essential regulator of pathogenicity of Cryptococcus neoformans

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J Clin Invest. 2006 June 1; 116(6): 1651–1659.

Published online 2006 June 1. doi: 10.1172/JCI27890.

2006 American Society for Clinical Investigation

Glucosylceramide synthase is an essential regulator of pathogenicity

of Cryptococcus neoformans

Philipp C. Rittershaus,1 Talar B. Kechichian,1 C. Allegood,2

Alfred H. Merrill,2 Mirko Hennig,3 Chiara Luberto,1 and Maurizio Del

Poeta1,4

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1466548

Discussion

In the present study the role of C. neoformans GCS in the virulence

of this pathogenic fungus was investigated. Our results show that C.

neoformans GCS was an essential factor in determining the success of

the fungal infection by regulating survival of C. neoformans during

the initial colonization of the lung. In addition, we found that GCS

guaranteed survival of C. neoformans in the lung extracellular space

by ensuring cell-cycle progression at neutral/alkaline pH and

physiologic CO2 concentration (Table 2).

The dramatic protection against pathogenicity imparted by loss of

GCS underlines the importance of the extracellular component of

fungal cells during C. neoformans infection. C. neoformans infects

the host through inhalation, and upon its arrival in the alveoli it

encounters 2 environments in which normally grows: the extracellular

space, characterized by a neutral/alkaline pH and a physiologic CO2

concentration of approximately 5.0% (compared to 0.04% present in

the air, ref. 29); and the intracellular environment of the

phagolysosome of alveolar macrophages, characterized by an acidic

pH. Our results suggest that GlcCer is critical for C. neoformans to

grow in the extracellular (alkaline) but not in the intracellular

(acidic) environment of the lung. Since during the infection most C.

neoformans cells are found in the host extracellular space (30), the

overall effect of the loss of GlcCer on C. neoformans pathogenicity

is significant. Indeed, when the gcs1 strain was introduced

intranasally, the overall growth of yeast cells in the alveoli was

arrested (Supplemental Figure 2); even if the mutant grew

intracellularly (Supplemental Figure 4), the host responded with the

formation of well-organized granulomas (Figure 4 and Supplemental

Figure 3), which efficiently contained gcs1 cells in the lung. This

allowed the host to survive the cryptococcal infection without any

evident clinical sign of disease (Figure 3A).

When the gcs1 strain was introduced intravenously, mice survived

longer than those infected with the WT or reconstituted strains

(Figure 3B). In the blood, gcs1 also finds a hostile environment

because the pH is approximately 7.4 and the CO2 concentration is

approximately 5% (partial pressure of CO2 [pCO2], ~40 mmHg). Thus

growth of gcs1 cells in the bloodstream was arrested until yeast

cells were either internalized by phagocytic cells or they left the

bloodstream and invaded the organs, in which they formed abscesses

such as those found in the brain (Supplemental Figure 3, F and H)

and the kidney (data not shown). In both the intracellular

environment and in abscesses, the pH was characteristically acidic,

and growth of gcs1 could resume. Indeed, it was interesting to note

that in the brain upon intravenous challenge of gcs1, yeast cells

were almost exclusively contained within abscesses (Supplemental

Figure 3, F and H), whereas in the WT strain, in addition to being

present in abscesses, cells were also present in the surrounding

tissue (data not shown). Thus we hypothesize that once the acidic

abscess is formed, gcs1 cells promptly restore growth, and the

consequent destruction of brain tissue will eventually manifest

abruptly with clinical sign of meningoencephalitis. The animal

infected intravenously with gcs1 would eventually die, although it

would take longer than mice infected with the control strains due to

the transient growth arrest that yeast cells undergo in the

bloodstream upon injection.

We believe the results from these studies identify GlcCer as a novel

virulence factor for C. neoformans because loss of this

glycosphingolipid does not affect other fungal characteristics, such

as capsule production, melanin formation, or growth at ambient CO2

and 37°C (Supplemental Figure 5), known to be required to cause

disease (1). Interestingly, a greater sensitivity of gcs1 mutant to

SDS was observed (Supplemental Figure 5), suggesting an alteration

of cell wall integrity. This hypothesis is supported by previous

observations of the corrleation of GlcCer density at the cell

surface with the cell wall thickness of C. neoformans (10). However,

the difference in cell growth of gcs1 in the presence of SDS

compared with control strains was not so dramatic to account for the

significant loss of virulence of the mutant. Instead, the growth of

gcs1 mutant was arrested in environments characterized by alkaline

pH and 5% CO2.

In budding yeasts such as C. neoformans, the S and G2 phases of the

cell cycle are characterized by bud formation and maturation (Figure

5C). Interestingly, previous studies have shown that GlcCer is

localized at the budding site of C. neoformans, and addition of

human antibodies against C. neoformans GlcCer to in vitro C.

neoformans culture inhibits cell budding and fungal cell growth

(10). Our studies showed that loss of GlcCer significantly prolonged

the S and the G2/M phases in alkaline environments, therefore

suggesting a mechanism whereby GlcCer regulates fungal cell growth

by allowing yeast cells to complete the cell cycle.

The role of GCS in alkaline tolerance is not restricted to C.

neoformans, as recent findings showed that a Kluyveromyces lactis

mutant defective in GlcCer cannot grow at pH 8.5 (31). On the other

hand, the molecular mechanism by which GlcCer regulates alkaline

tolerance at high but not low CO2 is not known. Studies have

proposed that fungal cells sense different concentrations of CO2

and, in conditions in which CO2 is elevated (e.g., 5%), fungi

respond by upregulating virulence factors. For instance, CO2

stimulates capsule formation in C. neoformans (32) and the

transformation of yeast cells to filamentous forms in C. albicans

through adenylyl cyclase (33). In other pathogenic fungi, such as

Coccidioides immitis, CO2 promotes the formation of endosporulating

spherules (34), which is a process required for virulence. These

studies suggest the existence of a conserved signaling pathway that

regulates fungal virulence through the sensing of CO2. C. neoformans

senses CO2 through 2 â-class carbonic anhydrases (Can1 and Can2),

which have been recently identified (35, 36), but whether GlcCer

regulates signaling events mediated by CO2-carbonic anhydrases at

different pHs awaits further investigation.

In conclusion, these studies showed that GlcCer plays a key role in

the pathogenicity of C. neoformans through a mechanism that ensures

the transition through the cell cycle of yeast cells in alkaline

environments with a physiological concentration of CO2. Since in

cases of cryptococcosis extracellular organisms predominate, the

function of GlcCer becomes clinically relevant. Interestingly, Gcs1

and GlcCer are found in a variety of pathogenic fungi, and because a

significant biochemical difference between human and C. neoformans

Gcs1 exists, compounds that would specifically inhibit the fungal

enzyme might represent a novel class of antifungal agents against

fungal infections (16, 25). Furthermore, since antibodies against

the fungal GlcCer inhibit C. neoformans growth in vitro and do not

interact with the human GlcCer (11), they may represent a new

therapeutic approach to control infections by fungal microorganisms

producing GlcCe.