Development of polymeric materials to inhibit bacterial quorum sensing

Bacterial infections are an increasing problem for human health. In fact, an increasing number of infections are caused by bacteria that are resistant to most antibiotics and their combinations. Therefore, the scientific community is currently searching for new solutions to fight bacteria and infectious diseases, without promoting antimicrobial resistance. One of the most promising strategies is the disruption or attenuation of bacterial Quorum Sensing (QS), a refined system that bacteria use to communicate. In a QS event, bacteria produce and release specific small chemicals, signal molecules - autoinducers (AIs) - into the environment. At the same time that bacterial population grows, the concentration of AIs in the bacterial environment increases. When a threshold concentration of AIs is reached, bacterial cells respond to it by altering their gene expression profile. AIs regulate gene expression as a function of cell population density. Phenotypes mediated by QS (QSphenotypes) include virulence factors, toxin production, antibiotic resistance and biofilm formation. In this work, two polymeric materials (linear polymers and molecularly imprinted nanoparticles) were developed and their ability to attenuate QS was evaluated. Both types of polymers should to be able to adsorb bacterial signal molecules, limiting their availability in the extracellular environment, with expected disruption of QS. Linear polymers were composed by one of two monomers (itaconic acid and methacrylic acid), which are known to possess strong interactions with the bacterial signal molecules. Molecularly imprinted polymer nanoparticles (MIP NPs) are particles with recognition capabilities for the analyte of interest. This ability is attained by including the target analyte at the synthesis stage. Vibrio fischeri and Aeromonas hydrophila were used as model species for the study. Both the linear polymers and MIP NPs, tested free in solutions and coated to surfaces, showed ability to disrupt QS by decreasing bioluminescence of V. fischeri and biofilm formation of A. hydrophila. No significant effect on bacterial growth was detected. The cytotoxicity of the two types of polymers to a fibroblast-like cell line (Vero cells) was also tested in order to evaluate their safety. The results showed that both the linear polymers and MIP NPs were not cytotoxic in the testing conditions. In conclusion, the results reported in this thesis, show that the polymers developed are a promising strategy to disrupt QS and reduce bacterial infection and resistance. In addition, due to their low toxicity, solubility and easy integration by surface coating, the polymers have potential for applications in scenarios where bacterial infection is a problem: medicine, pharmaceutical, food industry and in agriculture or aquaculture.

Infection mechanism of Diplodia corticola

Diplodia corticola is regarded as the most virulent fungus involved in cork oak decline, being able to infect not only Quercus species (mainly Q. suber and Q. ilex), but also grapevines (Vitis vinifera) and eucalypts (Eucalyptus sp.). This endophytic fungus is also a pathogen whose virulence usually manifests with the onset of plant stress. Considering that the infection normally culminates in host death, there is a growing ecologic and socio-economic concern about D. corticola propagation. The molecular mechanisms of infection are hitherto largely unknown. Accordingly, the aim of this study was to unveil potential virulence effectors implicated in D. corticola infection. This knowledge is fundamental to outline the molecular framework that permits the fungal invasion and proliferation in plant hosts, causing disease. Since the effectors deployed are mostly proteins, we adopted a proteomic approach. We performed in planta pathogenicity tests to select two D. corticola strains with distinct virulence degrees for our studies. Like other filamentous fungi D. corticola secretes protein at low concentrations in vitro in the presence of high levels of polysaccharides, two characteristics that hamper the fungal secretome analysis. Therefore, we first compared several methods of extracellular protein extraction to assess their performance and compatibility with 1D and 2D electrophoretic separation. TCA-Acetone and TCA-phenol protein precipitation were the most efficient methods and the former was adopted for further studies. The proteins were extracted and separated by 2D-PAGE, proteins were digested with trypsin and the resulting peptides were further analysed by MS/MS. Their identification was performed by de novo sequencing and/or MASCOT search. We were able to identify 80 extracellular and 162 intracellular proteins, a milestone for the Botryosphaeriaceae family that contains only one member with the proteome characterized. We also performed an extensive comparative 2D gel analysis to highlight the differentially expressed proteins during the host mimicry. Moreover, we compared the protein profiles of the two strains with different degrees of virulence. In short, we characterized for the first time the secretome and proteome of D. corticola. The obtained results contribute to the elucidation of some aspects of the biology of the fungus. The avirulent strain contains an assortment of proteins that facilitate the adaptation to diverse substrates and the identified proteins suggest that the fungus degrades the host tissues through Fenton reactions. On the other hand, the virulent strain seems to have adapted its secretome to the host characteristics. Furthermore, the results indicate that this strain metabolizes aminobutyric acid, a molecule that might be the triggering factor of the transition from a latent to a pathogenic state. Lastly, the secretome includes potential pathogenicity effectors, such as deuterolysin (peptidase M35) and cerato-platanin, proteins that might play an active role in the phytopathogenic lifestyle of the fungus. Overall, our results suggest that D. corticola has a hemibiotrophic lifestyle, switching from a biotrophic to a necrotrophic interaction after plant physiologic disturbances.This understanding is essential for further development of effective plant protection measures.