ABSTRACT
The virulence protein YopM of the plague bacterium Yersinia pestis has different dominant effects in liver and spleen. Previous studies focused on spleen, where YopM inhibits accumulation of inflammatory dendritic cells. In the present study we focused on liver, where PMN function may be directly undermined by YopM without changes in inflammatory cell numbers in the initial days of infection, and foci of inflammation are easily identified. Mice were infected with parent and ΔyopM-1 Y. pestis KIM5, and effects of YopM were assessed by immunohistochemistry and determinations of bacterial viable numbers in organs. The bacteria were found associated with myeloid cells in foci of inflammation and in liver sinusoids. A new in-vivo phenotype of YopM was revealed: death of inflammatory cells, evidenced by TUNEL staining beginning at d 1 of infection. Based on distributions of Ly6G(+), F4/80(+), and iNOS(+) cells within foci, the cells that were killed could have included both PMNs and macrophages. By 2 d post-infection, YopM had no effect on distribution of these cells, but by 3 d cellular decomposition had outstripped acute inflammation in foci due to parent Y. pestis, while foci due to the ΔyopM-1 strain still contained many inflammatory cells. The destruction depended on the presence of both PMNs in the mice and YopM in the bacteria. In mice that lacked the apoptosis mediator caspase-3 the infection dynamics were novel: the parent Y. pestis was limited in growth comparably to the ΔyopM-1 strain in liver, and in spleen a partial growth limitation for parent Y. pestis was seen. This result identified caspase-3 as a co-factor or effector in YopM's action and supports the hypothesis that in liver YopM's main pathogenic effect is mediated by caspase-3 to cause apoptosis of PMNs.
Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Caspase 3/metabolism , Liver/metabolism , Spleen/metabolism , Yersinia pestis , Animals , Caspase 3/genetics , Cell Death , Disease Models, Animal , Female , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Inflammation/immunology , Inflammation/metabolism , Liver/immunology , Liver/microbiology , Liver/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Neutrophils/immunology , Neutrophils/metabolism , Nitric Oxide Synthase Type II/metabolism , Plague/immunology , Plague/metabolism , Plague/microbiology , Plague/pathology , Spleen/microbiology , Virulence Factors , Yersinia pestis/pathogenicityABSTRACT
YadB and YadC are putative trimeric autotransporters present only in the plague bacterium Yersinia pestis and its evolutionary predecessor, Yersinia pseudotuberculosis. Previously, yadBC was found to promote invasion of epithelioid cells by Y. pestis grown at 37 °C. In this study, we found that yadBC also promotes uptake of 37 °C-grown Y. pestis by mouse monocyte/macrophage cells. We tested whether yadBC might be required for lethality of the systemic stage of plague in which the bacteria would be pre-adapted to mammalian body temperature before colonizing internal organs and found no requirement for early colonization or growth over 3 days. We tested the hypothesis that YadB and YadC function on ambient temperature-grown Y. pestis in the flea vector or soon after infection of the dermis in bubonic plague. We found that yadBC did not promote uptake by monocyte/macrophage cells if the bacteria were grown at 28 °C, nor was there a role of yadBC in colonization of fleas by Y. pestis grown at 21 °C. However, the presence of yadBC did promote recoverability of the bacteria from infected skin for 28 °C-grown Y. pestis. Furthermore, the gene for the proinflammatory chemokine CXCL1 was upregulated in expression if the infecting Y. pestis lacked yadBC but not if yadBC was present. Also, yadBC was not required for recoverability if the bacteria were grown at 37 °C. These findings imply that thermally induced virulence properties dominate over effects of yadBC during plague but that yadBC has a unique function early after transmission of Y. pestis to skin.
