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Summary

  1. Top of page
  2. Summary
  3. A G1 cyclin dedicated to hyphal morphogenesis is required for virulence
  4. Virulence gene expression is co-regulated with hyphal morphogenesis
  5. Co-regulation of virulence genes and morphogenesis genes by regulatory proteins
  6. Convergence of many regulatory pathways to a common set of genes
  7. How do hyphae contribute to pathogenesis?
  8. Conclusions
  9. Acknowledgements
  10. References

The fascinating ability of Candida albicans to undergo dramatic changes in cellular morphology has invited speculation that this plasticity in form contributes to the virulence of the organism. Molecular genetic analyses have confirmed this hypothesis and further demonstrated that genes that govern cellular morphology are co-regulated with genes encoding conventional virulence factors such as proteases and adhesins. The transcriptional regulatory networks of C. albicans thus ensure that hyphae are produced concomitantly with virulence factors, resulting in cells that are adapted for invading the tissues of an immunocompromised host. Hyphae are able to exert mechanical force, aiding penetration of epithelial surfaces, and hyphae damage endothelial cells, aiding escape of C. albicans from the host bloodstream into deeper tissue. Hyphal morphogenesis is thus an integral part of the overall virulence strategy of C. albicans.

Unlike highly specialized pathogens that express a single major virulence factor (e.g. Clostridium tetani ), the opportunistic fungal pathogen Candida albicans expresses a repertoire of activities that contribute to virulence. The sum of the effects of many Candida factors leads to the establishment of infection in a suitably compromised host. In some host niches, particular fungal proteins (e.g. adhesins) are important, while in other niches, these proteins are less important and other factors play a dominant role. By being adapted for growth in many possible host niches, C. albicans is able to cause the wide spectrum of clinical manifestations for which is it known.

Discussions of C. albicans virulence factors usually include dimorphism, the ability to grow in more than one morphological form. C. albicans grows either as budding yeast cells or as filamentous hyphae (chains of elongated, parallel-sided cells lacking constrictions at the septa) or pseudohyphae (chains of variably elongated cells with constrictions at the septa). Because of their similar elongated shapes and the many conditions and regulators that are shared in common, the hyphal and pseudohyphal forms are often not distinguished in the literature; the more general term, ‘filamentous forms’, is used to refer to both. The two morphologies, however, are fundamentally different and can be distinguished by a number of characteristics. The distinctions between the two forms have been elegantly reviewed by Sudbery et al. (2004).

Filamentation is believed to be important for virulence and thus, morphogenesis in this fungus has been a subject of considerable study. The potential roles of the hyphal form, in particular, in virulence have also been discussed frequently in the literature. While most mutants that fail to produce hyphae are compromised in virulence, several authors have pointed out that co-regulation of genes controlling hyphal morphogenesis with genes encoding virulence factors confounds the analysis (Kobayashi and Cutler, 1998; Brown, 2002; Gow et al., 2002; Liu, 2002). Thus, it has been difficult to determine unequivocably the contribution of the hyphal form per se to virulence. Recent studies support the conclusion that the hyphal form is important for virulence. It is also very clear that genes that control hyphal morphology are co-regulated with genes that encode more standard virulence factors such as proteases and adhesins (Fig. 1). This co-regulation ensures that the morphological conversion of yeast-form cells to hyphae occurs under conditions when degradative activities and adhesins that enhance virulence are also expressed. Thus, formation of hyphae is a component of the overall virulence strategy of C. albicans. This review will summarize findings that support a role for hyphae and co-regulated genes in virulence, and discuss models that illustrate how hyphae contribute to the pathogenicity of C. albicans.

image

Figure 1. Candida albicans hyphal morphogenesis coincides with the expression of virulence factors. Both the expression of virulence factors and the morphological transition from the budding yeast form (left) to the hyphal form (right) are regulated by a number of transcription factors. Among the better characterized of these factors are negative regulators, Tup1p, Nrg1p and Rfg1p, and positive regulators, Efg1p and Cph1p. Rfg1p has also been shown to act as a positive regulator of filamentous growth under certain conditions (not shown) (Kadosh and Johnson, 2001). The cytoplasmic protein Hgc1p (hypha-specific G1 cyclin) is a key determinant of cellular morphology. Virulence factors expressed by hyphal cells include adhesins (surface proteins involved in host cell recognition and attachment) and secreted proteases associated with tissue invasiveness.

