Cell wall composition in Cryptococcus neoformans is media dependent and alters host response, inducing protective immunity.

Introduction: Cryptococcus neoformans is a basidiomycete fungus that can cause meningoencephalitis, especially in immunocompromised patients. Cryptococcus grows in many different media, although little attention has been paid to the role of growth conditions on the cryptococcal cell wall or on virulence. Objective: The purpose of this study was to determine how different media influenced the amount of chitin and chitosan in the cell wall, which in turn impacted the cell wall architecture and host response. Methods: Yeast extract, peptone, and dextrose (YPD) and yeast nitrogen base (YNB) are two commonly used media for growing Cryptococcus before use in in vitro or in vivo experiments. As a result, C. neoformans was grown in either YPD or YNB, which were either left unbuffered or buffered to pH 7 with MOPS. These cells were then labeled with cell wall-specific fluorescent probes to determine the amounts of various cell wall components. In addition, these cells were employed in animal virulence studies using the murine inhalation model of infection. Results: We observed that the growth of wild-type C. neoformans KN99 significantly changes the pH of unbuffered media during growth. It raises the pH to 8.0 when grown in unbuffered YPD but lowers the pH to 2.0 when grown in unbuffered YNB (YNB-U). Importantly, the composition of the cell wall was substantially impacted by growth in different media. Cells grown in YNB-U exhibited a 90% reduction in chitosan, the deacetylated form of chitin, compared with cells grown in YPD. The decrease in pH and chitosan in the YNB-U-grown cells was associated with a significant increase in some pathogen-associated molecular patterns on the surface of cells compared with cells grown in YPD or YNB, pH 7. This altered cell wall architecture resulted in a significant reduction in virulence when tested using a murine model of infection. Furthermore, when heat-killed cells were used as the inoculum, KN99 cells grown in YNB-U caused an aberrant hyper-inflammatory response in the lungs, resulting in rapid animal death. In contrast, heat-killed KN99 cells grown in YNB, pH 7, caused little to no inflammatory response in the host lung, but, when used as a vaccine, they conferred a robust protective response against a subsequent challenge infection with the virulent KN99 cells. Conclusion: These findings emphasize the importance of culture media and pH during growth in shaping the content and organization of the C. neoformans cell wall, as well as their impact on fungal virulence and the host response.

Cryptococcus neoformans Cda1 and Cda2 coordinate deacetylation of chitin during infection to control fungal virulence.

Chitosan, a deacetylated form of chitin, is required for the virulence of Cryptococcus neoformans. There are three chitin deacetylase genes (CDA) that are essential for chitosan production, and deletion of all three genes results in the absence of chitosan, loss of virulence, and induction of a protective host response when used as a vaccine. Cda1 plays a major role in deacetylating chitin during pulmonary infection of CBA/J mice. Inoculation with the cda1Δ strain did not lead to a lethal infection. However, the infection was not cleared. The persistence of the fungus in the host suggests that chitin is still being deacetylated by Cda2 and/or Cda3. To test this hypothesis, we subjected strains deleted of two CDA genes to fungal virulence in CBA/J, C57BL/6 and BALB/c and found that cda1Δcda2Δ was avirulent in all mouse lines, as evidenced by its complete clearance. Consistent with the major role of Cda1 in CBA/J, we found that cda2Δcda3Δ was as virulent as its wild-type progenitor KN99. On the other hand, cda1Δcda3Δ displayed virulence comparable to that of cda1Δ. The virulence of each mutant correlates with the amount of chitosan produced when grown under host-mimicking culture conditions. In addition, the avirulence of cda1Δcda2Δ was followed by the induction of a protective immune response in C57BL/6 and CBA/J mice, when a live or heat-killed form of the mutant was used as a vaccine respectively. Taken together, these data imply that, in C. neoformans, coordinated activity of both Cda1 and Cda2 is essential for mediating fungal virulence.

Cryptococcus neoformans Evades Pulmonary Immunity by Modulating Xylose Precursor Transport.

