The Critical Role of Carbon Metabolism

1 THE CRITICAL ROLE OF CARBON METABOLISM IN THE VIRULENCE OF CRYPTOCOCCUS NEOFORMANS Introduction The Cryptococcus neoformans species complex consists of unicellular encapsulated fungi that can replicate with buds and share many common characteristics with Saccharomyces cerevisiae (Aslanyan et al., 2017). The obligate aerobe most commonly isolated from bird droppings, C. neoformans is a common environmental fungus found worldwide. Cryptococcal spores are generally in the air, and most individuals are frequently exposed to this fungus throughout their lives (Goldman et al., 2001). In healthy individuals, a functioning immune system is able to effectively eliminate spores and yeasts before the onset of disease with near-perfect efficiency. Cryptococcosis: Opportunistic Infections While C. neoformans rarely causes long-term complications or systemic disease in immunocompetent individuals, it sometimes results in fungal pneumonia that is overcome by a functioning immune system. However, cryptococcal infection (i.e. cryptococcosis) is the leading cause of meningoencephalitis in immunosuppressed patients worldwide (Dambuza et al., 2018). The highest incidence of disease occurs among individuals with HIV/AIDS, largely due to the lack of CD4+ T-helper cells present in the bloodstream. However, any individual with immunodeficiency can develop cryptococcosis, with at-risk groups including chemotherapy patients, transplant recipients with immunosuppressive anti-rejection drugs, and individuals using chronic corticosteroids for autoimmune disorders (Lin, Huang, Mitchell, & Heitman, 2006). Seven species of Cryptococcus can cause disease, although C. neoformans is the most clinically significant strain with effects ranging from localized skin infections to widespread systemic disease (Hagen et al., 2015). C. neoformans are invasive and difficult to treat, largely due to their ability to cross the blood-brain barrier and cause CNS infections. Antifungal drug options are limited and expensive, and although widely available in developed countries, these drugs often cause dangerous side effects (Fernandes, Martho, Tofik, Vallim, & Pascon, 2015). It is estimated that more than 223,000 individuals contract cryptococcal meningitis each year, with the prognosis highly dependent on access to sophisticated medical care (Rajasingham et al., 2017). Because the epidemiology of the disease is closely related to the AIDS pandemic, most cases are concentrated in sub-Saharan Africa due to the lack of antiretroviral therapy available to patients. Because of its mortality of more than 50%, scientists continue to work towards a better understanding of C. neoformans to develop new treatments to combat the disease (Rajasingham et al., 2017). Pathogenesis Due to the body's natural immune response, individuals with a functioning immune system are generally able to fend off C. neoformans infection prior to the development of systemic disease. Studies have shown that immunosaudience in healthy individuals is particularly effective, with respiratory epithelial cells playing an important role. These cells are separated by tight junctions, which help provide a physical barrier against invading microbes (Whitsett & Alenghat, 2015). In addition, T-helper 1 (Th1) cells are essential because they mediate the protective immune response through the production of various cytokines. Specific cell-signaling cytokines that play a role in the response to C. neoformans include interleukin-2 (IL-2), IL-12, interferon gamma, and alpha tumor necrosis factor that was originally produced by dendritic cells and macrophages (Whitsett & Alenghat, 2015). While important molecules and proteins have been isolated, the exact mechanism of these responses is still an active field of study. In immunosuppressed hosts, the ability of C. neoformans to spread systemically before infection is eradicated by an immune response leads to a serious, and often fatal, disease process. Although this fungus can travel to various locations in the body, it has a specific affinity for CNS. Research has shown that there are two main pathways for the dissemination of C. neoformans: intracellular mechanisms and extracellular mechanisms (Denham & Brown, 2018). Intracellular mechanism. The intracellular mechanism of C . neoformans spread uses the migration phagocyte machinery, especially macrophages, to move from the lungs to other areas of the body. Eventually, this method, also known as the Trojan Horse mechanism, allows fungal spores to enter macrophages, where they germinate into yeast that is carried to the CNS (Charlier et al., 2009; Tseng, et al., 2012). The spores or dry cells come into contact with macrophages and dendritic cells in the mucosal lining of the lungs. These macrophages, which can control infections in healthy individuals, inadvertently facilitate the spread of disease in immunosuppressed hosts. The mechanism is threefold. First, C. neoformans is engulfed by alveolar macrophages in the lungs (Denham & Brown, 2018; Bojarczuk et