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A G1 cyclin dedicated to hyphal morphogenesis is required for virulence

  1. Top of page
  2. Summary
  3. A G1 cyclin dedicated to hyphal morphogenesis is required for virulence
  4. Virulence gene expression is co-regulated with hyphal morphogenesis
  5. Co-regulation of virulence genes and morphogenesis genes by regulatory proteins
  6. Convergence of many regulatory pathways to a common set of genes
  7. How do hyphae contribute to pathogenesis?
  8. Conclusions
  9. Acknowledgements
  10. References

To determine the role of the hyphal form in virulence, researchers have sought a gene that is needed for production of hyphae but affects neither the expression of other hypha-co-regulated genes nor the growth of yeast-form cells. The recent studies of Zheng et al. demonstrate that HGC1 appears to have the properties of such a gene (Zheng et al., 2004). HGC1 encodes a G1 cyclin-related protein that is not expressed in yeast cells. However, when cells are shifted to hypha-promoting growth conditions (such as growth at 37°C in medium containing serum), HGC1 expression is rapidly induced. Unlike typical G1 cyclins, HGC1 expression does not vary during the cell cycle suggesting that the function of Hgc1p is distinct from that of more typical G1 cyclins that regulate cell cycle progression.

Deletion of hgc1 results in cells that grow normally as yeast-form cells but fail to produce hyphae when grown under several hypha-promoting conditions, including growth at 37°C in serum-containing liquid medium or other media, and growth on agar media. Although the mutant cells do not assume hyphal morphology, they were shown to express three hypha-associated genes. Therefore, HGC1 probably encodes a component of the hyphal formation machinery, rather than a regulator of hyphal gene expression. The hgc1 mutant is thus an ideal mutant to use in evaluating the role of hyphae in virulence.

Following intravenous inoculation of the mouse, hgc1 null mutant cells fail to result in the usual mortality that is observed when wild-type cells are intravenously inoculated (Zheng et al., 2004). This decreased lethality for mice demonstrates that the hgc1 mutant is dramatically attenuated for virulence. In addition, at an early stage of infection, the morphology of hgc1 mutant cells growing in the mouse kidney is non-filamentous, in contrast to cells of the wild-type strain that exhibit typical filamentous morphology.

Because the hgc1 mutation drastically affects hyphal morphogenesis with minimal effects on other cellular functions, the low virulence of the mutant provides strong support for the conclusion that hyphal morphology per se is important for virulence. Models that describe ways in which filamentous cells might contribute to host–pathogen interactions are discussed at the end of this review.

Virulence gene expression is co-regulated with hyphal morphogenesis

  1. Top of page
  2. Summary
  3. A G1 cyclin dedicated to hyphal morphogenesis is required for virulence
  4. Virulence gene expression is co-regulated with hyphal morphogenesis
  5. Co-regulation of virulence genes and morphogenesis genes by regulatory proteins
  6. Convergence of many regulatory pathways to a common set of genes
  7. How do hyphae contribute to pathogenesis?
  8. Conclusions
  9. Acknowledgements
  10. References

In addition to HGC1, expression of numerous genes is altered during hyphal morphogenesis (Lane et al., 2001; Nantel et al., 2002; Kadosh and Johnson, 2005). Some of these genes encode virulence factors that do not influence cellular morphogenesis (Table 1). These genes represent hypha-co-regulated genes whose function is unrelated to morphology.

