Anaplasma phagocytophilum AnkA secreted by type IV secretion system is tyrosine phosphorylated by Abl-1 to facilitate infection

Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, is an obligate intracellular bacterium of granulocytes. A. phagocytophilum specifically induces tyrosine phosphorylation of a 160 kDa protein (P160) in host cells. However, identity of P160, kinases involved, and effects of tyrosine phosphorylation on bacterial infection remain largely unknown. Here, we demonstrated through proteomic analysis that P160, an abundant and rapidly tyrosine-phosphorylated protein throughout infection, was AnkA of bacterial origin. Differential centrifugation and confocal microscopy revealed that AnkA was rarely retained within A. phagocytophilum or its inclusion, but localized mainly in the cytoplasm of infected cells. Using Cre recombinase reporter assay of Agrobacterium tumefaciens, we proved that AnkA could be secreted by VirB/D4-dependent type IV secretion (T4S) system. Yeast two-hybrid and coimmunoprecipitation analyses demonstrated that AnkA could bind to Abl-interactor 1 (Abi-1), an adaptor protein that interacts with Abl-1 tyrosine kinase, thus mediating AnkA phosphorylation. AnkA and Abl-1 were critical for bacterial infection, as infection was inhibited upon host cytoplasmic delivery of anti-AnkA antibody, Abl-1 knockdown with targeted siRNA, or treatment with a specific pharmacological inhibitor of Abl-1. These data establish AnkA as the first proven T4S substrate in members of obligate intracellular alpha-proteobacteria; furthermore, it demonstrated that AnkA plays an important role in facilitating intracellular infection by activating Abl-1 signalling pathway, and suggest a novel approach to treatment of human granulocytic anaplasmosis through inhibition of host cell signalling pathways.     

Anaplasma phagocytophilum PSGL-1-independent infection does not require Syk and leads to less efficient AnkA delivery

Anaplasma phagocytophilum is an obligate intracellular bacterium that infects neutrophils to cause granulocytic anaplasmosis in humans and mammals. P-selectin glycoprotein ligand-1 (PSGL-1) and the tetrasaccharide sialyl Lewis x (sLe^x), which caps the PSGL-1 N-terminus, are confirmed A. phagocytophilum receptors. A. phagocytophilum is capable of sLe^x-modified PSGL-1-dependent and -independent infection. PSGL-1 N-terminus-mediated entry is dependent on spleen tyrosine kinase (Syk). Here, we determined that PSGL-1-independent entry does not alter bacterial replication and investigated whether it involves Syk using NCH-1A2, an enriched subpopulation of A. phagocytophilum NCH-1 obtained through cultivation in a sLe^x-deficient HL-60 cell line, HL-60 A2. Pharmacological inhibition of Syk nearly abolishes NCH-1 infection, but does not alter NCH-1A2 invasion and only marginally reduces NCH-1A2 propagation. This phenomenon was confirmed by a competitive infection assay using PSGL-1-dependent and -independent A. phagocytophilum organisms transformed to express mCherry or green fluorescent protein respectively. We also assayed for delivery and tyrosine phosphorylation of the A. phagocytophilum effector, AnkA, following NCH-1or NCH-1A2 incubation with HL-60 or HL-60 A2 cells in the presence of PSGL-1 blocking antibody. PSGL-1 N-terminus recognition promotes optimal AnkA delivery while binding to sLe^x or the unknown receptor is comparably less important for this process.     

