FEMS Pathogens and Disease, 73, 2015, ftv092 doi: 10.1093/femspd/ftv092 Advance Access Publication Date: 15 October 2015 Minireview

MINIREVIEW

MARTX toxins as effector delivery platforms Hannah E. Gavin and Karla J. F. Satchell∗ Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA ∗

Corresponding author: Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, 303 E. Chicago Ave, Ward 6–205, Chicago, IL 60611, USA. Tel: +312-503-2162; E-mail: [email protected] One sentence summary: This review examines the multifunctional autoprocessing RTX (MARTX) toxins of bacteria and their role in delivery of diverse effector proteins into eukaryotic cells. Editor: Peter Sebo

ABSTRACT Bacteria frequently manipulate their host environment via delivery of microbial ‘effector’ proteins to the cytosol of eukaryotic cells. In the case of the multifunctional autoprocessing repeats-in-toxins (MARTX) toxin, this phenomenon is accomplished by a single, >3500 amino acid polypeptide that carries information for secretion, translocation, autoprocessing and effector activity. MARTX toxins are secreted from bacteria by dedicated Type I secretion systems. The released MARTX toxins form pores in target eukaryotic cell membranes for the delivery of up to five cytopathic effectors, each of which disrupts a key cellular process. Targeted cellular processes include modulation or modification of small GTPases, manipulation of host cell signaling and disruption of cytoskeletal integrity. More recently, MARTX toxins have been shown to be capable of heterologous protein translocation. Found across multiple bacterial species and genera—frequently in pathogens lacking Type 3 or Type 4 secretion systems—MARTX toxins in multiple cases function as virulence factors. Innovative research at the intersection of toxin biology and bacterial genetics continues to elucidate the intricacies of the toxin as well as the cytotoxic mechanisms of its diverse effector collection. Keywords: bacterial toxin; Vibrio; RTX; MARTX; effector; translocation; secretion

INTRODUCTION Bacteria have evolved not only to exist in diverse habitats, but also to manipulate their respective niches to support microbial outcomes. For microorganisms that associate with single or multicellular hosts, the ability to deliver ‘effectors’ to the eukaryotic cell cytosol is integral to strategic modulation of the cellular environment to benefit the bacterium. Two common methods of bacterial effector delivery have been well described. In one method, a complex secretion machinery is expressed on the bacterial membrane. Through the secretion machinery, a diverse array of protein cargos are delivered directly to host cell cytosol to modify cell biological processes to promote bacterial pathogenesis. This strategy is exemplified by the Salmonella Type 3 secretion systems (T3SS) (Moest and Meresse 2013), the Legionella Type 4 secretion system (T4SS) (So et al. 2015) and

the Type 6 secretion systems (T6SS) of Vibrio cholerae (Ma and Mekalanos 2010). Alternatively, effectors can be delivered as extracellularly secreted toxins, where an ‘A’ component is a single effector protein that bears an activity and a ‘B’ component is important for binding the target cell and delivering the effector. The A and B components can be separate secreted proteins, such as anthrax toxin or the clostridial binary toxins, or distinct functional domains of a single protein, such as the clostridial glucosylating toxins or diphtheria toxin. This strategy allows for extracellularly exported bacterial effectors to be transported into the target cell without necessitating direct cytosolic injection. The functional results of A–B toxins are similar to effectors of direct injection systems in that they often modulate cell biological pathways (Alouf and Popoff 2006).

Received: 31 August 2015; Accepted: 7 October 2015  C FEMS 2015. All rights reserved. For permissions, please e-mail: [email protected]

1

2

FEMS Pathogens and Disease, 2015, Vol. 73, No. 9

A unique hybrid of the two described approaches is the effector delivery mechanism of the multifunctional, autoprocessing repeats-in-toxins (MARTX) toxins. Almost exclusively expressed from organisms lacking T3SS or T4SS, MARTX toxins—like A–B toxins—encompass all necessary components for both effector activity and delivery within a large, singular, extracellularly secreted polypeptide. However, more like direct injection systems, MARTX toxins can deliver not just one, but multiple cytopathic bacterial effectors to target host cells. Further, via horizontal gene acquisition and genetic recombination, diverse combinations of effector domains can arise within the single polypeptide. Thus, MARTX toxins are multifunctional effector cargo translocation, processing and delivery machines. Similar to other effector delivery systems, MARTX toxins are key factors for pathogenesis within numerous organisms. MARTX toxins act as accessory toxins during V. cholerae infections, supporting bacterial colonization of the intestine (Olivier, Queen and Satchell 2009). MARTX toxins aid V. vulnificus in defense against predation by amoeba (Lee et al. 2013) and during host infection act as the primary virulence factor facilitating initial colonization, dissemination and lethality in mammals and eels (Lo et al. 2011; Jeong and Satchell 2012; Amaro et al. 2015). The fish pathogen V. anguillarum has also been shown to induce lethal infections in salmon dependent upon a MARTX toxin (Li, Rock and Nelson 2008). Thus, MARTX toxins contribute significantly to disruption of human and aquatic health. This short review will summarize our knowledge of the secretion of MARTX toxins, structure and function of the toxins, and the repertoire of effector domains delivered by the MARTX toxin platform.