Cryptococcus neoformans is a fungal pathogen that kills almost 200,000 people each year and is distinguished by abundant and unique surface glycan structures that are rich in xylose. A mutant strain of C. neoformans that cannot transport xylose precursors into the secretory compartment is severely attenuated in virulence in mice yet surprisingly is not cleared. We found that this strain failed to induce the nonprotective T helper cell type 2 (Th2) responses characteristic of wild-type infection, instead promoting sustained interleukin 12p40 (IL-12p40) induction and increased IL-17A (IL-17) production. It also stimulated dendritic cells to release high levels of proinflammatory cytokines, a behavior we linked to xylose expression. We further discovered that inducible bronchus-associated lymphoid tissue (iBALT) forms in response to infection with either wild-type cryptococci or the mutant strain with reduced surface xylose; although iBALT formation is slowed in the latter case, the tissue is better organized. Finally, our temporal studies suggest that lymphoid structures in the lung restrict the spread of mutant fungi for at least 18 weeks after infection, which is in contrast to ineffective control of the pathogen after infection with wild-type cells. These studies demonstrate the role of xylose in modulation of host response to a fungal pathogen and show that cryptococcal infection triggers iBALT formation.

Cryptococcus neoformans Chitin Synthase 3 Plays a Critical Role in Dampening Host Inflammatory Responses.

Cryptococcus neoformans infections are significant causes of morbidity and mortality among AIDS patients and the third most common invasive fungal infection in organ transplant recipients. One of the main interfaces between the fungus and the host is the fungal cell wall. The cryptococcal cell wall is unusual among human-pathogenic fungi in that the chitin is predominantly deacetylated to chitosan. Chitosan-deficient strains of C. neoformans were found to be avirulent and rapidly cleared from the murine lung. Moreover, infection with a chitosan-deficient C. neoformans strain lacking three chitin deacetylases (cda1Δcda2Δcda3Δ) was found to confer protective immunity to a subsequent challenge with a virulent wild-type counterpart. In addition to the chitin deacetylases, it was previously shown that chitin synthase 3 (Chs3) is also essential for chitin deacetylase-mediated formation of chitosan. Mice inoculated with the chs3Δ strain at a dose previously shown to induce protection with the cda1Δcda2Δcda3Δ strain die within 36 h after installation of the organism. Mortality was not dependent on viable fungi, as mice inoculated with a heat-killed preparation of the chs3Δ strain died at the same rate as mice inoculated with a live chs3Δ strain, suggesting that the rapid onset of death was host mediated, likely caused by an overexuberant immune response. Histology, cytokine profiling, and flow cytometry indicate a massive neutrophil influx in the mice inoculated with the chs3Δ strain. Mice depleted of neutrophils survived chs3Δ inoculation, indicating that death was neutrophil mediated. Altogether, these studies lead us to conclude that Chs3, along with chitosan, plays critical roles in dampening cryptococcus-induced host inflammatory responses.IMPORTANCECryptococcus neoformans is the most common disseminated fungal pathogen in AIDS patients, resulting in ∼200,000 deaths each year. There is a pressing need for new treatments for this infection, as current antifungal therapy is hampered by toxicity and/or the inability of the host's immune system to aid in resolution of the disease. An ideal target for new therapies is the fungal cell wall. The cryptococcal cell wall is different from the cell walls of many other pathogenic fungi in that it contains chitosan. Strains that have decreased chitosan are less pathogenic and strains that are deficient in chitosan are avirulent and can induce protective responses. In this study, we investigated the host responses to a chs3Δ strain, a chitosan-deficient strain, and found that mice inoculated with the chs3Δ strain all died within 36 h and that death was associated with an aberrant hyperinflammatory immune response driven by neutrophils, indicating that chitosan is critical in modulating the immune response to Cryptococcus.

Chitosan Biosynthesis and Virulence in the Human Fungal Pathogen Cryptococcus gattii.

