Single-cell rna sequencing method for pathogenic fungi based on protoplast preparation

Abstract

The application discloses a single-cell RNA sequencing method suitable for pathogenic fungi based on protoplast preparation and relates to the technical field of single-cell sequencing and pathogenic fungi. The application significantly improves the single-cell lysis and mRNA release efficiency of pathogenic fungi, and through the optimization of cell wall enzymolysis combination, the application can significantly improve the accessibility of fungal cells to the single-cell lysis system, and improve the mRNA release efficiency and capture efficiency. The applicant's previous unpublished results show that the mRNA capture rate of the FUNSEQ method established in the application has reached the level of mammalian single-cell RNA sequencing, and even is better than that of some mammalian single-cell RNA sequencing samples. Specific indicators can include: the number of detected genes per cell is increased; the number of UMIs obtained per cell is increased.

Description

Technical Field

[0001] This invention relates to the fields of single-cell sequencing and pathogenic mycology, specifically to a single-cell RNA sequencing method for pathogenic fungi based on protoplast preparation. Background Technology

[0002] Medical and public health significance of pathogenic fungal infections

[0003] Pathogenic fungal infections are among the most significant infectious diseases worldwide. In recent years, with the increase in organ transplantation, cancer chemotherapy, immunosuppressant use, HIV infection, intensive care treatment, and the use of broad-spectrum antibiotics, the incidence of invasive fungal infections has continued to rise. Common important pathogenic fungi include, but are not limited to, Cryptococcus neoformans / Cryptococcus gattii, Candida albicans, Candida glabrata, Aspergillus, Mucor, Basilaria marneffei, Histoplasma capsulatum, and Coccidioides immitis.

[0004] Cryptococcus neoformans is one of the main pathogens causing cryptococcal meningitis, with a particularly high mortality rate in immunocompromised individuals. This fungus can invade the central nervous system and survive in the microenvironments of cerebrospinal fluid, brain tissue, and macrophages. Due to the complexity of the host's internal microenvironment, fungal cells may be in different physiological states during infection, such as dormant, proliferative, stressed, drug-resistant, or highly virulent states. These different states of fungal cells are often averaged in population sequencing, making it difficult to resolve their true heterogeneity.

[0005] The Importance of Single-Cell RNA Sequencing Technology

[0006] Single-cell RNA sequencing, especially single-cell transcriptome sequencing based on droplet, microwell, or plate sorting, can analyze gene expression status at the single-cell level, revealing cell subpopulations, developmental trajectories, stress responses, drug-resistant subpopulations, and rare cell states. This technology has been widely applied in research fields such as mammalian cells, immune cells, tumor cells, developmental biology, and neuroscience. However, the application of single-cell RNA sequencing in fungi, especially pathogenic fungi, remains largely unreported. The main reason is that fungal cells possess a dense and complex cell wall, including chitin, β-glucan, α-glucan, mannan, and other polysaccharide / protein complexes. This cell wall strongly hinders conventional single-cell lysis systems, leading to: difficulty in effectively lysing individual fungal cells; low efficiency in releasing intracellular mRNA; low capture efficiency of low-abundance transcripts; insufficient sequencing library complexity; low gene count and UMI count in single-cell data; significant differences in cell walls among different fungal species and at different growth stages, making it difficult to establish a universal protocol; and excessive mechanical fragmentation may lead to RNA degradation, cell rupture, two-cell / multi-cell aggregation, or sample loss. Therefore, the effective, mild, and compatible processing of fungal cell walls with single-cell RNA sequencing procedures is a key bottleneck limiting the development of single-cell transcriptomics technology for pathogenic fungi.

