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ScienceDirect Antibacterial effector/immunity systems: it’s just the tip of the iceberg§ Juliane Benz and Anton Meinhart Bacteria do not live anchoretic; rather they are constantly in touch with their eukaryotic hosts and with other bacteria sharing their habitat. Therefore, bacteria have evolved sophisticated proteinaceous weapons. To harm other bacteria, they produce antibacterial effector proteins, which they either release into the environment or export via direct intercellular contact. Contact-dependent killing is mediated by two specialized secretion systems, the type V and VI secretion system, whereas contact-independent processes hijack other transport mechanisms. Regardless of the transport system, cells co-express immunity proteins to protect themselves from suicide and fratricide. In general, effector protein activities and secretion mechanisms differ between Gram-positive and Gram-negative bacteria and evidence is emerging that different effector/immunity systems act synergistically and thus extend the bacterial armory. Address Department of Biomolecular Mechanisms, Max-Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany Corresponding author: Meinhart, Anton ([email protected])
Current Opinion in Microbiology 2014, 17:1–10 This review comes from a themed issue on Host–microbe interactions: bacteria Edited by Olivia Steele-Mortimer and Agathe Subtil For a complete overview see the Issue and the Editorial Available online 8th December 2013 1369-5274/$ – see front matter, # 2013 The Authors. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mib.2013.11.002
Introduction Bacteria produce a multitude of effector proteins which they either export into their environment or directly into other cells. These effector molecules are targeted against eukaryotic host cells where they promote pathogenicity as virulence factors (reviewed in [1]). Additionally, effector proteins are also used to compete with or even to gain advantage over other species during bacterial warfare within the microbiome. In the latter case, cells co-produce § This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
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immunity proteins to protect themselves from suicide or fratricide. Export of effector proteins is accomplished by secretion systems (SSs) that are classified according to the transported effector molecules, their targets as well as the producing cell type (Gram-negative or Gram-positive) (reviewed in [2–4]). The general secretion (Sec) and the two-arginine translocation (Tat) pathways and the type IV secretion system (T4SS) are found in both bacterial cell types. In contrast, the T1SS, T2SS, T3SS, T5SS, and the recently discovered T6SS have only been identified in the genome of Gram-negative bacteria, whereas the T7SS has been exclusively found in Gram-positive Mycobacteria. Depending on the secretion system, the effector proteins are either exported into the environment (T1SS, T2SS, T7SS) or are directly transported into the target cell upon physical contact (T3SS, T4SS, T5SS, T6SS). Bacteria have evolved several different secretion systems but only three (T5SS, T6SS, T7SS) have been reported to be used in bacterial growth competition. The other secretion systems have been reported to exclusively target eukaryotic cells and transport virulence factors (reviewed in [2,5]). This article reviews the current knowledge of antibacterial effector/immunity systems, classified into released or injected effector proteins and discusses similarities and differences between antibacterial effector/immunity systems and the well-known toxin/antitoxin systems.
Contact-independent antibacterial systems — the bacteriocins Bacteriocins are effector proteins that are released by bacteria into their environment and are the best characterized antibacterial effector proteins to date (reviewed in [6,7]). They are widespread among all bacterial species and are synthesized by ribosomes together with a cognate immunity protein that protects bacteria from their own or their sibling’s bacteriocins [6,7]. In this respect, bacteriocins are different to antibiotics such as vancomycin, which are produced by non-ribosomal peptide synthetases. Bacteriocins have a narrow spectrum of activity and primarily act on closely related species. They are highly diverse in terms of their activities, amino acid sequences and three-dimensional structures [6,7]. Similarly, archaea produce archaeocins to gain growth advantages over their rivals (reviewed in [6]). Although archaeocins have been predicted to be a general feature of haloarchaea [8], only eight different halophilic strains (halocins) and Sulfolobus islandicus (sulfolobicins) have been reported to express such a system [6]. Current Opinion in Microbiology 2014, 17:1–10
2 Host–microbe interactions: bacteria
Bacteriocins produced by Gram-negative bacteria Gram-negative bacteriocins are named after the producing bacteria followed by the suffix -cin, with colicins from Escherichia coli being the founding and most extensively investigated members (reviewed in [7]). These bacteriocins are encoded from a single operon together with their cognate immunity protein. Whereas most operons also encode a lysis protein that induces autolysis for effector protein release (Figure 1a), only a few bacteriocins are released by cell wall leakage. Notably, the lysis proteins are highly conserved and functionally interchangeable between different strains [7]. Gram-negative bacteriocins generally consist of three domains; a translocation (N-terminal), a receptor binding (central) and a killing domain (C-terminal). The central domain hijacks specific receptor proteins that are usually responsible for nutrient uptake (Figure 1a). These receptors are highly diverse among bacteria leading to a narrow killing spectrum of bacteriocins [7]. Import of bacteriocins is mediated by the recipient’s Ton or Tol system (Figure 1a) [7,9,10]. These systems have been reported to make use of a proton motive force for dissociation of the tightly bound immunity proteins from the effector protein [10] and also for translocation of the bacteriocins into the target cell (reviewed in [9]). Except for colicin M, which hydrolyzes peptidoglycan precursors [11] and pesticin from Yersinia pestis, which possesses muramidase activity [12], all other Gram-negative bacteriocins have either pore forming or nuclease activity (Figure 1a) [7].
Bacteriocins produced by Gram-positive bacteria In contrast to their Gram-negative relatives, Grampositive bacteriocins have a broad killing spectrum, and some of them even target Gram-negative bacteria (reviewed in [6,13,14]). Since Gram-positive bacteriocins simply diffuse through their target’s cell wall, they have a very broad killing spectrum [14]. Furthermore, when compared to Gram-negative bacteriocins, they are found in large gene clusters and their production relies on specific regulatory and transport factors [13]. Thus, Gram-positive bacteria survive bacteriocin release, whereas most Gram-negative bacteria undergo autolysis for effector protein release [13,14]. Gram-positive bacteria, particularly lactic acid bacteria, produce a plethora of different bacterocins which are either small peptides or proteins with a molecular weight of more than 30 kDa and are grouped into four major classes: The well-known lantibiotics (class I), small heat-stable peptides (class II), heat-labile proteins that kill bacteria by either lytic or non-lytic mechanisms (class III) and cyclic peptides of which the mechanism of function is still elusive [6]. However, all these bacteriocins do not belong to the classical effector/immunity systems and are often referred to as bacteriocin-like effectors, since they are either small Current Opinion in Microbiology 2014, 17:1–10
peptides, or do not possess any enzymatic activity or lack an immunity protein when they are classical enzymes. The exception to the rule is the ribonuclease Barnase from Bacillus amyloliquefaciens [15]. Although Barnase and its cognate immunity protein Barstar [16] were identified 50 years ago, their putative antibacterial effector/immunity function is exclusively predicted based on similarities to bacteriocins and is still hypothetical. An antibacterial activity was suggested since E. coli cells expressing Barnase inhibit the growth of other bacteria and expression of Barstar confers immunity [17]. However, no direct evidence has been reported so far that B. amyloliquefaciens harms other bacteria using Barnase and the mechanism by which Barnase enters bacteria is also still elusive.
Contact-dependent antibacterial systems Apart from simple secretion of effector proteins into the environment, bacteria also kill others by establishing intercellular contacts. Two different contact-dependent systems have been described so far: the contact-dependent growth inhibition (CDI) and the T6SS effector/ immunity systems.
‘Toxin on a stick’ — contact-dependent growth inhibition Contact dependent growth inhibition (CDI) by the E. coli EC93 isolate is evoked by the cdiBAI operon [18]. CdiA is the effector protein consisting of two domains, a large conserved N-terminal domain (NtD) and a smaller Cterminal antibacterial effector domain (CdiA-CT) responsible for dissipating the proton motive force of the targeted cell [19]. The CdiI protein provides immunity by complex formation with CdiA effector molecules which have been secreted by siblings. Secretion of CdiA relies on CdiB, which has been predicted to be a channelforming protein (Figure 1b) [18,20]. The CdiA effector is a large protein (about 300 kDa) and was predicted to protrude from the surface by several nanometers. The Nterminal domain of CdiA binds to the outer membrane receptor BamA of the target cell [21] and thereby establishes a direct physical contact [18]. Subsequently, CdiACT is most likely autoproteolytically cleaved from the Nterminal domain and assimilated into the competitor cell [18]. However, CDI systems are much more prevalent in the genome of protebacteria than previously expected. A bioinformatic search for putative effector variants revealed an entire family of CDI operons with a conserved N-terminal BamA binding domain [22]. The toxic CdiA-CT domain, however, shows a significant degree of polymorphism with tRNase, DNase or pore forming activities [19,22]. Although these polymorphic CdiA proteins are generally conserved in their N-terminal domain, specificity for extracellular loop regions of the BamA receptor of different bacterial species is achieved www.sciencedirect.com
Antibacterial effector/immunity systems Benz and Meinhart 3
Figure 1
(a)
(b)
(c)
ribosome
5′ 5′
HC
CdiA
3′
3′ 5′
5′ 3′
3′
CdiA
? Ton
HIM
HP
ATP
HOM
Tol
ADP
ATP
??? ADP
CdiA
Receptor
BamA VgrG
CdiA ECM
Ions Protons etc.
Ions Protons etc. Hcp
OM
CdiB Lysis
P
IM
Sec Lysis ATP
Cdil
ADP
ClpV ATP ADP
Cdil Cdil
C Cdil
CdiA
CdiB Cdil Current Opinion in Microbiology
Secretion mechanism and mode of action of the different antibacterial effector/immunity systems. (a) Release of Gram-negative bacteriocins (purple) is a lethal event for the producer cell as it is accomplished by a lysis protein (red). Extracellular released bacteriocins are incorporated into the host cell by receptors (dark blue) usually responsible for nutrient uptake. Translocated into the host periplasm (HP), bacteriocins are either integrated into the host’s outer membrane (HOM) or further transported across the host’s inner membrane (HIM). Transport across the HIM is performed by the energy dependent Ton or Tol system (light blue). Furthermore, the immunity proteins (spheres colored in purple) are only released in the host cell before Ton/ Tol import. Gram-negative bacteriocins have RNase or DNase activity and degrade tRNAs, mRNAs or DNAs of the host cells. (b) T5SSs are composed of two components CdiA (magenta) and CdiB (green) that are exported into the periplasm of the producer cell via their Sec-system (yellow). CdiB is a b-barrel forming protein that forms a pore in the outer membrane (OM) of the producer cell and transports CdiA across the OM. CdiA is presented on the cell surface and binds to the BamA receptor (dark blue) of host cells upon direct cell–cell contact. BamA transports CdiA across the HOM into the periplasm where it is either integrated into the membrane or further transported into the host cytoplasm (HC) by a yet unknown mechanism. CidA proteins harbor nuclease activity and inhibit bacterial cell growth by degrading tRNA or DNA of the host cell. Furthermore, to prevent suicide and fratricide, the donor cells co-express CdiI immunity proteins (magenta spheres). (c) The T6SS transports effector proteins directly into competing bacterial cells. Hcp and VgrG are highly conserved among all T6SSs and homologous to the bacteriophage tip and tube components, respectively. Assembly and disassembly of the T6SS needle is driven by ATP hydrolysis performed by ClpV. Three main classes of antibacterial T6SS effector proteins have been identified, which are active on three distinct cellular compartments of the host cell: periplasm (red), lipid bilayer (orange) or the cytoplasm (blue). On the basis of these workplaces the cognate immunity proteins (spheres colored according to the corresponding effector) are also either localized in the periplasm, membrane or in the cytoplasm of the producer cell. Effector proteins that degrade the bacterial peptidoglycan scaffold show either amidase or glycoside hydrolase activity. Effector proteins with phospholipase activity lead to cell lysis by modifying the composition of the lipid bilayer. The mechanisms and targets of cytoplasmic active effector proteins are still elusive. C: cytoplasm, IM: inner membrane, P: periplasm, OM: outer membrane, ECM: extracellular milieu, HOM: host outer membrane, HP: host periplasm, HIM: host inner membrane, HC: host cytoplasm.
