Cryptococcal infection in the lungs begins following inhalation of infectious cells or spores (Giles et al. 2009). There are no documented cases of human-to-human spread of cryptococcosis and thus infections are thought to result almost exclusively from environmental exposure. Following inhalation, C. neoformans initially colonises the extracellular alveolar space, where it comes into contact with the host innate immune system. As part of the innate immune response, alveolar macrophages migrate to the site of infection and attempt to clear the infection by engulfing fungal cells via phagocytosis (see Fig. 5.2). Following phagocytosis, however, C. neoformans is able to survive and grow within the intracellular niche provided by the macrophage. Initial respiratory infection occurs in both healthy and immunocompromised individuals, but subsequent disseminated infection is far more likely if an immune deficiency is present.

Disease outcome following C. neoformans infection depends on the strength of the host immune response. Broadly there are three possible outcomes:

For C. neoformans, fatal disseminative disease (ii) occurs almost exclusively in individuals with pre-existing immune deficiencies whereas total clearance (i) and latent disease (iii) can manifest in individuals who are immune competent.

Epidemiological studies can never truly determine the extent of C. neoformans infection within immune competent populations because the majority of infections are asymptomatic and go unreported. To address this issue, studies have used anti-cryptococcal immunoglobulin titres within immune competent populations as a sign of past infection. These studies conclude that cryptococcal infection is common in the normal adult population (Deshaw and Pirofski 1995; Houpt et al. 1994) and most individuals are probably exposed to the fungus during childhood (Abadi and Pirofski 1999; Houpt et al. 1994). These conclusions seem reasonable considering the ubiquity of C. neoformans within the natural environment, thus it is likely that individuals encounter C. neoformans many times during their lifetime but are protected by a healthy immune system.

Some studies show that latent infection contained by the immune system may re-emerge if an immune deficiency later develops. Evidence for this phenomenon comes from a study by Garcia-Hermoso et al., which determined the genetic origin of clinical C. neoformans isolates taken from a cohort of cryptococcosis sufferers in France. This analysis found that a number of immunocompromised African immigrants in the study, who had lived in France for many years before developing immune deficiency, were infected with C. neoformans strains originating in Africa. Garcia-Hermoso et al. concluded that these patients may have developed a latent form of cryptococcosis before moving to France and that the infection re-emerged when immune deficiency later developed (Garcia-Hermoso et al. 1999).

Macrophages are innate immune phagocytic cells that patrol the body’s tissues searching for signs of infection. When a macrophage detects a foreign cell, such as a pathogenic microorganism, it becomes activated and will attempt to engulf the invader via a process called phagocytosis (see Fig. 5.3). Phagocytosis begins when phagocytic receptors on the cell surface of the macrophage encounter ligands that are only found on foreign cells. Following receptor ligation, signalling pathways are activated inside the macrophage, which trigger cytoskeletal actin rearrangements around the receptor, leading to internalisation of the particle into a membrane-bound compartment termed the phagosome. Over time, conditions within the phagosome are altered by the macrophage to create an environment that is destructive to microorganisms, a process called phagosomal maturation. Via a series of vesicle fusion events, membrane proteins such as NADPH oxidase, V-ATPase and NOS are delivered to the phagosome. Together, these create a highly antimicrobial environment, for instance via the production of reactive oxygen species (ROS) by the NADPH oxidase, or lowering of the phagosomal pH by the V-ATPase. Finally, the phagosome fuses with a specialised cytosolic vesicle called the lysosome, which contains an array of digestive enzymes. These digestive enzymes, which work best at low pH, kill any remaining organisms in the phagosome and then break up the dead cell into its constituent parts.

Phagocytosis can clear infection directly but it also triggers signals that enhance the immune response. Upon sensing infection, macrophages can release proinflammatory cytokines such as tumour necrosis factor (TNF)-α and interleukin (IL)-1β; the inflammation that these cytokines produce attracts other immune cells to the site of infection, ultimately engaging the adaptive immune system if the infection is not cleared rapidly.