Cryptococcus gattii R265 is a hypervirulent fungal strain responsible for the recent outbreak of cryptococcosis in Vancouver Island of British Columbia in Canada. It differs significantly from Cryptococcus neoformans in its natural environment, its preferred site in the mammalian host, and its pathogenesis. Our previous studies of C. neoformans have shown that the presence of chitosan, the deacetylated form of chitin, in the cell wall attenuates inflammatory responses in the host, while its absence induces robust immune responses, which in turn facilitate clearance of the fungus and induces a protective response. The results of the present investigation reveal that the cell wall of C. gattii R265 contains a two- to threefold larger amount of chitosan than that of C. neoformans The genes responsible for the biosynthesis of chitosan are highly conserved in the R265 genome; the roles of the three chitin deacetylases (CDAs) have, however, been modified. To deduce their roles, single and double CDA deletion strains and a triple CDA deletion strain were constructed in a R265 background and were subjected to mammalian infection studies. Unlike C. neoformans where Cda1 has a discernible role in fungal pathogenesis, in strain R265, Cda3 is critical for virulence. Deletion of either CDA3 alone or in combination with another CDA (cda1Δ3Δ or cda2Δ3Δ) or both (cda1Δ2Δ3Δ) rendered the fungus avirulent and cleared from the infected host. Moreover, the cda1Δ2Δ3Δ strain of R265 induced a protective response to a subsequent infection with R265. These studies begin to illuminate the regulation of chitosan biosynthesis of C. gattii and its subsequent effect on fungal virulence.IMPORTANCE The fungal cell wall is an essential organelle whose components provide the first line of defense against host-induced antifungal activity. Chitosan is one of the carbohydrate polymers in the cell wall that significantly affects the outcome of host-pathogen interaction. Chitosan-deficient strains are avirulent, implicating chitosan as a critical virulence factor. C. gattii R265 is an important fungal pathogen of concern due to its ability to cause infections in individuals with no apparent immune dysfunction and an increasing geographical distribution. Characterization of the fungal cell wall and understanding the contribution of individual molecules of the cell wall matrix to fungal pathogenesis offer new therapeutic avenues for intervention. In this report, we show that the C. gattii R265 strain has evolved alternate regulation of chitosan biosynthesis under both laboratory growth conditions and during mammalian infection compared to that of C. neoformans.

Induction of memory-like dendritic cell responses in vivo.

Dendritic cells (DCs), a vital component of the innate immune system, are considered to lack antigen specificity and be devoid of immunological memory. Strategies that can induce memory-like responses from innate cells can be utilized to elicit protective immunity in immune deficient persons. Here we utilize an experimental immunization strategy to modulate DC inflammatory and memory-like responses against an opportunistic fungal pathogen that causes significant disease in immunocompromised individuals. Our results show that DCs isolated from protectively immunized mice exhibit enhanced transcriptional activation of interferon and immune signaling pathways. We also show long-term memory-like cytokine responses upon subsequent challenge with the fungal pathogen that are abrogated with inhibitors of specific histone modifications. Altogether, our study demonstrates that immunization strategies can be designed to elicit memory-like DC responses against infectious disease.

An Automated Assay to Measure Phagocytosis of Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis, which kills 200,000 individuals worldwide each year. It is ubiquitous in the environment and is first inhaled into the lungs of the host, where it is taken up by phagocytes. The interaction of these fungal cells with host phagocytes, therefore, is a critical step in the pathogenesis of this disease. One characteristic of this initial step in host-pathogen interactions is the avidity with which fungal cells are taken up by phagocytes, described by the phagocytic index. In this chapter, we detail a high-throughput method of directly assessing the phagocytic index of fungal cells using an imaging-based paradigm. By automating image collection and processing, this method permits rapid assessment of this critical host interaction. © 2019 by John Wiley & Sons, Inc.

IFN-γ immune priming of macrophages in vivo induces prolonged STAT1 binding and protection against Cryptococcus neoformans.

Development of vaccines against opportunistic infections is difficult as patients most at risk of developing disease are deficient in aspects of the adaptive immune system. Here, we utilized an experimental immunization strategy to induce innate memory in macrophages in vivo. Unlike current trained immunity models, we present an innate memory-like phenotype in macrophages that is maintained for at least 70 days post-immunization and results in complete protection against secondary challenge in the absence of adaptive immune cells. RNA-seq analysis of in vivo IFN-γ primed macrophages revealed a rapid up-regulation of IFN-γ and STAT1 signaling pathways following secondary challenge. The enhanced cytokine recall responses appeared to be pathogen-specific, dependent on changes in histone methylation and acetylation, and correlated with increased STAT1 binding to promoter regions of genes associated with protective anti-fungal immunity. Thus, we demonstrate an alternative mechanism to induce macrophage innate memory in vivo that facilitates pathogen-specific vaccine-mediated immune responses.

