Non classical secretion systems

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Research in Microbiology xx (2013) 1e9www.elsevier.com/locate/resmic

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Non classical secretion systems

Roland Lloubes a,*, Alain Bernadac b, laetitia Houot a, Stephanie Pommier c

a Laboratoire d’Ingenierie des Systemes Macromoleculaires, Aix-Marseille Universite, CNRS e UMR7255, 31 chemin Joseph Aiguier,

13402 Marseille Cedex 20, Franceb Institut de Microbiologie de la Mediterranee, Aix-Marseille Universite, CNRS e UMR7255, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France

c INSERM, 101 rue de Tolbiac, 75654 Paris Cedex 13, France

Received 20 November 2012; accepted 27 February 2013

858687
88 Abstract 8990919293949596979899
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Bacteria use molecular machines or weapons to colonize, invade or fight other bacteria and eukaryotic cells. In addition to these varioussecretion systems, two different systems that release bacterial compounds have also been described. The first one corresponds to membranevesicle formation and to long distance delivery of membrane or soluble components. The second system is dependent of the expression of thecolicin lysis genes known for releasing cytoplasmic colicins as well as other soluble proteins. Both systems will be described thereafter.� 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Keywords: Membrane vesicle; Protein delivery; sE regulation; Tol system; SOS genes; Colicin release

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1. Bacterial membrane vesicles

Membrane vesicle production is a general processdescribed in Bacteria and Archae. Membrane vesicles (MVs)are ubiquitously released from laboratory, environmental,pathogenic and non-pathogenic strains. Electron microscopyhas been a most powerful tool to establish the presence of theMVs. First discovery of MVs came from Escherichia colicultures grown under lysine limited growth medium (Bishopand Work, 1965; Knox et al., 1966) or under normal growthconditions (Hoekstra et al., 1976) and from Salmonella andNeisseria (Devoe and Gilchrist, 1973). Differently, bacteriadeleted of two major proteins interconnecting the outermembrane and the peptidoglycan layers were shown to pro-duce extensive blebbing and to adopt spherical shapemorphology (Sonntag et al., 1978). MV formation wasdiscovered later in Gram-positive bacteria, Clostridium ther-mosulfurogenes (Specka et al., 1991) and was recently

* Corresponding author.

E-mail addresses: [email protected] (R. Lloubes), bernadac@

imm.cnrs.fr (A. Bernadac), [email protected] (laetitia Houot), stephanie.

[email protected] (S. Pommier).

Please cite this article in press as: Lloubes, R., et al., Non classical secret

j.resmic.2013.03.015

0923-2508/$ - see front matter � 2013 Institut Pasteur. Published by Elsevier Ma

http://dx.doi.org/10.1016/j.resmic.2013.03.015

demonstrated for other Gram-positive species and Archae(Ellen et al., 2009).

MV formation is observed from bacteria grown in liquidand in solid medium, bacteria isolated from ecological andclinical niches as well as in biofilm. Genetic analyses havebeen performed to find genes involved in MV production. TheMV content of pathogenic bacteria and the MV enrichment invirulence factors or in quorum sensing molecules, are part of aresearch domain that is in constant expansion. Thus, togetherwith membrane components some soluble molecules appear tobe more or less packaged and released into MVs. Moreover,the recent discovery on regulation of MV production and theMV-dependent molecular trafficking lead to propose MVs as ananoparticle delivery/secretion system. Here, we will focusour review on the non-classical secretion of proteins into MVconsidering the most studied Gram-negative bacterial MVs.

1.1. MV biogenesis
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Membrane vesicles are produced from bacteria, Archae aswell as from eukaryotic cells. Briefly, fungi or parasites formMVs by a regulated mechanism involving the conservedmembrane scission machinery. The release of the MVs occurs

ion systems, Research in Microbiology (2013), http://dx.doi.org/10.1016/

128129130

sson SAS. All rights reserved.

mailto:[email protected]









Fig. 1. OMV producing bacteria. Electron micrograph showing negatively

stained E. coli tolA cells producing OMVs. Bacteria were grown on agar plate

and suspended in Tris-buffered saline before staining (Bernadac et al., 1998).

