proteomics in gram-negative bacterial outer membrane...

PROTEOMICS IN GRAM-NEGATIVE BACTERIAL OUTERMEMBRANE VESICLES

Eun-Young Lee,1 Dong-Sic Choi,1 Kwang-Pyo Kim,2 and Yong Song Gho1*1Department of Life Science and Division of Molecular and Life Sciences,Pohang University of Science and Technology, Pohang, Republic of Korea2Institute of Biomedical Science and Technology, Department of MolecularBiotechnology, Konkuk University, Seoul, Republic of Korea

Received 12 December 2007; accepted 28 February 2008

Published online 17 April 2008 in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/mas.20175

Gram-negative bacteria constitutively secrete outer membranevesicles (OMVs) into the extracellular milieu. Recent researchin this area has revealed that OMVs may act as intercellularcommunicasomes in polyspecies communities by enhancingbacterial survival and pathogenesis in hosts. However, themechanisms of vesicle formation and the pathophysiologicalroles of OMVs have not been clearly defined. While it isobvious that mass spectrometry-based proteomics offers greatopportunities for improving our knowledge of bacterial OMVs,limited proteomic data are available for OMVs. The presentreview aims to give an overview of the previous biochemical,biological, and proteomic studies in the emerging field ofbacterial OMVs, and to give future directions for high-throughput and comparative proteomic studies of OMVs thatoriginate from diverse Gram-negative bacteria under variousenvironmental conditions. This article will hopefully stimulatefurther efforts to construct a comprehensive proteome data-base of bacterial OMVs that will help us not only to elucidatethe biogenesis and functions of OMVs but also to developdiagnostic tools, vaccines, and antibiotics effective againstpathogenic bacteria. # 2008 Wiley Periodicals, Inc., MassSpec Rev 27:535–555, 2008Keywords: outer membrane vesicles; gram-negative bacteria;communicasomes; antibiotics; proteomics; vaccines

I. INTRODUCTION

Communication between cells and the environment is anessential process in living organisms, and intercellular commu-nication is believed to be mediated mainly by the secretion ofsoluble factors, cell-to-cell contacts, and tunneling machinery

such as nanotubes (Ratajczak et al., 2006). Recently, amechanism mediated by membrane vesicles (MVs), which arespherical, bilayered proteolipids with an average diameter of0.03–1 mm, has drawn much attention (Beveridge, 1999;Ratajczak et al., 2006). The secretion of MVs is a universalcellular process occurring from simple organisms to complexmulticellular organisms, including humans (Thery, Zitvogel,& Amigorena, 2002; Mashburn-Warren & Whiteley, 2006).Throughout evolution, both prokaryotic and eukaryotic cellshave adapted to manipulate MVs for intercellular communi-cation via outer membrane vesicles (OMVs) in the case ofGram-negative bacteria and microvesicles in eukaryotic cells.Increasing evidence suggests that MVs act as potent communi-casomes, that is, nano-sized extracellular organelles that playdiverse roles in intercellular communication (Choi et al., 2007),and that the biogenesis and functions of MVs may share manyfeatures in different biological systems. Thus, the study of MVsprovides crucial keys to understanding the intercellular commu-nication network in living organisms and the evolutionaryconnections between prokaryotes and eukaryotes (Mashburn &Whiteley, 2005).

A wide variety of Gram-negative bacteria constitutivelysecrete OMVs during growth (Beveridge, 1999), includingEscherichia coli, Neisseria meningitidis, Pseudomonas aerugi-nosa, Shigella flexneri, and Helicobacter pylori (Devoe &Gilchrist, 1973; Hoekstra et al., 1976; Fiocca et al., 1999;Kadurugamuwa & Beveridge, 1999). OMVs are spherical,bilayered proteolipids with an average diameter of 20–200 nm;they are composed of outer membrane proteins, lipopolysac-charide (LPS), outer membrane lipids, periplasmic proteins,cytoplasmic proteins, DNA, RNA, and other factors associatedwith virulence (Horstman & Kuehn, 2000; Wai et al., 2003;Kuehn & Kesty, 2005; Bauman & Kuehn, 2006; Nevot et al.,2006; Lee et al., 2007). Studies of OMVs from diverse bacterialstrains suggest their roles in the delivery of toxins to host cells, thetransfer of proteins and genetic material between bacterial cells,cell-to-cell signals, and the elimination of competing organisms(Kuehn & Kesty, 2005; Mashburn-Warren & Whiteley, 2006).Because OMVs are essential to bacterial survival and patho-genesis in the host, modulation of vesicle formation and theirfunctions may be a useful objective in relation to the developmentof antibiotics (Henry et al., 2004; Lee et al., 2007).

Although recent research in this area has revealed the diversefunctions of OMVs, the mechanisms of vesicle formation and ofprotein sorting into OMVs, as well as the pathophysiological

Mass Spectrometry Reviews, 2008, 27, 535– 555# 2008 by Wiley Periodicals, Inc.

————Contract grant sponsor: National R&D Program for Cancer Control,

Ministry of Health & Welfare, Republic of Korea; Contract grant

number: 0320380-2; Contract grant sponsor: Korea Basic Science

Institute K-MeP; Contract grant number: T27021; Contract grant

sponsor: Korea Science and Engineering Foundation (KOSEF)

(MOST); Contract grant number: R15-2004-033-05001-0; Contract

grant sponsor: Brain Korea 21 fellowship.

*Correspondence to: Yong Song Gho, Department of Life Science,

Division of Molecular and Life Sciences, Pohang University of Science

and Technology, San31 Hyojadong, Pohang, Kyungbuk 790-784,

Republic of Korea. E-mail: [email protected]

roles of OMVs, have not been clearly defined. To address theseissues, vesicular proteins should be comprehensively identified.Proteomics offers a powerful approach to decode the proteincomponents of OMVs. Mass spectrometry (MS)-based proteo-mic studies have been used in human microvesicles to identifythousands of vesicle-associated proteins from diverse cancer celllines, immune cells, and human fluids including serum, urine, andbreast milk (Pisitkun, Shen, & Knepper, 2004; Jin et al., 2005;Yates et al., 2005; Admyre et al., 2007; Choi et al., 2007).However, only a few proteomic analyses of bacterial OMVs havebeen reported, although they are more ubiquitous and easier toobtain than human samples (Post et al., 2005; Bauman & Kuehn,2006; Nevot et al., 2006; Lee et al., 2007). These studies did notachieve high-throughput proteomics and identified only a smallnumber of well-known proteins, except for E. coli-derived OMVs(Lee et al., 2007). In contrast to native OMVs, several proteomicstudies have been performed on detergent-extracted OMVs(DOMVs), which are made from whole bacteria with a detergenttreatment (Nally et al., 2005; Ferrari et al., 2006; Uli et al., 2006;Vipond et al., 2006). Since outer membrane proteins and LPS ofOMVs can induce a host immune response, DOMVs frompathogenic strains are promising vaccine candidates. DOMVsderived from N. meningitidis are now in clinical trials (Girardet al., 2006). However, although DOMVs are clinically importantand similar in size and morphology to native OMVs (Ferrari et al.,2006), information on DOMV components does not provide anyclue to the biogenesis and functions of native OMVs in bacterialcommunities. Therefore, global proteomic studies of nativeOMVs derived from diverse nonpathogenic and pathogenicbacteria will help us not only elucidate the biogenesis andfunctions of OMVs but also develop diagnostics, vaccines, andantibiotics effective against pathogenic strains.

After an overview of previous biochemical and biologicalstudies on Gram-negative bacterial OMVs, this review will focuson current strategies used for proteomic analyses of OMVs,emphasizing the impact of those studies on this emerging field.Finally, future directions for high-throughput and comparativeproteomics studies of OMVs from diverse Gram-negativebacteria under various environmental conditions will be high-lighted in hopes of advancing both basic and clinical sciences.

