The morphotype heterogeneity in Cryptococcus neoformans

Abstract

Many environmental fungi have evolved exceptional abilities to overcome host defenses and to cause systemic infections. However, the evolutionary trajectory that gives rise to the remarkable pathogenic traits of otherwise saprophytic species is poorly understood. Recent studies suggest that social behaviors likely enhance fitness and augment virulence in the ubiquitous fungus Cryptococcus neoformans. In this regard, heterogeneity in morphotypes and the ability to switch morphotype offer flexibility and resilience for this fungus in disparate environmental and host niches. Here, we discuss the tradeoffs of different morphotypes, the complex intercellular communications that coordinate the transitions of diverse morphotypes, and how the resulting heterogeneity in morphotype provides a source of fitness.

Introduction

Wild microbes are subject to changes in environments teeming with competitors and natural stressors [1]. To cope with the biological and abiotic pressures, microbes develop complex social strategies to ensure their survival. Even within a sibling microbial community composed of genetically identical cells, there exists high heterogeneity in physiology or morphology of its sub-populations [2]. Such heterogeneity creates a source of variation/diversity that can maximize microbial survival in unpredictable environments [3].

In many facultative microbial pathogens, social behaviors are critical for both their saprophytic and pathogenic lifestyles [3-6]. The ability to cause diseases by these microbes that do not require the host to complete their normal life cycle is likely derived from their sophisticated social strategies evolved to adapt to a plethora of natural stressors [6-9], as unveiled in many environmental fungal pathogens [3,10,11]. For these pathogens, an encounter with the host is more of a nuisance than a necessity. Cryptococcus neoformans is an important environmental fungal pathogen and cryptococcal meningitis alone claims half a million deaths annually worldwide [12,13]. This fungus can exist in multiple morphotypes and there is remarkable morphological heterogeneity within a mating community or during disease progression (Figure 1) [14-17]. Cells of different morphologies differ in their virulence potential and in their resistance to a given natural stress [15^•,16,18,19]. Consequently, morphotype transition is controlled by coordinated cell-cell communications in response to environmental or host stimuli [20-23]. Here, we evaluate the tradeoffs offered by multiple morphotypes and their relevance to cryptococcal mating and infection. We also discuss how morphotype transition and heterogeneity might account for the success of C. neoformans by coordinating its dual lifestyles as a saprophyte and as an opportunistic pathogen.

Figure 1.

The unique features and tradeoffs of different Cryptococcus morphotypes. The tradeoff choices are linked with multiple morphotypes, which occur during differentiation of mating community and/or during infection.

Morphotype transition and heterogeneity during mating community differentiation and sexual reproduction

C. neoformans is typically a haploid organism with two opposite mating-types, a and α [24]. This fungus can undergo two forms of sexual reproduction, α- a bisexual mating and unisexual mating (mostly α) [24,25^•]. It is hypothesized that the predominance of the α mating type in nature (>99%) is attributable to or a result of the α unisexual reproduction [24,26].

In C. neoformans, sexual reproduction is coupled with the development of a mating community, where successive events of transition between different morphotypes occur. These transitions create remarkably heterogeneous morphotypes within a mating community, which coordinate the progression of sexual reproduction and natural adaptation (Figure 1) [22^••,25^•]. For instance, upon the mating stimulation, a subpopulation of yeast cells in the community quickly switch to shmoo cells [15^•] (Figure 1). In Saccharomyces cerevisiae, shmoo cells are mating-competent cells that conduct early mating behaviors, such as the perception of the mating partner and cell-cell fusion [27]. Similarly, this shmoo morphotype is observed during mating and is controlled by the pheromone sensing pathway in Cryptococcus [23,28]. Overexpression of Mat2, the pheromone pathway output regulator, is sufficient to drive cells to differentiate into the shmoo morphotype, even under conditions extremely unfavorable to mating [29]. Thus, the shmoo morphotype likely specializes in the early mating events in C. neoformans.

C. neoformans is predominantly considered to be yeasts in nature. Occasionally, it is also observed as pseudohyphae, especially in a mating community or when cells are confronted with a protozoan predator like soil amoeba (Figure 1) [17-19,30]. Pseudohyphal morphology is widely adopted by non-mobile yeasts across the fungal kingdom. Pseudohyphae enable S. cerevisiae to forage for nutrients unavailable at a short distance [31], suggesting that this morphotype provides an alternative approach for cell migration in the environment. In addition, pseudohyphae confer C. neoformans resistance to amoeba, a natural predator of C. neoformans [32].

