Many toxins have been characterized that disrupt the eukaryotic actin cytoskeleton. These include fungal and marine small molecule toxins that bind to actin to prevent actin polymerization [1,2]. Other bacterial proteins toxins and effectors can alter the normal regulation of cytoskeletal assembly, by directly or indirectly modifying the activity of small Rho family GTPases [3]. Finally, toxins have been identified that covalently modify actin itself, for example by the addition of an ADP-ribosyl group that then prevents its incorporation into F-actin [4,5].

Studies over the past ten years have been directed toward characterizing a distinct mechanism to disrupt actin filament assembly by covalently crosslinking actin monomers. The crosslinks can be introduced repetitively to form long chains of dysfunctional actin that cannot be incorporated into F-actin filaments, resulting in irreversible destruction of the host cell cytoskeleton [6].

An actin crosslinking toxin was first discovered in current pandemic strains of the human diarrheal pathogen Vibrio cholerae [6]. When added to mammalian cells in culture, V. cholerae was shown to secrete a factor that induced rapid cell rounding, ultimately resulting in loss of all polymerized actin [6,7]. When actin in V. cholerae-treated cells was monitored by western blotting as a potential target for covalent modification, it was noted that all the monomeric actin in the cell disappeared within 180 min of addition of bacteria and actin was detected as higher weight molecular forms. The change in the electrophoretic mobility of actin corresponded to the formation of dimers, trimers, and higher order oligomers, revealing that actin in V. cholerae-treated cells had been crosslinked into chains [6].

This actin crosslinking activity depended upon the gene rtxA, the largest gene of the V. cholerae genome [6,7] and actin crosslinking activity was found to be widely present among both clinical and environmental isolates of V. cholerae [8,9,10,11]. The protein encoded by rtxA, now referred to as the Multifunctional, Autoprocessing RTX toxin of V. cholerae (MARTX_Vc), is the founding member of a new family of toxins [12]. The MARTX toxins are a grouping of toxins that are all large in size (>350 kDa) and are secreted from the bacteria by dedicated Type I secretion systems. Conserved features of all MARTX toxins include N- and C-terminal glycine rich repeats and an autoprocessing cysteine protease. These conserved structural elements are proposed to function in toxin translocation to deliver one to five effector domains found in the central region of the toxins to the eukaryotic cell cytosol [12].

To identify the specific effector of MARTX_Vc responsible for actin crosslinking, subfragments of V. cholerae rtxA gene were ectopically expressed in epithelial cells as fusions to GFP and GFP-positive cells were visualized for rounding. Cells transfected with DNA corresponding to amino acids 1963-2375 of the rtxA gene (according to the original annotation by Lin et al. [7]) resulted in cell rounding and cells were confirmed to contain crosslinked actin by western blotting. This region was thus designated the actin crosslinking domain, or ACD [13], and it is now recognized as an effector delivered by autoprocessing of the large MARTX_Vc toxin [14].

Subsequent to the discovery of the ACD within MARTX_Vc, two other MARTX toxins have been identified by protein sequence analyses that also carry ACD effectors. Genomic sequencing of Aeromonas hydrophila strain ATCC 7966T revealed this pathogen associated with disease in invertebrates and an emerging pathogen in humans has a gene for a MARTX toxin that includes an ACD effector [12,15]. By contrast, genomic sequencing of human pathogenic Biotype 1 V. vulnificus strains revealed they also encode a MARTX toxin [16,17], but this toxin was not associated with actin crosslinking and did not have an ACD [13,18]. However, an environmental V. vulnificus Biotype 2 strain isolated from a diseased eel was found to carry a gene for a second MARTX toxin on the pR99 virulence plasmid (MARTX_R99) and this toxin included an ACD [19] (Figure 1). At this time, neither of these ACDs have been confirmed biochemically to be functional for actin crosslinking.

A second protein encoded by V. cholerae was also found to have an ACD [13]. This protein, VgrG-1, is a bacterial effector that is secreted from V. cholerae dependent upon a Type 6 secretion (T6S) system [20]. The first domain of the protein belongs to the VgrG family, a group of proteins required to form a structural element of the T6S apparatuses [21]. VgrG-1 is unusual among the Vgr proteins because it is an “evolved VgrG” with an attached second domain that is delivered to the cell cytosol and carries effector function, in this case, an ACD that has been demonstrated experimentally to have actin crosslinking activity [22] (Figure 1).

