Purification and Characterization of a Mycelial Catalase from Scedosporium boydii, a Useful Tool for Specific Antibody Detection in Patients with Cystic Fibrosis

Abstract

bScedosporium boydii is an opportunistic filamentous fungus which may be responsible for a wide variety of infections in immunocompetent and immunocompromised individuals. This fungus belongs to the Scedosporium apiospermum species complex, which usually ranks second among the filamentous fungi colonizing the airways of patients with cystic fibrosis (CF) and may lead to allergic bronchopulmonary mycoses, sensitization, or respiratory infections. Upon microbial infection, host phagocytic cells release reactive oxygen species (ROS), such as hydrogen peroxide, as part of the antimicrobial response. Catalases are known to protect pathogens against ROS by detoxification of the hydrogen peroxide. Here, we investigated the catalase equipment of Scedosporium boydii, one of the major pathogenic species in the S. apiospermum species complex. Three catalases were identified, and the mycelial catalase A1 was purified to homogeneity by a three-step chromatographic process. This enzyme is a monofunctional tetrameric protein of 460 kDa, consisting of four 82-kDa glycosylated subunits. The potential usefulness of this enzyme in serodiagnosis of S. apiospermum infections was then investigated by an enzyme-linked immunosorbent assay (ELISA), using 64 serum samples from CF patients. Whatever the species involved in the S. apiospermum complex, sera from infected patients were clearly differentiated from sera from patients with an Aspergillus fumigatus infection or those from CF patients without clinical and biological signs of a fungal infection and without any fungus recovered from sputum samples. These results suggest that catalase A1 is a good candidate for the development of an immunoassay for serodiagnosis of infections caused by the S. apiospermum complex in patients with CF. Scedosporium boydii (formerly known as Pseudallescheria boydii) is one of the major pathogenic species within the Scedosporium apiospermum complex, which comprises four other species, namely, Scedosporium apiospermum sensu stricto, Scedosporium aurantiacum, Scedosporium minutisporum, and Scedosporium dehoogii, Scedosporium prolificans having been reassigned recently to the genus Lomentospora (Lomentospora prolificans) (1-4). These filamentous fungi are soilborne fungi that may cause a wide range of infections in humans, including subcutaneous mycetomas and ocular, bone, or joint infections resulting from traumatic inoculation of some fungal elements and infections of the respiratory tract (i.e., sinusitis and lung fungus ball), which are thought to be due to the inhalation of some airborne conidia (5-7). However, these fungi have gained attention during the past 2 decades mainly because of their recognition as common agents of colonization of the airways in patients with cystic fibrosis (CF). When appropriate culture media are used, the S. apiospermum species complex ranks second among the filamentous fungi recovered from respiratory specimens, with a prevalence ranging from 4.5 to 11.6% in patients (8-12). Although usually asymptomatic, this fungal colo...

Identification of Scedosporium boydii catalase A1 gene, a reactive oxygen species detoxification factor highly expressed in response to oxidative stress and phagocytic cells

“…This protein sequence revealed the presence of six potential N-glycosylation sites and eleven O-glycosylated sites which confirms the presence of glycosylated residues demonstrated previously on the purified enzyme by affinity chromatography and Western-blotting (Mina et al 2015). The N-terminal region of S. boydii catalase A1 comprised a 26 amino acid-motif corresponding to a signal peptide responsible for the membrane translocation of the protein and its secretion into the culture medium, which is in agreement with detection of the enzyme in culture supernatants (Mina et al 2015).…”

“…Analysis of deduced protein sequences suggests that there are enough epitope differences between S. boydii catalase A1 and its ortholog in A. fumigatus. Indeed, our previous study showed that a specific serological test for scedosporiosis could be developed using biochemically purified S. boydii catalase A1 (Mina et al 2015). Moreover, anti-Lomentospora prolificans catalase immunoglobulins A were detected in the saliva from healthy immunocompetent individuals .…”

“…Catalases and superoxide dismutases are well known to provide efficient systems to evade the oxidative stress generated by the host phagocytic cells and to thwart the deleterious effects of ROS. Recent biochemical characterization of the major catalase produced by S. boydii (Mina et al 2015) allowed us to identify the encoding gene and to evaluate its possible role against oxidative stresses induced chemically or generated by macrophages derived from THP-1 monocytes or by neutrophils.…”

“…Scedosporium boydii IHEM 15155 originally isolated from human sputum in Angers University Hospital (France) was used throughout this study Mina et al 2015). This strain was maintained by weekly passages on homemade yeast extract-peptone-dextrose-agar (YPDA) medium (containing in g L À1 : yeast extract, 5; peptone, 10; glucose, 20; and agar, 20) supplemented with chloramphenicol 0.5 %.…”

“…In addition, hydrolytic enzymes thought to be involved in the host tissue invasion by the degradation of host protein barriers such as serine proteases (Larcher et al 1996), metalloproteases (Pereira et al 2009;Silva et al 2011), and phosphatases (Kiffer-Moreira et al 2007) have been identified, as well as enzymes possibly involved in evading the host oxidative response such as a cytosolic Cu,Zn-superoxide dismutase (SOD) (Lima et al 2007). In addition, we showed recently that S. boydii produces three distinct catalases and one of these enzymes called catalase A1 (Cat A1) was purified and characterized (Mina et al 2015). Nevertheless, only three genes encoding putative virulence factors had been sequenced, i.e.…”