Hyphae also confer fungal ability to forage nutrients in distance [3,33] and resistance to amoeba predation [34]. In Cryptococcus, the transition to hyphal growth can be initiated in response to the stimuli triggering sexual reproduction (Figure 1) [11]. Both hyphal initiation and extension require the zinc-finger regulator Znf2 that functions downstream of pheromone signaling pathway or other filamentation-inducing stimuli [15^•,35]. Activation of ZNF2 drives hyphal growth in vitro and in vivo [15^•]. During sexual reproduction, Znf2 is activated and bisexual dikaryotic or unisexual monokaryotic hyphae are produced. Some of these hyphae differentiate into a specialized terminal structure termed basidium where meiosis takes place. Meiosis followed by multiple rounds of mitosis results in the production of four chains of spores, which are proposed as major infectious propagules for Cryptococcus [36]. Sexual reproduction generated hyper-virulent and drug resistant strains [37,38^••] and sex may have been an important driving force for cryptococcal evolution and the long-term lineage fitness [25^•,38^••]. Sexual reproduction and hyphal differentiation are genetically bridged [25^•] as hyphae are developmentally primed for sexual reproduction [38^••]. Overall, the yeast-hypha transition is critical for Cryptococcus to coordinate its interactions with other species, its natural adaptation, and evolution. This morphotype transition helps balance the short term adaptive fitness and the long term lineage success (Figure 1).

Impact of morphotype transition and heterogeneity for cryptococcal pathogenicity

Non-yeast morphotypes such as shmoo, pseudohypha, and hypha are rarely detected in host tissues, consistent with the idea that they might be more critical for cryptococcal fitness in the environment. Indeed, the pseudohypha morphology is unfit for cryptococcal virulence, due to its temperature sensitivity [32]. Interestingly, pseudohyphae were able to trigger protective immune-responses in the host, indicating that this morphotype could exert strong anti-virulence effects [39]. Similarly, the hypha form is also unsuited for cryptococcal infections, and the host physiological condition is extremely inhibitory to the yeast-to-hypha transition [3,40]. Forced expression of ZNF2 in vivo attenuates cryptococcal virulence [15^•]. The ZNF2 overexpression cells, either viable or heat killed, can serve as vaccine to provide host protection against subsequent lethal yeast infections (Zhai et al., under review). The inverse relationship between hyphae and virulence in Cryptococcus is similar to what have been observed in other environmental fungal pathogens like Histoplasma capsulatum, Blastomyces dermatitidis, and Penicillium marneffei, suggesting that the anti-virulence property of hyphae might be conserved in these environmental pathogens [3]. This is in striking contrast to Candida albicans [41], likely because the latter has evolved as a human commensal [42]. Thus, the pseudohypha and hypha morphotypes in Cryptococcus exhibit a clear-cut tradeoff between environmental adaptation and pathogenicity in warm blooded mammals.

During lung infection, spores will convert to yeast cells [43] and a subpopulation of yeast cells become considerately enlarged (Figure 1) [21,44]. The formation of these giant cells, also known as titan cells, is concomitant with a remarkable increase in ploidy [21,44]. In fungi, ploidy change is an important adaptive strategy against stressors (Gerstein and Berman, this issue). In agreement with this notion, titan cells are resistant to phagocytosis and oxidative and nitrosative stresses [21,45]. The titan morphology can also be observed in vitro in response to poor nutrients, suggesting that it is cryptococcal inherent adaptive behavior against starvation and other environmental stressors [45]. Consistently, the ability to form titan cells is considerably impaired in the absence of functional cAMP-PKA pathway [45], which is a core pathway in fungi for sensing environmental factors. In fact, this pathway is also adopted by other fungi to control morphotypes/phenotypes in response to environmental stimuli. The importance of this pathway in morphologic plasticity is evidently shown in Candida albicans where it regulates transitions between white, opaque, gray, and gut phenotypes (Scaduto and Bennett, this issue).

Cryptococcus infection initiates in the lung and eventually it spreads to the brain, causing the fatal cryptococcal meningitis [46]. Although titan cells are observed in the lungs, these cells fail to disseminate to the brain, likely due to their size. Thus, the titan morphology likely specializes for a specific infection stage in the lung tissue [21]. Consistently, smaller size appears to expedite extrapulmonary dissemination [21]. By live imaging, Shi et al demonstrated that yeast cells switch to an ovoid-shaped morphology after being trapped in mouse brain capillaries (Figure 1) [14]. This morphotype switch might facilitate cryptococcal cells to transmigrate from the capillaries into the brain [14]. Collectively, these data suggest that engaging in the morphotype transition is coordinated with the disease progression.

Intercellular communications modulate cryptococcal morphotype heterogeneity and transition.

Microbial community is dynamic. The populations can undergo considerable reconstruction during microbial development or in response to environmental changes [2]. Cell-cell communication, through a “secrete-to-sense” circuit, can specialize subpopulations or synchronize the whole population via its control of gene expression [1,47].

