Interaction of pathogenic fungi with host cells: Molecular and cellular approaches Free

Abstract

This review provides an overview of several molecular and cellular approaches that are likely to supply insights into the host-fungus interaction. Fungi present intra- and/or extracellular host-parasite interfaces, the parasitism phenomenon being dependent on complementary surface molecules. The entry of the pathogen into the host cell is initiated by the fungus adhering to the cell surface, which generates an uptake signal that may induce its cytoplasmatic internalization. Furthermore, microbial pathogens use a variety of their surface molecules to bind to host extracellular matrix (ECM) components to establish an effective infection. On the other hand, integrins mediate the tight adhesion of cells to the ECM at sites referred to as focal adhesions and also play a role in cell signaling. The phosphorylation process is an important mechanism of cell signaling and regulation; it has been implicated recently in defense strategies against a variety of pathogens that alter host-signaling pathways in order to facilitate their invasion and survival within host cells. The study of signal transduction pathways in virulent fungi is especially important in view of their putative role in the regulation of pathogenicity. This review discusses fungal adherence, changes in cytoskeletal organization and signal transduction in relation to host-fungus interaction.

1 Adhesion

Adhesion of microorganisms to host cells and tissues represents a critical step in the process of infection [1]. In mycology, the fundamental work in this field goes back to that on Candida albicans[2] and these experiments were extended to Aspergillus fumigatus[3–5], Blastomyces dermatitidis[6], Coccidioides immitis[7], Cryptococcus neoformans[8], Histoplasma capsulatum[9], Paracoccidioides brasiliensis[10–13], Pneumocystis carinii[14–16], Penicillium marneffei[17,18] and Sporothrix schenckii[19]. Excellent reviews on cell-wall biomolecules and putative adhesins of Candida spp. have appeared in recent years [2,20–28]. The majority of studies emphasizes biochemical characterization and in some cases molecular studies carried out with the isolation of some genes [29–36]. Table 1 summarizes some adhesins found in some pathogenic fungi. Lately, four structurally related adhesins, Ala1p/Als5p, Als1p, Hwp1 from C. albicans and Epa1p from C. glabrata, have been studied in depth. They are members of a class of proteins termed glycosylphosphatidylinositol-dependent cell-wall proteins (GPI-CWP) and have N-terminal signal peptides and C-terminal features that mediate the addition of a glycosylphosphatidylinositol (GPI) membrane anchor, as well as other determinants leading to attachment to cell-wall glucan. Als1p [36] and Ala1p [34] also called Als5p [37], encoded by members of the large ALS (agglutinin-like sequence) gene family [27], were reported to adhere to endothelial and epithelial cells, and ECM proteins, respectively [23]. Comparison of mutant strains showed that the als3/als3 strain was defective in adhesion to both human umbilical vein endothelial cells (HUVEC) and buccal epithelial cells (BEC), but not to fibronectin (FN)-coated plastic plates. Adhesion data suggested that loss of Als3p affects C. albicans adhesion more than loss of Als1p [38]. Hwp1 is found on surfaces of germ tubes in vitro and in vivo in specimens of oral pseudomembranous candidiasis [33,39,40]. The similarity of Hwp1 to small proline-rich proteins that are expressed only in stratified squamous epithelium [41] suggests that Hwp1 may be more important for mucosal than systemic candidiasis. Indeed, its importance in mucosal candidiasis was recently demonstrated in germfree, beige athymic and transgenic-26 mice having combined defects in innate and cell-mediated immunity [42]. In C. albicans, the gene EAP1 was isolated as a putative cell-wall adhesin and sequence analysis shows that it also contains a signal peptide, a glycosylphosphatidylinositol ancr site, and possesses homology with many other yeast genes encoding cell-wall proteins. In addition to increasing adhesion to polystyrene, heterologous expression of EAP1 in Saccharomyces cerevisiae and autologous expression of EAP1 in a C. albicans efg1 homozygous null mutant significantly enhanced attachment to HEK293 kidney epithelial cells [43]. On the other hand, C. glabrata also adhered avidly to human epithelial cells in culture and deletion of the adhesin encoded by the EPA1 gene reduced this adherence. This adhesin is probably a glucan-cross-linked cell-wall protein and binds to host-cell carbohydrates, specifically recognizing asialo-lactosyl-containing carbohydrates [44].

