA)4 was used as a random single primer for RAPD, and 11 strains of Cryptococcus neoformans and 14 strains of Cryptococcus neoformans were analyzed. The results showed that the PCR fingerprints generated by (GACA) 4 primers were in the species of Cryptococcus neoformans. There are distinct characteristic differences in serotype and strain levels. The experiment also found a case of cryptococcal recurrence, the initial isolate and the relapsed isolate had the same PCR fingerprint, indicating that it was the same strain, suggesting that the recurrence of cryptococcus was caused by the re-ignition of the original strain, rather than the infection of the new strain. . The authors believe that RAPD analysis can be developed as a tool for the classification, identification and epidemiological investigation of Cryptococcus neoformans.
Although sequence determination takes a lot of time, special gene sequence analysis has been used to detect polymorphisms in Cryptococcus neoformans and to distinguish between different strains. It has been established that DNA sequence changes exist between Cryptococcus neoformans strains. For example, a single copy of the URA5 gene has extensive base pair substitutions between a single strain and two variants, up to 6% of the gene coding sequence. Substituent substitutions usually occur in the third nucleotide of the codon or in the intron, so the structure of the protein does not change. The recombinant C. neoformans topoisomerase I gene has a base sequence of 3.6% difference between the serum type A strains T and the type strains. Experiments have shown that Cryptococcus neoformans has the ability to replace genes. In a complementary investigation of the C. neoformans multi-gene family, two ubiquitin genes, UBI1 and UBI2, were found on different chromosomes. Sequence analysis revealed that the nucleotide sequences of the repeats of the two genes were greatly different (about 15%), and there were also differences in the number of coding sites and introns. The exact mechanism of such high frequency recombination of repetitive sequences has not yet been elucidated. In fact, it is not known whether there is or is there a degree of methylation that may affect the gene expression of Cryptococcus neoformans and/or alter the RFLP banding pattern. However, it has been confirmed from the study of sequence data that there are many gene transformation and/or recombination events in Cryptococcus neoformans, and thus a sensitive method for identifying strains at the level of gene sequences can be established.
As a result of the final analysis, it is wise to identify Cryptococcus neoformans with several different molecular biology techniques. Brandt and C. cryptus were used to identify 33 Cryptococcus, using multi-site enzyme electrophoresis, electrophoresis karyotype, RADP technology and CNRE-1 probe, etc., and found that there is a certain relationship between the technical methods and isolated strains. The correlation, the difference that occurs in one method can be compensated for by another method. It must be emphasized that the selection of different technical methods requires attention to be able to truly detect the balance between the method by which infection leads to micro-evolution of the strain and the lack of methods to identify differences in strains. In short, a combination of multiple analytical techniques can be used to identify Cryptococcus neoformans.
Fourth, molecular diagnosis
The early detection methods were mainly DNA probe technology. It is customary to always try to select a sequence with a higher copy number in the genome as a probe. Such a probe has higher sensitivity, so the repeat fragment is selected as a DNA probe, such as an rDNA probe. Since the birth of PCR technology, direct DNA-DNA hybridization with DNA probes has been eliminated. In theory, PCR technology can detect a single bacterial cell. It is now common practice to use a DNA probe as a target DNA in a PCR reaction, and to directly establish a PCR diagnostic method through primer design.