II. PROTEIN SECRETION INGRAM-NEGATIVE BACTERIA

A. Conventional Protein Secretion Pathways inGram-Negative Bacteria

Gram-negative bacteria are enclosed within two lipid bilayers,consisting of a phospholipid-rich inner membrane and aphopholipid- and LPS-rich outer membrane. The periplasmicspace separates these membranes and contains peptidoglycans(Beveridge, 1999). In contrast to nucleated eukaryotic cells,bacterial cytoplasm is not compartmentalized, no large organ-elles are present, and no active transport mechanisms such asmolecular motors are known (Howard, Rutenberg, & de Vet,2001). However, protein secretion is a basic cellular functionfound in all living organisms. Gram-negative bacteria have

evolved several secretion pathways, including some mechanismscommon among human and plant pathogens (Lory, 1992).Approximately 20% of the polypeptides synthesized by bacteriaare located partially or completely outside of the cytoplasmfollowing secretion (Pugsley, 1993; Kostakioti et al., 2005).

Previously, six major protein secretion pathways in Gram-negative bacteria were known; they can be classified by thepresence or absence of a required signal sequence, Sec (Table 1).Sec-dependent pathways include the type II, IV, and V secretionsystems, which utilize cleavable N-terminal signal peptidesfor protein transport across the inner membrane (Kostakiotiet al., 2005). The type II secretion system, also known as thegeneral secretory pathway, is responsible for the secretion ofseveral toxins and utilizes the Tat signal motif in addition to Sec(Voulhoux et al., 2001). The type IV secretion system allows thetransfer of DNA and multi-subunit toxins, including pertussistoxin, by conjugation machinery (Cascales & Christie, 2003).Depending on the bacterial strain, both Sec-dependent and Sec-independent secretion have been observed in the type IV system(Desvaux et al., 2004). Proteins using type V secretion, alsoknown as the autotransporter pathway, are translocated acrossthe outer membrane via a transmembrane pore formed by aself-encodedb-barrel structure (Desvaux, Parham, & Henderson,2004).

Sec-independent pathways include the type I, III, andVI secretion systems, which are one-step mechanisms that do notinvolve periplasmic intermediates (Kostakioti et al., 2005). In thetype I secretion system, an ATP-binding cassette transporter-likechannel transports various molecules from ions and drugs toproteins (Binet & Wandersman, 1995). The type III system isspecific for the transport of factors by pathogenic bacteria andallows the direct injection of a protein into a eukaryotic host cell(Galan & Collmer, 1999). Recently, the secretion of severalproteins via the type VI pathway was reported in Vibrio choleraeand P. aeruginosa (Mougous et al., 2006; Pukatzki et al., 2006).

B. OMVs as a Novel Protein Secretion Pathway inGram-Negative Bacteria

Protein secretion via OMVs in Gram-negative bacteria hascome of age by defining a distinct type that is independent ofthe type I–VI secretory systems (Kuehn & Kesty, 2005). Thedynamic feature of Gram-negative cell wall is that it constantlydischarges OMVs from the cell surface (Fig. 1), which is notobserved in Gram-positive bacteria (Beveridge, 1999). Growingevidence suggests that hundreds of proteins, lipids, and geneticmaterial might be secreted via OMVs (Dorward, Garon, & Judd,1989; Horstman & Kuehn, 2000; Mashburn-Warren & Whiteley,2006; Lee et al., 2007). For example, export of cytolysin A(ClyA) into the extracellular milieu, which does not follow thesix known protein secretion mechanisms (Wai et al., 2003), ismediated by OMVs (Bendtsen et al., 2005). ClyA is a pore-forming cytotoxin protein expressed by E. coli and some otherenterobacteria. Therefore, shedding of bacterial OMVs is a novelprotein secretion mechanism in Gram-negative bacteria andthis system performs a variety of important ‘‘remote-control’’functions for bacterial growth and survival, as well as patho-genesis in hosts.

& LEE ET AL.

536 Mass Spectrometry Reviews DOI 10.1002/mas

III. GRAM-NEGATIVE BACTERIAL OMVS

A. History of Gram-Negative Bacterial OMVs

The discovery of OMVs as the universal secretory machinery inGram-negative bacteria dates back almost 40 years, owing to theelectron microscope (EM). In the 1960s, active research on theultrastructure of bacteria revealed the presence of OMVs andthey were named blebs, membranous elements, or channels(Bladen & Waters, 1963; Bayer & Anderson, 1965). Thin section

EM studies of Gram-negative bacteria in that era suggestedthat secretory vesicles constantly emanate from bacteria underlysine-limited, phosphate-limited, and normal growth conditions(Knox, Vesk, & Work, 1966; Mergenhagen, Bladen, & Hsu,1966; Ingram, Cheng, & Costerton, 1973; Lindsay et al., 1973).

Moreover, OMVs have been found in every environment inwhich Gram-negative bacteria reside, from laboratory cultures,including planktonic and surface-attached biofilm, to naturalenvironments such as domestic water drains, sewage, andriverbeds (Beveridge, 1999; Schooling & Beveridge, 2006).The identification of OMVs from a variety of Gram-negativestrains including E. coli, Veillonella, V. cholerae, P. aeruginosa,Salmonella typhimurium, and N. meningitidis implies thatvirtually all Gram-negative bacteria produce OMVs as an activeand essential process (Bladen & Mergenhagen, 1964; Bayer &Anderson, 1965; Chatterjee & Das, 1967; Devoe & Gilchrist,1973; Beveridge, 1999).

B. Current Research Trends on Gram-NegativeBacterial OMVs

Although bacterial OMVs have long been studied, previousreports have focused primarily on pathogenic strains, and manyquestions remain to be answered before attaining an integratedview of the biogenesis and the pathophysiological functions ofOMVs from both nonpathogenic and pathogenic bacteria (Kuehn& Kesty, 2005). Recently, biochemical analyses and a fewproteomics applications revealed that bacterial OMVs includeproteins, lipids, and genetic material (Kadurugamuwa &

TABLE 1. Conventional protein secretion pathways in Gram-negative bacteria

FIGURE 1. Discharge of OMVs by Gram-negative bacteria. (A) Thin-

section EM of E. coli DH5a, showing the formation of OMVs (arrows)

on the cell surface. Bar¼ 100 nm (B) Magnified EM image of OMVs.

The membrane bilayer (arrow) is easily visible. Bar¼ 50 nm. [Reprinted

with permission from Proteomics 7: 3143-3153, 2007, Lee EY et al.,

Global proteomic profiling of native outer membrane vesicles derived

from Escherichia coli, with permission from Wiley-VCH Verlag GmbH

& Co. KGaA. Weinheinm, Germany. Copyright 2007.]

PROTEOMICS IN BACTERIAL OUTER MEMBRANE VESICLES &

Mass Spectrometry Reviews DOI 10.1002/mas 537

Beveridge, 1995; Horstman & Kuehn, 2000, 2002; Post et al.,2005; Bauman & Kuehn, 2006; Nevot et al., 2006; Lee et al.,2007). Moreover, genetic studies examining some candidategenes that modulate the level of vesiculation suggest possiblebiogenesis models for bacterial OMVs (Mashburn-Warren &Whiteley, 2006; McBroom et al., 2006; McBroom & Kuehn,2007). In addition, the pathophysiological roles of OMVs inthe interspecies world, as well as polymicrobial communities,are being gradually elucidated. Some groups are studying thephysiological relevance of stress responses, including heat-shockand antibiotic treatment, which alter the generation of OMVs(Katsui et al., 1982; Kadurugamuwa & Beveridge, 1995).

In the following subsections, we summarize previousbiochemical and biological research on Gram-negative bacterialOMVs.