MARTX TOXIN PRODUCTION AND SECRETION MARTX toxin rtx gene loci are present across multiple bacterial genera including Aeromonas, Photorhabdus, Proteus, Vibrio and Xenorhabdus, and are best characterized in the Vibrio species V. cholerae, V. vulnificus and V. anguillarum (Table 1). Each rtx locus consists of two divergently transcribed operons: rtxHCA and rtxBDE (Lin et al. 1999; Boardman and Satchell 2004; Lee et al. 2007; Li, Rock and Nelson 2008). The rtxHCA operon is regulated by the anti-H-NS protein HlyU (Liu et al. 2007; Liu, Naka and Crosa 2009; Li, Mou and Nelson 2011; Wang et al. 2015). The first gene in the operon, rtxH, encodes a conserved hypothetical protein of as-yet unexplored function. The adjacent rtxC gene encodes for a putative acyltransferase (Lin et al. 1999). Fatty acid post-translational modification by homologous acyltransferases is a well-established mechanism of activation among members of the repeats-in-toxins (RTX) toxin family (Stanley, Koronakis

and Hughes 1998; Basar et al. 2001). Yet, the contribution of rtxC to MARTX-associated virulence is still debatable and does not appear to be essential for cytotoxicity (Liu et al. 2007; Gulig et al. 2009; Cheong et al. 2010). Further, acylation of any MARTX toxin has not yet been reported. The last gene in the operon, the rtxA gene (sometimes designated rtxA1), encodes the MARTX toxin itself. The rtxA open reading frames can exceed 15 kb, with rtxA often being the largest gene in the entire bacterial genome. The genes in the adjacent rtxBDE operon are inverted relative to the rtxHCA operon (Lin et al. 1999). These genes, in cooperation with the unlinked tolC gene, form a dedicated MARTX toxin Type 1 secretion system (T1SS) (Fig. 1) (Bina and Mekalanos 2001; Boardman and Satchell 2004). In a manner analogous to the hly-encoded hemolysin secretion system of Escherichia coli, RtxD is the presumed transmembrane protein linking with the outer membrane porin TolC. Together these proteins form a narrow ‘chunnel’ through which each MARTX toxin is secreted as an unfolded protein directly from bacterial cytoplasm to the extracellular environment without a periplasmic intermediate (Andersen, Hughes and Koronakis 2000). MARTX toxin secretion, again similar to that of E. coli hemolysin, is energized by ATPases peripherally associated with the inner membrane (Fig. 1) (Bielaszewska et al. 2014). Atypically, the MARTX toxin secretion systems require two different ATPase proteins, RtxB and RtxE (Boardman and Satchell 2004; Lee et al. 2008). Both ATPases are essential for secretion and are proposed to work as a heterodimer, although the functional consequence of this unusual T1SS is not known (Boardman and Satchell 2004). Upon secretion, MARTX toxin-associated activity can be detected from supernatant fluids (Fullner and Mekalanos 2000; Li, Rock and Nelson 2008; Lee et al. 2008; Dolores et al. 2015; Kim, Gavin and Satchell 2015). However, observable toxin activity can be short-lived due to degradation by secreted proteases (Fan et al. 2001; Boardman, Meehan and Satchell 2007) and induction of toxin autoprocessing by bacterial culture media (Shen et al. 2009; Kim et al. 2013). Given the relative instability of supernatantborne MARTX toxins, delivery may share similarities with the Bordetella pertussis adenylate cyclase toxin, where bacterial-host proximity is beneficial for freshly synthesized product to intoxicate target cells (Gray et al. 2004) and where toxin is captured in outer membrane vesicles (OMVs) (Hozbor et al. 1999). The MARTX toxin secreted by V. cholerae is stabilized by association with outer membranes and with OMVs (Boardman, Meehan and Satchell 2007). However this does not appear to be a universal phenomenon, as the same association between MARTX toxin and OMVs has not been observed with V. vulnificus (Kim et al. 2010).

Table 1. Distribution of rtxA loci among bacterial species1 . Human pathogens

Aquatic pathogens

Nematode symbionts/Insect pathogens

Other

Aeromonas hydrophila2 Proteus mirabilus Vibrio cholerae Vibrio vulnificus

Aeromonas hydrophila2 (fish) Moritella viscosa (fish) Vibrio anguillarum (fish) Vibrio ordalli (fish) Vibrio vulnificus BT2 (eels)

Photorhabdus asymbiotica Photorhabdus luminescens Photorhabdus temperata Xenorhabdus bovenii Xenorhabdus doucetiae Xenorhabdus nematophila Xenorhabdus poinarii

Vibrio caribbenthicus (coral) Vibrio nigripulchritudo (shrimp) Vibrio splendidus (seawater)

1

Alphabetical list limited to MARTX toxins previously annotated (Satchell 2011, 2015) or newly identified by TBLASTN search of 1544 representative or 3976 complete genomes deposited at National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) using V. cholerae MARTX toxin sequence without effector domains as query. 2 Although early genomics supported that human clinical isolates lack the rtxA gene (Grim et al. 2014), a TBLASTN search of deposited draft genomes identified rtxA in at least 12 human clinical samples (unpublished) and four isolates from fish (Pang et al. 2015)

Gavin and Satchell

3

Figure 1. Expression at the rtx gene locus leads to MARTX toxin secretion. Expression of the rtxBDE operon and unlinked tolC genes results in formation of a T1SS ‘chunnel’ spanning the bacterial inner and outer membranes. The MARTX toxin, product of the rtxA (or rtxA1) gene, is secreted from bacterium to extracellular space without a periplasmic intermediate. Secretion requires calcium (Ca2+ ), which likely participates in the folding of the RTX-like tandem aa repeats present in the MARTX C-terminus.

MARTX TOXIN DELIVERY OF EFFECTOR DOMAINS Regions of the toxin necessary for effector domain translocation MARTX toxins, once expressed, are unmistakably massive proteins with deduced primary sequences from 3500–5300 amino acid (aa). The toxins are comprised of four distinct regions: the N-terminal arm, the effector domains, the cysteine protease domain (CPD) and the C-terminal arm (Fig. 2). They are classified as members of the broader RTX protein family by virtue of characteristic RTX tandem aa repeat sequences in the C-terminus. However, it is the presence of the N-terminal arm, the CPD and the C-terminal arm—similar in size and sequence across all MARTX toxins—that together define the core structures essential to be designated within the MARTX toxin subfamily (Fig. 2) (Satchell 2007). The C-terminal region of a MARTX toxin is dominated by 15 tandem copies of a ‘C’ repeat sequence. The first nine aa of each C-repeat match classical glycine and aspartate-rich RTX sequences known to form a beta-barrel with calcium ions at the turn at the end of each beta strand. Each repeat also contains nine additional aa. Together these two portions comprise the larger 18-aa non-canonical RTX motif of the C-repeat sequence (Lin et al. 1999; Satchell 2011).

