Inducible expression of beta defensins by human respiratory epithelial cells exposed to Aspergillus fumigatus organisms

<p>Abstract</p> <p>Background</p> <p><it>Aspergillus fumigatus</it>, a saprophytic mould, is responsible for life-threatening, invasive pulmonary diseases in immunocompromised hosts. The role of the airway epithelium involves a complex interaction with the inhaled pathogen. Antimicrobial peptides with direct antifungal and chemotactic activities may boost antifungal immune response.</p> <p>Results</p> <p>The inducible expression of defensins by human bronchial epithelial 16HBE cells and A549 pneumocyte cells exposed to <it>A. fumigatus </it>was investigated. Using RT-PCR and real time PCR, we showed an activation of hBD2 and hBD9 defensin genes: the expression was higher in cells exposed to swollen conidia (SC), compared to resting conidia (RC) or hyphal fragments (HF). The kinetics of defensin expression was different for each one, evoking a putative distinct function for each investigated defensin. The decrease of defensin expression in the presence of heat-inactivated serum indicated a possible link between defensins and the proteins of the host complement system. The presence of defensin peptide hBD2 was revealed using immunofluorescence that showed a punctual cytoplasmic and perinuclear staining. Quantification of the cells stained with anti hBD2 antibody demonstrated that SC induced a greater number of cells that synthesized hBD2, compared to RC or HF. Labelling of the cells with anti-hBD-2 antibody showed a positive immunofluorescence signal around RC or SC in contrast to HF. This suggests co-localisation of hBD2 and digested conidia. The HBD2 level was highest in the supernatants of cells exposed to SC, as was determined by sandwich ELISA. Experiments using neutralising anti-interleukine-1β antibody reflect the autocrine mechanism of defensin expression induced by SC. Investigation of defensin expression at transcriptional and post-transcriptional levels demonstrated the requirement of transcription as well as new protein synthesis during <it>A. fumigatus </it>defensin induction. Finally, induced defensin expression in primary culture of human respiratory cells exposed to <it>A. fumigatus </it>points to the biological significance of described phenomena.</p> <p>Conclusion</p> <p>Our findings provide evidence that respiratory epithelium might play an important role in the immune response during <it>Aspergillus </it>infection. Understanding the mechanisms of regulation of defensin expression may thus lead to new approaches that could enhance expression of antimicrobial peptides for potential therapeutic use during aspergillosis treatment.</p

Alternative TSS use is widespread in <i>Cryptococcus</i> fungi in response to environmental cues and regulated genome-wide by the transcription factor Tur1

Alternative transcription start site (TSS) usage regulation has been identified as a major means of gene expression regulation in metazoans. However, in fungi, its impact remains elusive as its study has thus far been restricted to model yeasts. Here, we first re-analyzed TSS-seq data to define genuine TSS clusters in 2 species of pathogenic Cryptococcus. We identified 2 types of TSS clusters associated with specific DNA sequence motifs. Our analysis also revealed that alternative TSS usage regulation in response to environmental cues is widespread in Cryptococcus, altering gene expression and protein targeting. Importantly, we performed a forward genetic screen to identify a unique transcription factor (TF) named Tur1, which regulates alternative TSS (altTSS) usage genome-wide when cells switch from exponential phase to stationary phase. ChiP-Seq and DamID-Seq analyses suggest that at some loci, the role of Tur1 might be direct. Tur1 has been previously shown to be essential for virulence in C. neoformans. We demonstrated here that a tur1Δ mutant strain is more sensitive to superoxide stress and phagocytosed more efficiently by macrophages than the wild-type (WT) strain

DNA polymorphism among Fusarium oxysporum f.sp. elaeidis populations from oil palm, using a repeated and dispersed sequence "Palm".

International audienceA worldwide collection, of 76 F. oxysporum f.sp. elaeidis isolates (Foe), and of 21 F. oxysporum isolates from the soil of several palm grove was analysed by RFLP. As a probe, we used a random DNA fragment (probe 46) from a genomic library of a Foe isolate. This probe contains two different types of sequence, one being repeated and dispersed in the genome "Palm", the other being a single-copy sequence. All F. oxysporum isolates from the palm-grove soils were non-pathogenic to oil palm. They all had a simple restriction pattern with one band homologous to the single-copy sequence of probe 46. All Foe isolates were pathogenic to oil palm and they all had complex patterns due to hybridization with "Palm". This repetitive sequence reveals that Foe isolates are distinct from the other F. oxysporum palm-grove soils isolates. The sequence can reliably discriminate pathogenic from non-pathogenic oil palm isolates. Based on DNA fingerprint similarities, Foe populations were divided into ten groups consisting of isolates with the same geographic origin. Isolates from Brazil and Ecuador were an exception to that rule as they had the same restriction pattern as a few isolates from the Ivory Coast, suggesting they may originated from Africa

Testing the efficacy of RNA interference constructs in Aspergillus fumigatus.

