In vitro and in vivo screening for novel essential cell-envelope proteins in Pseudomonas aeruginosa.

Regina Fernández-Piñar,Francesco Imperi,Alice Rossi,Alessandra Bragonzi,Serena Ranucci,Alessandra Lo Sciuto

doi:10.1038/srep17593

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.

Highlights

LPS transport protein LptA Conserved hypothetical protein localization of this putative lipoprotein was predicted by ref. 30
With the aim of characterizing novel P. aeruginosa proteins involved in cell envelope biogenesis and/or homeostasis, we focused our attention on eight genes that were previously proposed to be essential due to the Genotype and/or relevant characteristics
The recent discovery of new promising antimicrobials with a completely new mechanisms of action[22,23] indicates that we have only explored a fraction of the microbial targets that could be used for antibiotic drug discovery

Summary

Introduction

LPS transport protein LptA Conserved hypothetical protein localization of this putative lipoprotein was predicted by ref. 30. A probabilistic calculation of 60,000 random insertions over 6.5 megabases (i.e. the average size of the P. aeruginosa genome) suggested that a gene 327 bp in length should have a 95% probability of being disrupted by random transposon insertion[15], indicating that undisrupted genes larger than 327 bp are good candidates as potential essential genes Based on this information, we retrieved eight candidate essential genes of P. aeruginosa (undisrupted in the two transposon mutagenesis studies described above) that (i) are longer than 327 bp, (ii) have not previously been confirmed as essential genes in P. aeruginosa, (iii) are present in all P. aeruginosa genomes sequenced so far, and (iv) encode proteins with a predicted cell-envelope localization (Table 1). The role of the selected proteins in P. aeruginosa growth in vitro, cell envelope stability and pathogenicity in different animal models of infection was investigated in order to evaluate their suitability as potential targets for the development of novel anti-P. aeruginosa drugs

Methods

Results

Conclusion