LysMD3 is a type II membrane protein without an in vivo role in the response to a range of pathogens.

Germline-encoded receptors recognizing common pathogen-associated molecular patterns are a central element of the innate immune system and play an important role in shaping the host response to infection. Many of the innate immune molecules central to these signaling pathways are evolutionarily conserved. LysMD3 is a novel molecule containing a putative peptidoglycan-binding domain that has orthologs in humans, mice, zebrafish, flies, and worms. We found that the lysin motif (LysM) of LysMD3 is likely related to a previously described peptidoglycan-binding LysM found in bacteria. Mouse LysMD3 is a type II integral membrane protein that co-localizes with GM130+ structures, consistent with localization to the Golgi apparatus. We describe here two lines of mLysMD3-deficient mice for in vivo characterization of mLysMD3 function. We found that mLysMD3-deficient mice were born at Mendelian ratios and had no obvious pathological abnormalities. They also exhibited no obvious immune response deficiencies in a number of models of infection and inflammation. mLysMD3-deficient mice exhibited no signs of intestinal dysbiosis by 16S analysis or alterations in intestinal gene expression by RNA sequencing. We conclude that mLysMD3 contains a LysM with cytoplasmic orientation, but we were unable to define a physiological role for the molecule in vivo.

Induction of Broad-Spectrum Protective Immunity against Disparate Cryptococcus Serotypes.

Cryptococcosis is a fungal disease caused by multiple Cryptococcus serotypes; particularly C. neoformans (serotypes A and D) and C. gattii (serotypes B and C). To date, there is no clinically available vaccine to prevent cryptococcosis. Mice given an experimental pulmonary vaccination with a C. neoformans serotype A strain engineered to produce interferon-γ, denoted H99γ, are protected against a subsequent otherwise lethal experimental infection with C. neoformans serotype A. Thus, we determined the efficacy of immunization with C. neoformans strain H99γ to elicit broad-spectrum protection in BALB/c mice against multiple disparate Cryptococcus serotypes. We observed significantly increased survival rates and significantly decreased pulmonary fungal burden in H99γ immunized mice challenged with Cryptococcus serotypes A, B, or D compared to heat-killed H99γ (HKH99γ) immunized mice. Results indicated that prolonged protection against Cryptococcus serotypes B or D in H99γ immunized mice was CD4+ T cell dependent and associated with the induction of predominantly Th1-type cytokine responses. Interestingly, immunization with H99γ did not elicit greater protection against challenge with the Cryptococcus serotype C tested either due to low overall virulence of this strain or enhanced capacity of this strain to evade host immunity. Altogether, these studies provide "proof-of-concept" for the development of a cryptococcal vaccine that provides cross-protection against multiple disparate serotypes of Cryptococcus.

RNA Interference Screening Reveals Host CaMK4 as a Regulator of Cryptococcal Uptake and Pathogenesis.

Cryptococcus neoformans, the causative agent of cryptococcosis, is an opportunistic fungal pathogen that kills over 200,000 individuals annually. This yeast may grow freely in body fluids, but it also flourishes within host cells. Despite extensive research on cryptococcal pathogenesis, host genes involved in the initial engulfment of fungi and subsequent stages of infection are woefully understudied. To address this issue, we combined short interfering RNA silencing and a high-throughput imaging assay to identify host regulators that specifically influence cryptococcal uptake. Of 868 phosphatase and kinase genes assayed, we discovered 79 whose silencing significantly affected cryptococcal engulfment. For 25 of these, the effects were fungus specific, as opposed to general alterations in phagocytosis. Four members of this group significantly and specifically altered cryptococcal uptake; one of them encoded CaMK4, a calcium/calmodulin-dependent protein kinase. Pharmacological inhibition of CaMK4 recapitulated the observed defects in phagocytosis. Furthermore, mice deficient in CaMK4 showed increased survival compared to wild-type mice upon infection with C. neoformans This increase in survival correlated with decreased expression of pattern recognition receptors on host phagocytes known to recognize C. neoformans Altogether, we have identified a kinase that is involved in C. neoformans internalization by host cells and in host resistance to this deadly infection.

