Structure, function, and regulation of Pseudomonas aeruginosa exotoxin A.

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STRlrCTURE, FUNCTION, AND Annu. Rev. Microbiol. 1990.44:335-363. Downloaded from www.annualreviews.org Access provided by University of California - San Francisco UCSF on 12/18/14. For personal use only.

REGlJLATION OF PSEUDOMONAS AERlJGINOSA EXOTOXIN A M. J. Wick Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

D. W. Frank Department of Microbiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

D. G. Storey Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada T2N-4Nl

1 B. H. Iglewski KEY WORDS:

bacterial toxin, ADP-ribosyl transferase, iron regulation , protein domains, eukaryotic cell internalization

CONTENTS INTRODUCTION . . . . .

. . . . . . . . . . . . . . .

336

THE REGULATION OF EXOTOXIN A PRODUCTION . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . .

337 337 338 341 344 347

. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . .

Environmental Factors that bifluence Exotoxin A Yields . .... . . . . . . . ...... . . . . . . . .... . . . . Genes Involved in Exotoxin A Synthesis . . ..... . . . . . . . . . . . . . .... . . . . . . . , . .. . . . . . .. . . . . .. . . . . . Regulation of Exotoxin A Transcription ... . . ... . . . .. . . . . . ... . . . . . . . . . . . . ..... . . . . . . . . .. . . . . . . Regulation of the regA Promoters . . . . . .... . . . . . . . . . . . . ...... . . . . . ....... . . . ... . . . . . . . . . . . ..... Summary of Regulatory Studies . . . . . . . . . . . . ...... . . . . . . . ..... . . . . . . . . . . . . . . . . . . . . . . , . . .. . . . . . . .

'Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

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0066-4227/90/1001-0335$02. 00

Annu. Rev. Microbiol. 1990.44:335-363. Downloaded from www.annualreviews.org Access provided by University of California - San Francisco UCSF on 12/18/14. For personal use only.

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MOLECULAR ANALYSIS OF EXOTOXIN A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Secretion of Exotoxin A from P. aeruginosa. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . ... . . . ... Expression of Exotoxin A Enzymatic Activity . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .. . . . .. . . . . . Crystallographic Analysis of Exotoxin A . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Functional Analysis of Exotoxin A Domains ... . . .. . . .. . . .. ..... . . .. . . . . . . . . ...... . ..... . . ..

349 349 350 350 351

SIGNIFlCANCE AND PERSPECTIVES. . . . . . . .. . .... . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. ... . . .. . . . ..

357

INTRODUCTION Pseudomonas aeruginosa is a nonfennentative, gram-negative bacillus that

inhabits soil and water environments (83). It can survive on minimal nutrients and grows at temperatures as high as 42°C. The metabolic diversity of pseudomonads in general is well documented. P. aeruginosa can utilize over 80 organic compounds for growth. In nature, these organisms play an impor tant ecological role in the breakdown of organic matter. The medical importance of P. aeruginosa is underscored by the fact that the organism causes severe and often fatal infections in individuals that have been compromised by disease, design, or accident (9, 86, 95). Patients with chronic conditions such as leukemia, cancer, cystic fibrosis, diabetes, or heart disease are especially susceptible to infection with P. aeruginosa as well as other opportunistic pathogens. Immunodeficiency caused by drug treatment of transplant or cancer patients also predisposes individuals to P. aeruginosa infection. Wound infections following surgery, catheterization, tracheos tomy, corneal abrasion, or severe bums opens a portal of entry and can lead to life-threatening sepsis or severe tissue damage. Because of the intrinsic antibiotic resistance' of this organism, its widespread distribution, ability to survive under a variety of conditions, and range of susceptible hosts, it is not surprising that P. aeruginosa is the most common pathogen isolated from patients who have been hospitalized for more than one week (9). Virulence factors associated with P. aeruginosa include a number of secreted protein products (57, 61, 80, 95, 107). Two proteases, elastase and alkaline protease, increase the organism's ability to invade tissue, obtain nutrients (both amino acids and iron from transferrin), and evade the immune response by inhibiting neutrophil chemotaxis and by destruction of host immunoglobulin and complement components (4, 24, 30, 47, 77, 86). Phos pholipase C and rhamnolipid are thought to act in concert to release phos phorylcholine and to solubilize phospholipids from lung tissue (57, 61, 95). A number of pigments produced by P. aeruginosa function in iron acquisition from the environment and appear to inhibit the growth of other bacterial species (21, 22, 70, 95). P. aeruginosa produces two distinct ADP ribosyltransferase toxins, exotoxin A (ETA) (49) and exoenzyme S (51). Exoenzyme S appears to play a pathogenic role in lung and bum infections, causing tissue damage and aiding the dissemination of the organism (80,


P. AERUGINOSA EXOTOXIN A

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106). Its primary eukaryotic substrates are vimentin, a structural component of cells (19), and GTP binding proteins, including p21c-H-ras (20). ETA, the most toxic substance produced by P. aeruginosa (57), acts to halt eukaryotic protein synthesis at the level of polypeptide chain elongation by covalently modifying elongation factor 2 (EF2) (49, 50). The addition of ADP-ribose from nicotinamide adenine dinucleotide (NAD) to EF2 by ETA is identical to the reactiolll catalyzed by diphtheria toxi� (18, 49, 50). The severity of P. aeruginosa infections and the evidence that implicates ETA as an important virulence factor (61, 80, 81) has been the impetus for investigation of the molecular properties of ETA. Such studies have contrib uted to a partial understanding of the regulatory events governing ETA transcription. In addition, a combination of biochemical, crystallographic, and genetic: analyses have led to the division of ETA into functional domains. This review summarizes the current knowledge of the molecular basis for ETA intoxication of eukaryotic cells by describing what is known about the individuallsteps in this process. We begin by examining the regulatory events leading to production of ETA by the bacterium and the secretion of the mature protein into the environment. A discussion follows of our current understand ing of the mechanism whereby ETA binds, enters, and carries out the enzymatic reaction that ultimately leads to eukaryotic cell death.

THE REGULATION OF EXOTOXIN A PRODUCTION Environmental Factors that Influence Exotoxin A Yields

Optimization of strain and culture conditions was a necessary step in the study of ETA synthesis. Liu (56) originally selected strain PA103 because it has less extracellular protease activity than the prototypical P. aeruginosa strain PAOl. Thiis strain selection was fortuitous since PA103 was later found to differ at the positive regulatory locus for toxA transcription (103). These genetic distinctions contribute to the tenfold enhancement of extracellular ETA yields in the hypertoxigenic strain PA103 when compared to ETA yields of the prototypical strain PAOI (81, 103). Optimal yields of ETA from strain PA103 depend on specific growth conditions (56) . ETA production is not constitutive and maximal yields occur late in the growth phase of P. aeruginosa cultures. Aeration, growth at 32°C instead of :\7°C, the presence of glycerol as a carbon source, and the addition of certain amino acids such as glutamic acid to the basal trypticase soy broth enhance ETA yields (56). Heat treated nucleic acids or a nondialyzable fraction of trypicase soy broth inhibit ETA production but enhance cell growth (56). Cation concentration in the growth medium is also an important factor in ETA production. Growth of P. aeruginosa in medium containing low con-

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centrations of Fe3+ or moderate concentrations of Co2+ , Cu2+, or Mn2+ reduces the final yield of ETA (5, 7, 8). Growth in medium containing a high concentration of Ca2+ (0.5 M) enhances ETA production (7, 8). The effect of the physiologically important cation iron on ETA production has been studied in detail. The optimal Fe3+ concentration in culture medium for ETA produc tion is 1 JLM. However, at Fe3+ concentrations of 5 JLM or above, ETA production is repressed to less than 10% of the optimal yield (5). Other exoproducts of P. aeruginosa including pigments, elastase, alkaline protease, pyochelin, and pyoverdin are similarly affected by iron levels in the growth medium (5, 6). The variation in ETA production when P. aeruginosa cells are cultivated under different growth conditions and the temporal expression of ETA indicate that its synthesis is highly regulated.

