Evaluation of 3D cell culture systems for host-pathogen interaction studies

Traditional cell culture models have limitations in extrapolating functional mechanisms that underlie strategies of microbial virulence. Indeed during the infection the pathogens adapt to different tissue-specific environmental factors. The development of in vitro models resembling human tissue physiology might allow the replacement of inaccurate or aberrant animal models. Three-dimensional (3D) cell culture systems are more reliable and more predictive models that can be used for the meaningful dissection of host–pathogen interactions. The lung and gut mucosae often represent the first site of exposure to pathogens and provide a physical barrier against their entry. Within this context, the tracheobronchial and small intestine tract were modelled by tissue engineering approach. The main work was focused on the development and the extensive characterization of a human organotypic airway model, based on a mechanically supported co-culture of normal primary cells. The regained morphological features, the retrieved environmental factors and the presence of specific epithelial subsets resembled the native tissue organization. In addition, the respiratory model enabled the modular insertion of interesting cell types, such as innate immune cells or multipotent stromal cells, showing a functional ability to release pertinent cytokines differentially. Furthermore this model responded imitating known events occurring during the infection by Non-typeable H. influenzae. Epithelial organoid models, mimicking the small intestine tract, were used for a different explorative analysis of tissue-toxicity. Further experiments led to detection of a cell population targeted by C. difficile Toxin A and suggested a role in the impairment of the epithelial homeostasis by the bacterial virulence machinery. The described cell-centered strategy can afford critical insights in the evaluation of the host defence and pathogenic mechanisms. The application of these two models may provide an informing step that more coherently defines relevant molecular interactions happening during the infection.

Apple latent infection caused by Neofabraea alba: host-pathogen interaction and disease management

Apple latent infection caused by Neofabraea alba: host-pathogen interaction and disease management Bull’s eye rot (BER) caused by Neofabraea alba is one of the most frequent and damaging latent infection occurring in stored pome fruits worldwide. Fruit infection occurs in the orchard, but disease symptoms appear only 3 months after harvest, during refrigerated storage. In Italy BER is particularly serious for late harvest apple cultivar as ‘Pink Lady™’. The purposes of this thesis were: i) Evaluate the influence of ‘Pink Lady™’ apple primary metabolites in N. alba quiescence ii) Evaluate the influence of pH in five different apple cultivars on BER susceptibility iii) To find out not chemical method to control N. alba infection iv) Identify some fungal volatile compounds in order to use them as N. alba infections markers. Results regarding the role of primary metabolites showed that chlorogenic, quinic and malic acid inhibit N. alba development. The study based on the evaluation of cultivar susceptibility, showed that Granny Smith was the most resistant apple cultivar among the varieties analyzed. Moreover, Granny Smith showed the lowest pH value from harvest until the end of storage, supporting the thesis that ambient pH could be involved in the interaction between N. alba and apple. In order to find out new technologies able to improve lenticel rot management, the application of a non-destructive device for the determination of chlorophyll content was applied. Results showed that fruit with higher chlorophyll content are less susceptible to BER, and molecular analyses comforted this result. Fruits with higher chlorophyll content showed up-regulation of PGIP and HCT, genes involved in plant defence. Through the application of PTR-MS and SPME GC-MS, 25 volatile organic compounds emitted by N. alba were identified. Among them, 16 molecules were identified as potential biomarkers.