Binding of the host tissue is considered a crucial stage for the establishment of the infection and seems to be mediated by the recognition of some components of the ECM, (i.e.), types I and IV collagens, fibrinogen, FN, and basement membrane laminin and tenascin. Some of the fungal receptors involved in these interactions have been identified at the molecular level [4,45–50]. The establishment of metastatic sites of infection throughout the body in disseminated infection presumably occurs after adherence to the endothelial basement membrane and/or subendothelial ECM. A great number of molecules with receptor-like characteristics implicated in laminin binding have been described for C. albicans[51–53], and some for A. fumigatus[54,55], Histoplasma capsulatum[9], P. brasiliensis[10–13] and Penicillium marneffei[17,18]. In C. albicans, various cell-surface receptors were identified in yeast or germinated cells (68, 62, and 60 kDa), exhibiting multiple affinities for laminin, fibrinogen, and C3d, while in ungerminated blastoconidia, a 37-kDa laminin-binding receptor was found, which did not bind to other mammalian proteins, such as fibrinogen, FN, and type IV collagen [52,53]. In A. fumigatus, a 72-kDa cell-wall receptor was described [3], as well as one of 37 kDa that bound laminin. The latter was detected only in a cytoplasmic extract and it may be a precursor of the former [46]. A protein of 20 kDa was identified in cell-wall extract of Penicillium marneffei and later characterized as a ligand for laminin and FN, suggesting that these two proteins share the same receptor [17,18]. A protein of 33 kDa, product of the P. carinii receptor gene binds both laminin and fibronectin in vitro [56]. In H. capsulatum, a 50-kDa laminin-binding protein is present in cell-wall components and a possible mechanism was suggested for the yeast to recognize and traverse the basement membrane [9]. In Blastomyces dermatitidis, the adhesin best studied is a glycoprotein of 120 kDa (WI-1), characterized as BAD1, capable of promoting adhesion of the yeast cells to the host cells or ECM proteins [6,57–60]. Hung and co-workers [7] reported the isolation of a Coccidioides immitis gene (SOWgp), which encodes a spherule outer-wall glycoprotein component specific to the parasitic phase. The recombinant polypeptide (rSOWp) bound to mammalian ECM proteins in an in vitro assay (laminin > FN > collagen type IV) and deletion of the SOWgp gene resulted in partial loss of the ability of spherules to bind to ECM proteins and a reduction of virulence. P. brasiliensis had the capacity to invade Vero cells [61] and the adherence phenomenon was variable, depending on the strain [11]. The glycoprotein of 43 kDa has been implicated as a probable adhesin, by inhibition either gp43 or anti-gp43 serum [10,11]. Recently, an adhesin of 30 kDa and pI 4.9 from P. brasiliensis was also capable of binding to laminin, but not to the other ECM components, fibronectin, type I or type IV collagen and may play a role in the virulence of this fungus [12]. P. brasiliensis also presents on its surface two proteins with molecular masses of 19 and 32 kDa that interact with different ECM proteins such as laminin, fibronectin, and fibrinogen [13]. Other fungus such as S. schenckii could interact with human umbilical vein endothelial cells (HUVECs) and surface ligand of approximately 90 and 135 kDa were described [62].