Bar corresponds to 100 nm.

2 R. Lloubes et al. / Research in Microbiology xx (2013) 1e9

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after fusion of the endosome containing MVs with the cellmembrane (Deatherage and Cookson, 2012). Similar ma-chinery is required by the Archae to release MVs (Makarovaet al., 2010). Gram-positive bacteria produce MVs that havebeen detected between the cytoplasmic membrane and the cellwall layer (Specka et al., 1991). The MV diameter iscomprised between 50 and 150 nm and these MVs containsoluble and membrane cytoplasmic components (Lee et al.,2009). Beside this, little is known about MV production andformation in Gram-positive bacteria.

Gram-negative MVs (called OMVs), size comprised be-tween 10 and 300 nm, are formed by outer membrane (OM)and periplasmic components. Unusually, cytoplasmic proteinsand DNA have been found associated with MVs(Kadurugamuwa and Beveridge, 1995; Lee et al., 2007) while2D proteomic analyses of OMV indicate the absence ofcytoplasmic proteins (Berlanda Scorza et al., 2008). Largeamounts of OMVs have been detected in Neisseria meningi-tidis (Devoe and Gilchrist, 1973) and later in Bacteroidesgingivalis strains (Grenier and Mayrand, 1987). The obser-vations from Neisseria indicated that OMVs were producedduring the exponential phase of growth while Bacteroidesresults indicated that membrane protein content of OMVs wassomewhat different from the OM fraction. The OMV pro-ducing bacteria are difficult to compare since various tech-niques used for OMV purification turn in imprecisequantifications. However, only few direct observations ofOMV bacterial production have been shown while most resultscorrespond to protein detections from purified and concen-trated OMV fractions (see native OMV purification figure inKulp and Kuehn, 2010). Thus, removal of bacteria, filtrationand concentration of OMVs are two steps often performedusing variable culture volumes that depend on OMV abun-dance. Moreover, besides the possible adhesion of OMVs tothe cell envelope, the filtration and sedimentation steps alsogive variable yields. Levels of bacterial OMV productions arethus questionable. Accordingly, direct observations of OMVsfrom bacterial cultures may concern highly producing strains.These major OMV producers correspond to the two humanpathogenic strains mentioned above, N. meningitidis and B.gingivalis, and also to Vibrio cholerae (Chatterjee and Das,1967), Helicobacter pylori (Keenan et al., 2000) and to mu-tants that affect membrane integrity (see “Regulation of OMVproduction” section) such as lpp, ompA and tolepal mutants inE. coli and Salmonella thyphimurium (Bernadac et al., 1998;Deatherage et al., 2009; Sonntag et al., 1978; Webster, 1991).It is noticeable to precise that bacterial growth on solid agarmedium had been used to directly analyze and compare OMVbacterial production in absence of any separation treatment.Simply, the picked colony was suspended in physiologicalbuffer and the suspension was deposited directly on carbongrid further visualized by EM after negative staining (Fig. 1;Bernadac et al., 1998).

However, differences are observed between S. thyphimu-rium and E. coli tolepal mutants considering the size of theOMVs. Large OMVs were found at the septum of S. thyphi-murium while their size was found similar from tolepal or lpp


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mutants (Bernadac et al., 1998; Deatherage et al., 2009).Different growth conditions (liquid or solid medium) andsample preparation (buffer osmolarity and sample fixation)have been used making EM results difficult to compare and itcould be suspected that osmotic shock has enhanced the sizeof the OMVs present at the septum.

More recently, direct observation of OMV formation duringbacterial growth was performed using fluorescence micro-scopy looking at tol mutants producing periplasmic GFP(Gerding et al., 2007, Fig. 2A).