Genes Involved in Exotoxin A Synthesis Cloning and nucleotide sequence analysis of the structural gene for ETA, toxA, made possible expansion of both regulatory and structure-function studies. Oligonucleotide probes gener ated from an amino-terminal amino acid sequence of purified ETA were used THE EXOTOXIN A STRUCTURAL GENE

to s elect toxA clones from a pUC9/PA103 chromosomal library (39). The

nucleotide sequence of the 2760 base pair (b p ) toxA locus revealed that the gene consists of a single open reading frame with a typical Shine-Dalgarno sequence upstream of the initiation codon and a rho-independent terminator downstream of the termination codon (39). The toxA transcript is monocistronic (38) with a relatively long half life of 8 to 10 minutes (58). Linker scanning and Ba131 deletion analysis suggest that the minimum region required for toxA transcription and regulation encompasses the 160 bp region 5' of the initiation codon (93). S l nuclease mapping analyses located trans criptional start sites 88 (16) or 89 and 62 (38) base pairs upstream of the toxA initiation codon. Homology to the consensus region of prokaryotic or Pseudo monas promoter regions (Tol plasmid, xylABC operon, xyLDEFG operon, and xylR gene) was not found in the -10 and -35 areas of either toxA transcrip tional start site (16, 38). In Escherichia coli hosts, the cloned toxA gene is not expressed unless the upstream region is replaced with a promoter that is recognized by E. coli RNA polymerase (38, 39, 60). Computer-assisted searches of the toxA upstream region indicate the presence of multiple direct repeat sequences (16, 38). The lack of homology to established promoter regions in either Pseudomonas or other prokaryotes, the presence of direct repeat sequences, and the failure of E. coli RNA polymerase to initiate transcription suggest that additional regulatory factors may be required for toxA mRNA synthesis (37, 88). IDENTIFICATION

augments ETA

OF THE

REGA GENE

A positive regulatory gene that by complementation of a

production has been cloned



339

hypotoxigenic mutant of P. aeruginosa, PA103-29 (44). When this cloned genetic region is provided in trans to strains with a functional taxA gene, ETA yields increase approximately tenfold (44). Deletion analysis of the cloned region suggests that this positive regulatory gene, regA, [also referred to as toxR (108)] resides on a 1. 9 kb fragment (46, 108). Nucleotide sequence and deletion analyses showed that the region responsible for complementation of the hypotoxigenic phenotype of PA103-29 contains an open reading frame encoding a protein with a predicted molecular weight of 28,824 (45). REGA EXPRESSION IN P. AERUGINOSA Expression of RegA was achieved in pKK223-3, a vector in which the regA gene is under the control of the tac promoter and is provided with a ribosomal binding site and transcription termination signals (111). Under these conditions, RegA is overproduced as a 29,OOO-dalton protein and sequestered in cytoplasmic inclusion bodies in E. coli (111). RegA obtained from inclusion bodies was used in the production of specific antibody for expression and localization studies in P. aeruginosa. Antiserum generated against isolated RegA reacts with a 29,OOO-dalton pro tein in the cell lysate from strain PA103 containing a single chromosomal copy of a functional regA gene. In trans, higher amounts of RegA are detectable in strain PA103 containing multiple copies of the regA gene. The antiserum does not react with lysate material from the regA mutant strain PA103-29. Localization of this protein in P. aeruginosa depends on the level of expression. In strains that overproduce RegA, as is the case when P. aeruginosa harbors a multicopy plasmid with the cloned regA gene, the protein is found in approximately equal quantities in the cytoplasmic and membrane fractions. On the other hand, when RegA is expressed in P. aeruginosa from a single chromosomal copy, the protein is predominantly localized in the membrane fraction (111) . The precise role that RegA has in the enhancement of ETA production remains to be elucidated. Evidence presented to date suggests that RegA is not a global regulatory molecule. The regA mutant strain, PA103-29, differs from the parental strain, PA103, only in ETA yields (82). In addition, multiple copies of the regA gene appear to enhance only ETA production because elastase and alkaline protease yields do not change (44). The potentiation of ETA production by regA is clearly linked to the transcription of toxA (33, 35, 92, 108; see following sections). However RegA composite proteins pro duced in E. coli do not bind to a taxA upstream probe in gel retardation assays (41). One explanation for negative DNA binding results could be that RegA requires association to, or modification by, an additional molecule. Such a factor may be present only in the natural host, P. aeruginosa. On the other hand, RegA may be a sensorlike molecule and act indirectly to enhance ETA transcription. Other possibilities for RegA function include: (a) a sigma factor that alters promoter recognition specificity; (b) a stimulator of toxA transcrip-

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tion via an antitermination mechanism; (c) a protein that covalently modifies other regulatory or sensory components; or (d) a specific RNA polymerase (37, 52, 88, 89, 102). A thorough structural and functional analysis of the RegA protein as well as the identification of other factors that may be influencing taxA transcription are required before the precise role of RegA in the regulation of ETA production is understood. IDENTIFICAnON OF THE REGR GENE Various isolates of P. aeruginasa produce different levels of extracellular ETA (6, 81). In particular, two genetically well characterized strains, the hypertoxigenic strain PAI03 and the prototypical strain PA0 1 , differ in their extracellular ETA yields by approximately tenfold (81 ) . Duplication of the taxA structural gene (96) or the regA gene (44), mutations in the promoter or structural regions of taxA (16, 93, 97, 103, 104), or translational efficiency of taxA (43) do not appear to be responsible for the observed tenfold difference in extracellular ETA yields between PA01 and PA 1 03. These observations combined with data concern ing the regulation of regA and taxA transcription (see following sections) indicate that the difference in ETA yields may result from differences in the regA gene or in the regulation of regA expression. To test this hypothesis, the regA locus from the prototypical P. aeruginasa strain PA01 was cloned and compared to the same region cloned from the hypertoxigenic strain PA103 ( 103). Comparison of ETA yields in the regA mutant strain PA103-29 containing the cloned PA0 1 or PA 1 03 regA locus indicates that the PAOl locus is five to seven times less efficient in enhancing ETA production than the PAl 03 regA locus (103). Nucleotide sequence comparison of the two regA regions re vealed several differences, including an amino acid substitution at position 144 in RegA (Thr in PA01 and Ala in PA l O3) and the absence in the PAO! sequence of a start codon for a small open reading frame located downstream of the PA103 regA coding region (1 03). Recombinant molecules were con structed to determine the contribution of each of these changes in nucleotide sequence on extracellular ETA yields. Extracellular ETA yields are identical when the PA103 wild-type regA gene (Ala 144) and a recombinant PA103 regA gene containing Thr at position 144 are compared. These data suggest that the amino acid difference within RegA does not affect extracellular ETA yields. To test the contribution of the downstream open reading frame, a hybrid protein was constructed that consisted of the PA103 regA open reading frame and the PA0 1 downstream region that lacks the second open reading frame. The amounts of extracellular ETA measured from this clone are similiar to those from the entire PAOl regA locus. This result indicates that the downstream open reading frame, regB, contributes to ETA synthesis. The absence of the regB open reading frame in PA01, and hence the RegB -