al., 2016). Because a low number of CD4+ helper T cells delays the immune response, macrophages in immunosuppressed individuals are able to take up C. neoformans, but they have a very difficult time destroying them once they do. Second, microbes must be able to survive inside macrophages or phagolysosomes for long periods of time, allowing enough time to travel to distant organs and body systems. Studies have shown that this is possible due to some of the main characteristics of C. neoformans. These fungi are able to survive at very low pH, which is essential for intracellular survival (Vogel, 1969). This adaptation likely stems from the fact that bird droppings, the main environmental host of C. neoformans, are usually quite acidic. Unfortunately, C. neoformans is actually able to replicate a little faster when it is an acidic environment, making it more difficult to remove in macrophages (Diamond & Bennet, 1973). The third and final step in the development of intracellular disease is the process of macrophage escape (Tucker & Casadevall, 2002). In simple terms, C. neoformans is able to increase macrophage/phagolysosome permeability by altering phospholipase expression to increase permeability; this is achieved either by physical disruption of the membrane or through modulation of cell signaling pathways (Djordjevic, 2010). At the same time, C. neoformans temporarily alters the host actin network which has a cage-like structure. These cage-like apparatus are capable of limiting phagosomes, but allow C. neoformans to exit through small gaps in actin tissues (Johnston & May, 2010). This same mechanism can be used for lateral macrophage transfer or for escape during macrophage fission or fusion (Johnston & May, 2010). Through a series of interrelated steps, the Trojan Horse method allows C. neoformans to enter the CNS and cause lifethreatening meningitis. Extracellular mechanisms. The second mechanism of disease spread is especially extramobile. This depends on the process of transcytosis (Chang et al., 2004). Transcytosis uses vesicular transport to capture particles or microbes on the luminal side of the cell, transport them through the inside of the cell, and release particles on the basal side. In the case of inhaled C. neoformans, the organism moves through the epithelial cells of the respiratory tract before being deposited into the bloodstream. From there, C. neoformans can travel systemically. GXM, the main polysaccharide in C. neoformans capsules, is directly responsible for this process (Wang, Aisen, & Casadevall, 1995). GXM mediates C. neoformans adherence to CD14, a protein found on epithelial cell membranes (Denham & Brown 2018). Once adhered to, C. neoformans uses a separate mechanism to enter epithelial cells and cause damage that allows the cells to pass through. To date, scientists are still determining the exact biochemical interactions that facilitate this process. Virulence Factor Among microorganisms, and pathogenic fungi in particular, there are several common indicators of virulence. Specifically for C. neoformans, capsule production, melanin production, and host temperature all play a big role (Haynes et al., 2011; Gomez & Nosanchuk, 2003). In healthy individuals, frontline phagocytes, neutrophils, and dendritic cells from the innate immune response recognize and destroy cryptoctic cells. However, in immunosuppressive patients, this process is disrupted; in the regulation of inefficient or ineffective immune responses, the virulence factor C. neoformans prevents infection control and causes widespread disease (Campuzano & Wormley, 2018). Melanin production has long been recognized as a source of pathogenicity of opportunistic fungi such as C. neoformans (Gomez & Nosanchuk, 2003). These darkcolored pigments are synthesized from a variety of biological molecules, including several neurotransmitters (Wang & Casadevall, 1994). Because it is composed of insoluble polymers, melanin cannot be easily degraded by macrophages in the host, making the immune system less effective against the cells that produce it (Mednick, Nosanchuk, & Casadevall, 2005). Thus, melanization plays a key role in protecting against degradative enzymes, and has also been shown to protect against phagocytes and reactive oxygen species that are part of the immune response (Wang & Casadevall, 1994). While melanin production is often used as a common indicator of fungal pathogenicity, capsule production is often considered the most important virulence factor for C. neoformans. Polysaccharide capsules, made up of a variety of polysaccharides, function both offensively and defensively in the host environment (Agustinho, Miller, Li, & Doering, 2018). In vivo, C. neoformans can also differentiate into larger titan cells, which have thicker polysaccharide capsules and contain more chitin that provides increased strength and protection (Mukaremera et al., 2018). This external cell structure helps modulate interactions between immune cells while protecting yeast against phagocyte cells from the host (Garcia-Carnero, Perez-Garcia, Martinez-Alvarez, ReyesMartinez, & Mora-Montes, 2018). As one of the most important virulence indicators in C. neoformans, capsule production is often used as a marker in determining pathogenicity in experimental studies (Casadevall et al., 2018). Carbon Metabolism in C. neoformans In order to cause disease in the host organism, microbes must obtain the basic building blocks of life from their environment. As a result, one of the biggest challenges facing microbes is finding efficient metabolic strategies in a rapidly changing environment (Fernandes et al., 2015). Typically, C. neoformans lives in soil, decaying wood, and bird droppings, where it can obtain sufficient amounts of carbon and nitrogen from the environment (Barkal, Walsh, Botts, Beebe, & Hull, 2016). Because the ability to obtain and metabolize nutrients is directly related to the fitness of pathogens in their respective environments, the metabolism of a key nutrient, namely carbon, is necessary for virulence in C. neoformans (Ramachandra et al., 2014). Not a member of the normal human flora, C. Neoformans usually enter the human host through inhalation of dried spores, where it must adjust its metabolic strategy before systemic dispersal and entry into the SSP (Diamond & Bennett, 1973) dan (Guess et al., 2018). Understanding carbon metabolism has long been recognized as an important strategy for developing effective drug targets for specific organisms and disease processes. For example, it has previously been established that cancer cells rely on functional glycolytic pathways to grow, as carbon metabolism allows cells to multiply and increase tumor size (Vander Heiden, Cantley, & Thompson, Thus, researchers have begun to develop glycolysis inhibitors to target specific types of cancer cells and limit their growth. While glycolytic enzymes are nearly identical across species and cell types, slight differences in nucleotide sequences provide potential species-specific targets for drug development. Proliferation is not only important for the development of malignant diseases, but strong similarities are seen between neoplastic processes and eukaryotic pathogens during the invasion (Vander Heiden et al., 2009). Thus, understanding the complicated role of carbon metabolism in the pathogenesis of C. neoformans infection provides valuable insights into potential disease treatment methods. Effects of Microenvironmental Changes Throughout the pathogenesis of the cryptococcus, C. neoformans must adapt to various microenvironments in order to survive and cause disease in the host. As mentioned earlier, C. neoformans It lives and thrives in a variety of ecological settings, but is most commonly isolated from decaying bark and soils containing bird droppings. In the environment, nitrogen and atmospheric oxygen are abundant. Additional nutrients such as carbon are easily obtained from decaying organic matter in the soil. While C. neoformans is a known human pathogen and the majority of individuals are exposed to this relatively common fungus during childhood, it is not a member of the normal human flora (Perfect, 2006; Diamond & Bennet, 1973). Metabolism during early lung infection. In addition to facilitating breathing and gas exchange within the human body, the lungs also provide a vital mucosal barrier that serves to protect against millions of microbes found in the air at any given moment. Contact with such microbes is inevitable, and C. neoformans are no exception (Denham & Brown, 2018). Thus, cryptococcal infections generally start in the lungs through inhalation of dried cells or spores. After the initial infection, the spores can germinate into yeast that spreads throughout the body, exist indefinitely as granulomas in the lungs, or can be eradicated by the body's immune response. Because the microenvironment of mammalian lungs is fundamentally different from the soil where C. neoformans Most commonly, these mushrooms have to adapt to changes in nutrient availability. While nitrogen and oxygen are still present in the lung tissues, the availability of carbon elements is significantly reduced compared to the external environment (Barelle et al., 2006). Thus, C. neoformans altering the transcription of genes that control carbon metabolism in a way that shifts the focus of metabolism to carbon procurement from alternative sources. Glyoxylate shunt, citric acid cycling, and gluconeogenesis are all regulated at the same time that glycolysis is derived (Barelle et al., 2006). While glycolysis is certainly the most efficient way to obtain energy from environmental carbon, increased regulation of gluconeogenesis provides C. neoformans with an alternative carbon source that can be used to survive in the lungs of mammals. Glycolysis and gluconeogenesis during early lung infection. Present in all eukaryotic organisms, the glycolytic pathway is one of the most frequently studied metabolic processes in biology. It plays a central role in carbon metabolism, breaking down glucose into pyruvate which can be diverted into the citric acid cycle. The energy