Anaplasma phagocytophilum AnkA binds to granulocyte DNA and nuclear proteins

Human granulocytic anaplasmosis (HGA) is caused by the obligate intracellular bacterium Anaplasma phagocytophilum. The bacterium infects, survives, propagates in, and alters neutrophil phenotype, indicating unique survival mechanisms. AnkA is the only known A. phagocytophilum component that gains access beyond neutrophil vacuoles and is transported to the infected host cell nucleus. The ability of native and recombinant AnkA to bind DNA and nuclear proteins from host HL-60 cells was assessed by the use of immunoprecipitation after cis-diamminedichloroplatinum (cis-DDP) DNA-protein crosslinking, by probing uninfected HL-60 cell nuclear lysates for AnkA binding, and by recovery and sequence analysis of immunoprecipitated DNA. AnkA binds HL-60 cell DNA as well as nuclear proteins of approximately 86, 53 and 25 kDa, whereas recombinant A. phagocytophilum Msp2 or control proteins do not. DNA immunoprecipitation reveals AnkA binding to a variety of target genes in the human genome, including genes that encode proteins with ATPase, tyrosine phosphatase and NADH dehydrogenase-like functions. These data indicate that AnkA could exert some effect on cells through binding to protein:DNA complexes in neutrophil nuclei. Whether AnkA binding leads to neutrophil functional alterations, and how such alterations might occur will depend upon definitive identification of binding partners and associated metabolic and biochemical pathways.     

Anaplasma phagocytophilum specifically induces tyrosine phosphorylation of ROCK1 during infection

Anaplasma phagocytophilum, an obligate intracellular pathogen that persists within polymorphonuclear leucocytes, is the second most common tick-borne agent in North America. We now show that infection of a promyelocytic cell line and neutrophils with A. phagocytophilum results in pathogen-specific tyrosine phosphorylation of ROCK1. Phosphorylation is associated with PSGL-1 and Syk, because PSGL-1 blocking antibodies and siRNA targeting Syk interfere with ROCK1 phosphorylation in A. phagocytophilum-infected cells. Knockdown of either Syk or ROCK1 also markedly impaired A. phagocytophilum infection. These data demonstrate a role for A. phagocytophilum-mediated ROCK1 phosphorylation in infection, and suggests that inhibiting this pathway may lead to new, non-antibiotic strategies to treat human granulocytic anaplasmosis.     

ASC/PYCARD and caspase-1 regulate the IL-18/IFN-gamma axis during Anaplasma phagocytophilum infection

Anaplasma phagocytophilum is an obligate intracellular pathogen that resides within neutrophils and can cause fever, pancytopenia, or death. IFN-gamma plays a critical role in the control of A. phagocytophilum; however, the mechanisms that regulate IFN-gamma production remain unclear. In this study, we demonstrate that apoptotic specklike protein with a caspase-activating recruiting domain (ASC)/PYCARD, a central adaptor molecule in the Nod-like receptor (NLR) pathway, regulates the IL-18/IFN-gamma axis during A. phagocytophilum infection through its effect on caspase-1. Caspase-1- and asc-null mice were more susceptible than control animals to A. phagocytophilum infection due to the absence of IL-18 secretion and reduced IFN-gamma levels in the peripheral blood. Moreover, caspase-1 and ASC deficiency reduced CD4+ T cell-mediated IFN-gamma after in vitro restimulation with A. phagocytophilum. The NLR family member IPAF/NLRC4, but not NALP3/NLRP3, was partially required for IFN-gamma production in response to A. phagocytophilum. Taken together, our data demonstrate that ASC and caspase-1 are critical for IFN-gamma-mediated control of A. phagocytophilum infection.     

Tyrosine phosphatases SHP-1 and SHP-2 are associated with distinct tyrosine-phosphorylated proteins.

SHP-1 and SHP-2 are two SH2 domain-containing tyrosine phosphatases. They share significant overall sequence identity but their functions are often opposite. The mechanism underlying this is not well understood. In this study, we have investigated the association of SHP-1 and SHP-2 with tyrosine-phosphorylated proteins in mouse tissues and in cultured cells treated with a potent tyrosine phosphatase inhibitor, pervanadate. Pervanadate was introduced into mice by intravenous injection. It induced robust tyrosine phosphorylation of cellular proteins in a variety of tissues. Both SHP-1 and SHP-2 were phosphorylated on tyrosyl residues upon pervanadate treatment, and they became associated with distinct tyrosine-phosphorylated proteins in different tissues and cells. Among these proteins, PZR and PECAM were identified as major SHP-2-binding proteins while LAIR-1 was shown to be a major SHP-1-binding protein. A number of other proteins are to be identified. We believe that the different binding proteins may determine the distinct physiological functions of SHP-1 and SHP-2. The present study also provides a general method to induce tyrosine phosphorylation of cellular proteins and to study protein-protein interactions involving tyrosine phosphorylation in vivo and in vitro.     

