Pseudomonas aeruginosa LptE is crucial for LptD assembly, cell envelope integrity, antibiotic resistance and virulence

Abstract

Lipopolysaccharide (LPS) is an essential structural component of the outer membrane (OM) of most Gram-negative bacteria. In the model organism Escherichia coli, LPS transport to the OM requires seven essential proteins (LptABCDEFG) that form a continuous bridge across the cell envelope. In Pseudomonas aeruginosa the recently-demonstrated essentiality of LptD and LptH, the P. aeruginosa LptA homologue, confirmed the crucial role of the Lpt system and, thus, of LPS in OM biogenesis in this species. Surprisingly, independent high-throughput transposon mutagenesis studies identified viable P. aeruginosa insertion mutants in the lptE gene, suggesting that it might be dispensable for bacterial growth. To test this hypothesis, we generated an lptE conditional mutant in P. aeruginosa PAO1. LptE depletion only slightly impairs P. aeruginosa growth in vitro. Conversely, LptE is important for cell envelope stability, antibiotic resistance and virulence in an insect model. Interestingly, the maturation and OM localization of LPS is only marginally affected in LptE-depleted cells, while the levels of the OM component LptD are strongly reduced. This suggests that P. aeruginosa LptE might not be directly involved in LPS transport, although it is clearly essential for the maturation and/or stability of LptD. While poor functionality of LptD caused by LptE depletion is somehow tolerated by P. aeruginosa, this has a high cost in terms of cell integrity, drug resistance and virulence, highlighting LptE function(s) as an interesting target to weaken P. aeruginosa defenses and reduce its infectivity.

Defects in Efflux ( oprM ), β-Lactamase ( ampC ), and Lipopolysaccharide Transport ( lptE ) Genes Mediate Antibiotic Hypersusceptibility of Pseudomonas aeruginosa Strain Z61

“…Downregulation of LptE did lead to a defect in membrane permeability, which is consistent with our observations here for the defect in Z61 LptE. The membrane defect resulting from LptE downregulation appeared to result at least in part from depletion of the LptD protein, which forms a complex with LptE, reflecting a compromised chaperone function of LptE in the formation of this active LptE-LptD complex rather than a direct role for LptE in LPS transport (47). This was proposed to lead to drug sensitivity based on the indirect effect of reduced LptD levels on LPS transport and cell division, as well as the possibility that nonstabilized LptD transporter barrel proteins lacking inserted LptE may circumvent the OM permeability barrier (47).…”

“…The LptD protein component of the LptE-LptD translocon that mediates the final steps of LPS transport (7) is essential for growth in P. aeruginosa, as clearly demonstrated genetically and via specific chemical inhibition (44)(45)(46). It has recently been questioned whether LptE is also essential in P. aeruginosa (47) as it is in E. coli (48). Early studies using saturation transposon mutagenesis suggested an inability to inactivate lptE in P. aeruginosa PAO1 or PA14 (49,50), whereas more recent experiments using transposon sequencing indicated the existence of insertions in lptE (51,52).…”

“…Early studies using saturation transposon mutagenesis suggested an inability to inactivate lptE in P. aeruginosa PAO1 or PA14 (49,50), whereas more recent experiments using transposon sequencing indicated the existence of insertions in lptE (51,52). The lptE gene could not be deleted from the genome of PAO1 strain ATCC 15692 but a derivative of this strain with controlled expression of LptE was viable in the absence of induction, prompting further speculation that LptE may indeed be dispensable for growth (47). However, it was not clear whether a small amount of uninduced expression of LptE may have been sufficient to support growth in that study.…”

Changes in physiology and virulence during the selection of resistant Yersinia ruckeri mutants under subinhibitory cefotaxime concentrations

“…This protein, together with LptD, is involved in the assembly of lipopolysaccharide (LPS) at the surface of the outer membrane. Alterations in lipopolysaccharide have been previously linked to virulence (Lo Sciuto et al, ). Results indicated clear alterations on LPS of Y. ruckeri 150 CTX.…”

“…Interestingly, and opposite what happens in Salmonella Typhimurium, this change in metabolism resulted in a total attenuation of virulence in Y. ruckeri.The second protein upregulated in Y. ruckeri CTX 150 was LptE. This protein, together with LptD, is involved in the assembly of lipopolysaccharide (LPS) at the surface of the outer membrane.Alterations in lipopolysaccharide have been previously linked to virulence(Lo Sciuto et al, 2018). Results indicated clear alterations on LPS of Y. ruckeri 150 CTX.…”