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

Staphylococcus aureus seroproteomes discriminate ruminant isolates causing mild or severe mastitis

Staphylococcus aureus is a major cause of mastitis in ruminants. In ewe mastitis, symptoms range from subclinical to gangrenous mastitis. S. aureus factors or host-factors contributing to the different outcomes are not completely elucidated. In this study, experimental mastitis was induced on primiparous ewes using two S. aureus strains, isolated from gangrenous (strain O11) or subclinical (strain O46) mastitis. Strains induced drastically distinct clinical symptoms when tested in ewe and mice experimental mastitis. Notably, they reproduced mild (O46) or severe (O11) mastitis in ewes. Ewe sera were used to identify staphylococcal immunoreactive proteins commonly or differentially produced during infections of variable severity and to define core and accessory seroproteomes. Such SERological Proteome Analysis (SERPA) allowed the identification of 89 immunoreactive proteins, of which only 52 (58.4%) were previously identified as immunogenic proteins in other staphylococcal infections. Among the 89 proteins identified, 74 appear to constitute the core seroproteome. Among the 15 remaining proteins defining the accessory seroproteome, 12 were specific for strain O11, 3 were specific for O46. Distribution of one protein specific for each mastitis severity was investigated in ten other strains isolated from subclinical or clinical mastitis. We report here for the first time the identification of staphylococcal immunogenic proteins common or specific to S. aureus strains responsible for mild or severe mastitis. These findings open avenues in S. aureus mastitis studies as some of these proteins, expressed in vivo, are likely to account for the success of S. aureus as a pathogen of the ruminant mammary gland

Molecular Basis of Virulence in Staphylococcus aureus Mastitis

S. aureus is one of the main pathogens involved in ruminant mastitis worldwide. The severity of staphylococcal infection is highly variable, ranging from subclinical to gangrenous mastitis. This work represents an in-depth characterization of S. aureus mastitis isolates to identify bacterial factors involved in severity of mastitis infection

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

Inhibition of [i]Staphylococcus aureus[/i] invasion into bovine mammary epithelial cells by contact with live Lactobacillus casei

Staphylococcus aureus is a major pathogen that is responsible for mastitis in dairy herds. S. aureus mastitis is difficult to treat and prone to recurrence despite antibiotic treatment. The ability of S. aureus to invade bovine mammary epithelial cells (bMEC) is evoked to explain this chronicity. One sustainable alternative to treat or prevent mastitis is the use of lactic acid bacteria (LAB) as mammary probiotics. In this study, we tested the ability of Lactobacillus casei strains to prevent invasion of bMEC by two S. aureus bovine strains, RF122 and Newbould305, which reproducibly induce acute and moderate mastitis, respectively. L. casei strains affected adhesion and/or internalization of S. aureus in a strain-dependent manner. Interestingly, L. casei CIRM-BIA 667 reduced S. aureus Newbould305 and RF122 internalization by 60 to 80%, and this inhibition was confirmed for two other L. casei strains, including one isolated from bovine teat canal. The protective effect occurred without affecting bMEC morphology and viability. Once internalized, the fate of S. aureus was not affected by L. casei. It should be noted that L. casei was internalized at a low rate but survived in bMEC cells with a better efficiency than that of S. aureus RF122. Inhibition of S. aureus adhesion was maintained with heat-killed L. casei, whereas contact between live L. casei and S. aureus or bMEC was required to prevent S. aureus internalization. This first study of the antagonism of LAB toward S. aureus in a mammary context opens avenues for the development of novel control strategies against this major pathogen

Monoclonal antibodies against human translation termination factor eRF3 and their utilization for sub-cellular localization of eRF3.

International audienceEukaryotic translation termination is triggered by peptide release factors eRF1 and eRF3. eRF1 recognizes the stop codon and promotes nascent peptide chain release, while eRF3 facilitates this peptide release in a GTP-dependent manner. In addition to its role in termination, eRF3 is involved in normal and nonsense-mediated mRNA decay. Despite extensive investigation, the complete understanding of eRF3 function have been hampered by the lack of specific anti-eRF3 monoclonal antibodies (Mabs). The purpose of the study was production of recombinant eRF3a/GSPT1, development of anti-eRF3a/GSPT1 Mabs and their utilization for eRF3a/GSPT1 sub-cellular localization. Plasmid encoding C-terminal part of human GSPT1/eRF3a was constructed. Purified protein, which was predominantly present in the inclusion bodies, was used for the development of Mabs. Characterization of the regions recognized by Mabs using GSPT1/eRF3a mutants and its visualization in the 3D space suggested that Mabs recognize different epitopes. Consistent with its function in translational termination, immunostaining of the cells with developed Mabs revealed that the endogenous GSPT1/eRF3a localized in endoplasmic reticulum. Taking into account the important role of eRF3 for the fundamental research one can suggests that developed Mabs have great prospective to be used as a research reagent in a wide range of applications