1. Components of Gram-Negative Bacterial OMVs

Although bacterial OMVs were believed to consist of proteinsand lipids from the outer membrane and periplasm but notfrom either the inner membrane or cytoplasmic components(Horstman & Kuehn, 2000), growing evidence suggests thatvirulence factors including LPS, cytoplasmic proteins, andgenetic material such as DNA and RNA are components ofOMVs (Dorward, Garon, & Judd, 1989; Kolling & Matthews,1999; Ferrari et al., 2006; Lee et al., 2007).

The presence of vesicular proteins has been analyzed bysodium dodecyl sulfate–polyacrylamide gel electrophoresis(SDS–PAGE) with Coomassie or silver staining, as well asWestern blotting with in-house antibodies (Horstman & Kuehn,2000; Ferrari et al., 2006). Several outer membrane proteinsincluding OmpA, OmpC, and OmpF have been identified, whichrepresent the most abundant proteins; they have been found in allstrains of E. coli studied to date (Kesty et al., 2004). Biochemicalanalysis has detected periplasmic proteins such as alkalinephosphatase and AcrA in OMVs, supporting the hypothesis thatsome periplasmic proteins are sorted into OMVs by encapsula-tion during vesicle formation (Horstman & Kuehn, 2000).

Pathogenic bacteria secrete OMVs that contain severalvirulence factors, including toxins, adhesins, invasins, and otherrelated enzymes (Kesty & Kuehn, 2004; Kesty et al., 2004;Kuehn & Kesty, 2005). Toxins are the best characterized factorsthat might be involved in OMV-mediated bacterial pathogenesis.For example, LPS, which are critical components of allGram-negative bacteria, can activate host immune responsesvia production of various cytokines. The large number ofvesicular toxins that contribute to the pathogenesis of infectionwas summarized in a recent report (Kuehn & Kesty, 2005). Theability of OMVs to adhere to and invade host cells via adhesinsand invasins is important in initiating vesicle-mediated patho-genesis. For example, Ail, IpaB, IpaC, and IpaD play importantroles in interactions with and invasion of host cells (Kaduruga-muwa & Beveridge, 1998; Kesty & Kuehn, 2004).

Thin layer chromatography revealed the presence ofglycerophospholipids, phosphatidlyethanolamine, phosphatidyl-glycerol, and cardiolipin in enterotoxigenic E. coli-derivedOMVs (Horstman & Kuehn, 2000). The lipid profiles of OMVsare similar to those of the outer membrane, but their specific types

and ratios have not been determined. Therefore, systematicstudies on the lipid composition of OMVs that might governvesicular fate and function under physiological and pathologicalconditions are a challenge for lipidomics.

The presence of DNA within OMVs was identified in N.gonorrhoeae, Haemophilus influenzae, P. aeruginosa, and E. coliO157:H7 (Mashburn-Warren & Whiteley, 2006). The fact thatvesicular DNA is resistant to DNase treatment suggests that it ispresent in the lumen of OMVs. Therefore, DNA within OMVs isexpected to be protected from nucleases, thereby increasing theefficiency of vesicle-mediated DNA delivery into a recipient cell.DNA within OMVs can originate by one of two mechanisms:DNA exists in the periplasm, and along with other periplasmiccomponents, becomes encapsulated, or DNA in the extracellularenvironment, potentially derived from lysed bacteria, is incorpo-rated into OMVs by the ‘‘opening and closing’’ phenomenon(Renelli et al., 2004). RNA is also a vesicular component,although in lesser amounts than DNA (Dorward, Garon, & Judd,1989).

While multiple sources of vesicular components obviouslycontribute to the biogenesis and functions of OMVs, the exactvesicular composition of OMVs derived from different bacterialstrains should not be identical. These differences in vesicularcomponents may define unique physiological and pathologicalfunctions of OMVs derived from specific strains of Gram-negative bacteria. However, limited data are available on strain-specific OMV components, a problem that must be solved in thenear future.

2. Biogenesis of Gram-Negative Bacterial OMVs

The mechanisms by which Gram-negative bacteria shed OMVsand sort vesicle-targeted proteins have not been fully determined.However, genetic mutant studies, biochemical experiments, andmicroscopic observations suggest three plausible models forOMV formation, as shown in Figure 2 (Mashburn-Warren &Whiteley, 2006). The first model suggests that vesicles aregenerated by the loss of cell envelope integrity that occurs whenthe outer membrane expands more quickly than the underlyingpeptidoglycan layer (Wensink & Witholt, 1981). The secondmodel is that the formation of OMVs is linked to the turgorpressure of the cell envelope, which changes with theaccumulation of peptidoglycan fragments in the periplasm (Zhouet al., 1998). A recent study in P. aeruginosa proposed the thirdmodel, in which quinolone signal molecules enhance the anionicrepulsion between LPS by destabilizing the Mg2þ and Ca2þ saltbridges in the outer membrane, thereby causing membraneblebbing (Mashburn & Whiteley, 2005). These three mechanismsmay not be mutually exclusive and may contribute collectively tothe biogenesis of bacterial OMVs. Further study is needed, sincerecent studies suggest that OMV production is independent ofmembrane instability (McBroom et al., 2006) and controversialresults have come from the mutation of lipoproteins (Bernadacet al., 1998; Rolhion et al., 2005).

Currently, little is known about the mechanisms by whichbacteria sort proteins into OMVs. However, analysis of vesicularproteins by SDS–PAGE have shown different banding patterns inthe OMVs compared to the outer membrane, periplasm, and other

& LEE ET AL.


cellular fractions, suggesting that specific protein sortingmechanisms are in effect when OMVs are produced (Fig. 3)(Horstman & Kuehn, 2000; Lee et al., 2007). Although furtherstudy of this issue is needed, these findings imply the presence of‘‘hot spots’’ for vesicle budding: the outer membrane becomesloosely attached to the bacterium at specific sites and forms

OMVs that are shed into the extracellular milieu. Budding ofOMVs from the limiting membrane can be inferred from humancell-derived microvesicles that are reportedly associated withspecific membrane sites called lipid rafts (Rajendran & Simons,2005).

3. Physiological and Pathological Functions ofGram-Negative Bacterial OMVs

Since OMVs bear diverse proteins, LPS, outer membrane lipids,genetic material, other factors associated with virulence, andconjugational machinery to target other bacteria or host cells,they should play diverse roles as intercellular communicasomesnot only in bacterial communities but also in interspecies worlds.Gram-negative bacterial OMVs might play a role in the transferof proteins and genetic material between bacterial cells, in theelimination of competing organisms, in cell-to-cell signaling andbacterial survival, and in the delivery of toxins to host cells(Kuehn & Kesty, 2005; Mashburn-Warren & Whiteley, 2006).

OMVs are involved in the transfer of proteins as well asgenetic material in polymicrobial communities. OMVs derivedfrom P. aeruginosa showed beneficial effects to their own groupby transferring an antibiotic resistance protein, b-lactamase,to increase survival (Mashburn-Warren & Whiteley, 2006).Predatory roles of OMVs have been proposed, in which E. coli-and P. aeruginosa-derived vesicles can kill competing bacteria bypeptidoglycan degradation or cell lysis via vesicular componentssuch as murein hydrolases (Kadurugamuwa & Beveridge, 1996;Li, Clarke, & Beveridge, 1998). Furthermore, OMVs packagechromosomal, plasmid, and phage DNA as well as RNA, whichmay increase genetic diversity by transforming neighboringbacteria (Kuehn & Kesty, 2005).

FIGURE 3. Proteins present in OMVs derived from E. coli DH5a.

Coomassie blue-stained SDS–PAGE comparison of the proteins from

whole-cell lysates (WC), periplasmic proteins (PP), outer membrane

proteins (OMP), and OMVs showing specific protein sorting into

vesicles. Molecular weight standards are indicated on the left (kDa).

