The Power of Asymmetry: Architecture and Assembly of the … · The gram-negative cell envelope and...

MI70CH14-Trent ARI 10 August 2016 15:5

The Power of Asymmetry:Architecture and Assembly ofthe Gram-Negative OuterMembrane Lipid BilayerJeremy C. Henderson,1 Shawn M. Zimmerman,2

Alexander A. Crofts,1 Joseph M. Boll,1 Lisa G. Kuhns,2

Carmen M. Herrera,2 and M. Stephen Trent2

1Department of Molecular Biosciences, The University of Texas at Austin, Texas 787122Department of Infectious Diseases, The University of Georgia, Athens, Georgia 30602;email: [email protected]

Annu. Rev. Microbiol. 2016. 70:255–78

First published online as a Review in Advance onJune 24, 2016

The Annual Review of Microbiology is online atmicro.annualreviews.org

This article’s doi:10.1146/annurev-micro-102215-095308

Copyright c© 2016 by Annual Reviews.All rights reserved

Keywords

phospholipids, lipid A, lipopolysaccharide, LPS, commensals, outermembrane vesicles, vaccines

Abstract

Determining the chemical composition of biological materials is paramountto the study of natural phenomena. Here, we describe the composition ofmodel gram-negative outer membranes, focusing on the predominant as-sembly, an asymmetrical bilayer of lipid molecules. We also give an overviewof lipid biosynthetic pathways and molecular mechanisms that organize thismaterial into the outer membrane bilayer. An emphasis is placed on thepotential of these pathways as targets for antibiotic development. We dis-cuss deviations in composition, through bacterial cell surface remodeling,and alternative modalities to the asymmetric lipid bilayer. Outer membranelipid alterations of current microbiological interest, such as lipid structuresfound in commensal bacteria, are emphasized. Additionally, outer membranecomponents could potentially be engineered to develop vaccine platforms.Observations related to composition and assembly of gram-negative outermembranes will continue to generate novel discoveries, broaden biotech-nologies, and reveal profound mysteries to compel future research.

255

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ANNUAL REVIEWS Further

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Glycerophospholipid(PL): a membranelipid with aglycerol-3-phosphatebackbone with fattyacyl chains esterifiedto positions 1 and 2

Lipopolysaccharide(LPS): a glycolipidlocalized to the outerleaflet of the outermembrane comprisinglipid A, core-oligosaccharide, andO-antigen domains

Contents

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256BIOSYNTHESIS OF A DIVERSE GLYCEROPHOSPHOLIPID

REPERTOIRE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258PL TRANSPORT—THE UNKNOWN OUTER MEMBRANE

ASSEMBLY APPARATUS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259Tracking Anterograde PL Transport to the Outer Membrane. . . . . . . . . . . . . . . . . . . . . 260Outer Membrane PL Removal via Retrograde Transport . . . . . . . . . . . . . . . . . . . . . . . . . 260

GRAM-NEGATIVE BIOSYNTHESIS OF ENDOTOXICKDO–LIPID A DOMAINS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262

FINAL ASSEMBLY AND TRANSPORT OF LPS TO THE OUTERMEMBRANE BILAYER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264

BIOLOGICAL SIGNIFICANCE OF CHEMICALLYALTERED KDO–LIPID A DOMAINS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264

GRAM-NEGATIVE BACTERIA WITHOUT ENDOTOXIN . . . . . . . . . . . . . . . . . . . . 266ENGINEERING OUTER MEMBRANE COMPONENTS

FOR APPLIED TECHNOLOGIES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267Detoxified LPS—A Classic Adjuvant Refurbished for Modern Vaccines . . . . . . . . . . . 267Design of Enhanced Vaccine Platforms—Outer Membrane Vesicles . . . . . . . . . . . . . . 268Outer Membrane Vesicles as Effective Glycoconjugate Vaccines . . . . . . . . . . . . . . . . . . 268Outer Membrane Vesicles Engineered to Present Proteinaceous

Vaccine Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269

INTRODUCTION

A defining feature of diderm, gram-negative bacteria is a second membrane bilayer that surroundsthe peptidoglycan layer (Figure 1). Unlike the inner bacterial membrane, this outer membrane isan asymmetrical lipid bilayer (66), with the periplasmic leaflet composed of glycerophospholipid(PL) and the surface-exposed outer leaflet consisting of lipopolysaccharide (LPS) (Figure 1). Thisunique membrane organization affords gram-negative organisms protection not only from largepolar molecules restricted by a typical membrane bilayer but also from lipophilic compounds.Evidence for the asymmetrical organization of the outer membrane was reported by Kamio &Nikaido (66) in the mid-1970s. They demonstrated that PLs of intact bacteria are not susceptibleto degradation by phospholipases or to chemical labeling by macromolecular reagents unable tocross the outer membrane. Overall this suggested PLs were confined to the inner leaflet of theouter membrane.

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→Figure 1The gram-negative cell envelope and major lipids of Escherichia coli. (a) The typical inner membrane and outer membrane bilayers areseparated by the periplasmic compartment, which contains the peptidoglycan layer. The inner membrane is a bilayer ofglycerophospholipids, whereas the outer membrane is an asymmetrical bilayer, with glycerophospholipids found in the innerperiplasmic leaflet and lipopolysaccharide localized to the outer-surface-exposed leaflet. (b) The major lipids of E. coli K12 are3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)2–lipid A, phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. The lengthand composition of fatty acyl chains of each major lipid type, as well as the number of molecules within a bacterial cell duringexponential growth phase, are indicated.

256 Henderson et al.

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OO

Core

Lipid A

Phospholipid

Outermembrane

Cytoplasm

Innermembrane

Lipo

poly

sacc

harid

es

Peptidoglycan

Kdo2–lipid A Phosphatidyl-ethanolamine

1214

1414

~2 × 106

Lipoprotein

a

b

Periplasm

O-antigen

~107

Kdo sugars

O

OO

HO

OO

OOOO

OO

OO

NH2NH2

Phosphatidyl-glycerol~2 × 106

O

OO

HO

OO

OO

OO

OO

OHOH

OHOH

PP

16 18:1

16 18:1

OO

Cardiolipin~4 × 105

O

OO

HO

OO

OO

OO

P OO

OO

OO

OO

OHOH

16 18:1

OO

OO

HO

OO

OO

OO

OO

16 18:1

OO

OOHOHOHOHO

HOHOOO

OO

OOHOHO

HOHO

OO

OO

OOOO

HOHO

P

OO

OO

OOHOHO

OO

OO

OO

OO

OO

NHNH

OHOH

14

OOOO

HOHO

14

OO

HOHO

NHNHOO

OHOH

OHOH

OHOH

OHOH

OHOH

PP PPPP

OHOH

β-barrelprotein

Integralmembrane

protein

www.annualreviews.org • The Power of Asymmetry: Gram-Negative Outer Membrane 257

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Given that the outer membrane is essential for bacterial growth, protects the bacterium fromenvironmental stresses (e.g., antibiotics), contains components that activate the innate immunesystem, and interfaces directly with the surrounding environment, it is of no surprise that it hasbeen the subject of intense research. Over the last two decades, there has been enormous progresstoward understanding outer membrane assembly and maintenance of bilayer asymmetry. One keyquestion has always been how lipophilic components, assembled at the inner face of the cytoplasmicmembrane, are guided through the crowded, aqueous environment of the periplasm for assemblyinto the outer membrane bilayer. Other areas of intense focus include biosynthesis and remod-eling of key outer membrane lipid components, development of antimicrobials targeting outermembrane assembly, and manipulation of outer membrane components in biotech applications.

