bacteria produce a number of Yops with different biochemical functions and cellular targets which disable the host immune response. In general, deletion of a single Yop reduces the severity of infection but does not completely attenuate the pathogen, whereas deletion of all the Yops severely abrogates virulence, indicating that each Yop plays a unique role in infection (29
). Each Yop has been studied in a variety of cell culture systems, and most Yops have multiple protein targets and several cellular phenotypes which could be important for some or all stages of infection (55
). To understand the functions of each Yop during infection, a combination of cell biological, bacterial genetics, and animal infection models are required to determine whether a specific biochemical function, protein target, and cellular phenotype are relevant during infection. Previous studies have shown that YopM is an essential virulence factor in Y. pestis
and Y. enterocolitica
) and that YopM localizes to the nucleus and cytoplasm in eukaryotic cells, binds to RSK1 in cultured HEK293 and J774.A1 cells, and increases the in vitro
kinase activity of RSK1 (27
). Here, we have detected two YopM-RSK1 complexes in infected macrophages (YMC1 and YMC2), defined sites of YopM that are required for its interaction with RSK1 in each of these complexes, and found that mutants unable to form detectable YMC1 complexes are attenuated for virulence in mice. In particular, the C-terminal tail of YopM is necessary for interaction with RSK1 and virulence, since alanine substitution of the last and penultimate set of three amino acids of YopM prevented both its ability to form detectable levels of YMC1 and YMC2 and its ability to promote colonization of spleens and lungs of mice. However, strains expressing YopM mutants that failed to bind detectably to RSK1 caused more pathology and inflammation than did a ΔyopM
deletion mutant, suggesting that binding to RSK1 is not the only consequential action of YopM during infection. Combined, these data support the hypothesis that RSK1 is a relevant, but not the only, target of YopM during infection of mice. Alternatively, the C-terminal mutants may bind to RSK1 at low levels, which may in turn cause the intermediate pathology observed with these mutants.
Previous work demonstrated that YopM interacts with both RSK1 and PRK2 in HEK293 cells transfected with a FLAG-YopM fusion; however, PRK2 was not detected after immunoprecipitation of YopM in infected J774A.1 cells (32
). While RSK1 was seen in both complexes in infected RAW 264.7 cells, PRK2 was not detected in either complex. The complete composition of these complexes is unknown, but based on their migration during native electrophoresis, the smaller complex could be composed of a monomer of YopM with RSK1 while the larger could be multimers of one of both proteins. Alternatively, both might contain one or more unidentified targets. In fact, YMC2 could consist of more than one complex, because multiple high-molecular-weight complexes may not be resolved on these gels. McDonald et al. used Nonidet P-40 (NP-40) to prepare lysates for coimmunoprecipitation of FLAG-YopM and potential targets in HEK293 cells (32
); however, we observed that lysis with NP-40 disrupts the higher-molecular-weight complex in our native binding assay (data not shown). Thus, the possibility that there are novel targets in these complexes is consistent with the experiments carried out by these authors. Since no studies have yet identified putative target proteins from the nuclear compartment of cells and since our lysis method should release proteins from the nuclear compartment of cells, this remains an open area of investigation.
We found that deletion of LRRs 8 and 9, 10 and 11, 12 and 13, and 14 and 15 reduced the amount of YopM detected in the nuclei of macrophages, while deletions of the other LRRs or the 24 C-terminal amino acids had only modest effects on nuclear localization of YopM, suggesting that LRRs 8 to 15 are important for nuclear localization. Mutants with mutations in LRRs 8 to 15 also did not form detectable levels of YMC2, raising the possibility that the formation of the higher-molecular-weight complex might depend on translocation to the nucleus. Mutants C7 and C8 were detected in the nucleus even though they were unable to bind to RSK1, suggesting that YopM is not shuttled to the nucleus through its association with RSK1. Other studies have examined the requirements for nuclear localization of YopM by fusing YopM to reporter proteins and/or by detecting the presence of YopM proteins by microscopy (5
). Skrzypek et al. showed by indirect immunofluorescence that YopM mutants from Y. pestis
lacking LRRs 4 to 7 or 7 to 10 were detected in the nuclei of infected HeLa cells and that the N-terminal half of YopM fused to enhanced GFP (EGFP; yEGFP-YopM ΔLRR8-end) localized to the nucleus of yeast (52
). While these results appear to conflict with ours, immunofluorescence is not quantitative and our Y. pseudotuberculosis
YopM mutants with deletions in LRRs 8 and 9 through 14 and 15 were detectable in the nuclear fraction of macrophages, albeit at much-reduced levels. In addition, there may be differences between nuclear trafficking of YopM in HeLa and yeast cells and that in macrophages and/or between that in Y. pseudotuberculosis
- and that in Y. pestis
-infected cells. Benabdillah et al. showed that trafficking of an EGFP-YopM fusion protein into the nucleus of transfected HEK293T cells was almost completely abolished by specific mutations within the last six amino acids of YopM to alanines (5
). In addition, these authors found that the last 32 amino acids of YopM act as a nuclear localization sequence for LexA-AD and GFP fusion proteins in yeast, as well as an EGFP-LexA-AD fusion protein in transfected HEK293T cells. While our YopM C7 and C8 mutants were not identical to those of Benabdillah et al., C7 and C8 trafficked efficiently to the nuclei of macrophages, and ΔC did so only slightly less. In agreement with our data, Skrzypek et al. also found that the 32-residue tail of Y. pestis
YopM was not sufficient to concentrate yEGFP in the nucleus of yeast cells and that YopM lacking the tail (yEGFP-YopM-NT) efficiently localized to the nucleus (52
). Finally, if the C terminus of YopM were sufficient to direct YopM to the nucleus, one would expect that the mutants with deletions in LRRs 8 and 9, 10 and 11, 12 and 13, and 14 and 15 would traffic to the nuclei. The failure of these mutants to efficiently localize to the nucleus indicates that in the context of cell culture infection, the C terminus of YopM is not sufficient to promote normal levels of nuclear localization.