[Reprinted with permission from Proteomics 7: 3143–3153, 2007, Lee

EY et al., Global proteomic profiling of native outer membrane vesicles

derived from Escherichia coli, with permission from Wiley-VCH Verlag

GmbH & Co. KGaA. Copyright 2007.]

FIGURE 2. Proposed models for biogenesis of Gram-negative bacterial

OMVs. Model 1: OMVs are liberated from specific regions on the cell

surface where peptidoglycan-associated lipoproteins are missing due

to the faster expansion of the outer membrane than the underlying

peptidoglycan layer. Model 2: Accumulation of peptidoglycan frag-

ments in the periplasm causes increased turgor pressure, thereby increas-

ing blebbing of the outer membrane. Model 3: In the P. aeruginosa outer

membrane, PQS sequesters the positive charge of Mg2þ, which results in

enhanced anionic repulsion between LPS molecules and membrane

blebbing. OM, outer membrane; PG, peptidoglycan; IM, inner mem-

brane; PQS, Pseudomonas quinolone signal. [Reprinted with permission

from Molecular Microbiology 61: 839–846, 2006, Mashburn-Warren

and Whiteley, Special delivery: vesicle trafficking in prokaryotes, with

permission from Blackwell Publishing Ltd Oxford, UK. Copyright

2006.]



Bacterial OMVs also play protective roles that contribute tobacterial survival by reducing toxic compounds and antibiotics,and by facilitating the release of attacking phages (Loeb &Kilner, 1978; Kobayashi et al., 2000). Recently, McBroomand Kuehn (2007) performed a genetic mutant study understress conditions by treatment with 10% ethanol or an outermembrane-damaging antimicrobial peptide and found that over-vesiculating mutant strains had enhanced survival due to therelease of misfolded proteins via increased shedding of OMVs(McBroom & Kuehn, 2007).

Some bacteria have coevolved in a symbiotic relationshipwith their hosts, although infectious pathogens may cause someproblems in hosts. Therefore, examining the roles of OMVsin the interspecies community is important to understandingthe mechanisms of symbiosis and pathogenesis. Diverse Gram-negative pathogens have exploited potent virulence strategies byvesicle-mediated toxin delivery to host cells (Kuehn & Kesty,2005). In the case of enterotoxigenic E. coli-derived heat-labileenterotoxins (LT), most (�95%) are secreted via OMVs (Horst-man & Kuehn, 2002). Some toxins, including LTand leukotoxin,are more active when they are associated with vesicles rather thanin a free form (Kuehn & Kesty, 2005). Moreover, LPS and theouter membrane proteins present in vesicles can activate hostimmune responses via Toll-like receptors. These LPS andsurface-localized antigens from pathogenic bacteria-derivedvesicles can cause overstimulated inflammatory responses orseptic shock in hosts (Namork & Brandtzaeg, 2002).

IV. CURRENT PROTEOMICS IN GRAM-NEGATIVEBACTERIAL OMVS

Although previous biochemical, biological, and genetic studieshelp us to understand the vesicular components, biogenesis,and diverse roles of OMVs in polyspecies communities, theinformation provided by those studies does not afford a com-prehensive understanding of the emerging biology of bacterialOMVs. MS-based proteomic studies offer a powerful way toclarify the mechanisms of vesicle formation and the pathophy-siological roles of OMVs by drawing a global map of diversebacterial OMVs.

Proteomic studies have been successfully used to studywhole cellular proteins from diverse bacteria and to studybacterial adaptation to various stress situations (Washburn &Yates, 2000; Hecker & Volker, 2004; Bandow & Hecker, 2007).However, complexity at the whole-cell level has a limited abilityto provide systematic insights into cell biology and often failsto identify low-abundance proteins, including outer membraneproteins, which are inevitably masked by high-abundanceproteins (Brunet et al., 2003). Because OMVs may representnano-sized extracellular organelles of bacteria, the applicationof organelle proteomics to bacterial OMVs will define newbiological processes for interactions among bacterial cells,symbiosis, and pathogenesis in hosts.

Regardless of the importance of native OMVs, which mayact as key mediators for intercellular communications, previousproteomic research has focused on DOMVs (Nally et al., 2005;Ferrari et al., 2006; Uli et al., 2006; Vipond et al., 2006). Since

DOMVs are produced under artificial conditions, DOMVs andnative OMVs may have different protein components. As shownin Table 2, a few studies have been published regarding theproteomics of native OMVs. These include OMVs isolated frompathogenic bacteria such as N. meningitidis and P. aeruginosa(Post et al., 2005; Bauman & Kuehn, 2006), nonpathogenicbacteria including Pseudoalteromonas antarctica NF3, andE. coli (Nevot et al., 2006; Lee et al., 2007), and peculiar mutantforms of N. meningitidis and extraintestinal pathogenic E. coli(Ferrari et al., 2006; Berlanda Scorza et al., 2007).

In the following subsections, we summarize previousproteomic studies on native OMVs derived from Gram-negativebacteria and discuss in detail clues provided by proteomics thatelucidate the biogenesis and functions of bacterial OMVs.

A. Preparation of Gram-Negative Bacterial OMVs

Efficient OMV preparation without any contamination by non-vesicular components is a critical prerequisite for proteomicanalysis. Generally, bacterial OMVs are isolated from the culturesupernatant using a combination of differential centrifugationto remove cells and cell debris, and ultracentrifugation to pelletthe OMVs (Wai et al., 2003). However, ultracentrifugationalone does not discriminate between OMVs and other membranedebris or large protein aggregates. Filtration of the cell-culturesupernatant through 0.22–0.45 mm filters before ultracentrifu-gation may reduce contamination (Horstman & Kuehn, 2000;Ferrari et al., 2006; Berlanda Scorza et al., 2007). Recent progressin the biology of OMVs shows that density gradient centrifuga-tion is one of the best separation methods to remove OMVs fromcontaminating protein aggregates, pili, and flagella (Bauman &Kuehn, 2006; Lee et al., 2007). Gel filtration chromatographyis an alternative method to isolate OMVs with high purity.The beads in a gel filtration chromatography column containpores that can fractionate vesicles on the basis of differentialdiffusion and size exclusion. Post et al. (2005) purifiedOMVs from N. meningitidis using a Sephacryl S500 column(Post et al., 2005). Gel filtration chromatography is an effectivemethod for purifying OMVs that are relatively homogeneousin size.

Recently, we reported the proteomic profile of native OMVsderived from representative strains of nonpathogenic E. coliDH5a (Lee et al., 2007). With some modifications of previouslydescribed purification methods (Horstman & Kuehn, 2000; Waiet al., 2003; Rolhion et al., 2005), we obtained highly pureOMVs secreted by DH5a cells using two sequential steps(Fig. 4A). In the first step, OMVs were isolated from the culturesupernatant using a combination of differential centrifugation toremove cells and cell debris; filtration through a 0.45 mm filter;pre-centrifugation at 20,000g and 40,000g to remove any largevesicles, vesicle aggregates, and cell debris; and then ultra-centrifugation at 150,000g. In the second purification step, theenriched OMVs were further purified using sucrose densitygradients to remove any remaining contaminants. EM of purifiedOMVs revealed that almost all were small, closed vesiclesranging from 20 to 40 nm in diameter, and no membrane whorls,fragments of lysed vesicles, large vesicles, or pili were detected(Fig. 4B).

