Pseudomonas aeruginosa exoenzyme S, a bifunctional type-III secreted cytotoxin

IJMM

Int.J. Med. Microbiol. 290,381-387 (2000) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/ijmm

Pseudomonas aeruginosa exoenzyme S, a bifunctional type-III secreted cytotoxin J. T. Barbieri Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701 Watertown Plk. Rd., Milwaukee, WI 53226, USA

Key words: Pseudomonas aeruginosa - exoenzyme S - type-III secretion - cytotoxin - ADPribosyltransferases

Introduction Studies on the cytotoxins produced by Yersinia have led to the recognition of a new class of bacterial toxins, termed type-III secreted cytotoxins (Comelis and WolfWatz, 1997). Type-III secreted cytotoxins differ from bacterial exotoxins. First, bacterial exotoxins are cytotoxic when added to eukaryotic cells at low concentrations, while type-III secreted cytotoxins are not cytotoxic when added to eukaryotic cells. Second, bacterial exotoxins possess leader sequences, which mediate cotranslational secretion across the bacterial cell membrane, while type-III secreted cytotoxins are not posttranslationally processed upon secretion across the bacterial cell membrane. Third, bacterial exotoxins often elicit pathological effects distal to the site of infection, while type-III secreted cytotoxins elicit pathological effects at the site of infection, which is due to the delivery of type-III secreted cytotoxins into eukaryotic cells via the direct contact of the bacterium with the eukaryotic cell. Type-III secreted cytotoxins are synthesized in the bacterial cytoplasm and often appear to be complexed to a chaperone (Comelis and Wolf-Watz, 1997; Frank, 1997). This places the cytotoxin in a conformation that is competent for translocation through the type-III secretion apparatus and into the cytosolic compartment of the eukaryotic cell. YopE of Yersinia is the prototypic type-III secreted cytotoxin (Frank, 1997;

Forsberg et aI., 1994). Intracellular delivery of YopE into the cytoplasm of eukaryotic cells elicits actin rearrangement. Originally, the type-III secretion pathway was thought to represent a novel delivery system. However, recent studies have identified type-III secretion pathways in Escherichia coli, Shigella, Salmonella, Pseudomonas, Bordetella, and others, which are reviewed in (Frank, 1997). Exoenzyme S of Pseudomonas aeruginosa is delivered into the cytoplasm of eukaryotic cells via a type-III secretion pathway.

Molecular properties

of Pseudomonas aeruginosa P. aeruginosa is a ubiquitous, Gram-negative, opportunistic pathogen of compromised patients (Bodey et aI., 1983). Especially susceptible are individuals with severe burn wounds, eye complications, neutropenia, and cystic fibrosis and individuals subjected to catheterization and surgery. P. aeruginosa pathogenesis is facilitated by the ability of the organism to grow in hospital environments (Kazama et aI., 1998) and its resistance to antibiotics (Kazama et aI., 1998; Finlan, 1972; Mendelson et aI., 1994). P. aeruginosa produces a number of virulence determinants, which are either cell-surface components or are secreted. Cell-surface components allow P. aeruginosa to colonize the site of infection, subvert the host innate and

Corresponding author: Dr. Joseph T. Barbieri, Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701 Watertown Plk. Rd., Milwaukee, WI 53226, USA, Fax: ++ 4144566535, E-mail: [email protected] 1438-4221100/290/4-5-381 $ 15.00/0

