CN113444158B

CN113444158B - Important role of B-type cyclin Cbc1 and coding gene thereof in pathogenicity of cryptococcus neoformans

Info

Publication number: CN113444158B
Application number: CN202010227474.5A
Authority: CN
Inventors: 王琳淇; 田秀云; 柯维鑫
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2020-03-27
Filing date: 2020-03-27
Publication date: 2022-11-18
Anticipated expiration: 2040-03-27
Also published as: CN113444158A

Abstract

The invention relates to an important role of B-type cyclin Cbc1 and an encoded protein thereof in pathogenicity of cryptococcus neoformans. The invention provides a cryptococcus pathogenic protein which is cyclin Cbc1. Also provided is the use of cyclin Cbc1 in the preparation of a medicament for the treatment of cryptococcus-induced diseases. The reduction of the expression level of the cyclin Cbc1 can cause the reduction of the pathogenicity of pathogenic bacteria, so that the protein can be used as a drug target and applied to the research and development of new drugs for treating diseases caused by cryptococcus.

Description

Important role of B-type cyclin Cbc1 and coding gene thereof in pathogenicity of cryptococcus neoformans

Technical Field

The invention relates to the field of biotechnology and medicine, in particular to an important role of B-type cyclin Cbc1 and an encoded protein thereof in pathogenicity of cryptococcus neoformans.

Background

Cryptococcus neoformans is the most important human pathogenic fungus in Basidiomycota, is one of important fungal pathogenic fungi causing death of people with immunodeficiency, and can cause death of more than 60 million people every year. Cryptococcus neoformans usually enters the lungs of a host through the respiratory system, spreads through the blood to different visceral organs, and can eventually cross the blood-brain barrier into the brain causing meningitis. The meningitis fatality/disability rate caused by the bacterium is high, and even in countries and regions with advanced diagnosis and treatment such as Europe and America, the infection fatality rate still exceeds 20%. In addition, cryptococcus neoformans is relatively unique in infection in China, has a tendency of infecting immunocompromised people, becomes a main pathogen of infectious encephalitis in some areas, and poses a more serious challenge to public health safety in China.

At present, three main antifungal drugs are used as treatment drugs for cryptococcus neoformans, and the antifungal drugs are respectively polyene drugs such as amphotericin B and nystatin, triazole drugs such as voriconazole and itraconazole, and nucleoside drugs such as caspofungin and 5-fluorocytosine. However, the selectivity and toxicity of these different types of drugs have limited the use of these drugs. For example: triazole drugs have the advantages of broad spectrum, high efficiency, light toxic and side effects and easy blood brain barrier passing, but cryptococcus neoformans is easy to have point mutation under the pressure of azole drugs, so that drug action targets, drug transporters and the like are changed, and drug-resistant strains are generated clinically. In order to exert the maximum effect of the medicine and reduce toxic and side effects, the 2010 American society for infectious diseases proposes that after the treatment of the cryptococcus neoformans infected by the central nervous system or severe patients is carried out by combination treatment of amphotericin B deoxycholate and 5-flucytosine for at least four weeks, fluconazole is used for continuous treatment; only mild to moderate patients with lung infections can be treated with fluconazole only. Although the toxic and side effects of the drugs on human bodies are reduced by the combined drug, the drugs still have extremely high selection pressure on fungi after long-term use, so that high-toxicity and drug-resistant strains in clinic continuously emerge, and the drug treatment is challenged.

Further discovery and improvement is needed for the diagnosis and treatment of cryptococcus neoformans.

Disclosure of Invention

The present invention aims to solve at least to some extent one of the technical problems existing in the prior art. The invention provides more target points aiming at cryptococcus therapy by digging the cyclin related to cryptococcus neoformans pathogenesis, thereby being applied to the therapy of diseases caused by cryptococcus neoformans.

Cells undergo a cell cycle from the completion of one division to the end of the next. The cell cycle comprises a pre-DNA synthesis phase (G1 phase), a DNA synthesizer phase (S phase), a post-DNA synthesis phase (G2 phase) and a division phase (M phase). In this process, the cell doubles the chromosome in the S phase and divides the chromosome equally in the M phase into two daughter cells. In a complete cell cycle, the cyclin and its corresponding protein kinase act together to promote and coordinate the smooth progress of the cell cycle. The cell cycle is an important basic process for controlling the growth and developmental differentiation of different organisms including fungi, and in the case of human pathogenic fungi, the pathogenicity of the pathogenic fungi is directly related to the developmental differentiation of the pathogenic fungi, so the cell cycle is closely related to the pathogenicity of the pathogenic fungi.

At present, few researches on the influence of cryptococcus neoformans cyclin on pathogenicity are carried out, the discovered cyclin has insufficient effect on the pathogenicity of cryptococcus neoformans, and no reports of the cyclin capable of completely losing the pathogenicity of cryptococcus neoformans exist. Among a plurality of cyclin members, the cyclin related to the pathogenicity of cryptococcus neoformans is found, the important role of the cyclin in the pathogenicity of the cryptococcus neoformans is explored, and more effective drug targets can be provided for the treatment of the cryptococcus neoformans.

The invention takes the cell cycle protein of cryptococcus neoformans as a research object, and analyzes an element regulated and controlled by a known RNA binding protein Pum1 (the deletion of which can cause the obvious reduction of the pathogenicity of cryptococcus neoformans) playing an important role in each development stage of cryptococcus neoformans, thereby finding a CBC1 coding product with obvious up-regulation expression from a plurality of cell cycle proteins, deleting and complementing the coding gene thereof, finding that the pathogenicity of the deletion mutant thereof is completely lost, indicating that the CBC1 gene and the coded protein thereof are necessary for the pathogenicity of cryptococcus neoformans, and taking the protein and the coded gene thereof as new targets to develop a therapeutic drug for cryptococcosis.