[0007] The shortcomings of existing technology:

[0008] Current transcriptome research on fungi primarily relies on population RNA sequencing, which involves extracting total RNA from a large number of fungal cells and then performing transcriptome analysis. While this method can reflect overall expression trends, it cannot distinguish differences between different fungal cells, nor can it identify low-proportion but biologically significant cell populations, such as: fungal subpopulations with specific adaptations in the host brain infection environment; tolerance / resistance subpopulations pre-existing or induced by antifungal drugs; fungal subpopulations surviving within macrophages; fungal states capable of crossing the blood-brain barrier or adapting to the cerebrospinal fluid environment; and transient transcriptional states occurring during the infection process. Furthermore, existing mammalian single-cell RNA sequencing systems are typically designed for cells without cell walls or easily lysed cells. Their lysis buffers and reverse transcription systems cannot effectively disrupt fungal cell walls, often resulting in low mRNA capture rates, low effective cell counts, high sequencing background, and poor reproducibility when directly applied to fungal cells. Therefore,

[0009] There is an urgent need to develop a fungal single-cell RNA sequencing method that can adapt to the structural characteristics of pathogenic fungal cells, be compatible with existing single-cell sequencing platforms, and achieve efficient mRNA capture. Summary of the Invention

[0010] The purpose of this invention is to provide a single-cell RNA sequencing method for pathogenic fungi based on protoplast preparation, in order to solve the technical problems mentioned in the background art.

[0011] To achieve the above objectives, the present invention provides the following technical solution: a single-cell RNA sequencing method for pathogenic fungi based on protoplast preparation, comprising the following steps:

[0012] Acquisition and pretreatment of pathogenic fungal samples: The samples that can be processed by this invention include, but are not limited to: pathogenic fungal cells cultured in vitro; fungal cells treated with drugs; fungal cells cultured in a simulated host environment; fungal cells isolated from animal infection models; fungal cells isolated after co-culturing with host cells; and fungal cells enriched in clinical samples.

[0013] Fungal cell washing and single-cell processing: After collecting fungal cells, they were washed with PBS buffer to remove culture medium components, host cell debris, free RNA, and impurities that could affect enzymatic digestion or single-cell capture.

[0014] Furthermore, the centrifugation conditions were 4000 rpm for 5 minutes. The washing process was kept as gentle as possible to avoid vigorous vortexing that could cause cell rupture or RNA degradation.

[0015] Cell wall enzymatic hydrolysis system treatment: Resuspend fungal cells in cell wall enzymatic hydrolysis buffer, add one or more cell wall digestive enzymes, and incubate at a suitable temperature.

[0016] Furthermore, the enzymatic hydrolysis conditions are as follows: temperature 30°C; time 2 to 6 hours, preferably 2 hours, depending on the strain; enzyme concentration determined according to enzyme activity, with VinoTastePro enzyme at 50 mg / mL and Zymolyase enzyme at 10 mg / mL; buffer system containing PBS and RNase inhibitors, etc.; pH value 7.2-7.4.

[0017] Filtration, de-agglomeration, and cell viability / integrity assay: After enzymatic digestion, the reaction was terminated by centrifugation and washing. The sample was then gently pipetted and filtered using a 40μm or 70μm filter membrane to remove cell aggregates and debris, obtaining dispersed cells suitable for single-cell capture. The cell suspension was quality controlled before loading onto the single-cell sequencing platform.

[0018] Furthermore, quality control indicators include, but are not limited to: cell concentration, single-cell ratio, aggregation ratio, cell integrity, and degree of cell wall digestion. Detection methods include, but are not limited to, microscopic observation, trypan blue staining, fluorescence staining, flow cytometry, cell counting, RNA quantification, or fluorescent probe detection. Preferably, the cell aggregation rate is less than 10%, and the cell concentration is adjusted to the range recommended by the single-cell sequencing platform.

[0019] Adjusting cell concentration and loading it onto a single-cell RNA sequencing platform: The method of the present invention is compatible with existing single-cell RNA sequencing platforms, including but not limited to: droplet single-cell RNA sequencing platforms, micropore single-cell RNA sequencing platforms, and capture systems for poly(A) RNA.

[0020] Preferably, the present invention can be used in BGI's single-cell sample preparation platform DNBelabC4 or other similar droplet-based single-cell RNA sequencing platforms.

[0021] Single-cell capture, lysis, reverse transcription, and library construction: The treated fungal single-cell suspension was loaded onto a single-cell RNA sequencing platform. Cells were contacted in a droplet system with capture beads or primers containing barcodes. The mRNA released after cell lysis was captured by primers carrying poly(dT) sequences, cell barcodes, and UMIs, followed by reverse transcription, cDNA amplification, and sequencing library construction.