BIOSYNTHESIS OF A DIVERSE GLYCEROPHOSPHOLIPIDREPERTOIRE

Eugene Kennedy and members of his laboratory combined molecular genetics and biochemicalapproaches to identify and characterize the genes responsible for PL metabolism in Escherichia coliand Saccharomyces cerevisiae over 40 years ago (41, 67, 103). E. coli PL biosynthesis is a paradigmfor most prokaryotes (Figure 2), whereas the S. cerevisiae pathway generally typifies related fungal

CdsA

CTP P P

PssA

PgsA

P Cyt

L-Serine

CMP

Gly3- P

CO2

Psd

CMP

CDP-DAG

PhosphatidylserinePhosphatidyl-ethanolamine

PgpAPgpBPgpC

P Gly P

PhosphatidylglycerolPGP

ClsAClsB

ClsC

Cardiolipin

Gly

PGorPE

EtNCytGlySer

EthanolamineCytidineGlycerolSerine

Headgroups

Phosphatidic acid

O

OO

HO

OHOH

OOOO

OO

Gly

Ser EtN

Gly

OO

PP

Figure 2The Kennedy pathway for glycerophospholipid biosynthesis in Escherichia coli. Eugene Kennedy and coworkers discovered the criticalrole of cytidine nucleotides as activating groups for subsequent phosphatidyl-transfer reactions in glycerophospholipid biosynthesis.Enzymes involved in these pathways are either intrinsic membrane proteins or membrane associated. Phosphatidic acid, de novosynthesized on the cytoplasmic surface of the inner membrane, is converted to cytidine diphosphate diacylglycerol (CDP-DAG) byCDP-DAG synthase (CdsA) in a reaction that utilizes cytidine triphosphate (CTP) with release of pyrophosphate. CDP-DAGfunctions as a donor of phosphatidyl moieties for the biosynthesis of phosphatidylserine and phosphatidylglycerol-3-phosphate (PGP)with release of cytidine monophosphate (CMP). The PS formed by phosphatidylserine synthase (PssA) is rapidly decarboxylated byphosphatidylserine decarboxylase (Psd) to generate the predominant glycerophospholipid (∼70%) found in E. coli,phosphatidylethanolamine. In phosphatidylglycerol (PG) synthesis, PGP is quickly dephosphorylated by one of several innermembrane phosphatases (e.g., PgpA) to form PG. Lastly, cardiolipin is synthesized through the condensation of two PG molecules bycardiolipin synthase A, B, or C (ClsA, ClsB, or ClsC) (ClsA being the major cardiolipin synthase). Enzyme names are in blue, lipidspecies in brown, and cofactors in black.


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PL headgroup:the chemical moietybound to thephosphate group atposition 3 on theglycerol backbone ofphosphatidic acid

Lipoprotein: amembrane proteinthat may be anchoredto the inner or outermembrane by a lipidmoiety

Kdo: 3-deoxy-D-manno-oct-2-ulosonicacid

Lipid A: thehydrophobic anchor oflipopolysaccharide

Coreoligosaccharide:an oligosaccharideattached to a lipid Adomain, with the innercore Kdo sugar as theattachment site

species. Both model organisms contain elements of more complex lipid anabolism observed inhigher-order eukaryotes. Today, genomic information combined with unprecedented sensitivityin detection of lipid species by means of mass spectrometry has enabled identification of previouslyunknown genes involved in lipid metabolism, even in well-trekked model organisms. Excitingdiscoveries in E. coli include a third phosphatidylglycerol-3-phosphate (PGP) phosphatase andtwo novel cardiolipin synthases (Figure 2); in yeast and humans a new PGP phosphatase and amitochondrial cytidine diphosphate diacylglycerol synthetase have been identified (80, 97, 125,126, 152). Mutations in these eukaryotic genes lead to severe mitochondrial dysfunction, as aconsequence of cardiolipin loss. However, despite the existence of three differentially regulatedcardiolipin synthase homologs in E. coli, the biological role of cardiolipin in this model bacteriumremains poorly understood.

Cardiolipin’s enigmatic biological function in bacteria is not exceptional. The physiologicalimportance of bacterial PL headgroup heterogeneity remains ill defined; however, a few consistentthemes emerge from various insightful reports. The topology of integral membrane proteins isaffected by interaction with specific lipid partners whereas, perhaps not surprisingly, numerous pe-ripheral membrane proteins associate preferentially with anionic PLs—such as phosphatidic acid,phosphatidylglycerol (PG), and cardiolipin—rather than zwitterionic phosphatidylethanolamine(PE) (12, 42, 84, 142, 153). Protein interactions with anionic PL have been implicated as neces-sary for SecA-dependent protein translocation and oriC-dependent DNA replication (20, 35, 54,79, 148). It is also reported that PLs serve as precursors in the chemical modification of sugars,proteins, and even other lipids. In many organisms, the diacylglycerol moiety of PG is used inthe posttranslational modification of lipoproteins, a chemical modification required for properinsertion of lipoproteins into the membrane (16, 59, 88, 130, 147). During processing, lipopro-teins are also aminoacylated at an N-terminal cysteine residue, where PLs serve as substrate acylchain donors (88, 116). Membrane-derived oligosaccharide, an enigmatic osmoregulatory com-ponent found in the periplasm of some gram-negative bacteria, can be modified with PG and PEheadgroups (13, 63, 64, 86). The Kdo–lipid A domain and core oligosaccharide of LPS can alsobe modified with PL headgroups or acyl chains (11, 92, 99, 105). Bacteria with these membranemodifications show increased resistance to cationic antimicrobial peptides, especially polymyxinsthat bind Kdo–lipid A specifically, but also an ever-growing list of other phenotypes (92) (see“Biological Significance of Chemically Altered Kdo–Lipid A Domains”).

In efforts to structurally characterize bacterial membrane lipids, lipidomics across known bac-terial taxa have uncovered a panoply of lipid species (an area of research covered in greater depthelsewhere; 118). This array of molecular composition somewhat undermines the universality ofthe E. coli paradigm. It has become increasingly evident that lipid diversity in nature is exquisiteand complex. Study of this diversity, and how chemical composition informs biological function,will surely continue to yield unique insights into our natural world.

