PERSPECTIVE
When There Is a Fungus Among Us, What Makes it Virulent?

Ling Ling Ma and Christopher H. Mody

Departments of Medical Sciences, Microbiology and Infectious Disease, and Internal Medicine, University of Calgary, Calgary, Alberta, Canada

Pathogenic microbes often have an overwhelming number of proposed virulence factors and mechanisms. These factors perform a complex choreography that determines whether the microbe or the host lives or dies. The factors can be inducible or constitutive, the direct product of genetic elements (proteins), or the products of complex biosynthetic pathways such as polysaccharides or lipid mediators. Thus, there are tremendous challenges for investigators who attempt to prove that an individual genetic feature is responsible for production of a virulence factor and thus the gene that makes an organism a pathogen. One such pathogen is Cryptococcus neoformans, which has proven to be an invaluable model to study the immune response and host defense in the lung. Although many factors arm this organism to be a pathogen, the polysaccharide capsule of C. neoformans has been implicated as one of the most important. The study performed by Wilder and colleagues in this issue (1) provides the proof that the cryptococcal capsule is a virulence factor in the lung.

	History and Classification

Cryptococcus neoformans was first described in 1894. The German pathologist Buss recovered a yeast-like organism from the bony lesion of a woman (2). Subsequently, the Italian scientist Sanfelice made the first environmental recovery from peach juice and called the organism Cryptococcus neoformans (3). C. neoformans, like C. albicans, contains half of the full complement of DNA (i.e., it is haploid). The perfect, or sexual state, which contains the full complement of DNA, is produced by the combination of compatible mating types and is called Filobasidiella neoformans (4).

Cryptococcus neoformans is ~ 5 µm in diameter and surrounded by a glucan-based cell wall (5). Pathogenic strains typically possess an additional polysaccharide capsule outside the cell wall. Although the ability of this capsule to confer virulence is the subject of the paper by Wilder and coworkers, the capsule also provides the antigens that confer the serotype of different strains. There are four serotypes (A, B, C, and D) that are divided into two varieties, var. neoformans and var. gattii. The variety neoformans, which consists of serotypes A and D, is prevalent worldwide and can be recovered from soil and avian excreta (6). Both of these serotypes are responsible for cryptococcosis in immune-compromised patients such as acquired immunodeficiency syndrome (AIDS) patients and normal hosts, although serotype A is the more common pathogen. The variety gattii, which consists of serotypes B and C, is geographically restrained to tropical or subtropical climates, associated with eucalyptus trees, and often causes diseases in immunocompetent individuals (6).

	Cryptococcosis and the Lung

The encapsulated yeast C. neoformans commonly enters the body by inhalation (7). Environmental organisms that are lightly encapsulated rapidly synthesize a thicker capsule upon exposure to the higher concentrations of CO₂ in the lung (8). The infection can then disseminate from the lung to other organs, particularly the central nervous system, although other targets include the eyes, prostate, and skin. The most common and devastating consequence of dissemination is cryptococcal meningitis, which is often fatal if not treated. Cryptococcal disease has emerged as an important cause of illness and death in persons infected with human immunodeficiency virus (HIV), and it is now the most common cause of meningitis at many large hospitals where AIDS patients are treated.

The development of Th1 type cell-mediated immunity (CMI) leads to leukocyte recruitment to the local site of C. neoformans infection, activation of the leukocytes for fungicidal activity, and clearance of the infection (9). The leukocytic infiltrate in the lungs contains both myeloid and lymphoid cells, all of which are capable of inhibiting or killing C. neoformans (10, 11). T lymphocytes, both CD4+ and CD8+ T cells, are required for maximal leukocyte recruitment, pulmonary clearance, and protection against dissemination of the infection (10, 12). When CD4+ and CD8+ T cells are absent, leuckocyte recruitment is minimal and the patient dies from disseminated cryptococcosis. In addition to the response to cognate antigens, which result in the development of cell-mediated immunity, C. neoformans also stimulates an innate T cell response that results in lymphocyte expansion and the development of lymphocyte-mediated antifungal activity (18, 19). T cell responses are associated with the production of some cytokines, such as tumor necrosis factor- (TNF-), interleukin (IL)-12, interferon- (IFN-), granulocyte macrophage-colony stimulating factor (GM-CSF) (20, 24), chemokines, macrophage inflammatory protein (MIP-1), monocyte chemotactic protein (MCP-1) (21), and the induction of nitric oxide (NO) (24). The above events are all critical in the efferent phase of host defense against C. neoformans.