Several putative receptors for FN on C. albicans have been also identified, including homologs of mammalian integrins [63,64] and 60- and 105-kDa glycoproteins [65]. Recombinant and proteolytic fragments of FN were used to map the domain of FN involved. At least three have been reported to bind to C. albicans and the cell was bound by the main, but not by the RGD domain. Proteins of 55 and 105 kDa from this yeast were shown to have FN-binding activity [66,67] and both proteins have cell-binding domain of FN, but the latter partially require RGD sequence, suggesting that apparently this protein is different. Gozalbo and co-workers [68] have demonstrated that the enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is also a FN and laminin binding protein and could be involved in the adhesion of fungal cells to host tissues, thus playing a role in its virulence. A C. albicans gene (ALA1) that confers adherence properties upon S. cerevisiae for ECM proteins, including laminin and FN, has been characterized [46] and the presence of a minimum of four contiguous threonine residues in a peptide was required for maximal adherence [69]. The importance of the outer cell wall layer in the adhesive properties of the conidia of Aspergillus fumigatus was further emphasized [70,71]. Two polypeptides with apparent molecular masses of 23 and 30 kDa specifically interact with fibronectin and they are presented at the conidial surface [71]. Merkel and Scofield [8] described the interaction of Cryptococcus neoformans with a human lung epithelial cell line (A549) and the acapsular strain was the most adherent. Few reports demonstrate the interaction between C. neoformans and proteins from the ECM [72]. Human monocytes were more effective in inhibiting C. neoformans growth when they were cultured on a FN-coated surface than on a plastic surface, but negative results were obtained when direct adhesion of encapsulated C. neoformans to FN was tested. Furthermore, Rodrigues and co-workers [73] demonstrated this fungus does not bind significantly to this ECM glycoprotein and the binding to soluble FN was also very poor in comparison with other fungal models. Both conidia and yeast cells of S. schenckii similarly adhered to proteolytic fragments of FN [74]. In addition to laminin, ECM components such as FN, and types I and IV collagens participate in the P. brasiliensis adherence process. A characteristic distribution of bound ECM components on the surface of yeast cells was detected, indicating the presence of specific binding sites for each one [70].

Another type of interaction that could provide a mechanism for the attachment of the fungus to the host cells may be the specific recognition of glycoconjugates on the epithelial cell membrane by lectins present on the surface of the fungal-wall. Few lectins have been described in human pathogenic fungi, including C. albicans[75,76], C. glabrata[44], Histoplasma capsulatum[70], as well as some dermatophytes and related keratinolytic fungi [77,78]. In C. albicans, depending on the strain, adherence was partially blocked by either fucose or N-acetyl-d-glucosamine or by lectins that recognize either of these two sugars. In A. fumigatus, a 32-kDa fucose-specific lectin, concentrated in conidia rather than in mycelium, has been identified [70]. Adhesin of 50 kDa was isolated from Fonsecaea pedrosoi and characterized as a lectin binding to mannose and N-acetyl-d-glucosamine [79].

Pathogenic microorganisms may disorganize the host molecules to which they adhere, with the aim of gaining entry and thus promoting their own survival and dissemination.

