Degradation of keratin substrates by keratinolytic fungi

Background: The hydrolysis of keratin wastes by microorganisms is considered a biotechnological alternative for recycling and valorization through keratinolytic microorganisms. Despite their resistant structure, keratin wastes can be efficiently degraded by various microorganisms through the secretion of keratinases, which are promising enzymes for several applications, including detergents, fertilizers, and leather and textile industry. In an attempt to isolate keratinolytic microorganisms that can reach commercial exploitation as keratinase producers, the current work assesses the dynamics of keratin biodegradation by several keratinolytic fungal strains isolated from soil. The activity of fungal strains to degrade keratin substrates was evaluated by SEM, FTRIR-ATR spectra and TGA analysis. Results: SEM observations offered relevant information on interactions between microorganism and structural elements of hair strands. FTIR spectra of the bands at 1035­1075 cm-1 assigned to sulfoxide bond appeared because of S­S bond breaking, which demonstrated the initiation of keratin biodegradation. According to TGA, in the second zone of thermal denaturation, where keratin degradation occurs, the highest weight loss of 71.10% was obtained for sample incubated with Fusarium sp. 1A. Conclusions: Among the tested strains, Fusarium sp. 1A was the most active organism in the degradation process with the strongest denaturation of polypeptide chains. Because keratinolytic microorganisms and their enzymes keratinases represent a subject of scientific and economic interest because of their capability to hydrolyze keratin, Fusarium sp. 1A was selected for further studies.

Differential effects on caspase mediated apoptosis of hela cells induced by different pseudomonas aeruginosa culture fractions.

The aim of this work was to investigate intracellular survival of Pseudomonas aeruginosa clinical strains in non-phagocytic, epithelial cells and the capacity to induce apoptosis of the host cell using qualitative (evaluation of virulence factors expression, adherence to HeLa cells) and quantitative assays (invasion assay, qRT-PCR). Taken together the results demonstrated that all tested strains adhered to HeLa cells, exhibiting a diffuse, aggregative or mixed diffuse-aggregative pattern with 20-70% adherence rates. The assays confirmed that P. aeruginosa had the ability to enter and invade the epithelial, non-phagoytic cells, probably due to the pores forming enzymes (hemolysins, lecithinase, lipase) released by the analyzed strains. Two strains proved the ability to induce apoptosis of HeLa cells, inducing an increased expression of caspase 3 and Bax genes, correlated with a decreased level of the anti-apoptotic factor Bcl-2. The apoptotic gene expression levels were also analyzed for HeLa cell treated with cell free culture supernatants, responsible for decreased expression levels of caspase 3 and caspase 9 genes. The results corroborate well with other reported data, proving the ability of these bacteria to penetrate non-phagocytic cells and to induce changes at molecular level, including apoptosis. The cell free culture supernatants did not demonstrate the ability to induce apoptosis by caspase – mediated pathways, leading us to the hypothesis that the host cells apoptosis is requiring cell to cell contact, probably implicating the activation of a type III secretion system responsible for the intracellular release of pro-apoptotic factors.

In vitro modulation of adherence and invasion ability of enteroinvasive escherichia coli by different viruses.

Studies on the relationship between viral and bacterial infections showed that in the context of viral infections the immunity of host organism is reduced temporarily, increasing the incidence of bacterial infections, like faster bacterial colonization of immunocompromised bodies, by increasing the level of expression of epithelial cell receptor for bacterial adesins. Modulation of viruses infected host cells signaling may also induce changes in the cytoscheleton, which may result in the increase / decrease invasion capacity of bacterial cells. Enteroinvasive Escherichia coli causes intestinal infections exploiting host cell function, which include the invasion into non-phagocytic eukaryotic cells such as epithelial and endothelial cells and associated host cell actin cytoskeletal rearrangements. One of our aims was to investigate the in vitro adherence and invasion capacity induced by an diarhhoeal enteroinvasive Escherichia coli strain in the presence of different viral strains: Vaccinia virus (Poxviridae), measles virus (Paramyxoviridae II); echovirus 32 (Picornaviridae) and Herpes simplex virus 1 (Herpesviridae). The viral adsorption on HeLa cells was done for six hours at 370C, followed by the evaluation of bacterial adherence and invasion to HeLa cells performed by the adapted Cravioto’s method and gentamycin protection assay. Viral preinfection of the cellular substrate induced an increased bacterial adherence index, as well as changes in the adherence pattern from diffuse tu aggregative. In exchange, the general effect of viral infection on invasive bacterial capacity was the decrease of invasive ability. In conclusion, viral preinfection of the susceptible substrate influenced the adherence and invasion ability of enteroinvasive E. coli bacterial strain, as observed by the intensification of the adherence capacity, explaining the increased incidence of bacterial infections after viral infections, as well as faster bacterial colonization of immunocompromised hosts and by reducing the invasive capacity of epithelial cell by bacterial strains, pleading for increased incidence of extracellular pathogenic organisms in post-viral infections.

The effects of heat and osmotic stress on vibrio parahaemolyticus survival rates, cellular morphology and TDH production.

Vibrio parahaemolyticus is an emerging enteric pathogen often associated with the consumption of improperly cooked seafood. The major virulence factor of this pathogen is represented by the termostable direct hemolysin (TDH). When entering the host organism, the vibrion has to face a series of environmental changes (such as temperature and osmolarity) which determine various cellular responses among which the heat shock response has an important role. The purpose of this study was to analyze the survival profiles, the morphological changes and the TDH production in V. parahaemolyticus strains grown under sub-lethal heat and osmotic stress. Our study has demonstrated the thermolotolerance in V. parahaemolyticus strains and the absence of cross-protection between the osmotic and heat stress. We have also observed that the osmotic stress induced important changes to the bacterial cell morphology. The production of the thermostable direct hemolysin, the major virulence factor in V. parahaemolyticus, was increased in cells remained viable after thermal inactivation at 47oC, these results demonstrating the risk of virulence conservation in insufficiently heat treated food.