341

protein, accounts for some of the difference in ETA yields exhibited by these strains (103). P. AERUGINOSA The regB gene is 228 nucleotides in length and encodes a protein of 75 amino acids with a calculated molecular weight of 7,527 daltons (103). Probes located downstream of the regA open reading frame (internal to regB) hybridize to both early and late PA103 regA transcripts (D. W. Frank & B. H. Iglewski, unpublished observations; see later sections). The information on transcript size (33), location of initiation sites for transcription (35), and downstream dot blot hybridization analyses suggest that regA and regB constitute an operon under control of the regA promoters. Because sequences showing homology to a consensus ribosome binding site: are not apparent upstream of regB, RegB expression may depend on the reinitiation of translation after the regA stop codon. The regA/regB locus in P. aeruginosa (103) appears to be structurally analogous to the toxR/toxS locus in Vibrio cholerae (72, 74). Both sets of genes are aITanged in tandem with the augmenting gene, regB or toxS, located immediately downstream from the respective transcriptional activator, regA or toxR. Both loci appear to form a regulatory operon and both produce an unstable message (33, 35, 74). The function of regB or toxS is not clear. ToxS has been postulated to modify ToxR to make it competent for transcriptional activation (74). RegB may be involved in the transcription of the regA locus and indirectly enhance toxA transcription by enhancing early regA transcrip tion (see following sections). ToxR has been shown to function as a trans membrane protein with a domain responsible for sensing environmental changes and a DNA binding domain that interacts with a repeated sequence in the upstream region of several genes involved not only in toxin production, but also in colonization (72, 75, 85). As discussed previously, RegA function has not been clearly assessed. Thus, although the regA and toxR loci appear similar, they may not be functionally analogous. Further comparisons be tween these: genes and other similarly organized virulence-related regulatory loci (73) await clarification of RegA and RegB function.


REGB EXPRESSION IN

Regulation of Exotoxin A Transcription IRON REPRESSION OF EXOTOXIN A TRANSCRIPTION Iron availablility within the mammalian host is low owing to the complexing of iron by molecules such as transferrin and lactoferrin (1). This nonspecific host mech anism discourages the growth of invading bacteria (11, 101). The production of several bacterial toxins (shiga toxin, diphtheria toxin, ETA) as well as other factors associated with a virulent phenotype, such as elastase or iron uptake systems, depend on growth in an iron-limited environment. Thus, examination of the regulatory pathway that governs iron repression of ETA

342

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Figure 1

Summary of dot blot transcript accumulation studies. The top panel shows a map of

the PA103 regA locus. Open boxes denote the open reading frames of regA and regB, which are separated by six base pairs (103 ) and the closed box represents Tn5 sequences from the original subcloning experiments (46). Arrows indicate transcriptional initiation sites, which are located 164 (Tl initiation site) and 75 (T2 initiation site) base pairs upstream of the ATG codon for regA (35) . Proposed promoter regions are depicted as PI and P2. DNA probes used for transcript accumulation studies are shown below the regA locus. Panels A and C used the upstream AvaI probe and panels B and D used the internal Sail probe. Transcripts of regA were monitored throughout the bacterial growth curve from strain PA lO3 (A and B) or from strain PA lO3 containing the regA locus on a multicopy plasmid (C, D). Open and closed symbols represent RNA samples from cells grown in low or high iron medium , respectively. CPM is counts per minute of bound probe and time in hours represents the time in secondary culture at which cells were harvested and total RNA was extracted (33, 3 5 ) .

synthesis may lead to a general understanding of the mechanisms required for bacterial survival within the host. Bjorn et al (5) first demonstrated that increasing iron concentration in the growth medium results in diminished yields of ETA. Because the addition of iron did not alter enzymatic activity or mouse toxicity, they concluded that iron may be decreasing the rate of production or increasing the rate of degradation of ETA (5). Analysis of taxA transcripts by several laboratories confirmed that growth in iron-rich medium reduces the amount of detectable


343


toxA mRNA (16, 34, 38, 46, 58). Iron added to cultures actively engaged in transcription results in a slow decrease of toxA mRNA (58). The lag time between the addition of iron and the first detectable effect on toxA mRNA is considerablly long (approximately one hour), indicating that factors, most likely invollved in sensing the change in iron status of the medium, need to be synthesized (58). The studies described above did not ehminate the possibility that the reduction in toxA transcription observed when cells are grown in high iron medium is mediated by a reduction iin the expression of the positive regulatory gene, regA. This question was directly assessed by following the accumulation of regA tran scripts during the bacterial growth cycle. For these experiments, PA 1 03 cells with only one regA gene (Figure 1, panels A and B) and PA103 cells containing the cloned regA locus on a multicopy plasmid (Figure 1, panels C and D) were grown under conditions for maximal toxA transcription (open symbols) or maximal repression of toxA transcription (closed symbols) (33, 35). In addition , two probes were used to analyze regA transcripts. An upstream AvaI fragment hybridizes to regA mRNA produced before the five to seven hour time points (early phase of mRNA accumulation) (Figure 1 , panels A and C) and an internal SaLI fragment recognizes both early and late phases of regA mRNA accumulation (Figure 1 , panels B and D). Northern blot analysis of the peaks of regA transcript accumulation indicate that each peak corresponds to a different size class of regA mRNA. The size of regA message during the early phase of accumulation is 1200-1500 bp (Tl tran script). The late phase of accumulation is characterized by a smaller 700-800 bp mRNA called T2 (33). Comparison of transcript accumulation from cells grown in low and high iron medium indicates that the two phases of regA transcription are differen tially regulated (35). When the regA locus is represented by a single copy in cells grown in high iron medium, the early phase is reduced and the second phase of accumulation does not initiate (Figure 1, panel B). When the regA locus is pr,esent in multiple copies, the initial phase of accumulation is no longer repr essed by growth in high iron medium (Figure 1, panels C and D) but the second phase is still fully iron repressible (Figure 1, panel D). Primer extension studies of regA mRNA isolated at different points in the growth cycle and under different iron growth conditions show that each phase of regA transcript accumulation corresponds to a different point of transcriptional initiation (35). Taken together, these studies suggest that regA transcription is controlled by two independently regulated promoter regions. The PI promoter region conlrols the production of the Tl transcript. Tl is expressed early in the growth cycle and is not regulated by iron when the regA locus is present in

IRON REPRESSION OF REGA TRANSCRIPTION


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WICK ET AL

multiple copies. The P2 promoter region controls the expression of the T2 transcript. P2 seems unable to initiate transcription unless the cells are growing in a low-iron environment regardless of the copy number of the regA locus. Thus, the iron regulation of toxA transcription appears to result from the failure of transcriptional initiation at the regA P2 promoter region (35). Experiments in which the regA gene is under the control of a foreign promoter support these results (97). If the iron regulation of taxA transcription is totally due to the iron repression of regA transcription late in the growth cycle, one would predict that the cloning of regA under the lac promoter would uncouple iron repression of taxA transcription. Results from this type of experiment correlate well with predictions based on direct measurements of regA and toxA transcripts. That is, substitution of the lac promoter for the regA promoter renders toxA transcription (measured as ,B-galactosidase units from a protein fusion) unresponsive to iron repression (97). Regulation of the regA Promoters

Studies of the regA and toxA transcripts suggest that the transcription of toxA depends on the expression of the regA locus. In addition, when the regA locus is present in multiple copies, production of the early Tl regAlregB transcript is no longer inhibitable by growth in high-iron medium (35). These results indicate that components of the regA locus not only affect toxA transcription but may also affect the regulation of early regAlregB transcription (D. G. Storey, D. W. Frank, & B. H. Iglewski, unpublished observation). Clearly, the next stage in the investigation of the regulation of toxA transcription requires an understanding of the regulation of the regA promoters. To analyze the regulation of the individual regA promoters, the sequences were subcloned using information from deletion analyses of the regA locus (46). Storey et al (92) used the subcloned promoter regions to construct transcriptional and translational fusions to two reports genes, J3-galactosidase and chloramphenicol acetyltransferase (CAT). The four regA-reporter gene fusions constructed for these expression studies were: pRL88, a translational fusion of both (PI and P2) promoters to the lacZ gene; pRLX5, a translational fusion of the P2 promoter to the lacZ gene; pPII, a transcriptional fusion of the PI promoter to the cat gene; and pP2I , a transcriptional fusion of the P2 promoter to the cat gene (92). The growth conditions used to examine �-galactosidase or CAT activity from strains containing the fusion constructions differed from those used in transcript accumulation experiments. In these studies, P. aeruginosa cells transformed with various constructs were grown in high iron medium, washed, and subcultured into either low- or high-iron medium. The net result of this type of cultivation is that one can follow expression from the regA