Downstream of the RTX repeats is a small region of less than 200 aa. A stop codon just 13 aa from the end of the toxin entirely disrupts the toxin function (Dolores and Satchell 2013). This result is consistent with other RTX-family toxins where the C-terminus carries information for secretion by the designated T1SS (Linhartova et al. 2010). Interestingly, elimination of upstream C-terminal repeats, even when the putative C-terminal secretion signal is retained, abolishes toxin secretion from the bacterium. Additionally, MARTX toxin secretion is calcium dependent (Kim, Gavin and Satchell 2015). Based on these data, it is proposed that MARTX C-repeats, similar to RTX calcium binding repeats, remain unstructured in the relatively low-calcium bacterial cytoplasm and fold only upon reaching the calciumrich extracellular environment (Kim, Gavin and Satchell 2015) (Fig. 2). This transition might drive MARTX toxin export from Cto N- terminus by a calcium-dependent molecular ratchet mechanism, as has been shown for other RTX toxins (Chenal et al. 2009; Blenner et al. 2010; Thomas et al. 2014; Sotomayor-Perez, Ladant and Chenal 2015). Distinguishing MARTX toxins from all other RTX-family proteins is the presence of conserved repeat sequences at the N-terminus of the protein (Fig. 2). The ∼1960 aa N-terminus is dominated by a total of 52 aa repeats sequences that fall into two groups: 14 copies of the 19-aa ‘A’ repeats and 38 copies of the 20aa ‘B’ repeats. While still glycine-rich, these are not classified as

4

FEMS Pathogens and Disease, 2015, Vol. 73, No. 9

Figure 2. MARTX toxins deliver effector domains to target cell cytosol. MARTX toxins exhibit modular organization with characteristic N- and C-terminal repeat regions flanking a central core consisting of the CPD and arrayed effector domains. Following toxin expression and secretion from a bacterium, MARTX repeat regions facilitate pore formation in target cell membranes. Once translocated through the repeat region pore, the CPD is activated for autoproteolysis by inositol hexakisphosphate (InsP6). Cleavage at susceptible leucine converts the MARTX effector domain cargo to finite bacterial effectors in the eukaryotic cytosol.

RTX repeats and—lacking aspartate residues in key positions— are not thought to bind calcium ions. Nonetheless, each is predicted to form a beta-barrel structure consistent with a function in pore formation (Satchell 2011, 2015). Indeed, the N-terminus is required for pore formation in host target cell membranes (Kim, Gavin and Satchell 2015). The MARTX toxin repeat region pore can in some cases lead to necrotic cytotoxicity (Kim et al. 2013; Kim, Gavin and Satchell 2015). However, some MARTX toxins induce cytopathic effects in the absence of cell lysis (Fullner and Mekalanos 2000; Li, Rock and Nelson 2008). While the pore-forming repeat regions of the MARTX toxins from V. vulnificus and V. cholerae, for example, are identical in size and 93% identical in aa sequence, only the toxin from V. vulnificus induces pore-dependent cell lysis (Dolores et al. 2015; Kim, Gavin and Satchell 2015). Though thus far unexplained, functional variances between MARTX toxins of different bacterial species may reflect differences in pore structure conferred by subtle alterations in the otherwise highly conserved repeat regions. Alternatively, V. vulnificus may simply produce much more toxin due to gene induction in the presence of cells (Kim et al. 2008; Lee et al. 2008). Whether or not the pore formed by the repeat regions causes cell lysis, the pore is for all MARTX toxins essential for translocation of the central toxin region of the toxin to the cytosol (Dolores et al. 2015; Kim, Gavin and Satchell 2015). Even in cases where the MARTX toxin acts as both cytolysin and effector delivery platform, the cytopathic activity of the toxin long

precedes pore-induced necrosis (Kim, Gavin and Satchell 2015). This suggests that the translocation of effector domains is the primary function of the MARTX toxin pore. Very little is known about the mechanistic process of effector domain translocation. The MARTX toxin pore has been estimated at 1.63 nm (Kim et al. 2008). The narrow diameter implies that the protein’s central region must unfold for its transfer across the eukaryotic membrane. Indeed, thermodynamic studies of domains within the central core suggest that it is their natural structural instability that allows for their translocation (Prochazkova et al. 2009; Kudryashova et al. 2014). Moreover, it has been suggested that the drive toward a more stable structural state may even provide energetic impetus for effector domain translocation from extracellular space to cytosol (Kudryashova et al. 2014; Sotomayor-Perez, Ladant and Chenal 2015). Within the translocated central portion of all MARTX toxins is the CPD that confers toxin autoprocessing activity, a core element that defines MARTX toxin (Fig. 2) (Boardman, Meehan and Satchell 2007; Satchell 2007; Sheahan, Cordero and Satchell 2007). After its translocation, the CPD is activated in the cell cytosol by the eukaryotic-specific small molecule inositol hexakisphosphate. Upon activation, the CPD proteolytically cleaves the MARTX toxin at susceptible leucine residues to release discrete portions of the MARTX toxin into the target cell cytosol (Prochazkova and Satchell 2008; Prochazkova et al. 2009; Shen et al. 2009; Egerer and Satchell 2010). This CPD autocleavage is

Gavin and Satchell

essential for cytopathic activity of the toxin to be observed, likely due to a role for subcellular localization for maximum effector function (Sheahan, Cordero and Satchell 2007; Dolores et al. 2015). CPD activity thus converts the large MARTX holotoxin into finite, bona fide bacterial effectors in a eukaryotic-specific context. A unique feature of MARTX toxins is that while the repeat regions and CPD are highly conserved in size and sequence across a multitude of Gram-negative bacteria, the delivered effector cargo content can vary quite dramatically. Thus, while MARTX toxins are classified as large single polypeptide toxins, they function more directly as effector delivery platforms wherein the effector domain cargo is part of the same polypeptide but can vary in size and content across bacterial species and strains.

