Contact-Dependent Interbacterial Antagonism Mediated by Protein Secretion Machines

TIMI 1776 No. of Pages 14

Trends in Microbiology Special Issue: Infection Biology in the Age of the Microbiome

Review

Contact-Dependent Interbacterial Antagonism Mediated by Protein Secretion Machines Timothy A. Klein,1,2,4 Shehryar Ahmad,1,2,4 and John C. Whitney

1,2,3,

*

To establish and maintain an ecological niche, bacteria employ a wide range of pathways to inhibit the growth of their microbial competitors. Some of these pathways, such as those that produce antibiotics or bacteriocins, exert toxicity on nearby cells in a cell contact-independent manner. More recently, however, several mechanisms of interbacterial antagonism requiring cell-to-cell contact have been identified. This form of microbial competition is mediated by antibacterial protein toxins whose delivery to target bacteria uses protein secretion apparatuses embedded within the cell envelope of toxin-producing bacteria. In this review, we discuss recent work implicating the bacterial Type I, IV, VI, and VII secretion systems in the export of antibacterial ‘effector’ proteins that mediate contactdependent interbacterial antagonism.

Highlights Interbacterial competition is an important determinant of microbial community composition in both environmental and human health contexts. Recent work demonstrates that bacteria use four distinct protein secretion machines to mediate antagonistic interbacterial interactions. Despite their unique molecular architecture, these protein secretion systems all export antibacterial effector proteins to intoxicate susceptible target bacteria.

Importance of Antagonistic Interbacterial Interactions Antibacterial effector proteins act by targeting conserved essential components of bacterial cells but can be neutralized if the target possesses the appropriate immunity proteins.

Bacteria rarely exist in isolation and instead are more typically found within diverse microbial communities [1]. Within these communities, different bacterial species compete with one another for nutrients and space, and these interactions have led to the evolution of a diverse array of pathways involved in interbacterial antagonism [2,3]. Over the past two decades, our knowledge of both the molecular mechanisms and the ecological significance of interbacterial antagonism has grown substantially. In plants and animals, pathways involved in interbacterial competition are used by both commensals and pathogens to promote colonization resistance and pathogenesis, respectively. For example, in humans, commensal strains of Staphylococcus lugdunensis secrete antibiotics that prevent colonization by pathogenic Staphylococcus aureus [4]. By contrast, the plant-protective bacterium Pseudomonas protegens secretes antibacterial toxins to outcompete gut commensals within the cabbage pest Pieris brassicae, markedly enhancing the ability of P. protegens to invade and kill this insect pest [5]. Antagonistic behaviors among bacteria are also important for microbial ecosystem maintenance. For example, sustained competition among species of human gut Bacteroidales results in greater community stability and resilience to perturbation over time [6–9]. In recent years, many different pathways involved in interbacterial antagonism have been identified. One way these pathways can be classified is by their two spatially distinct modes of action: (i) small diffusible molecules that function in a contact-independent manner and can therefore act between physically separated bacteria or (ii) antibacterial protein toxins that rely on protein secretion apparatuses to facilitate their contact-dependent delivery to target cells. Examples of contact-independent antibacterial molecules include secondary metabolite antibiotics and antimicrobial peptides. The specificity and target range of these molecules are highly variable with some exhibiting broad-spectrum activity across multiple bacterial phyla, whereas others have a much narrower target range and only act against members of the same or highly similar species. For example, the Abp118 bacteriocin produced by Lactobacillus salivarius is effective against many target bacteria, whereas the antibacterial activity of enterococcal bacteriocin 21 is restricted to Enterococcus faecalis and Enterococcus faecium [10,11]. Trends in Microbiology, Month 2020, Vol. xx, No. xx

An increased understanding of the molecular determinants that define microbial community composition may eventually allow for targeted manipulation of these populations.

1

Michael DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, ON, Canada L8S 4K1 2 Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada L8S 4K1 3 David Braley Centre for Antibiotic Discovery, McMaster University, Hamilton, ON, Canada L8S 4K1 4 These authors contributed equally

*Correspondence: [email protected] (J.C. Whitney).

https://doi.org/10.1016/j.tim.2020.01.003 © 2020 Elsevier Ltd. All rights reserved.

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In contrast to diffusible small-molecule antimicrobials, many antibacterial protein toxins are transported into target cells in a contact-dependent manner. This mechanism of toxin delivery requires the use of multicomponent protein secretion machines and may have evolved because toxins delivered in this way are less prone to dilution effects that can reduce the efficacy of diffusible antibacterial molecules in aqueous environments. The injection of protein toxins across multiple cellular membranes is a multifaceted process and to date, four bacterial protein secretion apparatuses known as the Type I, IV, VI, and VII secretion systems (T1SS, T4SS, T6SS, and T7SS) have been shown to facilitate the transport of toxins between bacteria [12–15]. Examples of the involvement of these protein secretion systems in interbacterial competition are found in a diverse range of bacteria with the most widespread being T6SSs, which have been found in many species of Proteobacteria and more recently, in the phylum Bacteroidetes [16]. It should be noted that these four secretion systems are distinct from bacterial two-partner secretion systems, which also play a role in bacterial competition via a process known as contact-dependent inhibition (CDI). Unlike the aforementioned protein secretion systems, CDI toxin export is not dependent on a molecular complex that spans the cell envelope and therefore will not be discussed further here. The reader is instead referred to several comprehensive reviews on this topic [17,18]. It should also be noted that T1SSs, T4SSs, and T7SSs have only recently been implicated in interbacterial antagonism and thus our current knowledge of these systems as mediators of bacterial competition likely underestimates their widespread use [13–15,19,20]. Despite the limitations in our understanding of these systems, they all have in common the ability to export antibacterial toxins referred to as ‘effectors’ to the cell surface. Furthermore, these effectors are always encoded by genes in proximity to genes encoding cognate immunity proteins that neutralize their toxic effects. This is an essential feature of these systems that prevents self-intoxication and protection from kin cells [13–15,20,21]. In the following section, we provide an overview of what is currently known about the secretion mechanism and effector repertoire used by each of these protein toxin secretion pathways to inhibit the growth of competitor bacteria.

Type I Secretion Systems Are Widespread Protein Export Pathways T1SSs were one of the first transenvelope protein secretion system to be described in Gram-negative bacteria and are involved in nutrient acquisition, pathogenesis, and bacterial competition [22,23]. Structurally, T1SSs form a tripartite translocation apparatus composed of (i) an ATP-binding cassette (ABC) transporter, (ii) a membrane fusion protein (MFP), and (iii) an outer membrane protein (OMP). The ABC transporter component recognizes substrate proteins in the cytoplasm and mediates their export across the inner membrane in a process that is powered by the energy generated from the binding and hydrolysis of ATP. T1SS ABC transporters interact with MFPs, which are inner membrane proteins with a large periplasmic region that comprises a significant portion of a periplasmic channel that links the ABC transporter to an OMP. MFPs are typically associated with a single ABC transporter and each MFP–ABC transporter pair tends to be highly specific for a single or closely related group of substrate proteins. By contrast, the OMP component of T1SSs often displays substrate promiscuity and because of this, a single OMP is often associated with more than one ABC transporter–MFP complex. When an OMP binds to a substrate–ABC transporter–MFP complex, the ensuing conformational change(s) triggers the transport of the substrate from the cytoplasm to the extracellular environment in a single step. Substrate proteins of T1SSs are often toxins that compromise host cell integrity, with α-hemolysin (HlyA) from Escherichia coli being a well-characterized example [24,25]. Other T1SSs have been shown to secrete proteins involved in nutrient acquisition, such as the HasA hemophore exported by Serratia marcescens, or release diffusible antibacterial peptides such as colicin V [26]. In all these examples, the T1SS-exported proteins act in a cell contact-independent manner. However, a recently identified T1SS termed Cdz (contact‐dependent inhibition by glycine zipper proteins) has been shown to differ from canonical T1SSs because it 2

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requires cell-to-cell contact to mediate interbacterial antagonism [13,27]. This discovery is a major paradigm shift for the T1SS field as it exemplifies new functionality for a well-studied system.