from this process is then used to induce oxidative fermentation in mitochondria, which eventually uses the proton gradient to produce cellular energy in the form of ATP (Hu, Cheng, Sham, Perfect, & Kronstad, 2008). ATP provides energy for various cellular processes and is necessary for any organism to survive, including C. neoformans. However, it has been shown that pyruvate kinase and phosphofructokinase, two enzymes that catalyze irreversible steps in glycolysis, are derived in C. neoformans cells that grow in mammalian lung tissue (Hu et al., 2008). This indicates a poor environment, and in particular glucose. Interestingly, two knockout strains of pyruvate kinase and hexokinase separate from C. neoformans showed attenuated virulence in the murin inhalation model but satisfactory persistence within the host lungs (Price et al., 2011). Because these two enzymes function as irreversible steps in the glycolytic pathway, glycolysis can be discarded as a means of carbon acquisition during early lung infections. In addition, transcription factors known to regulate glycolysis are lowered during early lung infections, which further corroborates this point (Kronstad et al., 2011). Alternatively, gluconeogenesis, an anabolic process that uses various forms of carbon to synthesize glucose, is regulated (Hu et al., 2008). Gluconeogenesis uses many precursors depending on the availability of different carbon sources, although some of the most common include lactate, glycerol, and amino acids such as alanine and glutamine. Surfactants (especially phosphatidylcholine and phosphatidylglycerol) are also present in the lungs of mammals and are potential substrates for gluconeogenesis. Several different processes are used to form pyruvate from each of these molecules, which accumulate in the mitochondria of hepatocytes and other specialized cell types. In mitochondria, pyruvate carboxylase converts pyruvate to oxaloacetate; A series of ten reactions that the additional enzymes catalyze then followed, leading to the formation of glucose. While glycolysis and gluconeogenesis are opposite metabolic processes, three different enzymes in gluconeogenesis are used to catalyze irreversible steps in the glycolytic pathway. One such enzyme, phosphoenolpyruvate carboxycinase I (PCK1) is a key regulatory step in gluconeogenesis and catalyzes the formation of phosphoenolpyruvate from oxaloacetate. A study looking at differential expression of PCK1 transcripts between C. neoformans cells grown in mammalian lung tissue versus in vitro cultures showed increased regulation of this enzyme during early infection (Hu et al., 2008). Subsequent studies showed significantly attenuating persistence in rabbit models of CNS disease, further corroborating that gluconeogenesis plays a central role in the early pathogenesis of cryptococcal infections (Price et al., 2011). Use of glyoxylate shunts during early lung infections. In addition, C. Neoformans are able to catabolize lipids and other complex carbon compounds for use as alternative energy sources in early infections. Fluid collected from mammalian lung tissue by bronchoalveolar rinsing is rich in phospholipids (Rooney, Young, & Mendelson, 1994). Transcriptional profiles showed temporarily elevated lipid and fatty acid catabolism genes in lung cultures infected with C. neoformans when compared to CNS samples or in vitro laboratory cultures (Hu et al., 2008). Combined with upregulation of PCK1, this indicates significant glucose limitation during early lung infection within the host (Hu et al., 2008). Shunt glycyrrhagic is a metabolic process similar to the citric acid cycle that plants, protists, and fungi use to produce macromolecules from alternative carbon sources. Specifically, this anabolic process uses a series of enzymatic reactions to convert 2-carbon compounds such as acetate into glucose, which can then be used for a variety of cellular functions. Since acetate is one of the compounds produced in the βoxidation of fatty acids, glyoxylate shunt is closely related to lipid catabolism. Five citric acid cycle enzymes are used in this process, with the first two steps identical. The acetate produced by the oxidation of fatty acids β enters a cycle bound to the tiol group CoA, where it becomes citrate, cis-aconitate, and then isocitrate. During the glyoxylate shunt, isocithate is then broken down into glyoxate and succinate by the enzyme isocitrate lyase. It differs from the citric acid cycle, in which isocithate dehydrogenase serves to produce α-ketoglutarate while releasing a single molecule of carbon dioxide (CO2). Both processes eventually produce oxaloacetate which can be converted into phosphoenolpyruvate for use in gluconeogenesis. In summary, the benefit of glyoxylate shunts is that they allow organisms to take advantage of the oxidation β fatty acids through a process that bypasses the CO2 release steps in the citric acid cycle. Because carbon, and specifically the carbon found in the form of glucose, is essential for many cellular functions, upregulation of the glyoxylate