345

promoters with minimum contamination of regA or toxA products from the previous cell cycle (92). Expression studies indicate that when the two promoters are tandemly cloned (pRL88), they function in concert to yield two phases of expression in strain PAlO3 (92). The early phase of expression is not affected by the iron content of the medium. In contrast, the second phase, which comes later in the growth cycle, does not occur in high-iron medium. These phases parallel the two phases of regA transcript accumulation detected using Northern and dot blot analyses (33, 35). REGULATION OF THE p2 PROMOTER PAI03 cells transformed with con structions containing only the P2 promoter region (pRLX5 or pP21) express ,B-galactosidase or CAT late in the growth cycle in low-iron medium (92). Expression from PAlO3/pRLX5 or PAlO3/pP21 is not observed early after subculture and does not occur in high-iron medium (92). Clearly, the P2 promoter is tightly iron regulated. An iron-regulated inducer likely controls the iron regulation of the P2 promoter. However, this inducer has not yet been identified. Recently, studies using strains deficient in expression of either regA, regB, or toxA have shown that the proteins encoded by these genes do not play a role in the regulation of the P2 promoter (D. G. Storey, D. W. Frank, M. J. Wick & B. H. Iglewski, manuscript in preparation). The iron regulation and lack of involvement of regA, regB, and toxA in the expression from the )02 promoter correlates with transcript accumulation results. In addition, analysis of the regulation of the P2 promoter provides further evidence that the iron regulation of regA, and hence· toxA, is primarily mediated through the P2 promoter of regA. REGULATION OF THE pI PROMOTER Northern blot, dot blot, and primer extension mapping indicate that early expression of regA transcription is mediated by the PI promoter (35). Fusions pRL88 (PI + P2) and pPII (PI) (92) were used to examine the expression of the regA PI promoter region in different genetic backgrounds. Expression of the PI promoter in strain PAlO3 occurs early after subculture and is not repressed by growth in high-iron medium (92). However, when pRL88 is expressed in PAOI (a prototypical ETA producer) or PAlO3-29 (a regA and regB mutant) no early phase of {3-galactosidase synthesis is observed (Table 1; D. G. Storey, D. W. Frank, M. J. Wick, & B. H. Iglewski, manuscript in preparation). These results indicate that the PI promoter is not functioning in either PADI or PAI03-29. The lack of PI expression in PAlO3-29 and PAOI was confusing until the respective regA genes were cloned and sequenced (103, D. G. Storey, D. W.


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Frank, M. J. Wick, & B. H. Iglewski, manuscript in preparation). Sequence analysis of the PA103-29 regA reveals two mutations (D. G. Storey, D. W. Frank, M. J. Wick, & B. H. Iglewski, manuscript in preparation). One mutation occurs 183 base pairs upstream of the T1 transcriptional start site. The other mutation introduces a premature translational stop codon into the PAI03-29 regA open reading frame. Since neither TI nor T2 transcripts are detectable from PA103-29 (D. W. Frank & B. H. Iglewski, unpublished observation), the most likely explanation of these results is that the mutation within the coding region of regA allows the rapid degradation of mRNA. As a result, RegA (111) and presumably, RegB, are not produced in PAI03-29. As discussed in previous sections, the nucleotide sequence of the PAOI regA locus reveals a mutation in the start codon for regB (103). The absence of RegB in PAOI accounts for some of the reduction in ETA yields observed when this strain is compared to PAI03 (103). The only common mutation between PAOI and PA103-29 that could account for the lack of expression from the PI promoter and the reduction in ETA yields in PAOI is the lack of RegB in both strains. Thus, regB must be required for expression from the regA PI promoter early in the bacterial growth cycle. The temporal expression and intricate control of regAltoxA transcription (33, 92) and ETA secretion (42, 62) suggests that ETA may play an intracellular role as a regulatory protein. The role of ETA in its own regulation was explored using a PAlO3 toxA - strain as a host for analysis of expression from the two regA promoters. Analysis of expression from pRL88 (PI + P2) in PAlO3 toxA - compared with expression in strain PAlO3 reveals that the early phase of f3galactosidase expression is identical in both strains (Table 1). In low iron medium, however, the level of expression from the regA promoters late in the growth cycle is approximately twofold higher in PAlO3 toxA - than in PAlO3 (Table I). This pattern of expression indicates that ETA is acting on one of the regA promoters. Since the effect occurs late in the growth cycle and only in iron-limiting conditions, ETA apparently influences regulation of the regA P2 promoter. However, the pattern of expression from pRLX5 (P2) is identical in both toxA+ and toxA - genetic backgrounds (Table 1). The alternative possibility is that ETA influences the regulation of the regA PI promoter. To examine this hypothesis, expression from the PI promoter was compared in strains PAI03/pP11 and PAlO3 toxA-/pPl l. In iron limiting growth medium, no difference in expression between the two strains early in the growth cycle is observed (Table 1). In contrast, a much higher level of expression from PAlO3 toxA-/pPl l is observed late in the growth FEEDBACK INHIBITION BY EXOTOXIN A ON REGA TRANSCRIPTION

347

P. AERUGINOSA EXOTOXIN A Table 1

PI and P2 promoter function in P. aeruginasa strains with different genetic backgrounds Relative expression measured early or late across a growth curveb

Genetic make up pRL88


of the strains' Strains

regA

regB

taxA

Early

Late

PA 1 0 3

+ +

+

+ +

+

+

+

+ + + ++

PAOI

+

PA 1 0 3-29 PA103 taxA-a

+

pRLX5 Early

Late

pPl l Early

pP2 1

Late

Early

Late

+

+

+

ND

ND

ND

ND

ND

ND

ND

ND +

++

ND

ND

+

+ +

(+) Strain can transcribe the gene. (-) Strain cannot transcribe the gene_

media only: (+) expression from either calor la,Z reporter gene; (-) n o expres:;ion from reporter gene; (ND) not determined. b Results are shown for growth in iron limiting

cycle (Table 1). This higher level of expression from pP11 and PAlO3 toxA is similar in timing and quantity to expression from pRL88 in this strain. These results suggest that ETA inhibits continued expression from the regA PI promoter through feedback control.

Summary of Regulatory Studies Studies of the regulation of ETA production in P. aeruginosa have led to the discovery of several regulatory events that allow or modulate toxA transcrip tion. These events center on the expression of a positive activator protein, RegA, that is required for enhanced toxA transcription. RegB is an additional regulatory factor that appears to act synergistically with RegA to ultimately increase ETA yields. ETA, RegA, and RegB form a regulatory loop that apparently allows P. aeruginosa to fine tune ETA production in response to environm{�ntal stimuli (iron) or to the stage in cell growth. The temporal regulation of regAlregB and toxA transcription may be coordinated with post-transcriptional events such as translation, processing, and secretion of ETA. Clearly, complete understanding of the regulation of ETA synthesis requires investigation into events that occur after regA, regB, and toxA transcription. Figure 2 is a working model incorporating data on the regulation of the regA locus and the transcription of the toxA structural gene. Early in cell growth (panel A), transcription initiated at the PI promoter results in the production of a long mRNA encoding both regA and regB. The presence of RegB appears to have a positive effect on transcription initiated from the PI promoter. RegA and RegB may be needed synergistically for autoregulation to occur. JRegA production (or RegA + RegB) results in a small enhancement of toxA transcription. ETA that is produced during this period is cell associated! and is not -detected in the supernatant.

