Candida albicans morphology: still in focus

KEYWORDS:

• Candida albicans
• dimorphism
• hyphal formation
• quorum sensing
• inhibition
• hyphal associated genes
• hyphal extension
• compound screening

The yeast-to-hyphal transition (dimorphism) of the most common human pathogenic yeast Candida albicans remains in the focus of molecular and medical mycology [1–4]. Since our review on ‘C. albicans dimorphisms as a therapeutic target’ in 2012 [5], over 100 studies and reviews focusing on the morphology of this yeast have been published, dealing with the multiple aspects of this crucial virulence attribute.

The studies include diverse topics such as large-scale functional screening studies [6–8], interaction with bacteria and their products, interaction with other fungi, new regulatory factors, the role of the vacuole and trafficking, hyphal-associated genes and their functions, hyphal tip and directed growth, hyphal extension and branching, the role of hyphal formation during interaction with macrophages, and finally, large-scale compound screening to identify inhibitors and characterization of selected inhibitors.

As one of the new emerging topics, clinically relevant interactions of C. albicans with bacteria were investigated by several groups with fungal morphology as a crucial element. In most studies, bacteria were found to inhibit fungal filamentation, often by secreted compounds that are linked to bacterial quorum sensing, virulence factors, or metabolism [9–12]. In the case of Fusobacterium nucleatum, however, hyphal inhibition was contact dependent [13]. Interestingly, both probiotic and facultative pathogenic bacteria were shown to negatively affect hyphal formation [9,10,12–15]. It, therefore, appears possible that the interaction with bacteria, rather than distinct environmental conditions or transcriptional programs, is responsible for the predominance of C. albicans yeast morphology in the gastrointestinal tract. Depletion of bacteria by antibiotics might alleviate bacteria-induced suppression of filamentation and thereby contribute to the shift of C. albicans from a commensal to pathogen. This would be a significant new finding as it may explain at least in part what triggers the shift to pathogenicity.

Not all Candida–bacterial interactions are antagonistic; synergistic interactions have been described, for example, for streptococci [16], favoring mixed biofilm formation, and Staphylococcus aureus. For S. aureus, the role of C. albicans hyphal formation in the interaction appears to be niche specific: While filamentation is dispensable for enhanced virulence in peritoneal coinfection [17], S. aureus uses attachment to C. albicans hyphae to invade across mucosal barriers and to cause systemic infections [18]. Similarly, it has been shown that C. albicans hyphae can pave the way for another pathogenic nonfilamentous Candida species, C. glabrata [19]. However, it should be noted that C. glabrata can be highly pathogenic for man, even without a coinfection with C. albicans [20].

Although the regulation of the morphological transition is already well investigated [1–4,21], multiple new regulatory factors directly or indirectly involved in hyphal formation and new insights into regulatory mechanisms have been reported.

For example, it has been shown that activation of Ras1, a key regulator of morphogenesis, requires proteolysis, mitochondrial activity, and the adenylate cyclase Cyr1 [22,23]. Furthermore, signaling for hyphal formation is associated with cell cycle-independent phospho-regulation of Fkh2 [24] and mediated by septins [25]. A genetic approach to dissect hyphal development identified a novel function for the transcription factor Ace2 [26], and diminished expression of the protein kinase Cak1 was shown to bypass filamentation [27]. A similar subtle level of regulation was shown when Hog1 basal activity was reduced [28]. A further link in the complex regulatory network was the identification of a novel feedback circuit formed by the known regulators Brg1 and Nrg1 form [29]. In addition, ceramide synthases [30] and regulation of actin organization [31] play important roles during filamentous growth. A novel link between phenotypic switching and yeast-to-hyphal transition was also found by demonstrating that white and opaque cells undergo distinct programs of filamentous growth [32]. These studies nicely complete our understanding of the already complex picture of the regulation of dimorphism.

Three further subtopics in regulation of hyphal development were in the highlights: (1) the role of oxygen concentrations, (2) the role of the mediator complex, and (3) quorum sensing. Clearly, in vivo hyphal formation is mediated by a mix of environmental signals in the host, which includes hypoxia, temperature, CO₂ concentrations, and nutrient conditions, which in turn require a complex network of signaling factors and modules [33,34]. An interesting connection between filament regulation and the general transcription machinery has been found by the discovery that the mediator complex is directly linked to transcriptional regulation of hyphal regulators and hypha-associated genes [35–37]. Farnesol is the dominant quorum-sensing molecule and acts via different pathways. Novel discoveries of its mode of action include inhibition of Cup9 degradation, which in turn leads to repressed expression of SOK1, encoding a kinase required for degradation of Nrg1 to facilitate initiation of hyphal growth [38]. Farnesol-mediated cell death was found to depend on Efg1 and Cph1, thereby possibly explaining the lower resistance of opaque cells to farnesol [39]. A new discovery was that farnesol affects not only hyphal initiation but also hypha-to-yeast transition [40], which might partially explain the hyphal maintenance defect of an EED1 deletion mutant. This mutant is the first identified to be hypersensitive to farnesol, via unknown mechanisms distinct from pathways known to be targeted by farnesol, and at the same time produces increased amounts of this molecule [41]. After over a decade of intensive research on farnesol, many open questions remain regarding the existence of receptors and transporters and regulation of its synthesis, recently summarized in a commentary by Nickerson and Atkins [42]. Finally, recent evidence demonstrates that it negatively affects the host immune response [43,44], thereby reducing its suitability for therapeutic approaches.

