Risk biomarker of cryptococcus meningitis and application thereof

Abstract

The invention specifically discloses a risk biomarker for cryptococcus meningitis and application thereof, and relates to the technical field of biological medicines. The invention provides a brand new cryptococcus brain infection antivirulence factor mitochondrial citric acid transporter: by closing the mitochondrial citric acid transporter, glucose metabolism hijacking in the brain is realized, and the colonization advantage is obtained.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to risk biomarkers for cryptococcal meningitis and their applications. Background Technology

[0002] Cryptococcus neoformans is a widespread opportunistic pathogenic fungus in nature, listed first on the WHO's list of "priority fungal pathogens." With the increase in immunodeficiency, malignant tumors, and diabetes, the incidence and mortality rates of cryptococcosis are rising annually, infecting approximately 220,000 to 1 million people worldwide each year and causing 180,000 to 600,000 deaths, posing a significant threat to public health, especially in resource-limited areas. Cryptococcus neoformans can enter the human body through the respiratory tract, spreading from the lungs to other tissues and organs via the bloodstream, including the liver and kidneys. Cryptococcal pneumonia and cryptococcal meningitis (CM) are the most common. Cryptococcal meningitis has the highest mortality rate and is the focus and challenge in the treatment of cryptococcosis; even in developed countries using antifungal treatment, the mortality rate can reach 10% to 30%. Furthermore, the prognosis for cryptococcal meningitis is poor, and long-term neurological damage, such as hearing loss, visual impairment, and cognitive dysfunction, may still occur after treatment.

[0003] The treatment and long-term management of cryptococcal meningitis imposes a significant economic burden on patients, families, and the healthcare system. Treatment for cryptococcosis primarily involves the combination of fluconazole and amphotericin B, but the blood-brain barrier leads to low drug delivery efficiency and strong drug toxicity, limiting the effectiveness of intracranial infection treatment. Although liposomal amphotericin B preparations have improved local drug concentration and safety, the treatment of cryptococcal meningitis still faces serious challenges such as high recurrence rates, long treatment cycles, high drug costs, and drug resistance. Statistics from an HIV-positive population in Africa show that despite receiving 10 weeks of fluconazole treatment and ART after a diagnosis of cryptococcal antigenemia, 8% of patients still progress to cryptococcal meningitis within the following 6 months. There are currently no effective drugs to completely block the dissemination of cryptococcus to the central nervous system. Adding to the challenge, we still do not understand why cryptococcus has such a strong affinity for the brain. Therefore, the key to treating cryptococcosis should focus on elucidating the colonization strategies of Cryptococcus neoformans in the brain. By deeply exploring the adaptive regulatory mechanisms of Cryptococcus neoformans in the brain microenvironment, we can develop brain-tissue-specific therapies to improve treatment efficacy and reduce side effects.

[0004] Fungal infections involve complex interactions between pathogens and their hosts. Fungi evolve organ-adaptive regulatory mechanisms to cope with tissue-specific immune systems or nutritional microenvironments. Recent research has also offered new insights into the influence of the brain microenvironment on drug sensitivity, but the specific adaptive mechanisms of Cryptococcus in the brain microenvironment remain incompletely understood. Previous research by the applicant found that differences in the microenvironment between lung and brain tissues significantly affect the pathogenic mechanism of Cryptococcus, but the key metabolic regulatory nodes for its colonization in the brain remain unclear. Elucidating the colonization strategies of Cryptococcus in the brain microenvironment will not only fill gaps in basic scientific understanding but also provide a theoretical basis for developing specific antifungal therapies for brain infections. Summary of the Invention

[0005] (a) Technical problems to be solved Therefore, one of the main objectives of this invention is to provide risk biomarkers for cryptococcal meningitis and their applications. This invention provides a novel antiviral factor in cryptococcal brain infection: the mitochondrial citrate transporter protein. By "shutting down" the mitochondrial citrate transporter protein, glucose metabolism in the brain is hijacked, resulting in colonization advantage.

[0006] (II) Technical Solution To achieve the above objectives, the present invention provides a susceptibility / risk biomarker for cryptococcal meningitis, wherein the diagnostic biomarker comprises mitochondrial citrate transporter (SLC25A1) and / or nucleic acid encoding mitochondrial citrate transporter.

[0007] In another aspect, the present invention provides a diagnostic biomarker for cryptococcal meningitis, said diagnostic biomarker comprising mitochondrial citrate transporter and / or nucleic acid encoding mitochondrial citrate transporter.