An important conclusion that can be drawn from the fitness of the Δ6-7rec, Δ8-9rec, and Δ10-11rec mutants is that YopM can undergo a decrease of two LRRs in length without being affected in its contribution to colonization of the tissues that we have studied here. Results from the work of Hines et al. support this conclusion. When LRRs 4 to 7 and LRRs 8 and 9 were deleted, YopM could be cross-linked to thrombin. However, if LRRs 4 to 9, 6 to 9, or 7 to 10 were deleted, cross-linking was reduced (Δ6-9) or abolished (Δ4-9 and Δ7-10), and a deletion of 3 LRRs resulted in a 1,000-fold increase in the LD50
of Y. pestis
after i.v. infection of mice (23
Deletion of LRRs 8 and 9, 10 and 11, 12 and 13, or 14 and 15 disrupted YMC2 and reduced nuclear localization of YopM; however, Δ8-9, Δ10-11, and Δ12-13 had no effect on colonization of Y. pseudotuberculosis. The simplest conclusion from these data is that YMC2 and wild-type levels of nuclear localization are not essential for YopM's contribution to tissue colonization and virulence. Alternatively, YMC2 formation by Δ8-9, Δ10-11, and Δ12-13 might occur at an undetectable but sufficient level for tissue colonization, or colonization 4 days postinfection may be the wrong readout for detecting a phenotype of these mutants; time-to-death or LD50 assays for virulence might reveal differences between these strains and the wild type. An observation which lends support to the possibility that Δ8-9 and Δ10-11 mutations did attenuate the virulence of Y. pseudotuberculosis in other ways is that the mice infected with the Δ6-7rec strain, which expresses a form of YopM that can take part in both complexes with RSK1, consistently showed greater signs of pathology, including a more scruffy appearance, bleeding in various organs, and cases of premature death, than did those infected with the Δ8-9rec or Δ10-11rec strain.
The histopathological and flow cytometric results for the C-terminal mutants also highlight the fact that a phenotype in one assay does not necessarily describe all aspects of a mutant. Intriguingly, infection with the C7rec and C8rec strains caused more pathology and inflammation in spleens than did infection with the yopM
deletion strain, although the bacterial loads were similar. This suggests that YopM may have multiple functions in cells in the spleen and that the C7 and C8 mutants retain some of this activity. Alternatively, YopM itself may trigger an immune response regardless of its ability to interact with RSK1, or the C-terminal mutants may retain RSK1 binding activity which was below our limit of detection but which was sufficient to cause increases in pathology. In either case, the WTrec strain caused an increase in the percentage of GR1high
cells in the spleens of infected animals, and this change was partially dependent on YopM. While the total numbers of splenocytes were not determined, spleens from WTrec-infected mice were not different in size or weight from spleens from yopM
mutant-infected mice, and so it is likely that the changes in percentages of GR1high
cells reflect changes in total numbers of GR1high
cells. This is an interesting result in light of the recent finding that a yopM
mutant of Y. pestis
was rescued by depletion of GR1+
). With this in mind, one interpretation of our results is that WT Y. pseudotuberculosis
can tolerate higher levels of neutrophil recruitment because YopM functions to inhibit neutrophil functions.
Our flow cytometry results revealed out that while infection with wild-type Y. pseudotuberculosis
results in a decrease of CD49bhigh
NK cells, this effect is not dependent on YopM, since mice highly colonized with a ΔyopM
mutant also showed a decrease in NK cells. Decreased numbers of NK cells were also observed in mice highly infected with ΔyopE
mutants, adding further support to the notion that high bacterial CFU counts, similar to WT-like levels, are the cause of the depletion of NK cells. This result displays how the uncoupling of bacterial burden from the effect of removal of proteins can reveal more about the direct functions of proteins by removing the secondary effects of changes in colonization levels. However, these results do not rule out the possibility that YopM contributes to a decrease in NK cells during Y. pestis
infection and/or in C57BL/6 mice, as previously reported (25
This study also reveals some of the differences between the Y. pseudotuberculosis
strains YPIII and IP2666. After oral infection, wild-type IP2666 grew to slightly higher levels than did wild-type YPIII in all tissues examined, and differences between the wild-type and yopM
strains were more apparent in the less virulent YPIII strain (43
). An overall reduction in virulence of YPIII could render YopM more valuable for survival of YPIII. Another difference between the strains was that the IP2666 strain was consistently found in the spleen and liver of orally infected mice, whereas the YPIII strain often did not reach these systemic tissues.
In conclusion, the demonstration that YopM binding with RSK1 correlates with virulence in mice should prove useful in the continuing goal of understanding the mechanism by which YopM works. Future experiments include identifying the differences between the two YopM/RSK1 complexes, analysis of the downstream effects of complex formation, and further characterization of the cell types affected by the wild-type and yopM mutant strains during infection.