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B. Proteomic Analysis of Gram-NegativeBacterial OMVs

In addition to the purity of OMVs, reduction of proteomecomplexity by protein separation is the key step to identifying alarge number of vesicular proteins via proteomic analysis. Two-dimensional gel electrophoresis (2-DE) is a powerful tool for

protein separation. However, the limitations of this approachfor membrane proteins are well-known. The major obstacleis poor solubility of membrane proteins in the non-detergentisoelectric focusing buffer that causes the precipitation ofproteins at their isoelectric points (Wu & Yates, 2003).Moreover, 2-DE cannot properly resolve high molecularweight, very basic, or hydrophobic proteins (Wu & Yates,

TABLE 2. Summary of proteomic studies on native bacterial OMVs

FIGURE 4. Methods of preparation for OMVs derived from E. coli DH5a. A: Procedure for preparing

OMVs from DH5a. B: Negative-staining transmission EM of purified OMVs after sucrose density gradient

centrifugation, showing a homogeneous size of 20–40 nm. Bar¼ 50 nm. [Reprinted with permission from

Proteomics 7: 3143–3153, 2007, Lee EY et al., Global proteomic profiling of native outer membrane

vesicles derived from Escherichia coli, with permission from Wiley-VCH Verlag GmbH & Co. KGaA.

Copyright 2007.]



2003). The fact that outer membrane proteins, the majorcomponents of OMVs, are highly basic implies that majorcomponents of vesicular proteins are incompletely resolvedin 2-DE (Post et al., 2005).

The combination of one-dimensional (1-D) SDS–PAGEand liquid chromatography (LC)–MS/MS provides a powerfulalternative to 2-DE-based proteomic analysis (Aebersold &Mann, 2003). Although 1-D SDS–PAGE can efficientlyseparate proteins, even membrane proteins, the limitation ofthis approach in high-throughput mass analysis is the increasedprotein complexity in each gel fraction. This problem caneasily be overcome by using LC to separate the extractedpeptides based on hydrophobicity. Several groups have used thisstrategy (Post et al., 2005; Nevot et al., 2006), but they onlyexamined prominent protein bands of interest from gels, whichmight result in the identification of less than 50 vesicularproteins because of missing the less abundant and unknownproteins (Table 2). Because the molecular weights of vesicularproteins are different and OMVs also contain less abundantproteins, our group separated vesicular proteins by 1-D SDS–PAGE, cut the gel into five slices of equal size, and subjected itto trypsin digestion. From two independent nano-LC electro-spray ionization (ESI)–MS/MS analyses of the extractedpeptides, we identified 2,606 and 2,816 proteins with high-confidence peptide sequences, with an error rate less than 1% (Fscore> 2.17). Since peptides that are shared by multipleproteins are less informative than unique peptides, we usedthe protein hit score (PHS) for reliable protein identification(Park et al., 2006). Our analysis showed that proteins withPHS> 1 were identified by multiple peptides that are uniqueand shared with only a few proteins. Using a highly stringentfilter allowing only proteins with PHS> 1 that were filteredagain to reduce any repeated or homologous proteins, we finallyidentified a total of 141 proteins, including 127 previouslyunknown vesicular proteins, with high confidence, and reprodu-cibility (Lee et al., 2007).

C. Proteins Identified by Proteomic Analyses ofGram-Negative Bacterial OMVs

The available proteomic studies on bacterial OMVs have definedmore than 200 vesicular proteins from four native bacterial andtwo mutant strains (Table 2) (Post et al., 2005; Bauman &Kuehn, 2006; Ferrari et al., 2006; Nevot et al., 2006; BerlandaScorza et al., 2007; Lee et al., 2007). Although the names ofbacterial proteins are different in each species, they can beclassified into protein families based on both their sequencehomology and function. When the identified vesicular proteinswere categorized by protein family, several protein familieswere common in OMVs derived from several species of Gram-negative bacteria (Table 3). Porins (Omps, PorA, PorB, andOprF), abundant outer membrane proteins, are found in mostOMVs. Murein hydrolases (Mlt and SLT) are responsible for thehydrolysis of certain cell wall glycopeptides, particularlypeptidoglycans. Multidrug efflux pumps (Mtr, Mex, and TolC)function in the release of toxic compounds (Kobayashi et al.,2000). Moreover, most OMVs derived from different strainscontain ABC transporters (LamB and FadL), protease/chaper-

one proteins (DegQ/SurA), and motility proteins related tofimbriae (FliC) or pilus (PilQ). These conserved vesicularproteins provide an integrated view of the biogenesis andfunction of OMVs in nonpathogenic and pathogenic bacteria,which will be discussed in the following subsections. Forpathogenic strains, virulence factors including hemolysin, IgAprotease, and macrophage infectivity potentiator were alsoidentified (Post et al., 2005; Ferrari et al., 2006).

Many researchers believe that OMVs are composed solely ofouter membrane and periplasmic proteins, whereas cytoplasmicproteins are excluded (Horstman & Kuehn, 2000). However,although it is still debated, proteomic analyses have shown thatnative OMVs and DOMVs contain cytoplasmic proteins as well(Molloy et al., 2000; Henry et al., 2004; Ferrari et al., 2006; Weiet al., 2006; Xu et al., 2006; Lee et al., 2007). Among vesicle-associated cytoplasmic proteins, highly abundant proteins likeEF-Tu, GroEL, DnaK, and two ribosomal proteins (S1 andL7/12) have also been detected from cell supernatants or outermembrane fractions (Ferrari et al., 2006). Moreover, the fact thatvesicles carry DNA and RNA, and that translation of outermembrane proteins might occur simultaneously with theirintegration into the membrane, suggest that transcriptional andribosomal proteins can be sorted into vesicles during theinformational process (Dorward, Garon, & Judd, 1989; Kadur-ugamuwa & Beveridge, 1995; Kolling & Matthews, 1999; Yaronet al., 2000). Determining whether cytoplasmic proteins areindeed components of native OMVs should be a goal of futurestudies.

D. Proteins Involved in Biogenesis of Gram-NegativeBacterial OMVs

Although the mechanism of OMV formation has not yet beenelucidated, several vesicular proteins identified by proteomicanalyses support the first and second models (Fig. 2). Omps, Tol-Pal, YbgF, and Lpp lipoproteins found in the OMV proteomeshould be involved in outer membrane integrity and mighthelp liberate OMVs from the bacterial cell surface by initiatingfaster expansion of the outer membrane than the underlyingpeptidoglycan layer (Bernadac et al., 1998). Related tothe second model, murein hydrolases, including MltA, MipA,MltE, and SLP, may lead to the accumulation of peptidoglycanfragments in the periplasmic space, resulting in increased turgorpressure and causing the discharge of OMVs (Lommatzschet al., 1997).

When OMV proteomes are annotated according to theirsubcellular distribution, OMVs are highly enriched in outermembrane and periplasmic proteins, whereas inner membraneproteins are excluded (Post et al., 2005; Lee et al., 2007).For example, of 141 proteins identified in E. coli-derived OMVs,65 (46.1%), 16 (11.3%), 7 (5.0%), 52 (36.9%), and 1 (0.7%)proteins were derived from the outer membrane, periplasm, innermembrane, cytoplasm, and extracellular space, respectively(Fig. 5) (Lee et al., 2007). In contrast, from the EchoBASEdatabase of all 4,345 E. coli proteins, 149 (3.4%), 350 (8.1%),974 (22.4%), 2,862 (65.9%), and 10 (0.2%) are distributed inthe outer membrane, periplasm, inner membrane, cytoplasm, andextracellular space, respectively, suggesting that outer membrane

& LEE ET AL.


TABLE 3. Protein families identified by proteomic analyses of Gram-negative bacterial OMVs

(Continued )



TABLE 3. (Continued )

(Continued )

& LEE ET AL.


and periplasmic proteins are more commonly sorted into OMVs(Misra et al., 2005). Moreover, the inclusion of particular proteinsin OMVs does not appear to be a strict function of theirabundance. As shown in Table 4, several low-abundance outermembrane proteins, including FimD, FecA, FhuE, and FepA,and periplasmic proteins, including YddB, SLT, MalM, andPRC, were identified, whereas the most abundantly expressedperiplasmic proteins, such as OppA, FimA, HdeA, and LivJ, werenot (Corbin et al., 2003). These results further support the

hypothesis that special sorting mechanisms are in effect and/orthe OMVs bud at specific vesiculation sites.