Subject(s)
Adhesins, Bacterial/biosynthesis , Monocytes/immunology , Monocytes/microbiology , Yersinia pestis/radiation effects , Animals , Bacterial Load , Cells, Cultured , Disease Models, Animal , Mice , Phenotype , Plague/microbiology , Plague/pathology , Skin/microbiology , Skin/pathology , Temperature , Yersinia pestis/isolation & purification , Yersinia pestis/physiologyABSTRACT
YopM is one of the six "effector Yops" of the human-pathogenic Yersinia, but its mechanism has not been defined. After delivery to J774A.1 monocyte-like cells, YopM can rapidly bind and activate the serine/threonine kinases RSK1 and PRK2. However, in infected mice, effects of Y. pestis YopM have been seen only after 24-48 h post-infection (p.i.). To identify potential direct effects of YopM in-vivo we tested for effects of YopM at 1 h and 16-18 h p.i. in mice infected systemically with 10(6) bacteria. At 16 h p.i., there was a robust host response to both parent and ΔyopM-1 Y. pestis KIM5. Compared to cells from non-infected mice, CD11b(+) cells from spleens of infected mice produced more than 100-fold greater IFNγ. In the corresponding sera there were more than 100-fold greater amounts of IFNγ, G-CSF, and CXCL9, as well as more than 10-fold greater amounts of IL-6, CXCL10, and CXCL1. The only YopM-related differences were slightly lower CXCL10 and IL-6 in sera from mice infected 16 h with parent compared to ΔyopM-1 Y. pestis. Microarray analysis of the CD11b(+) cells did not identify consistent transcriptional differences of ≥4-fold at 18 h p.i. However, at 1 h p.i. mRNA for early growth response transcription factor 1 (Egr1) was decreased when YopM was present. Bone marrow-derived macrophages infected for 1 h also expressed lower Egr1 message when YopM was present. Infected J774A.1 cells showed greater expression of Egr1 at 1 h p.i. when YopM was present, but this pattern reversed at 3 h. At 6 h p.i., Cxcl10 mRNA was lower in parent-strain infected cells. We conclude that decreased Egr1 expression is a very early transcriptional effect of YopM and speculate that a pathway may exist from RSK1 through Egr1. These studies revealed novel early transcriptional effects of YopM but point to a time after 18 h of infection when critical transitional events lead to later major effects on cytokine gene transcription.
Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Early Growth Response Protein 1/antagonists & inhibitors , Plague/pathology , Virulence Factors/metabolism , Yersinia pestis/pathogenicity , Animals , Bone Marrow/immunology , Cells, Cultured , Cytokines/blood , Cytokines/metabolism , Disease Models, Animal , Early Growth Response Protein 1/biosynthesis , Female , Gene Expression Profiling , Leukocytes, Mononuclear/immunology , Mice , Mice, Inbred C57BL , Microarray Analysis , Plague/microbiology , Spleen/immunology , Time FactorsABSTRACT
We are using a systemic plague model to identify the cells and pathways that are undermined by the virulence protein YopM of the plague bacterium Yersinia pestis. In this study, we pursued previous findings that Gr1(+) cells are required to selectively limit growth of ΔyopM Y. pestis and that CD11b(+) cells other than polymorphonuclear leukocytes (PMNs) are selectively lost in spleens infected with parent Y. pestis. When PMNs were ablated from mice, ΔyopM Y. pestis grew as well as the parent strain in liver but not in spleen, showing that these cells are critical for controlling growth of the mutant in liver but not spleen. In mice lacking expression of the chemokine receptor CCR2, wild-type growth was restored to ΔyopM Y. pestis in both organs. In spleen, the Gr1(+) cells differentially recruited by parent and ΔyopM Y. pestis infections were CCR2(+) Gr1(+) CD11b(+) CD11c(Lo-Int) MAC3(+) iNOS(+) (inducible nitric oxide synthase-positive) inflammatory dendritic cells (iDCs), and their recruitment to spleen from blood was blocked when YopM was present in the infecting strain. Consistent with influx of iDCs being affected by YopM in spleen, the growth defect of the ΔyopM mutant was relieved by the parent Y. pestis strain in a coinfection assay in which the parent strain could affect the fate of the mutant in trans. In a mouse model of bubonic plague, CCR2 also was shown to be required for ΔyopM Y. pestis to show wild-type growth in skin. The data imply that YopM's pathogenic effect indirectly undermines signaling through CCR2. We propose a model for how YopM exerts its different effects in liver and spleen.
