C. Staerck

Microbial antioxidant defense enzymes

Free radicals are often described as chemical compounds characterized by unpaired electrons in their outer orbital rendering them highly reactive species. In mammalians, studies on free radicals were focused on reactive oxygen species (ROS) or reactive nitrogen species (RNS) due to their relative importance in physiological as well as in pathological processes. These cellular compounds are produced by different physiological systems such as the aerobic metabolism and play a major role in cell signaling pathways but also in the host immune defenses against pathogenic microorganisms. ROS and RNS are highly reactive species with potentially harmful effects on any cellular components (lipids, proteins and nucleic acids) when produced with a high level. To maintain ROS and RNS at a non-toxic concentration, enzymatic and non-enzymatic cellular antioxidants coordinate the balance between their production and their degradation. Superoxide dismutases, catalases, glutathione system, thioredoxin system, peroxidase systems, flavohemoglobins and nitrate or nitrite reductases represent the prominent enzymatic antioxidants used to scavenge excess of internal as well as external ROS and RNS. Bacteria, fungi and parasites also display similar enzymatic activities to escape the host oxidative defenses during the immune response against infectious processes. Here we summarize current knowledge on the enzymatic systems that allow microorganisms to fight against ROS and RNS, and shed light on the role that take some of them in microbial infections. Such microbial protective systems are considered as virulence factors, and therefore represent key targets for diagnosis of the infections or development of anti-infectious drugs.

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

Scedosporium boydii is an opportunistic filamentous fungus which may be responsible for a large variety of infections in both 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). Species of the S. apiospermum complex are able to chronically colonize the CF airways suggesting pathogenic mechanisms allowing persistence and growth of these fungi in the respiratory tract. Few putative virulence factors have been purified and characterized so far in the S. apiospermum complex including a cytosolic Cu,Zn-superoxide dismutase (SOD) and a monofunctional catalase (catalase A1). Upon microbial infection, host phagocytes release reactive oxygen species (ROS), such as hydrogen peroxide, as part of the antimicrobial response. Catalases are known to protect pathogens against ROS by degradation of the hydrogen peroxide. Here, we identified the S. boydii catalase A1 gene (CATA1) and investigated its expression in response to the environmental conditions encountered in the CF airways and to the oxidative stress. Results showed that S. boydii CATA1 gene expression is not affected by hypoxia, hypercapnia or pH changes. In contrast, CATA1 gene was overexpressed in response to a chemically induced oxidative stress with a relative gene expression 37-fold higher in the presence of 250 μM H(2)O(2), 20-fold higher with 250 μM menadione and 5-fold higher with 2 mM paraquat. Moreover, S. boydii CATA1 gene expression progressively increased upon exposure to activated THP-1-derived macrophages, reaching a maximum after 12 h (26 fold). Activated HL60-derived neutrophils and activated human peripheral blood neutrophils more rapidly induced S. boydii CATA1 gene overexpression, a maximum gene expression level being reached at 75 min (17 fold) and 60 min (15 fold), respectively. In contrast expression of the gene encoding the Cu,Zn-SOD (SODC gene) was not affected by H(2)O(2), menadione, paraquat or in co-culture with phagocytic cells. These results suggest that S. boydii CATA1 gene is highly stimulated by the oxidative burst response whereas SODC gene is constitutively expressed.

Varying susceptibility of clinical and environmental Scedosporium isolates to chemical oxidative stress in conidial germination

Scedosporium species are opportunistic pathogens causing a great variety of infections in both immunocompetent and immunocompromised individuals. The Scedosporium genus ranks the second among the filamentous fungi colonizing the airways of patients with cystic fibrosis (CF), after Aspergillus fumigatus, and most species are capable to chronically colonize the respiratory tract of these patients. Nevertheless, few data are available regarding evasion of the inhaled conidia to the host immune response. Upon microbial infection, macrophages and neutrophils release reactive oxygen species (ROS). To colonize the respiratory tract, the conidia need to germinate despite the oxidative stress generated by phagocytic cells. Germination of spores from different clinical or environmental isolates of the major Scedosporium species was investigated in oxidative stress conditions. All tested species showed susceptibility to oxidative stress. However, when comparing clinical and environmental isolates, differences in germination capabilities under oxidative stress conditions were seen between species as well as within each species. Among environmental isolates, Scedosporium aurantiacum isolates were the most resistant to oxidative stress whereas Scedosporium dehoogii were the most susceptible. Overall, the differences observed between Scedosporium species in the capacity to germinate under oxidative stress conditions could explain their varying prevalence and pathogenicity.