PL TRANSPORT—THE UNKNOWN OUTER MEMBRANEASSEMBLY APPARATUS

PL transport to the outer membrane presents a unique challenge to gram-negative bacteria.Lipoproteins and β-barrel outer membrane proteins contain specific amino acid signal sequencesfor export to the outer membrane, whereas LPS is strictly compartmentalized to the outer mem-brane surface by dedicated transport machinery. On the contrary, PLs appear randomly distributedbetween the inner bilayer and the periplasmic outer membrane leaflet, with no major observabledifference in PL composition. The process by which PLs are distributed between sites of synthesison the inner membrane to the periplasmic leaflet of the outer membrane is unknown. Transport


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MsbA: an integralmembrane transporterthat flips lipid A–coreoligosaccharide to theperiplasmic face of theinner membrane

Lipooligosaccharide(LOS): a form of LPSwith an extended coreoligosaccharide, butlacking O-antigen

Two-componentregulatory system:a system composed ofa sensor histidinekinase that receivesinput stimuli and thenphosphorylates aresponse regulator

PbgA: PhoPQ-barriergene protein A

machinery exists for other outer membrane components, so an analogous mechanism likely existsto distribute the cellular PL pool between inner and outer membranes. The rate of spontaneous,passive transfer of PLs between separated membranes in vitro is not sufficient to support bacterialgrowth (40); thus, an active mechanism is likely needed to facilitate movement of PLs across theperiplasmic compartment.

Tracking Anterograde PL Transport to the Outer Membrane

The most detailed account of PL transport from the inner to outer membrane was written byDonohue-Rolf & Schaechter in 1980 (40). To determine the in vivo translocation rate of newlysynthesized PLs, they used pulse-chase radiolabeling of cellular PL pools to track and comparePLs between inner and outer bilayers. The results indicated distinct rates of PL transportation,with rapid transport of PG or cardiolipin and slower rates of PE transport to the outer membrane(Figure 1). Depletion of cellular ATP and inhibition of lipid or protein synthesis did not appear toalter PE translocation rates, but inhibitors that target proton motive force significantly reduced thePE translocation rate. The authors postulated that zones of adhesion, or Bayer bridges, betweenthe outer membrane and inner membrane provide a route for phospholipid movement betweenmembranes and are controlled by the proton motive force (8). However, whether these zones existin vivo is controversial (70), and despite these works no essential anterograde transport mechanismshave been characterized.

Conditionally lethal mutants of MsbA accumulate LPS precursors and PLs in the inner mem-brane, leading some to conjecture that MsbA serves as a general flippase for both lipid molecules(37, 38). Further characterization has clearly shown MsbA is responsible for flipping nascent LPSintermediates from the cytoplasmic to periplasmic leaflet of the inner membrane. The role ofMsbA in general PL transport is less clear, complicated by the fact that accumulation of LPSprecursors could affect an unknown, independent PL transport mechanism. Evidence in supportof this idea came from a study in lipooligosaccharide (LOS)-deficient Neisseria meningitides thatconvincingly demonstrated that MsbA is not required for PL distribution to the outer membrane(128).

The study of proteins that alter wild-type ratios of PLs, specifically in the outer membrane com-partment, could prove beneficial in identifying putative PL transport mechanisms. Dalebroux et al.observed that activation of the PhoPQ two-component virulence regulatory system in Salmonellaenterica serovar Typhimurium increases relative concentrations of cardiolipin in the outer mem-brane (32, 33). This change in lipid content requires the membrane-tethered PbgA (YejM), whichcontains an integral membrane domain and a periplasmic cardiolipin-binding domain. PhoPQ ac-tivation in S. enterica promotes direct interaction of the PbgA periplasmic domain with the outermembrane, leading the authors to hypothesize that PbgA can serve as a cardiolipin transporterupon PhoPQ activation. Conservation of PbgA is limited even among proteobacteria; however,the existence of PbgA-like analogs cannot be ruled out. Broadened searches of genes responsiblefor perturbed PL content will continue to identify machinery bacteria employ to transit bulk PLsfrom the inner membrane to the outer membrane.

Outer Membrane PL Removal via Retrograde Transport

The Mla (maintenance of outer membrane lipid asymmetry) pathway is an intermembrane trans-port system proposed to function in the removal of accumulated PLs in the outer membrane bytransporting them to the inner membrane (81). The proteins that make up the Mla system containelements similar to those of the Lol lipoprotein transport apparatus (150) (Figure 3). Mla is made


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MlaEMlaE

MlaFMlaF

MlaD

ATP ADP + Pi ATP ADP + Pi

MlaC

a Mla system b Lol system

OmpC

?

?

??

LolA

LolB

LolD LolD

LolELolC

MlaA

BB

Figure 3Comparison of the Mla and Lol periplasmic transport systems. (a) The proposed model of the retrograde Mla (maintenance of outermembrane lipid asymmetry) system that is responsible for the removal of mislocalized glycerophospholipids from the gram-negativeouter membrane. It is unclear whether the Mla system removes excess glycerophospholipids (e.g., phosphatidylethanolamine) from theinner leaflet prior to migration to the cell surface or if the system removes glycerophospholipids directly from the outer leaflet. It hasbeen proposed that OmpC functions in a complex with the lipoprotein MlaA to extract phospholipids from the outer leaflet (22). Onceextracted, the targeted lipid is delivered to the soluble periplasmic transporter MlaC, which delivers the substrate to the MlaFEDBABC transporter complex. Using energy from ATP hydrolysis, the targeted lipid is reinserted into the inner membrane; however, thefate of the lipid is unknown. The biochemical mechanisms of substrate recognition, membrane extraction, and membrane insertionremain to be elucidated. (b) Schematic representation of the Lol (localization of lipoproteins) transport system. The LolCDE ABCtransporter complex utilizes ATP to direct the movement of mature (i.e., acylated) lipoproteins destined for the outer membrane byLolA. The LolA-lipoprotein complex transports target lipoproteins across the periplasm to LolB, which inserts lipoproteins into theouter membrane. It is unclear how surface-exposed lipoproteins are transported to the outer leaflet of the outer membrane.

ABC (ATP-bindingcassette) transporter:ubiquitous integralmembrane proteinsthat actively transportligands acrossmembranes utilizingenergy generated byATP hydrolysis

up of an ABC transporter (MlaFEDB), a periplasmic protein (MlaC), and a lipoprotein tetheredto the outer membrane (MlaA). Deletion of any Mla component leads to outer membrane defectsquantifiable by SDS-EDTA sensitivity. As reported by Malinverni & Silhavy (81), deletion of anyone Mla component results in identical sensitivity to SDS-EDTA, indicative that they comprisea single pathway. Shigella flexneri with mutations in Mla homologs fails to form plaques afterinvasion of human host cells, a proxy for intracellular spread (19). Like E. coli mla mutants, theseShigella mla mutants are sensitive to SDS-EDTA.

Suppressor mutations that repair the defective outer membrane phenotype (SDS-EDTA sen-sitivity) arise in the promoter region of pldA, leading to overexpression of the encoded outermembrane phospholipase A (81). PldA has long been thought to contribute to outer membranelipid asymmetry under certain growth conditions, by degrading PLs that have miscompartmen-talized to the outer leaflet of the outer membrane (10, 36, 117). PldA mutants do not accumulate


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Homeoviscousadaptation:maintenance ofmembrane fluiditythrough compositionaladaptation ofmembrane lipids

PLs in the outer membrane outer leaflet to the dramatic extent seen in Mla pathway mutants,indicating the necessity of Mla-based maintenance of outer membrane asymmetry. It is importantto note, however, that no biochemical evidence directly supports Mla retrograde PL transportfrom the outer membrane to the inner membrane.