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Current Opinion in Microbiology 2014, 17:1–10
4 Host–microbe interactions: bacteria
by sequence variations [23]. Thereby, CDI systems achieve specificity for their target cells and establish a narrow killing spectrum as observed for Gram-negative bacteriocins [20,22]. Furthermore, CDI operons always contain a small ORF located downstream of the CdiA locus. These ORFs have been predicted to encode for the cognate immunity proteins. For systems where the function has been experimentally verified, it became evident that the immunity proteins are also polymorphic and do not show any cross-reactivity with other CdiA effectors [22,24]. Downstream of many of these polymorphic CDI loci, potential genes of so called ‘orphan’ cdiA-CT/cdiI modules are found. Whereas the cdiA-CT gene is translationally silent, the cdiI ORF is expressed, but its function is still elusive. Fusion of such an orphan cdiA-CT gene to the NtD of the upstream and functional cdiA gene resulted in an active protein secreted by CdiB. Turning on such orphan cdiA-CT/cdiI genes by homologous recombination could enable bacteria to rapidly diversify their CDI systems and enable them to express a set of CdiI proteins for broad range immunity [25]. Strikingly, CDI loci have also been identified in rearrangement hotspot (rhs) elements, which are genomic regions that facilitate recombination in E. coli [25,26]. Rhs proteins have a conserved N-terminal region, which differs from the BamA receptor binding domain of CdiA proteins and a C-terminal, variable effector domain [25,26]. Similar to CdiI, a cognate RhsI immunity protein is encoded from the same operon but the channel-forming CdiB is missing [25]. Notably, CDI is not restricted to Gram-negative bacteria, and homologous proteins are also found in Gram-positive species like members of the genera Bacillus, Listeria, Clostridium and Streptococcus [27]. In fact, PF04740 proteins share conserved N-terminal domains and their C-terminal domains (CtDs) are homologous to the Gram-negative CdiA-CTs. The CtDs of B. subtilis and B. cereus have been experimentally shown to harbor RNase activity [27]. Furthermore, the ORFs downstream of the PF04740 locus encode small proteins which block growth inhibition, indicative for a functional effector/immunity pair. Similarly, the wall-associated proteins A (WapA) from various B. subtilis strains have variable C-terminal effector domains and an associated immunity protein WapI. In common, no ORF encoding for an orthologous channelforming CdiB protein has been identified for these Grampositive CDI-related effector/immunity systems and effector presentation at the surface must be accomplished by a different mechanism. Since the N-terminal domains of PF04740 proteins contain T7SS-specific secretion signals, export is most likely performed by this particular SS [27,28]. In contrast, WapA proteins carry an N-terminal signal sequence responsible for cell wall binding [27–29]. Current Opinion in Microbiology 2014, 17:1–10
The number of putative polymorphic effector proteins has been increased even more by a recent bioinformatic study which identified a plethora of C-terminal toxic domains with homologous large NtDs [30]. The toxic activities of the C-terminal domains vary and range from nuclease, deaminase, ADP-ribosyltransferase, peptidase and phospholipase to pore-forming activity. All of these putative polymorphic effector proteins are encoded from an operon together with a small ORF that most likely encodes the cognate immunity proteins [30]. However, it remains to be shown if and how these polymorphic putative toxins are secreted and what their role in bacterial warfare is.
‘Molecular syringes’ — effector proteins injected by the type VI secretion system Type VI secretion systems (T6SSs) are found in the genome of approximately 25% of the different bacterial species [31,32]. The T6SS injects a plethora of different effector proteins into target cells by an upside-down bacteriophage-like syringe (reviewed in [3,33]). Whereas anti-eukaryotic T6SS effector proteins and their role in pathogenesis have been extensively investigated (reviewed in [34–36]), the functional mechanisms and the regulation of antibacterial effector proteins are just starting to be resolved. The first antibacterial effector proteins secreted by a T6SS were found in the genome of P. aeruginosa and named Tse1-3 (type VI secretion exported 1–3) [37]. Tse1 and Tse3 act exclusively in the periplasm where they hydrolyze peptidoglycan and thereby provoke lysis of competing bacterial cells [38]. As with bacteriocins, carriers of an active T6SS co-express the immunity proteins Tsi1 and Tsi3 (type VI secretion immunity 1 and 3) from a common operon. Both immunity proteins harbor an N-terminal signal sequence for translocation into the periplasm to prevent fratricide [38]. In contrast, the third effector/immunity pair, Tse2 and Tsi2, is cytoplasmic localized and kills bacteria by an unknown mechanism (Table 1) [37,39].
T6SS effector proteins with amidase activity The discovery of the amidase activity of Tae1 (formerly referred as Tse1) led to the assignment of a plethora of previously hypothetical ORFs to an entire family of effector proteins that hydrolyse the muropeptide stems in the peptidoglycan scaffold [40] (Figure 2). On the basis of their cleavage specificity, these effectors were classified into four groups (type VI amidase effector Tae1-4) (Table 1). Structural characterization of these amidase effector proteins revealed a common N1pC/P60 peptidase core. Distinct structural differences clustering around the active site were observed, which probably reflect the different substrate specificities [41,42,43–49]. Whereas the amidase activity of these effector proteins is caused by a strictly conserved cysteine-histidine dyad in their active sites and www.sciencedirect.com
Antibacterial effector/immunity systems Benz and Meinhart 5
Table 1 Enzymatic activity, targets and immunity proteins of the different antibacterial T6SS effector proteins Effector
Enzymatic activity
Target
Immunity
Reference
Peptidoglycan (mDAP-D-Glu) Peptidoglycan (mDAP-D-Ala) Peptidoglycan (mDAP-D-Ala) Peptidoglycan (mDAP-D-Glu)
Tai1 (Tsi1)
[37,38,40]
Tai2
[40]
Tai3
[40]
Tai4
[40]
Tae1 (Tse1)
Amidase
Tae2
Amidase
Tae3
Amidase
Tae4
Amidase
Tse2
Unknown
Unknown
Tsi2
[37,39]
Tge1 (Tse3)
b-(1,4)-N-acetylmuramidase
Tgi1 (Tsi3)
[37,38,50]
Tge2
N-acetylglucosaminidase
Tgi2
[50]
Tge3
b-(1,4)-N-acetylmuramidase
Peptidoglycan (MurNAc-GlcNAc) Peptidoglycan (GlcNAc-MurNAc) Peptidoglycan (MurNAc-GlcNAc)
Tgi3
[50]
Tle1 Tle2 Tle3 Tle4 Tle5
Phospholipase A2 Phospholipase A1 Phospholipase Phopsholipase Phospholipase D
Plasma Plasma Plasma Plasma Plasma
Tli1 Tli2 Tli3 Tli4 Tli5
[54] [53,54] [54] [54] [54]
Vgr-3
Unknown
Peptidoglycan
TsaB
[53,56]
membrane membrane membrane membrane membrane
Former names of the T6SS effector proteins are given in brackets. GlcNAc: N-acetylglucosamine; MurNAc: N-acetylmuramic acid; D-Ala: D-alanine; D-Glu: g-D-glutamic acid; mDAP: meso-diaminopimelic acid.
the proteins are structurally homologous, their cognate immunity proteins are totally different and share no homology [41,42,43,46,47,49] (Figure 3).
T6SS effector proteins with glycoside hydrolase activity Furthermore, the identification of the muramidase activity of Tse3 from P. aeruginosa (renamed to Tge1PA/Tgi1PA) enabled the identification of three new classes of T6SS glycoside hydrolase effector (Tge) proteins and their cognate immunity proteins (Tgi) (Table 1) [50]. These effector proteins cleave the b1,4-glycosidic bond between MurNAc and GlcNAc in the peptidoglycan scaffold. On the basis of their sequence homology to other glycoside hydrolases, Tge1-3 were suggested to differ in their cleavage mechanism: lytic transglycosylase (Tge1), N-acetylglycosamidase (Tge2) and phage-type lysozyme (Tge3) (Figure 2). Crystal structures of Tge1 from P. aeruginosa [51,52] and Tge2 from Pseudomonas putida [50] in complex with their cognate immunity proteins have revealed how different effector proteins of this family acquire related glycoside hydrolase activity and are inhibited by non-related immunity proteins.
T6SS effector proteins with phospholipase activity Finally, antibacterial effector proteins that possess phospholipase activity were identified in P. aeruginosa, Burkholderia thailandensis and Vibrio cholerae [53,54]. These www.sciencedirect.com
enzymes modify the cell wall composition of competitors and were named Tle (type VI lipase effector) and their corresponding immunity proteins Tli (type VI lipase immunity). ORFs encoding for Tle proteins were identified in many other Gram-negative bacteria. On the basis of sequence homology and their phospholipase A1-like, A2-like and D-like activities, these proteins were grouped into 5 different families, Tle1-5 (Table 1) [53,54]. Notably, T6SS effector lipases harboring both antibacterial and anti-eukaryotic killing activities were reported, suggesting that they play an important role in bacterial pathogenicity [53,55].
T6SS scaffold proteins with antibacterial activity T6SSs not only secrete effector proteins, but also some of their structural components have killing potential (Table 1) [53,56]. Variations of the VgrG (valine-glycine repeat G) protein which is functionally related to the bacteriophage spike proteins (reviewed in [3]), were reported to be secreted and to harbor killing activity. For instance, the ‘evolved’ VgrG-3VC from V. cholera contains a Cterminal, lysozyme-like peptidoglycan binding domain and hydrolyzes the cell wall of Gram-negative competitors [53,56]. VgrG-3VC was suggested to be part of a functional effector immunity pair, since the downstream gene product encodes the cognate immunity protein TsaB [53,56]. Since VgrG proteins form the tip of the T6SS syringe, other functions apart from solely inducing cell lysis are also conceivable. For instance, peptidoglycan Current Opinion in Microbiology 2014, 17:1–10
6 Host–microbe interactions: bacteria
Figure 2
Pentapeptides Tge2
o
MurNAc
o
GlcNAc
o
Tetrapeptides Tge1/Tge3
MurNAc
o
GlcNAc
o
o
L-Ala D-Glu
o
MurNAc
Donor
D-Ala
D-Glu
mDAP
D-Ala
D-Ala
mDAP
D-Ala
mDAP
mDAP
D-Ala
D-Ala
D-Glu
D-Glu
Acceptor
L-Ala
L-Ala
GlcNAc
o
GlcNAc
o
o
MurNAc
o
GlcNAc
o
MurNAc
MurNAc
o
GlcNAc
o
L-Ala
L-Ala Tae2/3
Tge1/Tge3
Tge2
Tae4
D-Glu
Tae4
Tae2/3 Donor D-Ala
mDAP D-Ala Tae1 Acceptor
mDAP D-Glu L-Ala
o
o
GlcNAc
o
MurNAc
o
GlcNAc
o
MurNAc
o
Current Opinion in Microbiology
Peptidoglycan cleavage specificity of the different amidase and glycoside hydrolase effector protein families. Cleavage specificity of Tae1-4 on pentapeptide (left panel) and tetrapeptide (right panel) stems of bacterial peptidoglycan is illustrated according to [38,40]. Effector proteins that belong to the Tae2 and Tae3 family hydrolyze the cross-link between mDAP and D-Ala in tetrapeptides as well as in pentapeptides. In contrast, Tae1 and Tae4 effector proteins degrade the peptidoglycan by hydrolyzing the amide bond formed between g-D-Glu and mDAP. Whereas, Tae1 effector proteins, including the founding member Tse1 from Pseudomonas aeruginosa, specifically cleave the donor stem of cross-linked tetrapeptides, Tae4 effector proteins exclusively degrade acceptor stems of cross-linked as well as non-crosslinked tetrapeptides. Furthermore, pentapeptides can also be degraded by Tae4 family members but with a poor turnover rate. Additionally, bacteria express effector proteins with glycoside hydrolase activity and degrade the peptidoglycan scaffold by cleaving the bond between the sugar moieties. Classification of these Tge proteins is according to [50]. Tge1 (including Tse3 from P. aeruginosa) and Tge3 effector proteins cleave the b-(1,4) glycosidic bond between MurNAc and GlucNAc by their b-(1,4)N-acetylmuramidase activity. In contrast, Tge2 effector proteins possess N-acetylglucosaminidase activity and hydrolyze the b-(1,4) glycosidic bond between GlcNAc and MurNAc. Figure has been adapted from [47]. GlcNAc: N-acetylglucosamine; MurNAc: N-acetylmuramic acid; D-Ala: D-alanine; LAla: L-alanine; D-Glu: g-D-glutamic acid; mDAP: meso-diaminopimelic acid.