shunt during early lung infection allows C. neoformans to produce glucose through gluconeogenesis without sacrificing carbon in the form of CO2 (see Figure 1) (Hu et al., 2008). Glyoxylate shunts provide an efficient alternative means of carbon metabolism in Candida albicans and Aspergillus fumigatus that are necessary for survival (Lorenz & Fink, 2001; Olivas et al., 2008). However, functional glyoxylate shunts are not required for virulence in C. neoformans, making this species unique among pathogenic fungi (Rude, Toffaletti, Cox & Perfect, 2002). Metabolism during systemic infections. In higher eukaryotes such as mammals, Figure 1. The relationship between glyoxylate shunt and citric acid cycle. By eliminating the steps of the citric acid cycle that produce CO2, the glyoxylate shunt allows for the catabolism of fatty acids without carbon loss. Adapted from "The main role of isocitrate lyase and malat synthase in bacterial and fungal pathogenesis" by M. F. Dunn, J. A. Ramirez-Trujillo, and I. Hernandez-Lucas, 2009, Microbiology, 155(10), p. 3167. The CNS is made up of the brain and spinal cord. Often thought of as the control center of the human body, CNS is responsible for receiving incoming neuron signals from the peripheral nervous system (PNS), interpreting them, and sending the signals back to the efferent PNS to get a response. Due to its vital role in important functions such as higherorder thinking, controlling reflexes, and maintaining homeostasis, the transport of molecules in and out of the CNS is highly regulated. This poses new challenges for disseminated cryptococcal cells that have traveled from the lungs to the CNS, as they have to adapt to other microenvironments to survive (Barelle et al., 2006). While C. neoformans faces a nutrient-poor environment when inhaled in the lungs, oxygen and glucose are more abundant within the CNS and especially in the brain. However, because CNS cells quickly consume glucose and oxygen, these resources are not always easily accessible. Due to the high energy demand of actively multiplying cells, ATP production through oxidative phosphorylation in mitochondria C. neoformans is essential for survival (Ingavale et al., 2008). Indeed, isocitrate dehydrogenase is regulated in the rabbit cerebrospinal fluid (CSF) model of cryptococcal meningitis; NADH dehydrogenase components, cytochrome c subunits, and ATP synthase are also regulated, indicating an increase in energy demand in CNS (Steen et al., 2003). Glycolysis and gluconeogenesis. In contrast to the emphasis on gluconeogenesis seen in the mouse inhalation model of early lung infection, the persistence of C. neoformans within CNS is highly dependent on glycolysis. Again, this is demonstrated by using a strategic hexacase kinase double-elimination strain (hxk1∆ hxk2∆), which blocks the entry of glucose into the glycolytic pathway (Price et al., 2011). Hecose kinase activity is necessary not only for initial infection in the lungs, but also for persistence in the CNS (Price et al., 2011). In addition, the mutant knockout pyruvate kinase (PYK1) was used to block glucose escape from the glycolytic pathway, with the pyk1Δ strain showing significantly reduced virulence in rabbit CSF models. Mice infected with the pyk1Δ mutant survive for approximately 6 months, while mice infected with the hxk1Δ hxk2Δ mutant survive for approximately six weeks (Price et al., 2011). Both strains have impaired ability to utilize glucose, suggesting that glycolysis is a major player in C. neoformans' ability to metabolize carbon after the fungus reaches the CNS (Price et al., 2011). Interestingly, this evidence is corroborated by a similar central carbon metabolism pattern in C. albicans. The pyk1Δ strain of C. albicans showed significantly attenuated virulence and increased fungal load in a model of murin inhalation disease, consistent with the attenuated virulence seen in the model of C. neoformans (Barelle et al., 2006). While gluconeogenesis is essential for early lung infection and the spread of cryptoctous cells from the lungs to the CNS, functional gluconeogenesis pathways are not essential for persistence within the CNS itself. The researchers used a real-time PCRbased protocol to determine PCK1 expression in rabbit CSF versus ex vivo human CSF and in vitro cultures of C. neoformans.PCK1 expression increased in the rabbit CSF model in vivo compared to other samples, suggesting an increased rate of glucose synthesis in the CNS during cryptococcal pathogenesis. However, this same study created a knockout strain of pck1Δ that maintained its virulence despite lacking a functional glukoneogenic pathway. This serves to show that while gluconeogenesis is regulated in glucose-poor CSF environments, it is not the primary way C. neoformans utilizes alternative carbon sources during CNS infection (Price et al., 2011). Inositol as an alternative carbon source. In addition to glucose, C. neoformans is able to use an unfavored source of carbon as a means of obtaining energy and performing cellular functions within the CNS. Inositol, a