1.2. Regulation of OMV production

Variations in OMV production have been described inresponse to numerous environmental factors, as diverse astemperature (Katsui et al., 1982), nutrient abundance (Dutsonet al., 1971), general envelope stresses (McBroom and Kuehn,2007) or biofilm mode of growth (Yonezawa et al., 2009).Moreover, OMV content does vary depending of bacteriallifestyle. For example, Pseudomonas aeruginosa grown as abiofilm produces OMVs that contain more proteolytic en-zymes than when grown as planktonic cells (Schooling andBeveridge, 2006). Consequently, OMV synthesis is seen asan adaptive mechanism during niche colonization. In agree-ment with this theory, several regulatory cascades were foundto interfere with OMVs synthesis in Gram-negative bacteria,although the existence of a specific regulatory pathway stillremains to be found. Thus, proposed models for OMV for-mation are based on OM integrity defects, on the accumulationof misfolded proteins in the periplasm inducing turgor pres-sure in the periplasm and on the modification of the outermembrane content (Deatherage and Cookson, 2012; Kulp andKuehn, 2010).

Several studies resulted in the identification of OMV hyper-forming mutant strains (Table 1). A first category of thesemutants targets the multiprotein TolePal complex. These en-velope proteins are thought to form a structural bridge be-tween the inner membrane (IM) and the outer membrane


Fig. 2. A: Periplasmic tGFP is recovered into OMVs. Phase contrast (left

pannel) and superimposition of GFP fluorescence (right panel) of exponen-

tially growing tolA cells producing periplasmic tGFP (tGFP: GFP harboring

the Tat signal sequence of the trimethylamine N-oxide reductase, TMAO

reductase, Santini et al., 2001). The fluorescent OMVs are not visible under

phase contrast microscopy. Bar corresponds to 2 mm. B: GFP is entrapped into

the OMV lumen. Proteinase K digestions of tGFP isolated from OMVor OMV

free supernatant (S), (Incubation temperatures are indicated; e, no PK).

Addition of 0.01% Triton X100 to OMV confers tGFP accessible to PK

degradation (not shown). C: Purified outer membrane vesicles Electron

micrograph showing the negatively stained OMVs containing tGFP. Bar cor-

responds to 100 nm.

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(OM) of Gram-negative bacteria. A mutation in any of thecorresponding genes confers an increase in OMV release fromthe cell surface, as visualized by electron microscopy(Bernadac et al., 1998) or by fluorescence microscopy(Gerding et al., 2007). Because TolePal mutants also showhypersensitivity to detergents and increased periplasmicleakage (Webster, 1991), this sum of phenotypes is thought toresult from a general defect in OM integrity (Lloubes et al.,2001). In addition, the TolePal complex is involved in thecell division process and its precise role is suspected to enablethe OM constriction (Gerding et al., 2007).


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In relation with the different OMV sizes observed from tol,pal, lpp and ompA mutants, Deatherage and Cookson (2012)propose two different regions for OMV formation, onelocated at the septum and the second located on the bacteriallateral wall. Thus, large OMVs are observed at the septum oftolepal mutants while small OMV are present at the lateralcell wall of lpp and ompA mutants. This model of OMV for-mation suggests a decreased density of the Lpp and OmpAOM proteins in interaction with the peptidoglycan (PG) and adecreased density of the TolePal protein complex involved ina network of protein interactions linking the IM, the OM andthe PG. In E. coli and most of the Gram-negative bacteria, twospecific complexes are required for the biogenesis of the cellwall, one located at the lateral cell wall and the other at theseptum. These complexes contain one lipoprotein (LpoA orLpoB) and one Penicillin Binding Protein (PBP1A or PBP1B). Interestingly, the LpoB-PBP1B complex is working intandem with the TolePal system (Typas et al., 2010). Theseresults agree with those on the TolePal localization requiredfor cell division (Gerding et al., 2007). In addition, the Tol-ePal system is essential in Caulobacter that contains only onePBPeLpo complex and is essential in E. coli mutant devoid ofthe PBP1BeLpoB complex. As developed thereafter, regula-tion of the tolepal gene expression may be important to un-derstand its relation with OMV formation. Differently, N.meningitidis and B. gingivalis, are two rare genus devoid ofthe TolePal system (Sturgis, 2001) which have been shown toproduce large amounts of OMVs.