E. Proteins Involved in Biological Functions ofGram-Negative Bacterial OMVs

In addition to supporting previously known biological roles ofOMVs, vesicular proteins identified in proteomic studies suggest

TABLE 3. (Continued )

aAccession numbers of individual proteins originate from each reference.



novel functions, and we can specify what proteins are involvedin each physiological and pathological function. As shown inFigure 6, protein function can be largely classified by theinteracting partner, including bacteria and host cells. First, theorganic solvent tolerance protein (OstA), multidrug-resistantefflux pumps (Mex, Mtr, and TolC), and phage target receptors(FepA, LamB, and OmpA) in E. coli, N. meningitidis, andP. antarctica NF3-derived OMVs may contribute to bacterialsurvival by reducing levels of toxic compounds such as n-hexaneand antibiotics, and by facilitating the release of attacking phages(Post et al., 2005; Nevot et al., 2006; Lee et al., 2007). ABCtransporters for specific nutrients (LamB, BtuB, and FadL),

inorganic ions (FepA, FhuA, and Fiu), and nucleosides (Tsx)exert their roles as delivery systems in bacterial communities.In particular, TonB-dependent receptors (BtuB, FhuA, and FhuE)in OMVs have been suggested to be nutrient sensors andtransporters, and their presence has been postulated to representan alternative mechanism for survival in nutrient-limited systems(Nevot et al., 2006). Furthermore, murein hydrolases (e.g., MltA,SLT) in OMVs are involved not only in OMV biogenesis asdescribed above, but also in predatory activities by killingcompeting bacteria via cell wall degradation (Kadurugamuwa &Beveridge, 1996; Li, Clarke, & Beveridge, 1998).

In the host environment, effective pathological functions ofOMVs can be achieved by increased resistance to bactericidalfactors. OmpT in vesicles may degrade cationic antimicrobialpeptides produced by epithelial cells or macrophages, and Issmay increase serum survival of OMVs (Stumpe et al., 1998;Nolan et al., 2003). Moreover, in addition to pathogenic-specifictoxins, outer membrane porin proteins, including OmpA andOmpF, which are enriched in OMVs, have immunostimulatoryactivity and induce leuckocyte migration (Galdiero et al., 1999).Pathogenic-specific adherent/invasive proteins and outer mem-brane porins, including OmpA, OmpW, and OmpX, are involvedin targeting OMVs to host cells. Furthermore, OmpA enhancesthe uptake of LPS by macrophages and contributes to the invasionof brain microvascular endothelial cells (Korn et al., 1995;Prasadarao et al., 1996).

V. FUTURE DIRECTIONS FOR PROTEOMICSTUDIES IN GRAM-NEGATIVE BACTERIAL OMVS

Previous proteomic studies have established a proteome databaseof OMVs derived from four native and two mutant bacteria.These studies have provided important information about the bio-genesis, pathophysiological functions, and protein composition

TABLE 4. Sorting profiles of vesicular proteins in E. coli DH5a

OMP, outer membrane protein; PP, periplasmic protein.

aAbundance represents mRNA signal intensity on GeneChip.

FIGURE 5. Subcellular distribution of vesicular and cellular proteins

present in E. coli. When compared to the complete E. coli proteome,

DH5a-derived OMVs are highly enriched in outer membrane proteins,

whereas inner membrane proteins are excluded. [Adapted from Lee

et al. (2007).]

& LEE ET AL.


of bacterial OMVs (Post et al., 2005; Bauman & Kuehn, 2006;Ferrari et al., 2006; Nevot et al., 2006; Berlanda Scorza et al.,2007; Lee et al., 2007). However, compared to human micro-vesicles, for which significant progress has been made in definingthe proteomes of vesicular components (Yates et al., 2005),proteomic information on bacterial OMVs still remains scarceexcept for E. coli (Berlanda Scorza et al., 2007; Lee et al., 2007).Limited proteomic data make it difficult to elucidate the exactmechanism of vesicle formation and new functions of OMVs.Further studies are therefore necessary to construct a vesicularproteome for diverse Gram-negative bacteria under a variety ofconditions. Overall, the future directions for proteomic analyseson Gram-negative bacterial OMVs are summarized in Figure 7.

A. OMVs Derived from DiverseGram-Negative Bacteria

Diverse Gram-negative bacteria provide unlimited material forOMVs, as most have been reported to secrete vesicles. Valuablesamples can be obtained from host fluids and tissues (Table 5).The facts that Gram-negative bacteria are the main causes ofseptic shock, and that LPS and outer membrane proteins ofbacterial OMVs elicit a complex pattern of inflammatory

reactions suggest that bacterial OMVs are involved in theprogress of septic shock. Therefore, proteomic analysis of OMVsobtained from the serum of septic human patients or septic ratsshould increase our understanding about the pathological roles ofOMVs in septic shock (Brandtzaeg et al., 1992; Hellman et al.,2000). Moreover, vesicles shed by N. meningitidis have beenfound in the cerebrospinal fluid and blood of a patient withmeningitis (Stephens et al., 1982; Brandtzaeg et al., 1992;Namork & Brandtzaeg, 2002). In the case of Borreliaburgdorferi-infected mice, OMVs were detected in the urineand blood (Dorward, Schwan, & Garon, 1991). Furthermore,OMVs derived from Bacteroides (Porphyromonas) gingivalis,which causes oral cavities, inflammation, and bleeding atperitonitis sites, can be obtained from dental plaque samples(Grenier & Mayrand, 1987; Imamura et al., 1995).

B. Preparation of Gram-Negative Bacterial OMVs

As noted above, OMVs are usually prepared by ultracentrifuga-tion followed by density gradient centrifugation or gel filtration(Horstman & Kuehn, 2000; Wai et al., 2003; Post et al., 2005; Leeet al., 2007). Other methods such as free-flow electrophoresis(FFE) and capillary electrophoresis (CE) can be used to isolate

FIGURE 6. Proposed physiological and pathological functions of Gram-negative bacterial OMVs.

Functions of Gram-negative bacterial OMVs are predicted based on the available proteomes of OMVs

derived from nonpathogenic and pathogenic bacteria. [Color figure can be viewed in the online issue, which

is available at www.interscience.wiley.com.]



OMVs in intact form and with high purity. FFE is a powerful toolto separate human cellular organelles such as peroxisomes,lysosomes, endosomes, melanosomes, and Golgi vesicles, aswell as mitochondria (Morre, Morre, & Heidrich, 1983; Fuchs,Male, & Mellman, 1989; Marsh, 1989; Kushimoto et al., 2001;Mohr & Volkl, 2002; Zischka et al., 2006). This system allowspurification of bacterial OMVs in a native state and with high

purity by discriminating similar-density membrane fragments,which are difficult to remove solely by density gradientultracentrifugation.

CE is an analytical technique that employs narrowcapillaries for electric field-mediated separation of particles witha surface charge. Recently, CE was used to separate humanorganelles including mitochondria, acidic organelles, nuclei, and

FIGURE 7. Future directions for high-throughput and comparative proteomics in OMVs originating from

diverse Gram-negative bacteria under various environmental conditions.

