Cryptococcal Virulence

Virulence Factors

Virulence factors increase the degree of pathogenicity of a microbe. C. neoformans has a number of virulence factors; generally, the virulence of an isolate cannot be attributed to any single factor, but rather it is attributed to many working in unison to cause progressive disease. As virulence factors go, those of C. neoformans would be considered low-grade. We will discuss each virulence factor separately; however, the severity of the host's disease results from a combination of several virulence factors superimposed on the host's innate and immune resistance status. The virulence factors that will be discussed are capsule, cryptococcal products, melanin production, mannitol production, and potential factors such as superoxide dismutase, proteases, phospholipase B, and lysophospholipase. The polysaccharide capsule and the soluble extracellular constituents of C. neoformans (referred to here as cryptococcal products) are probably the dominant virulence factors.

Capsule

C. neoformans has a capsule composed primarily of a high molecular weight polysaccharide that has a backbone of -1,3-D-mannopyranose units with single residues of ß-D-xylopyranosyl and ß-D-glucuronopyranosyl attached. This polysaccharide is referred to as glucuronoxylomannan (GXM) and has four serotypes: A and D, produced by C. neoformans var. neoformans, and B and C, produced by C. neoformans var. gattii. The evidence indicates that the capsule is a key virulence factor for C. neoformans; acapsular mutants are typically avirulent, whereas encapsulated isolates have varying degrees of virulence. Similar observations have been made with the CAP64 gene. Although their biochemical functions have not been defined, it appears that the two genes are essential for capsule formation and virulence.

Chemotactic Effects on Leukocytes

Some properties of the C. neoformans capsule enable the host to more effectively clear the organism from tissues; however, others protect the organism from host defenses. The capsules of serotype A and D isolates are chemotactic for neutrophils. Moreover, complement is fixed by cryptococcal capsules by the alternative pathway, and this process produces chemotactic peptides such as C5a. Chemotaxis of leukocytes induced by either of these mechanisms would be advantageous to the host.

Effects of Complement Interactions

Complement fixing by C. neoformans in tissue would result in chemotactic factor production and attraction of leukocytes into the infection site. Once in the infected tissue, the leukocytes would interact with and kill the organism. As complement is fixed, C3b and C3bi are deposited on the surface of the cryptococci. The capsule can mask the C3b and C3bi deposits; however, if they are not completely masked, the deposited complement components facilitate binding of the cryptococci to CR3 receptors on leukocytes. Such binding interactions are advantageous to the host; they enhance the opportunity for the leukocytes to kill the cryptococci either extracellularly or after phagocytosis. The organism can also be opsonized by antibodies to GXM, but the capsule may block the Fc portion of the antibody and prevent it from binding to Fc receptors on the phagocytic host cells. Some of these functions of the capsule favor the host; however, if the capsule is very large, the organism is protected. The cryptococci could deplete complement in the host, creating an environment that favors the cryptococci.

Effects on Phagocytosis

All considered, the capsule is more beneficial to the organism than to the host. Encapsulated C. neoformans cells are not phagocytized or killed by neutrophils, monocytes, or macrophages to the same degree as acapsular mutants. Encapsulated C. neoformans have a stronger negatively charged surface than acapsular cells or Saccharomyces cerevisiae cells. The high negative charge could cause electrostatic repulsion between the organism and the negatively charged host effector cells and reduce intimate cell-cell interactions required for clearance of the cryptococci.

Altered Antigen Presentation

The inability of macrophages to ingest the encapsulated organisms could also diminish antigen presentation to T cells and consequently reduce immune responses. This speculation has been experimentally demonstrated by Collins and Bancroft. Other studies with human alveolar macrophages have confirmed that antigen presentation is not as effective with encapsulated cryptococci as with acapsular strains. Unlike acapsular cells, encapsulated isolates cannot stimulate proliferative responses in T cells because of the reduced secretion of interleukin-1 (IL-1) by the antigen-presenting cells stimulated with the encapsulated yeasts.