Direct physical interaction between the OM lipoprotein, MlaA, and the β-barrel protein OmpChas been observed, and it is speculated that OmpC may provide a β-barrel-based channel for MlaAto reach PLs in the outer leaflet (Figure 3) (22). This hypothetical protein-interaction model drawsparallels to the LptDE plug-and-barrel system used to span the outer membrane bilayer in exportof LPS to the bacterial surface (50, 56) (Figure 4). OmpC might instead function in interleafletPL transport, shuttling outer leaflet PLs to the inner leaflet, where they become accessible toperiplasmic MlaA. It is also possible that MlaA is deposited on the bacterial surface, or spansacross the bacterial outer membrane, as has been suggested with other lipoproteins (28, 100). Arecent mutation in MlaA was reported by Silhavy and colleagues, with a variety of interestingphenotypes that are bound to provide a more robust understanding of the Mla pathway uponfurther mechanistic examination (124). To determine the exact mechanism of MlaA, a moredefinitive elucidation of its outer membrane leaflet distribution is needed. Moreover, interactioncharacterization, of and between Mla proteins, will further improve our understanding as to howMla controls outer membrane PL composition.

GRAM-NEGATIVE BIOSYNTHESIS OF ENDOTOXICKDO–LIPID A DOMAINS

The genes and molecular intermediates along the near-completely conserved biosynthetic path-way for Kdo–lipid A assembly in gram-negative bacteria have been characterized over a span of30 years (Figure 4). Pathway enzymes have been comprehensively characterized through crys-tallography and classical enzymology. Nine enzymes are required for canonical (Kdo)2–lipid Abiosynthesis in E. coli (105) and are considered essential for growth in all gram-negative bacteria,with the exception of the late-step acyltransferases LpxL and LpxM and their various homologs(60, 133, 139). Although not all organisms contain high-identity homologs to each gene in theE. coli pathway, each enzymatic step is universally conserved by organism-specific functionalanalogs. For example, UDP-2,3-diacyl-GlcN hydrolase activity (Figure 4) is catalyzed by LpxHin most gamma- and betaproteobacteria, whereas LpxI performs this role in alphaproteobacteriaand other bacterial groups (145). LpxG, a third enzyme of distant homology to both LpxH andLpxI and capable of UDP-2,3-diacyl-GlcN hydrolase activity, was recently identified in Chlamydiatrachomatis (151). A very similar distribution pattern is seen in lipid A late acyltransferases, whereLpxJ substitutes for LpxM in many organisms (109) (Figure 4). Departures from the canonicalbiosynthetic structure include variations in acyl chain length and degree of saturation, impor-tant for homeoviscous adaptation, as controlled by selective hydrocarbon ruler domains of LpxAor LpxD and their functional equivalents (4, 7, 18, 104). Nuanced selectivity in a number ofKdo sugars, and modifications thereof (e.g., phosphorylation or hydroxylation), represent otherorganism-specific variations to the canonical biosynthetic route (23, 144).

Compounds that inhibit early steps in Kdo–lipid A domain synthesis are candidates for broad-spectrum antibacterials. However, impediments to the successful in vivo implementation of Raetzpathway inhibitors are problematic in antibacterial development in general, e.g., narrow-spectrumantibacterial activity, insolubility, and off-target protein binding. Compound screens have success-fully identified competitive inhibitors of early-step acyltransferases LpxA and LpxD (65), and asulfonyl piperazine inhibitor of LpxH was recently discovered (90). These compounds are effectiveagainst specific gram-negative taxa. Given that LpxC catalyzes the committed step for Kdo–lipid A


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Ac

UDP

LpxALpxCLpxD

LpxH

LpxBCore

glycosyl-transferases

LpxK LpxL,LpxM

KdtA (2×)

UDP-GlcNAc

Lipid IVA Kdo2–Lipid IVA Kdo2–Lipid A

Lipid X

LptF LptG

AcGlucosamine Acetyl groupPhosphateFatty acid

LptC

LptA

LptDC

LptA

LptE

ATP ADP + Pi ATP ADP + Pi

MsbA

UDP UDP Ac

UDP

LptB LptB

LptDN

Figure 4Biosynthesis and transport of lipopolysaccharide (LPS) in Escherichia coli. Biosynthesis of the canonical Kdo2–lipid A substructure ofLPS is required for the growth of most gram-negative bacteria. It is synthesized via the nine-step Raetz pathway. All reactions in thepathway are catalyzed by a single enzyme (red ) and occur at the interface between the cytosol and the inner membrane. Acyl-ACP (acylcarrier protein) is the preferred donor for each acylation event, and each acyltransferase utilizes an active site hydrocarbon rulerproviding acyl chain specificity. Although Kdo is technically part of the core oligosaccharide, Kdo transfer is required for lipid Abiosynthesis because the final two steps, catalyzed by LpxL and LpxM, require the presence of covalently attached Kdo. The remainingcore oligosaccharide is extended at the cytoplasmic face of the inner membrane, requiring various glycosyl transferases (not shown).MsbA, the core Kdo–lipid A domain flippase, functions as an ABC transporter, moving core Kdo–lipid A domains to the periplasmicface of the inner membrane. For simplicity, the addition of O-antigen that typically occurs in the periplasm is not shown. Theintermembrane translocation of mature LPS is carried out by the Lpt (LPS transport) system, which forms an envelope-spanningtranslocation machine. LPS is removed from the inner membrane via the ABC transporter LptBFG and delivered to LptC. Along withthe periplasmic domain of LptC, soluble LptA forms a periplasmic bridge with the N-terminal domain of the outer membrane proteinLptD. LptE sits within the β barrel formed by the C-terminal domain of LptD and promotes passage to the bacterial surface.

biosynthesis, and has no homology to any protein domain in humans, LpxC inhibitors remain themost promising broad-spectrum target (2, 78). For many years CHIR-090 has been explored as apowerful inhibitor of many bacterial LpxC homologs (5, 6, 74, 85). A fine-tuned understanding ofthe interaction between CHIR-090 and LpxC has led to the design of dozens of novel compoundswith differing specificity and increased potency to various LpxC homologs (17, 74–77, 82, 131).Compounds that inhibit the synthesis or transfer of Kdo to tetraacylated lipid A have also beendescribed; however, their in vivo efficacy is unclear (9, 24–26, 101, 149).


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O-antigen:a long-chainpolysaccharideattached to the lipid Acore oligosaccharidethat provides a majorcellular antigen

FINAL ASSEMBLY AND TRANSPORT OF LPS TO THE OUTERMEMBRANE BILAYER

Core oligosaccharide is assembled stepwise on nascently synthesized Kdo–lipid A domains by spe-cific glycosyltransferases that utilize activated sugar nucleotides. Upon completion MsbA translo-cates core Kdo–lipid A from the inner leaflet to the outer leaflet of the inner membrane, in anATP-dependent manner (Figure 4). As translocation from the inner to outer leaflet appears essen-tial, MsbA is a potential target for antimicrobial development. Characterization of MsbA in vitroalso suggests it may serve as a capable multidrug efflux pump (43, 107, 146). To our knowledgeno compounds that specifically block MsbA in vivo have been reported.

