Research topics

The Polissi Lab is interested in the problem of antibiotic resistance. To develop new approaches to treat resistant bacterial infections, we focus on the mechanisms underlying the assembly of the multilayered envelope of Gram-negative bacteria, an essential structure and a virulence determinant in pathogens. In particular research is focused on the Lpt protein machine that assembles lipopolysaccharide (LPS) at the outer membrane, the most external shell of Gram-negative bacteria that protects them from many toxic molecules including antibiotics. We have identified the components of the Lpt machine, and we currently elucidating how it functions and cooperates with the protein machineries that orchestrate growth of the underneath peptidoglycan layer.  A long-term goal is to discover molecules that interfere with envelope growth and assembly. 

In this context our specific objectives are:

-      Dissect the functioning of the Lpt protein machine that transport LPS to the outer membrane. LPS molecules are mainly responsible of the impermeability of Gram negative bacteria to antibiotics and are major virulence determinants in pathogens

-      Understand how outer membrane assembly is coordinated with peptidoglycan growth. Unbalance in envelope layers growth is lethal for the cell and therefore is a novel unexploited pathway that can be targeted by antibacterials

-      Develop smart biochemical and genetic screens to identify molecules that interfere with LPS and envelope assembly

 

Techniques (Methodologies)

Our research relies on a wide range of multidisciplinary techniques developed in our laboratory. 

Bacterial genetics
-      Mutant construction by allele replacement or P1 transduction, phenotypic and molecular characterization
-      Suppressor mutant identification and characterization by genetic mapping and whole genome sequencing

Molecular biology
-      Molecular cloning, site directed and random mutagenesis, transformation, gene expression analysis, immunofluorescence imaging, qualitative and quantitative biofilm assays

Biochemistry
-      Protein expression and purification, western blot analysis, protein-protein interaction by immunoprecipitation and co-purification, mapping of protein-protein interaction regions by UV-photocrosslinking, purification and analysis of membrane protein complexes
-      Subcellular fractionation, membrane separation by sucrose density gradient
-      LPS purification and analysis
-      Set up and development of biochemical and genetic screening assays 

 

Collaborators
Dr. Cristina Airoldi, Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Milano
Prof. Giberto Chririco, Dipartimento di Fisica, Università di Milano-Bicocca, Milano
Prof. Tanneke den Blaauwen, Swammerdam Institute for Life Sciences, University of Amsterdam (TheNetherlands)
Prof. Francesca Granucci, Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Milano
Prof. Francesco Imperi, Dipartimento di Scienze e Tecnologie Biomediche, Università di Roma Tre, Roma
Prof. Daniel Kahne, Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA (US)
Prof. Antonio Molinaro, Dipartimento di Scienze Chimiche, Università di Napoli Federico II, Napoli
Prof. Marco Nardini, Dipartimento di Bioscienze, Università di Milano
Dr. Jean Pierre Simorre, Institut de Biologie Structurale (IBS), CNRS, Grenoble (France)
Prof. Waldemar Vollmer, Institute for Cell and Molecular Biosciences, Newcastle University, (UK) 

 

Selected publications

1. Laguri C, Sperandeo P, Pounot K, Ayala I, Silipo A, Bougault CM, Molinaro A, Polissi A, Simorre JP. (2017) Interaction of lipopolysaccharides at intermolecular sites of the periplasmic Lpt transport assembly. Sci Rep. 2017 7:9715 
2. Santus W, Barresi S, Mingozzi F, Broggi A, Orlandi I, Stamerra G, Vai M, Martorana AM, Polissi A, Köhler JR, Liu N, Zanoni I, Granucci F. (2017) Skin infections are eliminated by cooperation of the fibrinolytic and innate immune systems. Sci Immunol. 2. pii: eaan2725.
3. Falchi FA, Maccagni EA, Puccio S, Peano C, De Castro C, Palmigiano A, Garozzo D, Martorana AM, Polissi A, Dehò G, Sperandeo P. (2018) Mutation and suppressor analysis of the essential LPS-transport protein LptA reveals strategies to overcome severe outer membrane permeability defects in Escherichia coli. J Bacteriol. 200: e00487-17.
4. Peters K, Pazos M, HugonnetJ-M, Martorana AM, Polissi A, VanNieuwenhze MS, Arthur M, Vollmer W. (2018) Copper inhibits peptidoglycan LD-transpeptidases suppressing β-lactam resistance due to by-pass of Penicillin-binding proteins. Proc Natl Acad Sci USA 115:10786-10791
5. Morè N, Martorana AM, Biboy J, Otten C, Winkle M, Montón Silva A, Atkinson L, Yau H, Breukink E, den Blaauwen T, Vollmer W, Polissi A. (2019) Peptidoglycan remodeling enables E. colito survive severe outer membrane assembly defect. mBio pii: e02729-18