degradation by VgrG-3 could facilitate penetration through the producer’s cell envelope during needle formation [56]. ‘Evolved’ VgrG proteins could act as an advance guard by increasing the cell wall permeability of other bacteria and thereby facilitating injection or infusion of other effector proteins. A stealthy penetration of effector proteins into the competitor cell by VgrG proteins is further supported by physical interaction between VgrG-3VC and the phospholipase effector Tle2VC [53]. Finally, it remains to be shown whether cell wall hydrolysis activity of ‘evolved’ VgrG proteins is also used to attack Gram-positive bacteria.
is strictly regulated and not activated by common mechanisms, even not in closely related species. Moreover, effector proteins might even not be secreted under laboratory conditions or are only activated under certain conditions. This hypothesis is further supported by the failure to detect any T6SS activity in re-cultured pandemic V. cholera strains under laboratory conditions [59]. Additionally, antibacterial T6SS activity was identified in various bacteria but no effector proteins could be assigned yet [57,60]. In other studies, putative effector proteins could be identified, but their killing activity and cellular targets are still elusive [37,39,61].
T66S effector proteins are commonly used antibacterial weapons
Effector/immunity systems and other toxins — an interdisciplinary assassination team?
Evidence is emerging that the T6SS effector/immunity proteins characterized so far are just the tip of the iceberg and that the bacterial armory is more extensive and complex than previously thought. For instance, different Acinetobacter baumannii strains contain conserved T6SS loci [57,58], however, not all of them exhibit killing activity towards other bacteria under the conditions tested [58]. This suggests that effector protein secretion Current Opinion in Microbiology 2014, 17:1–10
Effector/immunity and type II toxin/antitoxin (TA) systems are often spuriously mixed, as both induce cell death in bacteria and their genomic organization in bicistronic operons is related. However, there is a fundamental, functional difference between the two systems. Effector proteins are released and serve to attack competitors and the stable immunity proteins provide www.sciencedirect.com
Antibacterial effector/immunity systems Benz and Meinhart 7
Figure 3
(a)
long-term protection against siblings [39]. In contrast, toxin proteins rest inside the cell and the unstable antitoxin provides only temporary protection and thus toxin/ antitoxin systems induce bacterial suicide upon activation (reviewed in [62]).
(b)
Tae1 Ser109
Tae1 Tai1 Cys30 Cys30 His91
(c)
Ser109 His91
Tai1
(d)
(e)
Tae3
Tai3
Tai3
Tai3
Tai3
Tae3 His81
Tai3
Tae3
(f)
Cys23
Tai3
Cys23 His81
Tai3 Tai3
His81
Cys23
Tae3 (g)
Tae4
(h)
Ser98
Tai4
Tae4
Tai4
His126 Cys44 Ser98
Ser98
Tae4 Tai4 (i)
Cys44 His126
Tai4 Ser98 His126
Cys44
Tae4 Current Opinion in Microbiology
Structural overview of the effector/immunity system Tae1/Tai1 from Pseudomonas aeruginosa, Tae3/Tai3 from Ralstonia pickettii and Tae4/ Tai4 from Salmonella typhimurium. (a) Schematic representation of the www.sciencedirect.com
But evidence is emerging that both systems have common origins and/or overlapping functions and a strict separation is not always possible. For instance, both systems are often found within mobile genetic elements, pathogenicity islands and transposon-like regions (reviewed in [7,22,63,64]). It remains to be shown whether TA toxins are translocated in addition to effector proteins by SSs and directly used as weapons against competitors. Such a hijack of the T6SS by non-related T6SS toxins has been shown, for instance for the Rhs proteins of Serratia marcescens [61] and Dickeya dadantii [25,26]. As Rhs loci do not encode for any CdiB homologue nor do Rhs proteins harbor any leader sequence for CdiB-dependent transport they need to hijack other SSs [25]. Transport via the T6SS is rather likely, since Rhs Tae1/Tai1 effector/immunity complex from P. aerugionsa. Tae1 (red) and Tai1 (blue) form a heterodimer. The effector protein’s toxicity is inhibited by a conserved serine residue (Ser109) located in a loop region of Tai1 that prevents effector protein activation and substrate binding. (b) Crystal structure of the effector protein Tae1 (PDB code 4FGE) with the conserved NlpC/P60 catalytic core in gray. Secondary structure elements, which are specific for Tae1, are colored in red. The catalytic important Cys30 and His91 residues are depicted as stick model and highlighted with an orange rectangle. (c) Crystal structure of the Tae1 and Tai1 heterodimer (PDB code 4FGI) colored in red and blue, respectively. The inhibiting serine residue Ser109 as well as the catalytic important residues Cys30 and His91 are depicted as sticks and highlighted with an orange rectangle. (d) Schematic representation of the Tae3/Tai3 effector/immunity complex from R. pickettii. Tae3 (green) and Tai3 (yellow) form a heterohexamer. The effector protein’s toxicity is inhibited by blocking substrate binding. (e) Crystal structure of the effector protein Tae3 (PDB code 4HZ9) with the conserved NlpC/P60 catalytic core in gray. Secondary structure elements, which are specific for Tae3, are colored in green. The catalytic important Cys23 and His81 residues are depicted as stick model and highlighted with an orange rectangle. (f) Crystal structure of the Tae3 and Tai3 heterohexamer (PDB code 4FGI) formed by two Tae3 (green) and four Tai3 (yellow and orange) molecules. Similar to (e) the catalytic important Cys23 and His81 residues are depicted as sticks and the active site is highlighted with an orange rectangle. (g) Schematic representation of the Tae4/Tai4 effector/immunity complex from S. typhimurium. Tae4 (cyan) and Tai4 (orange) form a heterotetramer. The effector protein’s toxicity is inhibited by a conserved serine residue (Ser98) located in a loop region of Tai4 that prevents effector protein activation and substrate binding. (h) Crystal structure of the effector protein Tae4 with the conserved NlpC/ P60 catalytic core in gray (PDB code 4J32). Secondary structure elements, which are specific for Tae4, are colored in cyan. Cys44 and His126 that form the catalytically active dyad are depicted as stick model and highlighted with an orange rectangle. (i) Crystal structure of the Tae4/Tai4 heterotetramer (PDB code 4J32). Two symmetry related Tai4 molecules (orange) form a head-to-tail-dimer and inhibit two opposing Tae4 effector proteins (cyan). As with Tai1, Ser98 in Tai4 is located in a loop and is important for effector protein inhibition by blocking the active site of Tae4 and by preventing Cys44 activation. Residues in Tae4 as well as in Tai4 are depicted as sticks. The active site and the inhibiting serine residue are highlighted with an orange rectangle. Current Opinion in Microbiology 2014, 17:1–10
8 Host–microbe interactions: bacteria
loci are often found next to vgrG genes [25] and Rhsmediated bacterial growth inhibition relies on VgrG proteins [26]. However, this transport might not be accomplished through the injection needle, since the diameter of the Hcp tube (40 A˚) [65] seems to be too narrow for Rhs proteins. Most likely the conserved PAAR (proline-alanine-alanine-arginine) repeat domain of Rhs proteins localizes to the VgrG tip before assembly of the tube [66]. Notably, many uncharacterized PAAR-repeat proteins carry polymorphic C-terminal extensions with enzymatic functions like nuclease, peptidase or lipase activities and might fulfill similar functions [26,66].
Conclusion Effector proteins involved in bacterial competition are a highly polymorphic group of bacterial toxins that apparently form an interdisciplinary and synergistic team to kill competitors during warfare. It seems that just the tip of the iceberg of this arsenal has been discovered and many questions need to be answered before we fully understand bacterial competition and communication and its involvement in pathogenicity.
Acknowledgements We apologize to all scientific colleagues whose work has not been mentioned due to space limitations. We thank Y.-H. Dong for providing us with the PDB structure files for Tae3/Tai3. We are grateful to T. Barends, H. Mutschler, R.L. Shoeman and the Meinhart group for scientific exchange and suggestions for the manuscript and I. Schlichting for continuous support. A.M. is financially supported by the Chica and Heinz Schaller Foundation and the Max Planck Society.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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Antibacterial effector/immunity systems Benz and Meinhart 9
D. dadantii Rhs proteins and showed that VgrG proteins are required for induction of growth inhibition. 27. Holberger LE, Garza-Sanchez F, Lamoureux J, Low DA, Hayes CS: A novel family of toxin/antitoxin proteins in Bacillus species. FEBS Lett 2012, 586:132-136. 28. Sutcliffe IC: New insights into the distribution of WXG100 protein secretion systems. Antonie Van Leeuwenhoek 2011, 99:127-131. 29. Foster SJ: Molecular analysis of three major wall-associated proteins of Bacillus subtilis 168: evidence for processing of the product of agene encoding a 258 kDa precursor two-domain ligand-binding protein. Mol Microbiol 1993, 8:299-310. 30. Zhang DP, de Souza RF, Anantharaman V, Iyer LM, Aravind L: Polymorphic toxin systems: comprehensive characterization of trafficking modes, processing, mechanisms of action, immunity and ecology using comparative genomics. Biol Direct 2012:7. 31. Bingle LE, Bailey CM, Pallen MJ: Type VI secretion: a beginner’s guide. Curr Opin Microbiol 2008, 11:3-8. 32. Boyer F, Fichant G, Berthod J, Vandenbrouck Y, Attree I: Dissecting the bacterial type VI secretion system by a genome wide in silico analysis: what can be learned from available microbial genomic resources? BMC Genomics 2009, 10:104. 33. Silverman JM, Brunet YR, Cascales E, Mougous JD: Structure and regulation of the type VI secretion system. Annu Rev Microbiol 2012, 66:453-472. 34. Coulthurst SJ: The type VI secretion system — a widespread and versatile cell targeting system. Res Microbiol 2013, 164:640-654. 35. Pukatzki S, McAuley SB, Miyata ST: The type VI secretion system: translocation of effectors and effector-domains. Curr Opin Microbiol 2009, 12:11-17. 36. Jani AJ, Cotter PA: Type VI secretion: not just for pathogenesis anymore. Cell Host Microbe 2010, 8:2-6. 37. Hood RD, Singh P, Hsu F, Guvener T, Carl MA, Trinidad RR, Silverman JM, Ohlson BB, Hicks KG, Plemel RL et al.: A type VI secretion system of Pseudomonas aeruginosa targets a toxin to bacteria. Cell Host Microbe 2010, 7:25-37. This was the first study that identified an antibacterial activity of a T6SS. The authors characterized antibacterial T6SS effector proteins and their cognate immunity proteins in Pseudomonas aeruginosa. 38. Russell AB, Hood RD, Bui NK, LeRoux M, Vollmer W, Mougous JD: Type VI secretion delivers bacteriolytic effectors to target cells. Nature 2011, 475:343-347. 39. Li M, Le Trong I, Carl MA, Larson ET, Chou S, De Leon JA, Dove SL, Stenkamp RE, Mougous JD: Structural basis for type VI secretion effector recognition by a cognate immunity protein. PLoS Pathog 2012, 8:e1002613. 40. Russell AB, Singh P, Brittnacher M, Bui NK, Hood RD, Carl MA, Agnello DM, Schwarz S, Goodlett DR, Vollmer W et al.: A widespread bacterial type VI secretion effector superfamily identified using a heuristic approach. Cell Host Microbe 2012, 11:538-549. The authors developed a generally applicable method to identify T6SS substrates using a combination of mass spectrometry and bioinformatics and identified a diverse and widespread superfamily of amidase effector/ immunity pairs among different bacterial species.