carbocyclic-alcohol sugar made from glucose, serves as the structural basis for many important secondary messengers such as inositol triphosphate. Thus, these carbon-based compounds are present in high concentrations within the human brain, where many of the second messengers are synthesized and released. Interestingly, C. neoformans increases the regulation of inositol monophosphatase to provide an alternative means of carbon and energy acquisition in CNS (see Figure 2) (Steen et al., 2003). For the record, inositol production plays a direct role in intracellular proliferation and melanin production by C. neoformans (Luberto et al., 2001; Barnett 1976). Carbon Regulation in C. neoformans As previously established, C. neoformans' ability to adapt to a variety of host microenvironments is critical to its ability to cause disease in human hosts (Barelle et al., 2006). Specifically, these mushrooms have several molecular mechanisms that direct and regulate carbon metabolism depending on the nutrients available. In this way, C. neoformans are able to survive and reproduce despite environmental pressures and changes. The CCAAT binding complex (HAP) in S. cerevisiae is a complex of four separate proteins (Hap2/Hap3/Hap4/Hap5) that shift carbon metabolism from glucose utilization to production when environmental glucose has been depleted. These are Figure 2. The production of inositol through inositol monophosphatase (I-1-P phosphatase). Inositol monophosphatase catalyzes the conversion of inositol monophosphate to myoinositol, which can be used in the production of second messengers or as an alternative carbon source for pathogenic fungi residing within the CNS. Adapted from "Cloning and expression of inositol monophosphatase gene from Methanococcus jannaschii and enzyme characterization" by L. Chen and M. F. Roberts, 1998, Applied and Environmental Microbiology, 64(7), p. 2610. achieved by lowering the regulation of glycolysis and increasing the regulation of genes involved in the citric acid cycle and cytochrome subunits found in electron transport chains (Nehlin, Carlberg, & Ronne, 1991). Because Hap4 is the main regulatory subunit within the Hap Complex S . cerevisiae, HapX orthopedists, identified in C. neoformans (Caza, Hu, Price, Perfect, & Kronstad, 2016). MIG Mig1, a transcription factor originally found in S. cerevisiae, is a well-studied zinc Finger proteins are known for their role as global catabolite repressors. Because Mig1p is expressed at high levels when glucose is abundant, the Mig1p complex suppresses genes involved in alternative carbon acquisition and metabolism. Thus, Mig1p ensures that glucose is used as the main source of carbon when available. For the record, additional data suggest that Mig1p negatively affects the expression of oxidative phosphorylation and iron transport proteins in glucose-restricted environments (Pir et al., 2008). Mig1p orthopedists were identified in C. neoformans, with a new candidate gene identified as MIG1. It is interesting to note that the regulatory Hap4p subunit in S. cerevisiae acts as an activator of MIG1 transcription while the orthopedist C. neoformans, HapX, serves to suppress transcription. Although genomic analysis showed lower-than-expected conservation of nucleotides between these genes in S. cerevisiae and C. neoformans, the protein's zinc finger domain was highly conserved (Price et al., 2016). Several different methods were used to compare two orthopedists, which showed a highly preserved functional trait between the two organisms. The mutant strain of MIG1 deletion (mig1Δ) was then analyzed through microarray analysis in an attempt to determine the effect of MIG1 on transcriptome C. neoformants (Caza et al., 2016). As expected, mig1Δ cells showed increased expression of genes involved in alternative carbon metabolism when compared to wild type (WT). Interestingly, differential expression was also noted between cells grown on iron-rich and iron-poor media, suggesting a possible link between MIG1 function and iron availability. Further experiments linked the functional MIG1 gene to reactive oxygen species resistance (ROS), especially in cultures grown at 37°C (Caza et al., 2016). This is important to note, as the temperature of the human body generally ranges from 36.1°C to 37.2°C. The mig1Δ C. neoformans strain showed relatively unaffected persistence in a mouse macrophage model (Caza et al., 2016). Both the mig1Δ strain and the hapxΔ strain showed comparable fungal loads in lung tissue when compared to WT cells. However, when it lacked MIG1 and HAPX function, survival was noticeably reduced, and the fungal load was much smaller than that of WT or mig1Δ cells in all types of tissues examined except the brain. These data suggest that the interaction between the Hap complex, specifically the HapX subunit, and Mig1 is essential for nutrient sensing and virulence in cryptococcus pathogenesis. Importantly, the elimination of MIG1 in C. neoformans has been directly associated with increased susceptibility to antifungal compounds such as