Riassunti dei progetti del corso di dottorato di ricerca in biologia molecolare e cellulare
Project leader: GIOVANNI BERTONI (2) (giovanni.bertoni@unimi.it)
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- Project leader: MARTINO BOLOGNESI (martino.bolognesi@unimi.it)
- Project leader : FEDERICA BRIANI (federica.briani@unimi.it)
Project leader: GIOVANNI BERTONI (2) (giovanni.bertoni@unimi.it)Location: Department of Biosciences, University of Milan, Italy RESEARCH PROJECT SUMMARYCharacterization of novel essential functions in the opportunistic pathogen Pseudomonas aeruginosa The Gram-negative bacterium Pseudomonas aeruginosa is an important opportunistic pathogen in compromised individuals, such as patients with cystic fibrosis, severe burns or impaired immunity. The proponent lab aimed to screen novel essential functions of P. aeruginosa by shotgun antisense identification, a technique that was developed a decade ago for the Gram-positive bacterium Staphylococcus aureus and was under-used in Gram-negative bacteria for a considerable period of time. This approach in P. aeruginosa generated a panel of about 20 novel essential candidate proteins that are suggested to take part in disparate cellular functions, including protein secretion, biosynthesis of cofactors, prosthetic groups, and carriers, energy metabolism, central intermediary metabolism, transport of small molecules, translation, post-translational modification, non-ribosomal peptide synthesis, lipopolysaccharide synthesis/modification, and transcriptional regulation. The essential role of two of these proteins, TgpA [1] and Gcp, was validated by means of insertional and conditional mutagenesis. The main aim of this research project is to unravel the cellular role of both TgpA and Gcp and identify protein partners. 1. Milani, A., Vecchietti, D., Rusmini, R. and Bertoni, G. (2012) TgpA, a protein with a eukaryotic-like transglutaminase domain, plays a critical role in the viability of Pseudomonas aeruginosa. PLoS ONE 7(11): e50323. Project leader: MARTINO BOLOGNESI (martino.bolognesi@unimi.it)Location: Department of Biosciences, University of Milan, ItalyRESEARCH PROJECT SUMMARYStructure-based epitope discovery from B. pseudomallei antigens for vaccine development Structure-based antigen engineering is frequently used in the vaccine development process to specifically modify protein antigens of a pathogen to enhance their immunogenic properties, with the aim of improving their protective efficacy. Such approaches may entail engineering epitope-containing regions of the protein, or simply the epitope sequences themselves in the form of synthetic peptides. In this context, one of our lines of research regards structure-based epitope discovery, focusing on protein antigens from the Gram negative pathogen Burkholderia pseudomallei, which causes melioidosis, a severely debilitating, and often fatal disease, endemic in the subtropical and tropical regions of the word. Together with computational biologists and immunologists, we have constructed a structural vaccinology pipeline for epitope identification. Selected targets enter into a medium-throughput protein production pipeline involving bioinformatics (recombinant construct design), heterologous expression, purification and 3D structure determination (X-ray crystallography). 3D antigen structures form the basis for the application of in silico-based epitope predictions, combined with experimental validation and immunological testing. Our structural vaccinology network includes both national groups and international labs in the UK (immunologists), Spain (computational biologists) and Thailand (immunologists). The latter group carries out sera recognition tests with immune sera from melioidosis patients, to validate the reactivity of selected antigens and epitopes. Overall, we aim to connect the understanding of structural properties at atomic resolution, to the reactivity properties of the protein (or specific epitopes) in an immunological context, with the scope of identifying candidate epitopes to be considered in a potential vaccine. The success of our pipeline has been demonstrated for two known B. pseudomallei antigens (1, 2). Based on their crystal structures, consensus epitopes were successfully identified and, when synthesized as free peptides, were shown to possess interesting immunological properties in comparison with their full-length recombinant counterparts. Antibodies raised against one of the most reactive epitope peptides are presently being tested in passive immunization tests in mice. The proposed project forsees the use of molecular biology, protein biochemistry and structural biology techniques, for the design, cloning and heterologous expression of protein antigens as recombinant fusion proteins, biophysical analyses (dynamic light scattering, thermofluorimetry), protein crystallization screening and 3D structure determination. Diffraction data are collected at the European Synchrotron Research Facility (ESRF, Grenoble, France) on a routine basis. Subsequent computational data elaboration and structural determination is carried out in-house. Generated 3D structures will serve as the starting point for entry into the above-mentioned structural vaccinology pipeline. 1. Gourlay LJ, et al. Exploiting the Burkholderia pseudomallei Acute Phase Antigen BPSL2765 for Structure-Based Epitope Discovery/Design in Structural Vaccinology. (2013) Chem. Biol. 20, 1147-56. 2. Lassaux P, et al. A structure-based strategy for epitope discovery in Burkholderia pseudomallei OppA antigen. (2013) Structure. 21, 167-75. Project leader: FEDERICA BRIANI (federica.briani@unimi.it)Location: Department of Biosciences, University of Milan, ItalyRESEARCH PROJECT SUMMARYLooking for novel Pseudomonas aeruginosa inhibitors: S1 ribosomal protein as an unexploited target for new antibacterial molecules P. aeruginosa is a Gram negative, mesophilic bacterium, endowed with a noteworthy metabolic versatility reflected by a large genome. It can infect hosts as diverse as worms, flies and mammals. In humans it behaves as an opportunistic pathogen and it is responsible for a variety of serious nosocomial infections. Moreover, P. aeruginosa is the most common pathogen found in the lung of cystic fibrosis patients and the primary factor in pulmonary pathology. The diffusion of isolates of P. aeruginosa (and other pathogenic bacteria) multi-resistant to extant antibiotics makes urgently needed the development of new antibacterial molecules that may escape bacterial resistance. A strategy that can be applied to search for new antibiotics is to tackle factors participating in essential cellular processes. We propose to explore P. aeruginosa ribosomal protein S1 as a potential target for new antibacterials. S1 is a ribosomal protein widely conserved among Gram negative bacteria and absent in mammals. In vivo this protein is required for translation of most E. coli mRNAs. Conversely, the protein is dispensable for initiation complex formation on leaderless mRNAs (1,2,3). The general objective of this research will be pursued through the following activities: 1. Assessing S1 essentiality in P. aeruginosa Genes encoding functions essential for cell survival or pathogenesis may represent good antibiotics target. It is likely that rpsA is an essential gene in P. aeruginosa as it is in E. coli and M. tuberculosis, but a formal demonstration is lacking. We will aim at this objective by constructing a conditional allele of rpsA. If conversely, the gene will result to be non-essential, we will perform phenotypic analyses of the mutant both in vitro and in Galleria mellonella larvae infection model. 2. Identification and characterization of S1 and translation initiation inhibitors We have developed a whole-cell fluorescent screening for specific inhibitors of S1-dependent translation. The theoretical bases of our screening rest on differential S1 requirement exhibited by different transcripts in E. coli. This screening will be used to test a large collection of chemical compounds already available in our lab. Specific inhibitors of S1-dependent translation will be further characterized to elucidate their mechanism of action and their effect on P. aeruginosa.
2.Tedin,K., Resch,A. and Bläsi,U. (1997) Requirements for ribosomal protein S1 for translation initiation of mRNAs with and without a 5' leader sequence. Mol.Microbiol., 25, 189-199. 3. Delvillani,F., Papiani,G., Dehò,G. and Briani,F. (2011) S1 ribosomal protein and the interplay between translation and mRNA decay. Nucleic Acids Res., 39, 7702-7715.
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