Contact-Dependent Interbacterial Antagonism Mediated by a T1SS Cdz was discovered in Caulobacter crescentus and delivers a two-peptide bacteriocin to adjacent target bacteria (Figures 1A and 2A). The Cdz T1SS is composed of the ABC transporter CdzA and the MFP CdzB, whereas the OMP component of this system has yet to be identified. CdzC and CdzD interact to form a bacteriocin exported by the Cdz T1SS and together these proteins function to kill susceptible bacteria. Glycine-zipper motifs found within the N termini of CdzC/D promote their aggregation on the outer leaflet of the outer membrane of the attacking cell. The precise mechanism by which CdzC and CdzD maintain their cell surface association has yet to be determined, but it appears these proteins associate with fibrillar structures found on the surface of C. crescentus [13]. A genetic screen for CdzC/D-insensitive mutants revealed that mutations leading to an upregulation of the zerRAB operon were resistant to killing [27]. Further characterization of these mutants suggests that zerRAB plays a role in modulating cell envelope homeostasis corroborating experiments that indicate that bacterial killing by CdzC/D is caused by the formation of pores in the inner membrane. The remaining CdzC/D mutations mapped to perA, which encodes a surface-exposed OMP. Analyses of PerA loss-of-function

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Figure 1. Comparison of Effector Loci in Bacterial Secretion Systems with Eukaryote- and Bacteria-Targeting Capabilities. Representative genomic regions are shown for (A) Type I secretion systems (T1SSs), (B) Type IV secretion systems (T4SSs), (C) Type VI secretion systems (T6SSs), (D) and Type VII secretion systems (T7SSs). Species names for bacteria with loci containing bacteria-targeting effectors are colored in blue, whereas those with loci containing eukaryotetargeting effectors are colored in red. Immunity genes are only present in antibacterial pathways and are necessary to prevent self-intoxication by effectors and protection from attacking kin cells. Locus tags for indicated effector genes: cdzCD (CCNA_03932, CCNA_03933); tesG (PA4141); xac2609; cagA (HP0547); rhs1 (SL1344_0286); pdpC (FTN_1319); pdpD (FTN_1325); telA (SIR_0169); pe35 (Rv3872); ppe68 (Rv3873). Abbreviations: C. crescentus, Caulobacter crescentus; F. novicida, Francisella novicida; H. pylori, Helicobacter pylori; M. tuberculosis, Mycobacterium tuberculosis; P. aeruginosa, Pseudomonas aeruginosa; S. enterica, Salmonella enterica; S. intermedius, Streptococcus intermedius.

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Figure 2. Schematic of ContactDependent Antibacterial Secretion Systems and Their Proposed Mechanisms of Effector Export. (A)

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The CdzABCD T1SS from Caulobacter crescentus. Unfolded CdzCD effectors are recognized by the ABC transporter CdzA. An ATP-dependent conformational change facilitates effector entry into CdzB and presumably through a yetto-be-identified TolC-like transporter. Folded CdzCD exits the donor cell and is inserted into the membrane, forming large aggregates. Adhesion of CdzCD aggregates to a recipient cell may result in either (i) simultaneous penetration of both membranes by the toxin or (ii) discrete toxin delivery events into target cells. CdzCD aggregates cause ion efflux by forming pores, resulting in membrane depolarization. (B) The T4SS from Xanthomonas citri. The VirD4 ATPase binds the conserved C-terminal XVIPCD and transfers the effector to the VirB11 ATPase. The effector is then transported into the T4SS apparatus via an ATP-dependent process. Upon recipient cell contact by VirB5, the effector may be exported through the pilus or by a yet-to-bedetermined alternative mechanism. (C) A generic model for bacteria-targeting T6SSs. Upon detection of a recipient cell, a needle-like apparatus (Hcp tube, VgrG+PAAR spike, TssBC sheath) is assembled and loaded with effectors. Effectors either interact with Hcp hexamers or VgrG-PAAR complexes. Hcp-associated effectors can be found to (i) associate with the inside of the Hcp tube or (ii) form a C-terminal extension of an Hcp hexamer. VgrGassociated effectors are found as (i) a C-terminal extension of PAAR, (ii) a C-terminal extension of VgrG, or (iii) noncovalent interaction with VgrG. Sheath contraction ejects an Hcp tube directly into the target cell, where effectors are released and exert their toxic effects. (D) The T7SSb from S. intermedius. Effectors are recognized by EssC/EccC and effector export is thought to be an ATP-dependent process. Abbreviations: ABC, ATP-binding cassette; Hcp, hemolysin coregulated protein; MFP, membrane fusion protein; OMP, outer membrane protein; T1SS, Type I secretion system; T4SS, Type IV secretion system; T6SS, Type VI secretion system; T7SS, Type VII secretion system; VgrG, valine–glycinerich repeat protein G;

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mutants suggest the presence of a potential receptor for these toxins on target cells. PerA, like other bacteriocin receptors, is anchored to the outer membrane and likely facilitates the entry of Cdz toxins through a TonB-dependent import mechanism [27]. Despite evidence that CdzC/D act on a universally conserved cellular structure in recipient cells, C. crescentus cells expressing the Cdz system are only able to kill a narrow range of target bacteria [13]. This killing range includes other strains of C. crescentus and a limited number of other species of α-Proteobacteria. It seems probable that the inability of C. crescentus to kill more distantly related Proteobacteria is caused by either the absence of a PerA receptor or an inability of C. crescentus CdzC/D to interact with highly divergent PerA homologs present in the OMs of target bacteria. Cdz-like systems have been identified in many species of β- and γ-Proteobacteria, so it is possible that in these bacteria a distinct OMP is hijacked for toxin translocation across the outer membrane [13]. These putative Cdz-like systems await a more detailed characterization and future studies will provide a better understanding of the role played by antibacterial T1SSs in shaping bacterial communities.

Type IV Secretion Systems Are Functionally Diverse T4SSs are macromolecular complexes which, similar to T1SSs, span both the inner and outer membrane of Gram-negative bacteria. These systems were originally discovered in the 1950s as F plasmid conjugation machines in E. coli but have since been shown in other bacteria to transport protein effectors instead of nucleic acids [28–30]. T4SSs are encoded by 12 genes often named virB1 through virB11 and virD4 (Figure 1B). VirB2 and VirB5, respectively, form the major and minor adhesion pilin of these systems, although the role of the T4SS pilus is not yet fully understood and some T4SSs are functional even in the absence of a pilus [31]. VirB4, VirB11, and VirD4 are all ATPases that not only energize the system but also dictate effector recognition (Figure 2B) [32]. VirD4 is particularly interesting because it functions as a coupling protein that makes initial contact with protein effectors via a positively charged, C-terminal signal sequence [33]. After making contact, VirD4 then transfers effectors to VirB11, which in turn shuttles them into the inner membrane complex and through the secretion apparatus in a process that is driven by ATP binding and hydrolysis [32]. T4SSs are functionally diverse in terms of both the types of molecules they export and their biological roles. Some T4SSs facilitate conjugation by transporting DNA between bacterial cells, while others take up or release DNA from the environment [34,35]. A third group is involved in pathogenesis and in this context, T4SSs transport protein effectors or protein–DNA complexes into target cells, which lead to disease [36,37]. A well-characterized example of T4SSmediated pathogenesis is found in Agrobacterium tumefaciens, which uses a T4SS to cause crown gall disease in plants. The T4SS-mediated injection of transfer DNA is then integrated into the plant chromosome where A. tumefaciens genes encoding nutrient biosynthesis enzymes and plant growth hormones are expressed by the plant cell transcriptional machinery, causing the rapid development of tumors [37]. Human and animal pathogens also use T4SSs for virulence as is the case with the protein exporting T4SSs of Legionella pneumophila, Helicobacter pylori, and Brucella abortus [38–40]. For example, the T4SS of the agriculturally relevant pathogen B. abortus directly injects mammalian cells with effectors that subvert immune signaling and enable the B. abortus intracellular life cycle [40]. In the majority of cases, Type IV secretion is contact dependent with secretion events taking place after cell–cell contact has been established.

