title: In vitro and in vivo screening for novel essential cell-envelope proteins in Pseudomonas aeruginosa.
abstract: The Gram-negative bacterium Pseudomonas aeruginosa represents a prototype of multi-drug resistant opportunistic pathogens for which novel therapeutic options are urgently required. In order to identify new candidates as potential drug targets, we combined large-scale transposon mutagenesis data analysis and bioinformatics predictions to retrieve a set of putative essential genes which are conserved in P. aeruginosa and predicted to encode cell envelope or secreted proteins. By generating unmarked deletion or conditional mutants, we confirmed the in vitro essentiality of two periplasmic proteins, LptH and LolA, responsible for lipopolysaccharide and lipoproteins transport to the outer membrane respectively, and confirmed that they are important for cell envelope stability. LptH was also found to be essential for P. aeruginosa ability to cause infection in different animal models. Conversely, LolA-depleted cells appeared only partially impaired in pathogenicity, indicating that this protein likely plays a less relevant role during bacterial infection. Finally, we ruled out any involvement of the other six proteins under investigation in P. aeruginosa growth, cell envelope stability and virulence. Besides proposing LptH as a very promising drug target in P. aeruginosa, this study confirms the importance of in vitro and in vivo validation of potential essential genes identified through random transposon mutagenesis.
authors.affiliation: Laboratory of Molecular Microbiology, Department of Biology and Biotechnology "Charles Darwin", Sapienza University of Rome, Rome, Italy.
authors.forename: Regina
authors.initials: R
authors.lastname: Fernández-Piñar
authors.affiliation: Laboratory of Molecular Microbiology, Department of Biology and Biotechnology "Charles Darwin", Sapienza University of Rome, Rome, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Lo Sciuto
authors.affiliation: Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Alice
authors.initials: A
authors.lastname: Rossi
authors.affiliation: Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Ranucci
authors.affiliation: Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Bragonzi
authors.affiliation: Laboratory of Molecular Microbiology, Department of Biology and Biotechnology "Charles Darwin", Sapienza University of Rome, Rome, Italy.|Pasteur Institute-Cenci Bolognetti Foundation, Sapienza University of Rome, Rome, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Imperi
mesh_terms: D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D004195:Disease Models, Animal; D024901:Drug Resistance, Multiple, Bacterial / Q000502:physiology*; D011552:Pseudomonas Infections / Q000235:genetics / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D037521:Virulence Factors