In the second category of OMV hyper-producing strains fallgenes involved in envelope stress management and controlledby the sigma E stress factor (sE). First, the accumulation ofmisfolded proteins in the periplasm results in the induction ofthe sE regulon encoding periplasmic chaperones, foldingcatalysts, proteases as well as the major heat shock s32 factor,(Dartigalongue et al., 2001). These aberrant proteins are nor-mally degraded by specific periplasmic proteases such asDegP (E. coli) or MucD (P. aeruginosa), usually under thecontrol of the sE factor. Mutants targeting these periplasmicproteases or the anti-sE factor produced an increased level ofOMVs (Table 1). While OMVs remove the stressor molecules,the mechanism of OMV formation remains unknown(McBroom and Kuehn, 2007). Interestingly, the opposite resultis obtained in Vibrio fischeri, where a degP mutant decreasesOMV production, suggesting that in this bacterium, the role ofDegP homolog is distinct from its role in E. coli (Shibata andVisick, 2012). An additional link between sE regulon andOMV production has recently been identified through the OMprotein OmpA, also the role of this protein during OMV for-mation seems strictly species related. Thus, in Vibrio cholera,OmpA is repressed at the translation level by sE through asmall non-coding RNA, vrrA, leading to an increase in vesicleformation (Song et al., 2008).

But envelope stress might not be the unique trigger toproduce OMV. In P. aeruginosa, Pseudomonas QuinoloneSignal (PQS) and gentamicin induce OMV production inde-pendently of the sE pathway (Kadurugamuwa and Beveridge,1995; Mashburn-Warren et al., 2008). In V. fischeri,


Table 1

OMV high producing mutants.

Targeted genes Strain OMV-production References

Envelope integrity:

tolepal E. coli,

S. typhimurium

D Bernadac et al., 1998

lpp-ompA E. coli,

S. typhimurium

D Deatherage et al., 2009

D Sonntag et al., 1978

D Deatherage et al., 2009

ompA V. cholerae D Song et al., 2008

Envelope stress management:

rseA E. coli D McBroom et al., 2006

degPedegS E. coli D McBroom et al., 2006

degP V. cholerae La Shibata and Visick, 2012

mucD P. aeruginosa D Tashiro et al., 2009

Others:

pqs P. aeruginosa D Mashburn-Warren

et al., 2008

rscS V. cholerae Da Shibata and Visick, 2012

nlpI E. coli D McBroom et al., 2006

tatC E. coli D McBroom et al., 2006

a Increased levels of OMVs was dependent of the RscS overproduction.



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extracellular matrix production and thus biofilm formation isdependent of the syp locus, controlled by the membrane-associated sensor RscS histidine kinase. Cells overexpressingRscS have an increased OMV production. This increase cor-relates with enhanced biofilm formation and is strictlydependent of the presence of a functional sypK gene (Shibataand Visick, 2012). This suggests that in V. fischeri OMVrelease is not a consequence of general stress or toxicity but israther directly dependent on the synthesis of a biofilm extra-cellular matrix. Lastly in E. coli, McBroom et al. (2006)showed by transposon insertion that mutation in the tatCgene confers membrane instability and an increase in OMVformation probably in relation with PG metabolism since twoamidases substrate of the Tat (twin arginine translocation)export pathway could be mislocalized. Moreover authorsdiscovered that the nlpI gene disruption resulted in OMVoverproduction but in the absence of membrane instability orperiplasmic stress. The role of the nlpI gene product is still tobe determined. In addition with degS, degP and rseA genedisruptions, the results on nlpI demonstrate that in E. coliOMV formation may also be independent of membraneinstability.

Construction of mutant transposon libraries has been shownto be a powerful approach in identifying regulatory cascades.Surprisingly, there is only one example to date of such anapproach used for the identification of genes affecting OMVformation. This study conducted in E. coli identified 26 ORFsinvolved in OMV release (McBroom et al., 2006). It is strikingto notice that among these 26 ORFs, 21 are directly related tothe envelope structure or stress response pathway (Table 1),and that the screen failed to identify a regulatory pathway,apart members of the sE cascade. One explanation, beside thefact that the screen was not saturating, would be that regula-tor(s) involved in the control of OMV production is (are)master regulator(s) also impacting growth, which excludedthem from the screen as designed in this study.