The T4SS Is a Newly Characterized Interbacterial Antagonism Pathway In addition to the functions described earlier, new evidence has now implicated the T4SS as a mediator of contact-dependent interbacterial antagonism, which further adds to its versatility. This discovery was initially made in the bacterium Xanthomonas citri, a T4SS-containing plant Trends in Microbiology, Month 2020, Vol. xx, No. xx

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pathogen that encodes a unique set of VirD4-interacting proteins called Xanthomonas VirD4interacting proteins or XVIPs [15]. XVIPs lack the canonical T4SS C-terminal signal sequence that interacts with VirD4 and targets effectors to the membrane complex, and instead possess a unique C-terminal domain referred to as XVIPCD. In X. citri, this C-terminal domain is required for the T4SS-dependent secretion of a specific XVIP, termed X-TfeXAC2609 [15]. The N-terminal domain of X-TfeXAC2609 possesses peptidoglycan (PG) hydrolase activity that is thought to contribute to the ability of the X. citri T4SS to kill target bacteria. Each of the X-Tfe effectors identified thus far are encoded adjacent to immunity genes that encode proteins that likely bind to their cognate effectors to neutralize their toxicity. Other XVIP functions are anticipated based on bioinformatic analyses that identified domains with PG-binding, PG glycoside hydrolase, lytic transglycosylase, PG peptidase, and phospholipase activities [15]. A second T4SS involved in contact-dependent interbacterial antagonism has been identified in the opportunistic pathogen Stenotrophomonas maltophilia [19]. In this organism, 12 putative effector–immunity pairs were predicted, based on conservation of the XVIPCD sequence with that from X. citri effectors. Characterization of the representative effector–immunity pair, Smlt3024–Smlt3025, found that Smlt3024 secretion is dependent on a functional T4SS and that Smlt3025 confers immunity to Smlt3024-mediated toxicity [19]. A recent bioinformatic survey of T4SSs homologous to the aforementioned examples suggests that bacteria-targeting T4SSs exist broadly throughout the order Xanthomonadales and in some orders of Betaproteobacteria, implying that the use of this pathway as a mechanism for interbacterial competition may be widespread [41]. Future research is needed to determine the diversity of killing mechanisms employed by antibacterial T4SS effectors and if additional uncharacterized T4SSs can influence interbacterial competition.

Type VI Secretion Systems Mediate Competition between Diverse Species of Bacteria In contrast to the T1SS and T4SS pathways described earlier, the most commonly identified role of bacterial T6SSs is to mediate interbacterial antagonism. Early bioinformatic analyses of T6SS gene clusters indicated that some components of this pathway possess homology to subunits of T4SSs, leading to the speculation that this pathway, like many T4SSs, was involved in virulence [42–44]. However, there is now compelling evidence that the majority of T6SSs target bacteria with notable well-characterized exceptions including the host cell-targeting T6SSs from Francisella tularensis, Edwardsiella tarda, and several species of pathogenic Burkholderia (Figure 1C) [45–50]. Similar to T1SSs and T4SSs, T6SSs export effector proteins across both the inner and outer membranes of Gram-negative bacteria in a single step. The T6SS requires 14 structural components, 13 of which are labeled Type six secretion (Tss) A-M; the fourteenth derives its name PAAR from a proline–alanine–alanine–arginine repeat found within its sequence. These proteins constitute two subassemblies known as the membrane complex and the bacteriophage tail-like complex, the latter of which resembles an outward facing phage tail when completely assembled (Figure 2C) [51–53]. The membrane complex is critical for protein secretion by T6SS because it facilitates the assembly of the phage tail-like complex and acts as a conduit for effector delivery across the cell envelope. The bacteriophage tail-like subassembly consists of six components: the AAA+ ATPase ClpV, the phage tail sheath-like proteins TssB and TssC, hemolysin coregulated protein (Hcp), valine–glycine-rich repeat protein G (VgrG), and a PAAR repeat protein. Hcp protomers form hexameric rings that stack on top of one another to form a long nanotube [54]. This nanotube interacts with a VgrG trimer which possesses a flat surface that facilitates its interaction with a single PAAR repeat protein [55]. To deliver effectors to target cells, T6SSs undergo a phage tail-like contraction. Prior to this contraction event, the Hcp tube is capped with a single spike-shaped VgrG–PAAR complex [53,56]. This tube–spike complex is then surrounded by a phage tail sheath-like structure that upon contraction propels 6

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the spike-capped tube out of the bacterial cell [57]. The ClpV ATPase then disassembles the sheath following its contraction so that the individual sheath subunits can be reassembled for a subsequent firing event [58]. The phage tail-like assembly is the site of T6SS effector recruitment and there are currently five known ways in which effectors are delivered by this structure: (i) within the lumen of Hcp hexamers, (ii) as C-terminal extensions of Hcp, (iii) through interaction with VgrG proteins, (iv) as C-terminal extensions of VgrG proteins, and (v) as C-terminal extensions of PAAR repeat containing proteins [53,59–62]. The inner pore of the Hcp tube is approximately 40 Å in diameter and has a chaperone-like function, interacting with and promoting the stability of effectors below a specific size threshold (approximately 20 kDa) [59]. By contrast, effectors that interact noncovalently with or are C-terminal extensions of VgrG or PAAR proteins do not appear to have similar size restrictions with some effectors having a molecular weight in excess of 100 kDa [63].

Effectors That Transit the T6SS Target Diverse Molecules Required for Bacterial Growth In contrast to the Cdz T1SS and the aforementioned bacteria-targeting T4SSs, many T6SS effectors have been identified (Table 1). T6SSs that have been characterized to date deliver a ‘cocktail’ of effectors into target cells. Intriguingly, some bacteria, such as Pseudomonas aeruginosa, possess multiple evolutionarily distinct T6SSs that are differentially regulated, with each exporting their own repertoire of effectors [21,64–68]. Similar to bacteria-targeting effectors from other systems, T6SS effectors are typically encoded by genes adjacent to their cognate immunity genes, which allows for kin discrimination during interbacterial competition [12,21]. In general, members of T6SS effector families act on molecules found in either the periplasm or the cytoplasm that are essential for survival. Effectors that have been characterized biochemically are described in detail in the following sections.