	Polysaccharide Capsule is a Virulence Factor

There are several cryptococcal virulence factors that have been identified, which are the molecular products that enable the yeast to countermand immune responses and cause disease (Table 1). The capsule was the first cryptococcal virulence factor to be associated with the ability to produce disease and generally regarded as the most important. Many groups induced mutants that were unable to produce capsule and that were avirulent in mice (25). These investigators demonstrated that reversion of the mutants to capsule production re-establishes virulence. Jacobson and colleagues used a detailed classical genetic analysis to characterize several acapsular mutants (26, 27). Subsequent analysis of the acapsular mutants has implicated capsule formation as an independent virulence factor for the production of disseminated cryptococcosis in mice (28, 29). However, spontaneous or induced acapsular mutants may possess additional mutations that affect virulence and therefore more specific approaches are needed.

	Chemical Composition of the Capsule

The polysaccharide capsule that distinguishes C. neoformans from other pathogenic fungi is an impressive structure, with a radius that can be many times that of the cell itself. It can usually be demonstrated by light microscopy when cells are mixed with India ink. The capsule excludes the ink particles and yields a characteristic halo around the cell wall. The polysaccharide capsule contains two dominant polysaccharide components, glucuronoxylomannan (GXM) and galactoxylomannan (GalXM). Subtle changes in the distribution of these sugars confer serotype specificity, and the details of these structures have been reviewed recently (30). The capsule also contains mannoprotein, which provides some of the T cell antigens of C. neoformans (31).

	Biologic Activity of the Capsule

The cryptococcal polysaccharide has several biologic properties that may contribute to virulence (Table 2). One of the earliest identified properties, and perhaps the most important, is its ability to inhibit phagocytosis of the organism. Phagocytes, such as monocytes and neutrophils, readily ingest nonencapsulated cryptococci, whereas encapsulated yeast cells are relatively resistant. GXM is believed to mediate the inhibition of phagocytosis because incubation of nonencapsulated organisms with purified polysaccharide capsule makes them resistant to phagocytosis (32, 33). The cryptococcal capsule also activates the alternative complement system, leading to consumption of complement (34). Opsonins normally aid clearance of the yeast, and complement depletion may be an important factor in AIDS patients with cryptococcosis (35). The capsule also provides the antigens for the opsonic effects of anti-cell wall antibodies (36). It has also been shown to influence cell-mediated immunity. The polysaccharide capsule interferes with processing of the organism that is necessary for subsequent T cell activation (37), which is critical for secretion of cytokines. Cryptococcal polysaccharide reduces TNF- production, and nitric oxide synthase (NOS) induction in primed macrophages (41). It inhibits leukocyte adhesion to the endothelium and leukocyte migration into inflammatory sites (42). Finally, the cryptococcal capsule enhances the infectivity of HIV-1 in cell culture, the subsequent production of infectious HIV, and the formation of HIV-induced syncytia in vitro, which may be due to increased adherence of HIV to target cells (43).

	Capsule Genes and Their Relationship to Virulence

The first molecular evidence that the capsule was a virulence factor came from studies that introduced cryptococcal DNA into acapsular mutant strains (complemented by transformation). One of the transformed colonies expressed capsule and regained virulence (44). The DNA that was responsible for the transformation was found to contain a gene called CAP59. The studies went on to produce a mutant strain by specifically knocking out CAP59 (disrupted by homologous integration), which resulted in a strain that lacked virulence.