Anaplasma phagocytophilum Rab10‐dependent parasitism of the trans‐Golgi network is critical for completion of the infection cycle

Anaplasma phagocytophilum is an emerging human pathogen and obligate intracellular bacterium. It inhabits a host cell‐derived vacuole and cycles between replicative reticulate cell (RC) and infectious dense‐cored (DC) morphotypes. Host–pathogen interactions that are critical for RC‐to‐DC conversion are undefined. We previously reported that A. phagocytophilum recruits green fluorescent protein (GFP)‐tagged Rab10, a GTPase that directs exocytic traffic from the sphingolipid‐rich trans‐Golgi network (TGN) to its vacuole in a guanine nucleotide‐independent manner. Here, we demonstrate that endogenous Rab10‐positive TGN vesicles are not only routed to but also delivered into the A. phagocytophilum‐occupied vacuole (ApV). Consistent with this finding, A. phagocytophilum incorporates sphingolipids while intracellular and retains them when naturally released from host cells. TGN vesicle delivery into the ApV is Rab10 dependent, up‐regulates expression of the DC‐specific marker, APH1235, and is critical for the production of infectious progeny. The A. phagocytophilum surface protein, uridine monophosphate kinase, was identified as a guanine nucleotide‐independent, Rab10‐specific ligand. These data delineate why Rab10 is important for the A. phagocytophilum infection cycle and expand the understanding of the benefits that exploiting host cell membrane traffic affords intracellular bacterial pathogens.     

Recombination during early herpes simplex virus type 1 infection is mediated by cellular proteins

Homologous recombination was examined in cells infected with herpes simplex virus type I. Circular and linear DNA with directly repeated sequences was introduced as recombination substrates into cells. Recombination was measured either by origin-dependent amplification of recombination products or by recombination-dependent expression of luciferase from a disrupted gene. Homologous recombination in baby hamster kidney cells converted linear DNA to circular templates for DNA replication and luciferase expression in the complete absence of virus. The products of homologous recombination were efficiently amplified by the viral replication apparatus. The efficiency of recombination was dependent on the structure of the substrate as well as the cell type. Linear DNA with the direct repeats at internal positions failed to recombine in Balb/c 3T3 cells and induced p53-dependent apoptosis. In contrast, linear DNA with directly repeated sequences precisely at the ends recombined and replicated in 3T3 cells. Homologous recombination in baby hamster kidney cells did not depend on the position of the repeated sequences. We conclude that homologous recombination is independent of viral gene functions and that it is likely to be carried out by cellular proteins. We suggest that homologous recombination between directly repeated sequences in the linear herpes simplex virus type 1 chromosome may help to avoid p53-dependent apoptosis and to promote viral DNA replication.     

Anaplasma phagocytophilum Ats-1 Is Imported into Host Cell Mitochondria and Interferes with Apoptosis Induction

Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis, infects human neutrophils and inhibits mitochondria-mediated apoptosis. Bacterial factors involved in this process are unknown. In the present study, we screened a genomic DNA library of A. phagocytophilum for effectors of the type IV secretion system by a bacterial two-hybrid system, using A. phagocytophilum VirD4 as bait. A hypothetical protein was identified as a putative effector, hereby named Anaplasma translocated substrate 1 (Ats-1). Using triple immunofluorescence labeling and Western blot analysis of infected cells, including human neutrophils, we determined that Ats-1 is abundantly expressed by A. phagocytophilum, translocated across the inclusion membrane, localized in the host cell mitochondria, and cleaved. Ectopically expressed Ats-1 targeted mitochondria in an N-terminal 17 residue-dependent manner, localized in matrix or at the inner membrane, and was cleaved as native protein, which required residues 55–57. In vitro-translated Ats-1 was imported in a receptor-dependent manner into isolated mitochondria. Ats-1 inhibited etoposide-induced cytochrome c release from mitochondria, PARP cleavage, and apoptosis in mammalian cells, as well as Bax-induced yeast apoptosis. Ats-1(55–57) had significantly reduced anti-apoptotic activity. Bax redistribution was inhibited in both etoposide-induced and Bax-induced apoptosis by Ats-1. Taken together, Ats-1 is the first example of a bacterial protein that traverses five membranes and prevents apoptosis at the mitochondria.     

The Anaplasma phagocytophilum PleC histidine kinase and PleD diguanylate cyclase two-component system and role of cyclic Di-GMP in host cell infection

Anaplasma phagocytophilum, the etiologic agent of human granulocytic anaplasmosis (HGA), has genes predicted to encode three sensor kinases, one of which is annotated PleC, and three response regulators, one of which is PleD. Prior to this study, the roles of PleC and PleD in the obligatory intracellular parasitism of A. phagocytophilum and their biochemical activities were unknown. The present study illustrates the relevance of these factors by demonstrating that both pleC and pleD were expressed in an HGA patient. During A. phagocytophilum development in human promyelocytic HL-60 cells, PleC and PleD were synchronously upregulated at the exponential growth stage and downregulated prior to extracellular release. A recombinant PleC kinase domain (rPleCHKD) has histidine kinase activity; no activity was observed when the conserved site of phosphorylation was replaced with alanine. A recombinant PleD (rPleD) has autokinase activity using phosphorylated rPleCHKD as the phosphoryl donor but not with two other recombinant histidine kinases. rPleCHKD could not serve as the phosphoryl donor for a mutant rPleD (with a conserved aspartic acid, the site of phosphorylation, replaced by alanine) or two other A. phagocytophilum recombinant response regulators. rPleD had diguanylate cyclase activity to generate cyclic (c) di-GMP from GTP in vitro. UV cross-linking of A. phagocytophilum lysate with c-di-[(32)P]GMP detected an approximately 47-kDa endogenous protein, presumably c-di-GMP downstream receptor. A new hydrophobic c-di-GMP derivative, 2'-O-di(tert-butyldimethylsilyl)-c-di-GMP, inhibited A. phagocytophilum infection in HL-60 cells. Our results suggest that the two-component PleC-PleD system is a diguanylate cyclase and that a c-di-GMP-receptor complex regulates A. phagocytophilum intracellular infection.     

PILRalpha, a novel immunoreceptor tyrosine-based inhibitory motif-bearing protein, recruits SHP-1 upon tyrosine phosphorylation and is paired with the truncated counterpart PILRbeta

SHP-1-mediated dephosphorylation of protein tyrosine residues is central to the regulation of several cell signaling pathways, the specificity of which is dictated by the intrinsic affinity of SH2 domains for the flanking sequences of phosphotyrosine residues. By using a modified yeast two-hybrid system and SHP-1 as bait, we have cloned a human cDNA, PILRalpha, encoding a 303-amino acid immunoglobulin-like transmembrane receptor bearing two cytoplasmic tyrosines positioned within an immunoreceptor tyrosine-based inhibitory motif. Substrate trapping in combination with pervanadate treatment of 293T cells confirms that PILRalpha associates with SHP-1 in vivo upon tyrosine phosphorylation. Mutation of the tyrosine residues in PILRalpha indicates the pivotal role of the Tyr-269 residue in recruiting SHP-1. Surface plasmon resonance analysis further suggests that the association between PILRalpha-Tyr-269 and SHP-1 is mediated primarily via the amino-terminal SH2 domain of the latter. Polymerase chain reaction amplification of cDNA in combination with genomic sequence analysis revealed a second gene, PILRbeta, coding for a putative activating receptor as suggested by a truncated cytoplasmic tail and a charged lysine residue in its transmembrane region. The PILRalpha and PILRbeta genes are localized to chromosome 7 which is in contrast with the mapping of known members of the inhibitory receptor superfamily.     