Cell Wall-Targeting T6SS Effectors PG maintains bacterial cell shape and protects cells from osmotic lysis, making it essential for survival [69]. The polysaccharide component of PG is composed of repeating β-1,4-linked N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) subunits. The MurNAc groups in adjacent glycan strands are connected by peptide crosslinks resulting in a sacculus that encompasses the entirety of the cell. To date, two superfamilies of PG-targeting T6SS effectors have been identified: (i) the Type VI amidase effectors (Tae) and (ii) the Type VI glycoside hydrolase effectors (Tge) [70,71]. Tae effectors exert their toxicity by hydrolyzing the peptide crosslinks of PG. Four Table 1. Antibacterial Effector Proteins Involved in Contact-Dependent Interbacterial Antagonism Molecular target

Biochemical activity

Effectors

Secretion system

PDB codes

Refs

Cell wall

NlpC/P60 amidase

Tae1, Tae2, Tae3, Tae4

T6SS

4EOB, 4HZ9, 4HFK

[12,71,72,108,109]

Chitosanase or lysozyme-like glycoside hydrolase

XAC2609, VgrG3, Tge1, Tge2, Tge3

T4SS, T6SS

4M5F, 4KT3

[12,15,70,110,111]

Zinc metallopeptidase

Tpe1, VgrG2b

T6SS

6H56

[19,67,112]

Lipid II phosphatase

TelC

T7SS

5UKH

[20]

Membrane depolarizing toxin

CdzC, CdzD, VasX, Tse4, TspA, Ssp6

T1SS, T6SS, T7SS

–

[13,78,79,81]

Cell membranes

Phospholipase

Tle1, Tle2, Tle3, Tle4, Tle5

T6SS

5O5P, 4R1D

[68,75,76]

DNA

DNase

RhsA, RhsB, Rhs2, Tde1, Tde2, PoNe, EsaD

T6SS, T7SS

–

[14,63,85–87]

Nucleotides

NAD(P)+ hydrolase

Tne1, Tne2, TelB

T6SS, T7SS

4ZV0, 6B12

[20,88,89]

(p)ppApp synthetase

Tas1

T6SS

6OX6

[90]

ADP-ribosyl transferase

Tre1, Tse2?

T6SS

6DRE, 5AKO

[91,113]

Proteins

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sequence-divergent Tae families exist, and the PG peptidase activity of these families differs because they target different chemical bonds within the peptide stem. Tae1 and Tae4 effectors function as DL-endopeptidases, whereas Tae2 and Tae3 are DD-endopeptidases [71]. The best characterized Tae effector is the Type six exported effector 1 (Tse1) from P. aeruginosa, which belongs to the Tae1 family [12]. X-ray crystal structures of Tse1 show that this enzyme adopts an NlpC/P60 papain-like cysteine protease fold [72]. Tse1 possesses some structural similarity to housekeeping PG amidases involved in cell wall homeostasis; however, in contrast to these enzymes, Tse1 lacks structural motifs that regulate its PG hydrolase activity – a feature that likely contributes to its potent antibacterial activity. In contrast to Tae effectors, Tge effectors act on the glycan backbone of PG. The Tge superfamily includes enzymes targeting either the GlcNAc–MurNAc bond (glucosaminidases) or the MurNAc– GlcNAc bond (muramidases). The best characterized Tge muramidase is Tse3, the founding member of the Tge1 family [12]. Tse3 adopts a lysozyme-like fold and requires a calcium cofactor for its glycan hydrolase activity [73]. A Tge2 member from P. protegens has also been structurally characterized, revealing its resemblance to PG glucosaminadases [70]. There are several reports indicating that both Tae and Tge effectors can be secreted by the same T6SS which would enable the complete degradation of the PG matrix of the target cell [12,70,71].

Cell Membrane-Targeting T6SS Effectors There are several T6SS effectors known to target membranes and these can be classified as either (i) phospholipases or (ii) pore-forming toxins (Figure 3A). Similar to cell wall-targeting effectors, membrane-targeting effectors act in the periplasm and are neutralized by immunity proteins that localize to this compartment. Phospholipase effectors are widely distributed among many T6SS-containing bacteria and are composed of five sequence-divergent families designated Tle1–5 (Type VI lipase effector 1–5). The Tle1–4 families all contain a conserved GxSxG motif most commonly found in esterases, whereas Tle5 possesses dual HxKxxxD motifs characteristic of phospholipase D enzymes [68]. Structural analyses of Tle1, Tle4, and Tle5 enzymes showed that these effectors adopt folds that are similar to the catalytic domains of well-characterized phospholipases, but also revealed novel α-helical membrane anchoring domains that have been postulated to improve toxin efficacy through interactions with the inner membrane once inside a recipient cell [74–77]. In addition to enzymatic hydrolysis of membrane phospholipids, T6SS membrane-targeting effectors can act by forming pores that puncture membranes of target cells. Some of these effectors bear homology to pore-forming colicins while others are similar to CdzCD in that they possess glycine zipper motifs reminiscent of those found in multimeric membrane protein channels and amyloid-β peptide [78–80]. The best understood example is the Tse4 effector from P. aeruginosa which possesses glycine zipper motifs and exerts its toxicity in the periplasm of target bacteria by forming ion-selective membrane pores that facilitate potassium efflux, resulting in membrane depolarization and dissipation of the proton motive force [79]. Similarly, the Ssp6 effector from S. marcescens was recently shown to inhibit bacterial growth by forming cation-selective pores in membranes [81].

Nuclease and Nucleotide Targeting T6SS Effectors Although the mechanisms by which T6SS effectors that act in the cytoplasm reach this cellular compartment are incompletely understood, it is well established that molecules such as nucleic acids and nucleotides are targeted by these toxins [82–84]. For example, several families of nuclease toxins have been identified and shown to inhibit the growth of the target bacterial cells by degrading chromosomal DNA [85–87]. The molecular basis for this enzymatic activity is not well understood but current evidence suggests that many of 8

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Figure 3. Effectors Exported by Antibacterial Secretion Systems Inhibit Bacterial Growth through Diverse Mechanisms. (A) Antibacterial effectors targeting Gram-negative bacteria act in either the periplasm or cytoplasm to kill or hinder the growth of competitors. Cytoplasmic effector functions include DNase, NAD(P)+ hydrolase, ADP-ribosyl transferase, and (p)ppApp synthetase activities. Periplasmic effectors target one of two essential structures: (i) the cell wall or (ii) the cell membrane. Representative effectors for each category are indicated. For a full list of well-characterized effectors, see Table 1. (B) Antibacterial effectors targeting Gram-positive bacteria act in the inner wall zone or cytoplasm. The T7SS exports effectors with four known activities: membrane depolarization, lipid II phosphatase, NAD(P)+ hydrolase, and DNase. Abbreviation: T7SS, Type VII secretion system; Tae, Type VI amidase effectors; Tge, Type VI glycoside hydrolase effectors; Tle1–5, Type VI lipase effector 1–5; Tne1, Type VI NADase effector 1; Tne2, Type VI NADase effector 2; Tse4, Type six exported effector 4; Tse6, Type six exported effector 6;

these effectors act as nonspecific DNases [63,85,86]. By contrast, nucleotide-targeting effectors deplete cells of high-energy molecules required for cell viability. Thus far, two classes of these effectors have been implicated in interbacterial antagonism: (i) NAD+/NADP+ [NAD(P)+] hydrolases and (ii) (p)ppApp synthetases. NAD(P)+ hydrolases studied to date belong to one of two Type VI NADase effector (Tne) families [88]. The best characterized NAD(P)+ hydrolase and founding member of the Tne1 group is the Tse6 toxin from P. aeruginosa [89]. The structure of the Tse6 toxin domain resembles protein-targeting ADPribosyl transferases; however, in contrast to these transferases, Tse6 lacks an open active site that would permit transfer of ADP-ribose from NAD+ to a protein target. Instead, Tse6 degrades NAD (P)+ at a rate that is approximately 1000-fold higher than structurally similar enzymes with ADPribosyl transferase activity. A second family of NAD(P)+ hydrolase effectors was more recently described and a representative member from P. protegens was shown to possess enzymatic properties similar to Tse6 [88]. In more recent work, our group identified Tas1, a T6SS effector that depletes cells of the essential nucleotides ADP and ATP [90]. The structure of Tas1 resembles enzymes that synthesize the survival-promoting bacterial alarmones (p)ppGpp; however, instead of synthesizing (p)ppGpp, Tas1 consumes ATP to pyrophosphorylate ADP and ATP to generate (p)ppApp. The enzymatic rate of Tas1 is approximately two orders of magnitude higher than previously described (p)ppGpp synthetases and consequently, delivery of this potent enzyme into target bacteria depletes them of ADP and ATP. (p)ppApp also binds and inhibits the activity of the (p)ppGpp target PurF, which catalyzes the committed step in de novo purine biosynthesis. Thus, Tas1-catalyzed production of (p)ppApp not only depletes cells of ADP and ATP but also inhibits the ability of the target cell to regenerate these essential nucleotides.