Specifically, the invention provides the following technical scheme:

in a first aspect of the invention, the use of the cyclin Cbc1 for the preparation of a medicament capable of treating a cryptococcus-induced disease. The deletion or the down-regulation of the expression quantity of the cyclin Cbc1 can reduce or completely lose the pathogenicity of cryptococcus. The cyclin Cbc1 is taken as a target point, so that a medicine with an inhibitory effect on cryptococcus can be obtained, and the obtained medicine can treat diseases caused by cryptococcus. The diseases caused by cryptococcus include but are not limited to pneumonia, meningitis, organ infection and other diseases.

According to an embodiment of the present invention, the use of the cyclin Cbc1 described above for the preparation of a medicament may further comprise the following technical features:

according to an embodiment of the invention, the cyclin Cbc1 is the target of action of the drug. The drug can take cyclin Cbc1 as an action target, and the pathogenicity of cryptococcus is reduced by down-regulating the expression of cyclin Cbc1, so that the drug can be used for treating diseases caused by cryptococcus.

According to the embodiment of the present invention, the drug can reduce the protein expression amount of the cyclin Cbc1.

According to an embodiment of the invention, the cryptococcus is cryptococcus neoformans.

In a second aspect of the invention, there is provided a cryptococcus pathogenic protein which is the cyclin Cbc1.

According to an embodiment of the present invention, the cryptococcus pathogenic protein described above may further comprise the following technical features:

according to an embodiment of the invention, the cyclin Cbc1 comprises at least one of: a) Has an amino acid sequence shown as SEQ ID NO. 2; b) Compared with the amino acid sequence shown in SEQ ID NO. 2, the amino acid sequence is subjected to one or more amino acid substitutions, deletions and/or additions.

According to an embodiment of the invention, the coding sequence of the cyclin Cbc1 is selected from at least one of the following: c) Has a nucleic acid sequence shown as SEQ ID NO. 1; d) Compared with the nucleic acid sequence shown in SEQ ID NO. 1, the nucleotide sequence has homology of more than 80 percent; e) Hybridizing under stringent conditions with the nucleic acid sequence shown in c) or d).

In a third aspect of the invention, the invention provides an expression vector comprising at least one of the following nucleic acids: 1) Has a nucleic acid sequence shown as SEQ ID NO. 1; 2) Compared with the nucleic acid sequence shown in SEQ ID NO. 1, the nucleic acid sequence has homology of more than 80 percent; 3) Hybridizing under stringent conditions with the nucleic acid sequence shown in 1) or 2). Useful vectors include, but are not limited to, plasmid, cosmid, phage or viral vectors, and the like. The expression vector may further comprise an operational element operably linked to the nucleic acid. The operational elements include, but are not limited to, a promoter to initiate transcription of the CBC1 gene, a terminator to terminate transcription of the CBC1 gene, and enhancer sequences.

In a fourth aspect of the invention, the invention provides a recombinant cell having an expression vector according to the third aspect of the invention. The recombinant cells mentioned may be present as single cells or in multicellular organisms, for example yeasts, bacteria, algae or fungi, such as Agrobacterium or the like.

In a fifth aspect of the invention, there is provided a method of screening for a drug for use in the treatment of a cryptococcus-induced disease, the method comprising: the candidate drug is applied to the recombinant cell of the fourth aspect of the invention, and the drug which reduces the expression level of the cyclin Cbc1 of the recombinant cell is screened to be used as the drug for treating cryptococcus-induced diseases.

In a sixth aspect of the invention, the invention provides the use of cyclin Cbc1 as a drug target for screening drugs for the treatment of cryptococcus-induced diseases.

According to the embodiment of the invention, the application of the cyclin Cbc1 as a drug target in drug screening can further comprise the following technical characteristics:

according to an embodiment of the invention, the medicament is capable of inhibiting the growth of cryptococcus at 37 degrees celsius.

According to the embodiment of the invention, the medicament can reduce the expression amount of cryptococcus virulence factor capsular polysaccharide and/or melanin. According to the embodiment of the invention, the drug enables the expression level of cryptococcus virulence factor capsular polysaccharide to be reduced to below 10%, for example to below 8%, to below 7%, to below 6%, to below 5%, to below 4% compared with the wild type strain. According to the embodiment of the invention, the drug can reduce the expression level of cryptococcus virulence factor melanin to below 10%, such as below 9%, below 8%, below 7%, below 6%, below 5% compared with the wild strain.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 is a schematic diagram of a C-terminal Cyclin-containing domain of Cbc1 provided in accordance with an embodiment of the present invention.

FIG. 2 is a block diagram of Cryptococcus neoformans exhibiting G2 block due to deletion of the CBC1 gene provided in accordance with an embodiment of the present invention.

FIG. 3 is a graph showing the effect of CBC1 gene deletion on the growth of Cryptococcus neoformans under different temperature conditions, according to an embodiment of the present invention.

FIG. 4 is a graph showing the effect of CBC1 gene deletion on capsular polysaccharide of a virulence factor of Cryptococcus neoformans, provided in accordance with an embodiment of the present invention.

FIG. 5 is a graph showing the effect of CBC1 gene deletion on the cryptococcus neoformans virulence factor melanin provided by an embodiment of the present invention.

Fig. 6 is a graph showing the effect of the CBC1 gene deletion cryptococcus neoformans on mice according to an embodiment of the present invention, wherein fig. 6A is the result of the survival rate of the mice of different treatment groups, and fig. 6B is the result of the weight change of the mice of different treatment groups.