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One other large-scale mutant-transposon screen, firstdesigned to study factors involved in intestinal cell invasion byan Adherent-Invasive E. coli (AIEC) strain also identified aprotein affecting the OMV production. The screen was con-ducted in the clinical isolate LF82. The targeted gene, yfgL,encodes a lipoprotein involved in peptidoglycan turnover. Theauthors proposed that this phenotype could be related to PGamount in the envelope of the lipoprotein mutant strain, linkedto variations in turgor pressure in the periplasm. This regula-tion was found to be conserved in the K-12 E. coli laboratorystrain, as the yfgL mutant also showed a decrease in OMVdischarge from the cell surface. Interestingly, a concomitantdecrease in the ability of LF82 to invade intestinal cells in anin vitro model of infection was observed, which is coherentwith a role of the OMVs as a niche adaptation factor (Rolhionet al., 2005).

Conditions affecting the level of expression of the geneslisted above are expected to influence the amount of OMVs.Envelope proteins are usually well conserved among Gram-negative bacteria. However, vesicle-forming phenotype is notfrequently reported in bacterial studies. For example, tol geneexpression was found controlled by iron and temperature in P.aeruginosa (Lafontaine and Sokol, 1998), and by temperatureand RcsBC, a two-component system involved in the synthesisof capsular polysaccharides in E. coli (Clavel et al., 1996).Following this idea, available transcriptomic analysis mayrepresent a great resource for data mining. A quick look intopublished transcriptomes suggests that the tol operon isrepressed in high osmolarity conditions in V. cholerae(Shikuma and Yildiz, 2009) and Sinorhizobium meliloti(Dominguez-Ferreras et al., 2006). Thus, more is probably tobe found to correlate regulation of previously identified gene(homologs of tolepal, ompA, degP, vrra, sE regulon), OMVproduction, and bacterial adaptation to their environment.

1.3. OMV content

The protein content of most OMVs is similar to that of OM,essentially composed by large amounts of porins and OmpAbut with reduced amounts PG-bound lipoproteins. Accord-ingly, Wensink and Witholt (1981) suggested that OMV for-mation results from faster OM extend than PG layer or thatPG-bound lipoproteins prevent the release of OMVs. TheLPS present in the outer leaflet of the OMVs is occasionallyobserved with little changes in its composition while the innerleaflet, formed by phospholipids, is suspected to be identical tothat of the OM. The B-band LPS was found enriched in OMVcompared to the OM of P. aeuginosa (Kadurugamuwa andBeveridge, 1995). In association with OM and periplasmiccomponents, the presence of DNA has been reported in fewlaboratory bacterial species and in clinical isolates (Dorwardet al., 1989; Kadurugamuwa and Beveridge, 1995; Sharpeet al., 2011). In some cases virulence factors are associatedand/or enriched into OMVs or sorted under selective pack-aging as shown for the membrane associated enterotoxins(Devoe and Gilchrist, 1973; Middeldorp and Witholt, 1981;Horstman and Kuehn, 2000), the leukotoxin from


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Actinobacillus actinomycetemcomitans (Kato et al., 2002) andthe cytolethal distending toxin present only in the OMVfraction (Berlanda Scorza et al., 2008). Moreover, the ClyAcytotoxin, secreted to the OM by an unknown mechanism, ismaintained in an active form when released in the OMVs (Waiet al., 2003). In general, periplasmic proteins are recoveredinto the OMV lumen while also present in the periplasm or theexternal medium. In agreement with this, foreign proteins suchas the GFP addressed to the periplasm using the twin argininesignal-sequence (tGFP), were easily detected in the OMVs ofE. coli tol mutants (Gerding et al., 2007, Fig. 2A). Usingprotease accessibility experiments, we observed that tGFPassociated with the OMV purified fraction was resistant todegradation while tGFP present in the OMV-free supernatant(Fig. 2B) and in the OMV fraction treated with Triton X100(not shown) were degraded. In addition to the exported pro-teins, secreted proteins such as a-haemolysin, phospholipaseC, alcaline phosphatase and other proteases were also recov-ered associated with OMVs (Kadurugamuwa and Beveridge,1995; Balsalobre et al., 2006) while some virulence factorswere exclusively secreted into OMVs such as the PagK-homologous proteins of Salmonella enterica (Yoon et al.,2011). Recently, a preferential packaging of virulence fac-tors that excluded major OM proteins into OMVs wasdiscovered in B. gingivalis (Haurat et al., 2011).

