Pseudomonas aeruginosa Type III Secretory Toxin ExoU and Its Predicted Homologs

Pseudomonas aeruginosa ExoU, a type III secretory toxin and major virulence factor with patatin-like phospholipase activity, is responsible for acute lung injury and sepsis in immunocompromised patients. Through use of a recently updated bacterial genome database, protein sequences predicted to be homologous to Ps. aeruginosa ExoU were identified in 17 other Pseudomonas species (Ps. fluorescens, Ps. lundensis, Ps. weihenstephanensis, Ps. marginalis, Ps. rhodesiae, Ps. synxantha, Ps. libanensis, Ps. extremaustralis, Ps. veronii, Ps. simiae, Ps. trivialis, Ps. tolaasii, Ps. orientalis, Ps. taetrolens, Ps. syringae, Ps. viridiflava, and Ps. cannabina) and 8 Gram-negative bacteria from three other genera (Photorhabdus, Aeromonas, and Paludibacterium). In the alignment of the predicted primary amino acid sequences used for the phylogenetic analyses, both highly conserved and nonconserved parts of the toxin were discovered among the various species. Further comparative studies of the predicted ExoU homologs should provide us with more detailed information about the unique characteristics of the Ps. aeruginosa ExoU toxin.


Introduction
Bacterial pneumonia that occurs frequently in patients under artificial respiration is defined as ventilator-associated pneumonia (VAP) [1]. Pseudomonas aeruginosa (hereafter called Ps. aeruginosa) is one of the most frequent causative bacterial agents of VAP [2]. Additionally, when Ps. aeruginosa is the causative agent of pneumonia, acute lung injury and secondary sepsis can easily occur, leading to high fatality rates [3][4][5][6][7]. In animal models of Ps. aeruginosa pneumonia, once cytotoxic Ps. aeruginosa strains are administered to the lungs of an experimental animal, the animal immediately develops an acute lung injury followed by bacteremia and secondary sepsis [8][9][10]. Over the past 20 years, investigations to identify virulence factors and their related mechanisms of action in Ps. aeruginosa have led to the discovery of the type III secretory toxin, ExoU, as a major virulence factor responsible for acute lung injury and sepsis [10][11][12][13]. In addition, clinical associations with Ps. aeruginosa ExoU genotype isolates have recently been reported in various other infections, such as chronic otitis media, keratitis, burns and bloodstream infections, and skin infections in diabetic patients [14][15][16][17][18][19].
Until recently, there was no obvious homolog of ExoU, with the exception of Rickettsia prowazekii PR534 [20], and Ps. aeruginosa ExoU was considered to be virtually the sole patatin-like cytotoxin secreted by the type III secretory mechanism. However, recent advances in DNA sequencing technologies have facilitated identification of several predicted ExoU homologs in various bacterial species, and these homologs have been reported in the bacterial genome database of the National Amino acid sequence alignment of ExoU, patatins, and human cytosolic/calcium 2+ -independent phospholipase A2 (cPLA2/iPLA2). (d) ExoU, patatins, and human cPLA2/iPLA2 phylogenetic trees. Left, rooted tree with branch lengths; right, unrooted tree with branch lengths. The phylogenetic trees and the sequence alignments were made by ClustalW with the BLOSUM protein weight matrix. GenomeNet, Kyoto University Bioinformatics Center, Japan [35].

ExoU and PLP A2 Activity
Patatins, which account for about 40% of the soluble protein in potatoes (Solanum tuberosum) and are present in cucumbers (Cucumis sativus) and Para rubber trees (Hevea brasiliensis), are plant storage glycoproteins with acyl hydrolase activities [36,37]. Patatins are also known to be cross-reactive latex allergens that can cause latex allergies (natural rubber fruit syndrome, latex-fruit syndrome). Conversely, it has been found that many latex allergens contain Hev b7, a protein with a structure similar to patatin [38,39]. Patatin exhibits insecticidal activity against plant pest larvae; it is also considered to be an immune system protein of the plant kingdom, and has become known as an infection-specific pathogenesis-related protein in the plant pathology field [40].