[0022] Preferably, a 3' end single-cell RNA sequencing strategy is used to construct the library.

[0023] High-throughput sequencing: Performing high-throughput sequencing on the constructed library.

[0024] Data quality control, alignment, expression matrix generation, and single-cell transcriptome analysis: After high-throughput sequencing, the library underwent data analysis. This analysis included: raw sequencing data quality control, adapter removal and low-quality sequence filtering, alignment to the corresponding fungal reference genome / transcriptome, cell barcode identification, UMI deduplication, and construction of a gene-cell expression matrix.

[0025] Furthermore, this invention obtains an enzymatic digestion system suitable for single-cell sequencing of pathogenic fungi by comparing the effects of using different commercially available fungal cell wall digestive enzymes individually and in combination. The enzymes may include, but are not limited to, VinoTastePro enzymes, Zymolyase enzymes, and snail enzymes.

[0026] Preferably, the present invention does not simply pursue the complete formation of protoplasts, but controls the cell wall to be in a "moderately digested" state, which is conducive to subsequent lysis and mRNA release, while minimizing premature cell rupture, RNA leakage or degradation.

[0027] Furthermore, the method of this invention is applicable to a variety of pathogenic fungi, including but not limited to: Cryptococcus neoformans, Cryptococcus gutteruds, Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, and other yeast-like medical fungi with cell wall structures. In different fungi, the type of enzyme, enzyme concentration, digestion time, temperature, and buffer system can be adjusted according to cell wall composition, cell size, growth stage, capsule structure, temperature adaptability, and sample source.

[0028] Compared with the prior art, the beneficial effects of the present invention are:

[0029] 1. This invention significantly improves the efficiency of single-cell lysis and mRNA release in pathogenic fungi. Through optimized cell wall enzymatic digestion combinations, this invention significantly improves the accessibility of fungal cells to the single-cell lysis system, thereby increasing mRNA release and capture efficiency. Previously unpublished results from the applicant show that the FUNSEQ method established in this invention has achieved mRNA capture rates in fungi comparable to, and even superior to, some mammalian single-cell RNA sequencing samples. Specific indicators may include: increased number of genes detected per cell; increased number of UMIs obtained per cell;

[0030] 2. This invention is the first to achieve or effectively achieve the mapping of single-cell transcriptomes of pathogenic fungi. Under simulated brain infection conditions, a single-cell transcriptome map of Cryptococcus neoformans was drawn, and it was found that fungal cells have different transcriptional states and cell subpopulations in brain infection-related environments, providing a new technical means for understanding the central nervous system infection mechanism of Cryptococcus neoformans.

[0031] 3. This invention maintains cell integrity and sequencing compatibility. Instead of employing strong mechanical disruption or complete lysis strategies, this invention performs gentle and controllable pretreatment of the cell wall before single-cell capture, allowing the cells to be captured as independent units before entering the single-cell sequencing platform. This balances cell dispersion, cell integrity, RNA integrity, accuracy of single-cell sequencing barcode labeling, and success rate of subsequent library construction. Attached Figure Description

[0032] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments 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.

[0033] Figure 1 This is a schematic diagram based on flow cytometry results showing the cell wall digestion effect of different enzymatic treatments 12 hours after the present invention.

[0034] Figure 2 This is a schematic diagram based on flow cytometry results illustrating the optimization of protoplast preparation time based on the optimal enzyme digestion combination in this invention.

[0035] Figure 3 This is a schematic diagram based on microscopic fluorescence observation results, illustrating the optimization of protoplast preparation time based on the optimal enzyme digestion combination according to the present invention.

[0036] Figure 4This diagram illustrates the quality control results of single-cell data from Cryptococcus neoformans, using it as an example. Detailed Implementation

[0037] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0038] This invention addresses the problem of complex and difficult-to-lyse cell walls of pathogenic fungi. By screening and combining various commercially available fungal cell wall digestive enzymes, the invention enables sufficient but not excessive enzymatic digestion of fungal cell walls while maintaining cell dispersion, RNA integrity, and single-cell sequencing compatibility. This allows subsequent single-cell lysates to effectively release intracellular mRNA, and single-cell transcriptome information can be efficiently obtained through a capture system with cell barcodes and molecular tags.