Cell Host-Microbe Interactions: Turning Pathogen Mechanisms Into Cell's Advantages

Host-Pathogen Interaction is a very vast field of biological sciences, indeed every year many un- known pathogens are uncovered leading to an exponential growth of this field. The present work lyes between its boundaries, touching different aspects of host-pathogen interaction: We have evaluate the permissiveness of Mesenchimal Stem cell (FM-MSC from now on) to all known human affecting herpesvirus. Our study demonstrate that FM-MSC are full permissive to HSV1, HSV2, HCMV and VZV. On the other hand HHV6, HHV7, EBV and HHV8 are susceptible, but failed to activate a lytic infection program. FM-MSC are pluripotent stem cell and have been studied intensely in last decade. FM-MSC are employed in some clinical applications. For this reason it is important to known the degree of susceptibility to transmittable pathogens. Our atten- tion has then moved to bacterial pathogens: we have performed a proteome-wide in silico analy- sis of Chlamydiaceae family, searching for putative Nuclear localization Signal (NLS). Chlamy- diaceae are a family of obligate intracellular parasites. It’s reasonably to think that its members could delivered to nucleus effector proteins via NLS sequences: if that were the case the identifi- cation of NLS carrying proteins could open the way to therapeutic approaches. Our results strengthen this hypothesis: we have identified 72 protein bearing NLS, and verified their func- tionality with in vivo assays. Finally we have conceived a molecular scissor, creating a fusion protein between HIV-1 IN protein and FokI catalytic domain (a deoxyexonuclease domain). Our aim is to obtain chimeric enzyme (trojIN) which selectively identify IN naturally occurring target (HIV LTR sites) and cleaves subsequently LTR carrying DNA (for example integrated HIV1 DNA). Our preliminary results are promising since we have identified trojIN mutated version capable to selectively recognize LTR carrying DNA in an in vitro experiments.

Exploring host-pathogen interactions through protein microarray. Large-scale protein microarray analysis revealed novel human receptors for the staphylococcal immune evasion protein FLIPr and for the neisserial adhesin NadA

Adhesion, immune evasion and invasion are key determinants during bacterial pathogenesis. Pathogenic bacteria possess a wide variety of surface exposed and secreted proteins which allow them to adhere to tissues, escape the immune system and spread throughout the human body. Therefore, extensive contacts between the human and the bacterial extracellular proteomes take place at the host-pathogen interface at the protein level. Recent researches emphasized the importance of a global and deeper understanding of the molecular mechanisms which underlie bacterial immune evasion and pathogenesis. Through the use of a large-scale, unbiased, protein microarray-based approach and of wide libraries of human and bacterial purified proteins, novel host-pathogen interactions were identified. This approach was first applied to Staphylococcus aureus, cause of a wide variety of diseases ranging from skin infections to endocarditis and sepsis. The screening led to the identification of several novel interactions between the human and the S. aureus extracellular proteomes. The interaction between the S. aureus immune evasion protein FLIPr (formyl-peptide receptor like-1 inhibitory protein) and the human complement component C1q, key players of the offense-defense fighting, was characterized using label-free techniques and functional assays. The same approach was also applied to Neisseria meningitidis, major cause of bacterial meningitis and fulminant sepsis worldwide. The screening led to the identification of several potential human receptors for the neisserial adhesin A (NadA), an important adhesion protein and key determinant of meningococcal interactions with the human host at various stages. The interaction between NadA and human LOX-1 (low-density oxidized lipoprotein receptor) was confirmed using label-free technologies and cell binding experiments in vitro. Taken together, these two examples provided concrete insights into S. aureus and N. meningitidis pathogenesis, and identified protein microarray coupled with appropriate validation methodologies as a powerful large scale tool for host-pathogen interactions studies.

Analysis of the immunological and functional features of the Neisserial Heparin Binding Antigen (NHBA)

Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein ubiquitously expressed by genetically diverse Neisseria meningitidis strains and is an antigen of the multicomponent protein-based 4CMenB vaccine, able to induce bactericidal antibodies in humans and to bind heparin-like molecules. The aim of this study is to characterize the immunological and functional properties of NHBA. To evaluate immunogenicity and the contribution of aminoacid sequence variability to vaccine coverage, we constructed recombinant isogenic strains that are susceptible to bactericidal killing only by anti-NHBA antibodies and engineered them to express equal levels of selected NHBA peptides. In these recombinant strains, we observed different titres associated with the different peptide variants. These recombinant strains were then further engineered to express NHBA chimeric proteins to investigate the regions important for immunogenicity. In natural strains, anti-NHBA antibodies were found to be cross-protective against strains expressing different peptides. To investigate the functional properties of this antigen, the recombinant purified NHBA protein was tested in in vitro binding studies and was found to be able to bind epithelial cells. The binding was abolished when cells were treated specifically with heparinase III, suggesting that the interaction with the cells is mediated by heparan sulfate proteoglycans (HSPG). Mutation of the Arg-rich tract of NHBA abrogated the binding, confirming the importance of this region in mediating the binding to heparin-like molecules. In a panel of N. meningitidis strains, the deletion of nhba resulted in a reduction of adhesion with respect to each isogenic wild type strain. Furthermore, the adhesion of the wild-type strain was prevented by using anti-NHBA polyclonal sera, demonstrating the specificity of the interaction. These results suggest that NHBA could be a novel meningococcal adhesin contributing to host-cell interaction. Moreover, we analysed NHBA NalP-mediated cleavage in different NHBA peptides and showed that not all NHBA peptides are cleaved.

Influence of plant structure, cultural pratices and environmental conditions on the development of the bacterial canker of kiwifruits

Italy has a preeminent rank in kiwifruit industry, being the first exporter and the second largest producer after China. However, in the last years kiwifruit yields and the total cultivated area considerably decreased, due to the pandemic spread of the bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa). Several climatic conditions and cultural practices affect the development of the bacterial canker. This research work focused on the impact of agricultural practices and microclimate conditions on the incidence and epidemiology of Psa in the orchard. Therefore, the effect of fertilization, irrigation, use of bio-regulators, rootstock, training system and pruning were examined. The effect of different tunnel systems was analyzed as well, to study the plant-pathogen interaction. Considering the importance of insects as vectors in other pathosystems, the role of Metcalfa pruinosa in the spread of the bacterial canker was investigated in controlled conditions. In addition, quality and storage properties of fruits from infected plants were assessed. The study of all these aspects of the agronomic practices is useful to define a strategy to limit the bacterial diffusion in the orchard. Overall, excess nitrogen fertilization, water stress, stagnant water supplies, pruning before summer and the high number of Metcalfa pruinosa increased the Psa incidence. In contrast, tunnel covers may be useful for the control of the disease, with special attention to the kind of material.

Bifidobacterium - Human Host Interaction: Role of Human Plasminogen

Bifidobacterium is an important genus of the human gastrointestinal microbiota, affecting several host physiological features. Despite the numerous Bifidobacterium related health-promoting activities, there is still a dearth of information about the molecular mechanisms at the basis of the interaction between this microorganism and the host. Bacterial surface associated proteins may play an important role in this interaction because of their ability to intervene with host molecules, as recently reported for the host protein plasminogen. Plasminogen is the zymogen of the trypsin-like serine protease plasmin, an enzyme with a broad substrate specificity. Aim of this thesis is to deepen the knowledge about the interaction between Bifidobacterium and the human plasminogen system and its role in the Bifidobacterium-host interaction process. As a bifidobacterial model, B. animalis subsp. lactis BI07 has been used because of its large usage in dairy and pharmaceutical preparations. We started from the molecular characterization of the interaction between plasminogen and one bifidobacterial plasminogen receptor, DnaK, a cell wall protein showing high affinity for plasminogen, and went on with the study of the impact of intestinal environmental factors, such as bile salts and inflammation, on the plasminogen-mediated Bifidobacterium-host interaction. According to our in vitro findings, by enhancing the activation of the bifidobacterial bound plasminogen to plasmin, the host inflammatory response results in the decrease of the bifidobacterial adhesion to the host enterocytes, favouring bacterial migration to the luminal compartment. Conversely, in the absence of inflammation, plasminogen acts as a molecular bridge between host enterocytes and bifidobacteria, enhancing Bifidobacterium adhesion. Furthermore, adaptation to physiological concentrations of bile salts enhances the capability of this microorganism to interact with the host plasminogen system. The host plasminogen system thus represents an important and flexible tool used by bifidobacteria in the cross-talk with the host.