Table 1.  Hypha-co-regulated genes and transcription regulators.
GeneFunctionExpression in hyphae
HGC1G1 cyclin-related proteinEfg1p-dependent
SAP4Secreted aspartic proteaseEfg1p-dependent
SAP5Secreted aspartic proteaseEfg1p-dependent
SAP6Secreted aspartic proteaseEfg1p-dependent
ALS3AdhesinEfg1p-dependent
ALS1AdhesinEfg1p-dependent (not hypha-specific)
HWP1AdhesinEfg1p-dependent
RBT1Unknown (putative cell wall protein)Efg1p-dependent
RBT4Unknown (putative secreted protein)Efg1p-dependent
EFG1Transcription factorDownregulated
CPH1Transcription factor 
NRG1Negative regulator of transcriptionEfg1p-dependent downregulation

Well-studied examples of hypha-co-regulated virulence genes are the SAP4, SAP5 and SAP6 genes, which encode secreted aspartic proteases (SAPs). At least 10 different genes encode SAPs and the different genes are expressed under different laboratory conditions (reviewed in Naglik et al., 2003); SAP4, SAP5 and SAP6 are expressed during hyphal morphogenesis. Also, different SAPs contribute to virulence in different models of infection (Naglik et al., 2003) and the proteases encoded by SAP4, SAP5 and SAP6 are important for invasion of parenchymal tissue following intraperitoneal inoculation of the mouse (Felk et al., 2002). SAP proteases may degrade host proteins such as components of the extracellular matrix and proteins involved in host defence, enhancing C. albicans pathogenicity (Naglik et al., 2003). SAP4, SAP5 and SAP6 fit the definition of hypha-co-regulated virulence genes whose functions are unrelated to morphology.

Hypha-specific cell wall proteins include the adhesins Als3p and Hwp1p. The ALS gene family, comprised of eight members, encodes glycosylphosphatidylinositol (GPI)-modified cell wall glycoproteins. Proteins such as Hwp1p and the Als proteins are modified at the C-terminus by addition of a GPI moiety, which results in their initial localization to the cell membrane; subsequently the GPI anchor is cleaved and the protein is cross-linked to the cell wall (Sundstrom, 2002). Als3p promotes adherence to both human umbilical vein endothelial cells (HUVEC) and buccal epithelial cells (BEC) (Zhao et al., 2004). Als1p, another member of the Als family, is also expressed by filamenting cells and is important for adherence of C. albicans to HUVEC (endothelial cells) but not to BEC (epithelial cells) (Fu et al., 2002; Zhao et al., 2004); als1 mutants also display reduced virulence following intravenous inoculation (Fu et al., 2002).

The hypha-specific cell wall protein Hwp1p is another GPI-modified cell wall protein. Hwp1p plays a role in adherence to BEC (epithelial cells) (Staab et al., 1999; Sundstrom et al., 2002a) but is not required for adherence to HUVEC (endothelial cells) (Tsuchimori et al., 2000). Hwp1p is a substrate for keratinocyte transglutaminase and can be stably cross-linked to BEC (epithelial cells); therefore, Hwp1p mediates very tight binding of C. albicans cells to host cells (Staab et al., 1999). An hwp1 mutant strain is moderately compromised for virulence following intravenous inoculation of mice (Sundstrom et al., 2002b; Sharkey et al., 2005). The mutant is also defective in damaging the tongue and oesophagus of immunodeficient mice (Sundstrom et al., 2002a). These hypha-co-regulated adhesins represent additional factors that are important for C. albicans virulence.

RBT1 and RBT4 encode a hyphally expressed GPI-anchored cell wall protein and a hyphally expressed secreted protein respectively. The specific functions of these genes are unknown but their deletion results in lower virulence in two different animal models of infection (Braun et al., 2000).

These results demonstrate that hyphae express numerous proteins that enhance virulence. As a result, hyphae are adapted for adhering to host cells and for damaging host tissue through proteolysis. It is noteworthy that hyphae express an epithelial cell adhesin, Hwp1p, an endothelial cell adhesin, Als1p, and an adhesin for both cell types, Als3p. C. albicans hyphae are thus adapted for adherence to both epithelial tissue and endothelium. Being prepared to adhere to and colonize multiple types of tissues is probably a strategy that allows C. albicans to be a successful opportunistic pathogen.