Macrophages must detect the presence of foreign organisms before they can initiate phagocytosis. To facilitate detection, macrophages express an array of cell surface phagocytic receptors that bind to ligands present on foreign cells but not host cells. Phagocytic receptors can be categorised according to the nature of their ligands. Non-opsonic phagocytic receptors or pathogen recognition receptors (PRRs) bind to molecular ligands (pathogen-associated molecular patterns or PAMPs) such as polysaccharide residues or lipid species found on the surface of foreign cells. In contrast, opsonic receptors bind to opsonising proteins, such as antibodies or complement proteins, which coat foreign cells following activation of the humoral immune system.

Capsule expression has long been implicated in cryptococcal virulence since it was observed that Cryptococcus acapsular mutant strains are avirulent (Fromtling et al. 1982; Chang and Kwon-Chung 1994). The capsule has a number of properties that contribute to virulence during infection, but the most important seems to be its anti-phagocytic quality, which was first realised when acapsular strains were found to be phagocytosed by macrophages more readily than were capsular strains (Kozel and Gotschlich 1982; Cross and Bancroft 1995). Capsule growth can be stimulated by CO₂ levels and pH conditions similar to those found during host infection (Granger et al. 1985) and has also been observed to occur in vivo (Feldmesser et al. 2001). Synthesis of the capsule is controlled by at least four genes, designated CAP64, CAP59, CAP10 and CAP60 (Buchanan and Murphy 1998; Ma and May 2009). In a series of experiments, Chang et al. found that deletion of each CAP gene produced an acapsular mutant that lacked virulence until the gene was reconstituted (Chang et al. 1996; Chang and Kwon-Chung 1994, 1999, 1998). The capsule is composed of two key polysaccharide components: glucuronoxylomannan (GXM) and galactoxymannan (GalXM) (Bose et al. 2003), with a ratio of approximately 9:1 by mass in favour of GXM (Idnurm et al. 2005).

One of the main ways that the capsule protects C. neoformans against phagocytosis is by providing a shroud around the cell, which interferes with macrophage detection. This shrouding effect blocks uptake via both opsonic and non-opsonic phagocytic receptors although it appears to have a greater inhibitory effect on non-opsonic uptake because non-opsonised C. neoformans cells are almost completely protected from phagocytosis (Mukherjee et al. 1996).

To grow inside a macrophage following phagocytosis, an intracellular pathogen must successfully avoid the killing mechanisms that develop in the phagosome during phagosomal maturation. A number of mechanisms are employed by pathogens to do this, including inhibition of V-ATPase to prevent phagosomal acidification as seen with Mycobacterium tuberculosis infection (Huynh and Grinstein 2007) and escape from the phagosome into the cytoplasm as seen with Listeria monocytogenes infection (Hamon et al. 2006). In contrast to such pathogens, C. neoformans is able to survive within the phagosome seemingly without modifying the maturation process. Instead, it is able to resist and overcome the destructive conditions it encounters in the phagosome and can even grow within this harsh environment.

The first barrier to cryptococcal growth in mammalian macrophages is the higher temperature within the host than in the environment. The complete molecular mechanisms that allow C. neoformans to grow at 37 °C are not fully known, but a number of potential pathways have been implicated. For instance, higher temperatures induce upregulation of TSP1 and TSP2, which together lead to higher levels of the polysaccharide trehalose, protecting cellular proteins from denaturation (Petzold et al. 2006).

Inside the phagosome, the largest barrier to cryptococcal growth is phagosomal maturation, i.e. the production of oxygen free radicals accompanied by the acidification of the phagosome. During infection most C. neoformans strains synthesise melanin, which is a dark-coloured pigment and an antioxidant (Wang et al. 1995) that is thought to protect the fungi from reactive oxygen species. In addition to melanin, capsule enlargement during macrophage infection may also be protective against oxygen free radicals by creating a barrier between the cell body and phagosomal contents (Zaragoza et al. 2008); furthermore, GXM within the capsule may act as an antioxidant (Monari et al. 2006; Vecchiarelli et al. 1996). Interestingly, however, the phagosomal acidification that occurs during maturation may actually be beneficial to C. neoformans because neutralising phagosomal pH can block intracellular replication (Levitz et al. 1999).