In the periplasmic space, O-antigen is ligated onto core Kdo–lipid A to form fully mature LPS(not shown in Figure 4; reviewed in Reference 145). In E. coli, transport of LPS onto the sur-face of the outer membrane requires a seven-protein complex (Figure 4). Inner membrane ABCtransporter LptBFG is required for LPS extraction from the periplasmic leaflet (57, 89, 110).This protein complex interfaces with an LPS-binding protein, LptC, to transfer LPS from theperiplasmic leaflet of the inner membrane to a periplasmic transporter, LptA (89, 96, 132). LptAforms a periplasm-spanning filament bridging the inner to the outer membrane (21, 51). At theouter membrane, LptA transfers LPS to the third and final LPS-binding protein, LptE, whereinLptD and LptE form a plug-and-barrel structure for final deposition of LPS into the outer leafletof the outer membrane (50, 56). How large LPS structures are threaded through the LptDEcomplex is an interesting and unanswered question. This physical constraint is also seen withouter membrane β-barrel assembly machinery (BAM) that helps properly fold outer membraneβ-barrel proteins. Substrate β-barrel proteins are often larger than the β-barrel component ofBAM complexes. It has been suggested that LptDE, BAM, and related complexes contain a gatedlateral opening to allow intramembranous transit of bulkier substrate molecules, as supported byexperimental evidence (39, 58, 94). Given that components of the Lpt machinery are on the bac-terial surface, and appear essential for growth, LPS transport is a viable target in the developmentof new antibacterial agents. Compounds that specifically target LptD in Pseudomonas aeruginosa(122) or LptB of E. coli have already been identified (114, 134, 143).

BIOLOGICAL SIGNIFICANCE OF CHEMICALLYALTERED KDO–LIPID A DOMAINS

Most bacteria modify the basic Kdo–lipid A structure to optimize outer membrane integrity forsurvival in a given niche, and pathogens remodel their outer membrane in response to environ-mental cues so as to enhance virulence. In general, Kdo–lipid A modifications involve changes inthe number or composition of acyl chains, phosphate groups, or any of various covalently attachedfunctional groups (92, 105). The Kdo–lipid A domain from S. enterica serovar Typhimurium servesas an exemplary model of the full range of modifications one organism may contain (Figure 5).

Chemical modifications to LPS typically involve smaller chemical alterations of less than200 Da. Larger chemical substitutions have been discovered (>500 Da), such as the covalentattachment of hopanoids to very long chain fatty acids of the Kdo–lipid A domain of Bradyrhi-zobium LPS (Figure 6) (115). Biophysical experiments in liposomes show that hopanoid-linkedlipid A helps stabilize model asymmetric bilayers, whereas in nature hopanoid modification isnecessary for the favorable environmental association with the bacteria’s leguminous symbiont.Interestingly, strains of Bradyrhizobium that were deficient for general hopanoid biosynthesis wereless able to maintain healthy symbiosis with host Aeschynomene evenia legumes (115). A better un-derstanding of how hopanoid–lipid A affects symbiosis could inform agricultural bioengineers


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EptA

EptB

LpxR

PagL

PagP Palmitoylates (C16:0) the primary linked acyl chain at 2 position

Transfers phosphoethanolamineto lipid A phosphate groups

Transfers phosphoethanolamine toouter Kdo sugar of inner core

Removes acyl chain from 3 position

Periplasm

Periplasm

Outermembrane

Outermembrane

Outermembrane

Enzyme Activity Active sitetopology

Removes acyl chains from 3' position

ArnT Transfers L-4-aminoarabinose to lipid A phosphate groups

Periplasm

LpxO Cytoplasm Hydroxylates the 3' fatty acidmyristate

LpxT PeriplasmPhosphorylates lipid A, forming apyrophosphate group at 1 position

OOXX

OO

P

OOOOHOHO

OO

NHNHOO

OO

OO

OONHNH

OHOH

PP

OO

OOOO

OHOH

PP

OO

OHOH

OHOH

OO

HOHO

OO

OO

NH2NH2

NH2NH2

P

OO

OO

OO

PP

OO

OHOH

H2NH2NOO OO

HOHO

OO

OOHOHO

OHOH

OOHOHOHOHO OHOH

OHOH

OO

OHOHOO

HOHO

HOHO

OO

OHOH

OO

or

LpxR

LpxT

PagP

PagL

ArnT

EptB

EptA

LpxOX = OH or H

OO

OO

OO

OO

OO

1'2'3'

4' 5'

6'

123

46

5

Modified lipid A ofSalmonella enterica

Figure 5Chemical modifications of the Kdo2–lipid A domain of Salmonella enterica. The Kdo2–lipid A domain of Salmonella spp. can be highlymodified by highly regulated enzymatic machinery (reviewed in 92, 145). Arrows indicate either the addition or the removal of acylchains. Numbers indicate positions on the disaccharide portion of lipid A. Addition of free-amine-containing residues, 4-amino-4-deoxy-L-aminoarabinose (L-4-aminoarabinose) and phosphoethanolamine, or the fatty acid palmitate promotes resistance toantimicrobial peptides. The removal of acyl chains is associated with reduction of endotoxicity and TLR4/MD-2 activation.

TLR4/MD-2(Toll-like receptor4/myeloiddifferentiationfactor 2): functions asa pattern-recognitionreceptor recognizingLPS; initiates a robustsignal cascade inmammals

Pattern-recognitionreceptor: receptor ofthe innate immunesystem that bindMAMPs of infectingpathogens leading toan inflammatoryresponse

interested in reducing our dependence on the environmentally and economically unsustainableuse of chemical fertilizers.

Provocative studies reveal the dynamic relationship between surface features of human com-mensals and host immunity, especially within the gastrointestinal tract, which contains morethan 100 trillion bacteria. Of prime interest are preventive mechanisms that limit overstimulationof proinflammatory receptor-mediated pathways by resident gastrointestinal commensals. Onesuch mechanism involves only sensing bacteria that invade past the luminal epithelial barrier,where resident professional immune cells are most abundant. Expression of TLR4/MD-2, thepattern-recognition receptor that specifically binds to and is activated by Kdo–lipid A, is local-ized to the basolateral surface of associated enterocytes (136), limiting unnecessary inflammatoryresponses to bulk commensals in the gastrointestinal lumen. Similarly, Paneth cells residing inintestinal crypts direct antimicrobial responses to TLR4/MD-2 activation (135). Some commen-sals appear to disguise their surface; for example, Bacteroides thetaiotaomicron expresses a lipid Aphosphatase (LpxF). Dephosphorylation simultaneously provides resistance against host luminalantimicrobial peptides and produces a lipid A molecule that weakly stimulates TLR4/MD-2 (31)(Figure 6). Similar chemical remodeling of the cell surface may be a general commensal strategy,given that a modified lipid A species, dephosphorylated and underacylated, is displayed on the sur-face of the obligate, gastric bacterium Helicobacter pylori (30) (Figure 6). Further understanding of