Studies on biotic and abiotic elicitors inducing defense responses in tomato

Tomato (Lycopersicon esculentum Mill., Solanum lycopersicon L.) is one of the most popular vegetable throughout the world, and the importance of its cultivation is threatened by a wide array of pathogens. In the last twenty years this plant has been successfully used as a model plant to investigate the induction of defense pathways after exposure to fungal, bacterial and abiotic molecules, showing triggering of different mechanisms of resistance. Understanding these mechanisms in order to improve crop protection is a main goal for Plant Pathology. The aim of this study was to search for general or race-specific molecules able to determine in Solanum lycopersicon immune responses attributable to the main systems of plant defense: non-host, host-specific and induced resistance. Exopolysaccharides extracted by three fungal species (Aureobasidium pullulans, Cryphonectria parasitica and Epicoccum purpurascens), were able to induce transcription of pathogenesis-related (PR) proteins and accumulation of enzymes related to defense in tomato plants cv Money Maker,using the chemical inducer Bion® as a positive control. During the thesis, several Pseudomonas spp. strains were also isolated and tested for their antimicrobial activity and ability to produce antibiotics. Using as a positive control jasmonic acid, one of the selected strain was shown to induce a form of systemic resistance in tomato. Transcription of PRs and reduction of disease severity against the leaf pathogen Pseduomonas syringae pv. tomato was determined in tomato plants cv Money Maker and cv Perfect Peel, ensuring no direct contact between the selected rhizobacteria and the aerial part of the plant. To conclude this work, race-specific resistance of tomato against the leaf mold Cladosporium fulvum is also deepened, describing the project followed at the Phytopathology Laboratory of Wageningen (NL) in 2007, dealing with localization of a specific R-Avr interaction in transfected tomato protoplast cultures through fluorescence microscopy.

Rapporti fra fattori ambientali e proteine di parete in Bifidobacterium

The normal gut microbiota has several important functions in host physiology and metabolism, and plays a key role in health and disease. Bifidobacteria, which are indigenous components of gastrointestinal microbiota, may play an important role in maintaining the well-being of the host although its precise function is very difficult to study. Its physiological and biochemical activities are controlled by many factors, particularly diet and environment. Adherence and colonization capacity are considered as contributing factors for immune modulation, pathogen exclusion, and enhanced contact with the mucosa. In this way, bifidobacteria would fortify the microbiota that forms an integral part of the mucosal barrier and colonization resistance against pathogens. Bifidobacteria are not only subjected to stressful conditions in industrial processes, but also in nature, where the ability to respond quickly to stress is essential for survival. Bifidobacteria, like other microorganisms, have evolved sensing systems for/and defences against stress that allow them to withstand harsh conditions and sudden environmental changes. Bacterial stress responses rely on the coordinated expression of genes that alter various cellular processes and structures (e.g. DNA metabolism, housekeeping genes, cell-wall proteins, membrane composition) and act in concert to improve bacterial stress tolerance. The integration of these stress responses is accomplished by regulatory networks that allow the cell to react rapidly to various and sometimes complex environmental changes. This work examined the effect of important stressful conditions, such as changing pH and osmolarity, on the biosynthesis of cell wall proteins in B. pseudolongum subsp. globosum. These environmental factors all influence heavily the expression of BIFOP (BIFidobacterial Outer Proteins) in the cell-wall and can have an impact in the interaction with host. Also evidence has been collected linking the low concentration of sugar in the culture medium with the presence or absence of extracromosomal DNA.

