1 Membrane vesicle release in Bacteria, Eukaryotes and Archaea: a

35 Pages·2012·4.69 MB·English

IAI Accepts, published online ahead of print on 12 March 2012
Infect. Immun. doi:10.1128/IAI.06014-11
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
1 Membrane vesicle release in Bacteria, Eukaryotes and Archaea: a
2 conserved yet underappreciated aspect of microbial life
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4 Brooke L. Deatherage1,3 and Brad T. Cookson1,2*
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7 Running title: Membrane vesicles: key to physiology and pathogenesis lo
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9 1 Department of Microbiology and 2 Laboratory Medicine, University of ro
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10 Washington, Seattle, WA, 98195
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11 3 Present address: Pacific Northwest National Laboratory, Richland, WA, 99354 //
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12 * Corresponding author information: 1959 NE Pacific St. Box 357110, Seattle, s
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13 WA, 98195-7110, phone: [phone], fax: [phone], email: o
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14 [email protected] o
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15 Abstract
16 Interaction of microbes with their environment depends on features of the
17 dynamic microbial surface throughout cell growth and division. Surface
18 modifications, whether used to acquire nutrients, defend against other microbes,
19 or resist the pressures of a host immune system, facilitate adaptation to unique
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20 surroundings. The release of bioactive membrane vesicles (MVs) from the cell o
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21 surface is conserved across microbial life, in bacteria, archaea, fungi, and lo
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22 parasites. MV production occurs not only in vitro, but also in vivo during infection, e
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23 underscoring the influence of these surface organelles in microbial physiology o
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24 and pathogenesis through delivery of enzymes, toxins, communication signals,
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25 and antigens recognized by the innate and adaptive immune systems. Derived /
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26 from a variety of organisms that span kingdoms of life, and called by several s
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27 names (membrane vesicles, OMVs, exosomes, shedding microvesicles, etc.), o
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28 the conserved functions and mechanistic strategies of MV release are similar, o
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29 including use of ESCRT proteins and ESCRT protein homologues to facilitate p
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30 these processes in archaea and eukaryotic microbes. Although MV release by 3
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31 different organisms share similar visual, mechanistic, and functional features, 1
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32 there has been little comparison across microbial life. This underappreciated y

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33 conservation of vesicle release, and the resulting functional impact throughout e
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34 the tree of life, explored in this review, stresses the importance of vesicle-
35 mediated processes throughout biology.
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37
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38 Microbial Membrane Vesicles: Introduction
39 The production of spherical, membranous vesicles from microbial cell
40 surfaces is conserved among organisms from all three branches of the tree of
41 life, spanning both prokaryotes and eukaryotes: Gram-negative and Gram-
42 positive bacteria (16, 47, 58, 73), archaea (17, 18), fungi (3, 65-67) and parasites
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43 (82, 83). For consistency in this review, we will refer to bacterial and archaeal o
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44 structures as membrane vesicles (MVs) and fungal and parasitic vesicles as lo
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45 either exosomes or shedding microvesicles (two distinct populations referred to e
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46 collectively as microvesicles (55)). The microscopic observation of microbial o
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47 MVs spans more than 50 years, and numerous functions have been attributed to
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48 these extracellular vesicles by many investigators. The release of vesicles /
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49 provides flexibility to respond to environmental cues, secrete components s
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50 destined for the cell surface, virulence factors, and antigens, and interact with the o
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51 host in the case of pathogens. Because MV release is conserved across many o
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52 organisms, MV-mediated functions are likely to be critical to microbial life. p
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53 Both bacterial MVs and archaeal MVs are derived from the cell surface 3
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54 (Figure 1A, 2A). Early observation of Gram-negative bacterial MVs revealed the 1
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55 release of an antigenic complex of lipopolysaccharide (LPS) and lipoprotein into y