Subject(s)
Bacterial Outer Membrane Proteins/genetics , Plague/microbiology , Receptors, CCR2/metabolism , Receptors, Chemokine/metabolism , Yersinia pestis/growth & development , Animals , Antigens, Ly/genetics , Antigens, Ly/metabolism , Bacterial Outer Membrane Proteins/metabolism , CD11b Antigen/metabolism , Disease Models, Animal , Female , Gene Expression Regulation , Liver/microbiology , Mice , Mice, Inbred C57BL , Mutation , Neutrophils/physiology , Receptors, CCR2/genetics , Receptors, Chemokine/genetics , Skin/microbiology , Spleen/microbiology , Virulence , Yersinia pestis/genetics , Yersinia pestis/pathogenicityABSTRACT
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.
Subject(s)
Bacterial Outer Membrane Proteins/physiology , Monocytes/immunology , Plague/immunology , Yersinia pestis/growth & development , Animals , Antigens, Ly/physiology , Bacterial Outer Membrane Proteins/analysis , Dendritic Cells/immunology , Female , Immunity, Innate , Killer Cells, Natural/immunology , Liver/immunology , Macrophages/immunology , Mice , Mice, Inbred C57BL , Neutrophils/immunology , Plague/microbiology , Spleen/immunologyABSTRACT
Cells of wild-type Yersinia pestis exhibit a low-calcium response (LCR) defined as bacteriostasis with expression of a pCD-encoded type III secretion system (T3SS) during cultivation at 37 degrees C without added Ca(2+) versus vegetative growth with downregulation of the T3SS with Ca(2+) (>or=2.5 mM). Bacteriostasis is known to reflect cumulative toxicity of Na(+), l-glutamic acid and culture pH; control of these variables enables full-scale growth ('rescue') in the absence of Ca(2+). Several T3SS regulatory proteins modulate the LCR, because their absence promotes a Ca(2+)-blind phenotype in which growth at 37 degrees C ceases and the T3SS is constitutive even with added Ca(2+). This study analysed the connection between the LCR and Ca(2+) by determining the response of selected Ca(2+)-blind mutants grown in Ca(2+)-deficient rescue media containing Na(+) plus l-glutamate (pH 5.5), where the T3SS is not expressed, l-glutamate alone (pH 6.5), where l-aspartate is fully catabolized, and Na(+) alone (pH 9.0), where the electrogenic sodium pump NADH : ubiquinone oxidoreductase becomes activated. All three conditions supported essentially full-scale Ca(2+)-independent growth at 37 degrees C of wild-type Y. pestis as well as lcrG and yopN mutants (possessing a complete but dysregulated T3SS), indicating that bacteriostasis reflects a Na(+)-dependent lesion in bioenergetics. In contrast, mutants lacking the negative regulator YopD or the YopD chaperone (LcrH) failed to grow in any rescue medium and are therefore truly temperature-sensitive. The Ca(2+)-blind yopD phenotype was fully suppressed in a Ca(2+)-independent background lacking the injectisome-associated inner-membrane component YscV but not peripheral YscK, suggesting that the core translocon energizes YopD.
Subject(s)
Calcium/metabolism , Temperature , Yersinia pestis/growth & development , Yersinia pestis/metabolism , Bacterial Outer Membrane Proteins/metabolism , Bacterial Proteins/metabolism , Gene Deletion , Glutamic Acid/metabolism , Hydrogen-Ion Concentration , Membrane Proteins/metabolism , Molecular Chaperones/metabolism , Pore Forming Cytotoxic Proteins/metabolism , Secretory Pathway , Sodium/metabolism , Yersinia pestis/geneticsABSTRACT
In all Yersinia pestis strains examined, the adhesin/invasin yadA gene is a pseudogene, yet Y. pestis is invasive for epithelial cells. To identify potential surface proteins that are structurally and functionally similar to YadA, we searched the Y. pestis genome for open reading frames with homology to yadA and found three: the bicistronic operon yadBC (YPO1387 and YPO1388 of Y. pestis CO92; y2786 and y2785 of Y. pestis KIM5), which encodes two putative surface proteins, and YPO0902, which lacks a signal sequence and likely is nonfunctional. In this study we characterized yadBC regulation and tested the importance of this operon for Y. pestis adherence, invasion, and virulence. We found that loss of yadBC caused a modest loss of invasiveness for epithelioid cells and a large decrease in virulence for bubonic plague but not for pneumonic plague in mice.