Reverse genetic studies of Benyvirus - Polymyxa betae molecular interaction: Role of the RNA4-encoded protein in virus transmission

Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, is included within viruses transmitted through the soil from plasmodiophorid as Polymyxa betae. BNYVV is the causal agent of Rhizomania, which induces abnormal rootlet proliferation and is widespread in the sugar beet growing areas in Europe, Asia and America; for review see (Peltier et al., 2008). In this latter continent, Beet soil-borne mosaic virus (BSBMV) has been identified (Lee et al., 2001) and belongs to the benyvirus genus together with BNYVV, both vectored by P. betae. BSBMV is widely distributed only in the United States and it has not been reported yet in others countries. It was first identified in Texas as a sugar beet virus morphologically similar but serologically distinct to BNYVV. Subsequent sequence analysis of BSBMV RNAs evidenced similar genomic organization to that of BNYVV but sufficient molecular differences to distinct BSBMV and BNYVV in two different species (Rush et al., 2003). Benyviruses field isolates usually consist of four RNA species but some BNYVV isolates contain a fifth RNA. RNAs -1 contains a single long ORF encoding polypeptide that shares amino acid homology with known viral RNA-dependent RNA polymerases (RdRp) and helicases. RNAs -2 contains six ORFs: capsid protein (CP), one readthrough protein, triple gene block proteins (TGB) that are required for cell-to-cell virus movement and the sixth 14 kDa ORF is a post-translation gene silencing suppressor. RNAs -3 is involved on disease symptoms and is essential for virus systemic movement. BSBMV RNA-3 can be trans-replicated, trans-encapsidated by the BNYVV helper strain (RNA-1 and -2) (Ratti et al., 2009). BNYVV RNA-4 encoded one 31 kDa protein and is essential for vector interactions and virus transmission by P. betae (Rahim et al., 2007). BNYVV RNA-5 encoded 26 kDa protein that improve virus infections and accumulation in the hosts. We are interest on BSBMV effect on Rhizomania studies using powerful tools as full-length infectious cDNA clones. B-type full-length infectious cDNA clones are available (Quillet et al., 1989) as well as A/P-type RNA-3, -4 and -5 from BNYVV (unpublished). A-type BNYVV full-length clones are also available, but RNA-1 cDNA clone still need to be modified. During the PhD program, we start production of BSBMV full-length cDNA clones and we investigate molecular interactions between plant and Benyviruses exploiting biological, epidemiological and molecular similarities/divergences between BSBMV and BNYVV. During my PhD researchrs we obtained full length infectious cDNA clones of BSBMV RNA-1 and -2 and we demonstrate that they transcripts are replicated and packaged in planta and able to substitute BNYVV RNA-1 or RNA-2 in a chimeric viral progeny (BSBMV RNA-1 + BNYVV RNA-2 or BNYVV RNA-1 + BSBMV RNA-2). During BSBMV full-length cDNA clones production, unexpected 1,730 nts long form of BSBMV RNA-4 has been detected from sugar beet roots grown on BSBMV infected soil. Sequence analysis of the new BSBMV RNA-4 form revealed high identity (~100%) with published version of BSBMV RNA-4 sequence (NC_003508) between nucleotides 1-608 and 1,138-1,730, however the new form shows 528 additionally nucleotides between positions 608-1,138 (FJ424610). Two putative ORFs has been identified, the first one (nucleotides 383 to 1,234), encode a protein with predicted mass of 32 kDa (p32) and the second one (nucleotides 885 to 1,244) express an expected product of 13 kDa (p13). As for BSBMV RNA-3 (Ratti et al., 2009), full-length BSBMV RNA-4 cDNA clone permitted to obtain infectious transcripts that BNYVV viral machinery (Stras12) is able to replicate and to encapsidate in planta. Moreover, we demonstrated that BSBMV RNA-4 can substitute BNYVV RNA-4 for an efficient transmission through the vector P. betae in Beta vulgaris plants, demonstrating a very high correlation between BNYVV and BSBMV. At the same time, using BNYVV helper strain, we studied BSBMV RNA-4’s protein expression in planta. We associated a local necrotic lesions phenotype to the p32 protein expression onto mechanically inoculated C. quinoa. Flag or GFP-tagged sequences of p32 and p13 have been expressed in viral context, using Rep3 replicons, based on BNYVV RNA-3. Western blot analyses of local lesions contents, using FLAG-specific antibody, revealed a high molecular weight protein, which suggest either a strong interaction of BSBMV RNA4’s protein with host protein(s) or post translational modifications. GFP-fusion sequences permitted the subcellular localization of BSBMV RNA4’s proteins. Moreover we demonstrated the absence of self-activation domains on p32 by yeast two hybrid system approaches. We also confirmed that p32 protein is essential for virus transmission by P. betae using BNYVV helper strain and BNYVV RNA-3 and we investigated its role by the use of different deleted forms of p32 protein. Serial mechanical inoculation of wild-type BSBMV on C. quinoa plants were performed every 7 days. Deleted form of BSBMV RNA-4 (1298 bp) appeared after 14 passages and its sequence analysis shows deletion of 433 nucleotides between positions 611 and 1044 of RNA-4 new form. We demonstrated that this deleted form can’t support transmission by P. betae using BNYVV helper strain and BNYVV RNA-3, moreover we confirmed our hypothesis that BSBMV RNA-4 described by Lee et al. (2001) is a deleted form. Interesting after 21 passages we identifed one chimeric form of BSBMV RNA-4 and BSBMV RNA-3 (1146 bp). Two putative ORFs has been identified on its sequence, the first one (nucleotides 383 to 562), encode a protein with predicted mass of 7 kDa (p7), corresponding to the N-terminal of p32 protein encoded by BSBMV RNA-4; the second one (nucleotides 562 to 789) express an expected product of 9 kDa (p9) corresponding to the C-terminal of p29 encoded by BSBMV RNA-3. Results obtained by our research in this topic opened new research lines that our laboratories will develop in a closely future. In particular BSBMV p32 and its mutated forms will be used to identify factors, as host or vector protein(s), involved in the virus transmission through P. betae. The new results could allow selection or production of sugar beet plants able to prevent virus transmission then able to reduce viral inoculum in the soil.