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56 the surrounding medium following amino acid deprivation of an E. coli lysine e
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57 auxotroph (40), which was initially proposed to be derived from the LPS-
58 containing outer membrane (OM) of the bacteria (29). Since these early
59 investigations, many groups have developed methodologies to isolate and
60 analyze bacterial MVs. Although reconciling these differences in experimental
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61 design often make it difficult to draw generalized conclusions, it is well accepted
62 that Gram-negative bacterial MVs range from 10-300nm in diameter and contain
63 OM and periplasmic constituents, including proteins, lipoproteins, phospholipids,
64 and LPS (43, 58). Contents of the inner membrane and cytoplasm were
65 generally thought to be excluded from MVs, although recent analyses of the
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66 bacterial MV proteome suggest that some proteins typically annotated as having o
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67 cytoplasmic localization consistently appear in MVs (15, 45, 92, 94). In addition lo
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68 to bacterial membrane proteins, toxins and signaling molecules can be e
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69 incorporated into the membrane or lumen of the MV; MV release then serves as o
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70 a secretion mechanism (42, 54, 95). Although Gram-negative bacterial MVs
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71 have been most rigorously studied, recent observation of Gram-positive MV /
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72 release has demonstrated that this is a function more widely conserved across all s
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73 bacteria. MVs derived from Gram-positive bacteria, such as Bacillus spp., are o
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74 similarly sized (50-150nm in diameter (47, 73)), and are rich in membrane lipids o
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75 as well as toxins (including the anthrax toxin). p
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76 Archaeal MVs, such as those released by Sulfolobus species, range from 3
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77 90-230nm in diameter and contain membrane lipids and S-layer proteins also 1
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78 derived from the archaeal cell surface (17, 69). A common functional theme y

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79 begins to emerge: these MVs can also transport toxic compounds into the e
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80 surrounding milieu (69), although toxin production is not required for vesicle
81 release, as non-toxin producing strains and other archaea such as Ignococcus
82 naturally release MVs as well (71).
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83 Eukaryotic microbial vesicles, derived from fungi and parasites, include at
84 least 2 vesicle populations (Figure 3) (25, 66). Exosomes (40-100nm in
85 diameter) are derived from multi-vesicular bodies (MVBs) within the cell, and are
86 typically homogenously shaped (Figure 3B) (55). Shedding microvesicles
87 (SMVs; 100-1000nm in diameter) bud directly from the cell surface, resulting in
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88 more heterogeneous vesicle morphology (Figure 3C) (13). Vesicles derived from o
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89 eukaryotic microbes contain characteristic lipids and proteins that reflect both lo
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90 surface constituents and secreted cellular components. While these two e
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91 processes are visually similar when observed microscopically, it is likely that the o
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92 cellular machinery participating in formation and the downstream functions of
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93 these MV populations are distinct. The presence of multiple active vesicle /
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94 secretion mechanisms is supported by work in Saccharomyces cerevisiae, in s
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95 which extracellular MVs were released even in the absence of known secretory o
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96 pathways (67). o
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97 The release of vesicles has been demonstrated for both pathogenic and p
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98 non-pathogenic microbes in a range of growth conditions, including in liquid broth 3
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99 and on agar plates in the laboratory (15, 82, 90), in biofilms (6, 78), upon 1
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100 infection with bacteriophage (50), and by pathogenic organisms growing within y