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Protein-Targeting T6SS Effectors The majority of protein toxins that target bacteria function by degradation or depletion of molecules essential for cell viability. Less common are antibacterial toxins that act by modulating the function of a target protein, presumably because it is easier for intoxicated cells to evolve resistance to this type of activity. The first example of a T6SS effector to act in this way is the recently described ADP-ribosylating toxin Tre1 from Serratia proteamaculans [91]. This unique effector causes growth cessation by inhibiting cell division, which it accomplishes via ADP-ribosylation of the prokaryotic tubulin homolog FtsZ. FtsZ protomers polymerize to form a contractile ‘Z-ring’ that plays an essential role in cytokinesis during cell division. ADP-ribosylation of FtsZ prevents Z-ring formation causing a defect in cell division, resulting in filamentation and the eventual death of targeted cells. Remarkably, the Tre1-specific immunity protein, Tri1, can protect cells from Tre1 intoxication via its ADP-ribosylhydrolase activity. In contrast to all other characterized effector–immunity pairs, this mechanism of immunity is not entirely reliant on a highly specific protein–protein interaction and can therefore confer broad immunity to diverse interbacterial ADP-ribosyltransferases.

Role of Chaperones in T6SS Effector Delivery The Hcp component of T6SSs possesses chaperone-like properties that promote the stability of small, single-domain effectors prior to their export [59]. By contrast, large multidomain effectors, which interact with VgrG proteins to facilitate their export, often require an additional protein for secretion. Three domain of unknown function (DUF) protein superfamilies, DUF4123, DUF1795, and DUF2169, have been demonstrated to interact with and promote the secretion of VgrG-interacting effectors [63,89,92,93]. For example, the DUF1795 chaperone EagT6 from P. aeruginosa binds transmembrane segments of its cognate PAAR domain-containing effector Tse6 and facilitates complex formation with its cognate VgrG [83]. The precise roles of DUF2169 and DUF4123 chaperones have not been established; however, a DUF4123 chaperone from P. aeruginosa was shown to require interaction with a co-chaperone to bind its cognate effector and together, these proteins facilitate loading of a PAAR domain-containing effector onto a VgrG spike [94]. Further structural and biochemical analyses of these chaperone families are needed to better understand their function in effector delivery.

Type VII Secretion Systems Are Protein Export Pathways Found in Gram-Positive Bacteria The T7SS was originally identified in Mycobacterium tuberculosis and is required for the pathogenesis of this bacterium [95–97]. A comparative genomic analysis between the Bacillus Calmette– Guérin (BCG) vaccine strain Mycobacterium bovis and virulent M. tuberculosis identified a region of difference 1 (RD1) locus that is not present in BCG. RD1 was subsequently shown to encode a protein secretion apparatus termed ESX-1 or more recently, T7SS [96,98]. A genetically distinct system in Firmicutes bacteria, sometimes denoted as T7SSb, was later discovered and has been implicated in the pathogenesis of S. aureus [99]. The only two components of T7SSs conserved between T7SSa and T7SSb pathways are a membrane-embedded ATPase referred to as either EccC (T7SSa) or EssC (T7SSb) and small secreted effectors belonging to the WXG100 protein family (Figure 1D). EssC/EccC is thought to energize effector transport, whereas WXG100 effectors are small, α-helical proteins required for T7SS function and in M. tuberculosis, facilitate disruption of phagosomal membranes (Figure 2D) [100]. Although WXG100 effectors typically do not contain toxin domains, their integral role in T7SS function makes them essential for the secretion of other T7SS-exported toxins [101].

Type VII Secretion Systems Mediate Competition between Gram-Positive Bacteria In addition to their pathogenic role, several T7SSb pathways have been implicated in mediating antagonistic interactions between bacteria by secreting effectors with toxic enzymatic activities [14,20]. An interesting example of this is EsaD/EssD, a nuclease toxin found in S. aureus. A growing 10

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body of evidence suggests that EsaD/EssD plays a dual role in pathogenesis and interbacterial antagonism [14,102]. Similar to Gram-negative bacteria that produce and export antibacterial effectors, S. aureus protects itself from the toxicity of EsaD/EssD by producing an immunity protein termed EsaG [14]. Export of EsaD/EssD requires a chaperone, EsaE (DUF5081), that promotes its stability and secretion. EsaE–EsaD–EsaG form a ternary complex with EsaE interacting with the N terminus of EsaD while EsaG binds the C-terminal toxin domain. Recent data suggest that EsaE targets this ternary complex to the EssC ATPase, which may be involved in the dissociation of the complex prior to the export of EsaD/EssD out of the cell [14].

Outstanding Questions

The T7SSb of Streptococcus intermedius has also been linked to both pathogenesis and interbacterial competition [20,103]. The T7SSb of this organism exports three effector proteins, TelA, TelB, and TelC, which belong to the LXG family of polymorphic toxins. LXG proteins were first identified by Aravind and colleagues who predicted that these proteins were T7SS effectors involved in interbacterial antagonism between Gram-positive bacteria [104]. This prediction was substantiated by our biochemical characterization of the S. intermedius LXG effector TelC, which functions as a phosphatase that cleaves the PG precursor molecule, lipid II (Figure 3B). Like EsaD/EssD, Tel effector toxicity can be neutralized by immunity proteins [105]. Furthermore, each effector interacts with a WXG100-like protein (DUF5082) that may act as an intracellular chaperone [20]. A fourth LXG effector, TspA, has been reported as a T7SSb toxin exported by S. aureus [106]. Using a zebrafish infection model, this effector was shown to be involved in both interbacterial antagonism and pathogenesis, and likely exerts its toxicity by depolarizing cell membranes. Beyond the aforementioned T7SSb effectors, there exists thousands of predicted LXG toxins throughout the genomes of Firmicutes bacteria. Characterization of these proteins will improve our currently limited knowledge of the mechanisms used by Gram-positive bacteria to antagonize one another and may highlight vulnerabilities in the physiology of this group of bacteria that is distinct from their Gram-negative counterparts.

Which cellular compartments are T4SS, T6SS, and T7SS effectors delivered to by the secretion apparatus? If effectors are deposited on the cell surface (Gram positive) or delivered to the periplasm (Gram negative), how do effectors that act in the cytoplasm reach this compartment?

How do the recognition motifs within T1SS, T4SS, and T7SS antibacterial effectors facilitate their recruitment to their respective secretion systems? Are there receptor proteins on the surface of recipient cells that impart and/or increase susceptibility to T4SS-, T6SS-, and T7SS-mediated attack?

In instances where bacteria possess many contact-dependent pathways involved in interbacterial antagonism, which is dominant against specific competitors and why? Many bacteria possess multiple copies of the same contact-dependent interbacterial antagonism pathway. Are these pathways differentially expressed and if so, what are the environmental conditions that regulate their expression?