MARTX toxin effector domains Any singular MARTX toxin boasts a repertoire of one to five of ten defined effectors, organized in a characteristic modular fashion (Fig. 2) (Satchell 2011, 2015). Once translocated and autoprocessed by the CPD, MARTX effectors act on a variety of eukaryotic cellular targets (Table 2), generally to modulate key cellular pathways regulating cellular energy balance, cytoskeletal integrity and survival/proliferation. Eukaryotic proteins are in some cases targeted for direct molecular modification by MARTX effector domains. A key example is demonstrated by the proteolytic activity of the Ras/Rap1-specific peptidase (RRSP) that site-specifically cleaves the Ras and Rap1 GTPases to control activation of mitogen

5

activated kinases and thereby control cell survival. This highly potent toxic activity rapidly and completely abolishes proliferation of cells treated either with just the toxin domain or with bacteria expressing a MARTX toxin (Antic, Biancucci and Satchell 2014; Antic et al. 2015). Another example of direct modification is the MARTX-dependent catalysis of G-actin crosslinking by the actin crosslinking domain (ACD) (Sheahan, Cordero and Satchell 2004; Kudryashov et al. 2008b; Suarez et al. 2012). While the crosslinking of actin in the presence of bacteria is 100% efficient, resulting in crosslinking of all actin within 90 minutes (Fullner and Mekalanos 2000; Cordero et al. 2006; Dolores et al. 2015), dramatic cell rounding due to the ACD can occur ahead of completion of crosslinking by the binding of crosslinked actin to formin on the growing end of actin filaments (Heisler et al. 2015). In other cases, MARTX effector activity manipulates cell signaling. The ExoY-like adenylate cyclase effector converts ATP to cyclic AMP (Ziolo et al. 2014), presumably affecting cell biological processes controlled by cytosolic levels of cyclic AMP. The DUF1 effector domain (referred to also as RtxA1-D2) has recently been shown to bind and upregulate host protein prohibitin on the cytoplasmic membrane via the ERK pathway, putatively increasing MARTX-mediated cytotoxicity (Kim et al. 2015). Signaling through small GTPases other than Ras/Rap1 is also manipulated. The Rho Inactivation domain (RID) inactivates GTPases RhoA and CDC42 (Sheahan and Satchell 2007; Dolores et al. 2015). The alpha/beta hydrolase (ABH) effector domain, a phospholipase A1, reduces phosphatidylinositol-3-phosphate levels resulting in inhibition of endocytic trafficking and autophagy while also

Table 2. MARTX toxin effector domains. Abbreviationa

Namea

Detailed description

Functionally characterized in:

ABH

Alpha–beta hydrolase

Phosphatidylinositol-3-phosphate specific lipase Activation of small GTPase CDC42

V. cholerae (Agarwal et al. 2015c; Dolores et al. 2015)

ACD

Actin crosslinking domain

Formation of isopeptide linkage between actin monomers resulting in cell rounding, inhibition of phagocytosis, and blocking actin filament elongation

V. cholerae (Fullner and Mekalanos 2000; Sheahan et al. 2004; Kudryashov et al. 2008a; Dolores et al. 2015; Heisler et al. 2015); V. vulnificus (Kwak et al. 2011) A. hydrophila (Suarez et al. 2012)

DmX

Domain X (formerly PasyHD1)

Domain of no known function

Not characterized in any species

DUF1

Domain of unknown function in the first position

Binds prohibitin, increases prohibitin expression on cell surface

V. vulnificus (Kim et al. 2015a)

ExoY

Adenylate cyclase

Conversion of ATP to cyclic AMP similar to Pseudomonas aeruginosa ExoY

V. vulnificus (Ziolo et al. 2014)

MCF

Makes caterpillars floppy-like

Cellular-induced cysteine autoprotease that induces the intrinsic pathway of apoptosis

V. vulnificus (Agarwal et al. 2015a; Agarwal et al. 2015b) A. hydrophila (Agarwal et al. 2015a)

PasyHD2

P. asymbiotica homology domain 2

Domain of no known function with a predicted lipid raft localization motif (French et al. 2009)

Not characterized in any species

RID

Rho-inactivation domain

Inactivation of small GTPases Rho and CDC42 resulting in cellular actin depolymerization

V. cholerae (Sheahan and Satchell 2007; Ahrens et al. 2013; Dolores et al. 2015)

RRSP

Ras/Rap1 specific peptidase (formerly DUF5)

Site-specific processing of small GTPases Ras and Rap resulting in loss of phospho-ERK, cell rounding, and inhibition of cell proliferation

V. vulnificus (Antic et al. 2014; Antic et al. 2015) A. hydrophila (Antic et al. 2015)

VIP2

VIP2-like protein

Putative ADP-ribosylating activity with homology to Bacillus cereus VIP2 and other VIP2-like toxins (Stiles et al. 2014)

Not characterized in any species

a

Names, presence and layout in MARTX toxins of various bacterial strains and species detailed in previous reviews (Satchell 2011, 2015).

6

FEMS Pathogens and Disease, 2015, Vol. 73, No. 9

indirectly stimulating CDC42 activity (Agarwal et al. 2015b; Dolores et al. 2015). Finally, effector domains have been found to trigger intrinsic apoptotic cell death (Lee, Choi and Kim 2008; Murciano, Hor and Amaro 2015), in particular through action of the Makes caterpillars floppy-like (MCF) effector domain (Agarwal et al. 2015a,c). In addition to these effectors where the function is partially or fully characterized, the function of at least three other uncharacterized effectors—Domain X (PasyHD1 or DmX), Photorhabdus asymbiotic homology domain 2 (PasyHD2), and the vegetative insecticidal protein 2 homology domain (VIP2)—remain to be investigated. Sequence analysis reveals little about the function of DmX and PasyHD2, but similarities between the VIP2 domain and other VIP2-like toxins predict this region could have ADP-ribosylation activity (Dolores et al. 2015; Satchell 2015). However, the activities of this effector, and particularly the two effectors lacking homology to any known protein, remain a mystery until experimentally explored.