In addition to OMV protein secretion, Pseudomonas putidaOMVs confer protective properties against toluene sincebacteria became toluene-tolerant and released tolueneenriched OMVs suspected to detoxify the OM (Kobayashiet al., 2000). Moreover, several studies have highlighted thespecific release of hydrophobic quorum sensing molecules intoOMVs (PQS: Pseudomonas quinolone signal, Mashburn andWhiteley, 2005). More recently PQS molecules were foundinvolved in OMV formation as the result of their directinsertion in the outer leaflet of the membrane (Schertzer andWhiteley, 2012). The role of three murein-bound proteins onthe PQS dependent OMV formation has been investigated.

Fig. 3. A: Time lapses study of OMV formation. Time lapses study of tolA cells p

sequence) strap up in 0.2% agar, observed by fluorescent microscopy. B: Periplasm

strain: cell (C), OMV supernatant (S) or OMV fractions (lanes 1 to 6 correspon

(OMV) � 109 cells).


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The oprI (lpp) and oprF (ompA) mutants were shown toinduce the PQS dependent OMV formation while oprL ( pal )mutant had the same phenotype as the wild type strain (Wesselet al., 2013). To pursue with non-protein secretion, antibioticshave been successfully released into OMVs and delivered toeukaryotic cells (Kadurugamuwa and Beveridge, 1998).

Similarly, Gram-positive MVs have shown importantpathogenic functions. Major determinants have been detectedsuch as the anthrax toxins in Bacillus anthracis MVs (Riveraet al., 2010) while the Staphylococcus aureus MVs wereshown to induce apoptotic cell death through the interactionswith the lipid raft machinery (Gurung et al., 2011).

1.4. Delivery/secretion of OMV associated components

Delivery of secreted molecules or virulence factors char-acterizes the non-classical secretion mechanism mediated byOMVs. In addition, results presented above indicate that OMVsecretion prevents the dilution of soluble proteins. Thesenanoparticles can deliver components to neighboring bacteria(Kadurugamuwa and Beveridge, 1999) or to eukaryotic cells(Kesty and Kuehn, 2004). Toxins are often found membraneassociated while hydrophobic compounds (such as PQS orgentamicin) are inserted in the outer leaflet of these nano-particles. In a recent example, the communication of Bacter-oides fragilis with host cell was found dependent on thedelivery of its capsular polysaccharides through OMV release(Shen et al., 2012). Thus, pathogens interact through directcell contact with host using injection machines belonging tothe type III, type IV and type VI secretion systems and alsocommunicate through the OMV delivery system (Hodges andHecht, 2012). Lastly, nanopod structures have been discoveredin soil bacteria (Shetty et al., 2011). Under non-hydratedconditions these tubular structures contain small OMVs (size�1 nm) able to be delivered at long distance and to transmitpeptidase or hydrolase activities.

roducing periplasmic tGFP (tGFP: GFP harboring the TMAO reductase signal

ic protein distribution. Immunodetections of b-lactamase and tGFP from tolA

ding respectively to the equivalent of 0.4e0.1 (C), 5.0e1.0 (S) and 10-2.0


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OMV secretion of exported GFP was demonstrated in E.coli laboratory and pathogenic strains (Kesty and Kuehn,2004). However, the OMV associated GFP was not directlyobserved using GFP fluorescence. Taking advantage of thehigh level of OMVs production of tol cells and of the directdetection of tGFP present in the OMV using fluorescencemicroscopy (Gerding et al., 2007, Fig. 2A), we were able toperform time-lapse experiments of OMV formation (Fig. 3A).In first event, the periplasm became fluorescent according tothe export of tGFP. The formation of fluorescent OMVsappeared 4e8 min later with a concomitant decrease of theperiplasmic fluorescence. The results of periplasmic release ofb-lactamase and tGFP (Fig. 3B) suggested that tol cells,defective in OM integrity, are able to maintain or enrich tGFPconcentration into the OMV lumen. It is noteworthy that the E.coli tolA mutant has also been used for biotechnologicalpurposes using an engineered ClyA cytotoxin fused with GFP(Kim et al., 2008).