International audienceWe recently developed a silencing vector in Aspergillus fumigatus which carries a hygromycin resistance marker and a transcriptional unit for hairpin RNA expression under the control of the inducible glucoamylase promoter (pGla) (Mouyna et al. in FEMS Microbiol Lett 237:317-324, 2004). We showed previously that this vector can be used for the RNA interference application of two genes ALB1 and FKS1 of which reduced mRNA levels occurred for both, with phenotypic consequences resembling disruptions of genes involved in melanin (ALB1) and beta(1-3)glucan biosynthesis (FKS1). We reported here the silencing of KRE6 and CRH1, two other genes putatively involved in cell wall biosynthesis using a similar construction under the control of the constitutive promoter glyceraldehyde-3-phosphate dehydrogenase (pgpdA). Silencing of the expression of these two genes was obtained. Further analysis of the transformants showed however that (1) a 100% loss of expression was never achieved for all genes tested (2) the vector used for RNAi is lost or modified over successive transfers resulting in an inhibition of the silencing. These disadvantages of RNAi indicate that classical gene disruption by gene replacement remains the most efficient method for a molecular analysis of gene function in A. fumigatus

Members of Glycosyl-Hydrolase Family 17 of A. fumigatus Differentially Affect Morphogenesis

Cell wall biosynthesis and remodeling are essential for fungal growth and development. In the fungal pathogen Aspergillus fumigatus, the β(1,3)glucan is the major cell wall polysaccharide. This polymer is synthesized at the plasma membrane by a transmembrane complex, then released into the parietal space to be remodeled by enzymes, and finally incorporated into the pre-existing cell wall. In the Glycosyl-Hydrolases family 17 (GH17) of A. fumigatus, two β(1,3)glucanosyltransferases, Bgt1p and Bgt2p, have been previously characterized. Disruption of BGT1 and BGT2 did not result in a phenotype, but sequence comparison and hydrophobic cluster analysis showed that three other genes in A. fumigatus belong to the GH17 family, SCW4, SCW11, and BGT3. In constrast to Δbgt1bgt2 mutants, single and multiple deletion of SCW4, SCW11, and BGT3 showed a decrease in conidiation associated with a higher conidial mortality and an abnormal conidial shape. Moreover, mycelium was also affected with a slower growth, stronger sensitivity to cell wall disturbing agents, and altered cell wall composition. Finally, the synthetic interactions between Bgt1p, Bgt2p, and the three other members, which support a functional cooperation in cell-wall assembly, were analyzed. Our data suggest that Scw4p, Scw11p, and Bgt3p are essential for cell wall integrity and might have antagonistic and distinct functions to Bgt1p and Bgt2p

Innate immunity and the role of epithelial barrier during aspergillus fumigatus infection

Fungi are the most important eukaryotic infective agents in Europe which largely overpass parasite infections. Total number of people dying of fungal infection is increasing and this trend is likely to continue due to the increase in immunosuppressive treatments. The opportunistic pathogen Aspergillus fumigatus (Af) is a saprophytic filamentous fungus that can cause invasive pulmonary diseases in immuno-compromised hosts. In veterinary medicine aspergillosis is also a recurrent problem since it infects various species, birds are particularly susceptible. It propagates through airborne conidia (spores), which are inhaled into the small airways where they may germinate and initiate an infection. The host epithelium has permanent contact with the environment and a multitude of diverse microorganisms, resulting in a network of the host’s defense mechanisms. Pathogens use various strategies to invade epithelial barriers, to exploit eukaryotic host function to their own benefit and disseminate throughout the host using the epithelium as a reservoir. The current revue will discuss the ways how epithelial and innate immunity cells can contlol Af infection. We will focus on Af strategies for the host’s invasion, antifungal innate immune response and antimicrobial activities of the respiratory epithelial cells

Gene silencing with RNA interference in the human pathogenic fungus Aspergillus fumigatus

International audienceAspergillus fumigatus is an opportunistic pathogenic fungus which causes fatal invasive aspergillosis among immunocompromised patients. To obtain a better understanding of the key elements involved in A. fumigatus virulence and to identify possible drug targets, it is necessary to be able to generate gene-deletion strains. Unfortunately, the molecular techniques available do not include a rapid method to disrupt and identify essential genes. RNA interference, a process in which the presence of double-stranded RNA homologous to a gene of interest results in specific degradation of the corresponding message, has been successfully tested on A. fumigatus. We have shown that expression of double stranded RNA corresponding to portions of the ALB1/PKSP and FKS1 genes results in reduced mRNA levels for those genes, with phenotypic consequences similar to that of gene disruption. The two genes could also be subjected to simultaneous interference through expression of chimeric double-stranded RNA. Use of RNA interference in Aspergillus will allow easier examination of the phenotypic consequences of reducing expression of a gene of interest, especially for essential genes

Molecular characterization of a cell wall-associated beta(1-3)endoglucanase of Aspergillus fumigatus.

International audienceA 74 kDa beta(1-3)endoglucanase of Aspergillus fumigatus was recently isolated from a cell wall autolysate and biochemically characterized. In this study, we report the cloning and the disruption of the ENGL1 gene encoding this beta(1-3)endoglucanase. ENGL1 contains an open reading frame of 2181 bp encoding a polypeptide of 727 amino acids. Sequence analysis showed that ENGL1 is the first characterized member of a new family of beta(1-3)glucanases. Disruption of ENGL1, however, did not lead to a phenotype distinct from the parental strain, indicating that this cell wall-associated beta(1-3)endoglucanase does not play an essential role in constitutive cell growth