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Hopanoid

CC

CH3CH3 OHOH

OHOH

OHOH

CH3

CH3CH3

CH3H3C

CH3 or H CH3

O

BradyrhizobiumjaponicumHelicobacter pylori

OOHOHOHOHO

OO

26–33

1412

P OO

OO OOHOHO

HOHOOOOO

HOHOHOHO

OOHOHOHOHO

OO

OOOO

OO

OOHOHO

OO

OO

OO

OO

OO

NHNH

14

OOOO

HOHO HOHO

14

OONHNH

OO

OHOH

OO

OO

OHOH

**RO**RO

32

*RO*RO

n = 11–18 ( )

( ) n = 17

R* and R** =Hopanoid or H

R* and R** =Hopanoid or H

OHOH

OHOHOHOH

OHOH

OOHOHO

HOHO

HOHOOO

OHOH

OHOH

OHOH

Bacteroidesthetaiotaomicron

OOOO

OOHOHOHOHO

OO

OO

OOHOHO

P

OO

OO

OOHOHO

OO

OO

OO

NHNH

OO

OOOO

HOHO

NH2NH2OO

HOHO

HOHO

OHOH

OHOH

18

18

16

OHOH

OONHNH

18

HOHO

OOHOHOHOHO

OO

OO

OO

P

OOOOHOHO

OO

OO

OO

NHNH

OHOHOO OONHNH

OO

HOHO

OHOH

OHOH

1517

16

17

16

OHOH

P

OO

OOHOHO

OHOH

OOOO

HOHO

HOHO HOHO

OO

OO

OHOH

Figure 6Kdo–lipid A domains of Bacteroides thetaiotaomicron, Helicobacter pylori, and Bradyrhizobium japonicum. The major Kdo–lipid A speciespresented on the surface of each organism is shown. Numbers indicate the length of acyl chains. The red box around the hopanoidhydroxyl group indicates the attachment site to indicated acyl chains on the Kdo–lipid A domain of the organism.

microbial-host homeostasis in the gastrointestinal tract will inform treatment options for inflam-matory bowel syndromes due to diet, pathogen attack, or chemotherapeutic/antibiotic protocols.Other human niches prone to disease caused by perturbations in microbiome populations (suchas the mouth, skin, and respiratory tract) also warrant deeper investigation (27, 44).

GRAM-NEGATIVE BACTERIA WITHOUT ENDOTOXIN

Biosynthesis of the Kdo–lipid A domain and subsequent transport of LPS/LOS onto the bacterialsurface were thought to be essential for survival of gram-negative bacteria (14, 15, 110, 119–121).The first challenge to the notion of essentiality was mounted in 1998, when lpxA in N. meningitidiswas inactivated by directed mutagenesis to produce a viable LOS-deficient mutant (123). Sevenyears later, Peng et al. (98) engineered an lpxA-deficient strain of Moraxella catarrhalis, also devoid


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Endotoxin:a synonym of LPS;introduced in thenineteenth century todescribe a heat-stabletoxin associated withgram-negative bacteria

Adjuvant: a vaccinecomponent thatincreases the immuneresponse to an antigen

of endotoxin. Without employing directed mutagenesis, Moffatt et al. (87) isolated LOS-deficientAcinetobacter baumannii using high concentrations (>10 μg/mL) of colistin (polymyxin E). Alter-ations in the composition and compartmentalization of PLs and outer membrane lipoproteinsappear to be emergent properties of LOS-deficient bacteria, but more sophisticated comparativeanalyses are needed. In addition to these organisms, the obligate intracellular pathogen C. tra-chomatis appears to survive inactivation of the Kdo–lipid A biosynthetic pathway when treated withan LpxC inhibitor (93). Loss of LOS correlates with an inability of the bacterium to transitioninto the infectious stage of the chlamydial life cycle. It is worth noting that all these pathogenssynthesize a smaller LOS glycoform that lacks the O-antigen domain found in canonical LPSstructures.

Whereas some organisms survive inactivation of Kdo–lipid A biosynthetic genes, reports indi-cate other gram-negative bacteria naturally lack LOS/LPS altogether. Sphingomonas paucimobilisdoes not appear to produce LOS/LPS but instead produces glycosphingolipids, which are typi-cally found in eukaryotes (68). Glycosphingolipid compartmentalization to the outer membranewas found on fractionation of S. paucimobilis membranes, suggesting that these glycolipids couldsubstitute as divergent chemical analogs to LOS/LPS (69). Pathogenic spirochetes like Borreliaburgdorferi and Treponema pallidum also lack the necessary genes for Kdo–lipid A biosynthesis(48, 49). Work to characterize the surface of these pathogens, which naturally lack outer mem-brane β-barrel proteins, has shown an enrichment of lipoproteins in the outer membrane, similarto what transcriptomic data suggest for A. baumannii (29). It appears that LPS/LOS is not essentialfor some gram-negative bacteria wherein diverse, organism-specific compensatory mechanismsare required for survival.

LPS-deficient gram-negative bacteria have enormous potential for biotechnology applications,including (a) endotoxin-free production of recombinant proteins or antibiotics, (b) bioengineeringof more effective probiotics and (c) production of enhanced vaccines.

ENGINEERING OUTER MEMBRANE COMPONENTSFOR APPLIED TECHNOLOGIES

Detoxified LPS—A Classic Adjuvant Refurbished for Modern Vaccines

Because of their immunostimulatory properties, Kdo–lipid A domains have been evaluatedas potential vaccine adjuvants; however, inherent toxicity of the canonical hexaacylated bis-phosphorylated structure precludes its use in commercial vaccines without prior detoxification.This barrier was first overcome by Ribi and coworkers, who subjected S. enterica serovar MinnesotaR95 to successive acid and base hydrolysis to detoxify LPS, a process that yields monophospho-rylated lipid A (MPL) (102). The current clinical grade MPL adjuvant approved and licensed bythe US Food and Drug Administration is a chemically heterogeneous mixture of several lipid Aspecies (53). The primary lipid A species in the MPL adjuvant, 3-O-deacyl-4′-monophosphoryl A,induces a robust adaptive immune response with bias toward a less inflammatory TLR4-mediatedsignaling pathway, rendering the adjuvant mixture 100-fold less toxic than wild-type LPS, and isthe first adjuvant used that is capable of activating effector T cell responses (62, 83, 95, 111, 129).

Production of the MPL adjuvant relies on costly, harsh chemical treatments. Alternatives toLPS detoxification protocols include engineering bacterial strains to produce MPL in vivo, orKdo–lipid A domains with similarly desirable adjuvant properties (3, 34, 91). A combinatorialrecombinant library of 61 E. coli strains was recently generated to produce homogenous mixturesof Kdo–lipid A domains, which display a gamut of TLR4/MD-2 agonist functions, as well asgraded effects on immune system models in mice (91). Libraries of this nature have tremendous


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DESIGNER OUTER MEMBRANE VESICLES

Outer membrane vesicles (OMVs) are spherical, nonreplicating nanostructures (20–250 nm) naturally produced inlow numbers by gram-negative bacteria. They are formed through blebbing of the outer membrane and periplasmiccompartments. Because OMVs are small and extracellular, ultracentrifugation protocols allow for easy purificationof OMVs from associated source bacteria. There has been renewed interest in developing OMVs as vaccine plat-forms because they naturally contain, or can be designed to package, microorganism-associated molecular patterns(MAMPs) that are recognized by the innate immune system (e.g., lipopolysaccharide). This could make it possibleto formulate vaccines with a combinatorial display of adjuvant and antigen. In addition to nonpathogenicity, onebenefit of OMVs is the near native presentation of antigens normally found on pathogen surfaces. Equally importantis the potential to improve presentation of antigens not normally associated with membranes, through presentationon host-cell membrane vesicles that double as delivery vehicle and adjuvant. Protective OMV vaccines have been inuse since the 1980s, and many others have been investigated. However, their use in vaccine formulations has beenlimited because of hurdles discussed in this manuscript. Certain bacterial strains are well known for preferentiallyenriching OMVs with specific proteins; there is also a precedent for engineering multivalent OMV vaccines.