Effects on Cytokine Production

In addition to being antiphagocytic, more resistant to killing, and less able to stimulate T-cell proliferation, highly encapsulated isolates of C. neoformans opsonized with a heat-labile serum component, presumably complement, do not stimulate monocytes and macrophages to produce proinflammatory cytokines such as TNF, IL-1ß, and IL-6 as effectively as similarly opsonized nonencapsulated or weakly encapsulated isolates. The signal for induction of cytokine production can be a direct result of the attachment of the monocyte or macrophage to the acapsular cryptococci or can be an outcome of the phagocytic process, induced by acapsular cryptococci. Since the capsule blocks phagocytosis, any cytokines induced by the phagocytic process would not be induced by the encapsulated C. neoformans cells. If the cryptococcal cell wall materials must be exposed to induce cytokine production, the capsule would block the direct induction of cytokine production. Regardless of the mechanism of stimulation, the lack of production of proinflammatory cytokines could have a bearing on protection. TNF is necessary for the induction of the protective immune response against C. neoformans. Consequently, the lack of or reduced production of TNF in infections with highly encapsulated isolates of C. neoformans would prevent the induction of protective immunity, resulting in progressive disease. The roles of IL-1ß and IL-6 in protecting against C. neoformans have not been defined, but it is highly probable that the lack of these two cytokines could compromise the protective responses of the host and give cryptococci the advantage.

In contrast to the reduced TNF, IL-1ß, and IL-6 produced by stimulating monocytes and macrophages with highly encapsulated cryptococci, IL-10 produced by these host cells increases after interaction with encapsulated strains. Neutralization of IL-10 with anti-IL-10 in cocultures of human peripheral blood mononuclear cells and encapsulated cryptococci increased the amounts of TNF and IL-1ß produced, which indicates that the induction of IL-10 production by stimulating macrophages with encapsulated C. neoformans downregulates the production of the proinflammatory cytokines TNF and IL-1ß. One might predict that the induction of high levels of IL-10 would also preferentially stimulate the induction of a T-helper 2 (Th2) response rather than a Th1 response in the T cells. Since the Th1 response is associated with protection against C. neoformans, increased levels of IL-10 would dampen induction of the protective immune response.

Encapsulated cryptococcal cells do not affect the different types of leukocytes in the same manner. Although encapsulated isolates do not stimulate macrophages to produce proinflammatory cytokines, they do stimulate neutrophils to produce proinflammatory cytokines and the chemotactic factor IL-8 more effectively than weakly encapsulated or acapsular organisms. As with the stimulation of macrophages by acapsular cryptococci to produce proinflammatory cytokines, serum is required for the encapsulated organisms to induce neutrophils to produce cytokines. In the case of cytokine production by neutrophils in response to encapsulated C. neoformans, it appears that the opsonization process releases a factor into the supernatant that induces the neutrophils to produce the cytokines. How these opposing in vitro observations with macrophages and neutrophils relate to the in vivo system or pathogenicity is yet to be determined.

Cryptococcal Products

In disseminated cryptococcosis, measurable levels of cryptococcal products are present in the body fluids of the patients. Although GXM is the major cryptococcal component in body fluids, it is highly probable that the organism also sheds galactoxylomannan (GalXM) and mannoproteins (MP) in vivo. This speculation is based on indirect evidence from in vivo studies and on the fact that GalXM and MP are in culture medium when the organism is grown in vitro. Cryptococcal antigens in serum or spinal fluid are diagnostic for cryptococcosis. Furthermore, if disseminated cryptococcosis patients have high cryptococcal antigen titers at the onset of therapy, they are less likely to respond to therapy or more likely to die before therapy is completed than patients with low cryptococcal antigen titers. The direct relationship of cryptococcal antigen levels in body fluids and the severity of disease suggests that the cryptococcal antigens in the host's circulatory system or spinal fluid may have adverse effects on host defenses.