Antifungal Activity of Plasmacytoid Dendritic Cells against Cryptococcus neoformans In Vitro Requires Expression of Dectin-3 (CLEC4D) and Reactive Oxygen Species.

Conventional dendritic cells (cDCs) are critical for protection against pulmonary infection with the opportunistic fungal pathogen Cryptococcus neoformans; however, the role of plasmacytoid dendritic cells (pDCs) is unknown. We show for the first time that murine pDCs have direct activity against C. neoformans via reactive oxygen species (ROS), a mechanism different from that employed to control Aspergillus fumigatus infections. The anticryptococcal activity of murine pDCs is independent of opsonization but appears to require the C-type lectin receptor Dectin-3, a receptor not previously evaluated during cryptococcal infections. Human pDCs can also inhibit cryptococcal growth by a mechanism similar to that of murine pDCs. Experimental pulmonary infection of mice with a C. neoformans strain that induces protective immunity demonstrated that recruitment of pDCs to the lungs is CXCR3 dependent. Taken together, our results show that pDCs inhibit C. neoformans growth in vitro via the production of ROS and that Dectin-3 is required for optimal growth-inhibitory activity.

Cryptococcus and Phagocytes: Complex Interactions that Influence Disease Outcome.

Cryptococcus neoformans and C. gattii are fungal pathogens that cause life-threatening disease. These fungi commonly enter their host via inhalation into the lungs where they encounter resident phagocytes, including macrophages and dendritic cells, whose response has a pronounced impact on the outcome of disease. Cryptococcus has complex interactions with the resident and infiltrating innate immune cells that, ideally, result in destruction of the yeast. These phagocytic cells have pattern recognition receptors that allow recognition of specific cryptococcal cell wall and capsule components. However, Cryptococcus possesses several virulence factors including a polysaccharide capsule, melanin production and secretion of various enzymes that aid in evasion of the immune system or enhance its ability to thrive within the phagocyte. This review focuses on the intricate interactions between the cryptococci and innate phagocytic cells including discussion of manipulation and evasion strategies used by Cryptococcus, anti-cryptococcal responses by the phagocytes and approaches for targeting phagocytes for the development of novel immunotherapeutics.

STAT1 signaling within macrophages is required for antifungal activity against Cryptococcus neoformans.

Cryptococcus neoformans, the predominant etiological agent of cryptococcosis, is an opportunistic fungal pathogen that primarily affects AIDS patients and patients undergoing immunosuppressive therapy. In immunocompromised individuals, C. neoformans can lead to life-threatening meningoencephalitis. Studies using a virulent strain of C. neoformans engineered to produce gamma interferon (IFN-γ), denoted H99γ, demonstrated that protection against pulmonary C. neoformans infection is associated with the generation of a T helper 1 (Th1)-type immune response and signal transducer and activator of transcription 1 (STAT1)-mediated classical (M1) macrophage activation. However, the critical mechanism by which M1 macrophages mediate their anti-C. neoformans activity remains unknown. The current studies demonstrate that infection with C. neoformans strain H99γ in mice with macrophage-specific STAT1 ablation resulted in severely increased inflammation of the pulmonary tissue, a dysregulated Th1/Th2-type immune response, increased fungal burden, deficient M1 macrophage activation, and loss of protection. STAT1-deficient macrophages produced significantly less nitric oxide (NO) than STAT1-sufficient macrophages, correlating with an inability to control intracellular cryptococcal proliferation, even in the presence of reactive oxygen species (ROS). Furthermore, macrophages from inducible nitric oxide synthase knockout mice, which had intact ROS production, were deficient in anticryptococcal activity. These data indicate that STAT1 activation within macrophages is required for M1 macrophage activation and anti-C. neoformans activity via the production of NO.

STAT1 signaling is essential for protection against Cryptococcus neoformans infection in mice.

