Adherence to Host Cells and Extracellular Matrix

Cell-cell adhesion and adhesion to extracellular matrix (ECM) proteins play an important role in invasive fungal disease. The first glycoprotein described to be involved in P. brasiliensis adherence to ECM proteins was gp43, as described previously. These data would place gp43 as a virulence factor but it is clear that binding to laminin, which can be demonstrated in vitro, highly depends on the model used for fungal infection in vivo. Another report claimed that gp43 displayed an inhibitory effect on peritoneal macrophages but no clear definition of the cell types present was done in this study (Popi et al., 2002). B1-b cells that are abundant in the peritoneal and pleural cavities were shown by the same group to be antigen presenting cells expressing high levels of MHC class II and inducing an efficient proliferation of gp43-sensitized T lymphocytes (Vigna et al., 2002). Furthermore, activated B1-b cells significantly produce IL-10 that can inhibit macrophage functions. In an in vitro model of granuloma assembly it was shown that granulomas were formed when isolated B-1 cells were added to macrophage cultures and that gp43 strongly stimulated this response (Vigna et al., 2006). These results and the intense humoral and cellular immune responses to gp43, which combined, are protective in infected mice, do not support a role for gp43 as a virulence factor in the human or experimental lung infection. The same happened with gp43-plasmid vaccination (Pinto et al., 2000). Different responses, however, probably related to the genetic background of mice have been observed. Protection experiments by gp43 and P10 were mainly obtained in Balb/c mice. By using B10.A susceptible mice, it was shown that administration of dendritic cells incubated with gp43 (10 |g/mL) and LPS caused an increase in lung CFUs of infected animals (Ferreira & Almeida, 2006). Macrophages from susceptible mice, however, produced high and persistent levels of NO throughout the fungal infection and incubation of resident macrophages with P. brasiliensis also resulted in NO production. Over expression of NO can induce T cell immunosuppression during infection, inhibition of TNFa production, and less killing of the fungus (Nascimento et al., 2002). It is still unclear whether gp43 acts in B10.A mice as a stimulant of NO production beyond the level that is needed for anti-fungal activity as observed in A/Sn resistant mice. Gp43 and most certainly the high molecular weight glycoconjugate of P. brasiliensis (Matsuo et al., 2006) may, however, stabilize the serine-thiol proteinase activity of yeast cells which degrade components of the ECM, conceivably related to fungal dissemination.

An in vitro assay for the adhesion of P. brasiliensis to cultured mammalian epithelial cells has established a correlation between adherence and virulence, with virulent strain (Pb18) having a greater capacity to adhere. Yeast cells were shown to attach to Vero cells via a narrow tube similar to a germ tube, which subsequently led to the yeast cells making close and extensive contact with the membrane of the host cell, inducing cytoskeletal changes before being taken up or invading the cell (Hanna et al., 2000). Both adhesion that depended on gp43 and cell invasion induced apoptosis demonstrated by TUNEL, DNA fragmentation and Bak and Bcl-2 expression (Mendes-Giannini et al., 2004).

Dectin-1 is a major P-glucan receptor highly expressed in alveolar monocyte/ macrophages and particularly in inflammatory cells (Taylor et al., 2002). A. fumigatus stimulated alveolar macrophages to produce IFN-y and mediate a Th-1 response that was dectin-1 dependent. Curiously, IL-10 synthesis was also dectin-1 dependent (Steele et al., 2005). Macrophages from mice transgenic for pentraxin-3 expression had improved phagocytosis of zymosan particles and yeast forms of P. brasiliensis. Increased microbicidal activity and nitric oxide production was observed. Blockade of dectin-1 receptor inhibited phagocytosis in this system (Diniz et al., 2004), suggesting that P. brasiliensis yeast forms expose P-glucan despite the fact that a-1,3-glucan predominates in this morphological phase. As provocative hypothesis conidia reaching the alveoli could bind to alveolar macrophages via dectin-1, be internalized and differentiate into infective yeast forms similarly to the model of early pathogenesis of H. capsulatum (Procknow et al., 1960; Newman et al., 1990). Adhesion and invasion of nonprofessional phagocytes (epithelial cells) by P. brasiliensis depends on signal-transduction involving protein tyrosine kinase (PTK). Treatment with genistein significantly inhibited fungal invasion (Monteiro da Silva et al., 2006).

The adhesion of yeasts to Vero cells (Hanna et al., 2000) was inhibited by anti-gp43 antiserum and a pool of sera from patients with PCM. Mendes-Giannini et al. (2006) showed that ECM proteins bind to the surface of P. brasiliensis yeast cells in distinct qualitative patterns. Extracts from Pb18 isolate (virulent), exhibited differential adhesion to ECM components, before and after animal inoculation. After animal inoculation, Pb18 had a higher capacity to adhere to ECM proteins. Laminin was the most adherent component followed by type I collagen, fibronectin (fragment of 120 kDa), and type IV collagen. In addition to gp43 that reacted with both laminin and fibronectin, type I collagen was recognized by 47 and 80 kDa proteins. A peptide derived from gp43 (NLGRDAKRHL) contributed most to the adhesion of P. brasiliensis to Vero cells and synthetic peptides derived from peptide YIGRS of laminin or from RGD of both laminin and fibronectin inhibited the adhesion of gp43 to Vero cells.

Other molecules in P. brasiliensis were found to interact with laminin, fibrinogen, and fibronectin. Two peptides of 19 and 32 kDa bound to all three proteins and the purified 32 kDa component as well as a mAb against it inhibited adherence of conidia to the ECM proteins in a dose dependent manner (Gonzalez et al., 2005a).

Another 30 kDa protein, with pI 4.9, was described with properties of an adhesin. Laminin but none of the other ECM components bound specifically to this fungal protein (Andreotti et al., 2005).

The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from P. brasiliensis, was identified by immunoproteomic sequencing and microsequencing of peptides in two isoforms of 36 kDa (pIs 6.8 and 7.0). The protein is expressed in the cytoplasm and on the cell wall of the yeast phase, as an adhesin. Recombinant GAPDH bound to fibronectin, laminin, and type I collagen in ligand far-Western blotting assays. Treatment of yeast cells with anti-GAPDH polyclonal antibody and incubation of pneumocytes with the recombinant protein inhibited adherence and internalization of P. brasiliensis to those in vitro-cultured cells (Barbosa et al., 2006).

The ability of microorganisms to adhere to host cells and to ECM components, resist the immune response, grow and invade tissues and the blood stream for a disseminated infection, are hallmarks of microbial pathogenicity. The research on P. brasiliensis virulence has made a significant advance towards the understanding of all these interactions and events at the molecular level.

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