Subject(s)
Adhesins, Bacterial/physiology , Plague/microbiology , Virulence Factors/physiology , Yersinia pestis/pathogenicity , Adhesins, Bacterial/biosynthesis , Adhesins, Bacterial/genetics , Animals , Bacterial Adhesion/genetics , Cell Line , Female , Gene Deletion , Gene Expression Regulation, Bacterial , Humans , Lethal Dose 50 , Mice , Mice, Inbred C57BL , Virulence/genetics , Virulence Factors/genetics , Yersinia pestis/geneticsABSTRACT
Current subunit vaccines provide partial protection against pneumonic plague if the infecting Y. pestis strain is encapsulated (F1+). Here we describe YadC, a novel Y. pestis outer membrane protein that provides partial protection against a F1(-) Y. pestis strain. Swiss-Webster mice were immunized subcutaneously with glutathione S-transferase (GST) or His6-tagged (HT) purified fusion proteins (GST-YadC137-409 or HT-LcrV) or buffer emulsified with Alhydrogel. Intravenous challenge with 1 x 10(4) F1(-) Deltapgm Y. pestis CO99-3015 revealed no protection for those mice immunized with GST-Alhydrogel alone, full protection for HT-LcrV-immunized mice, and partial protection for GST-YadC137-409-immunized mice. Similarly, C57BL/6 mice were immunized with GST-YadC137-409, HT-LcrV, or GST all with Alhydrogel adjuvant. After intranasal challenge with 3 x 10(3) F1(-) Y. pestis CO99-3015, 87% of GST-YadC137-409-immunized mice survived pneumonic plague. This is compared to the GST control group (0 surviving mice) and the LcrV-immunized group where 50% survived the challenge. This protection was correlated with a predominantly IgG1 response in LcrV-immunized mice and an IgG1/IgG3 antibody response in YadC-immunized mice. Additionally, we report the cytokine response from HT-LcrV- and GST-YadC137-409-stimulated peripherally derived macrophages. YadC-stimulated cells demonstrated a predominant pro-inflammatory cytokine production. This mixed Thl/Th2 response suggests that YadC's protection may involve a different adaptive immune response than the LcrV protein that currently is part of plague vaccines.
Subject(s)
Plague Vaccine/genetics , Plague Vaccine/immunology , Yersinia pestis/genetics , Yersinia pestis/immunology , Animals , Antibodies, Bacterial/biosynthesis , Antigens, Bacterial/genetics , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/immunology , Base Sequence , Cytokines/biosynthesis , DNA Primers/genetics , DNA, Bacterial/genetics , Female , Genes, Bacterial , Humans , Immunoglobulin G/biosynthesis , Macrophages/immunology , Macrophages/microbiology , Mice , Operon , Plague/immunology , Plague/prevention & control , Plasmids/genetics , Th1 Cells/immunology , Th1 Cells/microbiology , Th2 Cells/immunology , Th2 Cells/microbiology , Vaccines, Subunit/genetics , Vaccines, Subunit/immunology , Virulence/genetics , Virulence/immunology , Yersinia pestis/pathogenicityABSTRACT
This study identified major surface proteins of the plague bacterium Yersinia pestis. We applied a novel surface biotinylation method, followed by NeutrAvidin (NA) bead capture, on-bead digestion, and identification by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The use of stachyose during biotinylation focused the reaction to the surface. Coupled with NA pulldown and immunoblot analysis, this method determined whether a protein was accessible to the surface. We applied the method to test the hypothesis that the catalase KatY is a surface protein of the plague bacterium Y. pestis. A rabbit serum recognized the catalase KatY as a major putative outer membrane-associated antigen expressed by Y. pestis cells grown at 37 degrees C. Similar findings by other groups had led to speculations that this protein might be exposed to the surface and might be a candidate for evaluation as a protective antigen for an improved plague vaccine. KatY was obtained only in the total membrane fraction, and stachyose greatly reduced its biotinylation as well as that of the periplasmic maltose binding protein, indicating that KatY is not on the bacterial surface. LC-MS-MS analysis of on-bead digests representing ca. 10(9) cells identified highly abundant species, including KatY, Pal, and OmpA, as well as the lipoprotein Pcp, all of which bound in a biotin-specific manner. Pla, Lpp, and OmpX (Ail) bound to the NA beads in a non-biotin-specific manner. There was no contamination from abundant cytoplasmic proteins. We hypothesize that OmpX and Pcp are highly abundant and likely to be important for the Y. pestis pathogenic process. We speculate that a portion of KatY associates with the outer membrane in intact cells but that it is located on the periplasmic side. Consistent with this idea, it did not protect C57BL/6 mice against bubonic plague.