Co-regulation of virulence genes and morphogenesis genes by regulatory proteins

  1. Top of page
  2. Summary
  3. A G1 cyclin dedicated to hyphal morphogenesis is required for virulence
  4. Virulence gene expression is co-regulated with hyphal morphogenesis
  5. Co-regulation of virulence genes and morphogenesis genes by regulatory proteins
  6. Convergence of many regulatory pathways to a common set of genes
  7. How do hyphae contribute to pathogenesis?
  8. Conclusions
  9. Acknowledgements
  10. References

Studies of the regulatory proteins that control hyphal morphogenesis demonstrate that co-regulation of virulence factors and morphogenesis genes is accomplished through the activity of specific transcription factors (Table 1). The molecular mechanisms that control cellular morphogenesis in C. albicans have been extensively investigated and numerous genes that are important for formation of hyphae have been identified. Recent reviews have comprehensively described the molecular details of signalling pathways that regulate hyphal morphogenesis as well as molecules that are important for forming a hyphal structure (Brown, 2002; Liu, 2002; Whiteway and Oberholzer, 2004).

The best-characterized regulator of hyphal morphogenesis is the transcription factor, Efg1p, a member of the APSES family of basic helix–loop–helix transcriptional regulators (Stoldt et al., 1997). The putative transcription factor Cph1p, homologous to Saccharomyces cerevisiae Ste12p, also plays a role in regulating hyphal morphogenesis (Liu et al., 1994). Both EFG1 and CPH1 were identified by virtue of their ability to promote filamentous growth in the non-pathogenic yeast S. cerevisiae. The ability of C. albicans EFG1 and CPH1 to promote filamentation in S. cerevisiae reflects the conserved nature of some of the pathways that regulate filamentation in both organisms.

Candida albicans deletion mutants lacking either EFG1 or CPH1 exhibit defects in hypha formation, although the effects of deleting cph1 are minor compared with those of deleting efg1. In combination, the efg1 cph1 double mutant is extremely defective for filamentous growth under most laboratory conditions tested (Lo et al., 1997). Not surprisingly, expression of the HGC1 gene (hypha-specific G1 cyclin) during hyphal morphogenesis is dependent on Efg1p (Zheng et al., 2004) and the inability of Efg1p mutants to form hyphae is probably related, at least in part, to the failure to express this critical gene.

Lo et al. (1997) demonstrated that the extremely defective efg1 cph1 double null mutant fails to cause mortality following intravenous inoculation of mice and is thus completely avirulent in the systemic model. This dramatic result implicates both hyphal morphology, and the EFG1 and CPH1 pathways, in the virulence of C. albicans.

Interestingly, the efg1 cph1 double null mutant (lacking transcription regulators) is more defective in virulence than the hgc1 mutant (lacking the hyphal-dedicated G1 cyclin). Injection of efg1 cph1 double null mutant cells resulted in no mouse mortality (Lo et al., 1997) while injection of hgc1 mutant cells resulted in 30% lethality (Zheng et al., 2004). These results suggest that Efg1p is needed for the expression of genes other than the HGC1 gene that are important for virulence. In previous reviews, Kobayashi and Cutler (1998) and many others have raised the possibility that Efg1p is a direct regulator of virulence factors as well as a regulator of hyphal morphogenesis. It is now clear that Efg1p regulates the expression of numerous genes, including many that encode known virulence factors (Lane et al., 2001; Nantel et al., 2002; Sohn et al., 2003; Doedt et al., 2004; Harcus et al., 2004). Efg1p regulates functions and processes as diverse as cell wall dynamics and metabolism.

For example, during growth within a host or within a model of human tissue (reconstituted human epithelium), efg1 mutant cells fail to express several SAP genes, including SAP4, SAP5 and SAP6, while wild-type cells express multiple SAP genes (Staib et al., 2002; Korting et al., 2003). In addition, Efg1p regulates the expression of the SAP4, SAP5 and SAP6 proteases during hyphal growth in the laboratory (Schroppel et al., 2000). Thus, as proposed, efg1 mutants are deficient in expressing this important group of virulence factors.