348

WICK ET AL Earfy Events In Low or High Iron Medium

A.

•

+

pl Pz

T1


'pxA ------- ---- �

Late Eventa

� C. P1 P2 I

rppA

-Fe

loean

reaA

utUe or no regA or regB mRNA

toIA

---------------------.- � High Iron Medium

Figure 2

p

I

tqxA

I�

-- Low Iron Medium

secreted

Model for the regulation of regAlregB and toxA transcription. Panel A represents

transcriptional and regulatory events that occur during logarithmic growth. Panels B (cells grown in high iron medium) and C (cells grown in low iron medium) are events that occur in late logarithmic or early stationary phase. Horizontal arrows located below genetic regions depict transcripts. Solid or dashed lines indicate relative amounts of mRNA as determined by dot blot experiments using different probes. Arrows between protein products or between protein products and promoter regions indicate net positive (+) or net negative (-) interactions that affect either regAlregB or toxA transcription. Question marks refer to areas in which several mechanisms may be used to explain the net result. IND is a postulated inducer molecule that controls transcription from the regA P2 promoter region .

At the end of logarithmic growth (Figure 2, panels B and C), PI promoter function is reduced. Preliminary experiments indicate that this reduction is mediated by ETA. This type of feedback inhibition may keep the intracellular level of ETA relatively low during early stages of growth. If cells are growing in an iron-sufficient environment (Figure 2, panel B), the P2 promoter of regA fails to initiate transcription. The shut down of PI and the failure of P2 to initiate results in a 90-95% reduction of toxA transcription. Failure of P2 initiation suggests that an additional regulatory factor, a P2 inducer (IND), controls regAlregB transcription late in bacterial growth. The synthesis of IND may depend on growth under iron stress conditions. Alternatively, iron may bind to this factor and inactivate it. The transition of cells from late logarithmic phase to stationary phase in an iron-limited environment results in great enhancement of ETA production (Figure 2, panel C). Under iron stress, as the cells shut off PI activity, the P2 promoter is activated. The initiation of transcription of the P2 promoter



349

appears to be mediated by IND that is either synthesized or functional under these growth conditions. A shorter T2 transcript is produced that may include regB seque:nces. The net result of these events is that taxA transcription is enhanced and ETA is now detected as a secreted product. Why the same regulatory factors produced early in the cell growth cycle have less of an effect on toxA transcription is not clear. Differences in specific activity could be explained by various mechanisms including the difference in translational efficiency between the regA Tl and T2 transcripts, covalent modification of RegA or RegB, and the postulation of additional regulatory molecules that act directly on the toxA promoter. Additional studies to address these questions, as well as the questions surrounding the structure and function of RegA and RegB, are required to fully understand the regulatory pathway of ETA synthesis. MOLECULAR ANALYSIS OF EXOTOXIN A The previous section described the events relating to the transcription of the ETA structural gene, taxA . Once taxA is transcribed, the mRNA is translated and ETA protein is secreted from the bacterium. This portion of the review focuses on the translated product of toxA and, specifically, the molecular details of the structure-function relationship of this protein. Secretion of Exotoxin A from P. aeruginosa

Nucleotide sequence analysis of the toxA gene cloned from P. aeruginasa strain PA103 indicates that ETA is produced as a 638-amino acid precursor protein with a 25-amino acid leader peptide (39). Examination of the amino terminal residues of the extracellular form of ETA reveals that the leader peptide is removed when ETA is secreted by the organism. This process releases a mature single polypeptide of 613 amino acids (66,583 daltons) to the enviroIllment (39). The exact mechanism by which ETA is secreted from P. aeruginasa is not clear. ETA apparently does not traverse through the periplasmic space of this organism (62). Lory et al (62) proposed a model for ETA secretion in which the protein is cotranslationally secreted by passing from the cytoplasm to the external medium through regions where the bacterial inner and outer mem branes are fused (Bayer junctions). Hamood et al (42) demonstrated that the leader peptide plus the first 30 amino acids are sufficient for the secretion of recombinant ETA molecules into the culture medium by P. aeruginosa. Current studies, which involve cloning and characterization of genes involved in protein secretion from P. aeruginosa (3, 31), may help elucidate the mechanism of ETA secretion.

350

WICK ET AL

Expression of Exotoxin A Enzymatic Activity


ETA is secreted from its natural host as an inactive proenzyme that is toxic to cultured cells and animals but does not exhibit significant enzymatic activity in vitro (18, 55, 59, 98). The predominant enzymatic reaction catalyzed by ETA is ADP-ribosyl transferase activity: NAD+

ETA +

EF2

�

ADP-ribose-EF2

+

Nicotinamide

+

H+.

This enzymatic activity results in the covalent modification of EF2 (50), a necessary component of eukaryotic translational machinery. ADP-ribosylated EF2 no longer functions in protein synthesis and intoxicated cells die as a result of their inability to elongate polypeptide chains (49). In the absence of the target protein EF2, ETA exhibits a weak NAD-glycohydrolase activity (18, 55): NAD+

ETA +

H20

�

ADP-ribose

+

Nicotinamide

+

H+.

Potentiation of both of these enzymatic activities can be achieved in vitro

either by physically disrupting intact ETA to release an enzymatically active 26,000-dalton peptide (18, 59, 98) or by treating the protein with a denaturing agent and a reducing agent simultaneously (18, 55, 59, 98). ETA contains four disulfide bonds (2, 59). At least two of these must be reduced for full expression of enzymatic activity (59). Thus, for ETA to be active in catalysis, the protein must undergo conformational alteration. This requirement suggests that the enzyme active site is buried within the native protein and becomes available to the reaction substrates only after constraints imposed on the catalytic center by the rest of the molecule have been removed (2). Although the requirements for expression of enzymatic activity are well defined in vitro, the structural alterations that occur in ETA for enzymatic activity to be expressed inside eukaryotic cells are unclear.

Crystallographic Analysis of Exotoxin A Allured and coworkers (2) solved the crystalline structure of the inactive proenzyme form of ETA to 3.0-A resolution. Using the deduced amino acid sequence (39) and the crystallographic data, they defined three structural domains within ETA. Domain I, which encompasses the amino terminal half of the protein, is composed primarily of antiparallel beta sheets and includes residues 1-252 and 365-404. Domain I is divided into two subdomains, Ia (1-252) and lb (365-404), that are adjacent to one another in the crystalline model but are separated in the linear amino acid sequence by the residues of domain II. The small, centrally located domain II (amino acids 253-364) is


351

composed of six consecutive alpha helices. Domain Ill, which includes residues 405-613, contains the carboxy-terminal portion of the protein. A prominent structural feature within domain III, an extended cleft, was identi fied as the most likely location for the catalytic center of ETA (2).