Linkage analysis excludes the involvement of the canine PKD2 homologue in bull terrier polycystic kidney disease

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Disruption of the sigS gene attenuates the local innate immune response to Staphylococcus aureus in a mouse mastitis model

Staphylococcus aureus (S. aureus) is a major pathogen involved in ruminant mastitis and present worldwide. Clinical signs of S. aureus mastitis vary considerably and are largely dependent on strain-specific factors. A comparison of two S. aureus strains that reproducibly induced either severe (O11) or mild (O46) mastitis in ewes revealed that the transcriptional regulator sigS was mutated in O46 (Le Maréchal et al., 2011. PLoS One. 6 (11) e27354. doi:10.1371/journal.pone.0027354). In the present paper, we analysed the sigS sequence in 18 other S. aureus strains isolated from goat or ewe mastitis and found a 4-bp deletion similar to that of the O46 sigS gene in three strains associated with subclinical ewe mastitis. This sigS gene was disrupted in strain O11 (O11ΔsigS), so our aim was to investigate its involvement in the severity of infections in the context of mastitis. The wild type (wt) and mutant strains were then characterized in vitro to determine the involvement of sigS in the response S. aureus under various stress conditions, and assess its influence on the cytotoxicity of the pathogen, its invasive capacity and biofilm formation. The strains were compared in vivo in an experimental mouse mastitis model in which clinical signs and cytokine production were evaluated at 24 h post-infection. While no significant differences in the effect on bacterial growth between O11 and O11ΔsigS were observed either in vitro or in vivo, a significantly weaker in vivo production of interleukin (IL)-1α, IL-1β, and Tumor Necrosis Factor (TNF)-α was measured in the mammary glands infected with the mutant strain, suggesting that infection with O11ΔsigS induced an attenuated local innate immune response. These results suggest an impact of sigS disruption on S. aureus pathogenesis in a ruminant mastitis context. This disruption is probably involved in, and may partly explain, the milder symptoms previously observed in S. aureus O46-induced mastitis in ewes

Susceptibility of mice to invasive aspergillosis correlates with delayed cell influx into the lungs

International audienceP>Ubiquitous fungus Aspergillus fumigatus (A. fumigatus) is involved in invasive pulmonary aspergillosis (IPA), a frequent infection in immunocompromized patients. Genetic differences are likely to play a role predisposing to IPA. This study was aimed to compare six genetically different mouse strains in their susceptibility to IPA and to determine possible mechanisms involved in the pathogenesis of this infection. Immunosuppressed BALB/c and C57BL/6 mice infected with A. fumigatus conidia were more resistant to IPA than DBA/1, DBA/2, CBA, and A/Sn strains. Phagocytosis of A. fumigatus conidia by blood polymorphonuclear neutrophils (PMN) or bone marrow derived dendritic cells showed no difference between strains. All IPA susceptible strains demonstrated decreased PMN influx into the lungs during infection compared with resistant strains. Flow cytometry analysis of the composition of lung infiltrating cells showed that IPA susceptible mice had a decreased number of phagocytes before the infection. After infection the numbers of Gr-1(+)CD11b(+) PMN cells in the lungs of immunosuppressed mice increased from 10-20% to 50-60% while the percentage of CD11(+)F4/80(+) resident macrophages was unchanged. Among susceptible strains DBA/2 and A/Sn have a defect in C5 component of complement. Injection of normal serum into complement deficient but not into complement sufficient CBA or DBA/1 mice significantly improved their survival. We showed that complement replacement significantly increased PMN homing to the lungs of complement deficient mice. Thus, defect in complement system can predispose to IPA. Our results demonstrated that early influx of PMN into the lungs of mice is important for the resistance to IPA

Bacteriocins as an alternative in the treatment of infections by Staphylococcus aureus

International audienceStaphylococcus aureus (S. aureus) is a highly versatile Gram-positive bacterium that is carried asymptomatically by up to 30% of healthy people, while being a major cause of healthcare-associated infections, making it a worldwide problem in clinical medicine. The adaptive evolution of S. aureus strains is demonstrated by its remarkable capacity to promptly develop high resistance to multiple antibiotics, thus limiting treatment choice. Nowadays, there is a continuous demand for an alternative to the use of antibiotics for S. aureus infections and a strategy to control the spread or to kill phylogenetically related strains. In this scenario, bacteriocins fi t as with a promising and interesting alternative. These molecules are produced by a range of bacteria, defi ned as ribosomally synthesized peptides with bacteriostatic or bactericidal activity against a wide range of pathogens. This work reviews ascertained the main antibiotic-resistance mechanisms of S. aureus strains and the current, informative content concerning the applicability of the use of bacteriocins overlapping the use of conventional antibiotics in the context of S. aureus infections. Besides, we highlight the possible application of these biomolecules on an industrial scale in future work