Novel effector arrangement impact on cell biology The MARTX toxins are different from most other toxins as they are evidently amenable to delivery of effector repertoires variable in size, arrangement and content. In fact, a wide spectrum of MARTX effector compositions is observed across bacterial species, and even among strains of the same bacterial species (Roig, Gonzalez-Candelas and Amaro 2011; Satchell 2011, 2015; Ziolo et al. 2014). This breadth and variety of the effector repertoire has arisen at least in part by inter- and intra-species homologous recombination, as further supported by homology between select MARTX and non-MARTX effectors. Further, while some species seem to have constant effector arrangements, other species show dramatic diversity strain to strain (Kwak, Jeong and Satchell 2011; Roig, Gonzalez-Candelas and Amaro 2011; Dolores and Satchell 2013). In most cases, the toxin genes acquire new effector domain sequences by homologous recombination to swap effector content (Kwak, Jeong and Satchell 2011; Ziolo et al. 2014). In other cases, effector domains sequences are inserted or recombine imperfectly, such that evidence of the previous DNA arrangement remains (Satchell 2015). Since many of the toxin effectors can manipulate cell biological processes, it has been intriguing to consider how different arrangements can affect the function of the holotoxin. MARTX toxins are unique among toxins as they can easily exchange their catalytic function(s) and in this way they are more similar to T3SS and T4SS where horizontal acquisition of an effector gene can alter pathogenesis. For example, in V. vulnificus, many strains have MARTX toxins with five effector domains, but many others have lost the RRSP effector domain that is responsible for processing of Ras (Kwak, Jeong and Satchell 2011; Roig, Gonzalez-Candelas and Amaro 2011; Antic et al. 2015). The loss of this single effector domain alone accounts for a 10–50 fold decreased virulence in strains lacking RRSP (Kwak, Jeong and Satchell 2011). Similarly, the biotype 3 strains of V. vulnificus acquired adenylate cyclase activity by exchanging in the ExoY domain, while other V. vulnificus strains acquired actin crosslinking activity by exchanging in the ACD (Kwak, Jeong and Satchell 2011; Roig, Gonzalez-Candelas and Amaro 2011; Ziolo et al. 2014). These changes very likely will be shown in the future to contribute to the differing virulence of these strains (Kwak, Jeong and Satchell 2011). While much focus in research has been directed at understanding the activity of individual effector domains, there is also potential for crosstalk to occur between effector activities, par-

ticularly those that control cell signaling. An example is seen in the V. cholerae MARTX toxin. Here, it has been shown that the RID contributes to the inactivation of CDC42. However, the copresent ABH effector domain counteracts this inactivation by stimulating the activity of CDC42 (Dolores et al. 2015). The net effect in cells treated with a holotoxin that has both domains is that CDC42 is barely changed (Sheahan and Satchell 2007). Thus, while knowledge of the function of independent effector domains is valuable, the context of the effector domains within the holotoxin also contributes to the overall cell biological changes induced by the toxin.

MARTX toxins in heterologous protein transfer As demonstrated by the variety of naturally occurring effector repertoires, MARTX toxins are capable of delivering cargo that ranges considerably in identity and size. This suggested that a MARTX toxin should also be capable of translocation of heterologous proteins. Beta-lactamase (Bla) has served as a useful tool for study of protein transfer for many bacterial translocation systems because its presence in the eukaryotic cytosol can be monitored via enzymatic cleavage of fluorogenic and colorimetric lactam substrates (Pechous and Goldman 2015). When a bla DNA sequence is cloned into rtxA in place of the native rtxA effector domain sequences, Bla activity is detectable in the supernatant of bacterial cultures. Thus, the heterologous MARTX proteins can be expressed and are secreted from bacteria in the context of the MARTX toxin (Fig. 3) (Dolores et al. 2015; Kim, Gavin and Satchell 2015). Moreover, Bla activity is detected after translocation into the target cell cytosol, indicating that the MARTX toxin is capable of transporting this heterologous protein across the eukaryotic plasma membrane (Fig. 3) (Agarwal et al. 2015c; Dolores et al. 2015; Kim, Gavin and Satchell 2015). Release of Bla from the MARTX holotoxin by CPD autoprocessing in the case of the V. cholerae MARTX toxin is required for Bla to cleave beta-lactam cytosolic substrate (Dolores et al. 2015). In contrast, CPD activity is not required for cytosolic substrate cleavage when delivered by V. vulnificus MARTX, although CPD autoprocessing does increase the level of substrate hydrolysis detected (Kim, Gavin and Satchell 2015). These inter-species variances may reflect differences in overall level of toxin delivered by the two organisms, as baseline levels of Bla activity are higher when delivered by V. vulnificus as opposed to the V. cholerae MARTX toxin. Importantly, the collective results from heterologous protein transfer studies in multiple MARTX-expressing organisms establish that specific effector domains are not required for toxin secretion, for eukaryotic cell targeting, or for translocation. Further, any cytopathic effect induced by a single MARTX effector domain can subsequently be easily monitored as a gain of function by returning a domain back to the toxin in the absence of the other domains (Agarwal et al. 2015c; Dolores et al. 2015). This gain-of-function approach has further demonstrated that the central portion of the toxins indeed encodes for effector domain cargo delivered to the eukaryotic cytosol by the N- and Cterminal toxin regions. Moreover, cytosolic translocation of the heterologous Bla highlights the robustness of the MARTX toxin as a platform for effector delivery.