FIG. 7 is a graph showing the effect of CBC1 gene deletion cryptococcus neoformans on mice according to an embodiment of the present invention, wherein FIG. 7A is a mouse lung cryptococcus load result, and FIG. 7B is a mouse brain cryptococcus load result.

Detailed Description

The following describes in detail embodiments of the present invention. The following examples are illustrative only and are not to be construed as limiting the invention.

As used herein, cryptococcus is a pathogenic bacterium that can cause disease. Diseases caused by cryptococcus are generally called cryptococcosis, and are pulmonary or disseminated infectious diseases caused by cryptococcus, mainly cause pneumonia and meningitis, and also cause infections of skin, bones, internal organs and the like. As an important pathogenic bacterium in cryptococcus, cryptococcus neoformans can exist in a large amount in soil, bird manure, particularly pigeon manure and the like, and also exist in the body surface, oral cavity and manure of a human body to cause cryptococcosis of human and animals. By using the cyclin Cbc1 as a drug target, the drug which is effective in treating cryptococcosis can be obtained and applied to drug production and cryptococcosis treatment.

The invention analyzes an element regulated and controlled by RNA binding protein Pum1 which plays an important role in each development stage of cryptococcus neoformans and obviously influences the pathogenicity of the cryptococcus neoformans after deletion, and discovers that a cyclin Cbc1 (coded by a gene CNAG _ 02095) which is very obviously up-regulated and expressed by the Pum1 contains a conserved cyclin domain at the C end. Therefore, on the basis of a wild-type strain of cryptococcus neoformans, a deletion mutant of a gene CBC1 encoding cyclin Cbc1 is constructed, and a complementation strain (namely, a CBC1 gene is introduced into the deletion strain) is constructed on the basis of the deletion mutant. Then, pathogenicity and the like of these strains were investigated.

Firstly, quantitative flow cytometric fluorescence detection is carried out on cell chromosomes of wild strains, deletion mutant strains and anaplerotic strains, and the deletion mutant strains show the retardation of the G2 phase relative to wild strains; the anaplerotic strain is similar to the wild type and no obvious cell cycle change is found. Further testing of their growth capacity and production of virulence factors (capsular polysaccharide and melanin) at human body temperature, i.e., 37 ℃, resulted in the finding that the growth of the deletion mutant at 37 ℃ was extremely hindered relative to the wild type (e.g., only very minimal growth was seen when a droplet containing about 1200 cells of the deletion mutant was dropped onto the surface of the medium, whereas the wild type strain grew to a normal lawn under the same conditions), and that the production of virulence factor capsular polysaccharide and melanin was also significantly reduced (e.g., to about 4.8% and 6.0% of the wild type strain, respectively), indicating a reduction in virulence of the deletion mutant, and that the anaplerosis strain also exhibited a phenotype similar to the wild type. Finally, to determine the function of the deletion of the gene in the actual infection process, C57BL/6 mice were used as a model for nasal infection with wild type and deletion mutants at an infection dose of 1X10 ⁵ At cell, mice infected with the wild-type strain all died within 23 days post infection. While mice infected with the CBC 1-deleted mutant strain remained normal 60 days after infection and continued to gain weight. The above results indicate that cyclin Cbc1 plays a very critical role in the pathogenicity of cryptococcus neoformans.

Herein, the cyclin Cbc1 is also commonly referred to in the art as B-type cyclin Cbc1.

To this end, in one aspect of the invention, the invention provides a cryptococcus pathogenic protein which is the cyclin Cbc1. The deletion of this protein can cause the cryptococcus neoformans to completely lose pathogenicity to mice. The cryptococcus pathogenic protein provided is selected from at least one of the following: a) Has an amino acid sequence shown as SEQ ID NO. 2; b) Compared with the amino acid sequence shown in SEQ ID NO. 2, the amino acid sequence is subjected to one or more amino acid substitutions, deletions and/or additions.

According to an embodiment of the present invention, the one or several amino acid substitutions, deletions and/or additions provided are substitutions, deletions and/or additions of not more than 8 amino acids, preferably of not more than 5 amino acids, of not more than 4 amino acids, of not more than 3 amino acids, more preferably of not more than 2 amino acids, of not more than 1 amino acid.

The cyclin Cbc1 can be directly artificially synthesized, namely, the corresponding amino acid sequence can be directly obtained by means of chemical reaction; or the coding gene can be synthesized first and then expressed biologically. To obtain the cyclin Cbc1 in vitro, a tag sequence may be attached to the carboxy-terminal and/or amino-terminal end of the cyclin Cbc1 as described above. According to an embodiment of the present invention, the tag sequences used may be as shown in table 1 below:

TABLE 1 tag sequences

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

According to an embodiment of the invention, the coding sequence of the cyclin Cbc1 is selected from at least one of the following: c) Has a nucleic acid sequence shown as SEQ ID NO. 1; d) Compared with the nucleic acid sequence shown in SEQ ID NO. 1, the nucleotide sequence has more than 75 percent of homology. According to an embodiment of the present invention, the coding sequence preferably has more than 80% homology, preferably more than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology, more than 98% homology, more than 99% homology to the nucleic acid sequence as shown in SEQ ID NO. 1. The homology sequences mentioned may be due to degeneracy of the codons encoding the amino acids, or may be due to substitution, deletion and/or addition of amino acids, etc.

