PhD research title: "A kinetic and structural investigation of class A Penicillin Binding Proteins from Pseudomonas aeruginosa and Acinetobacter baumannii to aid the discovery of new antimicrobials"
Antimicrobial resistance (AMR), especially bacterial resistance to antibiotics, has become one of the major threats to global health. Antibiotic misuse and overuse, together with a dramatic decline in antibacterial drug development, have led to a shortage of treatment options for the emerging multidrug-resistant bacteria, particularly the so-called “ESKAPE” pathogens responsible for the majority of nosocomial infections. In addition to regulatory and financial impediments, scientific challenges including lack of tools to dissect the molecular mechanisms of antibiotic resistance, and novel approaches to developing new antimicrobials, have contributed to the current AMR problem. In this project, Penicillin Binding Protein (PBP) 1a from the nosocomial pathogens Pseudomonas aeruginosa and Acinetobacter baumannii, is under structural and kinetic investigation. PBP1a is an important enzyme of the peptidoglycan biosynthetic pathway and a well-validated antibacterial target. To aid the structure-based design of new antimicrobials, structures of PBP1a in complex with known and new compounds supplied by collaborators, will be determined, and a fragment-based screening will be carried out, by X-ray crystallography. Moreover, in-house developed kinetic assays will provide important mechanistic insight into the activities and regulation of PBP1a that may help to better understand its function in vivo, and kinetic data that will complement the structural studies.
Research title: "Investigation of cutinase-like protein 2 gene overexpression effects on cell wall lipids of Mycobacterium smegmatis"
Cutinases are serine esterases whose primary substrate is cutin, the waxy exterior layer of plants. Mycobacterium tuberculosis has maintained seven cutinase-like proteins, but none of them has been found to hydrolyze cutin and, to date, their physiological functions still remain unknown. Because most of the cutinase-like proteins are found either associated with the cell wall or secreted to the medium, it is likely that the natural substrates of these lipases may include either lipids incorporated into the cell wall or environmental lipids encountered by the pathogen inside the host macrophages. The aim of this study was to assess the putative enzymatic activity of cutinase-like protein 2 (CUT2) on mycobacterial cell wall lipids, through overexpression of the gene in Mycobacterium smegmatis cells and analysis of the in vivo effects on cell wall lipids. The homologous gene expressed in M. smegmatis was also included in this investigation and analysed under the same experimental conditions.
Techniques: PCR, cloning, bacterial cell cultures, lipid extraction, TLC.
Research title: "Structural characterization of the putative channel protein ComEC in Gram-positive bacteria"
ComEC has been proposed to be a cytoplasmic membrane channel participating in extracellular DNA uptake in Gram-positive as well as Gram-negative transformable bacteria. While functional and membrane topology studies of ComEC have been reported in literature, mostly performed in Bacillus subtilis, significant structural information is still missing. One of the possible factors responsible for this lacking data might be the challenging determination of the optimal parameters for expression, extraction, and purification of membrane proteins. Knowledge of ComEC structure is highly desired, as it may clarify key mechanistic features of the natural transformation process, which is notoriously implicated in spread of antibiotic resistance and virulence factors, therefore of medical and pharmacological interest. This study was an attempt to express, purify, and crystallize ComEC structural components from Gram-positive bacteria, the ultimate aim being the structural determination by X-ray crystallography.
Techniques: bacterial cell cultures, protein expression, protein purification, protein crystallization.
Research title: "Design of potential Hsp90 and HDAC6 dual inhibitors: virtual screening on HDAC6"
HDAC6 is an attractive target in cancer research because it partecipates in the deacetylation of non-histone regulatory proteins implicated in cancer relevant processes such as Hsp90. HDAC6 and Hsp90 are both validated targets in cancer and they are intimately biologically linked. In fact, since Hsp90 is a substrate of HDAC6, inhibition of HDAC6 leads to the accumulation of acetylated Hsp90, resulting in loss of biological function of the chaperone Hsp90 and loss of its ability to bind client proteins implicated in signal transduction and cell-cycle control. Inhibitors of HDAC6 and inhibitors of Hsp90 are currently in clinical trials. Recent studies have evaluated combination therapies, and showed that a combination of Hsp90 and HDAC6 inhibitors lead to synergistic effects. Because of the strong relationship between Hsp90 and HDAC6 functions and the synergistic effects demonstrated by their respective inhibitors, the discovery of potential dual Hsp90/HDAC6 inhibitors is a highly desirable goal. The aim of this research was the design of dual inhibitors of Hsp90 and HDAC6 by means of virtual screening techniques. A selection of compounds with high scores and favourable active site interactions with both proteins was made. These compounds were tested at the National Cancer Institute (NCI, USA).
Techniques: homology modelling, structure-based virtual screening (docking), post-docking optimization.
Research title: "Effect of an inflammatory stimulus on NMDA receptor subunits mRNA levels in the mouse central nervous system"
Currently, experiments on pain rely on the use of laboratory animal models, mice and rats in particular, that are subjected to nociceptive assays. However, as reported in literature, clear strain differences exist in each assay resulting in warning about the careful choice of the strain as it can significantly affect the final result. For this reason, a greater insight into the effects of genetic background and its interaction with the environment in presence of exogenous substances or adverse circumstances or manipulations is desired, as it could help the pain reserach community in designing future experiments. In this work we assessed the thermal sensitivity of two widely used mouse strains, CD1(outbred) and C57BL/6J (inbred), to a radiant heat source both at basal levels and during an ongoing inflammation by injecting animals either with Complete Freund’s Adjuvant (CFA) or saline in their left hind paw. Moreover, we measured intrastrain differences and CFA induced interstrain effects on the expression of genes with a recognized role in pain and codifying for NMDA receptor subunits NR1, NR2A and NR2B, in the hippocampus and thalamus which are two areas especially involved in modulating pain perception and the central response to inflammation.
Techniques: RNA extraction, retrotranscription, Real-time PCR, statistical analysis.
University of Warwick
School of Biosciences
University of Birmingham
Institute for Research in Biomedicine
Life Sciences Department
University of Modena and Reggio Emilia
Life Sciences Department
University of Modena and Reggio Emilia