Gr1+ Cells Control Growth of YopM-Negative Yersinia pestis during Systemic Plague

YopM, a protein toxin of Yersinia pestis, is necessary for virulence in a mouse model of systemic plague. We previously reported YopM-dependent natural killer (NK) cell depletion from blood and spleen samples of infected mice. However, in this study we found that infection with Y. pestis KIM5 (YopM⁺) caused depletion of NK cells in the spleen, but not in the liver, and antibody-mediated ablation of NK cells had no effect on bacterial growth. There was no YopM-associated effect on the percentage of dendritic cells (DCs) or polymorphonuclear leukocytes (PMNs) in the early stage of infection; however, there was a YopM-associated effect on PMN integrity and on the influx of monocytes into the spleen. Ablation of Gr1⁺ cells caused loss of the growth defect of YopM⁻ Y. pestis in both the liver and spleen. In contrast, ablation of macrophages/DCs inhibited growth of both parent and mutant bacteria, accompanied by significantly fewer lesion sites in the liver. These results point toward PMNs and inflammatory monocytes as major cell types that control growth of YopM⁻ Y. pestis. Infection with fully virulent Y. pestis CO92 and a YopM⁻ derivative by intradermal and intranasal routes showed that the absence of YopM significantly increased the 50% lethal dose only in the intradermal model, suggesting a role for YopM in bubonic plague, in which acute inflammation occurs soon after infection.Yersinia pestis, the infective agent of bubonic and pneumonic plague, has caused widespread loss of human life during recurrent pandemics. Y. pestis still infects rodent populations in large geographic zones where Y. pestis is endemic, and there are cases of human plague reported annually (15, 19, 56). Y. pestis and the closely related food-borne pathogens Yersinia pseudotuberculosis and Yersinia enterocolitica share a 70-kb plasmid carrying genes that encode a major set of proteins involved in pathogenic properties that compromise the host immune system (60). These include a type 3 secretion system (T3SS) that at mammalian body temperature delivers a set of six Yersinia outer protein (Yop) effector proteins into host cells once the bacteria contact host target cells. Enzymatic and cell biological mechanisms of five of the Yops, YopH, YopE, YopT, YpkA/YopO, and YopJ, have been elucidated. YopJ interferes with signal transduction through acetyltransferase activity but is not required for virulence in either a mouse model of systemic plague (57) or mouse and rat models of bubonic plague (28, 65). In tissue culture infection models, YopH, YopE, YopT, and YopO have been shown to antagonize focal complex formation and activity of Rho family GTPases and synergistically inhibit phagocytosis by mammalian cells. YopH and YopE have been shown to be crucial for lethality in a mouse model of systemic plague (intravenous [i.v.] infection), and a ΔyopH strain is attenuated for both bubonic and pneumonic plague (9). In addition, Y. pestis virulence proteins, such as the surface fibrils F1 and PsaA, have antiphagocytic effects and also have been found to contribute to virulence in systemic plague (7, 31). Accordingly, Y. pestis is believed to exist predominantly in an extracellular location in vivo, although initially the bacteria might invade resting tissue macrophages (Mφs) and dendritic cells (DCs), based on assays of mouse spleens in the systemic phase of bubonic plague (33). The intracellular versus extracellular locations of Y. pestis during the peripheral phases of plague on skin or in the lung have not yet been studied.It is believed that tissue Mφs, DCs, and polymorphonuclear leukocytes (PMNs) are early target cells for Yop delivery in vivo, because these cells are present before or soon after infection begins and function to initiate the innate defenses that are undermined by Yops. Consistent with this hypothesis, Y. pestis has been found in association with alveolar Mφs early during lung infection of mice (6) and likewise in association with Mφs, DCs, and PMNs in the spleens of mice infected i.v., and YopM can be injected into these cells (34). However, it is becoming clear that spleens and lungs present distinctly different inflammatory environments when infected by Y. pestis, with PMNs migrating rapidly into spleens infected by the i.v. route but not appearing in lungs until 36 h after intranasal (i.n.) infection (27, 58). Accordingly, some virulence properties required for lethality of systemic plague are not required in pneumonic plague. Examples are the capsular fibril F1 and the antiphagocytic adhesin PsaA (7, 11, 12). There is no information yet on the target cells or relative importance of Yops other than YopH in pneumonic or bubonic plague.The sixth effector Yop, YopM, is essential for virulence in the mouse model of systemic plague: in C57BL/6 mice, a YopM⁻ strain of Y. pestis KIM5 is reduced in lethality by at least 4 orders of magnitude (29). However, the function of YopM has not been defined. YopM is a 46.2-kDa acidic protein made up almost entirely of 15 repeats of a 19-residue leucine-rich repeat motif (30). The YopM monomer is horseshoe shaped and has the potential to form tetramers in which the monomers stack together to form a hollow cylinder; however, the form that YopM assumes within the mammalian cell is not known (16). After delivery to the host cell cytoplasm, YopM localizes to the nucleus in a process that is facilitated by vesicular trafficking (53). YopM was reported to form a complex with the serine/threonine kinases PRK2 (protein kinase C-related kinase 2) and RSK1 (90-kDa ribosomal S6 kinase) in HEK293 cells infected with Y. pseudotuberculosis (36), leading to activation of both kinases. However, the biological significance of this complex is not known. There is no visible effect of delivery of YopM into cultured cells, and microarray analysis of Mφ-like cell lines infected with Y. enterocolitica having or lacking YopM also has not yielded any clue to YopM''s mechanism of action (21, 50).Because these and other in vitro approaches to defining the pathogenic mechanism of YopM have not been fruitful, we have begun to characterize YopM''s effects in vivo. Previously we found that YopM was still required for lethality in i.v. infected SCID mice, showing that YopM''s virulence mechanism does not require B or T cells and indicating that early in systemic plague, YopM''s main function is to counteract a component of innate immunity (25). A striking YopM-specific effect during systemic plague in wild-type C57BL/6 mice was the depletion of natural killer (NK) cells from the spleen and a reduction of NK cell numbers in blood, suggesting that YopM might cause a loss of the NK cell compartment during systemic plague. Correlated with this effect, there was a YopM-associated loss of mRNA for gamma interferon (IFN-γ) by NK cells in infected spleens and diminished expression of mRNAs in splenic Mφs for cytokines that are required for viability and activation of NK cells (interleukin 15 [IL-15], IL-18, and IL-12). These findings supported the hypothesis that YopM may function to inhibit IFN-mediated activation of Mφs through the depletion of NK cells (25).In this study, we tested the hypothesis that NK cells are critical for controlling Y. pestis pathogenesis early in plague and that NK cells are the primary target of YopM. We characterized infection dynamics and leukocyte populations in both the liver and spleen during systemic plague in mice ablated for Mφs/DCs or for Gr1⁺ cells. The data point to PMNs and inflammatory monocytes as critical cells affected by YopM and to Mφs/DCs as an important early reservoir for bacterial growth. Consistent with a role in undermining acute inflammation, YopM was found to be important for the lethality of bubonic plague, but not pneumonic plague.