2 Internalization of fungi

Many pathogenic microorganisms can enter eukaryotic cells and use this normally hostile environment as a niche within which to replicate and/or evade the host immune response. Both professional phagocytes and cells that are not normally phagocytic, such as epithelial or endothelial cells, may be invaded [80]. Among the fungi, there are several species known to enter mammalian host cells in vitro and in vivo, but there are few studies describing this invasion process [70,81–83]. More recently however, the molecular mechanisms of fungal internalization in mammalian cells have been explored. C. albicans can induce phagocytosis in endothelial cells through the polymerization of their microfilaments and microtubules [84,85]. Also, yeasts and hyphae were found attaching to the surface and within HEp2 cells. The disorganization of the actin cytoskeleton may result from the direct action of Candida-secreted factor (arcsf) fungal products upon actin or actin-associated proteins, followed by cellular actin rearrangement, reduced membrane ruffling and decreased cell motility [81,86]. The internalization of A. fumigatus in endothelial cells culture has been associated with the mechanism of escape from phagocytes, as well as the possibility of dissemination [87]. Wasylnka and Moore [82] studied the uptake of two different A. fumigatus strains into A549 lung epithelial cells, human umbilical vein endothelial cells (HUVEC), and J774 murine macrophages, in vitro. Internalization of conidia by A549 required rearrangement of the underlying host cell cytoskeleton; uptake was inhibited by cytochalasin D and colchicine. These data provide evidence that non-professional phagocytes in vitro can internalize significant numbers of A. fumigatus conidia and these cells may serve as reservoirs for immune cell evasion and dissemination throughout the host. In contrast, Kogan and co-workers [88] showed that the actin cytoskeleton alone of A549 cells undergoes major structural changes in response to infection, including loss of actin stress fibers, formation of actin aggregates, disruption of focal adhesion sites, and cell blebbing. These findings suggest that A. fumigatus breaches the alveolar epithelial cell barrier by secreting proteases that act together to disorganize the actin cytoskeleton and destroy cell attachment to the substrate by disrupting focal adhesions. Lopes-Bezerra and Filler [89] investigated the interactions of A. fumigatus conidia and hyphae with endothelial cells in vitro and found that both forms of the organism induced microfilament rearrangement and subsequent endocytosis. These results suggest that angioinvasion and thrombosis caused by A. fumigatus hyphae in vivo may be due in part to endothelial cell invasion, induction of injury, and stimulation of tissue-factor activity. C. albicans is another angioinvasive fungus, which is similar to A. fumigatus in that both living and killed hyphae are endocytosed by endothelial cells and that this process can be inhibited by cytochalasin D [84,89,90]. C. neoformans induced actin cytoskeletal reorganization in human brain microvascular endothelial cells (HBMEC). In addition, it was observed that the dephosphorylated form of cofilin increased during cryptococcal adherence to HBMEC, concomitantly with the actin rearrangement [91]. Newman and co-workers [92] showed that disruption of actin microfilaments with cytochalasin D inhibited both the attachment and digestion of H. capsulatum yeasts by macrophages. In contrast, nocodazole, which prevents polymerization of microtubules, did not inhibit binding or ingestion. Scott and Woods [93] showed that murine RAW 264.7 cells internalized H. capsulatum strain G217B yeasts more efficiently than human U937 cells. Both cell lines exhibited dependence upon actin, and, to a lesser degree, microtubules, in G217B uptake. The invasion of A549 and Vero epithelial cells by P. brasiliensis affected the cytoskeletal structure of the cells, interfering in morphological aspects of the actin, tubulin and cytokeratin components. Cytochalasin D and colchicine treatment substantially reduced invasion, indicating the functional participation of microfilaments and microtubules in this mechanism [83]. On the other hand, the degradation of cytokeratin components by P. brasiliensis may be due to the effect of specific enzymes [94,95] or of gp43, which caused the loss of its characteristic filament network. Cytokeratin could also play a role in either the invasion process in vitro or in the process by which P. brasiliensis crosses epithelial barriers in vivo. This new observation stresses the importance of studying more deeply the fungal cytoskeleton in greater depth.

3 Signaling

The study of signal transduction pathways in virulent fungi is especially important in view of their putative role in the regulation of pathogenicity. Table 2 shows the principal signaling events in fungus and during interaction with host cells.

3.1 Protein kinase pathway

Protein phosphorylation is generally accepted to play key part in transducing signals involved in several processes such as cell adhesion, internalization and killing of pathogens [96]. In eukaryotes, phosphorylation/dephosphorylation cycles represent a major mechanism for switching cellular pathways in response to changing circumstances, both internal developmental cues and external environmental stimuli [97]. Different classes of protein kinases (PKs) have major roles in transducing extracellular signals and regulating essential cell processes in mammalian cells, such as cell growth, differentiation, transcription, disease and death [97–100]. The phosphorylation process has recently been implicated in defense strategies against a variety of pathogens that have evolved mechanisms to alter host-signaling pathways in order to facilitate their invasion and survival within host cells.

