The LspA proteins of H. ducreyi
remain among the largest prokaryotic polypeptides described to date. Despite their very large size (>4,000 aa), only two regions within the LspA proteins were previously shown to have any significant homology with functional domains characterized in other bacterial proteins (46
). A region near the N terminus of the LspA proteins contains a predicted secretion signal which resembles that present in the FhaB protein of Bordetella pertussis
) (Fig. ). In the C-terminal half of the LspA proteins, there is a domain that has homology with the YopT proteins of pathogenic Yersinia
) (Fig. ). In the present study, we show that the LspA proteins also contain motifs that are targets for tyrosine phosphorylation by macrophages.
When either whole H. ducreyi bacteria or H. ducreyi CCS was incubated with J774A.1 macrophages, the LspA proteins were tyrosine phosphorylated. Tyrosine phosphorylation of the LspA proteins could also be accomplished by nonimmune cells. In macrophages, tyrosine phosphorylation of the LspA proteins was found to be dependent on Src family PTKs. Site-directed mutagenesis of LspA1 fusion proteins allowed identification of motifs that contained tyrosines that were targets for phosphorylation by macrophage-encoded kinases. It should be noted that the LspA1 fusion proteins used in these site-directed mutagenesis experiments may fold differently from the much larger, full-length LspA1 protein. Use of a full-length LspA1 protein in site-directed mutagenesis studies will be necessary to confirm the identity of these tyrosine phosphorylation sites. Interestingly, tyrosine phosphorylation of one of these LspA1 fusion proteins could also be accomplished by macrophage lysates in an ATP- and divalent cation-dependent manner.
There are only a few reports of bacterial effector molecules that are tyrosine phosphorylated by eukaryotic kinases, and the function of the individual phosphorylated bacterial proteins varies widely. The Tir protein of enteropathogenic E. coli
) and the CagA protein of H. pylori
) are the best-studied examples of these types of effector molecules (for a review see reference 6
). Tir and CagA are injected directly into the eukaryotic cell by type III (10
) and type IV secretion systems (5
), respectively. Similarly, with the other three bacterial effector molecules that have been reported to be tyrosine phosphorylated (9
), injection-type secretion systems (i.e., type III or type IV) have been demonstrated or inferred to be necessary for the subsequent phosphorylation event (9
). The lack of a type III secretion system in H. ducreyi
(Robert S. Munson, Jr., personal communication) precludes direct injection of LspA proteins into macrophages by this particular method. In addition, although H. ducreyi
contains the flp
gene cluster that encodes a secretion system which shares conserved sequence features with some type IV secretion systems (31
), inactivation of the tadA
gene in the flp
operon did not eliminate the ability of H. ducreyi
to reduce the level of active Src PTKs in macrophages (data not shown). In addition, the absence of the tadA
gene product did not prevent tyrosine phosphorylation of the LspA proteins by macrophages (data not shown).
The truly novel finding from the present study is the ability of the LspA1 fusion proteins to be tyrosine phosphorylated by macrophage lysates produced by detergent solubilization. In addition, incubation of wild-type H. ducreyi bacteria with macrophage lysates also resulted in tyrosine phosphorylation of the LspA proteins (data not shown). These results were unexpected because after lysis of the macrophages the concentration of ATP (necessary for kinase activity) would be very minimal, and it is generally accepted that little or no tyrosine phosphorylation of proteins can be accomplished by eukaryotic cell lysates in the absence of exogenously added ATP. These data raised the possibility that either the LspA proteins have a strong interaction with macrophage-encoded PTKs or that the LspA proteins can bind ATP, thereby concentrating it and making it available for PTK activity. In this regard, it should be noted that the LspA proteins each contain a putative ATP/GTP binding domain (Walker motif A; GINTKGKT, aa 2206 to 2213 in LspA1 and aa 2344 to 2351 in LspA2). However, efforts to directly demonstrate ATP binding by the LspA fusion protein HL8 have been unsuccessful to date.
We also looked for other potential domains that might explain why the HL8 and GSTL fusion proteins differed in their ability to be tyrosine phosphorylated by macrophage lysates even though both of these fusion proteins were readily tyrosine phosphorylated by cells and purified c-Src PTK. The presence of more SH2 domain binding motifs in HL8 (three) than in GSTL (one) (Fig. ) raises the possibility that HL8 may bind a macrophage PTK more effectively than does GSTL. Perhaps more significantly, the Pho81 minimum domain homology in HL8 (Fig. ) distinguishes HL8 again from GSTL. Pho81 is a 130-kDa eukaryotic protein that inhibits the cyclin-dependent kinase Pho80-Pho85 under low-phosphate conditions (16
). An 80-aa motif in Pho81 (the Pho81 minimum domain; aa 645 to 724) is sufficient to inhibit Pho80-Pho85 (16
), and a 22-aa region within this motif has been shown to interact with the signaling molecule diphosphoinositol heptakisphosphate (24
). How the Pho81 minimum domain-like region in LspA1 might affect tyrosine phosphorylation of this H. ducreyi
molecule is not immediately apparent. However, it must be noted that CLUSTAL W-based analysis of the other bacterial proteins (Tir, CagA, Tarp, BepD, and AnkA) known to be tyrosine phosphorylated by eukaryotic cells revealed that they all had less homology with the Pho81 minimum domain than LspA1. In addition, within the primary amino acid sequence of each protein, the proven or putative tyrosine phosphorylation sites were generally distant from the region with any homology to the Pho81 minimum domain (data not shown).
Taken together, these data suggest that a previously undescribed interaction between the LspA proteins and both eukaryotic PTKs and related regulatory factors may provide an explanation for the unusual ability of these H. ducreyi proteins to be tyrosine phosphorylated by macrophage lysates. At the very least, the ability of the LspA proteins to undergo tyrosine phosphorylation by cell lysates raises the interesting possibility that extracellular LspA proteins in chancroidal lesions could be tyrosine phosphorylated and then contribute at some level to the disease process in chancroid. Whether the LspA proteins have to be tyrosine phosphorylated to allow H. ducreyi to inhibit phagocytosis also remains to be determined.
The basis for the physical interaction between the LspA proteins and the macrophage at the molecular level remains to be elucidated. Even in the absence of specific injection systems, other large bacterial exoproteins including the Vibrio cholerae
RTX toxin (>450 kDa) (14
) and the enteroaggregative E. coli
plasmid-encoded toxin, Pet (104 kDa) (30
), can gain access to the cytoplasm of the eukaryotic cell, where they exert their effects. Pet has been shown to be taken up in apparently intact form by clathrin-mediated endocytosis (29
). With the V. cholerae
RTX toxin, recent evidence indicates that after translocation of part of the toxin across the eukaryotic membrane, a cysteine protease domain within the RTX holotoxin cleaves the toxin at several different sites, thereby releasing one or more other activity domains into the cell cytoplasm (38
). It is therefore interesting that a region in the LspA proteins (Fig. ) has similarity to the YopT cysteine protease (37
). Whether this YopT-like region in the LspA proteins plays any role in the ability of H. ducreyi
to inhibit phagocytosis is currently under investigation.