In the present study, identification and deletion of a VPS34
gene associated with the virulence factor laccase allowed for the analysis of the role of inositol phosphate signaling, autophagy, and starvation response in the pathogen Cn. This expands further the genetic relationships between an immune modulatory virulence factor, laccase (33
), and starvation response programs such as autophagy that play a central role during virulence. Previously, regulation of laccase activity has been genetically linked to processes important under nutrient-limiting conditions such as gluconeogenesis (5
) and high-affinity copper acquisition (6
). Such a genetic association between laccase and attributes such as autophagy, important to survival during the search for nutrients, recalls the organism’s environmental niche within the hollows of trees where laccase is involved in the generation of energy by the use of alternative substrates including degradation of wood lignins in the absence of glucose (35
). The existence of such strong genetic relationships between virulence determinants and environmental genetic programs are a testament to the success of opportunistic pathogens such as Cn that appear to effortlessly switch between an environmental saprophyte and a virulent pathogen. In addition, laccase’s extensive relationships to virulence determinants provides additional insights into why laccase and its product melanin has proven successful as a strong marker of pathogenic strains of Cryptococcus
Autophagy is an evolutionarily conserved response to stress and has been linked to important protective roles in mammalian cells such as lifespan extension and protection from diverse human diseases including cancer, muscular disorders, neurodegeneration (e.g., Huntington, Alzheimer, and Parkinson diseases) (37
). In the mammalian host-pathogen interaction, the role of autophagy has mostly favored the host, as induction of autophagy in macrophages has been found to be key in the control of replication of diverse prokaryotes such as Mycobacterium tuberculosis
and Legionella pneumophila
within autophagic vesicles (25
). However, the present studies indicate that PI3K-dependent induction of autophagy is also a tool used by the eukaryotic pathogen to ensure survival and effect host cell damage. Interestingly, the fungal pathogen Candida albicans
has not been found to be dependent on autophagy for virulence (41
), although a reduction in virulence was found by deletion of a vps34
Δ homolog (42
), which may suggest that the 2 pathogens encounter different host-cell environments leading to varied requirements for an autophagic response. Alternatively, this could be a reflection of pathogen-specific alterations in the host cell environment. For example, Cn liberates an extensive polysaccharide capsule within macrophages which has macrophage modulating properties that could alter the environment of the phagolysosome (43
). In addition, Candida
species may elicit specific protective properties that improve nutrient conditions similar to Mycobacterium
within the host environment (44
), making it less dependent on its own autophagosomal machinery. Regardless of mechanisms, these different requirements for autophagy between pathogens offer insights into the unique aspects of interactions between fungi of different species and the mammalian host.
Induction of autophagy in Cn was found to be dependent on the PI3K locus VPS34
, as previously described in ascomycetous yeast (8
). Interestingly, the almost WT growth of the cryptococcal vps34
Δ mutant at 37°C distinguishes it from the temperature sensitivity of the homologous mutation in ascomycetous yeast and may reflect the evolution of higher eukaryotes. However, in spite of robust growth at 37°C, the cryptococcal vps34
Δ mutant showed a remarkably fast clearance from lungs of mice, which was associated with a defect in tolerance of starvation and formation of autophagic bodies. Such extremely different survival patterns of the vps34
Δ mutant in tissue culture media containing glucose compared with survival patterns in the host environment suggest that the former is not representative of host conditions. This discrepancy in pathogen response between these 2 conditions may have implications for the selection of conditions used to mimic the host environment, such as the determination of antifungal minimum inhibitory concentrations (MICs).
These analyses, used to predict antifungal resistance during clinical infections, were conducted under nutrient-replete conditions as recommended by the National Committee for Clinical Laboratory Standards (45
). Unfortunately, cryptococcal fungal MICs have shown a remarkable inability to predict clinical endpoints (47
), which could be partly due to differences in antifungal resistance within differing nutrient environments.
In summary, while these data suggest that an effective response, such as PI3K signaling and autophagy, by the pathogen to starvation encountered during infection may be detrimental to the host, they also suggest how attention to fungal stress response pathways may lead to more effective preventative or treatment modalities tailored to the unique environment of the host.