The deletion of the CBC1 gene can lead the growth of the human cryptococcus to be extremely hindered under the condition of human body temperature, the production of the capsular polysaccharide and melanin of virulence factors to be greatly reduced, the pathogenicity is completely lost, and the CBC1 gene can be used as a potential target spot to develop a novel treatment method and a novel medicament.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are conventional products which are commercially available, and are not indicated by manufacturers.

Example 1

Through researching the cyclin regulated by the important RNA binding protein Pum1 of cryptococcus neoformans, the cyclin Cbc1 is found to be the most obviously up-regulated by the Pum1, and the cyclin Cbc1 is presumed to be closely related to pathogenicity due to the obvious reduction of the pathogenicity of the cryptococcus neoformans after the deletion of the PUM1 and the extremely low transcription level of the CBC1 gene. And the relevance of the cyclin Cbc1 and cryptococcus pathogenicity is researched.

1. Nucleotide sequence of CBC1 gene and amino acid sequence of cyclin Cbc1

The nucleotide sequence of CBC1 (gene number CNAG _02095 in Cryptococcus neoformans var. Grubii) gene is shown in SEQ ID NO. 1.

SEQ ID NO:1 (cDNA sequence of SEQ ID NO:1, 5 '-3') is as follows:

atggctt cccgaaaccc cgtcgtcacc cgccgagctg ctactcgttc tcgaaatgac gagaacgccg ctcctcaacc gtcagtacga accaagccct caatctccca cctcggccct gcttccaagg ctgcggttgt caatgcctct gtagtcgctg gcaagaagcc tgtcgctgtc aaggctggcg ccaagaggac agctttggga ggcgtggttg ttaacggtaa ggaagatact gaagccgcga agaagccttt gaaggcgaat ggcaagactg tcaccgaggc tagacagcct cttgcgtccc gacaaaacaa tgctcagccc acacgaccga ttgccgccat ccctcaccga tccaaagcgg ctcctgccaa cgtctatgct cctgtagagg ctcctactaa gcttactgtc gacgacgata tgcagatgga gatcgatact cgacgatctc agcccgtctc cagcgctgcc ggcttcgcga ctgtcgatga ggagcttctc gatgacgaat ccgaagagga tgacgtggaa gaggaggatg aagaagattg gctgaggatg tctgaagagg agatggtgaa ggcgcaagag caactggacg tcgtgcaggc gactttcaaa gatgatgttg acatgtttga caccactatg gttgccgagt acgcggacga gattttcgag cacatggagc gacttgagga gactgttatg cctaaccctc gctacatgga cttccagact gagattgaat ggaccatgag gacaacactt attgattggc ttcttcaagt gcatcttcgt taccacatgc tccccgagac tctttggatc gctgtcaaca ttgttgaccg attcctttcc accagagtgg tctcccttgt caagcttcaa cttgttggtg tcaccgccat gttcatcgct gccaagtatg aagagatcct cgctccttct gttgaggagt ttgtctacat gactgaaaat gggtacacca aggatgagat ccttaaggga gaaaggatta tccttcaaac cctcgatttc accatttcat cctactgttc cccgtactca tgggtcaggc gaatttctaa ggccgacgat tacgacgttc aaactcgaac attgagcaaa ttcttgatgg aggtcaccct tttggaccac aggttcctca ggtgtaagcc tagcatgatc gctgccatcg ggatgtatct tgccaggaag atgttgggcg gcgactggaa tgacgctttt atctactact ccaactttac tgaatctcaa ctcatcactg gcgcatctct tttgtgcgag cgacttattg agcctgactt tgagtccgtg tacgtttaca agaagtacgc caacaagaag tttttgcgag cgtctacctt tgcgcgagat tgggctttga ccaacgccgc caactcatca tga

the encoded cyclin is Cbc1, and the amino acid sequence of the Cbc1 is shown in SEQ ID NO. 2.

SEQ ID NO 2 (479 amino acid residues composition) is as follows:

MASRNPVVTRRAATRSRNDENAAPQPSVRTKPSISHLGPASKAAVVNASVVAGKKPVAVKAGAKRTALGGVVVNGKEDTEAAKKPLKANGKTVTEARQPLASRQNNAQPTRPIAAIPHRSKAAPANVYAPVEAPTKLTVDDDMQMEIDTRRSQPVSSAAGFATVDEELLDDESEEDDVEEEDEEDWLRMSEEEMVKAQEQLDVVQATFKDDVDMFDTTMVAEYADEIFEHMERLEETVMPNPRYMDFQTEIEWTMRTTLIDWLLQVHLRYHMLPETLWIAVNIVDRFLSTRVVSLVKLQLVGVTAMFIAAKYEEILAPSVEEFVYMTENGYTKDEILKGERIILQTLDFTISSYCSPYSWVRRISKADDYDVQTRTLSKFLMEVTLLDHRFLRCKPSMIAAIGMYLARKMLGGDWNDAFIYYSNFTESQLITGASLLCERLIEPDFESVYVYKKYANKKFLRASTFARDWALTNAANSS

2. sequence analysis

The upstream and downstream sequences of the CBC1 gene are extended by 2000bp respectively to be used as reference sequences, reads of a transcriptome are positioned on the reference sequences by using Snapgene Viewer 4.1.6 software, and the analysis shows that the CBC1 gene has the total length of 1747bp (shown as SEQ ID NO:3 below) from a start codon to a stop codon and comprises 5 introns with the sizes of 88, 55, 52, 61 and 51bp respectively.