fluconazole and tetracycline, the current frontline treatment for cryptococcal infections. Thus, MIG1 has been identified as a potential drug target. Strategic drugs that target MIG1, when given in conjunction with current treatments such as fluconazole and tetracycline, can have a positive impact on the effectiveness of current treatment options. SNF1 While Mig1 plays an important role in enforcing the use of preferential glucose where available, Snf1 is a well-characterized protein in the S. cerevisiae that has the orthodontist's prediction gene (SNF1) in C. neoformans. As a serine-threonine protein kinase, Snf1 selectively phosphorylates serine or threonine residues on its specific substrate; in this way, Snf1 reverses the repression of glucose-dependent genes involved in the metabolism of secondary carbon sources (see Figure 3). This allows C. neoformans to metabolize alternative carbon sources when necessary, such as in the lung environment of glucose-poor mammals in early infections. Carbon metabolism is a complex and integrated process, and the glucose sensing function of the Snf1/AMPK pathway plays a role in regulating the opposite function of Mig1. As mentioned earlier, the Mig1 system is activated in glucose-rich environments, and alternatively the Snf1/AMPK pathway is activated in nutrient-poor settings (Caza et al., 2016). Deletion of the SNF1 gene in C. neoformans yielded interesting results. Melanin production is significantly disrupted, virulence decreases, and the utilization of alternative carbon sources is altered. Since the production of capsules depends on the use of carbon, the regulation of this essential virulence factor is also disrupted. Cryptococcal cells are still capable of spreading to the CNS, although they cannot survive for a long time after they arrive (Yang et al., 2010). SNF1-targeted knockout strains (snf1Δ) were created that showed a significant decrease in virulence when compared to WT C strains. Neoformans. After being injected with snf1Δ, the mice had an average survival of 72 days. This is significantly longer than the average survival time of 20 days of mice injected with WT C. neoformans (Yang et al., 2010). A large amount of data suggests that Mig1 and Snf1 are essential for carbon acquisition and metabolism in cryptococcal pathogenesis. By guiding the use of glucose and alternative carbon sources, these two proteins allow C. neoformans to survive and multiply in a diverse range of microenvironments. Figure 3. The SNF1/AMPK pathway regulates the use of alternative carbon. When glucose is limited, the SNF1/AMPK pathway is used to increase the regulation of gene expression involved in the metabolism of alternative carbon sources. Or, when glucose is available, these same genes are suppressed so that cells can metabolize glucose preferably. Adapted from "SNF1/AMPK/SnRK1 kinase, a global regulator at the heart of energy control?" by C. Polge and M. Thomas, 2007, Trends in Plant Science, 12(1), p. Polysaccharide capsules In addition to playing a role in many normal cellular functions, glucose metabolism is essential for the creation of the typical polysaccharide capsules of C. neoformans. The capsule is the most studied and most significant virulence factor in this pathogenic fungus, and can grow to a width several times the diameter of the cell (See Figure 4; Srikanta, Santiago-Tirado, & Doering, 2014). It also has antiphagocytic properties that prevent the immune system of the host organism from recognizing its existence, providing an additional obstacle for doctors and scientists working to treat cryptococcal infections (Srikanta, Yang, Williams, & Doering, 2011). In addition, the dynamic properties of these capsules help C. neoformans to evade human immune cells (O'Meara et al., 2010). These capsules are mainly made of two malignant polysaccharides: glucuronoxylomannan (GXM) and glucuronoxylomannogalactan (GXMGal) (Wang et al., 1995). Monomers are synthesized in the cytoplasm of the cell before being transported close to the cell surface and formed into long polymers. These polymers are then transported through the cell wall via vesicular transport before being attached. Additional polymerization can occur outside the cell if the fungus is exposed to the inducing condition. GXM is a large polysaccharide that mainly consists of the UDPmannosa subunit and accounts for about ninety percent of the capsule. In contrast, the main structural subunit of GXMGal is UDP-galactose. In addition to UDP-mannose and UDP- Figure 4. Polysaccharide capsules are virulence factor C . Neoformans. The polysaccharide capsules of C . neoformans grow to several times the diameter of the cell and are essential for virulence. This image depicts JEC21, a strain of C. neoformans, after being exposed to urea as a source of nitrogen. Staining with Indian Ink is a commonly used technique for visualizing large capsules, as seen in this image (Personal communication, MS Price, October 23, 2019). Galactosa, UDP-glucuronic acid and UDP-xylose are important components of the carbohydrate backbone. Each