This publication together with Ding et al. presents the first X-ray crystal structure of a T6SS effector/immunity pair. Tse1 adopts the fold of N1pC/ P60 peptidases and harbors a conserved cysteine/histidine diad in its active site. Furthermore, the authors described the inhibition mechanism of Tse1 by its immunity protein Tsi1 in detail. Bioinformatic analysis completes the study on the Pseudomonas aeruginosa Tse1/Tsi1 effector/immunity pair. 43. Shang G, Liu X, Lu D, Zhang J, Li N, Zhu C, Liu S, Yu Q, Zhao Y, Zhang H et al.: Structural insight into how Pseudomonas aeruginosa peptidoglycanhydrolase Tse1 and its immunity protein Tsi1 function. Biochem J 2012, 448:201-211. 44. Chou S, Bui NK, Russell AB, Lexa KW, Gardiner TE, LeRoux M, Vollmer W, Mougous JD: Structure of a peptidoglycan amidase effector targeted to Gram-negative bacteria by the type VI secretion system. Cell Rep 2012, 1:656-664. 45. Zhang H, Gao ZQ, Su XD, Dong YH: Crystal structure of type VI effector Tse1 from Pseudomonas aeruginosa. FEBS Lett 2012, 586:3193-3199. 46. Zhang H, Zhang H, Gao ZQ, Wang WJ, Liu GF, Xu JH, Su XD, Dong YH: Structure of the type VI effector-immunity complex (Tae4-Tai4) provides novel insights into the inhibition mechanism of the effector by its immunity protein. J Biol Chem 2013, 288:5928-5939. 47. Benz J, Reinstein J, Meinhart A: Structural insights into the effector-immunity system Tae4/Tai4 from Salmonella typhimurium. PLoS One 2013, 8:e67362. 48. Dong C, Zhang H, Gao ZQ, Wang WJ, She Z, Liu GF, Shen YQ, Su XD, Dong YH: Structural insights into the inhibition of type VI effector Tae3 by its immunity protein Tai3. Biochem J 2013, 454:59-68. 49. English G, Trunk K, Rao VA, Srikannathasan V, Hunter WN, Coulthurst SJ: New secreted toxins and immunity proteins encoded within the Type VI secretion system gene cluster of Serratia marcescens. Mol Microbiol 2012, 86:921-936. 50. Whitney JC, Chou S, Russell AB, Biboy J, Gardiner TE, Ferrin MA, Brittnacher M, Vollmer W, Mougous JD: Identification, structure, and function of a novel type VI secretion peptidoglycan glycoside hydrolase effector-immunity pair. J Biol Chem 2013, 288:26616-26624. 51. Wang T, Ding J, Zhang Y, Wang da C, Liu W: Complex structure of type VI peptidoglycan muramidase effector and a cognate immunity protein. Acta Crystallogr D Biol Crystallogr 2013, 69:1889-1900. 52. Li L, Zhang W, Liu Q, Gao Y, Gao Y, Wang Y, Wang DZ, Li Z, Wang T: Structural insights on the bacteriolytic and selfprotection mechanism of muramidase effector Tse3 in Pseudomonas aeruginosa. J Biol Chem 2013, 288:30607-30613. 53. Dong TG, Ho BT, Yoder-Himes DR, Mekalanos JJ: Identification of T6SS-dependent effector and immunity proteins by Tn-seq in Vibrio cholerae. Proc Natl Acad Sci USA 2013, 110:2623-2628. 54. Russell AB, LeRoux M, Hathazi K, Agnello DM, Ishikawa T, Wiggins PA, Wai SN, Mougous JD: Diverse type VI secretion phospholipases are functionally plastic antibacterial effectors. Nature 2013, 496:508-512. This paper reports the discovery of a superfamily of T6SS substrates that possess phospholipase activity and provides an extensive in vitro and in vivo analysis of this activity as well as its involvement in bacterial growth competition. 55. Miyata ST, Kitaoka M, Brooks TM, McAuley SB, Pukatzki S: Vibrio cholerae requires the type VI secretion system virulence factor VasX to kill Dictyostelium discoideum. Infect Immun 2011, 79:2941-2949.
41. Ding J, Wang W, Feng H, Zhang Y, Wang DC: Structural insights into the Pseudomonas aeruginosa type VI virulence effector Tse1 bacteriolysis and self-protection mechanisms. J Biol Chem 2012, 287:26911-26920. This paper together with Benz et al. presents the first three-dimensional structure of a T6SS effector/immunity pair. The studies revealed that Tse1 belongs to the family of papain-like peptidases and hydrolyzes peptidoglycan by a conserved active site. Additionally, the authors identified the inhibition mechanism of the peptidase by Tsi1. In vivo and binding studies complete the structural analysis.
56. Brooks TM, Unterweger D, Bachmann V, Kostiuk B, Pukatzki S: Lytic activity of the Vibrio cholerae type VI secretion toxin VgrG-3 is inhibited by the antitoxin TsaB. J Biol Chem 2013, 288:7618-7625. This publication presents a comprehensive analysis of an ‘evolved’ VgrG protein and its cognate immunity protein TsaB in Vibrio cholerae.
42. Benz J, Sendlmeier C, Barends TR, Meinhart A: Structural insights into the effector-immunity system Tse1/Tsi1 from Pseudomonas aeruginosa. PLoS One 2012, 7:e40453.
57. Carruthers MD, Nicholson PA, Tracy EN, Munson RS Jr: Acinetobacter baumannii utilizes a type VI secretion system for bacterial competition. PLoS One 2013, 8:e59388.
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58. Weber BS, Miyata ST, Iwashkiw JA, Mortensen BL, Skaar EP, Pukatzki S, Feldman MF: Genomic and functional analysis of the type VI secretion system in Acinetobacter. PLoS One 2013, 8:e55142.
immunity pairs have been identified. Furthermore, the regulatory system of T6SS in S. marcescens has been analyzed in detail.
59. MacIntyre DL, Miyata ST, Kitaoka M, Pukatzki S: The Vibrio cholerae type VI secretion system displays antimicrobial properties. Proc Natl Acad Sci USA 2010, 107:19520-19524.
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60. Schwarz S, West TE, Boyer F, Chiang WC, Carl MA, Hood RD, Rohmer L, Tolker-Nielsen T, Skerrett SJ, Mougous JD: Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions. PLoS Pathog 2010, 6:e1001068. This paper examines the function and evolutionary relationship of the five T6SS loci in Burkholderia thailandensis in eukaryotic and bacterial cell interaction. 61. Fritsch MJ, Trunk K, Diniz JA, Guo M, Trost M, Coulthurst SJ: Proteomic identification of novel secreted antibacterial toxins of the Serratia marcescens type VI secretion system. Mol Cell Proteomics 2013, 12:2735-2749. The authors report a global secretome study to identify new substrates of the T6SS in Serratia marcescens. Within this study four novel effector proteins, which are not related to any previously reported T6SS effector/
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62. Gerdes K (Ed): Prokaryotic Toxin-Antitoxins. Springer; 2013.
64. Van Melderen L, Saavedra De Bast M: Bacterial toxin-antitoxin systems: more than selfish entities? PLoS Genet 2009, 5:e1000437. 65. Mougous JD, Cuff ME, Raunser S, Shen A, Zhou M, Gifford CA, Goodman AL, Joachimiak G, Ordonez CL, Lory S et al.: A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus. Science 2006, 312:1526-1530. 66. Shneider MM, Buth SA, Ho BT, Basler M, Mekalanos JJ, Leiman PG: PAAR-repeat proteins sharpen and diversify the type VI secretion system spike. Nature 2013, 500:350-353. The authors determined the three-dimensional structure of VgrG-like proteins in complex with two PAAR-repeat proteins. PAAR-repeat proteins form a sharp, conical spike at the tip of the VgrG tube. Furthermore, their bioinformatic analysis suggested novel PAAR proteins to function as effector proteins.
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ScienceDirect Antibacterial effector/immunity systems: it’s just the tip of the iceberg§ Juliane Benz and Anton Meinhart Bacteria do not live anchoretic; rather they are constantly in touch with their eukaryotic hosts and with other bacteria sharing their habitat. Therefore, bacteria have evolved sophisticated proteinaceous weapons. To harm other bacteria, they produce antibacterial effector proteins, which they either release into the environment or export via direct intercellular contact. Contact-dependent killing is mediated by two specialized secretion systems, the type V and VI secretion system, whereas contact-independent processes hijack other transport mechanisms. Regardless of the transport system, cells co-express immunity proteins to protect themselves from suicide and fratricide. In general, effector protein activities and secretion mechanisms differ between Gram-positive and Gram-negative bacteria and evidence is emerging that different effector/immunity systems act synergistically and thus extend the bacterial armory. Address Department of Biomolecular Mechanisms, Max-Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany Corresponding author: Meinhart, Anton ([email protected])
Current Opinion in Microbiology 2014, 17:1–10 This review comes from a themed issue on Host–microbe interactions: bacteria Edited by Olivia Steele-Mortimer and Agathe Subtil For a complete overview see the Issue and the Editorial Available online 8th December 2013 1369-5274/$ – see front matter, # 2013 The Authors. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mib.2013.11.002
Introduction Bacteria produce a multitude of effector proteins which they either export into their environment or directly into other cells. These effector molecules are targeted against eukaryotic host cells where they promote pathogenicity as virulence factors (reviewed in [1]). Additionally, effector proteins are also used to compete with or even to gain advantage over other species during bacterial warfare within the microbiome. In the latter case, cells co-produce § This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
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immunity proteins to protect themselves from suicide or fratricide. Export of effector proteins is accomplished by secretion systems (SSs) that are classified according to the transported effector molecules, their targets as well as the producing cell type (Gram-negative or Gram-positive) (reviewed in [2–4]). The general secretion (Sec) and the two-arginine translocation (Tat) pathways and the type IV secretion system (T4SS) are found in both bacterial cell types. In contrast, the T1SS, T2SS, T3SS, T5SS, and the recently discovered T6SS have only been identified in the genome of Gram-negative bacteria, whereas the T7SS has been exclusively found in Gram-positive Mycobacteria. Depending on the secretion system, the effector proteins are either exported into the environment (T1SS, T2SS, T7SS) or are directly transported into the target cell upon physical contact (T3SS, T4SS, T5SS, T6SS). Bacteria have evolved several different secretion systems but only three (T5SS, T6SS, T7SS) have been reported to be used in bacterial growth competition. The other secretion systems have been reported to exclusively target eukaryotic cells and transport virulence factors (reviewed in [2,5]). This article reviews the current knowledge of antibacterial effector/immunity systems, classified into released or injected effector proteins and discusses similarities and differences between antibacterial effector/immunity systems and the well-known toxin/antitoxin systems.