[0008] In one embodiment, the nucleic acid is the CNAG_02288 gene.

[0009] In one embodiment, the susceptibility / risk biomarker or susceptibility / risk biomarker is derived from Cryptococcus.

[0010] In one embodiment, the cryptococcus is Cryptococcus neoformans.

[0011] In one embodiment, the Cryptococcus was isolated from the subject's cerebrospinal fluid, peritoneal catheter, and / or peritoneal fluid.

[0012] In another aspect, the present invention also provides any of the following applications of the detection reagent for detecting the levels of the above-mentioned biomarkers: (1): Use in the preparation of products for the diagnosis of cryptococcal meningitis in subjects; or (2): Use in the preparation of products for screening subjects for cryptococcal meningitis; or (3): Use in the preparation of products for assessing the risk of cryptococcal meningitis in subjects; or (4): Application in the preparation of products for prognostic assessment of cryptococcal meningitis.

[0013] In one embodiment, the detection reagent includes reagents for detecting the expression level of mitochondrial citrate transporter and / or reagents for detecting the expression level of nucleic acid encoding mitochondrial citrate transporter.

[0014] In one embodiment, the detection reagent includes a detection reagent for detecting the biomarkers described in any one of claims 1-4 by flow cytometry, Western blotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, near-infrared spectroscopy, immunochemiluminescence, capillary gel electrophoresis, or colloidal gold immunoassay.

[0015] In one embodiment, the detection reagent comprises an antibody that binds to the mitochondrial citrate transporter and / or primers for specifically amplifying nucleic acids encoding the mitochondrial citrate transporter.

[0016] In another aspect, the present invention also provides a kit comprising a detection reagent for detecting the level of the aforementioned biomarker.

[0017] In another aspect, the present invention also provides any of the following applications of the kit: (1): Use in the preparation of products for the diagnosis of cryptococcal meningitis in subjects; or (2): Use in the preparation of products for screening subjects for cryptococcal meningitis; or (3): Use in the preparation of products for assessing the risk of cryptococcal meningitis in subjects; or (4): Application in the preparation of products for prognostic assessment of cryptococcal meningitis.

[0018] In one embodiment, the detection reagent includes reagents for detecting the expression level of mitochondrial citrate transporter and / or reagents for detecting the expression level of nucleic acid encoding mitochondrial citrate transporter.

[0019] In one embodiment, the detection reagent includes a detection reagent for detecting the biomarkers described in any one of claims 1-4 by flow cytometry, Western blotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, near-infrared spectroscopy, immunochemiluminescence, capillary gel electrophoresis, or colloidal gold immunoassay.

[0020] In one embodiment, the detection reagent comprises an antibody that binds to the mitochondrial citrate transporter and / or primers for specifically amplifying nucleic acids encoding the mitochondrial citrate transporter.

[0021] In another aspect, the present invention also provides the use of mitochondrial citrate transporter protein and / or nucleic acid encoding mitochondrial citrate transporter protein as targets in the preparation of medicaments for the treatment and / or prevention of cryptococcal meningitis.

[0022] (III) Beneficial Effects This invention provides risk biomarkers for cryptococcal meningitis and their applications. Compared with existing technologies, it has the following advantages: 1. The survival advantage of CNAG_02288Δ in the brain microenvironment was verified using animal models.

[0023] 2. In vitro culture demonstrated that CNAG_02288Δ has advantages in glucose utilization. CNAG_02288 reprograms glucose metabolism, which may alter the mitochondrial oxidative respiratory chain, thereby achieving growth advantages such as optimized mitochondrial function and reduced ROS. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a transcriptome map of Cryptococcus neoformans isolated from the brain and peritoneum.

[0026] Figure 2 This is a graph analyzing the effects of differentially knocked-out gene knockout strains related to sugar metabolism on the virulence of infection in mice.

[0027] Figure 3 This is a graph showing the effect of CNAG_02288 knockout on the colonization ability of Cryptococcus neoformans.

[0028] Figure 4 This is a diagram illustrating the citrate transport function of the mitochondrial citrate transporter protein in Cryptococcus neoformans.

[0029] Figure 5 This is a graph showing the effect of CNAG_02288 knockout on glycolysis and TCA metabolic flux in Cryptococcus neoformans.

[0030] Figure 6 This is a metabolic flux analysis diagram of glucose from CNAG_02288 knockout Cryptococcus neoformans.