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acquired immune systems, and replicate within the host. The outer membrane of P. aeruginosa contains an endotoxin, which is not as potent as the endotoxin of the enterobacteriaceae, but can elicit an inflammatory response (Hickling et ai., 1998). Fimbriae are filamentous appendages, which are utilized as colonization factors and contribute to adherence to host cells (Schweizer et ai., 1998). The presence of a single polar flagellum is characteristic of the genus Pseudomonas and allows migration through the host environment (Feldman et ai., 1998). Alginate is an exopolysaccharide capsule, which allows P. aeruginosa to evade the host immune response and to form biofilms in the lungs of cystic fibrosis patients (Takeda, 1998; Sakagawa, 1998; Gacesa, 1998). The production of alginate is especially evident in lung isolates of P. aeruginosa. The recent identification of a type III-secretion system within P. aeruginosa (Frank, 1997) presents several new classes of virulence factors, which appear to contribute to the subversion of the innate and acquired immune responses. Type-III secreted cytotoxins include ExoU, Exo Y, ExoS, and ExoT. ExoU is a potent cytotoxin with an undefined mode of action (Finck-Barbacon et ai., 1997). ExoU is preferentially expressed in strains of P. aeruginosa, which are isolated from ocular infections (Fleiszig et ai., 1997). Exo Y possesses adenylate cyclase activity that is activated by a eukaryotic protein (Yahr et ai., 1998). ExoS and ExoT are bifunctional cytotoxins, which reorganize the actin cytoskeleton and ADP-ribosylate host proteins. The characterization of the molecular aspects of ExoS/Exo T is the focus of this review and will be described in detail in later sections. P. aeruginosa also secretes several virulence factors, which contribute to its pathogenesis. These factors possess both specific and nonspecific activities and often elicit pathological effects distal to the site of infection. Secreted virulence factors provide nutrients for growth, enhance invasive potential, or directly damage host tissue. P. aeruginosa produces several proteases, including LasA and LasB (Rust et ai., 1996; McIver et ai., 1995). LasB, also termed elastase, is produced by most clinical isolates of P. aeruginosa. LasB degrades the elastin lining of blood vessels and contributes to necrotic lesions on the lung and skin that are characteristic of P. aeruginosa infections. LasA has not been characterized in the same detail as LasB, but possesses greater target specificity (Toder et ai., 1991). LasA cleaves the pentaglycine-bridge within the peptidoglycan of Gram-positive bacteria, including Staphylococcus aureus (Kessler et ai., 1993). One role for LasA may be to provide P. aeruginosa an effective mechanism to control the growth of Gram-positive bacteria, which may compete for colonization at the site of infection. Las A may contribute to the observed coloni-

zation of bacterial flora in the lung of cystic fibrosis patients, where colonization proceeds sequentially with S. aureus and then P. aeruginosa. Phospholipase C is secreted from P. aeruginosa, which causes local tissue damage (Vasil, et ai., 1991). P. aeruginosa produces two siderophores, pyoverdin and pyochelin, which sequester iron from host sources (Meyer et ai., 1999; Vasil et ai., 1998). P. aeruginosa produces one bacterial ADP-ribosylating exotoxin, Exotoxin A, which is the most toxic protein produced by P. aeruginosa (Iglewski et ai., 1977). Exotoxin A is a 613 amino acid single chain protein with defined AB structure-function properties. The amino terminus encodes the receptorbinding and translocation domains (B), while the carboxyl terminus comprises the ADP-ribosyltransferase domain (A). Exotoxin A binds to the LDL-like receptor and enters cells via receptor mediated endocytosis (Fitzgerald et ai., 1994). It is not clear where, during its retrograde transport, the A domain is translocated into the cytosol, but the presence of a KDEL-like sequence at its carboxyl terminus implicates a role for retrograde transport in the trafficking mechanism. Exotoxin A must undergo two processing events, including proteolysis and disulfide bond reduction. The ADPribosylation of eukaryotic elongation factor 2 by exotoxin A inhibits protein synthesis and causes cell death (Iglewski et ai., 1977). Iglewski and coworkers (1978) identified exoenzyme S as an ADP-ribosyltransferase produced by P. aeruginosa, which possessed unique biochemical and enzymatic activities relative to Exotoxin A. The ADPribosyltransferase activity of exoenzyme S purified from the culture supernatant of P. aeruginosa as an aggregate composed principally of two proteins with apparent molecular masses of 53 kDa and 49 kDa (Nicas and Iglewski, 1984). The 49-kDa form of exoenzyme S possessed ADP-ribosyltransferase activity and was termed the enzymatically active form of exoenzyme S, while the 53-kDa protein possessed little intrinsic ADPribosyltransferase activity. Subsequent genetic analysis showed the 53- and 49-kDa forms of exoenzyme S to be encoded by separate genes (Figure 1) (Kulich et ai., 1994; Yahr et ai., 1996). Thus, exoenzyme S, as initially purified by Iglewski and coworkers, is composed of two related proteins, ExoS (the 49-kDa form) and ExoT (the 53-kDa form). ExoS and ExoT share little overall primary amino acid homology with other members of the family of bacterial ADP-ribosylating exotoxins, a common theme among members of this family of exotoxins. Coburn et ai. (1991) reported that exoenzyme S possessed an absolute requirement for a eukaryotic protein to express ADP-ribosyltransferase activity, termed FAS, factor activating Exoenzyme FAS was cloned from a eukaryotic cDNA library and identified as a member of the 14-3-3 protein family (Fu