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101 an animal host (9, 20, 23, 52, 63, 88). In addition, modification of media e
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102 conditions, such as presence of serum (62), limitation of essential amino acids
103 (40), or treatment with sub-inhibitory concentrations of membrane active
104 antibiotics (33), stimulate MV production, suggesting vesicle release is both
105 dynamic and manipulable, essential characteristics for microbes subjected to
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106 ever-changing environments. Conservation of this process during both in vitro
107 and in vivo growth, and the difficulty (or inability (57, 58, 67)) of genetic
108 approaches to identify mutations that abrogate vesicle release reinforces the idea
109 that this process is integral to microbial life.
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111 Mechanisms of Vesicle Biogenesis o
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112 Bacteria. As this process in Gram-positive bacteria is only beginning to be lo
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113 explored (16, 47, 56, 73), we will focus our discussion on the understanding of e
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114 the process in Gram-negative bacteria. Gram-negative bacterial MV release, o
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115 observed microscopically over several decades (7, 40), has been proposed to
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116 occur by many different mechanisms (5, 29, 42, 54, 58, 60, 96). Reconciliation of /
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117 these mechanisms, however, is complicated by the variability under which these s
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118 studies were completed (i.e., using mutant bacterial strains with altered LPS o
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119 and/or nutrient requirements, or using varied growth conditions, such as amino o
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120 acid deprivation or the presence of antibiotics). Most models of MV release, p
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121 studied primarily in Proteobacteria, propose an “either-or” involvement of proteins 3
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122 or LPS in this process. However, it is likely that MV formation results from the 1
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123 contribution of multiple dynamic surface components (Figure 1), and there are y

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124 several proposed mechanisms of MV release. As detailed reviews exist on this e
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125 topic that are outside the scope of this review (43), we will limit our discussion
126 here and include recent developments in the field.
127 LPS is integral to the structure and physiology of Gram-negative bacteria.
128 Disruption of this molecule, therefore, impacts the stability and architecture of the
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129 microbial surface, including vesicle release. For example, in P. aeruginosa,
130 perturbation of the bacterial surface with subinhibitory concentrations of the
131 antibiotic gentamicin stimulates increased MV release. Gentamicin is thought to
132 interact uniquely with the two structurally distinguishable LPS species of P.
133 aeruginosa, based on charge attraction (44, 74). Not only does gentamicin
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134 treatment induce MV release, but it also results in enrichment of certain LPS o
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135 species in MVs (64). A similar phenomenon is found in Porphromonas gingivalis, lo
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136 which also expresses two differentially charged O-antigens. Significant decrease e
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137 in the abundance of one LPS species in the OM altered protein cargo o
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138 incorporated into MVs (27). Furthermore, treatment of the E. coli cell surface
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139 with Mg2+ resulted in decreased MV production, supporting the idea that charge //
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140 interactions on the cell surface (via the LPS O-antigen polysaccharide) influence s
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141 MV release (89), and that substances acting on the cell surface in different o
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142 environments may induce release of MVs with specific contents. Additionally, o
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143 stimuli from within the bacterial cell can also influence MV release. The p
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144 interaction of the lipid A portion of LPS with the P. aeruginosa-produced quorum 3
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145 sensing molecule pqs (54) alters membrane curvature (53), thereby inducing MV 1
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146 production (Figure 4B). y

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147 The role bacterial surface proteins play in MV release must also be e
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148 considered, as they are integral to both the structure and function of the cell, and
149 there are several non-mutually exclusive mechanisms discussed here in which
150 proteins may be involved. The first model considers MV release in the absence
151 of environmental stressors (like antibiotics) or mutations affecting nutrient
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152 requirements or LPS structure. A systematic and quantitative approach to
153 analyze the MVs produced by Salmonella enterica serovar Typhimurium (S.
154 Typhimurium) revealed that specific major envelope proteins modulate MV
155 release by wild-type (WT) bacteria (15). WT MV production occurred along the
156 cell body and at division septa (Figure 1A). When specific envelope proteins
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157 were deleted, cells exhibited an enrichment of MV release at either cell body o
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158 (Figure 1B) or septa (Figure 1C). This localization was due to the absence of lo
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159 important envelope interconnections normally present in envelope proteins of e
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160 interest; when interacting domains as small as one amino acid were removed, o
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161 the resulting phenotype mimicked a full deletion, underscoring the importance of
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162 these interactions. Specifically, proteins such as OmpA and LppAB are /
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163 anchored in the OM but interact with the peptidoglycan (PG) layer (Figure 1B, s
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164 purple OM-PG interconnections), while Pal, TolB, and TolA form a protein o
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165 complex that spans the OM-IM (Figure 1C, orange OM-PG-IM interconnections). o
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166 In WT cells, these tethers normally maintain MV production at minimal levels. p
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167 OM-PG linked proteins dampen the release of small MVs from the cell body of S. 3
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168 Typhimurium, whereas formation of the cell division plane requires movement of 1
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169 OM-PG-IM protein complexes, facilitating septal MV release (15). Migration of y