[0031] Figure 7 This diagram illustrates the regulatory role of mitochondrial citrate transporters in mitochondrial function.

[0032] Figure 8 This is a diagram illustrating the regulatory mechanism of mitochondrial citrate transporters in the brain. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Terms and Definitions As used herein, the term "susceptibility / risk biomarker" refers to a biomarker that is associated with an increased (or, in some cases, decreased) risk of an individual (who is not yet clinically ill) developing a certain disease or medical condition in the future. For example, genetic biomarkers may indicate an increased probability of developing cancer later in life. Susceptibility / risk biomarkers can be detected years (and in some cases decades) before the onset of clinical symptoms and signs, and are not associated with any specific treatment.

[0035] The primary clinical value of susceptibility / risk biomarkers lies in guiding prevention strategies. These biomarkers help determine whether lifestyle modifications, nutritional interventions, or other preventative measures are necessary, or guide high-risk populations to enhance disease surveillance.

[0036] In pharmaceutical product development, susceptibility / risk biomarkers can improve research efficiency through clinical trial enrichment strategies. In primary prevention scenarios, the low incidence of clinical endpoint events often makes it difficult to conduct clinical trials with statistical power. Therefore, screening high-risk populations for preventative clinical trials is particularly necessary, especially in the evaluation of targeted applications of chemopreventive therapies or vaccines. This strategy achieves a dual objective: 1) making trials feasible by enriching potentially high-risk populations; 2) enabling preventative interventions that may have side effects to be precisely applied to the population with the optimal risk-benefit balance.

[0037] Both susceptibility / risk biomarkers and prognostic biomarkers have the ability to predict the risk of future disease-related events, but the key difference lies in their applicability: prognostic biomarkers are applicable to individuals with a confirmed disease, while susceptibility / risk biomarkers can be used in seemingly healthy individuals. The boundary between the two may be blurred in some cases; nevertheless, screening and intervention strategies for seemingly healthy individuals and confirmed patients remain significantly different.

[0038] As used in this article, the term "diagnostic biomarkers" refers to a class of biomarkers used in clinical practice for the precise diagnosis of diseases and medical conditions. They play a central role in key decisions such as determining whether a patient has a specific disease requiring intervention, or determining whether an individual meets the inclusion criteria for a clinical trial for a specific disease.

[0039] The clinical value of diagnostic biomarker testing needs to be confirmed through clinical performance validation. Typically, clinical sensitivity (i.e., the proportion of positive tests in real patients) and specificity (i.e., the proportion of negative tests in healthy individuals) are calculated based on reference diagnostic criteria. Ideally, a perfect diagnostic biomarker should possess 100% sensitivity (no missed diagnoses) and 100% specificity (no false diagnoses), but in practical applications, no testing method simultaneously satisfies perfect clinical and analytical performance.

[0040] Defining the expected performance characteristics of diagnostic biomarker tests in the intended use scenarios is crucial. This requires a focus on the characteristics of the target diagnostic population and the implementation methods of the tests. The disease prevalence in the target population is the core parameter determining the test performance, specifically reflected in the positive predictive value (PPV, the proportion of people who test positive but actually have the disease) and the negative predictive value (NPV, the proportion of people who test negative but actually do not have the disease). PPV and NPV depend not only on the sensitivity and specificity of the test itself, but also on the prevalence in the population: achieving a high PPV is challenging when the prevalence is low; conversely, it is difficult to obtain a high NPV in a high-prevalence population.

[0041] In clinical practice, a trade-off must be struck between performance metrics such as sensitivity, specificity, PPV, and NPV. The decision is primarily based on the difference in harm caused by false positives versus false negatives. For example, in screening asymptomatic healthy individuals (where the prevalence of the target disease is extremely low), high-specificity and high-PPV testing protocols are typically prioritized to avoid unnecessary medical interventions and psychological harm caused by a large number of false positives. However, in diagnostic assessments of high-risk populations (where early intervention has proven clinical benefits), greater emphasis is placed on the sensitivity and NPV of the test.

[0042] In addition to clinical performance, diagnostic biomarker assays must meet reliable analytical performance standards. For example, accredited testing facilities and operators should obtain highly consistent results when performing the same assay. Significant deficiencies in analytical performance will inevitably lead to a substantial reduction in clinical performance.

[0043] The solute carrier family 25 (mitochondrial citrate transporter) disclosed in this invention can be a naturally purified product, a chemically synthesized product, or produced from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, higher animals, insects, and mammalian cells) using recombinant technology. Preferably, the mitochondrial citrate transporter disclosed in this invention is encoded by the Cryptococcus neoformans CNAG_02288 gene or its homologous gene or family gene.