Contact-independent antibacterial systems — the bacteriocins Bacteriocins are effector proteins that are released by bacteria into their environment and are the best characterized antibacterial effector proteins to date (reviewed in [6,7]). They are widespread among all bacterial species and are synthesized by ribosomes together with a cognate immunity protein that protects bacteria from their own or their sibling’s bacteriocins [6,7]. In this respect, bacteriocins are different to antibiotics such as vancomycin, which are produced by non-ribosomal peptide synthetases. Bacteriocins have a narrow spectrum of activity and primarily act on closely related species. They are highly diverse in terms of their activities, amino acid sequences and three-dimensional structures [6,7]. Similarly, archaea produce archaeocins to gain growth advantages over their rivals (reviewed in [6]). Although archaeocins have been predicted to be a general feature of haloarchaea [8], only eight different halophilic strains (halocins) and Sulfolobus islandicus (sulfolobicins) have been reported to express such a system [6]. Current Opinion in Microbiology 2014, 17:1–10
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Bacteriocins produced by Gram-negative bacteria Gram-negative bacteriocins are named after the producing bacteria followed by the suffix -cin, with colicins from Escherichia coli being the founding and most extensively investigated members (reviewed in [7]). These bacteriocins are encoded from a single operon together with their cognate immunity protein. Whereas most operons also encode a lysis protein that induces autolysis for effector protein release (Figure 1a), only a few bacteriocins are released by cell wall leakage. Notably, the lysis proteins are highly conserved and functionally interchangeable between different strains [7]. Gram-negative bacteriocins generally consist of three domains; a translocation (N-terminal), a receptor binding (central) and a killing domain (C-terminal). The central domain hijacks specific receptor proteins that are usually responsible for nutrient uptake (Figure 1a). These receptors are highly diverse among bacteria leading to a narrow killing spectrum of bacteriocins [7]. Import of bacteriocins is mediated by the recipient’s Ton or Tol system (Figure 1a) [7,9,10]. These systems have been reported to make use of a proton motive force for dissociation of the tightly bound immunity proteins from the effector protein [10] and also for translocation of the bacteriocins into the target cell (reviewed in [9]). Except for colicin M, which hydrolyzes peptidoglycan precursors [11] and pesticin from Yersinia pestis, which possesses muramidase activity [12], all other Gram-negative bacteriocins have either pore forming or nuclease activity (Figure 1a) [7].
Bacteriocins produced by Gram-positive bacteria In contrast to their Gram-negative relatives, Grampositive bacteriocins have a broad killing spectrum, and some of them even target Gram-negative bacteria (reviewed in [6,13,14]). Since Gram-positive bacteriocins simply diffuse through their target’s cell wall, they have a very broad killing spectrum [14]. Furthermore, when compared to Gram-negative bacteriocins, they are found in large gene clusters and their production relies on specific regulatory and transport factors [13]. Thus, Gram-positive bacteria survive bacteriocin release, whereas most Gram-negative bacteria undergo autolysis for effector protein release [13,14]. Gram-positive bacteria, particularly lactic acid bacteria, produce a plethora of different bacterocins which are either small peptides or proteins with a molecular weight of more than 30 kDa and are grouped into four major classes: The well-known lantibiotics (class I), small heat-stable peptides (class II), heat-labile proteins that kill bacteria by either lytic or non-lytic mechanisms (class III) and cyclic peptides of which the mechanism of function is still elusive [6]. However, all these bacteriocins do not belong to the classical effector/immunity systems and are often referred to as bacteriocin-like effectors, since they are either small Current Opinion in Microbiology 2014, 17:1–10
peptides, or do not possess any enzymatic activity or lack an immunity protein when they are classical enzymes. The exception to the rule is the ribonuclease Barnase from Bacillus amyloliquefaciens [15]. Although Barnase and its cognate immunity protein Barstar [16] were identified 50 years ago, their putative antibacterial effector/immunity function is exclusively predicted based on similarities to bacteriocins and is still hypothetical. An antibacterial activity was suggested since E. coli cells expressing Barnase inhibit the growth of other bacteria and expression of Barstar confers immunity [17]. However, no direct evidence has been reported so far that B. amyloliquefaciens harms other bacteria using Barnase and the mechanism by which Barnase enters bacteria is also still elusive.
Contact-dependent antibacterial systems Apart from simple secretion of effector proteins into the environment, bacteria also kill others by establishing intercellular contacts. Two different contact-dependent systems have been described so far: the contact-dependent growth inhibition (CDI) and the T6SS effector/ immunity systems.
‘Toxin on a stick’ — contact-dependent growth inhibition Contact dependent growth inhibition (CDI) by the E. coli EC93 isolate is evoked by the cdiBAI operon [18]. CdiA is the effector protein consisting of two domains, a large conserved N-terminal domain (NtD) and a smaller Cterminal antibacterial effector domain (CdiA-CT) responsible for dissipating the proton motive force of the targeted cell [19]. The CdiI protein provides immunity by complex formation with CdiA effector molecules which have been secreted by siblings. Secretion of CdiA relies on CdiB, which has been predicted to be a channelforming protein (Figure 1b) [18,20]. The CdiA effector is a large protein (about 300 kDa) and was predicted to protrude from the surface by several nanometers. The Nterminal domain of CdiA binds to the outer membrane receptor BamA of the target cell [21] and thereby establishes a direct physical contact [18]. Subsequently, CdiACT is most likely autoproteolytically cleaved from the Nterminal domain and assimilated into the competitor cell [18]. However, CDI systems are much more prevalent in the genome of protebacteria than previously expected. A bioinformatic search for putative effector variants revealed an entire family of CDI operons with a conserved N-terminal BamA binding domain [22]. The toxic CdiA-CT domain, however, shows a significant degree of polymorphism with tRNase, DNase or pore forming activities [19,22]. Although these polymorphic CdiA proteins are generally conserved in their N-terminal domain, specificity for extracellular loop regions of the BamA receptor of different bacterial species is achieved www.sciencedirect.com
Antibacterial effector/immunity systems Benz and Meinhart 3
Figure 1
(a)
(b)
(c)
ribosome
5′ 5′
HC
CdiA
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3′ 5′
5′ 3′
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CdiA
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HIM
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HOM
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CdiA ECM
Ions Protons etc.
Ions Protons etc. Hcp
OM
CdiB Lysis
P
IM
Sec Lysis ATP
Cdil
ADP
ClpV ATP ADP
Cdil Cdil
C Cdil
CdiA
CdiB Cdil Current Opinion in Microbiology
Secretion mechanism and mode of action of the different antibacterial effector/immunity systems. (a) Release of Gram-negative bacteriocins (purple) is a lethal event for the producer cell as it is accomplished by a lysis protein (red). Extracellular released bacteriocins are incorporated into the host cell by receptors (dark blue) usually responsible for nutrient uptake. Translocated into the host periplasm (HP), bacteriocins are either integrated into the host’s outer membrane (HOM) or further transported across the host’s inner membrane (HIM). Transport across the HIM is performed by the energy dependent Ton or Tol system (light blue). Furthermore, the immunity proteins (spheres colored in purple) are only released in the host cell before Ton/ Tol import. Gram-negative bacteriocins have RNase or DNase activity and degrade tRNAs, mRNAs or DNAs of the host cells. (b) T5SSs are composed of two components CdiA (magenta) and CdiB (green) that are exported into the periplasm of the producer cell via their Sec-system (yellow). CdiB is a b-barrel forming protein that forms a pore in the outer membrane (OM) of the producer cell and transports CdiA across the OM. CdiA is presented on the cell surface and binds to the BamA receptor (dark blue) of host cells upon direct cell–cell contact. BamA transports CdiA across the HOM into the periplasm where it is either integrated into the membrane or further transported into the host cytoplasm (HC) by a yet unknown mechanism. CidA proteins harbor nuclease activity and inhibit bacterial cell growth by degrading tRNA or DNA of the host cell. Furthermore, to prevent suicide and fratricide, the donor cells co-express CdiI immunity proteins (magenta spheres). (c) The T6SS transports effector proteins directly into competing bacterial cells. Hcp and VgrG are highly conserved among all T6SSs and homologous to the bacteriophage tip and tube components, respectively. Assembly and disassembly of the T6SS needle is driven by ATP hydrolysis performed by ClpV. Three main classes of antibacterial T6SS effector proteins have been identified, which are active on three distinct cellular compartments of the host cell: periplasm (red), lipid bilayer (orange) or the cytoplasm (blue). On the basis of these workplaces the cognate immunity proteins (spheres colored according to the corresponding effector) are also either localized in the periplasm, membrane or in the cytoplasm of the producer cell. Effector proteins that degrade the bacterial peptidoglycan scaffold show either amidase or glycoside hydrolase activity. Effector proteins with phospholipase activity lead to cell lysis by modifying the composition of the lipid bilayer. The mechanisms and targets of cytoplasmic active effector proteins are still elusive. C: cytoplasm, IM: inner membrane, P: periplasm, OM: outer membrane, ECM: extracellular milieu, HOM: host outer membrane, HP: host periplasm, HIM: host inner membrane, HC: host cytoplasm.
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Current Opinion in Microbiology 2014, 17:1–10
4 Host–microbe interactions: bacteria
by sequence variations [23]. Thereby, CDI systems achieve specificity for their target cells and establish a narrow killing spectrum as observed for Gram-negative bacteriocins [20,22]. Furthermore, CDI operons always contain a small ORF located downstream of the CdiA locus. These ORFs have been predicted to encode for the cognate immunity proteins. For systems where the function has been experimentally verified, it became evident that the immunity proteins are also polymorphic and do not show any cross-reactivity with other CdiA effectors [22,24]. Downstream of many of these polymorphic CDI loci, potential genes of so called ‘orphan’ cdiA-CT/cdiI modules are found. Whereas the cdiA-CT gene is translationally silent, the cdiI ORF is expressed, but its function is still elusive. Fusion of such an orphan cdiA-CT gene to the NtD of the upstream and functional cdiA gene resulted in an active protein secreted by CdiB. Turning on such orphan cdiA-CT/cdiI genes by homologous recombination could enable bacteria to rapidly diversify their CDI systems and enable them to express a set of CdiI proteins for broad range immunity [25]. Strikingly, CDI loci have also been identified in rearrangement hotspot (rhs) elements, which are genomic regions that facilitate recombination in E. coli [25,26]. Rhs proteins have a conserved N-terminal region, which differs from the BamA receptor binding domain of CdiA proteins and a C-terminal, variable effector domain [25,26]. Similar to CdiI, a cognate RhsI immunity protein is encoded from the same operon but the channel-forming CdiB is missing [25]. Notably, CDI is not restricted to Gram-negative bacteria, and homologous proteins are also found in Gram-positive species like members of the genera Bacillus, Listeria, Clostridium and Streptococcus [27]. In fact, PF04740 proteins share conserved N-terminal domains and their C-terminal domains (CtDs) are homologous to the Gram-negative CdiA-CTs. The CtDs of B. subtilis and B. cereus have been experimentally shown to harbor RNase activity [27]. Furthermore, the ORFs downstream of the PF04740 locus encode small proteins which block growth inhibition, indicative for a functional effector/immunity pair. Similarly, the wall-associated proteins A (WapA) from various B. subtilis strains have variable C-terminal effector domains and an associated immunity protein WapI. In common, no ORF encoding for an orthologous channelforming CdiB protein has been identified for these Grampositive CDI-related effector/immunity systems and effector presentation at the surface must be accomplished by a different mechanism. Since the N-terminal domains of PF04740 proteins contain T7SS-specific secretion signals, export is most likely performed by this particular SS [27,28]. In contrast, WapA proteins carry an N-terminal signal sequence responsible for cell wall binding [27–29]. Current Opinion in Microbiology 2014, 17:1–10
The number of putative polymorphic effector proteins has been increased even more by a recent bioinformatic study which identified a plethora of C-terminal toxic domains with homologous large NtDs [30]. The toxic activities of the C-terminal domains vary and range from nuclease, deaminase, ADP-ribosyltransferase, peptidase and phospholipase to pore-forming activity. All of these putative polymorphic effector proteins are encoded from an operon together with a small ORF that most likely encodes the cognate immunity proteins [30]. However, it remains to be shown if and how these polymorphic putative toxins are secreted and what their role in bacterial warfare is.