Functional Analysis of Exotoxin A Domains The available evidence suggests that ETA recognizes and binds to eukaryotic cells via a specific receptor (68), although the precise nature of this receptor remains to be defined. Several investigators have used molecular techniques to define the regions of ETA responsible for eukaryotic cell recognition. One strategy used was to clone and express fragments of taxA in E. coli. The biologic activity of these recombinant proteins is then examined with respect to the ability of these peptides to block the inhibition of protein synthesis in cultured cells by a lethal challenge of native ETA. Using this approach, Hwang et al (48) and Guidi-Rontani & Collier (40) presented data demonstrating that recombinant proteins encoding domain Ia protect cultured cells from a cytotoxic challenge with native ETA. However, peptides lacking domain Ia but containing do mains II and III are not protective. This approach was extended to an in vivo system that showed that mice receiving purified recombinant ETA lacking domain Ia but retaining intact domains II and III had increased survival over mice receiving native ETA (48). These studies suggest that the amino acids within domain Ia are responsible for recognizing the eukaryotic receptor for ETA. Chemical or"biological modification of ETA was used to identify molecules with decreased cytotoxicity but full enzymatic activity and to screen for modifications that alter the kinetics of ETA and target cell interaction. This approach is useful for the identification of residues within domain Ia that may directly interact with the eukaryotic cell receptor. Pirker et al (87) character ized ETA proteins that were chemically derivitized with reagents that react with free amino groups, such as those present on Lys residues, to identify modified proteins with parental enzymatic activity but decreased cytotoxicity. Because domain II contains no Lys residues and enzymatic activity of the derivitized molecules was unaltered, the observed decrease in cytotoxicity appears to result from alteration of domain I Lys residues. These amino acids may be important in the interaction of ETA with the eukaryotic cell receptor. To determine which lysines were important, each of the 12 domain I Lys residues was converted to Glu, either singly or in multiples, by oligonuc leotide-directed mutagenesis (53). Conversion of Lys-57 to GIu results in a mutant ETA protein with a 50- to 100-fold reduction in cytotoxicity, indicat ing that Lys-57 plays a role in the cytotoxicity of ETA. Furthermore, competi tion binding assays show that the Glu-57 substitution specifically affects


DOMAIN I: EUKARYOTIC CELL BINDING


352

WICK ET AL

binding to the eukaryotic cell (53). Thus, Lys-57 appears to be a domain Ia residue that is important in eukaryotic cell recognition. Chaudry et al ( I 5) modified the amino acid sequence of domain Ia by inserting hexanucIeotides that encode a specific dipeptide at designated posi tions and examined the resultant ETA proteins for reduced cytotoxicity. A Glu-Phe dipeptide inserted between residues 60 and 61 results in an ETA molecule that shows a great reduction in cytotoxicity but retains parental levels of enzymatic activity. They further characterized the ability of the purified mutant protein to interact with the eukaryotic cell receptor and demonstrated that the mutant protein dissociates more readily than does native ETA (15). This result indicates that the dipeptide insertion disrupts the ability of ETA to interact with the eukaryotic cell receptor. Analysis of the crystal line structure of domain la reveals that the dipeptide insertion lies within a concave area that encompasses three antiparallel beta sheets contained within residues 55-61, 63-69, and 72-80. Lys-57, which was identified as having a role in eukaryotic receptor binding as discussed above (53), also lies within this area and, in fact, lies on the same beta sheet as the Glu-Phe dipeptide insertion. Thus, Chaudry et al (15) proposed that the concave area including the three beta sheets between residues 55-80 may be important in the interac tion of domain Ia with the eukaryotic cell receptor. A third approach that has been used to define the role of domain Ia in eukaryotic cell recognition is through the creation of recombinant ETA proteins that have altered target specificity (84). This has been done by removing the DNA encoding domain la from the ETA structural gene and splicing in DNA encoding an alternate target cell specificity. For example, a fusion of sequences encoding T cell growth factor alpha and ETA domains ll-Ib-III has been shown to recognize cells bearing epidermal growth factor receptors (13, 91). Similarly, chimeric ETA molecules of interleukin 2, interleukin 4, interleukin 6, or the T cell CD4 determinant to ETA domains ll-Ib-III have been characterized (see 84 and references therein). Thus, strategies using deletion and subclone analysis of ETA, chemical or biological modification of domain la residues, and formation of hybrid toxins lacking the native recognition sequences of ETA clearly delineate the receptor binding portion of ETA to structural domain la. DOMAIN II: TRANSLOCATION After binding to susceptible eukaryotic cells, the toxin molecule enters by receptor-mediated endocytosis, is internalized through clathrin-coated pits, and enters prelysosomal vesicles called endo somes (32, 78, 79, 90, 1 05). To release the active form of ETA into the cell cytosol, the toxin must be able to escape the endosome, presumably through a translocation event. Endosomes are a major site for macromolecular sorting in the cell ( l 05). They rapidly become acidified due to the action of proton


P. AERUGINOSA EXOTOXIN

A

353

pumps contained within the membrane (69, 94, 1 05). Several investigators (32, 76, 79) have demonstrated that a critical step in the intoxication process of ETA is exposure of the toxin to an acidic environment. The acidification step appears to be required for efficient binding and insertion of ETA into a bilayer (27, 11 0). The low pH induces conformational changes in ETA (28) that exposl;! hydrophobic regions normally buried within the molecule and allows the toxin to efficiently bind , insert into, and subsequently translocate a lipid bilay�:r (27-29). Such a process could facilitate the binding and traversal of ETA across the organelle membrane, which in turn, could allow the active form of ETA to translocate into the cell cytosol where the target protein, EF2, resides. However, from which organelle ETA enters the cytosol is still unclear. Whether a conformationally altered form of intact toxin, or a frag ment of the molecule, is responsible for the in vivo ADP-ribosylation of EF2 is also not known. Regardless of the precise mechanism of ETA exit from the vesicle and entry into the cytosol, the toxin must traverse a membrane at some point. Initial evidence obtained by deletion and subclone analysis suggested that domain II plays a role in the translocation process (48). Characterization of an ETA peptide containing a deletion of half of domain II revealed that this molecule retains full ADP-ribosyl transferase activity but lacks cytotoxicity. This deletion derivative was able to block the cytotoxicity induced by native ETA in tissue culture. Together, these observations suggested that the eu karyotic receptor binding and ADP-ribosyl transferase moieties were intact. Thus, the authors (48) proposed that the recombinant molecule, which was missing part of domain II, was deficient in the translocation function of ETA. Subsequent studies have used techniques such as deletion analysis and oligonucleotide-directed mutagenesis to define specific residues within do main II that are required for the translocation function of ETA. Deletion analysis of residues within domain II reveals molecules that have normal levels of ADP-ribosyl transferase activity but decreased cytotoxicity (91). The role of the disulfide bridge between Cys-265 and Cys-287 , which links the A and B helices of domain II, has been studied by mutating both of these residues to Ser (67, 91). Examination of purified mutant Ser-265/Ser-287 ETA revealed that the protein retains full enzymatic activity but is sub stantially reduced in cytotoxicity (67, 91). In addition, Siegall et al (91) used competition binding assays to show that the binding function of this mutant protein is unaltered. These data indicate that ETA proteins with alterations of domain II residues are defective in a step of intoxication that occurs after ETA binds and is internalized into the cell but before ADP-ribosylation of the target protein occurs. The defect in these mutant proteins is therefore believed to occur at the translocation step when ETA exits the eukaryotic vesicle to reach the cell cytosol.