.s.

Pseudomonas aeruginosa exoenzyme S, a bifunctional type III secreted cytotoxin

et aI., 1993). 14-3-3 Proteins have been shown to be ubiquitous within the eukaryotic kingdom with homologues identified in a broad range of species from yeast to vertebrates (Aitken et aI., 1995a, b). 14-3-3 Proteins contribute to host cell physiology via the regulation of numerous signal transduction pathways (Fu et aI., 1993). Recent studies have shown that ExoS possesses intrinsic ADP-ribosyltransferase activity (Knight and Barbieri, 1999) and that FAS is an allosteric activator, which increases the affinity of ExoS for NAD and increases the catalytic turnover rate. The activation of ExoS by FAS appears to be kinetically similar to the activation of cholera toxin by ARF (Bobak et aI., 1990).

The amino terminus of ExoS stimulates Rho family-dependent actin reorganization To enhance the resolution of the effect of ExoS on the physiology of eukaryotic cells, a transient CMV expression vector was developed to transfect cultured cells with plasmids encoding specific forms of ExoS. Intracellular expression of the amino-terminal 234 amino acids of ExoS in CHO cells elicited actin reorganization via a Rho-dependent mechanism. An involvement of the Rho GTPases was implicated by the observation that intoxication with CNF1 reversed the rounding phenotype elicited by transfection with C234. Analysis of reporter protein expression and trypan blue staining showed that this actin reorganization was not cytotoxic to eukaryotic cells. Although the in vivo role of ExoS in pathogenesis remains to be elucidated, the amino terminus of ExoS shares limited homology with the YopE cytotoxin of Yersinia and the amino terminus of StpP of Salmonella (Yahr et aI., 1996; Collazo and Galan, 1997; Frithz-Lindsten et aI., 1997).

The carboxyl terminus of ExoS is an ADP-ribosyltransferase, which is cytotoxic to cultured cells Intracellular expression of the carboxyl-terminal 222 amino acids of ExoS, which included the ADPribosyltransferase domain, was cytotoxic to eukaryotic cells. Intracellular expression of ~N222 reduced the expression of two independent reporter proteins and decreased trypan blue exclusion. In contrast, intracellular expression of a noncatalytic form of ~N222, which expressed reduced ADP-ribosyltransferase activity, elicited little inhibition of reporter protein expression and little cytotoxicity to transfected cells. These data in-

Aggregation 1 and secreHon 99

ExoS (exoS) 233 H279

Actin Reorganizotion Aggregation 1and secretion 99

383

FAS-dependent ADP-ribosyl' activity

ExoT (exoT) 235 IGlu282, catalytic defect)

Fig.1. Molecular organization of exoenzyme SexoS (49-kDa form) and exoT (53-kDa form),

dicated that expression of the ADP-ribosyltransferase domain of ExoS is cytotoxic to cultured cells.