[0044] Depending on the host used in the recombinant production protocol, the mitochondrial citrate transporter disclosed in this invention may be glycosylated or non-glycosylated. The term also includes active fragments and active derivatives of the mitochondrial citrate transporter.

[0045] As used herein, the terms “CNAG_02288 gene,” “mitochondrial citrate transporter encoding gene,” or “nucleic acid molecule encoding mitochondrial citrate transporter” are used interchangeably and all refer to a nucleotide sequence encoding the mitochondrial citrate transporter disclosed in this invention, which may be, for example, the nucleotide sequence shown in CNAG_02288, a molecule that hybridizes to these sequences under stringent conditions, or a family gene molecule that is highly homologous to the above-mentioned molecules, and the expression function of the gene.

[0046] The full-length nucleotide sequence or fragment thereof of CNAG_02288 disclosed in this invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in this disclosure, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared using conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified from each amplification in the correct order.

[0047] It should be understood that CNAG_02288 disclosed in this invention is preferably obtained from Cryptococcus neoformans. Other genes from other animals that are highly homologous to Cryptococcus neoformans genes (e.g., having more than 50%, preferably more than 55%, 60%, 65%, 70%, 75%, 80%, more preferably more than 85%, such as 85%, 90%, 95%, 98%, or even 99% or more sequence identity) are also within the scope of this preferred consideration. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0048] As used herein, the term "antibody" refers to a polypeptide that includes canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen, such as mitochondrial citrate transporter.

[0049] Antibodies include antibody fragments. Antibodies also include, but are not limited to, polyclonal, monoclonal, chimeric dAb (domain antibody), single-chain, Fab, Fa, F(ab)2 fragments, scFv, and Fab expression libraries. Antibodies can be whole antibodies, immunoglobulins, or antibody fragments.

[0050] Recombinant mitochondrial citrate transport proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography, and various other liquid chromatography techniques, as well as combinations of these methods.

[0051] As used herein, the term "specific binding" means that an antibody or peptide preferentially binds to a binding partner in a competitive binding assay. Competitive binding assays and other methods for determining specific binding are well known in the art.

[0052] As used herein, "primer" refers to an oligonucleotide primer that specifically anneals to the CNAG_02288 gene and initiates DNA synthesis from it under appropriate conditions to produce the corresponding amplification product. Each of the primers discussed anneals to a target within or adjacent to the corresponding target nucleic acid molecule such that at least a portion of each amplification product contains a nucleic acid sequence corresponding to the target. If one or more of the CNAG_02288 gene nucleic acids are present in the sample, one or more amplification products are produced; therefore, the presence of one or more CNAG_02288 gene amplification products indicates the presence of a bacterial strain in the sample. The amplification product should contain a nucleic acid sequence complementary to one or more detectable probes of the target bacterial gene.

[0053] As used in this article, "probe" refers to an oligonucleotide probe that is specifically annealed to the nucleic acid sequence encoding the CNAG_02288 gene.

[0054] As used herein, the term "amplification" refers to the process of synthesizing a nucleic acid molecule complementary to one or both strands of a template nucleic acid molecule. Amplification typically involves denaturing the template nucleic acid, annealing the primers to the template nucleic acid at a temperature below the primer melting temperature, and enzymatically extending the primers to produce the amplification product. Amplification usually requires the presence of deoxyribonucleoside triphosphates, DNA polymerase (e.g., Platinum Taq), and appropriate buffers and / or cofactors for optimal polymerase activity (e.g., MgCl2 and / or KCl).

[0055] As used herein, the term "detection reagent" includes detection reagents for biomarkers detected by flow cytometry, Western blotting, immunofluorescence assay, enzyme-linked immunosorbent assay, mass spectrometry, near-infrared spectroscopy, immunochemiluminescence assay, capillary gel electrophoresis, or colloidal gold immunoassay.