354

WICK ET AL

If domain II is involved in the translocation step, it must be able to facilitate translocation of either intact ETA, or a fragment thereof, through the vesicle bilayer. Thus domain II may have the property of facilitating movement of proteins that are not normally secreted into or across a membrane. Chaudhary et al (14) examined such a feature of domain II. They showed that recom binant ETA molecules containing domain II alone, or domain II in combina tion with other portions of ETA, are secreted into the periplasm of E. coli. Furthermore, proteins that are not normally secreted, such as T cell growth factor alpha or alkaline phosphatase. can be altered by fusion to ETA domain II or domains II-Ib-III to result in molecules that are transported to the periplasm of E. coli. In some cases, these recombinant molecules were detectable in the growth medium (14). Certain structural features support the experimental evidence for the translocation function of domain II. For example, two of the domain II alpha helices, B and E, are 30 A long (2), a length sufficient to span a membrane. Although no extensive hydrophobic areas are evident within domain II, regions enriched in hydrophobic residues are present (2). One of these hydrophobic regions lies within the A helix, which is linked to the B helix by a disulfide bridge. Furthermore, the loop of residues that connects the A and B helices is located on the surface of ETA and contains three positively charged Arg residues (54). Because positively charged residues often flank hydrophobic transmembrane domains (99, 100), Jinno et al (54) used oligo nucleotide-directed lJ1utagenesis to examine the importance of these surface located domain II Arg residues. Mutation of either Arg-276 or Arg-279 to Lys, another basic amino acid, or to His, Glu, or Gly, results in molecules with negligible cytotoxicity without alteration of ADP-ribosyl transferase activity (54). This result suggests that Arg-276 and/or Arg-279 may be important for cytotoxicity. Further characterization of the mutant Gly-276 protein showed that this molecule is unaltered in its ability to bind and become internalized into cultured cells (54). Because of the apparent specific require ment for Arg at positions 276 and 279 for a fully cytotoxic ETA protein, Jinno and coworkers (54) hypothesized that either or both of these Arg residues may specifically interact with an intracellular component to allow translocation of ETA into the cytosol. They investigated the presence of a specific in tracellular site by noting that this site should be saturable in a concentration dependent fashion. They demonstrated that a chimeric ETA protein, which enters cultured cells primarily through a receptor other than the normal ETA receptor, could protect cells in a concentration-dependent manner against the cytotoxic activity of an ETA challenge. A similar chimeric ETA protein with a mutant residue (Gly) at position 276 did not exhibit this protective effect (54), which suggests the presence of a saturable step in intoxication that occurs at a level other than the receptor-binding or the enzymatic step.


P. AERUGINOSA EXOTOXIN

A

355

FurthermoTe, ETA molecules with a mutation at Arg-276 are defective at this stage of intoxication. Thus, the surface-located domain II Arg-276, and possibly Arg-279, may interact specifically with an intracellular eukaryotic cell component, perhaps within the vesicle containing internalized ETA, to facilitate translocation of this molecule into the cell cytosol. Although the translocation step of ETA intoxication is apparently a process that is difficult to characterize, studies such as these are expanding our understanding of this complex event. DOMAIN III: ENZYMATIC ACTIVITY After the translocation event, the active form of ETA enters the cytosol where it carries out its enzymatic reaction, the ADP-ribosylation of eukaryotic EF2. The enzymatic function of ETA resides in the carboxy-terminal portion of the molecule (39, 48). Deletion analysis from the amino- (48, 9 1 ) and carboxy- (17) terminal ends of ETA demonstrate that residUl�s 400-608 encompass the minimal region for stable expression of full enzymatic activity. This region includes structural domain III plus a few residues from the adjacent domain lb. Examination of the molecular model of ETA reveals an extended cleft within domain III that may function as the enzyme active center (2). Several different biochemical and genetic tech niques hav'e been used to delineate specific residues within domain III that are involved in catalysis. Such analyses have provided insight into how the reaction substrates, NAD + and EF2, interact with ETA. Several reports provide strong evidence that Glu-553 is an active site residue that is the nicotinamide subsite of NAD binding. In photoaffinity labeling studies using NAD preparations radioactively labeled at various positions, Carroll & Collier ( 1 2) showed that when the radiolabel is in the nicotinamide moiety of NAD, it is efficiently and specifically transferred to Glu-553 . Glu-553 is a domain III residue whose side chain carboxylate group extends into the cleft. Analysis of the photoproduct suggests that the side chain carboxylate of this residue is in close proximity to the nicotinamide ring of bound NAD in the enzyme-substrate complex (12). The use of several methodologies provided further evidence supporting an important role of Glu-553, or more specifically the side chain carboxylate of Glu-553, in the interaction of ETA with NAD. First, deletion of Glu-553 results in a stable, fully immunoreactive protein reduced in cytotoxicity and ADP-ribosyl transferase activity by > 1Q6-fold (66). Second, mutation of Glu-553 to Asp, the most conservative substitution possible, results in an ETA protein that is > 1Q4-fold less cytotoxic and > 1Q3 -fold less active in ADP-ribosyl transferase activity (25). Coupling the observation that the loss in enzymatic activity of the Asp-553 mutant toxin did not appear to result from a conformational change of the protein (25, 65) and the fact that the side chain carboxylate of Asp is shorter than that of Glu by a single carbon atom,


356

WICK ET AL

Douglas & Collier (25) hypothesized that the precise location of the position 553 carboxylate group within the cleft is an important factor in the catalytic function of ETA. Lukac & Collier (65) used oligonucleotide-directed mutagenesis to replace the wild-type Glu-553 with Cys to test the carboxylate-group hypothesis. This substitution results in a protein decreased in cytotoxicity and enzymatic activity by > 1Q4-fold (65). They then derivitized the Cys-553 mutant protein with iodoacetic acid, a carboxymethylation reagent, to produce an artificial carboxylate-containing side chain at position 553 that is approximately one A longer than the side chain when the native Glu is at position 553. This change results in a protein with > 1Q3 -fold increase in enzymatic activity (one-sixth the parental level) (65). Although these experiments provide strong evidence for a direct role of the carboxylate side chain of Glu-553 in the enzymatic reaction catalyzed by ETA, alternative explanations for the differences in the enzymatic activity observed between the Glu-553, Asp-553, and derivatized Cys-553 ETA proteins, such as differences in the pKas or steric differences in the side chains, have not yet been eliminated (65). Other residues involved in NAD binding in the cleft have been identified. By locating bound substrate fragments (adenosine, AMP, and ADP) crystal lographically, Brandhuber et al (10) put forth a model for the binding of NAD in the cleft. Their model implicates the involvement of several domain III residues in the interaction with NAD . For example, the model suggests that the nicotinamide ring of NAD would stack on the indole ring of Trp-466. Such an interaction could explain the earlier observation of tryptophan fluorescence quenching of an enzymatically active ETA fragment (18) . Brandhuber et al (10) also proposed that the positively charged side chain of Arg-458 and Arg-467 may interact with the negatively charged phosphates of NAD. Finally, the location of the phenolic side chain of Tyr-470 and Tyr48 1 , two residues that are located near the cleft, may be involved in the interaction of ETA with NAD. The role of both Tyr-481 and Tyr-470 in catalysis was investigated using oligonucleotide-directed mutagenesis. Lukac & Collier (64) made the con servative substitution of Phe for each of these Tyr residues independently and examined ADP-ribosyl transferase activity, NAD-glycohydrolase activity, and cytotoxicity of the Phe-470 and Phe-481 mutant proteins. They found that the Phe-470 mutation caused no significant change in any of these reactions and thus eliminated a direct role of the Tyr-470 phenolic side chain in catalysis (64). However, the mutant Phe-48I toxin shows a tenfold reduction in ADP-ribosyl transferase activity and cytotoxicity but was unaffected in NAD-glycohydrolase activity, suggesting that the Tyr-481 side group may be involved in the interaction of ETA with EF2 (64) . Genetic analysis of the taxA locus from a strain of P. aeruginasa that