ExoS ADP-ribosylates Ras at multiple arginine residues Ras is a central component of eukaryotic signal transduction, which makes its ADP-ribosylation by ExoS of potential physiological significance. Other bacterial exotoxins post-translationally modify Ras and other members of the Ras superfamily, resulting in the modulation of eukaryotic signal transduction (Just et aI., 1996). ExoS ADP-ribosylated Ras in vitro to a stoichiometry of 2 molecules of ADP-ribose incorporated per molecule ofRas, which suggested that ExoS could ADPribosylate Ras at more than one arginine residue. Utilizing in vitro transcribed/translated Ras, Arg41 was identified as the preferred site of ADP-ribosylation. While ExoS ADP-ribosylated Ras at a second site, it appeared that this second ADP-ribosylation did not occur at a specific arginine residue (Ganesan et aI., 1998). Subsequent studies showed that ExoS double ADPribosylated Ras at Arg41 and Arg128 (Ganesan et aI., 1999) (Figure 2). Analysis of the double mutant, RasArg41Lys, Arg128Lys, revealed an alternative site for ADP-ribosylation by ExoS as Arg13S. Since ExoS did not ADP-ribosylate Arg13S in wild-type Ras, it appears that the ADP-ribosylation at Arg128 sterically blocked the ADP-ribosylation of Arg13S. This indicated that ExoS could bind Ras in several orientations to facilitate ADP-ribosylation along the a-helix containing Arg128 and Arg13S. ExoS ADP-ribosylates Ras at Arg residues on two distinct surfaces of Ras, the ~-sheet containing Arg41 and the a-helix containing Arg128 and Arg13S. These data are consistent with the presence of two independent binding events to achieve double ADPribosylation. The plasticity of the ExoS-Ras interactions may explain the observed ability of ExoS to ADP-

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Arg41 Arg128 Arg135

Mechanism for the inhibition of Ras-mediated signal transduction by ExoS

Fig.2. Sites of ADP-ribosylation of Ras by ExoS.

Ragionl 2BO

29 0

310

300

32 0

ExeS

YTGIHYADLNRALRQGQELDAGQKL IDQGMSAAFEKSGQAE QVVKTFRGTRGGDAFNA

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I ::11

1:11 1

I I III

130 Region2 340 350

330

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140

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VEEGKVGHDDGYLSTSLNPGVARS- - F-- GQGTISTVFGRSGIDVSG I SNYKNEKEIL

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aligned more extensively with the vertebrate ADPribosyltransferases than the bacterial ADP-ribosyltransferases. Similarities extended from Region 1, included sequences surrounding the Ser-Thr-Ser motif (Region 2), through the catalytic glutamic acids of Region 3. tFASTA alignment between the catalytic portion of ExoS and the vertebrate ADP-ribosyltransferase RT6 is shown in Figure 3. In contrast, alignment of ExoS with the bacterial ADP-ribosyltransferases showed primary amino acid homology centered within the Ser-Thr-Ser sequence of Region 2 and did not extend to the catalytic glutamic acids of Region 3. These data suggest that ExoS may have an evolutionary link with the vertebrate ADP-ribosyltransferases.

: 1 90

I::

I I :1:1: I 200

21 0

Fig. 3. tFASTA alignment of ExoS and the eukaryotic mono-ADP-ribosyltransferase RT6 (31 .9% identity in 116 aa overlap).

ribosylate numerous small-molecular-weight GTPbinding proteins in vitro (Coburn et ai., 1989).

Functional and sequence relationships between ExoS and the vertebrate ADPribosyltransferases One unexpected observation that has been made during the characterization of ExoS is that it shares some functional and molecular properties with the vertebrate ADP-ribosyltransferases. To date, several vertebrate ADP-ribosyltransferases have been identified (Mironov et aI., 1997; Yost and Moss, 1983), including rabbit skeletal muscle ADP-ribosyltransferase (RNART), rat RT6, human ecto ADP-ribosyltransferase, and chicken ADP-ribosyltransferase type I and type II. Properties common to ExoS and the vertebrate ADP-ribosyltransferases include the ability to ADPribosylate multiple target proteins and the ability to ADP-ribosylate more than one arginine residue within a target protein. tFASTA algorithm showed that ExoS