OMVs have been described as nanoparticles acting asoffensive or defensive weapons (Macdonald and Kuehn,2012). OMVs can deliver hydrophilic, hydrophobic com-pounds and virulence factors at high concentration, canmediate cell interactions and contribute to the modulation ofthe host immune response (Deatherage and Cookson, 2012;Ellis and Kuehn, 2010; Unal et al., 2011). Last discovered roleof Myxococcus xanthus OMVs indicates that they are preda-tory agents able to kill other bacteria such as E. coli by fusionwith the OM (Evans et al., 2012). Beside numerous enzymesfound in M. xanthus OMVs, the alcaline phosphatase was

Fig. 4. T5 phages binding to OMVs containing FhuA. (From Stephanie Pommier

OMVs (indicated by “v”) incubated with head-tailed T5 phages, Bar corresponds


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quite exclusively recovered packaged into OMVs indicating anactive sorting process. Lastly, the defensive role of OMVs isan additional function that consists in the removal of toxic ormisfolded proteins or toxic components and in the protectionof bacteria against phage infection or killing molecules (seePerspectives).

1.5. Perspectives

The protective role of OMVs has also been recentlydemonstrated to neutralize environmental agents that target theOM and to decrease antimicrobial peptide activity or T4bacteriophage infection (Manning and Kuehn, 2011). Usingpurified OMVs enriched with the T5 phage receptor FhuA, wealso demonstrate the protective role of FhuA enriched OMVs.For this purpose, the FhuA protein containing an exposed His-tag was produced in E. coli tolA mutant. Purified OMVs werefurther separated using an additional metal affinity chroma-tography step to remove FhuA depleted OMVs. Incubation ofthe purified FhuA-OMVs with saturating amounts of T5phages resulted in the detection of single or multiple phagesbinding to one OMV (Fig. 4). This demonstrates that it ispossible to trap efficiently T5 bacteriophages using FhuAcontaining OMVs. The protecting role of OMVs against phageinfection may also be efficient against bacterial toxin killing(colicins, pyocins, pesticins) as shown with the antimicrobialpeptides (Manning and Kuehn, 2011).

The results on OMVs isolated from planktonic, soil, biofilmor clinic strains confirm the secretion role of these

, PhD manuscript, 2005). Electron micrograph of negatively stained purified

to 200 nm.


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nanoparticles and their connecting role with bacteria and hostcells. Lot of questions remains to be answered, the first onebeing on the mechanism of membrane budding and vesicleformation. According to the first model of defective lateral cellenvelope integrity, OMVs containing similar protein content tothe OM may be expected. The defect in TolePal componentslocated at the septum of dividing cells may induce the for-mation of OMVs containing variable amounts of membrane orsoluble proteins, in relation with the dynamic rearrangementoccurring during cell division. Protein sorting has beendiscovered in B. gingivalis (Haurat et al., 2011) that is a nat-ural tolepal mutant able to perform preferential packaginginto OMVs. This observation and those on OMVs containingtGFP (Fig. 3) indicate that OMVs formed by tolepal mutantsare not only the result of random OM instability.

In relation with their small size, the lipid composition of theinner leaflet of OMVs may be different from that of the OM.This hypothesis arises in accordance with the detection ofcardiolipin micro-domains localized inside membrane regionsof high curvatures (Renner and Weibel, 2011). Thereby, thecardiolipin-dependent membrane curvature suggests that car-diolipin participates in OM budding. Another membranedependent possibility may be related to the accumulation of ananchored component in the outer leaflet of the OM that wouldconfer same membrane properties than PQS molecules orother small hydrophobic compounds (Schertzer and Whiteley,2012) to induce OMV budding. Indeed some lipoproteins havesurface exposed location in the OM (Kovacs-Simon et al.,2011) and may be potential candidates for inserting in theOM and provoke OMV budding.

