Lipoprotein trafficking to the bacterial outer membrane

Emory University, Atlanta GA

Investigators

Abstract

ABSTRACT Gram-negative bacteria produce an outer membrane (OM) that envelopes the cell. The OM is an essential organelle and a potent permeability barrier that blocks the entry of many antibiotics, limiting clinical options. The OM is at a distance from the cytosol, separated from the inner membrane (IM) by a periplasmic space. Yet, all OM components are synthesized in the cytosol. The orchestrated actions of dedicated molecular machines and chaperones ensure efficient transport and assembly of each component into the OM bilayer. Lipoproteins (lipid- anchored proteins) are key to building the OM. Almost all essential OM assembly machines require at least one OM lipoprotein for function. After being secreted from the cytosol, nascent lipoproteins are modified with three acyl chains, anchoring them into the IM. The cell must subsequently extract, and traffic these highly hydrophobic lipoproteins through an unfavorable aqueous periplasmic environment to the OM for insertion into the bilayer. The Escherichia coli “Lol pathway” was the paradigm for how lipoproteins are trafficked: the periplasmic protein LolA triggers release of lipoproteins from the IM and chaperones them to the OM where LolB inserts them. We discovered that, while LolA and LolB are important for trafficking, the genes encoding both proteins can be deleted and yet efficient lipoprotein trafficking continues in their absence. Our finding reveals that additional factors can traffic essential OM lipoproteins to sustain viability. We aim to uncover the factors responsible for LolAB-independent lipoprotein trafficking and understand why multiple routes exist. We now know that the Cpx two-component system continuously monitors OM lipoprotein trafficking in E. coli. If defects arise, Cpx acts to restore homeostasis and preserve viability. We aim to define the key toxicities that occur and how these are mitigated by Cpx. We will expand our understanding of stress and homeostasis by investigating how distantly related bacteria that lack Cpx monitor and mitigate trafficking stress, identifying new cell envelope stress responses. Since we can now make LolAB conditionally non-essential, we are uniquely placed to thoroughly dissect the molecular mechanisms of lipoprotein trafficking. We will utilize deep mutational scanning alongside complementary biochemical studies to directly capture lipoprotein trafficking intermediates to shed light on their mechanisms. The E. coli paradigm falls short in many diverse Gram-negative species, and we have limited understanding of their OM assembly. We discovered that many LolB-lacking species produce a bifunctional LolA chaperone that also fulfils insertase function (delegated to LolB in E. coli). How such proteins achieve unidirectional trafficking to the OM is not known and we will dissect the proteins and processes involved. Our research program will move our understanding of trafficking beyond the E. coli model and into diverse pathogens (Acinetobacter baumannii and Campylobacter jejuni). Gram-negative species are now a major cause of deaths attributable to antibiotic resistance. The crucial roles that lipoproteins play in building the OM makes their trafficking a focus of ongoing novel antibiotic development. Our discoveries will further empower those efforts.