Recent studies have addressed the mechanism for the inhibition of Ras-mediated signal transduction by ExoS that was observed in pcn cells. In these experiments, Ras was double ADP-ribosylated by ExoS and the physiological properties of ADP-r-Ras were compared to native Ras. ADP-ribosylation of Arg41 could modulate several steps in Ras-mediated signal transduction, including interactions with Raf or nucleotide exchange. Initial experiments showed that ADP-ribosylated Ras possessed an identical binding capacity for Raf as native Ras, which indicated that ADP-ribosylation did not interfere with Ras-Raf interactions. Although ADP-ribosylated Ras and native Ras possessed similar capacities for intrinsic nucleotide exchange, ADP-ribosylated Ras possessed a slower rate of guanine nucleotide exchange factor-mediated nucleotide exchange, relative to native Ras. Thus, it appears that ADP-ribosylation at Arg41 interferes with growth factor-stimulated signal transduction in eukaryotic cells. This mechanism of inhibition of Rasmediated signal transduction by ExoS is unique from other exotoxins. Future studies will address whether or not this inhibition is due solely to the expression of ADP-ribosyltransferase activity of ExoS.

Summary Our recent studies have shown ExoS to be a bifunctional type-III secreted cytotoxin. Intracellular expression of the amino terminus of ExoS (C234) in eukaryotic cells stimulates actin reorganization without cytotoxicity, which involves small-molecular-weight GTPases of the Rho subfamily. Expression of the carboxyl terminus of ExoS comprises an ADP-ribosyltransferase domain,

Pseudomonas aeruginosa exoenzyme S, a bifunctional type III secreted cytotoxin

which is cytotoxic when expressed in cultured cells (Pederson and Barbieri, 1998). Rho and Ras are molecular switches, which control numerous cellular processes. Recent signaling studies suggest that there is crosstalk between Rho and Ras (Keely et aI, 1997). Ras and Rho also contribute to wound healing processes and tissue regeneration. Recent studies have shown that microinjection of endothelial cells with activated Ras stimulated their motility, while microinjection of Ras-blocking antibodies inhibited cellular motility that is a component of the wound healing process (Fox et al., 1994). In addition, hepatocyte growth factor/scatter factor (HGFI SF) and epidermal growth factor stimulate cellular motility through the Ras signal transduction pathway (Ridley et al., 1995). Rae and Rho are also involved in motility and tissue regeneration, since dominant negative Rae inhibits the cellular motility stimulated by HGF/SF (Santos et al., 1997) and inhibition of Rho by either C. difficile ToxA and ToxB or the C. botulinum C3 transferase inhibits wound healing (Santos et al., 1997). Inhibition of tissue regeneration and wound healing appear to play a role in the pathogenesis of C. difficile, since treatment of gastrointestinal mucosa with C. difficile ToxA and ToxB alone inhibits regeneration of the gastric mucosa. Thus, ExoS may contribute to the establishment of P. aeruginosa infections by inhibiting wound healing and tissue regeneration by two mechanisms. The amino terminus of ExoS could inhibit Rho function and wound healing in a manner similar to C. difficile. Alternatively, ExoS could inhibit the cellular motility and angiogenesis required for wound healing by ADP-ribosylating Ras. Through the inhibition of tissue regeneration and wound healing, ExoS may play a pivotal role in chronic disease by maintaining sites of colonization. Inhibition of Ras or Rho signaling may also interfere with both innate and acquired immunity. Smallmolecular-weight GTP-binding proteins of the Ras superfamily are required for cellular processes, such as phagocytosis, as Rho proteins contribute to phagocytosis (Caron and Hall, 1998). Since Ras functions upstream of Rho in cellular signaling processes (Ridley et al., 1995), ADP-ribosylation of Ras by ExoS or the inhibition of Rho function by C234 may inhibit phagocytosis of P. aeruginosa by macrophages. Other studies indicate that Ras plays a role in T cell activation (Cantrell, 1994). Thus, ExoS may inhibit acquired immunity by inhibiting T-cell activation. Acknowledgements. This study was supported by a grant from NlH-NIAID AI-320 162.

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