In addition to its defect in expressing aspartic proteases, the efg1 null mutant is defective for adherence to mammalian cells and to abiotic surfaces such as plastic or catheter material (reviewed in Kumamoto and Vinces, 2005), probably as a consequence of the widespread effects of Efg1p on expression of cell surface proteins (Sohn et al., 2003). Expression of the adhesins Als1p, Als3p and Hwp1p is dependent on EFG1 (Hoyer et al., 1998; Sharkey et al., 1999; Fu et al., 2002). Purified Efg1p protein has been shown to bind directly to the promoter of the ALS3 gene (previously denoted ALS8) in a sequence-specific manner, demonstrating that Efg1p is a direct regulator of this virulence factor (Leng et al., 2001). Rbt1p and Rbt4p (virulence factors of unknown function) expression is also dependent on EFG1 (Braun et al., 2000). Thus, an efg1 null strain is deficient in expression of all of these important virulence factors and this defect probably contributes to the avirulence of the efg1 cph1 mutant strains.

Although Efg1p acts as a positive regulator of filamentous growth under most laboratory conditions, certain conditions have been identified in which filamentation is negatively regulated by Efg1p (Sonneborn et al., 1999; Giusani et al., 2002). Efg1p acts as a repressor of gene expression (Doedt et al., 2004) and, during the yeast-to-hyphae transition, many genes are upregulated in the absence of Efg1p (Harcus et al., 2004). It may be that the activity of Efg1p is modulated depending on the growth conditions and stage of growth. EFG1 itself is downregulated upon the onset of serum-induced hyphal growth (Stoldt et al., 1997), and artificially maintaining high levels of EFG1 levels interferes with true hyphal formation (although not pseudohyphal growth) (Tebarth et al., 2003) suggesting that even in conditions where it positively regulates filamentous growth, EFG1 must be downregulated in order for hyphal morphogenesis to occur.

In summary, Efg1p regulates both hyphal morphology and the expression of several genes that have direct roles in virulence. The striking avirulence of the efg1 cph1 double null mutant reflects the combined effects of defective cellular morphology and defective expression of co-regulated virulence factors. It is therefore more accurate to think of Efg1p as a virulence regulator rather than simply a regulator of morphogenesis. Regulation of cellular morphology is only one facet of the physiological function of Efg1p.

Convergence of many regulatory pathways to a common set of genes

  1. Top of page
  2. Summary
  3. A G1 cyclin dedicated to hyphal morphogenesis is required for virulence
  4. Virulence gene expression is co-regulated with hyphal morphogenesis
  5. Co-regulation of virulence genes and morphogenesis genes by regulatory proteins
  6. Convergence of many regulatory pathways to a common set of genes
  7. How do hyphae contribute to pathogenesis?
  8. Conclusions
  9. Acknowledgements
  10. References

Thus far we have presented a relatively simple picture of the regulation of morphogenesis by two pathways, but increasingly, other factors have been shown to play a role in this regulation. Among these are Tup1, Nrg1p and Rfg1p, repressors of hyphal gene expression and filamentous growth. The nrg1 mutant exhibits derepressed filamentous growth and is avirulent (Murad et al., 2001a). Importantly, expression of NRG1 is strongly downregulated during hyphal development (Murad et al., 2001a) and constitutive expression of NRG1 prevents formation of hyphae (Braun et al., 2001). In S. cerevisiae, the homologue of Nrg1p interacts with S. cerevisiae Tup1p to repress gene expression, and the C. albicans homologues likely do the same. tup1 and nrg1 mutants are very similar, displaying derepressed filamentation, derepressed hyphal gene expression, overlapping transcript profiles and highly attenuated virulence (Murad et al., 2001a,b). An additional partner of ScTup1 is Rox1p, and the C. albicans ROX1 homologue, RFG1, also displays mutant phenotypes related to those of nrg1 and tup1 mutants (Kadosh and Johnson, 2001; Khalaf and Zitomer, 2001). Rfg1p, however, appears to also act as a positive regulator of filamentous growth under certain growth conditions (Kadosh and Johnson, 2001).