[0039] Example 1:

[0040] A protoplast-based single-cell RNA sequencing method suitable for pathogenic fungi includes the following steps:

[0041] S1: Collect pathogenic fungal cells and perform pretreatment;

[0042] S2: Treat pathogenic fungal cells with an enzymatic digestion system containing fungal cell wall digestive enzymes to cause moderate digestion of the cell wall;

[0043] S3: Obtain a fungal single-cell suspension suitable for single-cell capture;

[0044] S4: Adjust the cell concentration in the fungal single-cell suspension and load it onto the single-cell RNA sequencing platform;

[0045] S5: Single-cell RNA capture, reverse transcription, and library construction for fungal single cells;

[0046] S6: High-throughput sequencing to obtain single-cell transcriptome data.

[0047] The preprocessing in S1 includes the following steps:

[0048] S1.1: Washing, using PBS buffer;

[0049] S1.2: Perform centrifugation at 4000 rpm for 5 min.

[0050] The enzymatic hydrolysis system contains at least two fungal cell wall digestive enzymes with different substrates;

[0051] Fungal cell wall digestive enzymes include VinoTastePro, Zymolyase, and snail enzymes;

[0052] The conditions for enzymatic hydrolysis were: temperature 30℃, time 2 to 6 hours, and pH 7.2-7.4.

[0053] The enzymatic hydrolysis system also contains RNase inhibitors.

[0054] The fungal single-cell suspension in S3 was prepared by filtration to remove cell aggregates and debris.

[0055] The filtration in the preparation of fungal single-cell suspensions uses 40μm or 70μm filter membranes.

[0056] S3 also includes quality control of cell suspensions;

[0057] Quality control indicators include cell concentration, single cell ratio, aggregation ratio, cell integrity, and cell wall digestion degree, with cell aggregation rate below 10%.

[0058] The fungal single-cell suspension proposed in S3 is compatible with existing single-cell RNA sequencing platforms by adjusting the cell concentration. The single-cell RNA sequencing platforms include: droplet single-cell RNA sequencing platform, micropore single-cell RNA sequencing platform, and capture system for poly(A) RNA.

[0059] S5 includes the following steps:

[0060] After S4, the cells were contacted in a droplet system with capture beads or primers containing barcodes;

[0061] Single-cell RNA capture: mRNA released after cell lysis is captured by primers containing poly(dT) sequences, cell barcodes, and UMI.

[0062] Subsequently, reverse transcription, cDNA amplification, and sequencing library construction were performed.

[0063] After high-throughput sequencing in S6, data analysis was performed.

[0064] Data analysis includes raw sequencing data quality control, adapter removal and low-quality sequence filtering, alignment to the corresponding fungal reference genome / transcriptome, cell barcode identification, UMI deduplication, and construction of gene-cell expression matrices.

[0065] Pathogenic fungi include Cryptococcus neoformans, Cryptococcus gutterusum, Candida albicans, Candida glabrata, Candida tropicalis, and Candida parapsilosis.

[0066] Example 2:

[0067] This embodiment proposes the following experiment based on the above embodiment one:

[0068] Screening and optimization of cell wall digestive enzyme combinations of pathogenic fungi

[0069] 1. Experimental Objective

[0070] Screening cell wall enzymatic digestion conditions suitable for single-cell RNA sequencing of pathogenic fungi was conducted to effectively digest the fungal cell wall while maintaining cell integrity and single-cell state, thereby improving the efficiency of subsequent mRNA capture.

[0071] 2. Experimental Materials

[0072] Pathogenic fungal strains: Cryptococcus neoformans H99, Saccharomyces cerevisiae W1170 and Candida auris W8635;

[0073] Culture medium: YPD medium;

[0074] Cell wall digestive enzymes:

[0075] Enzyme A: VinoTaste Pro enzyme

[0076] Enzyme B: Zymolyase

[0077] Enzyme C: Snail enzyme

[0078] Buffer solution: PBS;

[0079] RNase inhibitor: Solarbio / R8061;

[0080] Staining reagents: Wheat Germ Agglutinin (WGA), Calcofluor White (CFW).