CONCLUSIONS / FUTURE OUTLOOK A major stumbling block in the study of MARTX toxin is that, likely due to a combination of traits including hefty size,

Gavin and Satchell

7

Figure 3. MARTX toxins are capable of heterologous protein transfer. Where a bla gene is cloned in place of rtxA effector domains, the heterologous protein is expressed, secreted and delivered to target cells in the context of the MARTX toxin. Bla activity can be detected by the cleavage of fluorogenic and colorimetric lactam substrates, such as CCF2 (Pechous and Goldman 2015). Heterologous protein transfer demonstrates the robustness of the MARTX toxin as a platform for effector delivery.

inherent structural flexibility and generally low expression, no MARTX toxin has been successfully purified in the 16 years since its discovery. Thus, understanding its mechanism of action has in many ways differed from traditional toxin biology approaches. Since the toxins are modular in nature, ectopic expression, intoxication, and microinjection have been quite productive in studying distinct domains in isolation. In addition, as bacteria that produce MARTX toxins are often genetically tractable, the employment of bacterial genetics has facilitated study via manipulation of the rtxA gene on bacterial chromosomes by deletion, exchange and insertion of effector domains. This has been successfully employed so far primarily in V. cholerae. Strains that produce one, two, or three effector domains have been engineered to assign independent function to each of the effectors and to study their additive effects (Sheahan and Satchell 2007; Dolores et al. 2015). Most recently, a strategy of adding a domain back into a toxin lacking effector domains has been utilized to map the V. vulnificus MARTX toxin induction of apoptosis to a single effector domain (Agarwal et al. 2015c). Altogether, this research has resulted in an ever-increasing understanding of MARTX toxin composition and function. In the future, through increasing deployment of genetic and cell

biological tools, myriad remaining interesting questions can be explored. This review highlights the MARTX toxin as a robust effector delivery platform, but it is worth noting that this role of the toxin is not yet universally accepted. Further support for this novel toxin paradigm requires additional research into the functional consequences of: (1) slight variations in otherwise highly conserved pore-forming repeat regions; (2) diverse effector repertoires; and (3) the presence of MARTX toxins in other rtx-containing organisms. Ongoing efforts by groups investigating toxin dynamics and pathogenesis promise complimentary gains in knowledge on the dual paths of investigation. Moreover, the presence of rtx loci in numerous species has been reported through bioinformatics, yet to date, only four have been investigate experimentally. Research into these other systems is likely to illuminate many exciting avenues of exploration in these organisms.

ACKNOWLEDGEMENTS HEG is funded by a Ruth L. Kirschstein National Research Service Award from the National Institute of General Medical Sciences

8

FEMS Pathogens and Disease, 2015, Vol. 73, No. 9

(5T32GM008061). KJS is the recipient of grants from the National Institute of Allergy and Infectious Diseases (R01AI092825 and R01AI098369). Conflict of interest. KJS holds patents associated with CPD autoprocessing application to recombinant protein autoprocessing and has filed a provisional patent related to RRSP.

REFERENCES Agarwal S, Agarwal S, Biancucci M, et al. Induced autoprocessing of the cytopathic Makes caterpillars floppy-like effector domain of the Vibrio vulnificus MARTX toxin. Cell Microbiol 2015a;17:1494–509. Agarwal S, Kim H, Chan R, et al. Autophagy and endosomal trafficking inhibition by Vibrio cholerae MARTX toxin phosphatidylinositol-3-phosphate specific phospholipase A1 activity. Nat Commun 2015b;6:8745. Agarwal S, Zhu Y, Gius D, et al. Makes Caterpillars Floppy (MCFVv )-like domain of Vibrio vulnificus induces mitochondrial-mediated apoptosis. Infect Immun 2015c; 83:4392–403. Ahrens S, Geissler B, Satchell KJ. Identification of a His-AspCys catalytic triad essential for function of the Rho inactivation domain (RID) of Vibrio cholerae MARTX toxin. J Biol Chem 2013;288:1397–408. Alouf JE, Popoff MR. The Comprehensive Sourcebook of Bacterial Protein Toxins. San Diego, CA: Academic Press, 2006, 1072. Amaro C, Sanjuan E, Fouz B, et al. The fish pathogen Vibrio vulnificus biotype 2: epidemiology, phylogeny, and virulence factors involved in warm-water vibriosis. Microbiol Spectr 2015;3:VE0005-2014. Andersen C, Hughes C, Koronakis V. Chunnel vision. Export and efflux through bacterial channel-tunnels. EMBO Rep 2000;1:313–8. Antic I, Biancucci M, Satchell KJ. Cytotoxicity of the Vibrio vulnificus MARTX toxin effector DUF5 is linked to the C2A subdomain. Proteins 2014;82:2643–56. Antic I, Biancucci M, Zhu Y, et al. Site-specific processing of Ras and Rap1 Switch I by a MARTX toxin effector domain. Nat Commun 2015;6:7396. Basar T, Havlicek V, Bezouskova S, et al. Acylation of lysine 983 is sufficient for toxin activity of Bordetella pertussis adenylate cyclase. Substitutions of alanine 140 modulate acylation site selectivity of the toxin acyltransferase CyaC. J Biol Chem 2001;276:348–54. Bielaszewska M, Aldick T, Bauwens A, et al. Hemolysin of enterohemorrhagic Escherichia coli: structure, transport, biological activity and putative role in virulence. Int J Med Microbiol 2014;304:521–9. Bina JE, Mekalanos JJ. Vibrio cholerae tolC is required for bile resistance and colonization. Infect Immun 2001;69:4681–5. Blenner MA, Shur O, Szilvay GR, et al. Calcium-induced folding of a beta roll motif requires C-terminal entropic stabilization. J Mol Biol 2010;400:244–56. Boardman BK, Meehan BM, Satchell KJF. Growth phase regulation of Vibrio cholerae RTX toxin export. J Bacteriol 2007;189: 1827–35. Boardman BK, Satchell KJ. Vibrio cholerae strains with mutations in an atypical type I secretion system accumulate RTX toxin intracellularly. J Bacteriol 2004;186:8137–43. Chenal A, Guijarro JI, Raynal B, et al. RTX calcium binding motifs are intrinsically disordered in the absence of