Biofilm formation by the anaerobic pathogen Clostridium difficile

Clostridium difficile is an obligate anaerobic, Gram-positive, endospore-forming bacterium. Although an opportunistic pathogen, it is one of the important causes of healthcare-associated infections. While toxins TcdA and TcdB are the main virulence factors of C. difficile, the factors or processes involved in gut colonization during infection remain unclear. The biofilm-forming ability of bacterial pathogens has been associated with increased antibiotic resistance and chronic recurrent infections. Little is known about biofilm formation by anaerobic gut species. Biofilm formation by C. difficile could play a role in virulence and persistence of C. difficile, as seen for other intestinal pathogens. We demonstrate that C. difficile clinical strains, 630, and the strain isolated in the outbreak, R20291, form structured biofilms in vitro. Biofilm matrix is made of proteins, DNA and polysaccharide. Strain R20291 accumulates substantially more biofilm. Employing isogenic mutants, we show that virulence-associated proteins, Cwp84, flagella and a putative quorum sensing regulator, LuxS, Spo0A, are required for maximal biofilm formation by C. difficile. Moreover we demonstrate that bacteria in C. difficile biofilms are more resistant to high concentrations of vancomycin, a drug commonly used for treatment of CDI, and that inhibitory and sub-inhibitory concentrations of the same antibiotic induce biofilm formation. Surprisingly, clinical C. difficile strains from the same out-break, but from different origin, show differences in biofilm formation. Genome sequence analysis of these strains showed presence of a single nucleoide polymorphism (SNP) in the anti-σ factor RsbW, which regulates the stress-induced alternative sigma factor B (σB). We further demonstrate that RsbW, a negative regulator of alternative sigma factor B, has a role in biofilm formation and sporulation of C. difficile. Our data suggest that biofilm formation by C. difficile is a complex multifactorial process and may be a crucial mechanism for clostridial persistence in the host.