The secreted polyketide boydone A is responsible for the anti-Staphylococcus aureus activity of Scedosporium boydii.

&NA; Usually living as a soil saprophyte, the filamentous fungus Scedosporium boydii may also cause various infections in human. Particularly, it is one of the major causative agents of fungal colonization of the airways in patients with cystic fibrosis (CF). To compete with other microorganisms in the environment, fungi have evolved sophisticated strategies, including the production of secondary metabolites with antimicrobial activity that may also help them to establish successfully within the respiratory tract of receptive hosts. Here, the culture filtrate from a human pathogenic strain of S. boydii was investigated searching for an antibacterial activity, mainly against the major CF bacterial pathogens. A high antibacterial activity against Staphylococcus aureus, including methicillin‐resistant strains of this species, was observed. Bio‐guided fractionation and analysis of the active fractions by nuclear magnetic resonance or by high‐performance liquid chromatography and high‐resolution electrospray ionization‐mass spectrometry allowed us to identify boydone A as responsible for this antibacterial activity. Together, these results suggest that this six‐membered cyclic polyketide could be one of the virulence factors of the fungus. Genes involved in the synthesis of this secreted metabolite are currently being identified in order to confirm the role of this polyketide in pathogenesis.

Scedosporium boydii CatA1 and SODC recombinant proteins, new tools for serodiagnosis of Scedosporium infection of patients with cystic fibrosis

Scedosporium species rank the second among the filamentous fungi colonizing the airways of patients with cystic fibrosis (CF), after Aspergillus fumigatus. In CF, these fungi may cause various respiratory infections similar to those caused by A. fumigatus, including bronchitis and allergic broncho-pulmonary mycoses. Diagnosis of these infections relies on the detection of serum antibodies using crude antigenic extracts. However, many components of these extracts are common to Scedosporium and Aspergillus species, leading to cross-reactions. Here, 5 recombinant proteins from S. apiospermum or S. boydii were produced, and their value in serodiagnosis of Scedosporium infections was investigated by enzyme-linked immunosorbent assay. Two of them, corresponding to the Scedosporium catalase A1 or cytosolic Cu,Zn-superoxyde dismutase, allowed the detection of Scedosporium infection, and the differentiation with an Aspergillus infection. These recombinant proteins therefore may serve as a basis for the development of a standardized serological test.

Transcriptional profiling of Scedosporium apiospermum enzymatic antioxidant gene battery unravels the involvement of thioredoxin reductases against chemical and phagocytic cells oxidative stress

Scedosporium species rank the second, after Aspergillus fumigatus, among the filamentous fungi colonizing the airways of patients with cystic fibrosis (CF). Development of microorganisms in the respiratory tract depends on their capacity to evade killing by the host immune system, particularly through the oxidative response of macrophages and neutrophils, with the release of reactive oxygen species (ROS) and reactive nitrogen species (RNS). This is particularly true in the airways of CF patients which display an exacerbated inflammatory reaction. To protect themselves, pathogens have developed various enzymatic antioxidant systems implicated in ROS degradation, including superoxide dismutases, catalases, cytochrome C peroxidases, chloroperoxidases and enzymes of the glutathione and thioredoxin systems, or in RNS degradation, that is, flavohemoglobins, nitrate reductases, and nitrite reductases. Here we investigated the transcriptional regulation of the enzymatic antioxidant gene battery in 24-h-old hyphae of Scedosporium apiospermum in response to oxidative stress induced chemically or by exposure to activated phagocytic cells. We showed that 21 out of the 33 genes potentially implicated in the oxidative or nitrosative stress response were overexpressed upon exposure of the fungus to various chemical oxidants, while they were only 13 in co-cultures with macrophages or neutrophils. Among them, genes encoding two thioredoxin reductases and to a lesser extent, a peroxiredoxin and one catalase were found to be overexpressed after chemical oxidative stress as well as in co-cultures. These results suggest that thioredoxin reductases, which are known to be virulence factors in other pathogenic fungi, play a key role in pathogenesis of scedosporiosis, and may be new drug targets.