calcium: implication for protein secretion. J Biol Chem 2009;284:1781–9. Cheong TG, Chan M, Kurunathan S, et al. Construction and characterization of rtxA and rtxC mutants of auxotrophic O139 Vibrio cholerae. Microb Pathogenesis 2010;48:85–90. Cordero CL, Kudryashov DS, Reisler E, et al. The actin crosslinking domain of the Vibrio cholerae RTX toxin directly catalyzes the covalent cross-linking of actin. J Biol Chem 2006;281:32366–74. Dolores J, Satchell KJ. Analysis of Vibrio cholerae genome sequences reveals unique rtxA variants in environmental strains and an rtxA-null mutation in recent altered El Tor isolates. MBio 2013;4:e00624. Dolores JS, Agarwal S, Egerer M, et al. Vibrio cholerae MARTX toxin heterologous translocation of beta-lactamase and roles of individual effector domains on cytoskeleton dynamics. Mol Microbiol 2015;95:590–604. Egerer M, Satchell KJ. Inositol hexakisphosphate-induced autoprocessing of large bacterial protein toxins. PLoS Pathog 2010;6:e1000942. Fan JJ, Shao CP, Ho YC, et al. Isolation and characterization of a Vibrio vulnificus mutant deficient in both extracellular metalloprotease and cytolysin. Infect Immun 2001;69:5943–8. French CT, Panina EM, Yeh SH, et al. The Bordetella type III secretion system effector BteA contains a conserved N-terminal motif that guides bacterial virulence factors to lipid rafts. Cell Microbiol 2009;11:1735–49. Fullner KJ, Mekalanos JJ. In vivo covalent cross-linking of cellular actin by the Vibrio cholerae RTX toxin. EMBO J 2000;19:5315–23. Gray MC, Donato GM, Jones FR, et al. Newly secreted adenylate cyclase toxin is responsible for intoxication of target cells by Bordetella pertussis. Mol Microbiol 2004;53:1709–19. Grim CJ, Kozlova EV, Ponnusamy D, et al. Functional genomic characterization of virulence factors from necrotizing fasciitis-causing strains of Aeromonas hydrophila. Appl Environ Microb 2014;80:4162–83. Gulig PA, Tucker MS, Thiaville PC, et al. USER friendly cloning coupled with chitin-based natural transformation enables rapid mutagenesis of Vibrio vulnificus. Appl Environ Microb 2009;75:4936–49. Heisler DB, Kudryashova E, Grinevich DO, et al. ACD toxinproduced actin oligomers poison formin-controlled actin polymerization. Science 2015;349:535–9. Hozbor D, Rodriguez ME, Fernandez J, et al. Release of outer membrane vesicles from Bordetella pertussis. Curr Microbiol 1999;38:273–8. Jeong HG, Satchell KJ. Additive function of Vibrio vulnificus MARTXVv and VvhA cytolysins promotes rapid growth and epithelial tissue necrosis during intestinal infection. PLoS Pathog 2012;8:e1002581. Kim BA, Lim JY, Rhee JH, et al. Characterization of Prohibitin 1 as a Host Partner of Vibrio vulnificus RtxA1 Toxin. J Infect Dis 2015. Published online 2015 Jul 1, DOI: 10.1093/infdis/jiv362. Kim BS, Gavin HE, Satchell KJ. Distinct roles of the repeatcontaining regions and effector domains of the Vibrio vulnificus multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin. MBio 2015;6:e00324–15. Kim YR, Kim BU, Kim SY, et al. Outer membrane vesicles of Vibrio vulnificus deliver cytolysin-hemolysin VvhA into epithelial cells to induce cytotoxicity. Biochem Bioph Res Co 2010;399:607–12. Kim YR, Lee SE, Kang IC, et al. A bacterial RTX toxin causes programmed necrotic cell death through calcium-mediated mitochondrial dysfunction. J Infect Dis 2013;207:1406–15.

Gavin and Satchell

Kim YR, Lee SE, Kook H, et al. Vibrio vulnificus RTX toxin kills host cells only after contact of the bacteria with host cells. Cell Microbiol 2008;10:848–62. Kudryashov DS, Cordero CL, Reisler E, et al. Characterization of the enzymatic activity of the actin cross-linking domain from the Vibrio cholerae MARTX Vc toxin. J Biol Chem 2008a;283:445–52. Kudryashov DS, Durer ZA, Ytterberg AJ, et al. Connecting actin monomers by iso-peptide bond is a toxicity mechanism of the Vibrio cholerae MARTX toxin. P Natl Acad Sci USA 2008b;105:18537–42. Kudryashova E, Heisler D, Zywiec A, et al. Thermodynamic properties of the effector domains of MARTX toxins suggest their unfolding for translocation across the host membrane. Mol Microbiol 2014;92:1056–71. Kwak JS, Jeong HG, Satchell KJ. Vibrio vulnificus rtxA1 gene recombination generates toxin variants with altered potency during intestinal infection. P Natl Acad Sci USA 2011;108:1645–50. Lee BC, Choi SH, Kim TS. Vibrio vulnificus RTX toxin plays an important role in the apoptotic death of human intestinal epithelial cells exposed to Vibrio vulnificus. Microbes Infect 2008;10:1504–13. Lee BC, Lee JH, Kim MW, et al. Vibrio vulnificus rtxE is important for virulence, and its expression is induced by exposure to host cells. Infect Immun 2008;76:1509–17. Lee CT, Pajuelo D, Llorens A, et al. MARTX of Vibrio vulnificus biotype 2 is a virulence and survival factor. Environ Microbiol 2013;15:419–32. Lee JH, Kim MW, Kim BS, et al. Identification and characterization of the Vibrio vulnificus rtxA essential for cytotoxicity in vitro and virulence in mice. J Microbiol 2007;45:146–52. Li L, Mou X, Nelson DR. HlyU is a positive regulator of hemolysin expression in Vibrio anguillarum. J Bacteriol 2011;193:4779–89. Li L, Rock JL, Nelson DR. Identification and characterization of a repeat-in-toxin gene cluster in Vibrio anguillarum. Infect Immun 2008;76:2620–32. Lin W, Fullner KJ, Clayton R, et al. Identification of a Vibrio cholerae RTX toxin gene cluster that is tightly linked to the cholera toxin prophage. P Natl Acad Sci USA 1999;96:1071–6. Linhartova I, Bumba L, Masin J, et al. RTX proteins: a highly diverse family secreted by a common mechanism. FEMS Microbiol Rev 2010;34:1076–112. Liu M, Alice AF, Naka H, et al. The HlyU protein is a positive regulator of rtxA1, a gene responsible for cytotoxicity and virulence in the human pathogen Vibrio vulnificus. Infect Immun 2007;75:3282–9. Liu M, Naka H, Crosa JH. HlyU acts as an H-NS antirepressor in the regulation of the RTX toxin gene essential for the virulence of the human pathogen Vibrio vulnificus CMCP6. Mol Microbiol 2009;72:491–505. Lo HR, Lin JH, Chen YH, et al. RTX toxin enhances the Survival of Vibrio vulnificus during infection by protecting the organism from phagocytosis. J Infect Dis 2011;203:1866–74. Ma AT, Mekalanos JJ. In vivo actin cross-linking induced by Vibrio cholerae type VI secretion system is associated with intestinal inflammation. P Natl Acad Sci USA 2010;107:4365–70. Moest TP, Meresse S. Salmonella T3SSs: successful mission of the secret(ion) agents. Curr Opin Microbiol 2013;16:38–44. Murciano C, Hor LI, Amaro C. Host-pathogen interactions in Vibrio vulnificus: responses of monocytes and vascular endothelial cells to live bacteria. Future Microbiol 2015;10:471–87. Olivier V, Queen J, Satchell KJ. Successful small intestine colonization of adult mice by Vibrio cholerae requires ketamine anesthesia and accessory toxins. PloS One 2009;4:e7352.