ExoU and PLP A2 Activity
Patatins, which account for about 40% of the soluble protein in potatoes (Solanum tuberosum) and are present in cucumbers (Cucumis sativus) and Para rubber trees (Hevea brasiliensis), are plant storage glycoproteins with acyl hydrolase activities [36,37]. Patatins are also known to be cross-reactive latex allergens that can cause latex allergies (natural rubber fruit syndrome, latex-fruit syndrome). Conversely, it has been found that many latex allergens contain Hev b7, a protein with a structure similar to patatin [38,39]. Patatin exhibits insecticidal activity against plant pest larvae; it is also considered to be an immune system protein of the plant kingdom, and has become known as an infection-specific pathogenesis-related protein in the plant pathology field [40].
Lipid-degrading enzymes generally possess the triad structure characterized by the presence of asparagine (or glutamic acid), serine, and histidine. However, unlike lipid-degrading enzymes, PLPs such as potato patatin B2, human cPLA 2 /iPLA 2 , and Ps. aeruginosa ExoU all generate their acyl hydrolase activities through the enzyme active site of the dyad formed by serine and asparagine [36]. There is a possibility that, during the evolutionary process, the gene encoding the patatin-like protein with insecticidal activity in plants was somehow incorporated into the genomes of pathogenic bacteria through a horizontal gene transfer mechanism. Indeed, R. prowazekii RP534-which was the only obviously recognized homolog of ExoU until recently [20]-has a PLP structure, and the recombinant protein of RP534 has PLA 2 and lysophospholipase activities. Therefore, there is a possibility that Rickettsia RP534 is the link between plant and bacterial patatins [20].

Conserved Domains in PLPs
The PLP protein family, which includes ExoU, contains four conserved domains (blocks I-IV) ( Figure 1c) [41,42]. Block I is a glycine-rich region containing an arginine or lysine residue and consisting of an oxyanion hole. Block II, which is located 10-20 amino acids away from block I, contains a GxSxG (or GxSxS) hydrolase motif sequence and a serine residue at the PLP enzymatic activity center. Block III contains several well-conserved proline residues in the group, and these are considered to be critical for maintaining the three-dimensional structure. In block III, the bacterial PLPs have an ASxxxP sequence, while the eukaryotic PLPs have an AAP sequence. Block IV contains an Asp-residue that is a pair component of a catalytic dyad with the Ser-residue of block I.
The patatin-like motif in ExoU is located at amino acid positions 110-354 in the amino-terminal side of the protein. First, the 110-125 position in block I of ExoU has an oxyanion hole (a glycine-rich nucleotide binding loop) comprising a GGGAxxG/A motif. The 139-145 position of ExoU block II has a hydrolase motif GxSxG (or GxSxS) with a catalytic center Ser 142 residue. ExoU block III contains a patatin-specific proline at position 320 within the 309-325 amino acid region. Position 341-352 of ExoU block IV contains a D-G/S-G/A motif containing the other catalytic center residue, Asp 344 . This block displays high sequence homology with the blocks of other PLPs (Figure 1c). In the phylogenetic diagram that was created based on the primary amino acid sequence of the region containing blocks I and II of the patatin-like domain, ExoU is located just between human cPLA 2 /iPLA 2 and a plant PLP group ( Figure 1d).
The Pat_ExoU_VipD_like domain family, which is one of seven patatin domain families, has a sequence cluster comprising ExoU of Ps. aeruginosa, VipD of Legionella pneumophila, several other L. pneumophila proteins, and proteins from Mycobacterium, Clostridium botulinum, Burkholderia xenovorans, and Salmonella enterica (Figure 2a). In addition, this family includes eukaryotic proteins from purple sea urchin, starlet sea anemone, Trichoplax adhaerens, and Florida lancelet. VipD was shown to have no phospholipase activity, even though it shares high sequence similarity with several functional regions of ExoU (e.g., oxyanion hole, active site serine, active site asparagine). Additionally, in the patatin-domain protein family, the closest homolog to Ps. aeruginosa ExoU is R. prowazekii PR534.