Concluding Remarks It has become increasingly apparent that bacteria employ a variety of protein secretion machines to deliver antibacterial toxins into competitor bacteria. Here we have discussed four distinct pathways and described how they mediate contact-dependent interbacterial competition. These protein secretion systems are found broadly within Gram-negative or Gram-positive bacteria and were initially all viewed through the lens of bacterial pathogenesis [37,44,97,107]. However, examination of these systems in both pathogenic and non-pathogenic bacteria has shown that the bacteria-targeting capability of these pathways is widespread. Although these protein secretion systems are found in different bacteria and differ in structure, several interesting commonalities have emerged. First, all systems export antibacterial proteins across the cell envelope using an ATP-dependent secretion event. Second, each antibacterial effector protein is encoded near an immunity gene that protects toxin-producing cells and kin cells. Finally, regardless of the system used for effector export, antibacterial effectors universally target essential cellular components such as nucleic acids, cell membranes, and PG. Despite great advances in our knowledge of secretion systems in recent years, there remain many unanswered questions about each system with respect to their regulation, secretion mechanism, and effector repertoire. Nevertheless, it is remarkable how much the field of interbacterial antagonism has advanced over the past decade. Studies using a combination of computational, structural, and biochemical approaches continue to provide substantial insights into the molecular mechanisms at play during interbacterial competition. Linking the findings from these reductionist approaches to trends observed in population-level studies will be imperative to increase our understanding of the significance and impact of contact-dependent interbacterial antagonism in complex microbial communities (see Outstanding Questions). Trends in Microbiology, Month 2020, Vol. xx, No. xx

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Acknowledgments The authors thank Nathan Bullen and Susan Cole for helpful discussions and feedback on the manuscript. T.A.K. and S.A. were supported by an Alexander Graham Bell Canada Graduate Scholarship and Ontario Graduate Scholarship, respectively. This work was supported by a CIHR project grant (PJT-156129) to J.C.W.

References 1.

2. 3.

4.

5.

6.

7.

8. 9.

10.

11.

12. 13.

14.

15. 16.

17.

18.

19.

20.

21.

22. 23.

24.

12

Little, A.E. et al. (2008) Rules of engagement: interspecies interactions that regulate microbial communities. Annu. Rev. Microbiol. 62, 375–401 Hibbing, M.E. et al. (2010) Bacterial competition: surviving and thriving in the microbial jungle. Nat. Rev. Microbiol. 8, 15–25 Garcia-Bayona, L. and Comstock, L.E. (2018) Bacterial antagonism in host-associated microbial communities. Science 361, eaat2456 Zipperer, A. et al. (2016) Human commensals producing a novel antibiotic impair pathogen colonization. Nature 535, 511–516 Vacheron, J. et al. (2019) T6SS contributes to gut microbiome invasion and killing of an herbivorous pest insect by plantbeneficial Pseudomonas protegens. ISME J. 13, 1318–1329 Mougi, A. and Kondoh, M. (2014) Stability of competitionantagonism-mutualism hybrid community and the role of community network structure. J. Theor. Biol. 360, 54–58 Wexler, A.G. et al. (2016) Human symbionts inject and neutralize antibacterial toxins to persist in the gut. Proc. Natl. Acad. Sci. U. S. A. 113, 3639–3644 Ross, B.D. et al. (2019) Human gut bacteria contain acquired interbacterial defence systems. Nature 575, 224–228 Verster, A.J. et al. (2017) The landscape of type VI secretion across human gut microbiomes reveals its role in community composition. Cell Host Microbe 22, 411–419.e4 Corr, S.C. et al. (2007) Bacteriocin production as a mechanism for the antiinfective activity of Lactobacillus salivarius UCC118. Proc. Natl. Acad. Sci. U. S. A. 104, 7617–7621 Kommineni, S. et al. (2015) Bacteriocin production augments niche competition by enterococci in the mammalian gastrointestinal tract. Nature 526, 719–722 Russell, A.B. et al. (2011) Type VI secretion delivers bacteriolytic effectors to target cells. Nature 475, 343–347 Garcia-Bayona, L. et al. (2017) Contact-dependent killing by Caulobacter crescentus via cell surface-associated, glycine zipper proteins. eLife 6, e24869 Cao, Z. et al. (2016) The type VII secretion system of Staphylococcus aureus secretes a nuclease toxin that targets competitor bacteria. Nat. Microbiol. 2, 16183 Souza, D.P. et al. (2015) Bacterial killing via a type IV secretion system. Nat. Commun. 6, 6453 Russell, A.B. et al. (2014) A type VI secretion-related pathway in Bacteroidetes mediates interbacterial antagonism. Cell Host Microbe 16, 227–236 Willett, J.L. et al. (2015) Contact-dependent growth inhibition (CDI) and CdiB/CdiA two-partner secretion proteins. J. Mol. Biol. 427, 3754–3765 Hayes, C.S. et al. (2014) Mechanisms and biological roles of contact-dependent growth inhibition systems. Cold Spring Harb. Perspect. Med. 4, a010025 Bayer-Santos, E. et al. (2019) The opportunistic pathogen Stenotrophomonas maltophilia utilizes a type IV secretion system for interbacterial killing. PLoS Pathog. 15, e1007651 Whitney, J.C. et al. (2017) A broadly distributed toxin family mediates contact-dependent antagonism between grampositive bacteria. eLife 6, e26938 Hood, R.D. et al. (2010) A type VI secretion system of Pseudomonas aeruginosa targets a toxin to bacteria. Cell Host Microbe 7, 25–37 Kanonenberg, K. et al. (2013) Type I secretion systems – a story of appendices. Res. Microbiol. 164, 596–604 Noegel, A. et al. (1979) Plasmid cistrons controlling synthesis and excretion of the exotoxin alpha-haemolysin of Escherichia coli. Mol. Gen. Genet. 175, 343–350 Wandersman, C. and Delepelaire, P. (1990) TolC, an Escherichia coli outer membrane protein required for

Trends in Microbiology, Month 2020, Vol. xx, No. xx

25.

26.

27.

28. 29.

30.

31.

32.

33.

34.

35.

36. 37.

38.

39.

40.

41. 42.

43.

44.

