Involvement of SecretedAspergillus fumigatusProteases in Disruption of the Actin Fiber Cytoskeleton and Loss of Focal Adhesion Sites in Infected A549 Lung Pneumocytes

Abstract

Aspergillus fumigatus is an opportunistic pathogenic fungus that predominantly infects the respiratory system. Penetration of the lung alveolar epithelium is a key step in the infectious process. The cytoskeleton of alveolar epithelial cells forms the cellular basis for the formation of a physical barrier between the cells and their surroundings. This study focused on the distinct effects of A. fumigatus on the actin cytoskeleton of A549 lung pneumocytes. Of the 3 major classes of cytoskeletal fibers--actin microfilaments, microtubules, and intermediate filaments--only the actin cytoskeleton was found to undergo 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 changes could be specifically blocked in wild-type strains of A. fumigatus by the addition of antipain, a serine and cysteine protease inhibitor, and were not induced by an alkaline serine protease-deficient strain of A. fumigatus. Antipain also reduced, by approximately 50%, fungal-induced A549 cell detachment from the plates and reduction in viability. Our 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.

Transcription Factor PrtT Controls Expression of Multiple Secreted Proteases in the Human Pathogenic Mold Aspergillus fumigatus

“…CF from the prtT deletion strain exhibits reduced killing of lung epithelial cells and lysis of erythrocytes. Our previous studies have shown that secreted A. fumigatus proteases disrupt A549 cell actin fibers and focal adhesions, leading to cell detachment and death (15). Those results suggested 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.…”

“…The following evidence implicates secreted A. fumigatus proteases in virulence: (i) secreted A. fumigatus proteases induce proinflammatory cytokine release in infected macrophages and epithelial cells, thereby alerting the immune system (14); (ii) infected lung epithelial cells also undergo protease-dependent changes to the actin cytoskeleton, leading to cell peeling and death (15,36); (iii) proteases are secreted in vivo during infection, and protease-specific antisera show labeling of mycelium in the lungs of patients and experimentally infected animals (22); (iv) loss of elastase protease activity in mutagenized strains of A. fumigatus has been correlated with decreased virulence in vivo (16); and (v) mice intratracheally injected with purified ALP1 protease showed a marked degree of lower respiratory tract destruction (10).…”

“…The colonies were grown for 48 h at 37°C and then transferred to room temperature for another 48 h. Azocasein assay. Azocasein (Sigma) was dissolved at a concentration of 5 mg/ml in assay buffer containing 50 mM Tris (pH 7.5), 0.2 M NaCl, 5 mM CaCl 2 , and 0.05% Triton X-100 as previously described (15). Supernatants were removed from Aspergillus cultures at various times and centrifuged to pellet the cells.…”

Interactions of Paracoccidioides brasiliensis with host cells: recent advances

“…This mechanism is not used by all microorganisms, so it is considered as an important virulence factor. Some fungal species are known to be capable of invading mammalian cells in vitro and in vivi, but few studies have described the invasion process [11,19,22,[63][64][65].…”

Methylcitrate synthase from Aspergillus fumigatus is essential for manifestation of invasive aspergillosis

“…The genome of A. fumigatus contains several genes most likely coding for proteases (Rementeria et al, 2005), leading to the assumption that the need for nutrients could be satisfied from protein degradation. Moreover, during lung infection it has been demonstrated that A. fumigatus indeed produces several proteases (Kogan et al, 2004).…”