Patogenesi molecolare delle osteomieliti associate all'impianto

L’osteomielite associata all’impianto è un processo infettivo a carico del tessuto osseo spesso accompagnato dalla distruzione dell’osso stesso. La patogenesi delle osteomieliti associate all’impianto si basa su due concetti fondamentali: l’internalizzazione del patogeno all’interno degli osteoblasti e la capacità dei batteri di formare il biofilm. Entrambi i meccanismi consentono infatti di prevenire l’eliminazione del batterio da parte delle difese immunitarie dell’ospite e di ostacolare l’azione della maggior parte degli antibiotici (che non penetrano e non agiscono pertanto su microrganismi intracellulari), così sostenendo ed alimentando l’infezione. Il saggio di invasione messo a punto su micropiastra ha consentito di investigare in modo approfondito e dettagliato il ruolo ed il peso dell’internalizzazione nella patogenesi delle infezioni ortopediche peri-protesiche causate da S. aureus, S. epidermidis, S. lugdunensis ed E. faecalis. Lo studio ha evidenziato che l’invasione delle cellule MG-63 non rappresenta un meccanismo patogenetico delle infezioni ortopediche associate all’impianto causate da S. epidermidis, S. lugdunensis ed E. faecalis; al contrario, in S. aureus la spiccata capacità invasiva rappresenta un’abile strategia patogenetica che consente al patogeno di sfuggire alla terapia sistemica e alla risposta immunitaria dell’ospite. È stato studiato inoltre il ruolo dell’immunità innata nella difesa contro il biofilm batterico. In seguito all’incubazione del biofilm opsonizzato di S. epidermidis con i PMN è stato possibile osservare la formazione delle NETs. Le NETs rappresentano ottime armi nella difesa contro il biofilm batterico, infatti le trappole sono in grado di limitare la diffusione batterica e quindi di confinare l’infezione. La comprensione del ruolo dell’internalizzazione nella patogenesi delle osteomieliti associate all’impianto e lo studio della risposta immunitaria innata a questo tipo di infezioni, spesso caratterizzate dalla presenza di biofilm, sono presupposti per identificare e affinare le migliori strategie terapeutiche necessarie ad eradicare l'infezione.

Aureobasidium pullulans as biological control agent: modes of action

The postharvest phase has been considered an environment very suitable for successful application of biological control agents (BCAs). However, the tri-interaction between fungal pathogen, host (fruit) and antagonist is influenced by several parameters such as temperature, oxidative stresses, oxygen composition, water activity, etc. that could be determining for the success of biocontrol. Knowledge of the modes of action of BCAs is essential in order to enhance their viability and increase their potentialities in disease control. The thesis focused on the possibility to explain the modes of action of a biological control agent (BCA): Aureobasidium pullulans, in particular the strains L1 and L8, control effective against fruit postharvest fungal pathogen. In particular in this work were studied the different modes of action of BCA, such as: i) the ability to produce volatile organic compounds (VOCs), identified by SPME- gas chromatography-mass spectrometry (GC-MS) and tested by in vitro and in vivo assays against Penicillium spp., Botrytis cinerea, Colletotrichum acutatum; ii) the ability to produce lytic enzymes (exo and endo chitinase and β-1,3-glucanase) tested against Monilinia laxa, causal agent of brown rot of stone fruits. L1 and L8 lytic enzymes were also evaluated through their relative genes by molecular tools; iii) the competition for space and nutrients, such as sugars (sucrose, glucose and fructose) and iron; the latter induced the production of siderophores, molecules with high affinity for iron chelation. A molecular investigation was carried out to better understand the gene regulation strictly correlated to the production of these chelating molucules. The competition for space against M. laxa was verified by electron microscopy techniques; iv) a depth bibliographical analysis on BCAs mechanisms of action and their possible combination with physical and chemical treatments was conducted.