Structurally distinct requirements for binding of P-selectin glycoprotein ligand-1 and sialyl Lewis x to Anaplasma phagocytophilum and P-selectin

Colonization of neutrophils by the bacterium Anaplasma phagocytophilum causes the disease human granulocytic ehrlichiosis. The pathogen also infects mice, its natural host. Like binding of P-selectin, binding of A. phagocytophilum to human neutrophils requires expression of P-selectin glycoprotein ligand-1 (PSGL-1) and alpha1-3-fucosyltransferases that construct the glycan determinant sialyl Lewis x (sLex). Binding of A. phagocytophilum to murine neutrophils, however, requires expression of alpha1-3-fucosyltransferases but not PSGL-1. To further characterize the molecular features that A. phagocytophilum recognizes, we measured bacterial binding to microspheres bearing specific glycoconjugates or to cells expressing human PSGL-1 and particular glycosyltransferases. Like P-selectin, A. phagocytophilum bound to purified human PSGL-1 and to glycopeptides modeled after the N terminus of human PSGL-1 that presented sLex on an O-glycan. Unlike P-selectin, A. phagocytophilum bound to glycopeptides that contained sLex but lacked tyrosine sulfation or a specific core-2 orientation of sLex on the O-glycan. A. phagocytophilum bound only to glycopeptides that contained a short amino acid sequence found in the N-terminal region of human but not murine PSGL-1. Unlike P-selectin, A. phagocytophilum bound to cells expressing PSGL-1 in cooperation with sLex on both N-and O-glycans. Moreover, bacteria bound to microspheres coupled independently with glycopeptide lacking sLex and with sLex lacking peptide. These results demonstrate that, unlike P-selectin, A. phagocytophilum binds cooperatively to a nonsulfated N-terminal peptide in human PSGL-1 and to sLex expressed on PSGL-1 or other glycoproteins. Distinct bacterial adhesins may mediate these cooperative interactions.     

Characterization of a sialic acid- and P-selectin glycoprotein ligand-1-independent adhesin activity in the granulocytotropic bacterium Anaplasma phagocytophilum

Anaplasma phagocytophilum, the aetiologic agent of human granulocytic anaplasmosis, is an obligate intracellular bacterium that colonizes neutrophils and neutrophil precursors. The granulocytotropic bacterium uses multiple adhesins that cooperatively bind to the N-terminal region of P-selectin glycoprotein ligand-1 (PSGL-1) and to sialyl Lewis x (sLe(x)) expressed on myeloid cell surfaces. Recognition of sLe(x) occurs through interactions with alpha2,3-sialic acid and alpha1,3-fucose. It is unknown whether other bacteria-host cell interactions are involved. In this study, we have enriched for A. phagocytophilum organisms that do not rely on sialic acid for cellular adhesion and entry by maintaining strain NCH-1 in HL-60 cells that are severely undersialylated. The selected bacteria, termed NCH-1A, also exhibit lessened dependencies on PSGL-1 and alpha1,3-fucose. Optimal adhesion and invasion by NCH-1A require interactions with the known determinants (sialic acid, PSGL-1 and alpha1,3-fucose), but none of them is absolutely necessary. NCH-1A binding to sLe(x)-modified PSGL-1 requires recognition of the known determinants in the same manners as other A. phagocytophilum strains. These data suggest that A. phagocytophilum expresses a separate adhesin from those targeting sialic acid, alpha1,3-fucose and the N-terminal region of PSGL-1. We propose that NCH-1A upregulates expression of this adhesin.     