The final putative actin crosslinking toxin is hypothetical protein AND4_00045 identified in the draft genome of environmental Vibrio sp. AND4 isolated directly from the Andaman Sea in Thailand. Unlike the MARTX toxins or VgrG-1, this protein is a stand-alone actin crosslinking protein with no other associated domain for translocation [23]. Thus, if it is an effector used for pathogenesis of aquatic animals, it must be translocated directly to the eukaryotic cell cytosol, possibly by a Type III or T6S system also identified in the draft genome.

Early models of the mechanism of actin crosslinking recognized that crosslinking could occur either directly, in which the ACD is the catalytic enzyme, or indirectly, by activation of an unknown host cell protein with this activity [6]. To demonstrate direct action, the ACD portion of MARTX_Vc was purified as a fusion with the N-terminus of Anthrax Toxin Lethal Factor (LF_N). LF_N has been shown previously to mediate cytosolic delivery of heterologous fusion proteins through pores formed by Anthrax Toxin Protective Antigen (PA) [25,26]. This approach facilitated pretesting the LF_NACD for biochemical activity in the cytosol of mammalian cells after delivery by PA, prior to initiating experiments to establish in vitro crosslinking [27].

Subsequent experiments showed that LF_NACD does crosslink actin both in vivo after delivery by PA, as well as in vitro when added to eukaryotic cell lysates. Further refinement of the crosslinking reaction revealed the in vitro reaction is catalytic and progresses to completion in less that 10 min. This crosslinking occurs under reaction conditions composed of only purified actin, ATP, and Mg²⁺, indicating that no other proteins or cellular co-factors are required for crosslinking of actin by the ACD; and thus, ACD itself is the catalytic enzyme [27].

Polymerization of monomeric G-actin into F-actin filaments is dependent upon hydrolysis of ATP [28] and occurs in the presence of 2 mM Mg²⁺. Hence, the requirement of Mg²⁺ and ATP during the in vitro crosslinking reaction was initially thought to indicate that actin must be polymerized to be crosslinked. However, it was shown that crosslinking in vitro progressed efficiently when actin was locked as monomeric G-actin by association with latrunculin or kabiramide C or by modification with tetramethyl red [29]. Similarly, actin crosslinking progressed in vivo after addition of MARTX_Vc-producing V. cholerae to cells that were pretreated with cytochalasin or latrunculin to inhibit actin polymerization [27,30]. By contrast, stabilization of F-actin in vitro by the addition of phalloidin or in vivo using dolastatin 11 inhibited the efficiency of actin crosslinking [27,29]. These data indicated that G-actin, not F-actin, is the substrate for ACD in the crosslinking reaction. Furthermore, these data support that the requirement for ATP and Mg²⁺ during ACD-mediated actin crosslinking is not to drive actin polymerization.

Interestingly, by contrast to studies with MARTX_Vc, crosslinking of actin in macrophages due to VgrG-1 was completely blocked by cytochalasin. However, this inhibition was not due to the ACD of VgrG-1 requiring polymerized actin instead of monomeric actin; rather it revealed a requirement for the bacteria to be phagocytosed before the VgrG-1 effector could be delivered to the cytosol by T6S [21]. Next, to account for the requirement of ATP and Mg²⁺ in the crosslinking reaction, it was considered if the ACD itself is an ATPase. During an in vitro crosslinking reaction, inorganic phosphate (P_i) is released in a dose dependent manner such that one molecule of ATP is used for each crosslink introduced. This ATP hydrolysis is not due to polymerization of actin, but is an essential aspect of the crosslinking reaction itself [29] (see Figure 2).

It is well-recognized that in the living cell, G-actin does not exist in a free form and is typically bound to one of the several actin binding proteins (ABPs) [31]. Remarkably, prior association of actin with the common ABPs profilin, thymosin-β4, and gelsolin did not have any noticeable effect on actin crosslinking by ACD. Among the ABPs tested, only DNaseI completely blocked the formation of actin oligomers, and cofilin partially inhibited. These data show that in vivo, crosslinking of actin by MARTX_Vc likely progresses without interference of most ABPs [29].

Furthermore, actin crosslinking can occur when actin is previously crosslinked to itself. As G-actin is the substrate for crosslinking, higher order oligomers could arise by continued addition of G-actin monomers to longer chains or by the joining of oligomers. It was found that actin dimers disappear during crosslinking at ~4 fold slower rate than the monomers. This result indicated that although ACD-crosslinked oligomers can be further joined to form higher order species, the efficiency of the reaction diminishes with increased size of oligomers [29]. Thus, the formation of long crosslinked actin chains may occur predominantly by addition of monomers to linked oligomers.