Proteomic profiling of Serratia marcescens by high-resolution mass spectrometry.

Introduction: Serratia marcescens, an opportunistic human pathogen, is reported as an important cause of nosocomial infection and outbreaks. Although the genome of S. marcescens (ATCC 13880) was completely sequenced by 2014, there are no studies on the proteomic profile of the organism. The objective of the present study is to analyze the protein profile of S. marcescens (ATCC 13880) using a high resolution mass spectrometry (MS). Methods: Serratia marcescens ATCC 13880 strain was grown in Luria-Bertani broth and the protein extracted was subjected to trypsin digestion, followed by basic reverse phase liquid chromatography fractionation. The peptide fractions were then analysed using Orbitrap Fusion Mass Spectrometry and the raw MS data were processed in Proteome Discoverer software. Results: The proteomic analysis identified 15 009 unique peptides mapping to 2541 unique protein groups, which corresponds to approximately 54% of the computationally predicted protein-coding genes. Bioinformatic analysis of these identified proteins showed their involvement in biological processes such as cell wall organization, chaperone-mediated protein folding and ATP binding. Pathway analysis revealed that some of these proteins are associated with bacterial chemotaxis and beta-lactam resistance pathway. Conclusion: To the best of our knowledge, this is the first high-throughput proteomics study of S. marcescens (ATCC 13880). These novel observations provide a crucial baseline molecular profile of the S. marcescens proteome which will prove to be helpful for the future research in understanding the host-pathogen interactions during infection, elucidating the mechanism of multidrug resistance, and developing novel diagnostic markers or vaccine for the disease.

Structural insights of metallo-beta-lactamase revealed an effective way of inhibition of enzyme by natural inhibitors.

Metallo-beta-lactamase (MBL) is a class of enzyme that catalyzes the hydrolysis of a broad range of beta-lactam antibiotics leading to the development of drug resistance in bacteria. Inhibition of MBL is therefore pursued as a potential way to increase the susceptibility of bacteria to beta-lactam antibiotics. In this study, MBL inhibitors from natural sources such as Eupalitin, Rosmarinic acid and Luteolin are used as a potential alternative to explore their effect. The crystal structure of MBL revealed a hydrolyzed Meropenem, which was undocked from the active center pocket to get the apo-protein. The apo-protein was re-docked with substrate, three known MBL inhibitors and natural compounds to prepare the starting structure in the current work and to draw conclusions. Further, to explore the efficiency of natural inhibitors, we analyzed the dynamic behavior of the enzyme over simulation time using molecular dynamics studies. Our results suggest that MBL enzyme adopted altered conformational state in the presence of natural inhibitor. This is because, the natural inhibitors were tried to occupy a different binding pocket in the enzyme by causing positional drift from the active center pocket. Here, the different binding pocket partly comprised of active site pocket and partly by a new region explored by ligand, making it inappropriate for substrate to occupy the active site. Thus natural inhibitors may be potential entities to target MBL. AbbreviationsADMEAbsorption, Distribution, Metabolism and ExcretionBBBBlood brain barrierCHARMMChemistry at Harvard Macromolecular MechanicsCOMCenter of MassCYP2D6Cytochrome P450 2D6DSDiscovery StudioESBLExtended Spectrum Beta-lactamasesFDAFood and Drug AdministrationGLASSGlobal antimicrobial resistance surveillance systemGROMACSGROningen MAchine for Chemical SimulationsKDEKernel Density Estimation PlotsMBLMetallo-beta-lactamaseMBL-CMetallo-beta-lactamase bound to L-CaptoprilMBL-EMetallo-beta -lactamase bound to EupalitinMBL-IMetallo-beta -lactamase bound to ImipenemMBL-LMetallo-beta -lactamase bound to LuteolinMBL-RMetallo-beta -lactamase bound to Rosmarinic acidMDMolecular DynamicsMMPBSAMolecular Mechanics Poisson - Boltzmann surface areaNPTNumber of atoms in the system, Pressure of the system and Temperature of the systemnsNano secondsNVTNumber of atoms in the system, Volume of the system, and Temperature of the systemPDBProtein Data BankRgRadius of GyrationRMSDRoot Mean Square DeviationRMSFRoot Mean Square FluctuationSASASolvent Accessible Surface AreaSPC/ESimple Point ChargeWHOWorld Health OrganizationCommunicated by Ramaswamy H. Sarma.