9

Pang M, Jiang J, Xie X, et al. Novel insights into the pathogenicity of epidemic Aeromonas hydrophila ST251 clones from comparative genomics. Sci Rep 2015;5:9833. Pechous RD, Goldman WE. Illuminating targets of bacterial secretion. PLoS Pathog 2015;11:e1004981. Prochazkova K, Satchell KJ. Structure-function analysis of inositol hexakisphosphate-induced autoprocessing of the Vibrio cholerae multifunctional autoprocessing RTX toxin. J Biol Chem 2008;283:23656–64. Prochazkova K, Shuvalova LA, Minasov G, et al. Structural and molecular mechanism for autoprocessing of MARTX toxin of Vibrio cholerae at multiple sites. J Biol Chem 2009;284:26557–68. Roig FJ, Gonzalez-Candelas F, Amaro C. Domain organization and evolution of multifunctional autoprocessing repeats-intoxin (MARTX) toxin in Vibrio vulnificus. Appl Environ Microb 2011;77:657–68. Satchell KJ. MARTX, multifunctional autoprocessing repeats-intoxin toxins. Infect Immun 2007;75:5079–84. Satchell KJ. Structure and function of MARTX toxins and other large repetitive RTX proteins. Annu Rev Microbiol 2011;65: 71–90. Satchell KJ. Multifunctional-autoprocessing repeats-intoxin (MARTX) toxins of Vibrios. Microbiol Spectr 2015;3. VE-0002-2014. Sheahan KL, Cordero CL, Satchell KJ. Identification of a domain within the multifunctional Vibrio cholerae RTX toxin that covalently cross-links actin. P Natl Acad Sci USA 2004;101: 9798–803. Sheahan KL, Cordero CL, Satchell KJ. Autoprocessing of the Vibrio cholerae RTX toxin by the cysteine protease domain. EMBO J 2007;26:2552–61. Sheahan KL, Satchell KJ. Inactivation of small Rho GTPases by the multifunctional RTX toxin from Vibrio cholerae. Cell Microbiol 2007;9:1324–35. Shen A, Lupardus PJ, Albrow VE, et al. Mechanistic and structural insights into the proteolytic activation of Vibrio cholerae MARTX toxin. Nat Chem Biol 2009;5:469–78. So EC, Mattheis C, Tate EW, et al. Creating a customized intracellular niche: subversion of host cell signaling by Legionella type IV secretion system effectors. Can J Microbiol 2015;61:617–35. Sotomayor-Perez AC, Ladant D, Chenal A. Disorder-to-order transition in the CyaA toxin RTX domain: implications for toxin secretion. Toxins 2015;7:1–20. Stanley P, Koronakis V, Hughes C. Acylation of Escherichia coli hemolysin: a unique protein lipidation mechanism underlying toxin function. Microbiol Mol Biol R 1998;62:309–33. Stiles BG, Pradhan K, Fleming JM, et al. Clostridium and bacillus binary enterotoxins: bad for the bowels, and eukaryotic being. Toxins 2014;6:2626–56. Suarez G, Khajanchi BK, Sierra JC, et al. Actin cross-linking domain of Aeromonas hydrophila repeat in toxin A (RtxA) induces host cell rounding and apoptosis. Gene 2012;506:369–76. Thomas S, Bakkes PJ, Smits SH, et al. Equilibrium folding of pro-HlyA from Escherichia coli reveals a stable calcium ion dependent folding intermediate. Biochim Biophys Acta 2014;1844:1500–10. Wang H, Ayala JC, Benitez JA, et al. RNA-seq analysis identifies new genes regulated by the histone-like nucleoid structuring protein (H-NS) affecting Vibrio cholerae virulence, stress response and chemotaxis. PLoS One 2015;10:e0118295. Ziolo KJ, Jeong HG, Kwak JS, et al. Vibrio vulnificus Biotype 3 multifunctional autoprocessing RTX toxin is an adenylate cyclase toxin essential for virulence in mice. Infect Immun 2014;82:2148–57.