Aeromonas Exoenzyme T of Aeromonas salmonicida Is a Bifunctional Protein That Targets the Host Cytoskeleton

Abstract

Type III protein secretion has been shown recently to be important in the virulence of the fish pathogen Aeromonas salmonicida. The ADP-ribosylating toxin Aeromonas exoenzyme T (AexT) is one effector protein targeted for secretion via this system. In this study, we identified muscular and nonmuscular actin as substrates of the ADP-ribosylating activity of AexT. Furthermore, we show that AexT also functions as a GTPaseactivating protein (GAP), displaying GAP activity against monomeric GTPases of the Rho family, specifically Rho, Rac, and Cdc42. Transfection of fish cells with wild type AexT resulted in depolymerization of the actin cytoskeleton and cell rounding. Point mutations within either the GAP or the ADP-ribosylating active sites of AexT (Arg-143 as well as Glu-398 and Glu-401, respectively) abolished enzymatic activity, yet did not prevent actin filament depolymerization. However, inactivation of the two catalytic sites simultaneously did. These results suggest that both the GAP and ADP-ribosylating domains of AexT contribute to its biological activity. This is the first bacterial virulence factor to be described that has a specific actin ADP-ribosylation activity and GAP activity toward Rho, Rac, and Cdc42, both enzymatic activities contributing to actin filament depolymerization.Aeromonas salmonicida subsp. salmonicida (A. salmonicida) is the causative agent of furunculosis, a systemic disease that affects salmonid fish (salmon, trout, and char). A. salmonicida expresses a variety of extracellular toxins, many of which have been implicated in virulence. Several of these factors, including the pore-forming toxin aerolysin, serine protease, and the phospholipase GCAT, are secreted into the environment via the well characterized type II or general secretory pathway (1, 2). However, like many Gram-negative pathogens, A. salmonicida also possesses a type III protein secretion system (3-5). Such secretion systems translocate toxins, or type III effector proteins, directly into the cytosol of eukaryotic cells. Here the effector proteins are able to modulate cell signaling pathways or, alternatively, disrupt the dynamics of the cytoskeleton (6, 7).To date, the following genes encoding four type III effector proteins have been identified in A. salmonicida: aexT, aopP, aopO, and aopH (8 -10). Although functional studies of AopO and AopH have not yet been carried out, sequence homology with type III effector proteins YopO and YopH of Yersinia sp. suggests that AopO and AopH modify the actin cytoskeleton of target eukaryotic cells. In contrast, AopP has been shown to interfere with the NF-B signaling pathway by inhibiting translocation of NF-B into the nucleus of target cells (10).AexT, the first type III effector of A. salmonicida to be characterized, has been shown to function as an ADP-ribosylating toxin (8). However, its substrate has never before been identified. AexT also displays significant sequence homology to the bifunctional type III effector proteins ExoS and ExoT, expressed by Pseudomonas aeruginosa. ...

Isolation and characterization of virulent Aeromonas veronii from ascitic fluid of oscar Astronotus ocellatus showing signs of infectious dropsy

“…aexT is a bifunctional protein with ADP-ribosylating activity and guanosine triphosphatase-activating protein (GAP) activity (Fehr et al 2007). aexT mediates ADP-ribosylation of both muscular and non-muscular actin in vitro (Fehr et al 2007), thereby preventing polymerization (Masignani et al 2006 (Fehr et al 2007, Vilches et al 2008.…”

“…aexT is a bifunctional protein with ADP-ribosylating activity and guanosine triphosphatase-activating protein (GAP) activity (Fehr et al 2007). aexT mediates ADP-ribosylation of both muscular and non-muscular actin in vitro (Fehr et al 2007), thereby preventing polymerization (Masignani et al 2006 (Fehr et al 2007, Vilches et al 2008. ascV is a gene that serves as an indicator for the presence of the TTSS machinery (Stuber et al 2003), which encodes an inner membrane component of the TTSS apparatus, and ascF-ascG encode the needle complex and a chaperone, respectively (Ghosh 2004).…”

Photox, a Novel Actin-targeting Mono-ADP-ribosyltransferase from Photorhabdus luminescens

“…SpvB of Salmonella enterica consists of a single domain, and is thought to gain cell entry via a type III secretion system (15). Likewise, Aeromonas salmonicida AexT uses a type III secretion system for invasion of host cells and carries a second functional domain with Rho-GAP activity (16,17), reminiscent of the well characterized ExoS expressed by Pseudomonas aeruginosa. Streptococcus pyogenes SpyA (18) is thought to be a single-domain mART with a 30-residue signal sequence for which the mechanism of cell entry is not yet understood and most recently, VgrG1 was found to enter host cells via a type six secretion system (19).…”

MOLECULAR IDENTIFICATION AND CLONAL RELATION OF ATYPICAL ISOLATE Aeromonas salmonicida USING RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP)

“…Materials for DNA Amplification and Electrophoresis: easy DNA mini column (Qiagen), PCR Kit (Promega) for PCR Amplification of 16S rDNA using the primers 1A (5'-CGTTGGATATGGCTCTTCCT-3') and 16S rDNA 2A (5'-CTCAAAACGGCTGCGTACCA-3') (Hiney et al, 1992); prime r MS1 (5'-AGGATGGCCTGCTCGTCACC-3') and MS2 (5'-GTGACG CCCAGGCCA TCCTC-3 ') (Fehr et al, 2007).…”