In contrast to the well-characterized hyphal initiation, the mechanisms of hyphal extension are poorly understood but are in the focus of more recent studies [1]. One of the critical factors for hyphal extension is Eed1, a regulator of Ume6. As mentioned above, Eed1 controls farnesol sensitivity, and farnesol resistance might contribute to hyphal maintenance [41]. The molecular mechanisms behind this are still elusive. An interesting finding was that phosphorylation of Exo84 by Cdk1–Hgc1 is necessary for efficient hyphal extension [45]. As part of the exocyst, this complex is essential for cell surface expansion. In Saccharomyces cerevisiae, phosphorylation of Exo84 leads to exocyst disassembly, demonstrating redirection of conserved pathways in C. albicans to facilitate hyphal maintenance. Clearly, the maintenance of hyphal extension is as important as the initiation of hyphal formation and should be further investigated in detail. Similarly, we still know very little about the hyphal-to-yeast transition, which is equally crucial for pathogenicity and commensal growth.

Critical for the pathogenic potential of hyphae is also the directed growth and extension at the hyphal tip. This requires, among others, the protein Cdc42 [46,47]. The dynamics of this GTPase and the local activity [48] control directional growth and promote thigmotropism, which contributes to invasion. Hyphal tip growth requires intracellular compartments, vesicles, and trafficking. For example, it has been shown that prevacuolar compartment RabGTPases impact hyphal growth [49]; that the protein Sec2p is physically associated with SEC2 mRNA on secretory vesicles [45]; and that vacuolar acidification is essential for morphology and virulence [50]. One new, but related, aspect of hyphal growth and pathogenicity is hyphal branching, which precise role still remains to be elucidated [51,52].

Of note, the virulence potential of hyphae is not mediated by the hyphal morphology per se, but by genes which expression is associated with the morphological program. For example, the hyphal wall protein 1 (Hwp1) is required for virulence in distinct niches [53] and Csa2, a member of the Rbt5 protein family, is involved in the utilization of iron from hemoglobin during hyphal growth [54]. However, one study showed that hyphal growth does not always require induction of hyphal-specific genes [55], and another study found that the core filamentation response network is restricted to only eight genes [56].

One of these genes, ECE1, was already discovered in the 1990s due to its high expression during hypha formation. However, its function remained unknown until last year [57]. In fact, Ece1 is a polyprotein consisting of eight short peptides, each separated by lysine–arginine residues. The polyprotein is sequentially processed at these dibasic amino acids by two serine proteases, resulting in peptide secretion. One of these peptides adopts an α-helical structure and permeabilizes host epithelial membranes causing cell lysis and was therefore named Candidalysin. Deletion of the Candidalysin-encoding region from the ECE1 gene abolished the ability of C. albicans to damage epithelial cells and significantly attenuated virulence. Therefore, the production of Candidalysin rather than hyphal formation per se is the mediator of host (epithelial) cell damage. These observations provide the elusive link between hyphal formation and host (epithelial) cell damage and explain why C. albicans filaments are the pathogenic morphology during mucosal infections.

Hyphal formation also plays a key role during interaction of C. albicans with macrophages. Here, significant progress has been made in elucidating some vital questions: How does C. albicans inhibit phagosome acidification? Which intracellular mechanisms cause hyphal formation? How is hyphal formation linked to host cell death? One study showed that hyphal formation and masking of β-glucan via mannan inhibit macrophage phagosome maturation and thus acidification [58]. However, active alkalinization of the phagosome seems to be the main mechanism, which triggers intracellular hyphal formation. This modulation of phagosomal pH requires Stp2p [59], a regulator of amino acid transport, and members of the ATO gene family for transport of ammonia into the phagosome, which in turn causes neutralization of the phagosomal pH [60]. Although it has long been thought that physical piercing of the macrophage membrane by hyphae is the only cause of macrophage cell death, two studies have shown that killing by intracellular C. albicans cells is a two-step mechanism. In fact, C. albicans triggers pyroptosis, an inflammasome-associated programmed cell death, before physical forces cause membrane rupture [61]. The associated interleukin-1β production, however, seems to be independent of hyphal formation [62]. The significance of intracellular hyphal formation within macrophages was also highlighted by a microevolution experiment, where the nonfilamentous C. albicans efg1∆/cph1∆ mutant was exposed to macrophages for several months [37]. Intriguingly, a single point mutation within a gene (SSN3) encoding a component of the Cdk8 module of the mediator complex, which links transcription factors with the general transcription machinery (see above), was sufficient to bypass Efg1/Cph1-dependent filamentation.

Finally, as predicted in our previous review, multiple new compounds have been identified in the last years, some based on large-scale compound screening [63–65], which inhibit hyphal formation and, as shown in some studies, the corresponding hyphal-associated properties and virulence. This included filastatin [66], ascorbic acid [66], mucins [67], gymnemic acids [68], and quinacrine [69] to name only a few. Of note, even known antifungals seem to have specific effects on the different morphological forms of C. albicans, and one azole was shown to force the fungus into the yeast growth mode, potentially due to reduced ergosterol content of the cell membrane [70]. In agreement with that information, one study identified ergosterol synthesis as a critical aspect in a large-scale functional screening to identify regulators of morphogenesis [7].

In summary, hyphal formation and maintenance remain an area of intensive research with exciting discoveries to be made that are both relevant for our understanding of fungal biology and with a great potential to lead to novel adjunctive therapies.

Declaration of interest

The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Additional information

Funding

The authors were supported by the Deutsche Forschungsgemeinschaft (TR/CRC FungiNet to IDJ, and BH; SPP1580 DFG 528/16 and DFG 528/17 to BH; and DFG JA 1960/1-1 to IDJ), the Infect ERA-NET Program (FunComPath; BMBF031L0001A) to BH; the Integrated Research and Treatment Center for Sepsis Control and Care (CSCC) to IDJ and BH; the Jena School for Microbial Communication (JSMC), and the International Leibniz Research School (ILRS) to BH and IJ.