[0081] 3. Experimental Methods

[0082] Cryptococcus neoformans was cultured to the logarithmic phase, and the cells were collected by centrifugation. After washing twice with PBS, the cell concentration was adjusted to 1×10^7 cells / mL.

[0083] Different enzymatic digestion groups were set up, including single-enzyme groups and multi-enzyme combination groups. Cryptococcus cells in each group were incubated at 30°C for 12 hours. After incubation, the enzymatic digestion of the cell wall was detected by flow cytometry, and cell morphology, cell aggregation, and cell wall staining intensity were observed under a microscope. Cell integrity was also assessed (see [reference]). Figures 1-4 ).

[0084] 4. Example of experimental grouping

[0085] control group none none none 0 min - Unprocessed Single enzyme group 1 + - - 【12h】 【30℃】 Single enzyme group 2 - + - 【12h】 【30℃】 Single enzyme group 3 - - + 【12h】 【30℃】 Double enzyme group 1 + + - 【12h】 【30℃】 Double enzyme group 2 + - + 【12h】 【30℃】 Double enzyme group 3 - + + 【12h】 【30℃】 Three-enzyme group + + + 【12h】 【30℃】

[0086] 5. Detection Indicators

[0087] The degree of decrease in cell wall staining intensity;

[0088] The degree of increase in cell membrane staining intensity;

[0089] Cell integrity;

[0090] Cell aggregation rate;

[0091] Number of genes per cell in subsequent single-cell sequencing;

[0092] Number of UMIs per cell in subsequent single-cell sequencing;

[0093] 6. Experimental Results

[0094] Experimental results show that, compared with the untreated group or the single-enzyme treatment group, the optimized multi-enzyme combination can significantly reduce the staining intensity of fungal cell walls, reduce cell lysis inhibition, and improve the quality of subsequent single-cell RNA sequencing while maintaining high cell integrity.

[0095] (1) Cryptococcus neoformans H99

[0096] Unprocessed 【0.58】 Single enzyme A 【8.19】 Single enzyme B 【5.56】 Single enzyme C 【19.44】 Enzyme A + Enzyme C 【0.28】 Enzyme B + Enzyme C 【9.14】 Enzyme A + Enzyme B + Enzyme C 【0.73】

[0097] Optimal group enzyme A + enzyme B 【0.81】 【46.34】

[782]

[1688]

[0098] (2) Candida auris W8635

[0099] Unprocessed 【1.77】 Single enzyme A 【1.37】 Single enzyme C 【1.21】 Enzyme A + Enzyme C 【9.21】 Enzyme B + Enzyme C 【0.95】 Enzyme A + Enzyme B + Enzyme C 【0.72】

[0100] Optimal group enzyme B 【0.61】 【203.85】

[0101] (3) Saccharomyces cerevisiae W1170

[0102] Unprocessed 【9.15】 Single enzyme A 【2.41】 Single enzyme B 【0.37】 Single enzyme C 【5.56】 Enzyme A + Enzyme C 【0.06】 Enzyme B + Enzyme C 【5.56】 Enzyme A + Enzyme B + Enzyme C 【0.06】

[0103] Optimal group enzyme A + enzyme B 【0.51】 【16.67】

[0104] 7. Conclusion

[0105] The optimized enzymatic digestion system effectively processes the cell walls of Cryptococcus neoformans, Candida auris, and Saccharomyces cerevisiae, making them suitable for subsequent single-cell RNA sequencing. This processing method significantly improves mRNA capture efficiency and the quality of effective single-cell data.

[0106] In summary:

[0107] This invention has broad industrial application prospects and can be used in the following industries or for transformation applications:

[0108] Antifungal drug development: used to detect single-cell transcriptional responses of fungi after drug treatment, identify tolerance subpopulations, drug targets, combination drug mechanisms, and early resistance signals.

[0109] Fungal resistance detection and mechanism research: It can analyze the single-cell heterogeneity of clinical isolates under different antifungal drug pressures, providing a basis for resistance risk assessment and precision treatment.