[0056] The terms “level of expression” or “expression level” are used interchangeably and generally refer to the amount of a biomarker in a biological sample. “Expression” generally refers to the process of converting information (e.g., gene-encoded and / or epigenetic information) into structures that are present and function in the cell. Therefore, as used herein, “expression” can refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and / or polypeptide modification (e.g., post-translational modification of a polypeptide). Fragments of transcribed polynucleotides, translated polypeptides, or polynucleotide and / or polypeptide modifications (e.g., post-translational modifications of a polypeptide) should also be considered expressed, regardless of whether they originate from transcripts generated through alternative splicing or degradation, or from post-translational processing of polypeptides (e.g., through proteolysis). “Expressed genes” include those transcribed into polynucleotides such as mRNA and then translated into polypeptides, as well as those transcribed into RNA but not translated into polypeptides (e.g., transfer RNA and ribosomal RNA). The “amount” or “level” of a biomarker (e.g., expression level) can be measured by methods known to those skilled in the art and disclosed herein. The “quantity” or “level” of a biomarker that is associated with an increased clinical benefit to an individual can be, for example, at a detectable level in a biological sample.

[0057] "Increased expression", "increased expression level", "increased level", "elevated expression", "elevated expression level" or "elevated level" refers to an increase in the expression or level of a biomarker in an individual relative to one or more individuals or internal controls such as those without a disease or condition (e.g., cancer) or a housekeeping biomarker.

[0058] "Decreased expression," "decreased expression level," "decreased level," "reduced expression," "decreased expression level," or "reduced level" refers to a decrease in the expression or level of a biomarker in an individual relative to one or more individuals or internal controls (e.g., housekeeping biomarkers) that do not have a disease or condition (e.g., cancer). In some respects, reduced expression means little or no expression.

[0059] As used herein, the presence and / or expression level / amount of the biomarkers can be analyzed by a variety of methods, many of which are known in the art and understood by those skilled in the art, including but not limited to immunohistochemistry (“IHC”), Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, flow cytometry, fluorescence activated cell sorting (“FACS”), MassARRAY, proteomics, blood-based quantitative assays (e.g., serum ELISA), biochemical enzyme activity assays, in situ hybridization (ISH), fluorescence in situ hybridization (FISH), DNA blot analysis, RNA blot analysis, whole genome sequencing, massively parallel DNA sequencing (e.g., next-generation sequencing), polymerase chain reaction (PCR) (including quantitative real-time PCR (qRT-PCR) and other amplification-type detection methods, such as branched DNA, SISBA, TMA, etc.), RNA-seq, microarray analysis, gene expression profiling and / or serial analysis of gene expression (“SAGE”), and any of a variety of assays that can be performed by protein, gene and / or tissue array analysis. Typical protocols for evaluating the status of genes and gene products can be found, for example, in *Current Protocols in Molecular Biology*, edited by Ausubel et al., 1995, Units 2 (RNA blotting), 4 (DNA blotting), 15 (immunoblotting), and 18 (PCR analysis). Multiplex immunoassays, such as those available from Rules Based Medicine or Meso Scale Discovery (“MSD”), can also be used.

[0060] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0061] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in this technical field.

[0062] Example 1: Brain microenvironment drives specific activation of cryptococcal glycogen metabolism pathway: Based on a rare case of multi-site Cryptococcal infection, a unique model was constructed to study the adaptive mechanisms of Cryptococcus to the brain microenvironment. The subject developed an infection following ventriculoperitoneal shunt surgery, with Cryptococcus colonization occurring simultaneously in both cerebrospinal fluid and the peritoneal cavity. Case analysis and genomic analysis confirmed that the peritoneal strain originated from cerebrospinal fluid dissemination, rather than an independent infection event. This homologous strain model provides ideal research material for elucidating the influence of the organ microenvironment on the adaptive phenotype of Cryptococcus.

[0063] Cryptococcus was isolated simultaneously from the subject's cerebrospinal fluid (CSF), the tip of the peritoneal duct, and the peritoneal fluid. The morphology of the strains differed at different isolation sites. Figure 1 A in the transcriptome shows a brain microenvironment-specific gene expression profile. Figure 1 In the B and D groups, the differentially expressed genes between cerebrospinal fluid strains and peritoneal strains (Catheter vs CSF / Drainage vs CSF) were 309 and 335, respectively. Figure 1 C in the text). KEGG enrichment showed that the bacterial strains in the brain significantly activated pyruvate metabolism, glyoxylate cycle and citrate transport pathway (C). Figure 1 The presence of E in the data suggests a close relationship between the glucose metabolism pathway of Cryptococcus neoformans and the brain infection process.