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170 these proteins along the dynamic cell surface promotes enrichment of a subset of e
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171 constituents into septal or cell body-derived MVs, suggesting that Gram-negative
172 bacteria may be able to control the distribution and abundance of envelope
173 interconnections such that the site and content of released MVs is
174 correspondingly regulated. This idea was confirmed by proteomic analysis of MV
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175 purified from septa and cell body, respectively. Based on these data, we
176 proposed the following model: MV production occurs at envelope regions where
177 the density of OM-PG and/or OM-PG-IM interconnections has been temporarily
178 decreased (Figure 1B, C) (15). While some organisms may have evolved distinct
179 means of modulating LPS structure (61) resulting in modifications of MV
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180 formation, the high degree of conservation among the protein domains facilitating o
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181 OM-PG and OM-PG-IM envelope interconnections across diverse Gram-negative lo
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182 bacteria (15) supports the idea that these connections have widespread e
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183 importance in the process of bacterial MV release. o
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184 Other proteins may also contribute to MV formation. Accumulation of over-
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185 expressed periplasmic proteins promotes increased MV release, possibly via /
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186 induction of outward budding of the membrane (59). However, these models s
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187 assume that MVs are released into an aqueous milieu where they are able to o
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188 freely travel. Although this may be true for pathogenic organisms that live in o
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189 association with host cells and tissues, it is not the case in partially-hydrated p
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190 environments like microbe-rich soil. Recent studies have suggested a novel 3
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191 mechanism by which organisms may release MVs in water-restricted conditions. 1
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192 The construction of nanopods allow for MVs to travel from bacterial cells such as y

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193 Delftia species (81). Nanopods are seemingly protective tubular structures made e
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194 of surface layer protein through which MV-like structures containing LPS and OM
195 proteins are released from cells. Interestingly, nanopod formation is conserved
196 in other organisms, may be facilitated by LPS-surface layer protein interactions
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197 (19), and may represent a conserved mechanism for MV release in aqueous-
198 poor environments.
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200 Fungi, parasites, and archaea. Although taxonomically distant, many of the basic
201 features of vesicle production by fungi, parasites, and archaea appear to be
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202 conserved, including surface release and the important protein homologues o
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203 regulating the mechanisms of release. Archaeal vesicle release shares similar lo
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204 features with both prokaryotic and eukaryotic mechanisms of MV production, e
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205 perhaps representing the most evolutionarily basic process upon which other o
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206 microbes have adapted additional mechanisms (Figure 2). Archaeal MVs are
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207 surface derived, and are released by ‘pinching off’ of the cell surface, a /
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208 phenomenon reminiscent of bacterial MVs and eukaryotic SMVs (Figures 1B, s
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209 3C). While microbial SMV release may be controlled via mechanisms used in o
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210 mammalian SMV release (via various enzymes, including calpain, flippase, o
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211 floppase, scramblase, and gelosin (68)), this process is currently p
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212 uncharacterized. However, evidence supports a mechanism in which surface 3
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213 MV release by archaea is controlled by a regulated mechanism involving the 1
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214 conserved membrane scission machinery endosomal sorting complex required y

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215 for transport (ESCRT-III) and vacuolar sorting protein (Vps4) homologues (Figure e
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216 2B, C), as these proteins are found to be released in vesicles (17, 51).
217 Interestingly, the absence of ESCRT-III-like homologues in some vesicle-
218 producing archaea (such as the Thermococcales) suggests multiple vesicle
219 release mechanisms may exist (BLAST,(86)). We focus our discussion to the
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