2. Colicin lysis proteins

Colicins are protein toxins produced and secreted in theextracellular medium by E. coli or related bacterial species.They are active against E. coli and other related species toprotect the bacterial niche against invaders. Colicins usedifferent ways to kill susceptible cells, by depolarization of thecytoplasmic membrane, by cytotoxic activity against cyto-plasmic nucleic acids, or by interfering with peptidoglycanbiosynthesis. To produce and secrete colicins, bacteria needtwo other genes, one encoding the immunity against thecolicin, and in most cases, a gene encoding the bacterialrelease protein (BRP) also name kil or colicin lysis gene.Colicin and lysis genes are organized in operons under thecontrol of a SOS promoter arising from DNA damage.Regulation of colicin operons by LexA repressor has beendescribed for a long time (see Cascales et al., 2007) butrecently, a second additional repressor IcsR (global regulatorof iron-sulfur clusters) has been shown to control colicinexpression. The colicin-lysis gene expression occurs notdirectly after DNA damage but later under nutrient depletionconditions (Butala et al., 2012). Thus, colicin and lysis genesare tightly regulated by three mechanisms, (i) the inducedDNA bending upon LexA binding (Lloubes et al., 1988), (ii)the cooperative binding of two LexA dimers to tandem over-lapping SOS operators resulting in higher binding affinity of


j.resmic.2013.03.015

LexA and increased repression (Lloubes et al., 1991; Gilloret al., 2008), (iii) and the additional locking with IcsR regu-lator ensuring delayed expression. Surprisingly, the replace-ment of the SOS promoter sequence of the colicin lysis geneby that of an IPTG inducible promoter was possible to obtainwithout modifying BRP maturation and functioning (Cavardet al., 1989).

Colicin lysis genes encode small lipoproteins that are ableto promote the extracellular release of colicins. The signalpeptides of most BRP are stable after cleavage and accumulatein the inner membrane. All the lysis proteins possess a Glnresidue at position þ2 indicating a final location in the OM.Colicin lysis genes may be exchanged without affecting thecolicin release. Colicins, together with soluble periplasmic andcytoplasmic proteins, are released in the extracellular mediumaccording to the expression the colicin lysis gene (Baty et al.,1987). In laboratory conditions, BRP have been used to secreteperiplasmic proteins and recombinant proteins in the extra-cellular medium (van der Wal et al., 1998). The optimizationof BRP expression has been recently performed for biotech-nological purposes (Sommer et al., 2010).

Colicin release differs from the other secretion mechanismssince its depends on the BRP and on the activation of OmpLA,the OM phospholipase A. OmpLA had been suspected topermeabilize the OM and futher the IM through the formationof lysophospholipids (for a review see Cascales et al., 2007).Colicin remains cytoplasmic when produced in a pldA strain(defective in OmpLA) while its extracellular release occurs ina pldA mutant suppressor harboring OM permeability defectsuch as tol mutant (Howard et al., 1991).

The activity of the OmpLA and the colicin production maybe involved in OMV formation and protein release since BRPhave been detected in the supernatant fraction of inducedcolicinogenic strains (Cavard, 2004). In addition, P. aerugi-nosa strains which produced pyocins and colicin like toxins,(Cascales et al., 2007; Barreteau et al., 2012), produced OMVsupon induction of the SOS response (Maredia et al., 2012).Thus, the analyses of OMV formation and protein sortingupon colicin expression remain a domain to explore.

Acknowledgments

We thank Marthe Gavioli, Xiang Zhang for technical help,Denis Duche for careful reading of the manuscript, and GuyLeegeli for encouragements. This work has been fundedthrough grants from the Agence National de la Recherche toR.L. (SODATOL [ANR-07-BLAN-067], and PEPGLYCOL[ANR-07-MIME-022]). L. H. is a recipient of a postdoctoralfellowship from the Fondation pour la Recherche Medicale.

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