[0064] Example 2: Brain tissue colonization specificity of the CNAG_02288 knockout strain: By constructing sugar metabolism gene knockout strains using CRISPR-Cas9 and combining them with carbon source utilization phenotypic experiments, seven potential toxicity genes related to sugar metabolism were identified. Figure 2 (A and B in the text). Among them, CNAG_02288Δ exhibits glucose-biased metabolism: it grows well in glucose medium and is severely restricted only in ethanol / glycerol; while the isocitrate dehydrogenase knockout strain idpΔ shows only impaired glucose utilization function and can be used as a reverse control for glucose utilization function studies.

[0065] Meanwhile, CNAG_02288Δ exhibited brain-specific virulence, with a significantly higher bacterial load in the mouse brain compared to the wild-type strain H99, while it was significantly lower in blood and peripheral organs. Figure 2 (C in the text). CNAG_02288 regulates virulence in the brain in a tissue-specific manner, thus identifying CNAG_02288 as a key metabolic regulatory gene for Cryptococcus colonization in the brain.

[0066] Example 3: The effect of mitochondrial citrate transporters on glucose utilization efficiency: The functional annotation of the mitochondrial citrate transporter gene (CNAG_02288) in Cryptococcus is unclear. Homology with human protein sequences was compared. Figure 4The A in the figure suggests that it may have the same function as the mitochondrial citrate transporter SLC25A1 (SoluteCarrier Family 25 member1).

[0067] Interpro functional domain prediction revealed that mitochondrial citrate transporters may be involved in the transport of energy transfer and glucose metabolism-related substrates (including ADP / ATP, 2-oxoglutarate / malate, phosphate, and citrate).

[0068] Raman spectroscopy analysis revealed a decrease in cytoplasmic citrate content in CNAG_02288Δ (CNAG_02288 knockout strain). Figure 4 The study of BD, H99 (a wild-type Cryptococcus neoformans strain) confirmed its mitochondrial citrate transport function.

[0069] Central carbon-targeted metabolomics revealed that mitochondrial citrate transporter knockout strains showed a significant accumulation of intracellular glycolytic (e.g., glucose-6-phosphate, fructose-6-phosphate, lactate) and TCA cycle intermediates (e.g., isocitrate, fumarate) products. Figure 5 This demonstrates its ability to reprogram glucose metabolism.

[0070] To demonstrate that the sugar source was glucose, C13-labeled glucose was used as a substrate to culture wild-type strain H99 and CNAG_02288 knockout strain. Metabolic flux analysis showed that CNAG_02288Δ glucose-derived glycolysis and TCA flux were increased. Figure 6 This indicates that CNAG_02288Δ has advantages in glucose utilization.

[0071] Furthermore, the complex III inhibitor Antimycin A (idp1Δ) can completely reverse the glucose metabolism dominance of CNAG_02288Δ. Figure 7 In A), CNAG_02288Δ shows increased intracellular ATP and decreased ROS content. Figure 7 The B and C values ​​suggest its role in regulating the mitochondrial respiratory chain. This suggests that the mitochondrial citrate transporter's reprogramming of glucose metabolism may alter the mitochondrial oxidative respiratory chain, thereby achieving a growth advantage through optimized mitochondrial function and reduced ROS.

[0072] Example 4: Active downregulation of mitochondrial citrate transporter by Cryptococcus neoformans in the brain: Further investigation into the reasons for the colonization advantage of CNAG_02288Δ in the brain revealed that its growth advantage may be based on the adequate glucose supply in the brain.

[0073] CNAG_02288Δ showed growth advantage in 2% glucose medium, but after 32 hours, glucose was depleted, and the growth advantage was surpassed by the wild type, consistent with the results after 12 hours of cerebrospinal fluid culture; while the growth advantage in 4% glucose medium could be maintained for up to 48 hours. Figure 8 (A in the middle).

[0074] In in vivo infection experiments, although glucose is the primary carbon source for Cryptococcus, insufficient glucose supply in peripheral organs necessitates the use of multiple carbon sources to maintain survival after glucose depletion. This explains why CNAG_02288Δ exhibits lower CFU levels than the wild-type strain in systemic organs. However, in the brain, where glucose supply is abundant, CNAG_02288Δ demonstrates a sustained growth advantage.

[0075] Furthermore, RT-PCR experiments revealed that the wild-type Cryptococcus strain H99 actively downregulated the expression of mitochondrial citrate transporter protein with increasing glucose concentration and duration of glucose treatment. Figure 8 (B, C in the middle).