Changes in the expression and dynamics of SHP-1 and SHP-2 during cerulein-induced acute pancreatitis in rats

Protein tyrosine phosphatases (PTPs) are important regulators of cell functions but data on different PTP expression and dynamics in acute pancreatitis (AP) are very scarce. Additionally, both c-Jun N-terminal kinases (JNK) and extracellular signal-regulated kinases (ERK1/2), together with intracellular cAMP levels in inflammatory cells, play an essential role in AP. In this study we have detected an increase in PTP SHP-1 and SHP-2 in the pancreas at the level of both protein and mRNA as an early event during the development of Cerulein (Cer)-induced AP in rats. Nevertheless, while SHP-2 protein returned to baseline levels in the intermediate or later phases of AP, SHP-1 protein expression remained increased throughout the development of the disease. The increase in SHP-2 protein expression was associated with changes in its subcellular distribution, with higher percentages located in the fractions enriched in lysosomes+mitochondria or microsomes. Furthermore, while the increase in SHP-2 protein was also observed in sodium-taurocholate duct infusion or bile-pancreatic duct obstruction AP, that of SHP-1 was specific to the Cer-induced model. Neutrophil infiltration did not affect the increase in SHP-1 protein, but favoured the return of SHP-2 protein to control levels, as indicated when rats were rendered neutropenic by the administration of vinblastine sulfate. Inhibition of JNK and ERK1/2 with SP600125 pre-treatment further increased the expression of both SHP-1 and SHP-2 proteins in the early phase of Cer-induced AP, while the inhibition of type IV phosphodiesterase with rolipram only suppressed the increase in SHP-2 protein expression during the same phase. Our results show that AP is associated with increases in the expression of SHP-1 and SHP-2 and changes in the dynamics of SHP-2 subcellular distribution in the early phase of Cer-induced AP. Finally, both JNK and ERK1/2 and intracellular cAMP levels are able to modulate the expression of these PTPs.     

Protein-protein interaction between caveolin-1 and SHP-2 is dependent on the N-SH2 domain of SHP-2

Src homology 2-containing protein tyrosine phosphatase 2 (SHP-2) is known to protect neurons from neurodegeneration during ischemia/reperfusion injury. We recently reported that ROS-mediated oxidative stress promotes phosphorylation of endogenous SHP-2 in astrocytes and complex formation between caveolin-1 and SHP-2 in response to oxidative stress. To examine the region of SHP-2 participating in complex formation with caveolin-1, we generated three deletion mutant constructs and six point mutation constructs of SHP-2. Compared with wild-type SHP-2, binding of the N-SH2 domain deletion mutant of SHP-2 to p-caveolin-1 was reduced greatly, using flow cytometric competitive binding assays and surface plasmon resonance (SPR). Moreover, deletion of the N-SH2 domain of SHP-2 affected H₂O₂-mediated ERK phosphorylation and Src phosphorylation at Tyr 419 in primary astrocytes, suggesting that N-SH2 domain of SHP-2 is responsible for the binding of caveolin-1 and contributes to the regulation of Src phosphorylation and activation following ROS-induced oxidative stress in brain astrocytes. [BMB Reports 2015; 48(3): 184-189]     

Differential expression of VirB9 and VirB6 during the life cycle of Anaplasma phagocytophilum in human leucocytes is associated with differential binding and avoidance of lysosome pathway

Anaplasma phagocytophilum, an obligate intracellular bacterium, is the aetiologic agent of human granulocytic anaplasmosis (HGA). A. phagocytophilum virB/D operons encoding type IV secretion system are expressed in cell culture and in the blood of HGA patients. In the present study, their expression across the A. phagocytophilum intracellular developmental cycle was investigated. We found that mRNA levels of both virB9 and virB6 were upregulated during infection of human neutrophils in vitro. The antibody against the recombinant VirB9 protein was prepared and immunogold and immunofluorescence labelling were used to determine the VirB9 protein expression by individual organisms. Majority of A. phagocytophilum spontaneously released from the infected host cells poorly expressed VirB9. At 1 h post infection, VirB9 was not detectable on most bacteria associated with neutrophils. However, VirB9 was strongly expressed by A. phagocytophilum during proliferation in neutrophils. In contrast, with HL-60 cells, approximately 80% of A. phagocytophilum organisms associated at 1 h post infection expressed VirB9 protein and were colocalized with lysosome-associated membrane protein-1 (LAMP-1), whereas, VirB9-undetectable bacteria were not colocalized with LAMP-1. These results indicate developmental regulation of expression of components of type IV secretion system during A. phagocytophilum intracellular life cycle and suggest that bacterial developmental stages influence the nature of binding to the hosts and early avoidance of late endosome-lysosome pathway.     