& LEE ET AL.


lipid vesicles (Gunasekera, Musier-Forsyth, & Arriaga, 2002;Duffy et al., 2002; Fuller & Arriaga, 2003; Owen, Strasters, &Breyer, 2005). The electrophoretic mobility of organelles isdetermined by the electrical charge on the surface of organelles,their morphology, and size (Owen, Strasters, & Breyer, 2005).Using this technique, a very small number of organelles can beseparated electrokinetically or hydrodynamically into a capillary(Fuller & Arriaga, 2004). Therefore, CE should be used toprepare OMVs with high purity.

C. Strategies for the Proteomic Analysis ofGram-Negative Bacterial OMVs

1. Protein Identification

For a comprehensive proteomic analysis of OMVs derived fromvarious Gram-negative bacteria, extensive prefractionation ofsamples on the protein and/or peptide levels before mass analysisshould be considered. Although 2-DE is a powerful tool forprotein separation, this method cannot properly resolve high-molecular-weight, very basic, or hydrophobic proteins (Wu &Yates, 2003), suggesting that protein separation by 2-DE maybe not suitable for a global proteomic analysis of OMVs.Combination of 1-D SDS–PAGE and LC–MS/MS provides apowerful alternative to 2-DE-based proteomic analysis, as notedabove (Aebersold & Mann, 2003). Alternatively, multidimen-sional protein identification technology (MudPIT) (Washburn,Wolters, & Yates, 2001) should be useful for proteome analysis ofOMVs. Because MudPIT separates peptides using a combinationof two different kinds of LC prior to MS analysis, this systemgreatly reduces the complexity of the proteome at the peptidelevel, resulting in the identification a large number of proteins.Using this process, 1,484 proteins were identified fromSaccharomyces cerevisiae (Washburn, Wolters, & Yates, 2001).Furthermore, MudPIT is suitable for identifying proteins with

extreme pI, integral membrane proteins, and low-abundanceproteins (Graham, Graham, & McMullan, 2007).

Furthermore, a combination of matrix-assisted laserdesorption/ionization time-of-flight (MALDI-TOF) and ESImass spectrometer can also increase coverage and the numberof identified proteins. ESI-MS/MS and MALDI-TOF-MSanalyses of the same sample usually identify different sets ofproteins (Bodnar et al., 2003). Therefore, high proteomecoverage for OMVs can be achieved by the combination ofmultidimensional protein and/or peptide fractionation methods,as well as by the combination of various MS methods.

2. Bioinformatics for Annotation of Vesicular Proteins

To clarify how bacteria shed vesicles and identify thephysiological and pathological functions of OMVs, annotationof identified vesicular proteins based on subcellular localizationand function is important. Previous proteomic analyses of OMVsshowed that vesicular proteins are derived from varioussubcellular locations in bacteria (Post et al., 2005; Ferrari et al.,2006; Lee et al., 2007). Annotation of vesicular proteins bysubcellular localization in bacteria can help us elucidate themechanism of OMV biogenesis, as well as identify possiblecontaminants in proteomic analyses. Currently, several bio-informatics tools are available for bacterial protein localizationincluding PSORTb (Gardy et al., 2005), EchoBASE (Misra et al.,2005), Proteome Analyst (Lu et al., 2004), and SubLoc (Hua &Sun, 2001). They predict the localization of bacterial proteins onthe basis of known motifs or cleavage sites. Furthermore, thecomputational prediction of subcellular localization of vesicularproteins offers numerous insights that, for example, can assistin functional analyses of OMVs.

For the functional analysis of vesicular proteins, severalbioinformatics tools are available (Ouzounis et al., 2003). Oneof the most influential classification schemes comes from ahierarchy of properties for the gene products of E. coli, which was

TABLE 5. OMVs found in host fluids and tissues



later extended to develop multifunctional classes (Riley, 1993;Serres & Riley, 2000). Inspired by this classification, theautomatic genome-annotation system GeneQuiz was developedbased on the keyword mapping of protein families to 14functional classes (Andrade et al., 1999). Gene ontologyclassification, which comprises the three categories of molecularfunction, biological process, and cellular component, is com-monly used for protein annotation (Ashburner et al., 2000). TheKyoto Encyclopedia of Genes and Genomes uses a differentmethod of classifying genes and proteins by their participationin or association with metabolic pathways (Kanehisa et al.,2002). Since each data mining method uses a different strategy topredict the function of bacterial proteins, a combination ofbioinformatics tools should be used to annotate the identifiedvesicular proteins to elucidate the numerous physiological andpathological functions of Gram-negative bacterial OMVs.

D. Comparative Proteomics in Gram-NegativeBacterial OMVs

In contrast to the static nature of the genome sequence, whichprovides the blueprint for all protein-based cellular buildingblocks, the proteome is highly dynamic (Bandow & Hecker,2007). The protein composition of bacteria is constantly adjustedto facilitate survival, growth, and reproduction in an ever-changing environment. Bacteria face highly variable growthconditions and stress situations with respect to temperature, pH,osmolarity, nutrient availability, and host infection, among otherfactors. Like the whole bacteria proteome, components of OMVsare influenced by environmental stress and bacterial status(Katsui et al., 1982; Horstman & Kuehn, 2002; McBroom &Kuehn, 2007). Some vesicular proteins are temporarily expressed,whereas other proteins are expressed after signal transductionfrom the extracellular milieu, or at certain bacterial growthphases (Kuehn & Kesty, 2005). Moreover, the release of OMVsincreases when bacteria are exposed to conditions such ashigh temperature, exposure to antibiotics or serum, or nutrientdeprivation (Post et al., 2005). Therefore, comparative proteomicanalyses of OMVs derived from diverse conditions are expectedto provide new functional insights into bacterial OMV biology,facilitate the identification of pathogenic markers, and contributeto the discovery of proteins as therapeutic targets.

Three approaches have been most commonly used togenerate quantitative profiles of complex protein mixtures(Zhang, Yan, & Aebersold, 2004). The first is a combination of2-DE and MS (Aebersold & Mann, 2003). Classical 2-DE is stillthe method of choice for quantitative analysis of the proteome,and difference gel electrophoresis (DIGE) is a commonly usedcomparative 2-DE technique (Marouga, David, & Hawkins,2005). In DIGE, proteins from different samples are labeled withdifferent fluorescent dyes, mixed equally, and resolved by 2-DE.The protein samples are visualized using fluorescence imaging toenable detection of differences in protein abundance betweensamples, and the proteins that differ in abundance can beidentified by MS (Marouga, David, & Hawkins, 2005). UsingDIGE, comparative proteomic studies were carried out onDOMVs and OMVs derived from N. meningitidis (Ferrariet al., 2006). However, in spite of the progress in 2-DE

technology, it is still not technically feasible to obtain a completeexpression profile from a single two-dimensional gel becausesome proteins are not well separated, such as those thatare extremely basic or acidic, small or large, or of low abundance(Wu & Yates, 2003).

The second method for comparative proteomics is based onstable isotope tagging of proteins and automated LC–MS/MSanalysis of peptides derived from complex protein mixtures(Conrads et al., 2001). Isotope-coded affinity tags (ICATs) andisobaric tags for relative and absolute quantification (iTRAQ) arecommonly used chemical isotopic labeling strategies thatdifferentially label sulfhydryls or primary amines of proteins orpeptides, respectively (Gygi et al., 2002; Choe et al., 2005). UsingICATs, several comparative analyses of bacterial proteomes havebeen reported, including the P. aeruginosa proteome duringanaerobic growth (Peng et al., 2005) and magnesium-limitedconditions (Guina et al., 2003). The major disadvantage of thistechnique is that cysteine is a relatively rare amino acid that is notpresent in 10–20% of bacterial proteins (Cordwell, 2006). Sinceall proteins and peptides have primary amines on their N-terminalamino acids or lysine, iTRAQ should be better for comparativeproteomics than using ICATs (Danielsen et al., 2007).