The endocytosis of all of these microbial pathogens is regulated by the phosphorylation of tyrosine containing host cell proteins [80]. Kaposzta and co-workers [101] observed the rapid recruitment of late endosome and lysosome compartments in macrophages, by invading C. albicans yeasts that developed germ tubes within phagolysosomes, distending their membranes and escaping from macrophage defenses. These mechanisms could be useful for survival and virulence of this pathogen. It seems that uptake of Candida yeasts had characteristics of phagocytosis, required intact actin filaments, and depended on the activity of protein kinase C (PKC). Belanger and co-workers [102] demonstrated that C. albicans wild-type endocytosis was accompanied by the tyrosine phosphorylation of two endothelial cell proteins, respectively of 80 and 82 kDa. The phosphorylation of these proteins was closely associated with the endocytosis of both live and killed yeasts. However, these proteins seemed not to be phosphorylated in endothelial cells infected with a strain of C. albicans that was poorly endocytosed. Genistein and tyrphostin 47 were able to block the phosphorylation of the two-endothelial cell tyrosine kinase proteins and this blockage significantly reduced C. albicans endocytosis. Therefore, C. albicans probably induces its own endocytosis, stimulating the tyrosine phosphorylation of these endothelial cell proteins. C. albicans seems to use at least two signal pathways to regulate its conversion from the yeast form to filamentous growth (hyphae). One of these two pathways is similar to the S. cerevisiae pseudohyphal/mating pathway, which utilizes the regulatory protein, Cphlp [103]. The other is not totally defined but seems to require a second regulatory protein, referred to as Efg1p [43].

In F. pedrosoi, the attachment to and invasion of epithelial cells and macrophages, observed by Limongi and co-workers [104], suggested the involvement of PKs especially by the pathogenic fungus. Pre-treatment of macrophages, epithelial cells or conidia with the PK inhibitors, stausporine, genistein and calphostin C, decreased cell invasion by F. pedrosoi significantly and this effect was overcome by treatment with okadaic acid, a phosphatase inhibitor. Immunofluorescence assays showed that tyrosine residues were phosphorylated in the first step of the interaction, while serine residues were phosphorylated in the subsequent step of entry of the parasite into the host cell. These results suggest that both host-cell and conidium PK activities are important in the interaction and play a significant part in cell invasion.

The signal-transduction networks involving PK and protein phosphatase activities can modulate crucial events during fungal infections. However, PK activity and fungus infection has been described for certain microorganisms and the study of kinases and phosphatases in pathogenic fungi has become an active area of research.

3.2 Cyclic AMP (cAMP)-dependent signaling pathway

cAMP signaling cascades regulate a variety of cellular processes in many pathogenic and non-pathogenic fungi. In C. albicans and A. fumigatus, the adenylyl cyclase-dependent signaling pathways control the elaboration of specific phenotypes that are required for virulence. C. albicans strains with mutations in the adenylyl cyclase gene (CaCDC35) fail to switch from a budding yeast to a polarized form, a morphological transition required for the full virulence of this organism [105]. Also, the transcription of EAP1 in C. albicans is regulated by the transcription factor Efg1p, suggesting that EAP1 expression is activated by the cyclic AMP-dependent protein kinase pathway [43]. Liebmann and co-workers [106] verified the importance of the cAMP signaling pathway for A. fumigatus virulence, by deleting adenylate cyclase gene acyA and gpaB. Both genes encode α subunits of heterotrimeric G proteins, and reduced conidiation was observed in both mutants (δacyA and δgpaB). The killing rate for conidia from δacyA and δgpaB strains was significantly higher than that for wild-type conidia, suggesting that cAMP triggers a system that protects the fungus from the effects of immune effector cells of the host. Rhodes and co-workers [107] developed a tissue culture model to mimic the interaction of A. fumigatus with the endothelium and, using differential display, compared gene expression in fungal cells grown on endothelial cells. Two of these up-regulated genes encoding the regulatory subunit of cyclic adenosine monophosphate (cAMP)-dependent protein kinase, involved in cAMP-mediated signaling, and a member of the ras gene family, were associated as potentially virulence-related genes induced in A. fumigatus by the interaction with the host cell.

The human fungal pathogen C. neoformans utilizes cAMP signaling to regulate morphological transitions, but it has also coopted this cascade for the regulation of cellular determinants involved in virulence. The conserved components of a cAMP signaling cascade have been characterized and involve genes encoding the Gα protein (Gpa1), adenylyl cyclase (Cac1), and protein kinase A catalytic subunit (Pka1). Mutant strains lacking these components do not increase capsule or melanin production in response to normal inducing conditions [108,109]. Bahn and co-workers [110] have identified and characterized adenylyl cyclase-associated protein, Aca1, which functions in parallel with the Gα subunit Gpa1 to control the adenylyl cyclase (Cac1). Aca1 was shown to play a critical role in mating by regulating cell fusion and filamentous growth in a cAMP-dependent manner. Aca1 also regulates melanin and capsule production via the Cac1-cAMP-protein kinase A pathway. A single gene (CAC1) encodes adenylyl cyclase, and CAC1 mutants failed to produce two inducible virulence factors: capsule and melanin. As a consequence, cac1 mutant strains were avirulent in animal models of cryptococcal meningitis [108]. Taken together, these studies further define the cAMP signaling cascade controlling virulence of this ubiquitous human fungal pathogen.