Capsularpolysaccharides:a thick layer ofpolysaccharides thatsurrounds a bacterialcell

potential in the design of rational adjuvants to improve vaccines, not only for humans but also forspecies throughout the animal kingdom, particularly livestock. To improve on rationally designedlibraries, metagenomic profiling and biochemical characterization of commensal organisms shouldhelp uncover additional mechanisms to produce LPS variants with a range of adjuvant potential(i.e., immunostimulatory but not toxic).

Design of Enhanced Vaccine Platforms—Outer Membrane Vesicles

Current barriers to the practical use of naturally derived outer membrane vesicles (OMVs) inhumans (see sidebar about Designer Outer Membrane Vesicles; Figure 7) will be overcome byinsights into mechanisms that drive OMV formation, improved commercial production pipelines,and clever application of bioengineering techniques derived from discoveries in basic science (1, 55,72, 73, 113). A general mechanism that contributes to OMV production in gram-negative bacteria,regulated by Mla components, may have been recently identified (108). Undesirable reactogenicityof naturally derived OMVs is another important obstacle to overcome. The commercial OMV-based meningococcal group B vaccine (Bexsero) requires LPS detoxification, a procedure that canlimit yields of presented antigenic molecules (45, 61, 127, 137, 138). Reformulation of this vaccine,and indeed future vaccines, could be improved through recombinant preparation of OMVs withless-toxic LPS mixtures, a strategy that has shown promise when applied to existing Neisseriavaccines (137, 138). Design principles employed in the aforementioned E. coli–based adjuvantdetection library could also prove useful in developing desirable OMV detoxification schemes inother target OMV-producing organisms.

Outer Membrane Vesicles as Effective Glycoconjugate Vaccines

The abundant distribution of glycans on the surface of pathogens has always made carbohydratesan attractive vaccine target. Yet pure carbohydrates tend to be poorly immunogenic becausethey elicit a T cell–independent immune response. Protein-linked glycoconjugate vaccines havehistorically been used to enhance immunogenicity of glycan targets (47). Capsular polysaccharides


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Adjuvant (lipid A)engineering

E. coli

Surface displayof protein antigen

Purification of OMVs(adjuvant/antigen)

Surface displayof glycoantigen

Increased OMVproduction

Surface display of antigen(s)

Figure 7Engineering outermembrane vesicles(OMVs) for vaccines.Numerous lipidA–modifying enzymeshave beencharacterized andcould be used toengineer the lipid Adomain oflipopolysaccharide toserve as an adjuvant.Furthermore, bothproteins andcarbohydrates can bepresented as antigenson the bacterialsurface.

and the LPS O-antigen are two such epitopes initially recognized by the host immune system(106, 145). Technology has now been developed to covalently display any of these glycan domainsheterogeneously onto the core Kdo–lipid A domain using the WaaL ligase responsible for nativeO-antigen addition (140) (Figure 7). With this and similar technologies it is feasible that glycanantigen targets from pathogenic eukaryotes, viruses, or even cancer cells can be conjugated toand surface displayed on OMVs. Modulating the adjuvanticity of associated Kdo–lipid A furtherimproves upon this promising design strategy.

Outer Membrane Vesicles Engineered to Present ProteinaceousVaccine Targets

Multiple methods can engineer bacterial OMVs to package recombinant proteins (Figure 7). InE. coli and similar organisms, soluble proteins can be sorted to the bacterial periplasm by includ-ing an N-terminal signal sequence, for either Sec translocase or the twin-arginine translocationapparatus. This would enable recombinant proteins to be packaged in the lumen of subsequentlyformed OMVs. A desired epitope can also be conjugated to the surface of an OMV via fusion to amembrane-anchored protein (46), or through hybridization to an autotransporter domain (112).

Many groups have demonstrated the potential of OMV platforms for protein antigen delivery.Yersinia enterocolitica OMVs containing chimeric fusions of green fluorescent protein (GFP), whichis usually not immunoreactive in humans, to the β-barrel outer membrane protein Ail generate animmune response (71). A similar strategy was developed in E. coli, where researchers fused GFPto enterobacterial ClyA hemolysin, a toxin that doubles as a potential adjuvant (52, 141). BALB/c


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mice vaccinated with the ClyA-GFP OMV formulation developed robust humoral immunity andan impressive effector T cell response to ClyA toxin and GFP. These reports substantiate thepotential of OMV delivery as a universal protein vaccine platform, with an adjustable degree ofantigen valency and the opportunity to include improved adjuvant Kdo–lipid A domains or otheradjuvant biomolecules.

SUMMARY POINTS

1. The gram-negative outer membrane is a complex macromolecular assembly typicallycomposed of lipoproteins, β-barrel outer membrane proteins, extracellular glycans, andan asymmetric lipid bilayer. PLs are compartmentalized to the periplasmic leaflet andLPS to the external leaflet of the outer membrane lipid bilayer.

2. Transport proteins assemble final outer membrane architecture from material built onor within the inner membrane. The separate translocation machinery for lipoproteins,β-barrel outer membrane proteins, or LPS are best understood, wherein a few minorcomponents require further elucidation or identification.

3. LPS is considered essential for viability of gram-negative bacteria, with few reportedexceptions. As such, LPS biosynthetic enzymes and transport proteins are valid targetsfor developing antibacterials, where protein structures of these components will informdiscovery of novel compounds.

4. Remodeling of LPS chemical structures promotes bacterial survival in a given environ-mental or host niche. The type of LPS modifications and how they are regulated acrossgram-negative bacteria are highly variable.

5. Novel biotechnological applications manipulating bacterial surface architecture, e.g.,OMV-based vaccines or probiotic therapies, will require more sophisticated methods fordetermining the structure of outer membrane biomolecules and new tools for under-standing assembly dynamics.

FUTURE ISSUES

1. How is LPS synthesis machinery organized and distributed within the bacterial cell?Given the essentiality and complexity of the Raetz pathway, one could predict that an“endotoxome” exists to ensure optimal flux of key intermediates along the biosyntheticpathway.

2. Why is Kdo–lipid A essential in nearly all gram-negative organisms? Understanding whyendotoxin is dispensable in some organisms, such as A. baumannii, will provide insightinto the general requirement of Kdo–lipid A and possibly allow for engineering of LPS-deficient E. coli.