[0076] Meanwhile, total RNA was extracted from the brain and blood of mice infected with Cryptococcus tail vein, and the expression of Cryptococcus mitochondrial citrate transporter was detected, confirming that the expression of mitochondrial citrate transporter is downregulated in the brain. Figure 8 (D in the text). This demonstrates that Cryptococcus can sense glucose sufficiency signals in the brain, thereby shutting down mitochondrial citrate transporters, reducing the transport of citrate from mitochondria to the cytoplasm, increasing the flux and rate of glucose glycolysis, hijacking and plundering glucose in the brain, and thus achieving brain colonization dominance.

[0077] Studies have shown that cytoplasmic citrate can inhibit the activity of PFK1 (phosphofructokinase), a key enzyme in glycolysis, through conformational binding, thereby inhibiting glycolysis. Supplementing the environment with citrate resulted in a decreased growth advantage for the CNAG_02288Δ glycogenotype.

[0078] Example 5: Brain tissue-specific colonization advantages of the CNAG_02288 knockout strain: Cultured separately using cerebrospinal fluid ( Figure 3 A) A competitive advantage model in the brain during mouse tail vein infection ( Figure 3 B) and mouse intracerebroventricular injection infection model ( Figure 3 C and D in the study verified the survival advantage of CNAG_02288Δ in the brain microenvironment.

[0079] Cerebrospinal fluid culture: After resuscitation of Cryptococcus standard strains H99 and CNAG_02288Δ, single colonies were picked and cultured overnight at 30°C and 220 rpm in 10 ml LYPD liquid medium. The cells were centrifuged at 4000 rpm for 3 minutes, and the bacterial cell pellet was recovered and washed three times with sterile PBS. The cells were resuspended in PBS, diluted with artificial cerebrospinal fluid, and the bacterial cell concentration was adjusted to OD600 = 0.02 (approximately 5). 10 5 The sample ( / mL) was cultured in a 96-well plate at 30°C, and the OD600 absorbance was recorded every half hour to plot the growth curve.

[0080] Mouse tail vein infection model: After recovery of Cryptococcus standard strains H99 and CNAG_02288Δ, single colonies were picked and cultured overnight at 30℃ and 220 rpm in 10 mL YPD liquid medium. The cells were centrifuged at 4000 rpm for 3 minutes, and the bacterial cell pellet was recovered and washed three times with sterile PBS. The cells were resuspended in PBS and the bacterial cell concentration was adjusted to 5%. 10 5 / mL (10 5 / 200uL). 4-6 week old female BALB / c mice were selected: 15 mice in each group were anesthetized, and 200 μL of the prepared bacterial solution was injected via the tail vein. After confirming that all the bacterial solution had entered the tail vein, the mouse tail was pressed to prevent spillage. Fungal load analysis: 7 days later, 5 mice from each group were dissected, and brain, lung, liver, and spleen tissues were collected. The tissues were homogenized in 1 ml of sterile PBS, and 100 μL of the homogenate was plated. The homogenate was serially diluted with PBS, and 100 μL was plated. 100 μL of mouse blood was also plated. The cultures were incubated on YPD solid medium at 30℃ for 2 days, and the colony count was recorded.

[0081] Survival curve analysis: Ten mice were in each group. Their body weight and vital signs were observed daily for 40 days. The time of death was recorded (physiological death or weight loss to 20% of initial body weight was considered the endpoint of death).

[0082] A competitive advantage model of intracerebral infection in mice with tail vein infection: After recovery of Cryptococcus standard strains H99 and CNAG_02288Δ, single colonies were picked and cultured overnight at 30°C and 220 rpm in 10 mL YPD liquid medium. The cells were centrifuged at 3000 rpm for 3 minutes, and the bacterial cell pellet was recovered and washed three times with sterile PBS. The cells were resuspended in PBS and the bacterial cell concentration was adjusted to 5%. 10 5 / mL (10 5 Prepare a 1:1 mixed bacterial solution, ensuring that the cell concentrations of the two bacterial strains are 2.5 μL / 200 μL. 10 7 / mL. The procedure for mouse tail vein infection is the same as before. Brain tissue fungal load analysis: After 7 days, 5 mice from each group were dissected, and brain tissue was collected in 1 ml of sterile PBS. After homogenization, 100 μL of the homogenate was plated. The homogenate was serially diluted with PBS, and 100 μL was plated on YPD basal solid medium and YPD solid medium containing 100 μg / mL norocin as a screening drug. 100 μL of mouse blood was also plated. The culture was carried out at 30℃ for 2 days, and the colony count was recorded. Because a NAT (norocin resistance gene) tag was inserted into the genome during the construction of the CNAG_02288Δ gene knockout, it could be cultured in the selection medium. The competitive advantage of the two bacteria in simultaneous infection was compared by comparing the colony counts on the basal medium (total number of both bacteria) and the selection medium (CNAG_02288Δ strain only).