Subject(s)
Biotinylation/methods , Catalase/genetics , Plague/prevention & control , Yersinia pestis/enzymology , Animals , Antigens, Bacterial/chemistry , Bacterial Outer Membrane Proteins/immunology , Bacterial Outer Membrane Proteins/metabolism , Catalase/chemistry , Catalase/isolation & purification , Catalase/metabolism , Membrane Proteins/chemistry , Mice , Mice, Inbred C57BL , Yersinia pestis/chemistryABSTRACT
We report that females of some substrains of inbred mouse strain 129 are resistant to systemic plague due to conditionally virulent Deltapgm strains of Yersinia pestis; however, fully virulent Y. pestis is not attenuated in these mice. Therefore, these mice offer a powerful system in which to map in parallel host resistance traits and opposing bacterial virulence properties for plague.
Subject(s)
Immunity, Innate/genetics , Plague/immunology , Plague/microbiology , Yersinia pestis/genetics , Yersinia pestis/immunology , Animals , Female , Genetic Predisposition to Disease , Male , Mice , Mice, Inbred Strains , Mutation , Plague/genetics , Plague/mortality , Yersinia pestis/pathogenicityABSTRACT
LcrV of Yersinia pestis is a major protective antigen proposed for inclusion in subunit plague vaccines. One way that anti-LcrV antibody is thought to protect is by inhibiting the delivery of toxins called Yops to host cells. The present study characterizes the relation between this inhibition and the phagocytosis of the bacteria. J774A.1 cells were infected with Y. pestis KIM5 in the presence of a protective polyclonal anti-LcrV antibody or a nonprotective polyclonal anti-YopM antibody, and delivery of YopH and YopE into the cytoplasm was assayed by immunoblotting. The ability to inhibit the delivery of these Yops depended upon having antibody bound to the cell surface; blocking conditions that prevented the binding of antibody to Fc receptors prevented the inhibition of Yop delivery. Anti-LcrV antibody also promoted phagocytosis of the yersiniae, whereas F(ab')(2) fragments did not. Further, anti-LcrV antibody could not inhibit the delivery of Yops into cells that were unable to phagocytose due to the presence of cytochalasin D. However, Yops were produced only by extracellular yersiniae. We hypothesize that anti-LcrV antibody does not directly inhibit Yop delivery but instead causes phagocytosis, with consequent inhibition of Yop protein production in the intracellular yersiniae. The prophagocytic effect of anti-LcrV antibody extended to mouse polymorphonuclear neutrophils (PMNs) in vitro, and PMNs were shown to be critical for protection: when PMNs in mice were ablated, the mice lost all ability to be protected by anti-LcrV antibody.