To synchronize microbial behavior, extracellular signals must easily spread across the community (Figure 2b). As expected, these signals are of low-molecular-weight and highly diffusible. Quorum sensing molecules (QSM) represent the most studied group of global signals [47]. In general, QSMs facilitate microbe to sense cell density and to implement a functional or phenotypic switch in a synchronized manner. In fungi, QSM or QSM-like molecules function in the control of inoculum size-dependent behaviors. In C. albicans, QS regulation plays a fine-tuning role rather than a switch in controlling the speed and robustness of morphological transition [48]. In C. neoformans, we found that the Znf2-driven yeast-to-hypha transition is negatively affected by high inoculum when the ZNF2 overexpression strain is cultured in YPD liquid medium. The QS-like factor is yet to be identified (Wang L and Lin X, unpublished data). Despite the highly diffusible feature of QSMs, cells in a community respond to QSMs asynchronously, which may reflect the inherent heterogeneity of individual fungal cells in sensing or responding to QSMs. Such heterogeneous responses may prevent potential disastrous outcomes, should shifting of morphotype be detrimental.

Figure 2.

The ECM signal Cfl1 might represent a local signal. (a) Cfl1 exclusively expresses in the hyphal subpopulation in a mating community. (b) A diagram indicates the different regulatory patterns of easily diffusible signals and geographically more restricted ECM signals. Highly diffusible signals exert global regulation for the community . By contrast, an ECM signal cannot diffuse efficiently across the community, resulting in stimulation limited to a subset of the population. The combination of signals of these two systems can create enormous heterogeneity in the microbial community.

Besides QSMs, pheromones (usually oligopeptides), represent another family of diffusible fungal signals. Generally, pheromones are dedicated to early mating behaviors through its complex patterns of autocrine and/or paracrine signaling. In Cryptococcus, the paracrine regulation of pheromone during bisexual mating leads to mating partner attraction, shmoo differentiation, and cell fusion [23]. The autocrine regulation of pheromone is more important for unisexual mating [23]. During both bisexual and unisexual mating, pheromone signaling creates considerable heterogeneity in morphotypes in a mating community, and some cells in the hypha morphotype further differentiate to aerial fruiting structures. The deletion of MAT2, the master regulator of the pheromone sensing and response pathway, abolishes the cell heterogeneity in the community [15^•,29]. Thus, sensing and production of pheromones link external stimulation with genetic programming for sexual reproduction.

In higher eukaryotes, signals that exert local activities are important for the formation of highly differentiated tissue pattern [49]. A well-studied case is extracellular matrix (ECM) signals, which play fundamental roles in regulating cell shape, motility, and tissue differentiation [49]. Two recent studies unveiled that ECM signals also exist in microbe and are involved in the control of community differentiation. In Pseudomonas aeruginosa, the major biofilm matrix polysaccharide Psl acts as an autoinduction signal to promote biofilm formation [50^••]. In C. neoformans, we recently showed that an ECM protein Cfl1 stimulates the yeast-to-hypha transition [22^••]. Extracellular Cfl1 drives the transition through its upstream regulator Znf2, forming a positive-feedback regulation that resembles the activity of an autoinducer. Accordingly, Cfl1 is highly expressed in the hyphal subpopulation located at the periphery of a mating community (Figure 2a). Unlike the aforementioned low-molecular-weight signals, Cfl1 is a protein over 20kD [22^••]. Thus, it is unlikely that Cfl1 synchronizes gene expression for the whole community under most conditions. Consistent with this hypothesis, Cfl1 is concentrated in the extracellular matrix layer covering the colony surface after prolonged incubation [22^••]. This suggests that Cfl1 may serve as a local signal regulating morphotype transition in the cells enclosed or adjacent to the extracellular matrix, in which Cfl1 is highly concentrated (Figure 2b) [22^••].

Conclusions

In a C. neoformans community, many morphotypes co-exist. These morphotypes differ in their pathogenicity, their development competency, or their stress resistance, thereby creating a population that can potentially adapt to conditions with unpredictable changes. This heterogeneity in morphotype mirrors the regulatory complexity created by multiple communication signals with distinct regulatory patterns. Morphotype-related tradeoffs are also observed during the progression of cryptococcal infection [14,21,44], despite the fact that cryptococcal encounter with a mammalian host is purely incidental. This is consistent with the absence of Cryptococcus neoformans from human microbiota in metagenomic studies [51]. Thus, morphotype switches related to pathogenic lifestyle may well have been evolved from strategies to survive environmental conditions that mimic different host niches. This again demonstrates the importance of the “dual use” of cryptococcal social biology.

Highlights.

• Both host and environmental signals entail cryptococcal morphotype heterogeneity.

• Diverse morphotypes are coordinated with saprophytic and pathogenic lifestyles.

• Morphotype heterogeneity is fine-tuned by various intercellular signals.