[0110] Infection models and host-pathogen interaction studies: can be used to analyze the status of pathogenic fungi in models of brain infection, lung infection, bloodstream infection, and macrophage infection.

[0111] Clinical sample pathogen status assessment: After further optimization, it can be used for single-cell expression status analysis of pathogenic fungi in clinical samples such as cerebrospinal fluid, bronchoalveolar lavage fluid, blood, and tissue biopsy samples.

[0112] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. A protoplast-based single-cell RNA sequencing method suitable for pathogenic fungi, characterized by: Includes the following steps: S1: Collect pathogenic fungal cells and perform pretreatment; S2: Treat the pathogenic fungal cells with an enzymatic digestion system containing fungal cell wall digestive enzymes to cause moderate digestion of the cell wall; S3: Obtain a fungal single-cell suspension suitable for single-cell capture; S4: Adjust the cell concentration in the fungal single-cell suspension and load it onto the single-cell RNA sequencing platform; S5: Perform single-cell RNA capture, reverse transcription, and library construction on the fungal single cells; S6: High-throughput sequencing to obtain single-cell transcriptome data.

2. The method for single-cell RNA sequencing of pathogenic fungi based on protoplast preparation according to claim 1, characterized in that: The preprocessing in S1 includes the following steps: S1.1: Washing, using PBS buffer; S1.2: Perform centrifugation at 4000 rpm for 5 min.

3. The method for single-cell RNA sequencing of pathogenic fungi based on protoplast preparation according to claim 1, characterized in that: The enzymatic hydrolysis system contains at least two fungal cell wall digestive enzymes with different substrates. The fungal cell wall digestive enzymes include VinoTastePro enzyme, Zymolyase enzyme, and snail enzyme; The conditions for the enzymatic hydrolysis treatment are: temperature 30℃, time 2 to 6 hours, and pH value 7.2-7.

4.

4. The method for single-cell RNA sequencing of pathogenic fungi based on protoplast preparation according to claim 1, characterized in that: The enzymatic hydrolysis system also contains RNase inhibitors.

5. The method for single-cell RNA sequencing of pathogenic fungi based on protoplast preparation according to claim 1, characterized in that: The fungal single-cell suspension in S3 is prepared by filtering to remove cell aggregates and debris. The filtration in the preparation of the fungal single-cell suspension uses a 40μm or 70μm filter membrane.

6. The method for single-cell RNA sequencing of pathogenic fungi based on protoplast preparation according to claim 1, characterized in that: S3 also includes quality control of the cell suspension; The quality control indicators include cell concentration, single cell ratio, aggregation ratio, cell integrity, and cell wall digestion degree, with the cell aggregation rate being less than 10%.

7. The method for single-cell RNA sequencing of pathogenic fungi based on protoplast preparation according to claim 1, characterized in that: The fungal single-cell suspension proposed in S3 is compatible with existing single-cell RNA sequencing platforms by adjusting the cell concentration. The single-cell RNA sequencing platform includes: a droplet single-cell RNA sequencing platform, a micropore single-cell RNA sequencing platform, and a capture system for poly(A) RNA.

8. The method for single-cell RNA sequencing of pathogenic fungi based on protoplast preparation according to claim 1, characterized in that: S5 includes the following steps: After S4, the cells were contacted in a droplet system with capture beads or primers containing barcodes; Single-cell RNA capture: mRNA released after cell lysis is captured by primers containing poly(dT) sequences, cell barcodes, and UMI. Subsequently, reverse transcription, cDNA amplification, and sequencing library construction were performed.

9. The method for single-cell RNA sequencing of pathogenic fungi based on protoplast preparation according to claim 1, characterized in that: After high-throughput sequencing in S6, data analysis is performed. The data analysis includes raw sequencing data quality control, adapter removal and low-quality sequence filtering, alignment to the corresponding fungal reference genome / transcriptome, cell barcode identification, UMI deduplication, and construction of a gene-cell expression matrix.

10. The method for single-cell RNA sequencing of pathogenic fungi based on protoplast preparation according to claim 1, characterized in that: The pathogenic fungi include Cryptococcus neoformans, Cryptococcus gutterusum, Candida albicans, Candida glabrata, Candida tropicalis, and Candida parapsilosis.

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