Predicted ExoU Homologs in Pseudomonas and Other Gram-Negative Bacteria
In our current search of the NCBI bacterial genome database we identified many predicted protein homologs of Ps. aeruginosa ExoU: 17 proteins from other Pseudomonas species and 8 proteins from other Gram-negative bacteria (Table 1, Figure 3a,b). Photorhabdus species are Gram-negative entomopathogenic bacteria of the family Enterobacteriaceae. Predicted ExoU homologs have been found in two Photorhabdus species. Among several members of this genus, Photorhabdus asymbiotica is a pathogen of both insects and humans. Ph. heterorhabditis, which was recently proposed to be a novel species of the genus Photorhabdus, was isolated from populations of the pathogenic insect nematode Heterorhabditis zealandica collected in South Africa [43]. It was reported that Ph. asymbiotica induces cellular death through the activation of caspases 8 and 9 and the executioner caspases 3 and 7 [44][45][46][47], and this suggests that the predicted ExoU homolog might be involved in the cell death mechanism.

Predicted ExoU Homologs in Pseudomonas and Other Gram-Negative Bacteria
In our current search of the NCBI bacterial genome database we identified many predicted protein homologs of Ps. aeruginosa ExoU: 17 proteins from other Pseudomonas species and 8 proteins from other Gram-negative bacteria (Table 1, Figure 3a,b). Photorhabdus species are Gram-negative entomopathogenic bacteria of the family Enterobacteriaceae. Predicted ExoU homologs have been found in two Photorhabdus species. Among several members of this genus, Photorhabdus asymbiotica is a pathogen of both insects and humans. Ph. heterorhabditis, which was recently proposed to be a novel species of the genus Photorhabdus, was isolated from populations of the pathogenic insect nematode Heterorhabditis zealandica collected in South Africa [43]. It was reported that Ph. asymbiotica induces cellular death through the activation of caspases 8 and 9 and the executioner caspases 3 and 7 [44][45][46][47], and this suggests that the predicted ExoU homolog might be involved in the cell death mechanism.
Aeromonas, a Gram-negative, facultative anaerobic and rod-shaped bacterium, is ubiquitous in fresh and brackish water [48]. Most Aeromonas species are known to be associated with human diseases. Predicted ExoU homologs were found in five Aeromonas species. Aeromonas jandaei is a Gram-negative bacterium isolated from human feces in Oregon, USA [49]. A. lacus is one of three groups of Aeromonas strains isolated from lakes in Finland [50], and while A. encheleia was isolated from European eels in Valencia, Spain [51][52][53], A. tecta is a strain isolated from the stool of a child with diarrhea, from a healthy patient, and from environmental sources [54]. Aeromonas, a Gram-negative, facultative anaerobic and rod-shaped bacterium, is ubiquitous in fresh and brackish water [48]. Most Aeromonas species are known to be associated with human diseases. Predicted ExoU homologs were found in five Aeromonas species. Aeromonas jandaei is a Gram-negative bacterium isolated from human feces in Oregon, USA [49]. A. lacus is one of three groups of Aeromonas strains isolated from lakes in Finland [50], and while A. encheleia was isolated from European eels in Valencia, Spain [51][52][53], A. tecta is a strain isolated from the stool of a child with diarrhea, from a healthy patient, and from environmental sources [54]. Paludibacterium yongneupense, a bacterium that falls within the family Neisseriaceae, was isolated from a Korean wetland area [55]. In the primary sequence of the predicted Pa. yongneupense ExoU, blocks I-IV of this ExoU homolog are a good match with those of Ps. aeruginosa ExoU. Figure 4 shows the amino acid sequences of eight predicted ExoU homologs from various species aligned against the Ps. aeruginosa ExoU sequence. The predicted ExoU homologs from the three non-Pseudomonas species (Paludibacterium, Photorhabdus, and Aeromonas) exhibit high sequence conservation in blocks I-IV of the ExoU patatin domain, implying that they should probably exhibit phospholipase A2 activities once they are properly secreted and activated in the targeted eukaryotic cells, although their functionalities have not been tested yet. Paludibacterium yongneupense, a bacterium that falls within the family Neisseriaceae, was isolated from a Korean wetland area [55]. In the primary sequence of the predicted Pa. yongneupense ExoU, blocks I-IV of this ExoU homolog are a good match with those of Ps. aeruginosa ExoU. Figure 4 shows the amino acid sequences of eight predicted ExoU homologs from various species aligned against the Ps. aeruginosa ExoU sequence. The predicted ExoU homologs from the three non-Pseudomonas species (Paludibacterium, Photorhabdus, and Aeromonas) exhibit high sequence conservation in blocks I-IV of the ExoU patatin domain, implying that they should probably exhibit phospholipase A 2 activities once they are properly secreted and activated in the targeted eukaryotic cells, although their functionalities have not been tested yet.    Conserved blocks in the PLP domain of predicted ExoU homologs from Pseudomonas aeruginosa and other bacterial species. The sequence alignment was made by ClustalW with the BLOSUM protein weight matrix (DNA Data Bank of Japan) [48].