Polacheck et al. constructed a serum D-type Cryptococcus neoformans gene library using pBluescript SK plasmid as a vector and Escherichia coli DH5 as a vector, and obtained a 1.0 kb species-specific moderate repeat sequence and a 300 bp specific DNA fragment. . Wu Jianhua et al. constructed a DNA clone library of serum type A standard strains using pUC18 as a vector and Escherichia coli JM103 as host, and selected three specific fragments: species specific, variety specific and type A specific fragments. Among them, the specific probe is a multi-copy sequence and is about 500 bp long, which is suitable for use in a PCR reaction template. Gu Julin et al. established a nested PCR assay for detection of Cryptococcus: a double-primer system for nested PCR amplification. The first step was to use the outer primers to diagnose common pathogenic fungi, and to amplify 25 medical fungi representing 17 genera and 17 species. The amplification of all bacterial and human DNA was negative; in the second step, the inner primer was only amplified for Cryptococcus neoformans, and 11 strains of Cryptococcus neoformans were amplified to produce a specific fragment of 136 bp in length, 21 non-newborns. Cryptococci were negatively amplified. The authors used the above method to retrospectively study the 37 CSF specimens deposited. The results showed that if the smear method or / and fungal culture positive was used as a "zero gold standard" for discriminating the presence or absence of Cryptococcus neoformans, PCR detection The sensitivity is 100%, while the sensitivity of fungal culture is only 74%. The specificity of PCR detection is 93%, which has a good coincidence with the traditional method.
V. Molecular phylogeny
As genomic research continues to mature, the use of DNA sequence information will more strongly illustrate the germline relationship between fungi. Highly conserved rRNA and rDNA are the focus of molecular phylogenetic studies, and these gene sequences of Cryptococcus neoformans have been studied. In Cryptococcus neoformans, 16s, 5.8s, and 23s genes were arranged in order in the 8 kb repeat DNA fragment, and transcribed in the same direction. Fan measured the 16s and 23s rRNA gene sequences of Cryptococcus neoformans and used these sequences to establish a phylogenetic tree diagram between Cryptococcus neoformans and other fungi. Gueho constructed a genetic tree of evolutionary relationship between Cryptococcus neoformans and other species of Cryptococcus in the genus by analyzing partial sequences of the most variable region of the rRNA large subunit. In this tree-generating dendrogram, it can be found that there is a slight evolutionary difference between the serotype A of the genus A. faecalis and the other three serotypes, and it is inferred that it may develop at different evolution rates.
Current research focuses on the study of other genes and the analysis of their ancestral origins. Cox et al. investigated the germline relationship of Cryptococcus neoformans by analyzing highly conserved actin genes. This gene sequence can be used to classify Cryptococcus neoformans into a separate branch of fungal evolution, but more needs to be determined. Other fungal actin gene sequences were used for in-depth subgroup comparisons and were extensively corrected using the rRNA database data.
Another interesting use of molecular evolution studies in Cryptococcus is to determine differences in specific genetic virulence factors between related species. Cryptococcus neoformans is the asexual generation of the new linear sputum bacterium, and is the only animal pathogenic strain in the genus Phytophthora, and the main human pathogen in basidiomycetes. As the existing research work, it was found that when the probe and other heterologous basidiomycetes were hybridized, the Cryptococcus neoformans capsular gene (CAP59) and the laccase gene (CnLAC1) could not be detected. Among the species capable of producing capsules, For example, Tremella fuciformis, Cryptococcus marinus, and no genes homologous to CAP59 were found. Similarly, the CnLAC1 gene of the calcitonin was used as a probe to hybridize with several other heterologous basidiomycetes, and homologous genes could not be found. These results suggest that the original genes encoding the capsule and laccase were either formed early in evolution or were a distinct branch of a group of related fungi that were adapted to different ecological stresses. In order to further understand the relationship between other heterologous basidiomycetes and pathogenic Cryptococcus neoformans, the evolution of virulence factors can be further studied.
Boekhout et al. used a series of molecular biology typing methods to study the environmental, animal and clinical isolates of two variants of Cryptococcus neoformans. As a result, there are significant differences between the two varieties in terms of chromosome number and size, RAPD, etc. These differences make Boekhout It is suspected that the two variants actually represent different species. At present, there are many arguments that the new and variant varieties have independent characteristics, and their genotypes do have significant differences. In-depth molecular biology studies also prove that there is a genetic cross between the two varieties.
Progress in molecular biology of cryptococcosis
First, the improvement of nucleic acid extraction technology