The signaling pathways that control the morphological changes in P. brasiliensis are poorly understood and incompletely studied, but the involvement of both cAMP and mitogen-activated protein kinase (MAPK) signal transduction pathways has been reported in other dimorphic fungi [111]. Exogenous cAMP inhibits the yeast-to-mycelium (YM) transition in P. brasiliensis, thus maintaining the pathogenic yeast form [112]. In C. albicans, on the other hand, the YM transition is triggered by cAMP and exogenous cAMP stimulates the formation of pseudohyphae [113].

3.3 Mitogen-activated protein kinase (MAPK) cascade

Two-component phosphorelay systems are minimally comprised of a histidine kinase, which autophosphorylates in response to an environmental stimulus, and a response regulator component, resulting in a signal transduction of MAPK [114]. Several reports have focused on the role of the different signal transduction pathways in the activation of phagocytic cells.

Ibata-Ombetta and co-workers [115] compared the effects of C. albicans and the non-pathogenic yeast S. cerevisiae on the response of macrophages. By exploring MAPK signaling pathways activated after ingestion, phagocytosis of both yeasts was shown to stimulate the ERK1/2 pathway in the absence of significant phosphorylation of p38MAPK and SAPK/JNK. This was accompanied by phosphorylation of MEK, the upstream activator of ERK1/2, and of p90RSK, one of the known downstream products activated by ERK1/2 [116]. Comparison of the signals induced by the two yeasts demonstrated that ingestion of C. albicans by macrophages led to a down-regulation of both ERK1 and p90RSK phosphorylation. This decreased activation paralleled the impairment of the cells' ability to kill ingested C. albicans blastoconidia. The existence of a relationship between the decreased ability of cells to kill ingested yeasts and reduced phosphorylation was confirmed by experiments using the MEK inhibitor PD98059. Treatment with this inhibitor before addition of the yeasts led to a significant decrease both in activation of MAPK and in the ability of cells to kill S. cerevisiae. In Cryptococcus neoformans, Song and co-workers [117] demonstrated that PI-3K and Ras/MEK/ERK pathways control phagocytosis and MIP-1α induction, respectively. In addition, active mitogen-activated protein kinase kinase (MEK) induced MIP-1α, while Ras dominant-negative (DN) inhibited IC-induced ERK phosphorylation and MIP-1α production.

3.4 Cytoskeleton and extracellular matrix

Extracellular signals can alter the activity of accessory proteins, changing the cytoskeleton and cell behavior [118]. For the actin cytoskeleton, global structural rearrangements in response to external signals are triggered through diverse cell-surface receptors. But all of these signals seem to converge inside the cell on a group of closely related monomeric GTPases that are members of the Rho protein family [119]. The Rho family of GTPases is present in all eukaryotic cells, from yeast to mammals, and their role as key regulators in the signaling pathways that control actin organization and the morphogenetic process is well known.