Subject(s)
Antibodies, Bacterial/pharmacology , Antigens, Bacterial/immunology , Bacterial Outer Membrane Proteins/antagonists & inhibitors , Membrane Transport Modulators , Membrane Transport Proteins/antagonists & inhibitors , Phagocytosis/immunology , Plague/immunology , Yersinia pestis/immunology , Animals , Bacterial Outer Membrane Proteins/immunology , Bacterial Outer Membrane Proteins/metabolism , HeLa Cells , Humans , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fab Fragments/pharmacology , Membrane Transport Proteins/metabolism , Mice , Neutrophils/immunology , Neutrophils/microbiology , Plague/metabolism , Pore Forming Cytotoxic Proteins , Yersinia pestis/metabolismABSTRACT
The V antigen (LcrV) of the plague bacterium Yersinia pestis is a potent protective antigen that is under development as a vaccine component for humans. LcrV is multifunctional. On the bacterial surface it mediates delivery of a set of toxins called Yops into host cells, and as a released protein it can cause production of the immunosuppressive cytokine interleukin-10 (IL-10) and can inhibit chemotaxis of polymorphonuclear neutrophils. It is not known how these mechanisms of LcrV operate, what their relative importance is, when they function during plague, and which are critical to protection by antibody. This study investigated several of these issues. C57BL/6 mice, mice unable to express IL-10, or mice with the macrophage lineage eliminated were treated with a protective anti-LcrV antibody or a nonprotective antibody against YopM and infected intravenously by Y. pestis KIM5 or a strain that lacked the genes encoding all six effector Yops. Viable bacterial numbers were determined at various times. The data indicated that Yops were necessary for Yersinia growth after the bacteria had seeded liver and spleen. Anti-LcrV antibody prevented this growth, even in IL-10-/- mice, demonstrating that one protective mechanism for anti-LcrV antibody is independent of IL-10. Anti-LcrV antibody had no effect on persistence in organs of Y. pestis lacking effector Yops, even though the yersiniae could strongly express LcrV, suggesting that Yops are necessary for building sufficient bacterial numbers to produce enough LcrV for its immunosuppressive effects. In vitro assays showed that anti-LcrV antibody could partially block delivery of Yops and downstream effects of Yops in infected macrophage-like J774A.1 cells. However, cells of the macrophage lineage were found to be dispensable for protection by anti-LcrV antibody in spleen, although they contributed to protection in liver. Taken together, the data support the hypothesis that one protective effect of the antibody is to block delivery of Yops to host cells and prevent early bacterial growth. The findings also identified the macrophage lineage as one host cell type that mediates protection.
Subject(s)
Antibodies, Bacterial/administration & dosage , Antibodies, Bacterial/immunology , Antigens, Bacterial/immunology , Bacterial Outer Membrane Proteins/metabolism , Plague/immunology , Yersinia pestis/growth & development , Animals , Antibodies, Bacterial/blood , Bacterial Outer Membrane Proteins/genetics , Cell Line , Humans , Interleukin-10/genetics , Macrophages/immunology , Mice , Mice, Inbred C57BL , Plague/microbiology , Pore Forming Cytotoxic Proteins , Rabbits , Virulence , Yersinia pestis/immunology , Yersinia pestis/pathogenicityABSTRACT
Yersinia pestis, the etiologic agent of plague, delivers six Yersinia outer proteins (Yops) into host cells upon direct bacterial contact. One of these, YopM, is necessary for virulence in a mouse model of septicemic plague, but its pathogenic function is unknown. We report here the immune processes affected by YopM during infection. To test whether the innate or adaptive immune system is targeted by YopM, C57BL/6 (B6) and B6 SCID mice were infected with either the conditionally virulent Y. pestis KIM5 or a yopM deletion mutant and evaluated for bacterial growth in spleen and liver. Both B6 and SCID mice succumbed to infection with Y. pestis KIM5, whereas both mouse strains survived infection by the YopM(-) mutant. These data showed that YopM counteracts innate defenses present in SCID mice. The YopM(-) strain grew more slowly than the parent Y. pestis during the first 4 days of infection in both mouse strains, indicating an early pathogenic role for YopM. In B6 mice, populations of cells of the immune system were not differentially affected by the two Y. pestis strains, with one major exception: the parent Y. pestis KIM5 but not the YopM(-) mutant caused a significant global decrease in NK cell numbers (blood, spleen, and liver), beginning early in infection. NK cells and macrophages isolated early (day 2) from livers and spleens of mice infected with either Y. pestis strain contained comparable levels of cytokine mRNA: interleukin (IL)-1 beta, IL-12, IL-15, IL-18, and tumor necrosis factor alpha in macrophages and gamma interferon in NK cells. However, by day 4 postinfection, cells from mice infected with the parent Y. pestis expressed lower levels of these messages, while those from mice infected with the mutant retained strong expression. Significantly, mRNA for the IL-15 receptor alpha chain was not expressed in NK cells from Y. pestis KIM5-infected mice as early as day 2 postinfection. These findings suggest that YopM interferes with innate immunity by causing depletion of NK cells, possibly by affecting the expression of IL-15 receptor alpha and IL-15.
Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Immunity, Innate , Killer Cells, Natural/pathology , Plague/immunology , Plague/physiopathology , Yersinia pestis/pathogenicity , Animals , Bacterial Outer Membrane Proteins/genetics , Cytokines/metabolism , Female , Killer Cells, Natural/immunology , Liver/immunology , Liver/microbiology , Macrophage Activation , Macrophages/immunology , Mice , Mice, Inbred C57BL , Mice, SCID , Plague/microbiology , Spleen/immunology , Spleen/microbiology , Th1 Cells , Virulence , Yersinia pestis/genetics , Yersinia pestis/growth & developmentABSTRACT
Transgenic mice expressing an inducible suicide gene, which allows systemic and reversible elimination of macrophages, were developed. A macrophage-specific c-fms promoter was used to express enhanced green fluorescent protein and a drug-inducible suicide gene that leads to Fas-mediated apoptosis in resting and cycling cells of the macrophage lineage. Transgenic mice were fertile, of normal weight, and showed no abnormal phenotype before drug exposure. The transgene was expressed constitutively in macrophages and dendritic cells (DC) but not significantly in T cells or B cells. Induction of the suicide gene led to depletion of 70-95% of macrophages and DC in nearly all tissues examined. Depletion reduced the ability to clear bacteria from the blood and led to increased bacterial growth in the liver. Depleted mice displayed several abnormalities, including splenomegaly, lymphadenopathy, thymic atrophy, extramedullary hematopoiesis, and development of peritoneal adhesions. This new, transgenic line will be useful in investigating the role of macrophages and DC.
Subject(s)
Apoptosis/genetics , Genes, Transgenic, Suicide/genetics , Macrophages/metabolism , Tacrolimus/analogs & derivatives , fas Receptor/genetics , Animals , Apoptosis/drug effects , Bacterial Infections/genetics , Bacterial Infections/immunology , Bacterial Infections/physiopathology , Cell Count , Cells, Cultured , Dendritic Cells/cytology , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Dimerization , Disease Models, Animal , Genes, Transgenic, Suicide/drug effects , Green Fluorescent Proteins , Immunity, Cellular/genetics , Luminescent Proteins , Macrophages/cytology , Macrophages/drug effects , Mice , Mice, Inbred C57BL , Mice, Transgenic , Promoter Regions, Genetic/genetics , Receptor, Macrophage Colony-Stimulating Factor/genetics , Receptors, Nerve Growth Factor/genetics , Tacrolimus/pharmacology , Tacrolimus Binding Proteins/geneticsABSTRACT
YopM is a leucine-rich repeat (LRR) virulence protein that is delivered into host cells when any of the three human-pathogenic species of Yersinia binds to mammalian cells. It exhibits heterogeneity of size and sequence among the yersiniae, but the functional consequences of this variability are not yet known. Yersinia pestis YopM was previously shown to accumulate in the nuclei of infected HeLa cells by a mechanism that requires vesicular trafficking. In this study, we characterized the trafficking of Y. pestis YopM in a Saccharomyces cerevisiae model previously found to support nuclear localization of YopM from an enteropathogenic Yersinia strain (C. F. Lesser and S. I. Miller, EMBO J. 20:1840-1849, 2001). Y. pestis YopM was N-terminally fused to the yeast enhanced green fluorescent protein (yEGFP) and inducibly expressed in the cytoplasm. yEGFP-YopM localized to the yeast nucleus, showing that this property is conserved for YopMs so far tested and that infection and the presence of other Yops are not required for its trafficking. When expressed in S. cerevisiae that is temperature sensitive for vesicular transport, YopM failed to accumulate in the nucleus at the nonpermissive temperature but did accumulate when the permissive temperature was restored. This shows that vesicular trafficking also is required in yeast for normal localization of YopM. YopM consists of a 71-residue leader sequence, 15 LRRs, and a 32-residue tail. Deletion analysis revealed that the leader sequence or tail is alone insufficient to direct YopM to the nucleus, showing that the LRR structure is required. Both the N-terminal and C-terminal halves of YopM localized to the nucleus, indicating the possible presence of two nuclear localization signals (NLSs) in YopM or domains in YopM where an NLS-containing protein might bind; this fits with the presence of two highly conserved regions among Yersinia YopMs. yEGFP-YopM lacking LRRs 4 to 7 or 7 to 10 accumulated in the nucleus in yeast, and YopM lacking these LRRs concentrated normally in the HeLa cell nucleus after delivery by Yersinia infection, showing that these LRRs are not essential for YopM trafficking in eucaryotic cells. However, because Y. pestis carrying either of these YopMs is strongly compromised in virulence in mice, these findings revealed that LRRs 4 to 10 map a region of YopM or support a conformation of YopM that is necessary for a pathogenic effect.