Downregulation of CXCL12 signaling and altered hematopoietic stem and progenitor cell trafficking in a murine model of acute Anaplasma phagocytophilum infection

Infection with a variety of bacterial pathogens results in hematopoietic stem and progenitor cell (HSPC) mobilization. The mechanism and kinetics of HSPC mobilization during infection are largely unknown. Previously, we found altered HSPC activity in bone marrow, spleen and blood during infection with Anaplasma phagocytophilum, the agent of granulocytic anaplasmosis. We hypothesized that altered CXCL12/CXCR4 signaling, a central pathway for HSPC homing to, and retention within, the bone marrow, plays a role in infection-induced alterations in HSPC number and trafficking. Mice were infected with A. phagocytophilum. Lineage-cKit+ HSPCs were enumerated and proliferation determined. CXCL12 and CXCR4 mRNA were quantified along with CXCL12 protein, and CXCR4 surface, intracellular and total protein expression in HSPCs was determined. Increased bone marrow proliferation of HSPCs began at 2 d post-infection followed by HSPC mobilization and splenic homing. Proliferation of resident HSPCs contributed to increased splenic HSPC numbers. Bone marrow CXCL12 mRNA and protein levels were decreased at 4-8 d post-infection concurrent with HSPC mobilization. CXCR4 protein parameters were decreased in bone marrow HSPCs throughout 2-6 d post-infection. Reduction of CXCL12/CXCR4 signaling simultaneously occurs with HSPC mobilization from bone marrow. Findings suggest that deranged CXCL12/CXCR4 signaling plays a causal role in HSPC mobilization during acute A. phagocytophilum infection.     

Characterization of phosphotyrosine binding motifs in the cytoplasmic domain of B and T lymphocyte attenuator required for association with protein tyrosine phosphatases SHP-1 and SHP-2

B and T lymphocytes express receptors providing positive and negative co-stimulatory signals. We recently identified a novel co-stimulatory molecule, B and T lymphocyte attenuator (BTLA), which exerts inhibitory effects on B and T lymphocytes. The cytoplasmic domain of murine and human BTLA share three conserved tyrosine-based signaling motifs, a Grb-2 recognition consensus, and two immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Phosphorylation of the cytoplasmic domain of BTLA induced the association with the protein tyrosine phosphatases SHP-1 and SHP-2. Association of SHP-1 and SHP-2 to other receptors can involve recruitment to either a single receptor ITIM or to two receptor ITIMs. Here, we analyzed the requirements of BTLA interaction with SHP-1 and SHP-2 in a series of murine and human BTLA mutants. For human BTLA, mutations of either Y257 or Y282, but not Y226, abrogated association with both SHP-1 and SHP-2. For murine BTLA, mutation of either Y274 or Y299, but not Y245, also abrogated association with both SHP-1 and SHP-2. These results indicate that for both murine and human BTLA, association with SHP-1 or SHP-2 requires both of conserved ITIM motifs and does not involve the conserved Grb-2 consensus. Thus, similar to the bisphosphoryl tyrosine-based activation motif (BTAM) by which the Grb-2 associated binder (Gab1), PDGF receptor, and PECAM-1 recruit SHP-2, BTLA also relies on dual ITIMs for its association with the phosphatases SHP-1 and SHP-2.