3. What molecules make up the orphan PL transport pathway? Given the similarity to trans-port protein machinery involved with other outer membrane components, moleculesdedicated to the flip of newly synthesized PLs from the cytosolic to periplasmic leaflet ofthe inner membrane are yet to be identified, as are translocation components that shuttlePLs across the periplasm to the outer membrane.


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4. Why are there multiple genes for the enzymes (Cls and Pgp proteins) required for PGand cardiolipin synthesis? Does this indicate compartmentalization of PL synthesis, ordifferential regulation? Are these isoenzymes important for different stages of bacterialgrowth?

5. Given the unfavorable energy barrier to the movement of large biomolecules acrossa membrane bilayer, how are so-called surface-displayed lipoproteins flipped from theperiplasmic to the surface leaflet of the outer membrane?

6. A more sophisticated, and dynamic, understanding of organisms with complex patterns ofKdo–lipid A domain modification will further inform how these alterations affect naturalprocesses such as general bacterial physiology, pathogenesis, and other symbioses.

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

The authors gratefully acknowledge support from the National Institutes of Health (grantsRO1AI064184, RO1AI76322, and R21AI119879 to M.S.T. and grant F32GM113488 to J.M.B.)and the Army Research Office (grant 61789-MA-MUR to M.S.T.). Space limitations precludecovering all the research in this field, and we sincerely apologize to colleagues whose exceptionalwork could not be included in this review.

LITERATURE CITED

1. Acevedo R, Fernandez S, Zayas C, Acosta A, Sarmiento ME, et al. 2014. Bacterial outer membranevesicles and vaccine applications. Front. Immunol. 5:121

2. Anderson MS, Robertson AD, Macher I, Raetz CR. 1988. Biosynthesis of lipid A in Escherichia coli:Identification of UDP-3-O-[(R)-3-hydroxymyristoyl]-α-D-glucosamine as a precursor of UDP-N2,O3-bis[(R)-3-hydroxymyristoyl]-α-D-glucosamine. Biochemistry 27(6):1908–17

3. Asensio CJA, Gaillard ME, Moreno G, Bottero D, Zurita E, et al. 2011. Outer membrane vesiclesobtained from Bordetella pertussis Tohama expressing the lipid A deacylase PagL as a novel acellularvaccine candidate. Vaccine 29(8):1649–56

4. Bainbridge BW, Karimi-Naser L, Reife R, Blethen F, Ernst RK, Darveau RP. 2008. Acyl chain speci-ficity of the acyltransferases LpxA and LpxD and substrate availability contribute to lipid A fatty acidheterogeneity in Porphyromonas gingivalis. J. Bacteriol. 190(13):4549–58

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MI70-FrontMatter ARI 28 July 2016 14:32

Annual Review ofMicrobiology

Volume 70, 2016 Contents

Strolling Toward New ConceptsKoreaki Ito � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Regulation of mRNA Decay in BacteriaBijoy K. Mohanty and Sidney R. Kushner � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �25

The Role of Microbial Electron Transfer in the Coevolution of theBiosphere and GeosphereBenjamin I. Jelen, Donato Giovannelli, and Paul G. Falkowski � � � � � � � � � � � � � � � � � � � � � � � � � � �45

Genetic Mapping of Pathogenesis Determinants in Toxoplasma gondiiMichael S. Behnke, J.P. Dubey, and L. David Sibley � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �63

The Phage Shock Protein ResponseJosue Flores-Kim and Andrew J. Darwin � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �83

Feedback Control of Two-Component Regulatory SystemsEduardo A. Groisman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 103

Metagenomics and the Human Virome in Asymptomatic IndividualsNicolas Rascovan, Raja Duraisamy, and Christelle Desnues � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 125

Kin Recognition in BacteriaDaniel Wall � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 143

Protists and the Wild, Wild West of Gene Expression: New Frontiers,Lawlessness, and MisfitsDavid Roy Smith and Patrick J. Keeling � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 161

Molecular Genetic Analysis of Chlamydia SpeciesBarbara S. Sixt and Raphael H. Valdivia � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 179

Xenogeneic Silencing and Its Impact on Bacterial GenomesKamna Singh, Joshua N. Milstein, and William Wiley Navarre � � � � � � � � � � � � � � � � � � � � � � � � 199

The Atacama Desert: Technical Resources and the GrowingImportance of Novel Microbial DiversityAlan T. Bull, Juan A. Asenjo, Michael Goodfellow, and Benito Gomez-Silva � � � � � � � � � � � 215

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MI70-FrontMatter ARI 28 July 2016 14:32

Evolution and Ecology of Actinobacteria and Their BioenergyApplicationsGina R. Lewin, Camila Carlos, Marc G. Chevrette, Heidi A. Horn,

Bradon R. McDonald, Robert J. Stankey, Brian G. Fox, and Cameron R. Currie � � � 235

The Power of Asymmetry: Architecture and Assembly of theGram-Negative Outer Membrane Lipid BilayerJeremy C. Henderson, Shawn M. Zimmerman, Alexander A. Crofts, Joseph M. Boll,

Lisa G. Kuhns, Carmen M. Herrera, and M. Stephen Trent � � � � � � � � � � � � � � � � � � � � � � � � � 255

The Modern Synthesis in the Light of Microbial GenomicsAustin Booth, Carlos Mariscal, and W. Ford Doolittle � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 279

Staphylococcus aureus RNAIII and Its Regulon Link Quorum Sensing,Stress Responses, Metabolic Adaptation, and Regulation ofVirulence Gene ExpressionDelphine Bronesky, Zongfu Wu, Stefano Marzi, Philippe Walter,

Thomas Geissmann, Karen Moreau, Francois Vandenesch, Isabelle Caldelari,and Pascale Romby � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 299

Insights into the Coral Microbiome: Underpinning the Healthand Resilience of Reef EcosystemsDavid G. Bourne, Kathleen M. Morrow, and Nicole S. Webster � � � � � � � � � � � � � � � � � � � � � � � � 317

Biological Diversity and Molecular Plasticity of FIC Domain ProteinsAlexander Harms, Frederic V. Stanger, and Christoph Dehio � � � � � � � � � � � � � � � � � � � � � � � � � � � 341

Riboswitch-Mediated Gene Regulation: Novel RNA ArchitecturesDictate Gene Expression ResponsesAnna V. Sherwood and Tina M. Henkin � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 361

Lessons from Digestive-Tract Symbioses Between Bacteriaand InvertebratesJoerg Graf � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 375

Gut Microbiota, Inflammation, and Colorectal CancerCaitlin A. Brennan and Wendy S. Garrett � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 395

Autophagy Evasion and Endoplasmic Reticulum Subversion: The Yinand Yang of Legionella Intracellular InfectionRacquel Kim Sherwood and Craig R. Roy � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 413

(Per)chlorate in Biology on Earth and BeyondMatthew D. Youngblut, Ouwei Wang, Tyler P. Barnum, and John D. Coates � � � � � � � � � 435

Genomics of Natural Populations of Staphylococcus aureusJ. Ross Fitzgerald and Matthew T.G. Holden � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 459

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The Power of Asymmetry: Architecture and Assembly of the … · The gram-negative cell envelope and...

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