hemolysin secretion. Proc. Natl. Acad. Sci. U. S. A. 87, 4776–4780 Balakrishnan, L. et al. (2001) Substrate-triggered recruitment of the TolC channel-tunnel during type I export of hemolysin by Escherichia coli. J. Mol. Biol. 313, 501–510 Hwang, J. et al. (1997) Interactions of dedicated export membrane proteins of the colicin V secretion system: CvaA, a member of the membrane fusion protein family, interacts with CvaB and TolC. J. Bacteriol. 179, 6264–6270 Garcia-Bayona, L. et al. (2019) Mechanisms of resistance to the contact-dependent bacteriocin CdzC/D in Caulobacter crescentus. J. Bacteriol. 201, e00538-18 Cavalli, L.L. et al. (1953) An infective factor controlling sex compatibility in Bacterium coli. J. Gen. Microbiol. 8, 89–103 Stachel, S.E. and Zambryski, P.C. (1986) Agrobacterium tumefaciens and the susceptible plant cell: a novel adaptation of extracellular recognition and DNA conjugation. Cell 47, 155–157 Winans, S.C. et al. (1996) Adaptation of a conjugal transfer system for the export of pathogenic macromolecules. Trends Microbiol. 4, 64–68 Yuan, Q. et al. (2005) Identification of the VirB4-VirB8-VirB5VirB2 pilus assembly sequence of type IV secretion systems. J. Biol. Chem. 280, 26349–26359 Atmakuri, K. et al. (2004) Energetic components VirD4, VirB11 and VirB4 mediate early DNA transfer reactions required for bacterial type IV secretion. Mol. Microbiol. 54, 1199–1211 Vergunst, A.C. et al. (2005) Positive charge is an important feature of the C-terminal transport signal of the VirB/D4translocated proteins of Agrobacterium. Proc. Natl. Acad. Sci. U. S. A. 102, 832–837 Dillard, J.P. and Seifert, H.S. (2001) A variable genetic island specific for Neisseria gonorrhoeae is involved in providing DNA for natural transformation and is found more often in disseminated infection isolates. Mol. Microbiol. 41, 263–277 Hofreuter, D. et al. (2001) Natural transformation competence in Helicobacter pylori is mediated by the basic components of a type IV secretion system. Mol. Microbiol. 41, 379–391 Seubert, A. et al. (2003) A bacterial conjugation machinery recruited for pathogenesis. Mol. Microbiol. 49, 1253–1266 Vergunst, A.C. et al. (2000) VirB/D4-dependent protein translocation from Agrobacterium into plant cells. Science 290, 979–982 Segal, G. et al. (1998) Host cell killing and bacterial conjugation require overlapping sets of genes within a 22-kb region of the Legionella pneumophila genome. Proc. Natl. Acad. Sci. U. S. A. 95, 1669–1674 Bourzac, K.M. et al. (2007) Helicobacter pylori CagA induces AGS cell elongation through a cell retraction defect that is independent of Cdc42, Rac1, and Arp2/3. Infect. Immun. 75, 1203–1213 Roux, C.M. et al. (2007) Brucella requires a functional Type IV secretion system to elicit innate immune responses in mice. Cell. Microbiol. 9, 1851–1869 Sgro, G.G. et al. (2019) Bacteria-killing type IV secretion systems. Front. Microbiol. 10, 1078 Das, S. and Chaudhuri, K. (2003) Identification of a unique IAHP (IcmF associated homologous proteins) cluster in Vibrio cholerae and other proteobacteria through in silico analysis. In Silico Biol. 3, 287–300 Pukatzki, S. et al. (2006) Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc. Natl. Acad. Sci. U. S. A. 103, 1528–1533 Mougous, J.D. et al. (2006) A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus. Science 312, 1526–1530

Trends in Microbiology

45.

46.

47.

48. 49.

50.

51.

52. 53.

54.

55.

56.

57. 58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

Nano, F.E. et al. (2004) A Francisella tularensis pathogenicity island required for intramacrophage growth. J. Bacteriol. 186, 6430–6436 Eshraghi, A. et al. (2016) Secreted effectors encoded within and outside of the Francisella pathogenicity island promote intramacrophage growth. Cell Host Microbe 20, 573–583 Rao, P.S. et al. (2004) Use of proteomics to identify novel virulence determinants that are required for Edwardsiella tarda pathogenesis. Mol. Microbiol. 53, 573–586 Schell, M.A. et al. (2007) Type VI secretion is a major virulence determinant in Burkholderia mallei. Mol. Microbiol. 64, 1466–1485 Schwarz, S. et al. (2010) Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions. PLoS Pathog. 6, e1001068 Rosales-Reyes, R. et al. (2012) The Type VI secretion system of Burkholderia cenocepacia affects multiple Rho family GTPases disrupting the actin cytoskeleton and the assembly of NADPH oxidase complex in macrophages. Cell. Microbiol. 14, 255–273 Rapisarda, C. et al. (2019) In situ and high-resolution cryo-EM structure of a bacterial type VI secretion system membrane complex. EMBO J. 38, e100886 Cherrak, Y. et al. (2018) Biogenesis and structure of a type VI secretion baseplate. Nat. Microbiol. 3, 1404–1416 Shneider, M.M. et al. (2013) PAAR-repeat proteins sharpen and diversify the type VI secretion system spike. Nature 500, 350–353 Ballister, E.R. et al. (2008) In vitro self-assembly of tailorable nanotubes from a simple protein building block. Proc. Natl. Acad. Sci. U. S. A. 105, 3733–3738 Renault, M.G. et al. (2018) The gp27-like hub of VgrG serves as adaptor to promote Hcp tube assembly. J. Mol. Biol. 430, 3143–3156 Cianfanelli, F.R. et al. (2016) VgrG and PAAR proteins define distinct versions of a functional type VI secretion system. PLoS Pathog. 12, e1005735 Basler, M. et al. (2012) Type VI secretion requires a dynamic contractile phage tail-like structure. Nature 483, 182–186 Bonemann, G. et al. (2009) Remodelling of VipA/VipB tubules by ClpV-mediated threading is crucial for type VI protein secretion. EMBO J. 28, 315–325 Silverman, J.M. et al. (2013) Haemolysin coregulated protein is an exported receptor and chaperone of type VI secretion substrates. Mol. Cell 51, 584–593 Flaugnatti, N. et al. (2016) A phospholipase A1 antibacterial type VI secretion effector interacts directly with the C-terminal domain of the VgrG spike protein for delivery. Mol. Microbiol. 99, 1099–1118 Pukatzki, S. et al. (2007) Type VI secretion system translocates a phage tail spike-like protein into target cells where it crosslinks actin. Proc. Natl. Acad. Sci. U. S. A. 104, 15508–15513 Ma, J. et al. (2017) The Hcp proteins fused with diverse extended-toxin domains represent a novel pattern of antibacterial effectors in type VI secretion systems. Virulence 8, 1189–1202 Alcoforado Diniz, J. and Coulthurst, S.J. (2015) Intraspecies Competition in Serratia marcescens is mediated by type VIsecreted Rhs effectors and a conserved effector-associated accessory protein. J. Bacteriol. 197, 2350–2360 Goodman, A.L. et al. (2004) A signaling network reciprocally regulates genes associated with acute infection and chronic persistence in Pseudomonas aeruginosa. Dev. Cell 7, 745–754 Marden, J.N. et al. (2013) An unusual CsrA family member operates in series with RsmA to amplify posttranscriptional responses in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U. S. A. 110, 15055–15060 Allsopp, L.P. et al. (2017) RsmA and AmrZ orchestrate the assembly of all three type VI secretion systems in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U. S. A. 114, 7707–7712 Wood, T.E. et al. (2019) The Pseudomonas aeruginosa T6SS delivers a periplasmic toxin that disrupts bacterial cell morphology. Cell Rep. 29, 187–201.e7 Russell, A.B. et al. (2013) Diverse type VI secretion phospholipases are functionally plastic antibacterial effectors. Nature 496, 508–512

69.

70.

71.

72.

73.

74. 75.

76.

77.

78.

79.

80.

81.

82.

83.

84.

85.

86. 87.

88.

89.

90. 91.

92.

93.