Subject(s)
Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , HeLa Cells/metabolism , Saccharomyces cerevisiae/metabolism , Yersinia pestis/metabolism , Bacterial Outer Membrane Proteins/chemistry , Biological Transport , Cell Nucleus/metabolism , Green Fluorescent Proteins , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Nuclear Localization Signals/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Yersinia pestis/geneticsABSTRACT
We present the complete genome sequence of Yersinia pestis KIM, the etiologic agent of bubonic and pneumonic plague. The strain KIM, biovar Mediaevalis, is associated with the second pandemic, including the Black Death. The 4.6-Mb genome encodes 4,198 open reading frames (ORFs). The origin, terminus, and most genes encoding DNA replication proteins are similar to those of Escherichia coli K-12. The KIM genome sequence was compared with that of Y. pestis CO92, biovar Orientalis, revealing homologous sequences but a remarkable amount of genome rearrangement for strains so closely related. The differences appear to result from multiple inversions of genome segments at insertion sequences, in a manner consistent with present knowledge of replication and recombination. There are few differences attributable to horizontal transfer. The KIM and E. coli K-12 genome proteins were also compared, exposing surprising amounts of locally colinear "backbone," or synteny, that is not discernible at the nucleotide level. Nearly 54% of KIM ORFs are significantly similar to K-12 proteins, with conserved housekeeping functions. However, a number of E. coli pathways and transport systems and at least one global regulator were not found, reflecting differences in lifestyle between them. In KIM-specific islands, new genes encode candidate pathogenicity proteins, including iron transport systems, putative adhesins, toxins, and fimbriae.
Subject(s)
Genome, Bacterial , Yersinia pestis/genetics , Bacteriophages , Biological Transport/genetics , Bodily Secretions , Chemotaxis/genetics , DNA Replication/genetics , DNA Transposable Elements/genetics , Energy Metabolism/genetics , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Genes, rRNA/genetics , Operon/genetics , Plasmids/genetics , Protein Biosynthesis/genetics , Repetitive Sequences, Nucleic Acid , Virulence , Yersinia pestis/metabolism , Yersinia pestis/pathogenicityABSTRACT
LcrQ is a regulatory protein unique to Yersinia. Previous study in Yersinia pseudotuberculosis and Yersinia enterocolitica prompted the model in which LcrQ negatively regulates the expression of a set of virulence proteins called Yops, and its secretion upon activation of the Yop secretion (Ysc) type III secretion system permits full induction of Yops expression. In this study, we tested the hypothesis that LcrQ's effects on Yops expression might be indirect. Excess LcrQ was found to exert an inhibitory effect specifically at the level of Yops secretion, independent of production, and a normal inner Ysc gate protein LcrG was required for this activity. However, overexpression of LcrQ did not prevent YopH secretion, suggesting that LcrQ's effects at the Ysc discriminate among the Yops. We tested this idea by determining the effects of deletion or overexpression of LcrQ, YopH and their common chaperone SycH on early Yop secretion through the Ysc. Together, our findings indicated that LcrQ is not a negative regulator directly, but it acts in partnership with SycH at the Ysc gate to control the entry of a set of Ysc secretion substrates. A hierarchy of YopH secretion before YopE appears to be imposed by SycH in conjunction with both LcrQ and YopH. LcrQ and SycH in addition influenced the deployment of LcrV, a component of the Yops delivery mechanism. Accordingly, LcrQ appears to be a central player in determining the substrate specificity of the Ysc.