Predicted Ps. aeruginosa ExoU homologs were found in the following 17 Pseudomonas species: Ps. fluorescens, Ps. lundensis, Ps. weihenstephanensis, Ps. marginalis, Ps. rhodesiae, Ps. synxantha, Ps. libanensis, Ps. extremaustralis, Ps. veronii, Ps. simiae, Ps. trivialis, Ps. orientalis, Ps. tolaasii, Ps. taetrolens, Ps. syringae, Ps. Viridiflava, and Ps. cannabina. The primary amino acid sequence alignment scores for these homologs against Ps. aeruginosa ExoU varied from 41% to 60%, as shown in Table 1. We previously investigated the polymorphism levels in four toxin genes, exoS, exoT, exoU, and exoY, of the P aeruginosa type III secretion system in several clinical isolates. We reported that ExoU, the largest of the toxin genes (2064 base pairs long encoding a 687 amino acid protein), is the most highly conserved of these toxins at the nucleic acid and amino acid sequence levels [56]. When compared with the PA103 reference strain, ExoU gene single nucleotide polymorphisms (SNPs) are scattered throughout the nucleotide sequence, but there are no changes in the 359 amino acid N-terminal sequence of the protein encoded by it. Furthermore, the five amino acid sequence alterations found in the C-terminal portion of the protein occur in only 16% of the strains that were sequenced. Therefore, the sequence variations in the ExoU homologs among Ps. aeruginosa ExoU and other Pseudomonad homologs are definitely much greater than those among clinical isolates of Ps. aeruginosa. However, as shown in Figure 5, the amino acid sequences of four key blocks in the predicted PLP domain of the 17 predicted ExoU homologs from Pseudomonas species are quite conserved, implying that these homologs should act as phospholipase A 2 molecules once they are properly activated via the type III secretion mechanism. The predicted ExoU homologs from Pseudomonas genera, with the exception of Ps. fluorescens, Ps. lundensis, Ps. weihenstephanensis, and Ps. taetrolens, are 43-55 amino acids shorter at their aminotermini than that of Ps. aeruginosa ExoU (Figure 6a). The amino termini of the predicted homologs from Ps. fluorescens, Ps. lundensis, and Ps. weihenstephanensis each have a leader sequence (MKIQS) that is quite similar to the type III secretory leader sequence (MHIQS) of Ps. aeruginosa ExoU ( Figure  6a). In contrast, the N-termini of the predicted homologs of other Pseudomonas species contain different leader sequence types such as MxVxx and MHxxS; however, we do not know whether they function as type III secretory leaders at this point in time. The predicted ExoU homologs from Pseudomonas genera, with the exception of Ps. fluorescens, Ps. lundensis, Ps. weihenstephanensis, and Ps. taetrolens, are 43-55 amino acids shorter at their amino-termini than that of Ps. aeruginosa ExoU (Figure 6a). The amino termini of the predicted homologs from Ps. fluorescens, Ps. lundensis, and Ps. weihenstephanensis each have a leader sequence (MKIQS) that is quite similar to the type III secretory leader sequence (MHIQS) of Ps. aeruginosa ExoU (Figure 6a). In contrast, the N-termini of the predicted homologs of other Pseudomonas species contain different leader sequence types such as MxVxx and MHxxS; however, we do not know whether they function as type III secretory leaders at this point in time. Notably, amino acid sequence alignments show that the N-termini of the predicted ExoU homologs of Photorhabdus and Aeromonas contain ExoU-like leader sequences (MQIxI or MQIQQ), suggesting that these predicted proteins are probably secreted through the type III secretion mechanism (Figure 6a). In their C-termini, some variations are apparent in the primary sequences of the predicted ExoU homologs among all 22 bacterial species, although conserved regions exist among these homologs. Because the carboxyl terminal of ExoU is reported to be associated with enzymatic activation, further comparative analyses will help to resolve questions about the activation mechanism of this protein (Figure 6b). Such analyses are important because, until recently, no ExoU homologs had been tested for their potential in vitro enzymatic or cytotoxic activities against Notably, amino acid sequence alignments show that the N-termini of the predicted ExoU homologs of Photorhabdus and Aeromonas contain ExoU-like leader sequences (MQIxI or MQIQQ), suggesting that these predicted proteins are probably secreted through the type III secretion mechanism (Figure 6a). In their C-termini, some variations are apparent in the primary sequences of the predicted ExoU homologs among all 22 bacterial species, although conserved regions exist among these homologs. Because the carboxyl terminal of ExoU is reported to be associated with enzymatic activation, further comparative analyses will help to resolve questions about the activation mechanism of this protein ( Figure 6b). Such analyses are important because, until recently, no ExoU homologs had been tested for their potential in vitro enzymatic or cytotoxic activities against eukaryotic cells. Therefore, in the absence of information about such activities, we will need to wait for cytotoxicity testing of the ExoU homologs to be conducted to learn more about these proteins.