Despite the evidence for several pathways, Northern blot and microarray analyses show that these various pathways converge to regulate a common set of genes, termed here the hyphal regulon, which includes the ALS3, HWP1, RBT1, RBT4, SAP4, SAP5 and SAP6 genes discussed above (Lane et al., 2001; Murad et al., 2001b; Nantel et al., 2002; Kadosh and Johnson, 2005). Other members of the hyphal regulon that are convergently regulated include ECE1, DDR48 and HYR1, encoding hyphally regulated genes of unknown function. In addition to its regulation by Efg1p, expression of HGC1 is derepressed by tup1 or nrg1 mutations, as expected for a critical member of the hyphal regulon (Zheng et al., 2004). These results demonstrate that the genes of the hyphal regulon are co-regulated by numerous transcription factors and signalling pathways.

The fact that the genes of the hyphal regulon are co-regulated by several transcription factors argues that the cell has designed its transcription networks to ensure that hyphae are produced concomitantly with appropriate virulence factors under circumstances that favour virulence. A particularly compelling demonstration that expression of the hyphal regulon favours virulence was provided by Saville et al. (2003) who engineered a strain with regulated expression of the repressor Nrg1p. Cells that express NRG1 and therefore neither form hyphae nor express co-regulated genes are avirulent following intravenous inoculation. Cells in which NRG1 expression is inhibited are able to form hyphae and probably express hypha-co-regulated genes. These cells exhibit the typical lethality of wild-type cells in the mouse model of systemic infection. When expression of NRG1 was inhibited subsequent to inoculation of the mouse, lethality occurred soon after the switch, suggesting that expression of hyphal genes and co-regulated virulence genes led to a more virulent phenotype.

The co-regulation of cellular form with expression of other virulence genes through Efg1p and other factors argues that morphological changes are a central part of the virulence strategy employed by C. albicans. By having this critical set of genes under the control of multiple pathways and transcription factors, C. albicans is able to express the hyphal regulon in numerous host niches and as a result can produce infection in a wide range of tissues. This flexibility in regulation allows C. albicans to be a versatile exploiter of its host.

How do hyphae contribute to pathogenesis?

  1. Top of page
  2. Summary
  3. A G1 cyclin dedicated to hyphal morphogenesis is required for virulence
  4. Virulence gene expression is co-regulated with hyphal morphogenesis
  5. Co-regulation of virulence genes and morphogenesis genes by regulatory proteins
  6. Convergence of many regulatory pathways to a common set of genes
  7. How do hyphae contribute to pathogenesis?
  8. Conclusions
  9. Acknowledgements
  10. References

At least two important functions for hyphae have been suggested previously (Gow et al., 2002; Whiteway and Oberholzer, 2004; Zheng et al., 2004; Kumamoto and Vinces, 2005). (i) The hyphal form may be important for penetrating tissue surfaces. (ii) The hyphal form may be important for escaping from host cells following internalization. These ideas are discussed below in the context of hyphal invasion of epithelial surfaces and endothelial surfaces (Fig. 2).

image

Figure 2. Illustration of tissue invasion by C. albicans. On an epithelial surface, invasion by C. albicans (Ca, indicated by arrows) is initiated by attachment to host tissue via surface adhesins; tissue is penetrated by filamentous fungal cells. Invasion of endothelium occurs via an endocytic process in which C. albicans cells (yeast or filaments) are taken in by host cells. Subsequent tissue damage is caused by secreted factors, mechanical force exerted by the hyphal form, or a combination of both. (Not drawn to scale.) Figure by M.D. and R.F. Vinces.