atggcttcccgagtaagtatctgcgacccactttatccatcttcacgacatatgcaaacatcacattatgaaggcccaggctcactcttatcttgcacagaaccccgtcgtcacccgccgagctgctactcgttctcgaaatgacgagaacgccgctcctcaaccgtcagtacgaaccaagccctcaatctcccacctcggccctgcttccaaggctgcggttgtcaatgcctctgtagtcgctggcaagaagcctgtcgctgtcaaggctggcgccaagaggacagctttgggaggcgtggttgttaacggtaaggaagatactgaagccgcgaagaagccttgtaagtttgtcgatcagtcaacgtacctttgtttggtattgactttccgcgctagtgaaggcgaatggcaagactgtcaccgaggctagacagcctcttgcgtcccgacaaaacaatgctcagcccacacgaccgattgccgccatccctcaccgatccaaagcggctcctgccaacgtctatgctcctgtagaggctcctactaagcttactgtcgacgacgatatgcagatggagatcgatactcgacgatctcagcccgtctccagcgctgccggcttcgcgactgtcgatgaggagcttctcgatgacgaatccgaagaggatgacgtggaagaggaggatgaagaagattggctgaggatgtctgaagaggagatggtgaaggcgcaagagcaactggacgtcgtgcaggcgactttcaaagatgatgttgacatgtttgacaccactatggttgccgagtacgcggacgagattttcgagcacatggagcgacttgaggagactgttatgcctaaccctcgctacatggacttccagactgagattgaatggtaagcgtcaaaatctctatcaatcaaacaacatccactgaattatttttaggaccatgaggacaacacttattgattggcttcttcaagtgcatcttcgttaccacatgctccccgagactctttggatcgctgtcaacattgttgaccgattcctttccaccagagtggtctcccttgtcaagcttcaacttgttggtgtcaccgccatgttcatcgctgccaagtatgaagagatcctcgctccttctgttgaggagtttgtctacatgactgaaaatgggtacaccaaggatgagatccttaagggagaaaggattatccttcaaaccctcgatttcaccatttcatcctactgttccccgtactcatgggtcaggcgaatttctaaggccgacgattacgacgttcaaactcgaacattgagcaaattcttgatggaggtcacccttttggaccacaggttcctcaggtgtaagcctagcatgatcgctgccatcgggatgtatcttgccaggaagatgttgggcggcgactgggtaagctggtttaacacagatatgttgcccttgatgcactaacctgaccgttctacctcagaatgacgcttttatctactactccaactttactgaatctcaactcatcactggcgcatctcttttgtgcgagcgacttattgagcctgactttgagtccgtgtacgtttacaagaagtacgccaacaagaagtttttgcgagcgtctacctttgcgcgagattgggctttgaccaacgccgccaactcgtaagtggcttctgttcatcttccattatgtgggctaaccttgggctgtagatcatga(SEQ ID NO:3)。

The amino acid sequences of NCBI (https:// www.ncbi.nlm.nih.gov /) and ExPASY (https:// www.expasy.org /) cyclin Cbc1 were used for analysis.

As a result, the Cyclin Cbc1 is a protein with a conserved Cyclin domain (Cyclin domain) at the C-terminal, and has a molecular weight of 54048.41Da and an isoelectric point of 5.07 as shown in FIG. 1.

3. Deletion of CBC1 Gene

The technology adopts a TRACE method to carry out the deletion of the CBC1 gene, and the specific operations are as follows:

the upstream and downstream sequences of CBC1 gene are extended by 2000bp respectively to be used as reference sequences, primers Left F, left R, right F and Right R (upstream fragment reverse primer Left R and forward primer Right F of downstream fragment carry about 20bp sequences of matched resistance fragments respectively to facilitate later overlap-PCR) are designed respectively to amplify the fragments of 1-1.5kb respectively at the upstream and downstream of CBC1 gene to be used as homologous fragments for gene deletion homologous recombination (the upstream and downstream fragments are respectively defined as Left fragment and Right fragment).

The primers used therein were as follows:

CBC1-Left-F:AGGTCTTTACCTGCGATGACCT(SEQ ID NO:4)

CBC1-Left R:CTGGCCGTCGTTTTACGGCTAACCTTGGGCTGTAGATC(SEQ ID NO:5)

CBC1-Right F:GTCATAGCTGTTTCCTGAGTGGGTCGCAGATACTTACTCG(SEQ ID NO:6)

CBC1-Right R:TAGCGGTAGGACGTTCAGGTCA(SEQ ID NO:7)。

these two fragments were subjected to overlap-PCR using NEO (neomycin) as a template. Then, deletion of CBC1 gene was performed by TRACE System using the overlap-PCR product as a template (refer to the method described in Yumeng Fan and Xiaorong Lin.2018 multiple Applications of a Transmission CRISPR-Cas9 Coupled with electric (TRACE) System in the Cryptococcus neoformans Specifications complete. Genetics.2018Apr;208 (4): 1357-1372). The resulting CBC1 deletion strain is denoted CBC1. DELTA.

The primers used are shown below:

CBC1-sgRNA-F:ACTTCTTGCCAGCGACTACAGGTTTTAGAGCTAGAAATAGCAAGTT(SEQ ID NO:8)

CBC1-sgRNA-R:CTGTAGTCGCTGGCAAGAAGTAACAGTATACCCTGCCGGTG(SEQ ID NO:9)。

4. construction of a CBC1 deletion mutant complementation Strain

And amplifying a promoter region and a coding region of the CBC1 gene by PCR, performing double enzyme digestion by NotI and PacI, and connecting to a vector pXL1 subjected to the same enzyme digestion to obtain a complementation vector. The complementation vector is linearized by NotI and then electrically transferred into CBC1 delta, and the constructed complementation strain is represented by CBC1 delta + CBC1.