Endocytosis, a process by which plasma-membrane proteins are internalized into the cell, is developed by a large number of proteins and lipids that coordinate the timing and location of internalization. Various molecules, including kinases and components of the actin cytoskeleton have been identified as regulators of the internalization step of endocytosis in yeast [120]. The study of Santoni and co-workers [121] provides evidence that p105 focal adhesion kinase (Fak)-like protein (CaFak) of C. albicans yeasts, antigenically related to the vertebrate p125Fak, is involved in integrin-like-mediated fungus adhesion to vitronectin (VN) in the EA.hy 926 human endothelial cell line. In addition, this study demonstrated that tyrosine kinase activation is a primary event required for C. albicans yeast-cell adhesion to VN and to the endothelial cell line, by using the PTK inhibitors genistein and herbimycin. Moreover, CaFak seems to be redistributed from the cytosol to focal adhesion sites and colocalizes with αv, β3, and β5 integrin-like receptors A. The pretreatment of C. albicans with herbimycin A completely nullified Candida adhesion-dependent stimulation of CaFak tyrosine phosphorylation. These results strongly resemble previous results with mammalian cells in which cell adhesion to VN or clustering of αvβ3 and αvβ5 integrins triggers p125 Fak tyrosine phosphorylation. However, the interaction between focal adhesion proteins and the tyrosine-kinase signal pathway in other fungi has apparently not been described at the moment. Cryptococcal binding to HBMEC (Human Brain Microvascular Endothelial Cells) was increased in the presence of Y27632, a Rho kinase (ROCK)-specific inhibitor. Since ROCK activates LIM kinase (LIMK), which phosphorylates cofilin (inactive form), this suggests the involvement of ROCK → LIMK → cofilin pathway. In contrast, the phosphatase inhibitor sodium orthovanadate decreased adherence of Cryptococcus to HBMEC, concomitantly increasing of phosphorylation of cofilin [91]. Furthermore, the tight junction marker protein occludin became Triton-extractable, indicating alteration of tight junctions in brain endothelial cells.

Kamran and co-workers [122] have demonstrated that the C. glabrata homolog of the S. cerevisiae transcription factor gene ACE2 encodes a function that modulates virulence; inactivation of C. glabrata ACE2 did not result in attenuation but, conversely, in a strain that was hypervirulent in a murine model of invasive candidiasis. These data demonstrate that in C. glabrata the ACE2 product plays a critical role in mediating the host–Candida interaction. It was the first virulence-moderating gene to be described in a Candida species.

Marques and co-workers [123] have identified four genes, RHO1, SEP1, FLB1 and PCK1, which encode proteins involved in cell signaling and polarity establishment in P. brasiliensis. Interestingly, RHO1 was expressed at 10- to 15-fold higher levels in minimal medium than in the complete media. De Carvalho and co-workers [124] reported that the Ca²⁺/calmodulin signaling pathway has pleiotropic intracellular effects, acting on systems such as the cytoskeleton and regulating nuclear transcription factors that may affect the expression level of other genes. In P. brasiliensis, this pathway could be involved in the regulation of genes differentially expressed in the yeast and mycelial forms. Single fragments were identified by Southern blot, suggesting that the P. brasiliensis calmodulin gene is probably a single copy, as in other fungi such as H. capsulatum[125] and C. albicans[126].

3.5 Apoptosis

Apoptosis is a highly coordinated cell suicide program crucial for metazoan health and diseases. Although its increasing importance in cancer, neurodegenerative disorders and AIDS have led to intense research and a better understanding of apoptosis, in infectious disease many details of its regulation or the apoptotic phenotypes are poorly understood. The complex regulatory network and the often-contradictory results obtained with human cell lines make the application of an easier model system desirable. Apoptosis in yeast promises to provide a better understanding of the genetics of apoptosis [127].

Recent studies of the bakers' yeast, S. cerevisiae, have revealed the existence of a programmed cell death response after weak acid stress, oxidative stress, salt stress, and UV irradiation. Physiological cell death has also been observed in aging yeast cells, after exposure to mating pheromone. Cells dying under these conditions display several markers characteristic of apoptosis. These include the rapid exposure of phosphatidylserine (PS) at the outer cell membrane (revealed by annexin binding), the margination of chromatin in nuclei, nuclear fragmentation, and the degradation of DNA [revealed by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assays]. Exposure of cells to the protein-translation inhibitor cycloheximide prevents these death-associated changes, indicating that the death response requires active protein synthesis [128]. Regarding fungal infection, strains of C. albicans isolated from HIV-infected individuals showed increased expression of virulence traits such as proteinase production, adherence, resistance to antifungal drugs and phenotypic variation [129–131]. These observations support the hypothesis that more virulent strains concomitantly with the lowering of the host's cellular defenses, may result in apoptosis of macrophages. Panagio and co-workers [132] observed that a strain of C. albicans isolated from an HIV-infected patient induced apoptosis in macrophages and that these phagocytic cells presented early signs of apoptosis but progressed to necrosis. These results suggest that yeast strains that readily switched to germ-tubes inside apoptotic cells would have a competitive advantage, because germ-tubes released by cell lysis are more resistant than yeast cells to further attack by macrophages. Although innate cellular immunity is of prime importance for the host defense against fungal infections, it is not known whether C. albicans and other fungi can exploit phagocyte apoptosis to their own advantage [132].