simple sugar is covalently attached when the polymerization process occurs. Once the backbone structure is complete, it is modified with unique parts that ensure proper folding and branching (O'Meara & Alspaugh, 2012). Once certain parts are added, the capsules are further modified with various different mechanisms. O-acetylation via the enzyme Cas1 glycosyltransferase occurs exclusively on mannosa residues and glucuronylated (Janbon, Himmelreich, Moyrand, Improvisation, & Dromer, 2001). While their exact functions are still under investigation, Pbx1 and Pbx2 are two B-helix proteins that modify the backbone by incorporating glucose residues (Liu, Kelly, Chow, & Madhani, 2007). In addition, the Csp1 protein is responsible for synthesizing hyaluronic acid, a structural component of the capsule that allows C. neoformans to cross the blood-brain barrier through facilitated transport. While the exact regulatory mechanism is still unclear, hyaluronic acid-deficient cells have decreased capsule diameter and altered structure (O'Meara & Alspaugh, 2012; Jong et al., 2007). Because the spread of fungal cells and the ability to cause meningitis depends on the ability of C. neoformans to enter the CNS, proper capsule synthesis is essential for virulence. Interestingly, studies have shown that while C. neoformans is surrounded by large polysaccharide capsules when taken from in vivo samples, in vitro cultures show varying degrees of encapsulation (Granger, Perfect, & Durack, 1985). Similarly, this fungus has the ability to change the composition of its capsules depending on the microenvironment in which it is located, and the induction of capsule synthesis often begins upon entry into the host organism (O'Meara & Alspaugh, 2012). Once synthesis is induced, the structure of the capsule remains dynamic as it moves through the host. This dynamic nature of C. neoformans allows it to adapt in various organs such as the lungs and brain, which can be seen from the heterogeneous nature of the capsules observed from various organs (McFadden, Fries, Wang, & Casadevall, 2007). In addition, biochemical analysis through mass spectrometry and NMR showed the composition of the variable side chains of GXM and GXMGal which allows the binding of variable antibodies depending on the environment. Various studies have been conducted to better understand the biochemical regulation of capsule synthesis. Although this is a very complex process that is still under investigation, several environmental parameters have been identified: iron concentration, CO2 levels, ambient pH, glucose availability, and nitrogen availability (O'Meara & Alspaugh, 2012). When the cell faces an unfavorable rate of any of these parameters, the production of capsules is induced. Studies have shown that mutated strains that cannot produce capsules are avirulent, confirming that polysaccharide capsules are essential for the survival of pathogens in the host (Wilder, Olson, Chang, Kwon-Chung, & Lipscomb, 2002). This has important implications for clinical management, as capsule inhibitors can serve as targeted drug therapies when treating these diseases. It is important to note that C. neoformans capsules currently serve as an important diagnostic tool. Effective treatment of C. neoformans depends on early detection and diagnosis, which can be achieved through highly sensitive antigen tests that recognize common capsule serotypes. In addition, Indian ink staining can be used to identify encapsulated cells from sputum cultures or from bronchoalveolar lavage fluid. Conclusion Cryptococcosis, an infection resulting from a complex of C. neoformans species, kills thousands of immunosuppressed individuals each year. While most of these patients have end-stage HIV/AIDS, other vulnerable populations include transplant patients with immunosuppressive drugs, cancer patients, and individuals with autoimmune disorders. The severity of the disease can range from local cutaneous cryptococci to widespread systemic infections with the involvement of CNS. Despite aggressive antifungal therapy, the prognosis of patients with C. neoformans infection continues to be poor; survival rates range around fifty percent in patients with systemic disease, even in western countries where patients have access to sophisticated health care. Nutrient gain, and in particular carbon metabolism, are essential for the persistence and proliferation of C. neoformans in the host. In addition, carbon is the main structural component in the polysaccharide capsule, the most well-studied virulence factor and arguably the most important of these destructive fungi. Although treatments such as fluconazole do exist, they are expensive and only somewhat effective for treating late-stage and disseminated diseases. Thus, a better understanding of the relationship between carbon metabolism and virulence is essential for the development of new targeted drug therapies in the near future. Reference Agustinho, D.P., Miller, L.C., Li, L.C., & Doering, T.L. (2018). 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