Primers used to amplify the anaplerotic fragments:

CBC1-F:ATAAGAATGCGGCCGCGTCGGGAGGTATAACAAACGAGA(SEQ ID NO:10)

CBC1-R:GGCGGTTAATTAACAATAGTCGTGGCATAAGAACAGC(SEQ ID NO:11)。

5. verification of function of Cbc1 as cell cycle protein

In a complete cell cycle, cyclin and corresponding protein kinase act together to promote and coordinate the smooth progress of the cell cycle, in the process, the cell doubles the chromosome through the S phase, and the chromosome is equally divided into two daughter cells after the M phase, so that a cycle is completed. When the function of the cyclin is changed or lost, the cell cycle cannot be normally carried out, and the change of the composition of cell chromosomes can be detected in a flow cytofluorescence mode, so that the function of the cyclin in the normal cell cycle function can be analyzed or verified.

Flow cytometry detection of cryptococcus neoformans wild type, CBC1 delta and CBC1 delta + CBC1 shows that CBC1 delta presents obvious G2 block, and the result of the anaplerotic strain is similar to that of the wild type, and the function of CBC1 as cyclin is verified, as shown in fig. 2.

Example 2

Example 2 the effect of CBC1 gene deletion on the growth of Cryptococcus neoformans at different temperatures was investigated.

The wild type strains, CBC1 Δ and CBC1 Δ + CBC1, were diluted in concentration gradient and cultured at 30 ℃ and 37 ℃ respectively, and it was found that there was no difference in growth of the three at 30 ℃, while the growth of CBC1 Δ was significantly inhibited at human body temperature, as shown in fig. 3.

The experimental results show that: the deletion of the CBC1 gene severely hampers the growth of Cryptococcus neoformans at human temperature (37 ℃). The results show that: the CBC1 gene is deleted, so that the growth of cryptococcus neoformans under the in-vivo temperature condition can be inhibited.

Example 3

Example 3 the effect of deletion of the CBC1 gene on the virulence factor capsular polysaccharide of cryptococcus neoformans was investigated.

The wild type strains, CBC1. Delta. And CBC1. Delta. + CBC1 were 5% CO in DME medium ₂ After incubation under the conditions, the capsular polysaccharide layer of CBC1 Δ was observed to be significantly thinner (as shown in fig. 4) relative to wild-type and anaplerotic strains, suggesting that CBC1 gene deletion could greatly reduce the production of virulence factor capsular polysaccharide from cryptococcus neoformans.

Example 4

The melanin can help cryptococcus neoformans to resist the damage of oxidizing agents released by inflammatory cells of organisms to thalli, and is one of important virulence factors. Example 4 the effect of deletion of the CBC1 gene on the virulence factor melanin of cryptococcus neoformans was investigated.

The wild type strains, CBC1. Delta. And CBC1. Delta. Plus CBC1, were cultured separately in DME medium, and then the melanin synthesis substrate L-DOPA was added to the culture media separately. As a result, it was found that: the wild type strain can produce a large amount of melanin, so that the whole bacterial colony is black; while cbc1. Delta. Has a greatly reduced melanin-producing ability, the anaplerotic strain has a similar melanin-producing ability to the wild type (as shown in FIG. 5). Experimental results show that the deletion of the CBC1 gene greatly influences the production of the cryptococcus neoformans virulence factor melanin.

In summary of examples 2,3,4, human body temperature conditions are important barriers to be overcome for the survival of pathogenic bacteria in the host, while the production of capsular polysaccharide and melanin, which are virulence factors, is an important component of the pathogenic capacity of cryptococcus neoformans, and the three defects of Cbc1. Delta. Indicate that Cbc1 plays a very important role in the pathogenicity of cryptococcus neoformans.

Example 5 deletion of the CBC1 Gene results in complete loss of toxicity of Cryptococcus neoformans to C57BL/6 mice

The wild type strain of Cryptococcus neoformans and cbc1. DELTA. Were set at 1x10 ⁵ The cells/dose infected 6-7 weeks old C57BL/6 mice.

The experimental result shows that: mice infected with wild-type cryptococcus neoformans all died at day 23, whereas by the end of the 60 day experiment, mice infected with cbc1 Δ did not experience a weight loss or death but exhibited normal weight gain, as shown in fig. 6A and 6B.

Consistent with this result, for the lung and brain cryptococcus neoformans counts of mice 1 and 14 days after cryptococcus neoformans infection, the cryptococcus neoformans load in the lung and brain of mice infected with cbc1 Δ was significantly lower than that of the wild-type strain, as shown in fig. 7A and 7B, where ND in fig. 7B represents no detection.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

SEQUENCE LISTING

<110> institute of microbiology of Chinese academy of sciences

<120> important role of B-type cyclin Cbc1 and coding gene thereof in pathogenicity of cryptococcus neoformans