Another widely applicable technique is stable-isotopelabeling of amino acids in cell culture (SILAC) (Mann, 2006;Ong et al., 2002). In the SILAC procedure, cells are grown in thepresence of an isotopically heavy amino acid for severalgenerations, thereby replacing essentially all of the naturallyoccurring light amino acid in all proteins (Gingras et al., 2007).The advantage of SILAC over ICATs and iTRAQ is that SILACcan efficiently label all proteins present in complex samples,whereas the labeling efficiency of ICATs and ITRAQ may dependon several factors, including the status of proteins (i.e., native vs.denatured) (Gruhler et al., 2005). SILAC has been successfullyused in several recent comparative proteomic studies, includingprofiling of the dynamic association of chaperonin-dependentprotein folding in E. coli (Kerner et al., 2005).

VI. APPLICATIONS OF PROTEOMIC STUDIES ONGRAM-NEGATIVE BACTERIAL OMVS

As described above, high-throughput and comparative proteo-mics on bacterial OMVs will decipher the mechanisms under-lying intercellular communication by elucidating the biogenesisand specialized functions of bacterial OMVs. In addition toimproving our understanding of the basic biology of bacterialOMVs, knowledge about the vesicular proteome can facilitate avariety of biotechnology applications of OMVs, especially inhuman medical research, by identifying specific biomarkers thatcan be used to develop diagnostic and therapeutic tools againstpathogenic organisms. Because bacterial OMVs can be dissemi-nated from infectious sites and circulated in fluids within the host(Brandtzaeg et al., 1992; Hellman et al., 2000), characterizationof vesicles from patients with bacterial-associated diseasesymptoms is an effective way to diagnose pathogenesis.

Moreover, particular interest has emerged in the use ofOMVs as vehicles for stimulation of host immune responses.These studies have already led to clinical trials with DOMVs,

& LEE ET AL.


which are made artificially from bacterial cell membranes (deMoraes et al., 1992; Drabick et al., 1999; Girard et al., 2006).However, based on previous proteomic reports, the ratio of outermembrane proteins present in DOMVs was relatively lowcompared to native OMVs and was not consistent from batchto batch, resulting in different and diverse immune responses inthe host depending on the manufacturer (Ferrari et al., 2006).Therefore, vaccination strategies should also be envisionedusing native OMVs, which carry the enriched and nativetopology of strain-specific outer membrane antigens. Creatingsuper-blebbing bacterial mutants that can produce several timesmore vesicles than wild-type strains and genetic mutants that cancontrol LPS amounts might reduce the side effects of septicsymptoms in patients and will provide a particularly usefulsource for vaccine materials (van der Ley et al., 2001; BerlandaScorza et al., 2007).

Another important application of OMVs comes in the area ofantibiotics. Adverse reactions in patients undergoing therapy,resulting from antibiotic-induced liberation of bacterial compo-nents, including OMVs, have been a long-standing concern(Kadurugamuwa & Beveridge, 1995; Morand & Muhlemann,2007). Moreover, treatment with broad-spectrum antibiotics candisturb natural and beneficial microflora, leaving the patient moresusceptible to infection by opportunistic pathogens. Therefore,modulating the production of OMVs presents an attractiveapproach for treating bacteria-associated diseases.

VII. CONCLUDING REMARKS

Growing evidence suggests that Gram-negative bacterial OMVsare essential for bacterial survival and pathogenesis in hosts byacting as intercellular communicasomes in polyspecies com-munities. In spite of recent progress in this emerging field,previous biochemical and biological studies are limited in theirability to provide comprehensive information for understandingthe mechanisms of vesicle formation and the pathophysiologicalroles of OMVs. In addition to previous proteomic studies, furtherhigh-throughput and comparative proteomics studies of OMVsoriginating from diverse Gram-negative bacteria under variousenvironmental conditions will take us one step closer to anintegrated view of bacterial OMVs with regard to their biogenesisand pathophysiological functions. Furthermore, these studieswill stimulate the development of diagnostic tools, novelvaccines, and antibiotics effective against clinically importantGram-negative bacteria. We hope this review encourages furtherstudies on proteomics in Gram-negative bacterial OMVs.

VIII. ABBREVIATIONS

1-D one-dimensional

2-DE two dimensional gel electrophoresis

CE capillary electrophoresis

ClyA cytolysin A

DIGE difference gel electrophoresis

DOMV detergent-extracted outer membrane vesicle

EM electron microscope

ESI electrospray ionization

FFE free-flow electrophoresis

ICAT isotope-coded affinity tags

iTRAQ isobaric tags for relative and absolutequantification

LC liquid chromatography

LPS lipopolysaccharide

LT heat-labile enterotoxins

MALDI-TOF matrix-assisted laser desorption/ionizationtime-of-flight

MS mass spectrometry

MudPIT multidimensional protein identificationtechnology

MV membrane vesicle

OMV outer membrane vesicle

SILAC stable-isotope labeling by amino acids in cellculture

SDS–PAGE sodium dodecyl sulfate–polyacrylamide gelelectrophoresis

ACKNOWLEDGMENTS

This work was supported by a grant of the National R&DProgram for Cancer Control, Ministry of Health & Welfare,Republic of Korea (0320380-2), supported by the Korea BasicScience Institute K-MeP (T27021), and supported by the KoreaScience and Engineering Foundation (KOSEF) grant funded bythe Korea government (MOST, No. R15-2004-033-05001-0) toYong Song Gho. Eun-Young Lee and Dong-Sic Choi wererecipients of Brain Korea 21 fellowship.

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Eun-Young Lee received a B.Sc. degree in the Division of Life Science from Korea

University, Republic of Korea (2005). Presently she is a Ph.D. candidate at Pohang

University of Science and Technology, Republic of Korea, with a focus on the diverse roles

of bacterial outer membrane vesicles and mammalian cell-derived microvesicles under the

supervision of Prof. Yong Song Gho.

Dong-Sic Choi received a B.Sc. degree in the Department of Life Science and Division of

Molecular and Life Sciences from Pohang University of Science and Technology, Republic

of Korea (2006). He is now a Ph.D. candidate at Pohang University of Science and

Technology and his research involves proteomic analysis of microvesicles and plasma

membrane proteins derived from eukaryotic cells under the supervision of Prof. Yong Song

Gho.

Kwang-Pyo Kim received his B.Sc. and M.Sc. in Chemistry from Seoul National

University, Republic of Korea in 1990 and 1992, respectively. After working for a

pharmaceutical company, CJ corp., he started a Ph.D. program in Chemistry from the

University of Illinois at Chicago. In 2002 he thereafter worked in Harvard Medical School

as a postdoctoral fellow. Since 2004, he joined the Department of Molecular Biotechnology

at the Konkuk University in the Republic of Korea as an Assistant Professor. His current

research areas focus on: development and application of proteomics technologies to

investigate post-translational modifications, discovery of biomarkers with comparative

proteomics technologies, and MALDI Mass Tissue imaging.

Yong Song Gho received his B.Sc. and M.Sc. degrees in Chemistry from Seoul National

University, Republic of Korea in 1987 and 1989, respectively. He obtained his Ph.D. degree

in Biochemistry and Biophysics from University of North Carolina at Chapel Hill, USA in

1997. During 1998–2000, he was a visiting fellow at NIDCR of National Institutes of

Health, USA. From 2000 to 2004, he was an assistant professor at Kyung Hee University,

Republic of Korea. Since 2004, he became an assistant professor in the Department of Life

Science and Division of Molecular and Life Sciences at Pohang University of Science and

Technology, Republic of Korea. His current research interests aim at elucidating the

biogenesis and pathophysiological functions of extracellular membrane vesicles derived

from bacteria and mammalian cells, as well as determining profiles of membrane vesicular

genes and proteins using microarray and mass spectrometry.



proteomics in gram-negative bacterial outer membrane...

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