Typas, A. et al. (2011) From the regulation of peptidoglycan synthesis to bacterial growth and morphology. Nat. Rev. Microbiol. 10, 123–136 Whitney, J.C. et al. (2013) Identification, structure, and function of a novel type VI secretion peptidoglycan glycoside hydrolase effector-immunity pair. J. Biol. Chem. 288, 26616–26624 Russell, A.B. et al. (2012) A widespread bacterial type VI secretion effector superfamily identified using a heuristic approach. Cell Host Microbe 11, 538–549 Chou, S. et al. (2012) Structure of a peptidoglycan amidase effector targeted to Gram-negative bacteria by the type VI secretion system. Cell Rep. 1, 656–664 Lu, D. et al. (2014) Structural insights into the T6SS effector protein Tse3 and the Tse3-Tsi3 complex from Pseudomonas aeruginosa reveal a calcium-dependent membrane-binding mechanism. Mol. Microbiol. 92, 1092–1112 Aloulou, A. et al. (2012) Phospholipases: an overview. Methods Mol. Biol. 861, 63–85 Hu, H. et al. (2014) Structure of the type VI secretion phospholipase effector Tle1 provides insight into its hydrolysis and membrane targeting. Acta Crystallogr. D Biol. Crystallogr. 70, 2175–2185 Lu, D. et al. (2014) The structural basis of the Tle4-Tli4 complex reveals the self-protection mechanism of H2-T6SS in Pseudomonas aeruginosa. Acta Crystallogr. D Biol. Crystallogr. 70, 3233–3243 Yang, X.Y. et al. (2017) Structural and SAXS analysis of Tle5-Tli5 complex reveals a novel inhibition mechanism of H2-T6SS in Pseudomonas aeruginosa. Protein Sci. 26, 2083–2091 Miyata, S.T. et al. (2013) Dual expression profile of type VI secretion system immunity genes protects pandemic Vibrio cholerae. PLoS Pathog. 9, e1003752 LaCourse, K.D. et al. (2018) Conditional toxicity and synergy drive diversity among antibacterial effectors. Nat. Microbiol. 3, 440–446 Kim, S. et al. (2005) Transmembrane glycine zippers: physiological and pathological roles in membrane proteins. Proc. Natl. Acad. Sci. U. S. A. 102, 14278–14283 Mariano, G. et al. (2019) A family of Type VI secretion system effector proteins that form ion-selective pores. Nat. Commun. 10, 5484 Ho, B.T. et al. (2017) Vibrio cholerae type 6 secretion system effector trafficking in target bacterial cells. Proc. Natl. Acad. Sci. U. S. A. 114, 9427–9432 Quentin, D. et al. (2018) Mechanism of loading and translocation of type VI secretion system effector Tse6. Nat. Microbiol. 3, 1142–1152 Vettiger, A. and Basler, M. (2016) Type VI secretion system substrates are transferred and reused among sister cells. Cell 167, 99–110.e12 Ma, L.S. et al. (2014) Agrobacterium tumefaciens deploys a superfamily of type VI secretion DNase effectors as weapons for interbacterial competition in planta. Cell Host Microbe 16, 94–104 Jana, B. et al. (2019) A modular effector with a DNase domain and a marker for T6SS substrates. Nat. Commun. 10, 3595 Koskiniemi, S. et al. (2013) Rhs proteins from diverse bacteria mediate intercellular competition. Proc. Natl. Acad. Sci. U. S. A. 110, 7032–7037 Tang, J.Y. et al. (2018) Diverse NADase effector families mediate interbacterial antagonism via the type VI secretion system. J. Biol. Chem. 293, 1504–1514 Whitney, J.C. et al. (2015) An interbacterial NAD(P)(+) glycohydrolase toxin requires elongation factor Tu for delivery to target cells. Cell 163, 607–619 Ahmad, S. et al. (2019) An interbacterial toxin inhibits target cell growth by synthesizing (p)ppApp. Nature 575, 674–678 Ting, S.Y. et al. (2018) Bifunctional immunity proteins protect bacteria against FtsZ-targeting ADP-ribosylating toxins. Cell 175, 1380–1392.e14 Liang, X. et al. (2015) Identification of divergent type VI secretion effectors using a conserved chaperone domain. Proc. Natl. Acad. Sci. U. S. A. 112, 9106–9111 Bondage, D.D. et al. (2016) VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor-effector complex. Proc. Natl. Acad. Sci. U. S. A. 113, E3931–E3940

Trends in Microbiology, Month 2020, Vol. xx, No. xx

13

Trends in Microbiology

94.

95.

96.

97.

98.

99.

100.

101.

102.

103.

14

Burkinshaw, B.J. et al. (2018) A type VI secretion system effector delivery mechanism dependent on PAAR and a chaperoneco-chaperone complex. Nat. Microbiol. 3, 632–640 Lewis, K.N. et al. (2003) Deletion of RD1 from Mycobacterium tuberculosis mimics bacille Calmette-Guerin attenuation. J. Infect. Dis. 187, 117–123 Mahairas, G.G. et al. (1996) Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. J. Bacteriol. 178, 1274–1282 Pym, A.S. et al. (2002) Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti. Mol. Microbiol. 46, 709–717 Stanley, S.A. et al. (2003) Acute infection and macrophage subversion by Mycobacterium tuberculosis require a specialized secretion system. Proc. Natl. Acad. Sci. U. S. A. 100, 13001–13006 Burts, M.L. et al. (2005) EsxA and EsxB are secreted by an ESAT-6-like system that is required for the pathogenesis of Staphylococcus aureus infections. Proc. Natl. Acad. Sci. U. S. A. 102, 1169–1174 Conrad, W.H. et al. (2017) Mycobacterial ESX-1 secretion system mediates host cell lysis through bacterium contactdependent gross membrane disruptions. Proc. Natl. Acad. Sci. U. S. A. 114, 1371–1376 Champion, P.A. et al. (2006) C-terminal signal sequence promotes virulence factor secretion in Mycobacterium tuberculosis. Science 313, 1632–1636 Ohr, R.J. et al. (2017) EssD, a nuclease effector of the Staphylococcus aureus ESS pathway. J. Bacteriol. 199, e00528-16 Hasegawa, N. et al. (2017) Characterization of the pathogenicity of Streptococcus intermedius TYG1620 isolated from a human brain abscess based on the complete genome sequence with transcriptome analysis and transposon

Trends in Microbiology, Month 2020, Vol. xx, No. xx

104.

105.

106.

107. 108.

109.

110.

111.

112.

113.

mutagenesis in a murine subcutaneous abscess model. Infect. Immun. 85, e00886-16 Zhang, D. et al. (2012) Polymorphic toxin systems: comprehensive characterization of trafficking modes, processing, mechanisms of action, immunity and ecology using comparative genomics. Biol. Direct 7, 18 Klein, T.A. et al. (2018) Molecular basis for immunity protein recognition of a type VII secretion system exported antibacterial toxin. J. Mol. Biol. 430, 4344–4358 Ulhuq, F.R. et al. (2018) A membrane-depolarising toxin substrate of the Staphylococcus aureus type VII protein secretion system targets eukaryotes and bacteria. bioRxiv. Published online October 16, 2018. https://doi.org/10.1101/443630 Delepelaire, P. (2004) Type I secretion in Gram-negative bacteria. Biochim. Biophys. Acta 1694, 149–161 Dong, C. et al. (2013) Structural insights into the inhibition of type VI effector Tae3 by its immunity protein Tai3. Biochem. J. 454, 59–68 Zhang, H. et al. (2013) Structure of the type VI effectorimmunity complex (Tae4-Tai4) provides novel insights into the inhibition mechanism of the effector by its immunity protein. J. Biol. Chem. 288, 5928–5939 Brooks, T.M. et al. (2013) Lytic activity of the Vibrio cholerae type VI secretion toxin VgrG-3 is inhibited by the antitoxin TsaB. J. Biol. Chem. 288, 7618–7625 Li, L. et al. (2013) Structural insights on the bacteriolytic and self-protection mechanism of muramidase effector Tse3 in Pseudomonas aeruginosa. J. Biol. Chem. 288, 30607–30613 Ringel, P.D. et al. (2017) The role of type VI secretion system effectors in target cell lysis and subsequent horizontal gene transfer. Cell Rep. 21, 3927–3940 Robb, C.S. et al. (2016) The structure of the toxin and type six secretion system substrate Tse2 in complex with its immunity protein. Structure 24, 277–284