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(i) Epithelial invasion using mechanical force

An intriguing micrograph of a human biopsy specimen demonstrates that during penetration of human epidermis by C. albicans hyphae, deformation of host corneocytes occurs (Scherwitz, 1982). It was suggested that hyphae produce mechanical force aiding penetration of the host epidermis. In order for mechanical force to be exerted on the host tissue surface, tight anchorage of C. albicans cells to host tissue is probably a prerequisite. The adhesin Hwp1p may play an important role in tight adhesion because this protein is a substrate for host transglutaminases and thus can be covalently cross-linked to the surface of host tissue (Staab et al., 1999). hwp1 mutants are defective in adhesion to epithelial cells and fail to cause oral or oesophageal lesions in immunodeficient mice (Sundstrom et al., 2002a) suggesting that expression of the Hwp1p adhesin on the surface of hyphae may be important for penetration of epithelial surfaces. HWP1 is a member of the hyphal regulon, ensuring that this important gene is expressed in cells that are involved in host tissue penetration.

The efg1p cph1 double null mutant, lacking two transcription factors, produces markedly diminished oral thrush in an immunosuppressed animal model (Riggle et al., 1999). Both decreased surface proliferation and decreased tissue invasion are seen with the mutant strain. The defective surface proliferation of the mutant probably reflects the drastic alteration in expression of cell surface proteins, including adhesins, that occurs in the absence of Efg1p (Lane et al., 2001; Nantel et al., 2002; Sohn et al., 2003; Harcus et al., 2004). Defective tissue invasion is probably secondary to defective adhesion, because Efg1p is not required for filamentation within tissue. In fact, filamentous cells are found in the few oral lesions produced by the efg1 cph1 double null mutant in the tongues of immunosuppressed animals (Riggle et al., 1999) and efg1 mutant cells are hyperinvasive in an agar matrix (a laboratory model for tissue invasion) (Kumamoto and Vinces, 2005).

To summarize, invasion of an epithelial tissue is proposed to involve mechanical force exerted by hyphae. Efg1p-dependent expression of appropriate adhesins allows tight adherence of C. albicans cells to the epithelial surface, aiding the application of force by hyphae. Thus, the components of the hyphal regulon act together to produce the characteristic invasive lesions of mucosal candidiasis.

(ii) Breaching of endothelial barriers through hypha-dependent endothelial cell damage

The mechanisms used by C. albicans to breach an endothelial surface are probably different from those used to breach epithelial surfaces because C. albicans cells stimulate endothelial cells to take them up via endocytosis. In this situation, hyphae and expression of the hyphal regulon promote uptake and subsequent damage of endothelial cells. Damage of endothelial cells may be mechanistically related to the ability of hyphae to injure phagocytic cells during escape from their interior.

In a study of the importance of fungal morphology for interactions with HUVEC (endothelial cells), Phan et al. (2000) showed that the efg1 null mutant does not produce filaments when incubated with HUVEC. Nevertheless, the mutant cells are internalized about half as efficiently as the wild-type strain. Thus, although wild-type strains that form hyphae are internalized most efficiently, there is significant internalization of yeasts. Other investigators, using different C. albicans strains, have observed internalization of yeast-form cells by human brain microvascular endothelial cells (Jong et al., 2001) and by bovine aortic endothelial cells (Zink et al., 1996). It is most likely that expression of a critical gene(s) that allows engagement of the correct host cell receptor (Phan et al., 2005), rather than the morphology of the cells per se, is the important factor in stimulating the internalization of C. albicans.

After being internalized, C. albicans cells damage endothelial cells. Endothelial cell damage by one C. albicans cell is believed to aid penetration and traversal of the endothelium by other C. albicans cells. The mechanism by which C. albicans cells damage endothelial cells may be related to the mechanism by which C. albicans cells damage cultured macrophages (Lo et al., 1997) and neutrophils (Korting et al., 2003). It has been observed that hyphal formation allows phagocytosed C. albicans to escape from the interior of phagocytic cells and to cause severe damage to the phagocytes. By analogy, filamentous growth within endothelial cells may allow escape and transcytosis of the endothelial cell layer by C. albicans concomitant with endothelial cell damage. Images showing filamentous outgrowths from internalized C. albicans cells that protrude outside of endothelial cells support this model (Zink et al., 1996; Jong et al., 2001).

efg1 null mutant cells (lacking a transcription regulator) are extremely defective in damaging HUVEC (endothelial cells) (Phan et al., 2000). This defect may reflect both an inability to express key genes of the hyphal regulon, such as SAP proteases, and an inability to form hyphae during or after internalization. Thus, hyphal formation is a key mechanism by which C. albicans damages the endothelial barrier, enhancing penetration by additional C. albicans cells.