In C. albicans, the activation of protein kinase A by cAMP inhibited U937 apoptosis. On the other hand, rp-cAMP, a blocker of cAMP-dependent signal transduction, restored and elevated DNA fragmentation levels, expressed the bcl-2 protein, but infection did not increase expression of this proto-oncogene [133]. Recent studies demonstrated that C. albicans is able to survive within macrophages, escaping the macrophage intracellular autophagosome. This escape was observed during phagocytosis, associated with qualitative differences in the sequential phosphorylation of MAP kinases, and deregulation of the ERK1/2/p90RSK signal transduction pathway by C. albicans was associated with downstream reduction in Bad phosphorylation, specifically at Ser-112, and disappearance of free bcl-2 [134]. After exposure to a range of stimuli, including treatment with the antifungal agent amphotericin B (AMB), changes in cell death were observed in cells infected with C. albicans; cells treated at higher doses of these compounds showed necrotic cell changes. The necrotic cells exhibited reduced TUNEL staining, lack of surface phosphatidylserine on the cell surface, limited reactive-oxygen species production, and an inability to exclude propidium iodide. Moreover, these cells lacked nuclear shape and showed extensive intracellular vacuolization [128].

Histoplasma capsulatum is a fungus found intracellularly in neutrophils and peripheral blood mononuclear cells (PBMCs), suggesting that it is capable of evading damage and surviving inside these cells. Medeiros and co-workers [135] report that neutrophils from H. capsulatum-infected mice, as well as infected neutrophils and mononuclear human cells, presented less apoptosis than those from uninfected animals or cells. Moreover, cells harvested from infected animals are resistant to apoptosis induced by dexamethasone, a proapoptotic stimulant, while neutrophils harvested from infected mice and PBMCs from humans exposed to the fungus had a greatly decreased Mac-1 expression. Thus, H. capsulatum induces an antiapoptotic state in leucocytes, which correlates with decreased cell-surface Mac-1 expression. These facts may represent an escape mechanism for the fungus by delaying cell death and allowing the fungus to survive inside leucocytes.

Mendes-Giannini and co-workers [83] observed that P. brasiliensis induced apoptosis in epithelial cells but remained alive inside the cells, long enough to continue budding. Preliminary evidence suggests that P. brasiliensis cells sporulate more frequently when free within the cytoplasm of the host cell, but less so when enclosed in vacuoles, and that these unrestrained cytoplasmic fungal cells may escape from the host cells. Recently, Cacere and co-workers [136] suggested that in PCM patients, apoptosis plays a role in the antigen-specific hyporesponsiveness of T cells to the main antigen of P. brasiliensis, the 43-kDa glycoprotein. As other fungi, P. brasiliensis can also exploit phagocyte apoptosis to its own advantage and its intracellular residence in epithelial cells could potentially elicit this type of cell death response, assisting P. brasiliensis evasion and, consequently, favoring further dissemination. The ability of pathogens to induce apoptosis of phagocytes might be an important virulence factor, for it would curtail the host's defense mechanisms.

4 Remarkable conclusions

The special challenges presented by the dynamic epidemiology of invasive fungal infections demand and attract considerable responses, in the fields of diagnosis and therapeutics. Current strategies need great improvement, yet ongoing collaborative efforts will have a positive impact on our understanding of the fungus–host interaction and ultimately our ability to offer better care for our patients with invasive mycoses. On the other hand, new results on molecules and mechanisms promise new therapeutic targets.

References