Candida albicans: The Ability to Invade Epithelial Cells and Survive under Oxidative Stress Is Unlinked to Hyphal Length.

In its hyphal form, Candida albicans invades epithelial and endothelial cells by two distinct mechanisms: active penetration and induced endocytosis. The latter is dependent on a reorganization of the host cytoskeleton (actin/cortactin recruitment), whilst active penetration does not rely on the host's cellular machinery. The first obstacle for the fungus to reach deep tissues is the epithelial barrier and this interaction is crucial for commensal growth, fungal pathogenicity and host defense. This study aimed to characterize in vitro epithelial HeLa cell invasion by four different isolates of C. albicans with distinct clinical backgrounds, including a C. albicans SC5314 reference strain. All isolates invaded HeLa cells, recruited actin and cortactin, and induced the phosphorylation of both Src-family kinases (SFK) and cortactin. Curiously, L3881 isolated from blood culture of a patient exhibited the highest resistance to oxidative stress, although this isolate showed reduced hyphal length and displayed the lowest cell damage and invasion rates. Collectively, these data suggest that the ability of C. albicans to invade HeLa cells, and to reach and adapt to the host's blood, including resistance to oxidative stress, may be independent of hyphal length.

Histoplasma capsulatum-Induced Cytokine Secretion in Lung Epithelial Cells Is Dependent on Host Integrins, Src-Family Kinase Activation, and Membrane Raft Recruitment.

Histoplasma capsulatum var. capsulatum is a dimorphic fungus that causes histoplasmosis, a human systemic mycosis with worldwide distribution. In the present work, we demonstrate that H. capsulatum yeasts are able to induce cytokine secretion by the human lung epithelial cell line A549 in integrin- and Src-family kinase (SFK)-dependent manners. This conclusion is supported by small interfering RNA (siRNA) directed to α3 and α5 integrins, and PP2, an inhibitor of SFK activation. siRNA and PP2 reduced IL-6 and IL-8 secretion in H. capsulatum-infected A549 cell cultures. In addition, α3 and α5 integrins from A549 cells were capable of associating with H. capsulatum yeasts, and this fungus promotes recruitment of these integrins and SFKs to A549 cell membrane rafts. Corroborating this finding, membrane raft disruption with the cholesterol-chelator methyl-ß-cyclodextrin reduced the levels of integrins and SFKs in these cell membrane domains. Finally, pretreatment of A549 cells with the cholesterol-binding compound, and also a membrane raft disruptor, filipin, significantly reduced IL-6 and IL-8 levels in A549-H.capsulatum cultures. Taken together, these results indicate that H. capsulatum yeasts induce secretion of IL-6 and IL-8 in human lung epithelial cells by interacting with α3 and α5 integrins, recruiting these integrins to membrane rafts, and promoting SFK activation.

Paracoccidioides brasiliensis induces recruitment of α3 and α5 integrins into epithelial cell membrane rafts, leading to cytokine secretion.

Paracoccidioides brasiliensis is one of the etiological agents of paracoccidioidomycosis, a human systemic mycosis, highly prevalent in Latin America. In the present work, we demonstrate that P. brasiliensis yeasts promote IL-6 and IL-8 secretion by the human lung epithelial cell line A549 in an integrin-dependent manner. In fact, small interfering RNA directed to α3 and α5 integrins decreased IL-6 and IL-8 levels in P. brasiliensis-infected A549 cell cultures. This fungus also led to an increase in the expression of α3 and α5 integrins in this epithelial cell line. In addition, P. brasiliensis yeasts promoted α3 and α5 integrins clustering into A549 cell membrane rafts. Furthermore, epithelial cell membrane raft disruption with nystatin decreased IL-6 and IL-8 levels in P. brasiliensis-A549 cell cultures. Therefore, by increasing host α3 and α5 integrins levels and clustering these receptors into membrane rafts, P. brasiliensis yeasts may modulate host inflammation.

Paracoccidioides brasiliensis induces secretion of IL-6 and IL-8 by lung epithelial cells. Modulation of host cytokine levels by fungal proteases.