For the molecular study of the polysaccharide capsule, several different acapsular mutants have been used. To complement the acapsular mutations, libraries of genomic DNA in a telomere-based vector, which dramatically increases the transformation efficiency, were constructed. With these approaches, four genes, CAP 10 (45), CAP 59 (44), CAP 60 (46), and CAP 64 (47) have been cloned. These genes were used to complement four different groups of acapsular mutants. Although the four genes are essential for capsule formation, the biochemical function of them in the pathway of capsule synthesis is unknown. However, several distinctive features of these genes have been observed: (i) they all contain multiple introns. CAP64, the subject of the paper in this issue, contains eight introns and encodes a 56-kD protein (44); (ii) all of them are closely linked to other unrelated genes, except for CAP10. CAP64 is located on chromosome III and is closely linked to a convergently transcribed gene, a putative proteasome subunit gene, PRE1 (44, 48).

A mouse model has been used for virulence studies that examine the pathogenic importance of these genes. The strains with disrupted CAP genes were all avirulent in the murine model (44, 46, 47). Acapsular mutants that were generated either by mutagenesis or gene deletion, and capsule-producing strains from either wild-type or complemented strains, were inoculated into the mice and mortality was monitored (44, 46). The capsule-deficient mutants could be complemented with the corresponding wild-type CAP gene. When this was done, the strains regained virulence, indicating that all four capsule genes isolated are essential for virulence of C. neoformans. The results of these studies showed that the presence of the capsule is a virulence factor because deletion of each capsule-related gene resulted in the loss of the yeast's virulence, and complementation of the acapsular phenotype restored its virulence. These results fulfill the molecular Koch's postulates defined by S. Falkow, which have been applied in investigating the potential role of genes and their products in the pathogenesis of infections and diseases (Table 3) (49).

Many studies on the role of capsule in virulence and the effect of the capsule on cell-mediated immunity have been performed with acapsular mutants of C. neoformans. One of the most widely-used strains is acapsular strain 602, which was originally obtained from a clinical isolate (50). However, the parental strain of this mutant is not available and this makes it difficult to perform comparative studies. Furthermore, the DNA fingerprint pattern of strain 602 is most compatible with that of a typical serotype A strain (51), although its serotype could not be determined immunologically due to the lack of capsule. Unfortunately, the mating partner for serotype A strains has never been identified (similar to the situation in all strains of C. albicans in which no mating partner has ever been identified), and therefore classical recombination cannot be performed. These investigators were therefore forced to use an alternate approach to solve this problem. Cloning of capsule-related genes (44, 47, 52) and the establishment of the transformation system provided an opportunity to investigate the role of polysaccharide capsule as a single variable in host-parasite interactions. Previous studies have shown that acapsular strain 602 complemented with gene CAP64 could restore capsule formation and cause fatal intravenous infection in mice (48).

The current study performed by Wilder and colleagues provides direct evidence that polysaccharide capsule plays an essential role in lung virulence. The authors compared different virulence indicators, including the burden of organisms, cytokines, and numbers of inflammatory cells, by using mice inoculated via the intratracheal route. Pulmonary inoculation provides a more natural route of entry than intravenous or intraperitoneal inoculation and thus provides important information about the relation of capsule and lung virulence.

	Summary

C. neoformans is an encapsulated yeast that commonly enters the host through the respiratory route and the lung is therefore the initial site of infection. The polysaccharide capsule is the most critical virulence factor of C. neoformans. The current studies have used a transformation system and cloning of capsule-related genes, which have allowed us to fulfill Koch's molecular postulates for virulence in the lung. Studies of virulence factors are fundamental to understanding the pathogenesis of microbial infections, and their identification may point the way to new strategies for controlling disease.

	Footnotes

Address correspondence to: Christopher H. Mody, Rm. 273 Heritage Medical Research Building, University of Calgary, Calgary, AB T2N 4N1 Canada.

(Received in original form January 28, 2002).

Acknowledgments: L.L.M. is supported by a studentship from Alberta Lung Association. C.H.M. is a scholar of the Alberta Heritage Foundation for Medical Research.

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