Predicted Homologs of the Ps. aeruginosa ExoU Chaperone, SpcU
Ps. aeruginosa possesses the ExoU-specific chaperone protein, SpcU [22]. The gene for SpcU (spcU) is located just downstream of exoU (Figure 1a). Therefore, detecting the presence or absence of specific chaperones for each predicted ExoU homolog might help to determine whether the homologs are type III secreted or not. In the NCBI bacterial genome database, eight predicted homologs of SpcU were found in Ps fluorescens, Ps. lundensis, Ph. heterorhabditis, Ph. asymbioitica, A. jandaei, A. encheleia, A. diversa, and Pa. yongneupense, (Table 2, Figure 7). SpcU homologs have well-conserved primary sequences, especially in the PLP blocks of the central regions. The positions corresponding to ExoU Lys 178 , which is a known ubiquitination region, are conserved in all Aeromonas species and four Pseudomonas species, but not in the remaining six Pseudomonas and Paludibacterium species. The region corresponding to the ExoU carboxyl terminal residues 679-683, which has also been reported as the region ubiquitinated for enzyme activation, is quite well-conserved among all the predicted homologs.

Conclusions
ExoU, a major virulence factor in cytotoxic Ps. aeruginosa, has gradually revealed its unique characteristics since its discovery. There are still many unsolved questions about the ExoU toxin, such as its evolutionary origin, its association with its chaperone, the activation mechanism underlying its PLP activity, and the association between its PLP activity and the cell death mechanism. In this review, we analyzed the primary sequences of eight predicted ExoU homologs from Gram-negative  Alignment scores (as based on SpcU amino acid sequence similarities according to the BLOSUM protein weight matrix) are from the ClustalW multiple sequence alignment tool with the BLOSUM protein weight matrix (DNA Data Bank of Japan) [48]. bp: base pairs, aa: amino acids.

Conclusions
ExoU, a major virulence factor in cytotoxic Ps. aeruginosa, has gradually revealed its unique characteristics since its discovery. There are still many unsolved questions about the ExoU toxin, such as its evolutionary origin, its association with its chaperone, the activation mechanism underlying its PLP activity, and the association between its PLP activity and the cell death mechanism. In this review, we analyzed the primary sequences of eight predicted ExoU homologs from Gram-negative bacterial species, and 17 predicted homologs from Pseudomonas species other than Ps. aeruginosa. Both conserved and nonconserved regions were identified in the multiple alignments of the predicted primary sequences of the putative homologs. At this moment in time, the knowledge gap concerning the enzymatic functionality and cytotoxicity of the predicted homologs of ExoU will remain in place until in vitro and/or in vivo analyses are conducted on these proteins. Nevertheless, in the near future, we can probably obtain more knowledge about the unique characteristics of ExoU from cloning the genes from the predicted ExoU homologs of Ps. aeruginosa, from gene complementation studies, and investigation of the characteristics of recombinant proteins that share sequence homology with ExoU.