357

produces a full size enzymatically inactive mutant form of ETA (CRM 66) (23) implicates another domain III residue in catalysis ( 104, 109). CRM 66 contains Tyr at position 426 whereas wild-type ETA has His at this position (104, 109). Substitution of Tyr-426 for His-426 results in loss of approx imately 901% of the parental ADP-ribosyl transferase activity (104). To further investigate the role of His-426 in catalysis, oligonucleotide-directed mutagenesis was carried out to change His-426 to several other residues, including Ala, Glu, Gly, Lys, or Pro. Examination of ADP-ribosyl trans ferase activity of recombinant ETA peptides encoding one of these residues at position 426 reveals that between 70-99.9% of this activity is lost depending on which residue is present (M. 1. Wick, 1. M. Cook, & B . H. Iglewski, manuscript in preparation). Quantitation of NAD-glycohydrolase activity using purified preparations of mutant proteins also shows a substantial reduc tion in this activity. The reduction in both ADP-ribosyl transferase and NAD-glycohydrolase activities suggests that ETA proteins with mutations at position 426 may be affected in their ability to effectively interact with NAD. However, alternative explanations for the observed reduction in these activi ties, such as conformational changes in the protein or alterations in the interaction with EF2, have not been eliminated. In summary, biochemical and genetic techniques have been valuable in providing insight into the interaction of domain III residues with the reaction substrates NAD and EF2. Glu-553 has been identified as the nicotinamide subsite of NAD binding. A proposed model for NAD binding in the cleft suggests that other residues, including Arg-458, Arg-467, and Trp-466 are involved in the interaction of domain III with NAD. Finally, genetic studies that specifically mutagenize Tyr-481 and His-426 provide evidence for the involvement of these residues in catalysis, although their precise role remains to be elucidated. SIGNIFICANCE AND PERSPECTIVES ADP-ribosyltransferase enzymes form a large and diverse family of proteins that function in eukaryotes, viruses, and prokaryotes (36 and references therein). In eukaryotic organisms, nuclear ADP-ribosylation reactions involve changes in DNA integrity and chromatin structure that occur during events such as sister chromatid exchange, repair of damaged DNA, gene rearrange ments, differentiation, and transformation. Cytosolic mono-ADP-ribosylation plays a regulatory role in cell metabolism by covalently modifying key factors involved in protein synthesis, the regulation of Ca2+ efflux, and cAMP production. ADP-ribosyltransferases have also been described in bacter riophage. For example, T4 encodes two ADP-ribosyltransferases that modify E. coli RNA polymerase, and N4 produces an enzyme that ADP-ribosylates a


358

WICK ET AL

variety of bacterial proteins. Bacterial systems that use this type of covalent modification to regulate their own metabolism have also been characterized. Rhodospirillum rubrum modifies the Fe-protein of the nitrogenase complex in response to the changing metabolic state of the bacterium (63). In addition, ADP-ribosylated bacterial substrates of approximately 20,000 and 32,000 daltons have been partially characterized from Pseudomonas maltophilia (26). Our studies (manuscript in preparation) suggest that ETA from P . aeruginosa regulates its own synthesis at early stages i n cell growth. The ubiquitous nature of ADP-ribosyltransferases and the variety of substrates used serves to emphasize the general biologic significance of these regulatory enzymes. Bacterial toxins form a subfamily of ADP-ribosyltransferases that use eukaryotic proteins as substrates (71). The modification of eukaryotic proteins generally results in loss of function, tissue damage, and at times death of the host. Bacterial ADP-ribosyltransferases have been useful tools to study the function of certain eukaryotic proteins. Without access to these enzymes, the characterization of such eukaryotic proteins as EF2, cytoskeletal components, and GTP binding proteins may have been significantly delayed. The in vestigation of the structure and function of prokaryotic toxins, and in particu lar ETA, has led to the development of chimeric molecules that can be targeted to specific cell populations. This development opens the possibility of creating therapeutic reagents to eliminate cancer or virus-infected cells (84). Bacterial toxins are also model systems to examine receptor-ligand interactions, translocation of proteins from one cellular compartment to an other, and catalysis in detail . Comprehending the synthesis, structure, and function of both eukaryotic and prokaryotic ADP-ribosyltransferases will remain an important field of study for many years. Literature Cited I. Aisen, P. , Listowski, I. 1980. Iron transport and storage proteins. Annu. Rev. Biochem. 49:357-93

2. Allured, V. S . , Collier, R. J . , Carroll, S. F . , McKay, D. B. 1 986. Structure of exotoxin A of Pseudomonas aeruginosa at 3 . 0-Angstrom resolution. Proc. Natl. Acad. Sci. USA 83:1 320-24 3. Bally, M . , Wretlind, B . , Lazdunski, A. 1989. Protein secretion in Pseudomonas aeruginosa : molecular cloning and characterization of the xcp-l gene. J. Bacteriol. 171 :4342-48 4. Bever, R. A . , Iglewski, B. H. 1 988. Molecular characterization and nucleo tide sequence of the Pseudomonas aeru ginosa elastase structural gene. J. Bac teriol. 170:4309--1 4

5. Bjorn, M . J . , Iglewski, B. H . , Ives, S . K . , Sadoff, J. C . , Vasil, M. L. 1978. Effect of iron on yields of exotoxin A in cultures of Pseudomonas aeruginosa PA- 103. Infect. Immun. 1 9:785-91 6. Bjorn, M. J . , Sokol, P. A . , Iglewski, B . H . 1979. Influence o f iron o n yields of extracellular products in Pseudomonas aeruginosa cultures. J. Bacteriol. 1 38: 1 93--200 7. Blumentals, I. I . , Kelly, R. M . , Gorzig lia, M . , Kaufman, J. B . , Shiloach, J. 1 987. Development of a defined medium and two-step culturing method for improved exotoxin A yields from

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Structure-activity relationships of an exotoxin of Pseudomonas aeruginosa.

Mouse liver contains a Pseudomonas aeruginosa exotoxin A-binding protein.

Temperature-dependent inactivating factor of Pseudomonas aeruginosa exotoxin A.

Toxicity of Pseudomonas aeruginosa exotoxin A for human macrophages.

Enzyme-linked immunosorbent assay for Pseudomonas aeruginosa exotoxin A.

Composition, function, and regulation of T6SS in Pseudomonas aeruginosa.

Exotoxin production by clinical isolates of pseudomonas aeruginosa.

Purification of Pseudomonas aeruginosa exotoxin by affinity chromatography.

Lipid interaction of Pseudomonas aeruginosa exotoxin A. Acid-triggered permeabilization and aggregation of lipid vesicles.

Production of exotoxin A by Pseudomonas aeruginosa in a chemically defined medium.

Effect of iron on yields of exotoxin A in cultures of Pseudomonas aeruginosa PA-103.

Protective efficacy of recombinant exotoxin A--flagellin fusion protein against Pseudomonas aeruginosa infection.

Properties of Pseudomonas aeruginosa exotoxin A ionic channel incorporated in planar lipid bilayers.

In vivo protective effect of lipopolysaccharide against Pseudomonas aeruginosa exotoxin A in mice.

Large-scale purification and characterization of the exotoxin of Pseudomonas aeruginosa.

Regulation of urea uptake in Pseudomonas aeruginosa.

Serum antibody to Pseudomonas aeruginosa exotoxin measured by a passive hemagglutination assay.

Role of exotoxin and protease as possible virulence factors in experimental infections with Pseudomonas aeruginosa.

The Frequency of Exotoxin A and Exoenzymes S and U Genes Among Clinical Isolates of Pseudomonas aeruginosa in Shiraz, Iran.

Conformational integrity of a recombinant toxoid of Pseudomonas aeruginosa exotoxin A containing a deletion of glutamic acid-553.

Solution structure and properties of AlgH from Pseudomonas aeruginosa.

Protective activity of antibodies to exotoxin A and lipopolysaccharide at the onset of Pseudomonas aeruginosa septicemia in man.

Antibodies to proteases and exotoxin A of Pseudomonas aeruginosa in patients with cystic fibrosis: Demonstration by radioimmunoassay.

Pseudomonas aeruginosa exotoxin: purification by preparative polyacrylamide gel electrophoresis and the development of a highly specific antitoxin serum.