‘Molecular syringes’ — effector proteins injected by the type VI secretion system Type VI secretion systems (T6SSs) are found in the genome of approximately 25% of the different bacterial species [31,32]. The T6SS injects a plethora of different effector proteins into target cells by an upside-down bacteriophage-like syringe (reviewed in [3,33]). Whereas anti-eukaryotic T6SS effector proteins and their role in pathogenesis have been extensively investigated (reviewed in [34–36]), the functional mechanisms and the regulation of antibacterial effector proteins are just starting to be resolved. The first antibacterial effector proteins secreted by a T6SS were found in the genome of P. aeruginosa and named Tse1-3 (type VI secretion exported 1–3) [37]. Tse1 and Tse3 act exclusively in the periplasm where they hydrolyze peptidoglycan and thereby provoke lysis of competing bacterial cells [38]. As with bacteriocins, carriers of an active T6SS co-express the immunity proteins Tsi1 and Tsi3 (type VI secretion immunity 1 and 3) from a common operon. Both immunity proteins harbor an N-terminal signal sequence for translocation into the periplasm to prevent fratricide [38]. In contrast, the third effector/immunity pair, Tse2 and Tsi2, is cytoplasmic localized and kills bacteria by an unknown mechanism (Table 1) [37,39].
T6SS effector proteins with amidase activity The discovery of the amidase activity of Tae1 (formerly referred as Tse1) led to the assignment of a plethora of previously hypothetical ORFs to an entire family of effector proteins that hydrolyse the muropeptide stems in the peptidoglycan scaffold [40] (Figure 2). On the basis of their cleavage specificity, these effectors were classified into four groups (type VI amidase effector Tae1-4) (Table 1). Structural characterization of these amidase effector proteins revealed a common N1pC/P60 peptidase core. Distinct structural differences clustering around the active site were observed, which probably reflect the different substrate specificities [41,42,43–49]. Whereas the amidase activity of these effector proteins is caused by a strictly conserved cysteine-histidine dyad in their active sites and www.sciencedirect.com
Antibacterial effector/immunity systems Benz and Meinhart 5
Table 1 Enzymatic activity, targets and immunity proteins of the different antibacterial T6SS effector proteins Effector
Enzymatic activity
Target
Immunity
Reference
Peptidoglycan (mDAP-D-Glu) Peptidoglycan (mDAP-D-Ala) Peptidoglycan (mDAP-D-Ala) Peptidoglycan (mDAP-D-Glu)
Tai1 (Tsi1)
[37,38,40]
Tai2
[40]
Tai3
[40]
Tai4
[40]
Tae1 (Tse1)
Amidase
Tae2
Amidase
Tae3
Amidase
Tae4
Amidase
Tse2
Unknown
Unknown
Tsi2
[37,39]
Tge1 (Tse3)
b-(1,4)-N-acetylmuramidase
Tgi1 (Tsi3)
[37,38,50]
Tge2
N-acetylglucosaminidase
Tgi2
[50]
Tge3
b-(1,4)-N-acetylmuramidase
Peptidoglycan (MurNAc-GlcNAc) Peptidoglycan (GlcNAc-MurNAc) Peptidoglycan (MurNAc-GlcNAc)
Tgi3
[50]
Tle1 Tle2 Tle3 Tle4 Tle5
Phospholipase A2 Phospholipase A1 Phospholipase Phopsholipase Phospholipase D
Plasma Plasma Plasma Plasma Plasma
Tli1 Tli2 Tli3 Tli4 Tli5
[54] [53,54] [54] [54] [54]
Vgr-3
Unknown
Peptidoglycan
TsaB
[53,56]
membrane membrane membrane membrane membrane
Former names of the T6SS effector proteins are given in brackets. GlcNAc: N-acetylglucosamine; MurNAc: N-acetylmuramic acid; D-Ala: D-alanine; D-Glu: g-D-glutamic acid; mDAP: meso-diaminopimelic acid.
the proteins are structurally homologous, their cognate immunity proteins are totally different and share no homology [41,42,43,46,47,49] (Figure 3).
T6SS effector proteins with glycoside hydrolase activity Furthermore, the identification of the muramidase activity of Tse3 from P. aeruginosa (renamed to Tge1PA/Tgi1PA) enabled the identification of three new classes of T6SS glycoside hydrolase effector (Tge) proteins and their cognate immunity proteins (Tgi) (Table 1) [50]. These effector proteins cleave the b1,4-glycosidic bond between MurNAc and GlcNAc in the peptidoglycan scaffold. On the basis of their sequence homology to other glycoside hydrolases, Tge1-3 were suggested to differ in their cleavage mechanism: lytic transglycosylase (Tge1), N-acetylglycosamidase (Tge2) and phage-type lysozyme (Tge3) (Figure 2). Crystal structures of Tge1 from P. aeruginosa [51,52] and Tge2 from Pseudomonas putida [50] in complex with their cognate immunity proteins have revealed how different effector proteins of this family acquire related glycoside hydrolase activity and are inhibited by non-related immunity proteins.
T6SS effector proteins with phospholipase activity Finally, antibacterial effector proteins that possess phospholipase activity were identified in P. aeruginosa, Burkholderia thailandensis and Vibrio cholerae [53,54]. These www.sciencedirect.com
enzymes modify the cell wall composition of competitors and were named Tle (type VI lipase effector) and their corresponding immunity proteins Tli (type VI lipase immunity). ORFs encoding for Tle proteins were identified in many other Gram-negative bacteria. On the basis of sequence homology and their phospholipase A1-like, A2-like and D-like activities, these proteins were grouped into 5 different families, Tle1-5 (Table 1) [53,54]. Notably, T6SS effector lipases harboring both antibacterial and anti-eukaryotic killing activities were reported, suggesting that they play an important role in bacterial pathogenicity [53,55].
T6SS scaffold proteins with antibacterial activity T6SSs not only secrete effector proteins, but also some of their structural components have killing potential (Table 1) [53,56]. Variations of the VgrG (valine-glycine repeat G) protein which is functionally related to the bacteriophage spike proteins (reviewed in [3]), were reported to be secreted and to harbor killing activity. For instance, the ‘evolved’ VgrG-3VC from V. cholera contains a Cterminal, lysozyme-like peptidoglycan binding domain and hydrolyzes the cell wall of Gram-negative competitors [53,56]. VgrG-3VC was suggested to be part of a functional effector immunity pair, since the downstream gene product encodes the cognate immunity protein TsaB [53,56]. Since VgrG proteins form the tip of the T6SS syringe, other functions apart from solely inducing cell lysis are also conceivable. For instance, peptidoglycan Current Opinion in Microbiology 2014, 17:1–10
6 Host–microbe interactions: bacteria
Figure 2
Pentapeptides Tge2
o
MurNAc
o
GlcNAc
o
Tetrapeptides Tge1/Tge3
MurNAc
o
GlcNAc
o
o
L-Ala D-Glu
o
MurNAc
Donor
D-Ala
D-Glu
mDAP
D-Ala
D-Ala
mDAP
D-Ala
mDAP
mDAP
D-Ala
D-Ala
D-Glu
D-Glu
Acceptor
L-Ala
L-Ala
GlcNAc
o
GlcNAc
o
o
MurNAc
o
GlcNAc
o
MurNAc
MurNAc
o
GlcNAc
o
L-Ala
L-Ala Tae2/3
Tge1/Tge3
Tge2
Tae4
D-Glu
Tae4
Tae2/3 Donor D-Ala
mDAP D-Ala Tae1 Acceptor
mDAP D-Glu L-Ala
o
o
GlcNAc
o
MurNAc
o
GlcNAc
o
MurNAc
o
Current Opinion in Microbiology
Peptidoglycan cleavage specificity of the different amidase and glycoside hydrolase effector protein families. Cleavage specificity of Tae1-4 on pentapeptide (left panel) and tetrapeptide (right panel) stems of bacterial peptidoglycan is illustrated according to [38,40]. Effector proteins that belong to the Tae2 and Tae3 family hydrolyze the cross-link between mDAP and D-Ala in tetrapeptides as well as in pentapeptides. In contrast, Tae1 and Tae4 effector proteins degrade the peptidoglycan by hydrolyzing the amide bond formed between g-D-Glu and mDAP. Whereas, Tae1 effector proteins, including the founding member Tse1 from Pseudomonas aeruginosa, specifically cleave the donor stem of cross-linked tetrapeptides, Tae4 effector proteins exclusively degrade acceptor stems of cross-linked as well as non-crosslinked tetrapeptides. Furthermore, pentapeptides can also be degraded by Tae4 family members but with a poor turnover rate. Additionally, bacteria express effector proteins with glycoside hydrolase activity and degrade the peptidoglycan scaffold by cleaving the bond between the sugar moieties. Classification of these Tge proteins is according to [50]. Tge1 (including Tse3 from P. aeruginosa) and Tge3 effector proteins cleave the b-(1,4) glycosidic bond between MurNAc and GlucNAc by their b-(1,4)N-acetylmuramidase activity. In contrast, Tge2 effector proteins possess N-acetylglucosaminidase activity and hydrolyze the b-(1,4) glycosidic bond between GlcNAc and MurNAc. Figure has been adapted from [47]. GlcNAc: N-acetylglucosamine; MurNAc: N-acetylmuramic acid; D-Ala: D-alanine; LAla: L-alanine; D-Glu: g-D-glutamic acid; mDAP: meso-diaminopimelic acid.
degradation by VgrG-3 could facilitate penetration through the producer’s cell envelope during needle formation [56]. ‘Evolved’ VgrG proteins could act as an advance guard by increasing the cell wall permeability of other bacteria and thereby facilitating injection or infusion of other effector proteins. A stealthy penetration of effector proteins into the competitor cell by VgrG proteins is further supported by physical interaction between VgrG-3VC and the phospholipase effector Tle2VC [53]. Finally, it remains to be shown whether cell wall hydrolysis activity of ‘evolved’ VgrG proteins is also used to attack Gram-positive bacteria.
is strictly regulated and not activated by common mechanisms, even not in closely related species. Moreover, effector proteins might even not be secreted under laboratory conditions or are only activated under certain conditions. This hypothesis is further supported by the failure to detect any T6SS activity in re-cultured pandemic V. cholera strains under laboratory conditions [59]. Additionally, antibacterial T6SS activity was identified in various bacteria but no effector proteins could be assigned yet [57,60]. In other studies, putative effector proteins could be identified, but their killing activity and cellular targets are still elusive [37,39,61].
