Less is more: Loss of outer membrane constituents in hyperpermeable Pseudomonas aeruginosa mutants improves resistance to certain antibiotics

Diplôme Thèse Less is more: Loss of outer membrane constituents in hyperpermeable Pseudomonas aeruginosa mutants improves resistance to certain antibiotics eng 2023 2023-04-04 author Trachsel Emilie 804711 Département de microbiologie et médecine moléculaire (MIMOL) director Mattenberger Yves Benoît 502615 Département de microbiologie et médecine moléculaire (MIMOL) director Viollier Patrick 0000-0002-5249-9910 706923 Département de microbiologie et médecine moléculaire (MIMOL) 152 p. 10.13097/archive-ouverte/unige:168429 urn:nbn:ch:unige-1684297 Sc. Vie - Bioméd. 217 Gram-negative bacteria have an envelope composed of three different layers: an inner membrane (IM), a peptidoglycan layer, and an outer membrane (OM). The inner leaflet of the OM is composed of the same types of phospholipids that compose both leaflets of the IM, while the outer leaflet is composed of lipopolysaccharides (LPS). The barrier function of the outer membrane is important to restrict the entry of detergents, hydrophobic antimicrobial agents and charged antibiotics that are capable of crossing a phospholipid bilayer. In E. coli, the biosynthesis of the outer membrane requires the Lpt machinery, which is composed of seven essential proteins (LptABCDEFG). LptD and LptE form a heterodimeric OM translocon with a plug-and barrel architecture, which recieves LPS from LptA and transports it to the OM surface. LptD is a b-barrel OM protein, and its insertion in the OM requires the b-barrel assembly machinery (BAM). The BAM complex is composed of the essential b-barrel OM protein BamA, and five OM lipoproteins, BamB to BamE, of which only BamD is essential. Sampson et al. discovered that in Escherichia coli K12, a specific small in frame deletion in lptD, called lptD4213, confers increased sensitivity to various antibiotics, detergents, and dyes. Although cell envelope biosynthesis is well studied in the Gram-negative model bacterium Escherichia coli K12, far less is known about this process in Pseudomonas aeruginosa, a pathogen with a particularly impermeable outer membrane. Therefore, we used three different approaches to dissect the OM biosynthesis in P. aeruginosa. In the first project, we engineered a hypomorphic lptD4213 allele in P. aeruginosa PA14. The lptD4213 mutation not only made PA14 more sensitive to large soluble antibiotics, such as vancomycin, but also unexpectedly made it more sensitive to high concentrations of various inorganic salts. To dissect P. aeruginosa PA14 envelope homeostasis, we selected salt and vancomycin suppressors of the lptD4213 mutant. Analysis of a NaCl suppressor mutant of P. aeruginosa PA14 lptD4213 highlighted the role of the Bam complex, while the vancomycin suppressor mutants pointed out the interaction between the LPS O-antigen and the peptidoglycan biosynthesis pathway. In the second project, we decided to start from the WT PA14 strain, and we selected genes whose disruption impaired the barrier function of the OM. In this respect, we used two transposon mutant libraries to identify strains with outer membrane permeability barrier defects by searching for mutants that are sensitive to vancomycin. We identified bamB, which codes for one of the non-essential subunits of the BAM complex and showed that the loss of BamB was sufficient to selectively increase the permeability of the OM to several large antibiotics. Further analyses revealed that this increase in permeability was dependent on the presence of porins in the OM of P. aeruginosa PA14. Finally, in the last project, we performed competition between the transposon mutagenized library of PA14 and Staphylococcus aureus. We identified a few genes in which transposon insertions consistently decreased after exposure to S. aureus. However, while Tn-mutants in several genes were underrepresented in the Tn-seq analysis of P. aeruginosa cultivated with S. aureus, we could not reproduce this effect with clean deletion of two of the identified genes. Dewey 616 iGE3 PhD salary awards 2020 54 Département de microbiologie et médecine moléculaire 21B false 554 Régulation du cycle cellulaire dans C.crescentus, une bactérie asymétrique 895 dd74f24c-406f-4baf-b8da-37e60a41aa50 Thesis Private file.pdf 27488173 application/pdf e6ec37416a523858191ef617d43cf633667d23e205374f6b08b8677e51ee8515 01b7b4aa-32dc-4efd-8653-e58888da3e2f Imprimatur Public file1.pdf 194309 application/pdf c4fc67bb71073a003080eb9cb735ab6ea34c4f40e87be4c8226ed13050046256 94631570-be3e-4a20-83ed-f19eebcb8dda Agreement Private Contrat_diffusion_these_v2022_DocuS.pdf 1252048 application/pdf 4dfadc8c11368da4d09ea44ce48eb64ef15c5b4d890807a0bdecb89120ba6bf2 emilie.trachsel@unige.ch Avenue du 24-janvier, 30 1020 Renens (VD) closedAccess Faculté de médecine / Section de médecine fondamentale / Département de microbiologie et médecine moléculaire

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