Phospholipid retention in the absence of asymmetry strengthens the outer membrane permeability barrier to last-resort antibiotics

Abstract

The outer membrane of Gram-negative bacteria is a critical barrier that prevents entry of noxious compounds. Integral to this functionality is the presence of lipopolysaccharide (LPS) or lipooligosaccharide (LOS), a molecule that is located exclusively in the outer leaflet of the outer membrane. Its lipid anchor, lipid A, is a glycolipid whose hydrophobicity and net negative charge are primarily responsible for the robustness of the membrane. Because of this, lipid A is a hallmark of Gram-negative physiology and is generally essential for survival. Rare exceptions have been described, including , which can survive in the absence of lipid A, albeit with significant growth and membrane permeability defects. Here, we show by an evolution experiment that LOS-deficient can rapidly improve fitness over the course of only 120 generations. We identified two factors which negatively contribute to fitness in the absence of LOS, Mla and PldA. These proteins are involved in glycerophospholipid transport (Mla) and lipid degradation (PldA); both are active only on mislocalized, surface-exposed glycerophospholipids. Elimination of these two mechanisms was sufficient to cause a drastic fitness improvement in LOS-deficient The LOS-deficient double mutant grows as robustly as LOS-positive wild-type bacteria while remaining resistant to the last-resort polymyxin antibiotics. These data provide strong biological evidence for the directionality of Mla-mediated glycerophospholipid transport in Gram-negative bacteria and furthers our knowledge of asymmetry-maintenance mechanisms in the context of the outer membrane barrier.

Structural insight into outer membrane asymmetry maintenance of Gram-negative bacteria by the phospholipid transporter MlaFEDB

“…On the other hand, the molecular mechanism of how PLs are trafficked between the IM and the OM to maintain the OM asymmetry remains unclear. The maintenance of lipid asymmetry (Mla) pathway is believed to be important for maintaining the asymmetry and integrity of the OM by trafficking PLs between the IM and the OM [35][36][37][38][39][40][41] . Mutants of the Mla pathway in Escherichia coli (E. coli) or other pathogenic species show OM permeability and integrity defects and increased susceptibility to antibiotics [36][37][38][39][40][41][42] .…”

“…The maintenance of lipid asymmetry (Mla) pathway is believed to be important for maintaining the asymmetry and integrity of the OM by trafficking PLs between the IM and the OM [35][36][37][38][39][40][41] . Mutants of the Mla pathway in Escherichia coli (E. coli) or other pathogenic species show OM permeability and integrity defects and increased susceptibility to antibiotics [36][37][38][39][40][41][42] . Six proteins are involved in the Mla pathway, including an ATP-binding cassette (ABC) transporter MlaFE associated with an auxiliary periplasmic membrane protein MlaD and an auxiliary cytoplasmic protein MlaB, a periplasmic protein MlaC and an OM protein MlaA associated with an osmoporin OmpF/C 36,43,44 (Figure 1A).…”

Evolution toward maximum transport capacity of the Ttg2 ABC system inPseudomonas aeruginosa

“…A countercurrent model would explain how asymmetries in lipid distribution in the two membranes might be achieved 54 . Although genetic and functional evidences have mainly suggested that the Ttg2/Mla pathway is a retrograde transport system 19, 55, 56 , recent studies in E. coli have shown that MlaD spontaneously transfers phospholipids to MlaC in vitro 31, 57 .…”

“…Interestingly, this system has been recently identified as a regulator of P. aeruginosa ’s ttg2 operon 32 . More recently, nucleotide polymorphisms conferring resistance to polymyxins have been detected in genes of the Mla pathway in A. baumannii 55 . Although data on the precise mechanisms of resistance are scant and appear to be dependent on specific regulatory systems 66, 68 , the activity of the Ttg2 system on membrane phospholipid homeostasis appears to be partly responsible for the lower basal susceptibility of P. aeruginosa to colistin.…”

Systematic Discovery of Pseudomonad Genetic Factors Involved in Sensitivity to Tailocins

“…6 of the 9 hits appear to have functions relevant to maintaining a sufficient density of LPS at the outer leaflet of the outer membrane (OM) (see bold genes in Fig. 3B) 41,42 . Among these, 4 of the hits (TK06_RS11430, TK06_RS16275, TK06_RS16280, TK06_RS16290) are in the mla (maintenance of lipid asymmetry) pathway, which constitute a retrograde phospholipid (PL) transport system involved in OM lipid homeostasis.…”