T66S effector proteins are commonly used antibacterial weapons
Effector/immunity systems and other toxins — an interdisciplinary assassination team?
Evidence is emerging that the T6SS effector/immunity proteins characterized so far are just the tip of the iceberg and that the bacterial armory is more extensive and complex than previously thought. For instance, different Acinetobacter baumannii strains contain conserved T6SS loci [57,58], however, not all of them exhibit killing activity towards other bacteria under the conditions tested [58]. This suggests that effector protein secretion Current Opinion in Microbiology 2014, 17:1–10
Effector/immunity and type II toxin/antitoxin (TA) systems are often spuriously mixed, as both induce cell death in bacteria and their genomic organization in bicistronic operons is related. However, there is a fundamental, functional difference between the two systems. Effector proteins are released and serve to attack competitors and the stable immunity proteins provide www.sciencedirect.com
Antibacterial effector/immunity systems Benz and Meinhart 7
Figure 3
(a)
long-term protection against siblings [39]. In contrast, toxin proteins rest inside the cell and the unstable antitoxin provides only temporary protection and thus toxin/ antitoxin systems induce bacterial suicide upon activation (reviewed in [62]).
(b)
Tae1 Ser109
Tae1 Tai1 Cys30 Cys30 His91
(c)
Ser109 His91
Tai1
(d)
(e)
Tae3
Tai3
Tai3
Tai3
Tai3
Tae3 His81
Tai3
Tae3
(f)
Cys23
Tai3
Cys23 His81
Tai3 Tai3
His81
Cys23
Tae3 (g)
Tae4
(h)
Ser98
Tai4
Tae4
Tai4
His126 Cys44 Ser98
Ser98
Tae4 Tai4 (i)
Cys44 His126
Tai4 Ser98 His126
Cys44
Tae4 Current Opinion in Microbiology
Structural overview of the effector/immunity system Tae1/Tai1 from Pseudomonas aeruginosa, Tae3/Tai3 from Ralstonia pickettii and Tae4/ Tai4 from Salmonella typhimurium. (a) Schematic representation of the www.sciencedirect.com
But evidence is emerging that both systems have common origins and/or overlapping functions and a strict separation is not always possible. For instance, both systems are often found within mobile genetic elements, pathogenicity islands and transposon-like regions (reviewed in [7,22,63,64]). It remains to be shown whether TA toxins are translocated in addition to effector proteins by SSs and directly used as weapons against competitors. Such a hijack of the T6SS by non-related T6SS toxins has been shown, for instance for the Rhs proteins of Serratia marcescens [61] and Dickeya dadantii [25,26]. As Rhs loci do not encode for any CdiB homologue nor do Rhs proteins harbor any leader sequence for CdiB-dependent transport they need to hijack other SSs [25]. Transport via the T6SS is rather likely, since Rhs Tae1/Tai1 effector/immunity complex from P. aerugionsa. Tae1 (red) and Tai1 (blue) form a heterodimer. The effector protein’s toxicity is inhibited by a conserved serine residue (Ser109) located in a loop region of Tai1 that prevents effector protein activation and substrate binding. (b) Crystal structure of the effector protein Tae1 (PDB code 4FGE) with the conserved NlpC/P60 catalytic core in gray. Secondary structure elements, which are specific for Tae1, are colored in red. The catalytic important Cys30 and His91 residues are depicted as stick model and highlighted with an orange rectangle. (c) Crystal structure of the Tae1 and Tai1 heterodimer (PDB code 4FGI) colored in red and blue, respectively. The inhibiting serine residue Ser109 as well as the catalytic important residues Cys30 and His91 are depicted as sticks and highlighted with an orange rectangle. (d) Schematic representation of the Tae3/Tai3 effector/immunity complex from R. pickettii. Tae3 (green) and Tai3 (yellow) form a heterohexamer. The effector protein’s toxicity is inhibited by blocking substrate binding. (e) Crystal structure of the effector protein Tae3 (PDB code 4HZ9) with the conserved NlpC/P60 catalytic core in gray. Secondary structure elements, which are specific for Tae3, are colored in green. The catalytic important Cys23 and His81 residues are depicted as stick model and highlighted with an orange rectangle. (f) Crystal structure of the Tae3 and Tai3 heterohexamer (PDB code 4FGI) formed by two Tae3 (green) and four Tai3 (yellow and orange) molecules. Similar to (e) the catalytic important Cys23 and His81 residues are depicted as sticks and the active site is highlighted with an orange rectangle. (g) Schematic representation of the Tae4/Tai4 effector/immunity complex from S. typhimurium. Tae4 (cyan) and Tai4 (orange) form a heterotetramer. The effector protein’s toxicity is inhibited by a conserved serine residue (Ser98) located in a loop region of Tai4 that prevents effector protein activation and substrate binding. (h) Crystal structure of the effector protein Tae4 with the conserved NlpC/ P60 catalytic core in gray (PDB code 4J32). Secondary structure elements, which are specific for Tae4, are colored in cyan. Cys44 and His126 that form the catalytically active dyad are depicted as stick model and highlighted with an orange rectangle. (i) Crystal structure of the Tae4/Tai4 heterotetramer (PDB code 4J32). Two symmetry related Tai4 molecules (orange) form a head-to-tail-dimer and inhibit two opposing Tae4 effector proteins (cyan). As with Tai1, Ser98 in Tai4 is located in a loop and is important for effector protein inhibition by blocking the active site of Tae4 and by preventing Cys44 activation. Residues in Tae4 as well as in Tai4 are depicted as sticks. The active site and the inhibiting serine residue are highlighted with an orange rectangle. Current Opinion in Microbiology 2014, 17:1–10
8 Host–microbe interactions: bacteria
loci are often found next to vgrG genes [25] and Rhsmediated bacterial growth inhibition relies on VgrG proteins [26]. However, this transport might not be accomplished through the injection needle, since the diameter of the Hcp tube (40 A˚) [65] seems to be too narrow for Rhs proteins. Most likely the conserved PAAR (proline-alanine-alanine-arginine) repeat domain of Rhs proteins localizes to the VgrG tip before assembly of the tube [66]. Notably, many uncharacterized PAAR-repeat proteins carry polymorphic C-terminal extensions with enzymatic functions like nuclease, peptidase or lipase activities and might fulfill similar functions [26,66].
Conclusion Effector proteins involved in bacterial competition are a highly polymorphic group of bacterial toxins that apparently form an interdisciplinary and synergistic team to kill competitors during warfare. It seems that just the tip of the iceberg of this arsenal has been discovered and many questions need to be answered before we fully understand bacterial competition and communication and its involvement in pathogenicity.
Acknowledgements We apologize to all scientific colleagues whose work has not been mentioned due to space limitations. We thank Y.-H. Dong for providing us with the PDB structure files for Tae3/Tai3. We are grateful to T. Barends, H. Mutschler, R.L. Shoeman and the Meinhart group for scientific exchange and suggestions for the manuscript and I. Schlichting for continuous support. A.M. is financially supported by the Chica and Heinz Schaller Foundation and the Max Planck Society.
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Current Opinion in Microbiology 2014, 17:1–10
cross-complementation between the Tol and Ton systems. Biochem Soc Trans 2012, 40:1480-1485. 10. Vankemmelbeke M, Zhang Y, Moore GR, Kleanthous C, Penfold CN, James R: Energy-dependent immunity protein release during tol-dependent nuclease colicin translocation. J Biol Chem 2009, 284:18932-18941. 11. El Ghachi M, Bouhss A, Barreteau H, Touze T, Auger G, Blanot D, Mengin-Lecreulx D: Colicin M exerts its bacteriolytic effect via enzymatic degradation of undecaprenyl phosphate-linked peptidoglycan precursors. J Biol Chem 2006, 281:22761-22772. 12. Patzer SI, Albrecht R, Braun V, Zeth K: Structural and mechanistic studies of pesticin, a bacterial homolog of phage lysozymes. J Biol Chem 2012, 287:23381-23396. 13. Riley MA, Wertz JE: Bacteriocins: evolution, ecology, and application. Annu Rev Microbiol 2002, 56:117-137. 14. Jack RW, Tagg JR, Ray B: Bacteriocins of gram-positive bacteria. Microbiol Rev 1995, 59:171-200. 15. Nishimura S, Nomura M: Ribonuclease of Bacillus subtilis. Biochim Biophys Acta 1958, 30:430-431. 16. Smeaton JR, Elliott WH: Isolation and properties of a specific bacterial ribonuclease inhibitor. Biochim Biophys Acta 1967, 145:547-560. 17. Ramos HJ, Souza EM, Soares-Ramos JR, Pedrosa FO: Antibiosis by Bacillus amyloliquefaciens ribonuclease barnase expressed in Escherichia coli against symbiotic and endophytic nitrogen-fixing bacteria. J Biotechnol 2006, 126:291-294. 18. Aoki SK, Pamma R, Hernday AD, Bickham JE, Braaten BA, Low DA: Contact-dependent inhibition of growth in Escherichia coli. Science 2005, 309:1245-1248. 19. Aoki SK, Webb JS, Braaten BA, Low DA: Contact-dependent growth inhibition causes reversible metabolic downregulation in Escherichia coli. J Bacteriol 2009, 191:1777-1786. 20. Aoki SK, Poole SJ, Hayes CS, Low DA: Toxin on a stick: modular CDI toxin delivery systems play roles in bacterial competition. Virulence 2011, 2:356-359. 21. Aoki SK, Malinverni JC, Jacoby K, Thomas B, Pamma R, Trinh BN, Remers S, Webb J, Braaten BA, Silhavy TJ et al.: Contactdependent growth inhibition requires the essential outer membrane protein BamA (YaeT) as the receptor and the inner membrane transport protein AcrB. Mol Microbiol 2008, 70:323-340. 22. Aoki SK, Diner EJ, de Roodenbeke CT, Burgess BR, Poole SJ, Braaten BA, Jones AM, Webb JS, Hayes CS, Cotter PA et al.: A widespread family of polymorphic contact-dependent toxin delivery systems in bacteria. Nature 2010, 468:439-442. The authors mapped growth inhibition activity of CdiA proteins to their Cterminal domains and showed that CdiI proteins exclusively inhibit their cognate but not heterologous CdiAs. They performed in vitro DNase and RNase assays using different CdiAs to confirm their catalytic activity. 23. Ruhe ZC, Wallace AB, Low DA, Hayes CS: Receptor polymorphism restricts contact-dependent growth inhibition to members of the same species. MBio 2013, 4:1-9. 24. Morse RP, Nikolakakis KC, Willett JL, Gerrick E, Low DA, Hayes CS, Goulding CW: Structural basis of toxicity and immunity in contact-dependent growth inhibition (CDI) systems. Proc Natl Acad Sci USA 2012, 109:21480-21485. 25. Poole SJ, Diner EJ, Aoki SK, Braaten BA, t’Kint de Roodenbeke C, Low DA, Hayes CS: Identification of functional toxin/immunity genes linked to contact-dependent growth inhibition (CDI) and rearrangement hotspot (Rhs) systems. PLoS Genet 2011, 7:e1002217. 26. Koskiniemi S, Lamoureux JG, Nikolakakis KC, t’Kint de Roodenbeke C, Kaplan MD, Low DA, Hayes CS: Rhs proteins from diverse bacteria mediate intercellular competition. Proc Natl Acad Sci USA 2013, 110:7032-7037. The potential role of Rhs proteins during contact-dependent growth inhibition has been analyzed using Dickeya dadantii as model organism. The authors identified DNase activity of the C-terminal domains of two www.sciencedirect.com