Nonprotective immune responses to highly virulent Cryptococcus neoformans strains, such as H99, are associated with Th2-type cytokine production, alternatively activated macrophages, and inability of the host to clear the fungus. In contrast, experimental studies show that protective immune responses against cryptococcosis are associated with Th1-type cytokine production and classical macrophage activation. The protective response induced during C. neoformans strain H99γ (C. neoformans strain H99 engineered to produce murine IFN-γ) infection correlates with enhanced phosphorylation of the transcription factor STAT1 in macrophages; however, the role of STAT1 in protective immunity to C. neoformans is unknown. The current studies examined the effect of STAT1 deletion in murine models of protective immunity to C. neoformans. Survival and fungal burden were evaluated in wild-type and STAT1 knockout (KO) mice infected with either strain H99γ or C. neoformans strain 52D (unmodified clinical isolate). Both strains H99γ and 52D were rapidly cleared from the lungs, did not disseminate to the CNS, or cause mortality in the wild-type mice. Conversely, STAT1 KO mice infected with H99γ or 52D had significantly increased pulmonary fungal burden, CNS dissemination, and 90-100% mortality. STAT1 deletion resulted in a shift from Th1 to Th2 cytokine bias, pronounced lung inflammation, and defective classical macrophage activation. Pulmonary macrophages from STAT1 KO mice exhibited defects in NO production correlating with inefficient inhibition of fungal proliferation. These studies demonstrate that STAT1 signaling is essential not only for regulation of immune polarization but also for the classical activation of macrophages that occurs during protective anticryptococcal immune responses.

Vaccine-mediated immune responses to experimental pulmonary Cryptococcus gattii infection in mice.

Cryptococcus gattii is a fungal pathogen that can cause life-threatening respiratory and disseminated infections in immune-competent and immune-suppressed individuals. Currently, there are no standardized vaccines against cryptococcosis in humans, underlying an urgent need for effective therapies and/or vaccines. In this study, we evaluated the efficacy of intranasal immunization with C. gattii cell wall associated (CW) and/or cytoplasmic (CP) protein preparations to induce protection against experimental pulmonary C. gattii infection in mice. BALB/c mice immunized with C. gattii CW and/or CP protein preparations exhibited a significant reduction in pulmonary fungal burden and prolonged survival following pulmonary challenge with C. gattii. Protection was associated with significantly increased pro-inflammatory and Th1-type cytokine recall responses, in vitro and increased C. gattii-specific antibody production in immunized mice challenged with C. gattii. A number of immunodominant proteins were identified following immunoblot analysis of C. gattii CW and CP protein preparations using sera from immunized mice. Immunization with a combined CW and CP protein preparation resulted in an early increase in pulmonary T cell infiltrates following challenge with C. gattii. Overall, our studies show that C. gattii CW and CP protein preparations contain antigens that may be included in a subunit vaccine to induce prolonged protection against pulmonary C. gattii infection.

Characterization of IL-22 and antimicrobial peptide production in mice protected against pulmonary Cryptococcus neoformans infection.

Cryptococcus neoformans is a significant cause of fungal meningitis in patients with impaired T cell-mediated immunity (CMI). Experimental pulmonary infection with a C. neoformans strain engineered to produce IFN-γ, H99γ, results in the induction of Th1-type CMI, resolution of the acute infection, and protection against challenge with WT Cryptococcus. Given that individuals with suppressed CMI are highly susceptible to pulmonary C. neoformans infection, we sought to determine whether antimicrobial peptides were produced in mice inoculated with H99γ. Thus, we measured levels of antimicrobial peptides lipocalin-2, S100A8, S100A9, calprotectin (S100A8/A9 heterodimer), serum amyloid A-3 (SAA3), and their putative receptors Toll-like receptor 4 (TLR4) and the receptor for advanced glycation end products (RAGE) in mice during primary and recall responses against C. neoformans infection. Results showed increased levels of IL-17A and IL-22, cytokines known to modulate antimicrobial peptide production. We also observed increased levels of lipocalin-2, S100A8, S100A9 and SAA3 as well as TLR4(+) and RAGE(+) macrophages and dendritic cells in mice inoculated with H99γ compared with WT H99. Similar results were observed in the lungs of H99γ-immunized, compared with heat-killed C. neoformans-immunized, mice following challenge with WT yeast. However, IL-22-deficient mice inoculated with H99γ demonstrated antimicrobial peptide production and no change in survival rates compared with WT mice. These studies demonstrate that protection against cryptococcosis is associated with increased production of antimicrobial peptides in the lungs of protected mice that are not solely in response to IL-17A and IL-22 production and may be coincidental rather than functional.