Conclusions

  1. Top of page
  2. Summary
  3. A G1 cyclin dedicated to hyphal morphogenesis is required for virulence
  4. Virulence gene expression is co-regulated with hyphal morphogenesis
  5. Co-regulation of virulence genes and morphogenesis genes by regulatory proteins
  6. Convergence of many regulatory pathways to a common set of genes
  7. How do hyphae contribute to pathogenesis?
  8. Conclusions
  9. Acknowledgements
  10. References

This body of work demonstrates that filamentation is an integral component of the overall virulence strategy of C. albicans. In order to ensure that all of the activities that favour virulence are expressed together at the appropriate time, proteins that control cellular form, such as the hypha-specific G1 cyclin Hgc1p, are co-regulated with adhesins and proteases. Co-regulation is achieved through the activity of transcriptional regulators, such as Efg1p and Nrg1p.

These studies have also begun to illuminate some of the features that make C. albicans a successful opportunist. For example, hyphae simultaneously express adhesins for multiple cell types, allowing C. albicans cells to be prepared for adherence to multiple types of tissues. This strategy allows C. albicans to establish infection in many organs of the host. Also, the hyphal regulon is regulated by several signalling pathways and several transcription factors. This strategy makes it possible for C. albicans to respond to many cues provided by the host. The design of its transcription networks allows C. albicans to monitor its environment closely and to initiate infection in numerous host tissues.

As a transcription regulator of the hyphal regulon, Efg1p is a key regulator of virulence. Efg1p controls adherence of C. albicans cells to tissue, escape from within host cells and expression of proteases. Given its central role, it is not surprising that there are several factors that regulate Efg1p activity including protein kinase A (Bockmuhl and Ernst, 2001) and the DNA-binding protein Czf1p (Giusani et al., 2002).

Hyphae are important for tissue invasion because they allow application of mechanical force and because they allow C. albicans cells to escape from and damage host cells following uptake by endocytosis. Although this discussion has focused on the importance of hyphae, it is important to note that mutants that are constitutively filamentous also exhibit attenuated virulence (reviewed in Gow et al., 2002). Optimal virulence is thought to require the ability to switch from one morphological form to the other. Presumably hyphae have specific functions, as noted above, and yeast-form cells have other functions. The ability to switch from one form to the other in diverse niches and in response to many host cues makes C. albicans a versatile and well-adapted pathogen.

Acknowledgements

  1. Top of page
  2. Summary
  3. A G1 cyclin dedicated to hyphal morphogenesis is required for virulence
  4. Virulence gene expression is co-regulated with hyphal morphogenesis
  5. Co-regulation of virulence genes and morphogenesis genes by regulatory proteins
  6. Convergence of many regulatory pathways to a common set of genes
  7. How do hyphae contribute to pathogenesis?
  8. Conclusions
  9. Acknowledgements
  10. References

We thank Jenifer Coburn, Igor Bruzual and our anonymous reviewers for careful reading of the manuscript and helpful suggestions that increased the clarity of our review. We are also grateful to Ramon F. Vinces for the artwork shown in Fig. 2. Our research on this project was supported by Grant AI38591 from the National Institute of Allergy and Infectious Diseases.

References

  1. Top of page
  2. Summary
  3. A G1 cyclin dedicated to hyphal morphogenesis is required for virulence
  4. Virulence gene expression is co-regulated with hyphal morphogenesis
  5. Co-regulation of virulence genes and morphogenesis genes by regulatory proteins
  6. Convergence of many regulatory pathways to a common set of genes
  7. How do hyphae contribute to pathogenesis?
  8. Conclusions
  9. Acknowledgements
  10. References