<130> PIDC3202270

<160> 11

<170> PatentIn version 3.5

<210> 1

<211> 1440

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of CBC1 gene

<400> 1

atggcttccc gaaaccccgt cgtcacccgc cgagctgcta ctcgttctcg aaatgacgag 60

aacgccgctc ctcaaccgtc agtacgaacc aagccctcaa tctcccacct cggccctgct 120

tccaaggctg cggttgtcaa tgcctctgta gtcgctggca agaagcctgt cgctgtcaag 180

gctggcgcca agaggacagc tttgggaggc gtggttgtta acggtaagga agatactgaa 240

gccgcgaaga agcctttgaa ggcgaatggc aagactgtca ccgaggctag acagcctctt 300

gcgtcccgac aaaacaatgc tcagcccaca cgaccgattg ccgccatccc tcaccgatcc 360

aaagcggctc ctgccaacgt ctatgctcct gtagaggctc ctactaagct tactgtcgac 420

gacgatatgc agatggagat cgatactcga cgatctcagc ccgtctccag cgctgccggc 480

ttcgcgactg tcgatgagga gcttctcgat gacgaatccg aagaggatga cgtggaagag 540

gaggatgaag aagattggct gaggatgtct gaagaggaga tggtgaaggc gcaagagcaa 600

ctggacgtcg tgcaggcgac tttcaaagat gatgttgaca tgtttgacac cactatggtt 660

gccgagtacg cggacgagat tttcgagcac atggagcgac ttgaggagac tgttatgcct 720

aaccctcgct acatggactt ccagactgag attgaatgga ccatgaggac aacacttatt 780

gattggcttc ttcaagtgca tcttcgttac cacatgctcc ccgagactct ttggatcgct 840

gtcaacattg ttgaccgatt cctttccacc agagtggtct cccttgtcaa gcttcaactt 900

gttggtgtca ccgccatgtt catcgctgcc aagtatgaag agatcctcgc tccttctgtt 960

gaggagtttg tctacatgac tgaaaatggg tacaccaagg atgagatcct taagggagaa 1020

aggattatcc ttcaaaccct cgatttcacc atttcatcct actgttcccc gtactcatgg 1080

gtcaggcgaa tttctaaggc cgacgattac gacgttcaaa ctcgaacatt gagcaaattc 1140

ttgatggagg tcaccctttt ggaccacagg ttcctcaggt gtaagcctag catgatcgct 1200

gccatcggga tgtatcttgc caggaagatg ttgggcggcg actggaatga cgcttttatc 1260

tactactcca actttactga atctcaactc atcactggcg catctctttt gtgcgagcga 1320

cttattgagc ctgactttga gtccgtgtac gtttacaaga agtacgccaa caagaagttt 1380

ttgcgagcgt ctacctttgc gcgagattgg gctttgacca acgccgccaa ctcatcatga 1440

<210> 2

<211> 479

<212> PRT

<213> Artificial Sequence

<220>

<223> cyclin Cbc1

<400> 2

Met Ala Ser Arg Asn Pro Val Val Thr Arg Arg Ala Ala Thr Arg Ser

1 5 10 15

Arg Asn Asp Glu Asn Ala Ala Pro Gln Pro Ser Val Arg Thr Lys Pro

20 25 30

Ser Ile Ser His Leu Gly Pro Ala Ser Lys Ala Ala Val Val Asn Ala

35 40 45

Ser Val Val Ala Gly Lys Lys Pro Val Ala Val Lys Ala Gly Ala Lys

50 55 60

Arg Thr Ala Leu Gly Gly Val Val Val Asn Gly Lys Glu Asp Thr Glu

65 70 75 80

Ala Ala Lys Lys Pro Leu Lys Ala Asn Gly Lys Thr Val Thr Glu Ala

85 90 95

Arg Gln Pro Leu Ala Ser Arg Gln Asn Asn Ala Gln Pro Thr Arg Pro

100 105 110

Ile Ala Ala Ile Pro His Arg Ser Lys Ala Ala Pro Ala Asn Val Tyr

115 120 125

Ala Pro Val Glu Ala Pro Thr Lys Leu Thr Val Asp Asp Asp Met Gln

130 135 140

Met Glu Ile Asp Thr Arg Arg Ser Gln Pro Val Ser Ser Ala Ala Gly

145 150 155 160

Phe Ala Thr Val Asp Glu Glu Leu Leu Asp Asp Glu Ser Glu Glu Asp

165 170 175

Asp Val Glu Glu Glu Asp Glu Glu Asp Trp Leu Arg Met Ser Glu Glu

180 185 190

Glu Met Val Lys Ala Gln Glu Gln Leu Asp Val Val Gln Ala Thr Phe

195 200 205

Lys Asp Asp Val Asp Met Phe Asp Thr Thr Met Val Ala Glu Tyr Ala

210 215 220

Asp Glu Ile Phe Glu His Met Glu Arg Leu Glu Glu Thr Val Met Pro

225 230 235 240

Asn Pro Arg Tyr Met Asp Phe Gln Thr Glu Ile Glu Trp Thr Met Arg

245 250 255

Thr Thr Leu Ile Asp Trp Leu Leu Gln Val His Leu Arg Tyr His Met

260 265 270

Leu Pro Glu Thr Leu Trp Ile Ala Val Asn Ile Val Asp Arg Phe Leu

275 280 285

Ser Thr Arg Val Val Ser Leu Val Lys Leu Gln Leu Val Gly Val Thr

290 295 300

Ala Met Phe Ile Ala Ala Lys Tyr Glu Glu Ile Leu Ala Pro Ser Val

305 310 315 320

Glu Glu Phe Val Tyr Met Thr Glu Asn Gly Tyr Thr Lys Asp Glu Ile

325 330 335

Leu Lys Gly Glu Arg Ile Ile Leu Gln Thr Leu Asp Phe Thr Ile Ser

340 345 350

Ser Tyr Cys Ser Pro Tyr Ser Trp Val Arg Arg Ile Ser Lys Ala Asp

355 360 365

Asp Tyr Asp Val Gln Thr Arg Thr Leu Ser Lys Phe Leu Met Glu Val

370 375 380

Thr Leu Leu Asp His Arg Phe Leu Arg Cys Lys Pro Ser Met Ile Ala

385 390 395 400

Ala Ile Gly Met Tyr Leu Ala Arg Lys Met Leu Gly Gly Asp Trp Asn

405 410 415

Asp Ala Phe Ile Tyr Tyr Ser Asn Phe Thr Glu Ser Gln Leu Ile Thr

420 425 430

Gly Ala Ser Leu Leu Cys Glu Arg Leu Ile Glu Pro Asp Phe Glu Ser

435 440 445

Val Tyr Val Tyr Lys Lys Tyr Ala Asn Lys Lys Phe Leu Arg Ala Ser