Mechanisms of dendritic cell lysosomal killing of Cryptococcus.

Cryptococcus neoformans is an opportunistic pulmonary fungal pathogen that disseminates to the CNS causing fatal meningitis in immunocompromised patients. Dendritic cells (DCs) phagocytose C. neoformans following inhalation. Following uptake, cryptococci translocate to the DC lysosomal compartment and are killed by oxidative and non-oxidative mechanisms. DC lysosomal extracts kill cryptococci in vitro; however, the means of antifungal activity remain unknown. Our studies determined non-oxidative antifungal activity by DC lysosomal extract. We examined DC lysosomal killing of cryptococcal strains, anti-fungal activity of purified lysosomal enzymes, and mechanisms of killing against C. neoformans. Results confirmed DC lysosome fungicidal activity against all cryptococcal serotypes. Purified lysosomal enzymes, specifically cathepsin B, inhibited cryptococcal growth. Interestingly, cathepsin B combined with its enzymatic inhibitors led to enhanced cryptococcal killing. Electron microscopy revealed structural changes and ruptured cryptococcal cell walls following treatment. Finally, additional studies demonstrated that osmotic lysis was responsible for cryptococcal death.

Protective immunity against pulmonary cryptococcosis is associated with STAT1-mediated classical macrophage activation.

Experimental pulmonary Cryptococcus neoformans infection in BALB/c mice is associated with polarized Th2-type cytokine production, alternative macrophage activation, and severe bronchopneumonia. In contrast, pulmonary infection with a C. neoformans strain that secretes IFN-γ, H99γ, elicits Th1-type cytokine production and classical macrophage activation. Additionally, mice infected with H99γ resolve the acute infection and are subsequently protected against challenge with wild-type C. neoformans. The present study characterizes macrophage activation during the protective response to wild-type C. neoformans in mice previously immunized with H99γ. We observed increased pulmonary Th1-type cytokine production in lung homogenates and classical macrophage activation as evidenced by enhanced expression of inducible NO synthase in the lungs of H99γ-immunized mice compared with mice given a nonprotective immunization with heat-killed C. neoformans (HKCn). Furthermore, macrophages isolated from H99γ-immunized mice on day 7 postchallenge and cultured in vitro were fungistatic against C. neoformans, whereas cryptococcal growth was uncontrolled within macrophages from HKCn-immunized mice. Th2-type cytokine production and induction of alternatively activated macrophages were also observed in lungs of HKCn-immunized mice during rechallenge. Gene expression arrays showed that classical macrophage activation during challenge infection in H99γ-immunized mice was associated with induction of the transcription factor STAT1 and its downstream targets IFN regulatory factor-1, suppressor of cytokine signaling-1, CXCL9, and CXCL10. These studies demonstrate that protective responses to C. neoformans challenge in immunized mice include classical macrophage activation and enhanced macrophage fungistasis of C. neoformans yeasts. Finally, the classical activation phenotype of protective anticryptococcal macrophages is likely mediated via STAT1 signal transduction pathways.

Vaccine and immunotherapeutic approaches for the prevention of cryptococcosis: lessons learned from animal models.

Cryptococcus neoformans and C. gattii, the predominant etiological agents of cryptococcosis, can cause life-threatening infections of the central nervous system in immunocompromised and immunocompetent individuals. Cryptococcal meningoencephalitis is the most common disseminated fungal infection in AIDS patients, and C. neoformans remains the third most common invasive fungal infection among organ transplant recipients. Current anti-fungal drug therapies are oftentimes rendered ineffective due to drug toxicity, the emergence of drug resistant organisms, and/or the inability of the host's immune defenses to assist in eradication of the yeast. Therefore, there remains an urgent need for the development of immune-based therapies and/or vaccines to combat cryptococcosis. Studies in animal models have demonstrated the efficacy of various vaccination strategies and immune therapies to induce protection against cryptococcosis. This review will summarize the lessons learned from animal models supporting the feasibility of developing immunotherapeutics and vaccines to prevent cryptococcosis.