The study of the SPATE proteins and their roles in virulence continues to be a focus of several laboratories. However, the emphasis heretofore has been on each individual SPATE, without the examination of the family as a whole. Here, we have addressed the divergence and conservation of sequence and function within the family. These studies will provide a foundation for comparative structure-function analyses.
Several SPATE proteins, namely Pet, Sat, EspP, and SepA, have previously been suggested to produce cytotoxic effects. Cellular effects have not been reported for other SPATEs. In our hands, only Pet and Sat were able to elicit cytopathic effects on HEp-2 cells, indicating that the SPATE proteins were functionally distinct. We addressed the basis for the inability of some SPATEs to damage cells. Although the mechanism of Pet-induced damage remains to be fully elucidated, previous data suggest that Pet must bind to the HEp-2 cell surface, be internalized, and exert a proteolytic attack on an intracellular host protein (18
). Villaseca and coworkers have suggested that this target protein is spectrin and have shown the cleavage of erythroid spectrin in vitro by Pet (18
). Spectrin, a heterodimeric cytoskeletal protein, serves to link actin filaments with other cytoskeletal proteins and the plasma membrane (1
). Proteolytic attack on spectrin, leading to rearrangements of associated actin microfilaments, is a plausible mechanism behind the effects observed when HEp-2 cells are treated with Pet. Based on this proposed model, it seems likely that the noncytopathic SPATEs may be deficient at one or more of these steps: binding, internalization, or catalysis.
When spectrin was incubated with each SPATE overnight, only Pet, Sat, and, surprisingly, EspC cleaved this protein. Whereas both EspC and Pet exhibited acid-dissociable binding to HEp-2 cells, EspC was internalized far less efficiently. Previous studies have shown that Brefeldin A inhibits the cytopathic effects of Pet by disrupting its trafficking within HEp-2 cells (18
). These data suggest that Pet may employ retrograde transport via the Golgi apparatus, as do several other bacterial toxins; however, no endoplasmic reticulum retrieval signature is apparent in Pet. The details of Pet trafficking are as yet unclear, but further comparisons with EspC may be illuminating.
In addition to spectrin, we assayed the cleavage of other proposed biological substrates that have been reported for the SPATEs. EspP has been shown to cleave pepsin (5
). Given that passage through the stomach is required of all enteric pathogens, we hypothesized that all the SPATEs should cleave pepsin. However, only EspC, EspP, and Pet were able to degrade pepsin, indicating that this function is not common to all SPATEs. Notably, it has yet to be proven that these proteins are able to cleave pepsin in vivo. Similarly, mucinases are a growing class of proteins that could be adaptive for all mucosal pathogens. Our lab has shown previously that the Pic protein hydrolyzes bovine submaxillary mucin (12
). When the other SPATEs were assayed in a similar manner, only Tsh, in addition to Pic, exhibited mucinolytic activity.
Brunder et al. also reported that EspP cleaved human coagulation factor V, a potentially important function for EHEC. Coupled with the actions of potent cytotoxins that can damage the colonic mucosa, cleavage of factor V could aggravate the hemorrhagic colitis characteristic of EHEC infection (5
). We hypothesized that this function could also be adaptive for Shigella
spp., which also produce bloody diarrhea. Interestingly, however, the ability to cleave factor V was widespread, as EspC, Pet, Pic, Sat, and Tsh were also able to cleave the purified protein. In whole sera, all but Tsh obliterated the protein. Importantly, cleavage of factor V has not yet been demonstrated in the setting of infection.
In light of these findings, we reexamined the phylogenetic relations for this family of proteins. Our lab has shown here and previously that, when the entire protein sequences are considered, the SPATEs can be divided into two groups (11
), exemplified by Pic and Pet. Interestingly, phylogenetic analysis of entire SPATE passenger domains did not reveal a correlation with the biological substrates but did show evidence of significant homologous recombination among family members. Indeed, highly similar proteins such as Pet and Sat (which are 53% identical overall) do not share oligopeptide specificities despite shared abilities to cleave spectrin and cause cytopathic effects in HEp-2 cells. In contrast, proteins that are less similar, such as Pic and Sat (30% identical), do share some specificities for oligopeptides despite their being classified into different groups and cleaving different biological substrates.
In light of these data, we asked whether the SPATE protease domains would correlate better with cleavage profiles. Accordingly, split decomposition analysis was performed on the N-terminal third of each SPATE amino acid sequence. This region was suggested to comprise the proteolytic triad in Hap, a related autotransporter in H. influenzae
). The resulting phylogram is shown in Fig. (14
). The original bifurcating phylogenetic pattern remains, with EspC, EspP, Pet, and Sat comprising one group and Pic, Tsh, and SepA forming the other (11
). The major difference in the second tree, however, is that EspC is now grouped closer with Pet and Sat than in the original analysis. This difference is consistent with our hypothesis in that EspC, Pet, and Sat are the SPATEs able to cleave spectrin. These data suggest that significant substrate specificity may be largely determined by protease domains.
However, the phylogenic groupings of the protease regions are not completely consistent with the oligopeptide cleavage profiles. For example, Pet, Sat, Pic, and Tsh show the preferential cleavage of short chains of small, hydrophobic amino acids such as Ala-Ala-Pro-Ala, Ala-Ala-Pro-Abu (2-aminobutyric acid), and Ala-Ala-Pro-Val, thus indicating a strong similarity to the elastase family of proteases. In contrast, both EspC and EspP, which the tree shows to be closely related, have a high affinity for the Arg-Arg oligopeptide. This observation indicates that the active site clefts of these proteases accommodate basic, positively charged amino acids, much like trypsin. SepA, on the other hand, is the most distantly related SPATE and uniquely cleaves large, hydrophobic amino acids (exemplified by cleavage of the oligopeptide Suc-Val-Pro-Phe) (8
). Of note, Hap has also been reported to accommodate bulky, hydrophobic residues in its active site (9
In summary, many pathogenic bacteria secrete proteases to serve multiple functions. These SPATE proteins have been found in a variety of human pathogens, but no common role in virulence has been demonstrated. We show here that these proteins have varying effects on HEp-2 cells and that their specificity extends not only to proposed biological substrates but also to oligopeptides, indicating diversity at the active site. These observations suggest a common ancestral SPATE protein that each pathogenic species has modified to permit adaptation to its specific niche. Further characterization of this diversity will greatly enhance the understanding of structure-function relationships in this family of proteins.