450 455 460

Thr Phe Ala Arg Asp Trp Ala Leu Thr Asn Ala Ala Asn Ser Ser

465 470 475

<210> 3

<211> 1747

<212> DNA

<213> Artificial Sequence

<220>

<223> CBC1 Gene

<400> 3

atggcttccc gagtaagtat ctgcgaccca ctttatccat cttcacgaca tatgcaaaca 60

tcacattatg aaggcccagg ctcactctta tcttgcacag aaccccgtcg tcacccgccg 120

agctgctact cgttctcgaa atgacgagaa cgccgctcct caaccgtcag tacgaaccaa 180

gccctcaatc tcccacctcg gccctgcttc caaggctgcg gttgtcaatg cctctgtagt 240

cgctggcaag aagcctgtcg ctgtcaaggc tggcgccaag aggacagctt tgggaggcgt 300

ggttgttaac ggtaaggaag atactgaagc cgcgaagaag ccttgtaagt ttgtcgatca 360

gtcaacgtac ctttgtttgg tattgacttt ccgcgctagt gaaggcgaat ggcaagactg 420

tcaccgaggc tagacagcct cttgcgtccc gacaaaacaa tgctcagccc acacgaccga 480

ttgccgccat ccctcaccga tccaaagcgg ctcctgccaa cgtctatgct cctgtagagg 540

ctcctactaa gcttactgtc gacgacgata tgcagatgga gatcgatact cgacgatctc 600

agcccgtctc cagcgctgcc ggcttcgcga ctgtcgatga ggagcttctc gatgacgaat 660

ccgaagagga tgacgtggaa gaggaggatg aagaagattg gctgaggatg tctgaagagg 720

agatggtgaa ggcgcaagag caactggacg tcgtgcaggc gactttcaaa gatgatgttg 780

acatgtttga caccactatg gttgccgagt acgcggacga gattttcgag cacatggagc 840

gacttgagga gactgttatg cctaaccctc gctacatgga cttccagact gagattgaat 900

ggtaagcgtc aaaatctcta tcaatcaaac aacatccact gaattatttt taggaccatg 960

aggacaacac ttattgattg gcttcttcaa gtgcatcttc gttaccacat gctccccgag 1020

actctttgga tcgctgtcaa cattgttgac cgattccttt ccaccagagt ggtctccctt 1080

gtcaagcttc aacttgttgg tgtcaccgcc atgttcatcg ctgccaagta tgaagagatc 1140

ctcgctcctt ctgttgagga gtttgtctac atgactgaaa atgggtacac caaggatgag 1200

atccttaagg gagaaaggat tatccttcaa accctcgatt tcaccatttc atcctactgt 1260

tccccgtact catgggtcag gcgaatttct aaggccgacg attacgacgt tcaaactcga 1320

acattgagca aattcttgat ggaggtcacc cttttggacc acaggttcct caggtgtaag 1380

cctagcatga tcgctgccat cgggatgtat cttgccagga agatgttggg cggcgactgg 1440

gtaagctggt ttaacacaga tatgttgccc ttgatgcact aacctgaccg ttctacctca 1500

gaatgacgct tttatctact actccaactt tactgaatct caactcatca ctggcgcatc 1560

tcttttgtgc gagcgactta ttgagcctga ctttgagtcc gtgtacgttt acaagaagta 1620

cgccaacaag aagtttttgc gagcgtctac ctttgcgcga gattgggctt tgaccaacgc 1680

cgccaactcg taagtggctt ctgttcatct tccattatgt gggctaacct tgggctgtag 1740

atcatga 1747

<210> 4

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 4

aggtctttac ctgcgatgac ct 22

<210> 5

<211> 38

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 5

ctggccgtcg ttttacggct aaccttgggc tgtagatc 38

<210> 6

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 6

gtcatagctg tttcctgagt gggtcgcaga tacttactcg 40

<210> 7

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 7

tagcggtagg acgttcaggt ca 22

<210> 8

<211> 46

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 8

acttcttgcc agcgactaca ggttttagag ctagaaatag caagtt 46

<210> 9

<211> 41

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 9

ctgtagtcgc tggcaagaag taacagtata ccctgccggt g 41

<210> 10

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 10

ataagaatgc ggccgcgtcg ggaggtataa caaacgaga 39

<210> 11

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 11

ggcggttaat taacaatagt cgtggcataa gaacagc 37

Claims

1. The use of cyclin Cbc1 for the preparation of a medicament for the treatment of diseases caused by cryptococcus neoformans,

the cyclin Cbc1 has an amino acid sequence shown as SEQ ID NO: 2;

the cyclin Cbc1 is an action target of the medicine;

the drug can reduce the protein expression level of the cyclin Cbc1.

2. The use according to claim 1, characterized in that the cyclin Cbc1 coding sequence is the nucleic acid sequence shown in SEQ ID No. 1.

3. A method of screening for a drug for treating a disease caused by cryptococcus neoformans, the method comprising:

administering the drug candidate to a recombinant cell, the recombinant cell being capable of being used to treat a cryptococcus-induced disease, the recombinant cell having an expression vector comprising a nucleic acid sequence as set forth in SEQ ID No. 1;

screening drugs which can reduce the expression level of the cyclin Cbc1 of the recombinant cell, and using the drugs as drugs for treating diseases caused by cryptococcus neoformans.