[0083] Mouse intracerebroventricular injection infection model: After recovery of Cryptococcus standard strains H99 and CNAG_02288Δ, single colonies were picked and cultured overnight at 30℃ and 220 rpm in 10 mL YPD liquid medium. The cells were centrifuged at 3000 rpm for 3 minutes, and the bacterial cell pellet was recovered and washed three times with sterile PBS. The cells were resuspended in PBS, and the bacterial cell concentration was adjusted to 3.33. 10 5 / mL (10 3 / 3uL). 4-6 week old female BALB / c mice were selected: 15 mice were anesthetized in each group, and the cerebral cortex was incised. Using a mouse ventricle locator, a puncture site was taken 1-2 cm anterior to the coronal suture and 2-3 cm lateral to the midline. A cone (or burr) was made to the dura mater, and the puncture needle was inserted vertically approximately 4-6 cm into the anterior horn of the lateral ventricle, where CSF was observed flowing out. The prepared bacterial solution was thoroughly vortexed, and 3 μL was injected micro-injected at a rate of 3uL / 10min. After confirming that all the bacterial solution had entered the ventricle, the needle was withdrawn after 1 minute to prevent spillage. The cerebral cortex was sutured, and the mice were observed to return to their cages after regaining consciousness. Brain tissue fungal load analysis: 7 days later, 5 mice from each group were dissected, and the brains were collected in 1 ml of sterile PBS. After homogenization, 100 μL of the homogenate was plated. The homogenate was serially diluted with PBS, and 100 μL was plated on YPD basal solid medium. The medium was incubated at 30℃ for 2 days, and the colony count was recorded. Survival curve analysis: Ten mice were in each group. Their body weight and vital signs were observed daily for 20 days. The time of death was recorded (physiological death or a decrease in body weight to 20% of the initial body weight was taken as the endpoint of death).

[0084] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0085] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A susceptibility / risk biomarker for cryptococcal meningitis, characterized in that, The susceptibility / risk biomarkers include mitochondrial citrate transporters and / or nucleic acids encoding mitochondrial citrate transporters.

2. A diagnostic biomarker for cryptococcal meningitis, characterized in that, The diagnostic biomarkers include mitochondrial citrate transporter and / or nucleic acids encoding mitochondrial citrate transporter.

3. The susceptibility / risk biomarker according to claim 1 or the diagnostic biomarker according to claim 2, characterized in that, The biomarker is derived from Cryptococcus.

4. The susceptibility / risk biomarker according to claim 3 or the diagnostic biomarker according to claim 2, characterized in that, The Cryptococcus was isolated from the subject's cerebrospinal fluid, peritoneal catheter, and / or peritoneal fluid.

5. Any of the following applications of the detection reagent for detecting the level of the biomarker according to any one of claims 1-4: (1): Use in the preparation of products for the diagnosis of cryptococcal meningitis in subjects; or (2): Use in the preparation of products for screening subjects for cryptococcal meningitis; or (3): Use in the preparation of products for assessing the risk of cryptococcal meningitis in subjects; or (4): Application in the preparation of products for prognostic assessment of cryptococcal meningitis.

6. The application according to claim 5, characterized in that, The detection reagents include reagents for detecting the expression level of mitochondrial citrate transporter and / or reagents for detecting the expression level of nucleic acid encoding mitochondrial citrate transporter.

7. The application according to claim 5, characterized in that, The detection reagents include those for detecting the biomarkers described in any one of claims 1-4 by flow cytometry, Western blotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, near-infrared spectroscopy, immunochemiluminescence, capillary gel electrophoresis, and / or colloidal gold immunoassay.

8. The application according to claim 5, characterized in that, The detection reagent includes antibodies that bind to mitochondrial citrate transporters and / or primers for specifically amplifying nucleic acids encoding mitochondrial citrate transporters.

9. A reagent kit, characterized in that, The reagents include those for detecting the levels of the biomarkers described in any one of claims 1-4.

10. The use of mitochondrial citrate transporter and / or nucleic acids encoding mitochondrial citrate transporter as targets in the preparation of drugs for the treatment and / or prevention of cryptococcal meningitis.

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