Paracoccidioides brasiliensis is a pathogenic, dimorphic fungus that causes paracoccidioidomycosis, a systemic human mycosis that is highly prevalent in Latin America. In this study, we demonstrated that P. brasiliensis yeasts induced interleukin (IL)-8 and IL-6 secretion by human lung epithelial A549 cells. However, tumor necrosis factor-α and interferon-γ were undetectable in these cultures. Moreover, P. brasiliensis yeasts induced activation of p38 mitogen-activated protein kinase (MAPK), c-Jun NH(2)-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 in A549 cells, and IL-8 and IL-6 secretion promoted by this fungus was dependent on activation of p38 MAPK and ERK 1/2. In addition, IL-8 and IL-6 levels were significantly higher in culture supernatants of A549 cells that were incubated with formaldehyde-fixed P. brasiliensis compared to cultures of cells that were infected with live yeasts. Our results indicate that the observed cytokine level differences were due to protease expression, in live yeasts, that degraded these cytokines. Degradation of human recombinant IL-8 and IL-6 by live P. brasiliensis was inhibited by AEBSF and aprotinin, suggesting that these proteases belong to a family of serine proteases. This is the first report showing that P. brasiliensis may modulate host inflammation by expressing proteases that degrade proinflammatory cytokines.

Role of host glycosphingolipids on Paracoccidioides brasiliensis adhesion.

Binding of yeast forms to human lung fibroblast cultures was analyzed, aiming to better understand the initial steps of Paracoccidioides brasiliensis infection in humans. A significant P. brasiliensis adhesion was observed either to fibroblasts or to their Triton X-100 insoluble fraction, which contains extracellular matrix and membrane microdomains enriched in glycosphingolipids. Since human lung fibroblasts express at cell-surface gangliosides, such as GM1, GM2, and GM3, the role of these glycosphingolipids on P. brasiliensis adhesion was analyzed by different procedures. Anti-GM3 monoclonal antibody or cholera toxin subunit B (which binds specifically to GM1) reduced significantly fungal adhesion to fibroblast cells, by 35% and 33%, respectively. Direct binding of GM1 to yeast forms of P. brasiliensis was confirmed using cholera toxin subunit B conjugated to AlexaFluor(®)488. It was also demonstrated that P. brasiliensis binds to polystyrene plates coated with galactosylceramide, lactosylceramide, trihexosylceramide, GD3, GM1, GM3, and GD1a, suggesting that glycosphingolipids presenting residues of beta-galactose or neuraminic acid at non-reducing end may act as adhesion molecules for P. brasiliensis. Conversely, no binding was detected when plates were adsorbed with glycosphingolipids that contain terminal residue of beta-N-acetylgalactosamine, such as globoside (Gb4), GM2, and asialo-GM2. In human fibroblast (WI-38 cells), GM3 and GM1 are associated with membrane rafts, which remain insoluble after treatment with Triton X-100 at 4°C. Taken together, these results strongly suggest that lung fibroblast gangliosides, GM3 and GM1, are involved in binding and/or infection by P. brasiliensis.

Interaction of epithelial cell membrane rafts with Paracoccidioides brasiliensis leads to fungal adhesion and Src-family kinase activation.

Membrane rafts are cholesterol- and sphingolipid-enriched cell membrane domains, which are ubiquitous in mammals and play an essential role in different cellular functions, including host cell-pathogen interaction. In this work, by using several approaches, we demonstrated the involvement of epithelial cell membrane rafts in adhesion process of the pathogenic fungus Paracoccidioides brasiliensis. This conclusion was supported by the localization of ganglioside GM1, a membrane raft marker, at P. brasiliensis-epithelial cell contact sites, and by the inhibition of this fungus adhesion to host cells pre-treated with cholesterol-extractor (methyl-beta-cyclodextrin, MbetaCD) or -binding (nystatin) agents. In addition, at a very early stage of P. brasiliensis-A549 cell interaction, this fungus promoted activation of Src-family kinases (SFKs) and extracellular signal-regulated kinase 1/2 (ERK1/2) of these epithelial cells. Whereas SFKs were partially responsible for activation of ERK1/2, membrane raft disruption with MbetaCD in A549 cells led to total inhibition of SFK activation. Taking together, these data indicate for the first time that epithelial cell membrane rafts are essential for P. brasiliensis adhesion and activation of cell signaling molecules.