Antibacterial effector/immunity systems Benz and Meinhart 9
D. dadantii Rhs proteins and showed that VgrG proteins are required for induction of growth inhibition. 27. Holberger LE, Garza-Sanchez F, Lamoureux J, Low DA, Hayes CS: A novel family of toxin/antitoxin proteins in Bacillus species. FEBS Lett 2012, 586:132-136. 28. Sutcliffe IC: New insights into the distribution of WXG100 protein secretion systems. Antonie Van Leeuwenhoek 2011, 99:127-131. 29. Foster SJ: Molecular analysis of three major wall-associated proteins of Bacillus subtilis 168: evidence for processing of the product of agene encoding a 258 kDa precursor two-domain ligand-binding protein. Mol Microbiol 1993, 8:299-310. 30. Zhang DP, de Souza RF, Anantharaman V, Iyer LM, Aravind L: Polymorphic toxin systems: comprehensive characterization of trafficking modes, processing, mechanisms of action, immunity and ecology using comparative genomics. Biol Direct 2012:7. 31. Bingle LE, Bailey CM, Pallen MJ: Type VI secretion: a beginner’s guide. Curr Opin Microbiol 2008, 11:3-8. 32. Boyer F, Fichant G, Berthod J, Vandenbrouck Y, Attree I: Dissecting the bacterial type VI secretion system by a genome wide in silico analysis: what can be learned from available microbial genomic resources? BMC Genomics 2009, 10:104. 33. Silverman JM, Brunet YR, Cascales E, Mougous JD: Structure and regulation of the type VI secretion system. Annu Rev Microbiol 2012, 66:453-472. 34. Coulthurst SJ: The type VI secretion system — a widespread and versatile cell targeting system. Res Microbiol 2013, 164:640-654. 35. Pukatzki S, McAuley SB, Miyata ST: The type VI secretion system: translocation of effectors and effector-domains. Curr Opin Microbiol 2009, 12:11-17. 36. Jani AJ, Cotter PA: Type VI secretion: not just for pathogenesis anymore. Cell Host Microbe 2010, 8:2-6. 37. Hood RD, Singh P, Hsu F, Guvener T, Carl MA, Trinidad RR, Silverman JM, Ohlson BB, Hicks KG, Plemel RL et al.: A type VI secretion system of Pseudomonas aeruginosa targets a toxin to bacteria. Cell Host Microbe 2010, 7:25-37. This was the first study that identified an antibacterial activity of a T6SS. The authors characterized antibacterial T6SS effector proteins and their cognate immunity proteins in Pseudomonas aeruginosa. 38. Russell AB, Hood RD, Bui NK, LeRoux M, Vollmer W, Mougous JD: Type VI secretion delivers bacteriolytic effectors to target cells. Nature 2011, 475:343-347. 39. Li M, Le Trong I, Carl MA, Larson ET, Chou S, De Leon JA, Dove SL, Stenkamp RE, Mougous JD: Structural basis for type VI secretion effector recognition by a cognate immunity protein. PLoS Pathog 2012, 8:e1002613. 40. Russell AB, Singh P, Brittnacher M, Bui NK, Hood RD, Carl MA, Agnello DM, Schwarz S, Goodlett DR, Vollmer W et al.: A widespread bacterial type VI secretion effector superfamily identified using a heuristic approach. Cell Host Microbe 2012, 11:538-549. The authors developed a generally applicable method to identify T6SS substrates using a combination of mass spectrometry and bioinformatics and identified a diverse and widespread superfamily of amidase effector/ immunity pairs among different bacterial species.
This publication together with Ding et al. presents the first X-ray crystal structure of a T6SS effector/immunity pair. Tse1 adopts the fold of N1pC/ P60 peptidases and harbors a conserved cysteine/histidine diad in its active site. Furthermore, the authors described the inhibition mechanism of Tse1 by its immunity protein Tsi1 in detail. Bioinformatic analysis completes the study on the Pseudomonas aeruginosa Tse1/Tsi1 effector/immunity pair. 43. Shang G, Liu X, Lu D, Zhang J, Li N, Zhu C, Liu S, Yu Q, Zhao Y, Zhang H et al.: Structural insight into how Pseudomonas aeruginosa peptidoglycanhydrolase Tse1 and its immunity protein Tsi1 function. Biochem J 2012, 448:201-211. 44. Chou S, Bui NK, Russell AB, Lexa KW, Gardiner TE, LeRoux M, Vollmer W, Mougous JD: Structure of a peptidoglycan amidase effector targeted to Gram-negative bacteria by the type VI secretion system. Cell Rep 2012, 1:656-664. 45. Zhang H, Gao ZQ, Su XD, Dong YH: Crystal structure of type VI effector Tse1 from Pseudomonas aeruginosa. FEBS Lett 2012, 586:3193-3199. 46. Zhang H, Zhang H, Gao ZQ, Wang WJ, Liu GF, Xu JH, Su XD, Dong YH: Structure of the type VI effector-immunity complex (Tae4-Tai4) provides novel insights into the inhibition mechanism of the effector by its immunity protein. J Biol Chem 2013, 288:5928-5939. 47. Benz J, Reinstein J, Meinhart A: Structural insights into the effector-immunity system Tae4/Tai4 from Salmonella typhimurium. PLoS One 2013, 8:e67362. 48. Dong C, Zhang H, Gao ZQ, Wang WJ, She Z, Liu GF, Shen YQ, Su XD, Dong YH: Structural insights into the inhibition of type VI effector Tae3 by its immunity protein Tai3. Biochem J 2013, 454:59-68. 49. English G, Trunk K, Rao VA, Srikannathasan V, Hunter WN, Coulthurst SJ: New secreted toxins and immunity proteins encoded within the Type VI secretion system gene cluster of Serratia marcescens. Mol Microbiol 2012, 86:921-936. 50. Whitney JC, Chou S, Russell AB, Biboy J, Gardiner TE, Ferrin MA, Brittnacher M, Vollmer W, Mougous JD: Identification, structure, and function of a novel type VI secretion peptidoglycan glycoside hydrolase effector-immunity pair. J Biol Chem 2013, 288:26616-26624. 51. Wang T, Ding J, Zhang Y, Wang da C, Liu W: Complex structure of type VI peptidoglycan muramidase effector and a cognate immunity protein. Acta Crystallogr D Biol Crystallogr 2013, 69:1889-1900. 52. Li L, Zhang W, Liu Q, Gao Y, Gao Y, Wang Y, Wang DZ, Li Z, Wang T: Structural insights on the bacteriolytic and selfprotection mechanism of muramidase effector Tse3 in Pseudomonas aeruginosa. J Biol Chem 2013, 288:30607-30613. 53. Dong TG, Ho BT, Yoder-Himes DR, Mekalanos JJ: Identification of T6SS-dependent effector and immunity proteins by Tn-seq in Vibrio cholerae. Proc Natl Acad Sci USA 2013, 110:2623-2628. 54. Russell AB, LeRoux M, Hathazi K, Agnello DM, Ishikawa T, Wiggins PA, Wai SN, Mougous JD: Diverse type VI secretion phospholipases are functionally plastic antibacterial effectors. Nature 2013, 496:508-512. This paper reports the discovery of a superfamily of T6SS substrates that possess phospholipase activity and provides an extensive in vitro and in vivo analysis of this activity as well as its involvement in bacterial growth competition. 55. Miyata ST, Kitaoka M, Brooks TM, McAuley SB, Pukatzki S: Vibrio cholerae requires the type VI secretion system virulence factor VasX to kill Dictyostelium discoideum. Infect Immun 2011, 79:2941-2949.
41. Ding J, Wang W, Feng H, Zhang Y, Wang DC: Structural insights into the Pseudomonas aeruginosa type VI virulence effector Tse1 bacteriolysis and self-protection mechanisms. J Biol Chem 2012, 287:26911-26920. This paper together with Benz et al. presents the first three-dimensional structure of a T6SS effector/immunity pair. The studies revealed that Tse1 belongs to the family of papain-like peptidases and hydrolyzes peptidoglycan by a conserved active site. Additionally, the authors identified the inhibition mechanism of the peptidase by Tsi1. In vivo and binding studies complete the structural analysis.
56. Brooks TM, Unterweger D, Bachmann V, Kostiuk B, Pukatzki S: Lytic activity of the Vibrio cholerae type VI secretion toxin VgrG-3 is inhibited by the antitoxin TsaB. J Biol Chem 2013, 288:7618-7625. This publication presents a comprehensive analysis of an ‘evolved’ VgrG protein and its cognate immunity protein TsaB in Vibrio cholerae.
42. Benz J, Sendlmeier C, Barends TR, Meinhart A: Structural insights into the effector-immunity system Tse1/Tsi1 from Pseudomonas aeruginosa. PLoS One 2012, 7:e40453.
57. Carruthers MD, Nicholson PA, Tracy EN, Munson RS Jr: Acinetobacter baumannii utilizes a type VI secretion system for bacterial competition. PLoS One 2013, 8:e59388.
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58. Weber BS, Miyata ST, Iwashkiw JA, Mortensen BL, Skaar EP, Pukatzki S, Feldman MF: Genomic and functional analysis of the type VI secretion system in Acinetobacter. PLoS One 2013, 8:e55142.
immunity pairs have been identified. Furthermore, the regulatory system of T6SS in S. marcescens has been analyzed in detail.
59. MacIntyre DL, Miyata ST, Kitaoka M, Pukatzki S: The Vibrio cholerae type VI secretion system displays antimicrobial properties. Proc Natl Acad Sci USA 2010, 107:19520-19524.
63. Cascales E: The type VI secretion toolkit. EMBO Rep 2008, 9:735-741.
60. Schwarz S, West TE, Boyer F, Chiang WC, Carl MA, Hood RD, Rohmer L, Tolker-Nielsen T, Skerrett SJ, Mougous JD: Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions. PLoS Pathog 2010, 6:e1001068. This paper examines the function and evolutionary relationship of the five T6SS loci in Burkholderia thailandensis in eukaryotic and bacterial cell interaction. 61. Fritsch MJ, Trunk K, Diniz JA, Guo M, Trost M, Coulthurst SJ: Proteomic identification of novel secreted antibacterial toxins of the Serratia marcescens type VI secretion system. Mol Cell Proteomics 2013, 12:2735-2749. The authors report a global secretome study to identify new substrates of the T6SS in Serratia marcescens. Within this study four novel effector proteins, which are not related to any previously reported T6SS effector/
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62. Gerdes K (Ed): Prokaryotic Toxin-Antitoxins. Springer; 2013.
64. Van Melderen L, Saavedra De Bast M: Bacterial toxin-antitoxin systems: more than selfish entities? PLoS Genet 2009, 5:e1000437. 65. Mougous JD, Cuff ME, Raunser S, Shen A, Zhou M, Gifford CA, Goodman AL, Joachimiak G, Ordonez CL, Lory S et al.: A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus. Science 2006, 312:1526-1530. 66. Shneider MM, Buth SA, Ho BT, Basler M, Mekalanos JJ, Leiman PG: PAAR-repeat proteins sharpen and diversify the type VI secretion system spike. Nature 2013, 500:350-353. The authors determined the three-dimensional structure of VgrG-like proteins in complex with two PAAR-repeat proteins. PAAR-repeat proteins form a sharp, conical spike at the tip of the VgrG tube. Furthermore, their bioinformatic analysis suggested novel PAAR proteins to function as effector proteins.
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