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Department of Cellular and Molecular Medicine and Howard Hughes Medical Institute, 9500 Gilman Drive MC0625, University of California—San Diego, La Jolla, California 92093,1 Departments of Molecular Biology and Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 920372
Received 18 February 2006/ Returned for modification 5 April 2006/ Accepted 19 April 2006
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ABSTRACT |
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/ß, Syk, and phospholipase C-
2 upon immune activation. Codeficiency with CD22 restores IgM antigen receptor half-life at the cell surface in addition to reversing alterations in membrane trafficking and immune signaling. Diminished immune responses due to ST6Gal-I deficiency further correlate with constitutive recruitment of Shp-1 to CD22 in unstimulated B cells independent of Lyn tyrosine kinase activity and prevent autoimmune disease pathogenesis in the Lyn-deficient model of systemic lupus erythematosus, resulting in a significant extension of life span. Protein glycosylation by ST6Gal-I restricts access of antigen receptors and Shp-1 to CD22 and operates by a CD22-dependent mechanism that decreases the basal rate of IgM antigen receptor endocytosis in altering the threshold of B-cell immune activation. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mammalian immune receptors of the Siglec family are transmembrane lectins that exhibit extracellular binding specificities for sialic acid linkages and often encode cytoplasmic immunoreceptor tyrosine inhibitory motif (ITIM) domains (8). These hallmarks of Siglec structure suggest that binding to cell surface glycan ligands may in some contexts modify intracellular signal transduction pathways involving protein tyrosine phosphorylation. The B-cell-specific CD22 (Siglec-2) glycoprotein contains cytoplasmic domain ITIMs that are tyrosine phosphorylated upon activation and which recruit the Shp-1 tyrosine phosphatase to dampen immune signaling (7, 9, 11, 30, 43). The extracellular domain of CD22 bears Siglec ligand binding activity toward 2-6-linked sialic acid to underlying ß1-4-linked galactose (33, 39). CD22 Siglec ligands are produced by ST6Gal-I in the Golgi apparatus and are expressed on the B-cell surface, where they typically occupy (or "mask") cell surface CD22 Siglec binding activity (5, 16, 36). The masking of CD22 Siglec binding activity is reduced among postactivated splenic and bone marrow B cells, and thus ST6Gal-I function may be regulated in some contexts (5, 10, 13, 36). These findings infer a role for ST6Gal-I in modulating immune function by constructing Siglec ligands for CD22. Indeed, previous studies have shown that mutant CD22 molecules bearing mutations in the Siglec binding domain impart altered B-cell activation responses in vitro and in vivo (18, 19, 32). More recently, increased colocalization of cell surface IgM with CD22 and clathrin domains was observed among mice lacking ST6Gal-I, while restoration of normal IgM colocalization frequency with clathrin occurred coincident with elevated B-cell immune activation responses in the absence of both CD22 and ST6Gal-I (6).
We have further investigated the mechanisms and glycoprotein specificity by which ST6Gal-I modulates B-cell immune function among mice lacking combinations of ST6Gal-I, CD22, and the Lyn protein tyrosine kinase. Our findings are consistent with those recently published (6) and further show that ST6Gal-I function normally restricts Shp-1 recruitment to CD22 independent of Lyn activity. We also find that ST6Gal-I deficiency selectively increases the rate of IgM BCR and CD22 endocytosis, reducing the cell surface BCR half-life on unstimulated B cells by a CD22-dependent mechanism, and further attenuates autoimmune disease pathogenesis, yielding a significant increase in the life span of Lyn-deficient mice.
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MATERIALS AND METHODS |
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Protein half-lives and endocytosis. Isolated B cells were incubated with EZ-Link Sulfo-NHS-biotin (Pierce) for 30 min at 4°C to label cell surface proteins. Following two washes in ice-cold phosphate-buffered saline (PBS) with 100 mM glycine to quench excess biotin, 1 x 106 aliquots of cells were incubated at 37°C in RPMI 1640 containing 0.1 mM 2-mercaptoethanol, 10% fetal calf serum (FCS), and 2 mM L-glutamine. Cells were harvested at indicated times and lysed at 4°C in lysis buffer containing 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.25% deoxycholate, 1% NP-40, and protease inhibitor cocktail and then sonicated for two 30-s pulses. Cells were incubated for the indicated times in the absence or presence of methyl-ß-cyclodextrin (10 mM) as described previously (41). Lysates were incubated with monoavidin-agarose (Pierce) for 4 h. at 4°C. Precipitates were washed in lysis buffer, and biotinylated proteins were eluted by boiling in SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer. Total B-cell membrane proteins comprising 1 x 106 cell equivalents were loaded in each lane of 5 to 15% gradient SDS-PAGE gels, and the resulting gel blots were probed with antibodies including horseradish peroxidase (HRP)-conjugated anti-IgM and goat antibodies recognizing CD22 (clone Y19), Shp-1 (clone C19), and CD45 (clone M20), followed by HRP-conjugated anti-goat IgG (Santa Cruz). Blots were visualized by enhanced chemiluminescence (Amersham Biosciences), and the signals were quantified (UVP; BioImaging Systems).
Colocalization measurements. Isolated B cells (1 x 106 cells/ml) maintained at 4°C in RPMI 1640 with 2% FCS were treated with 2% paraformaldehyde prior to antibody labeling. Identical results were obtained when cells were instead maintained at 4°C and incubated with antibodies prior to treatment with 2% paraformaldehyde. No effect of cell isolation was observed as B cells among total splenocytes and lymph node cells treated as described above gave identical results. The antibodies used included CD22-fluorescein isothiocyanate (FITC), CD45-FITC (Pharmingen), anti-IgM-Texas red (Jackson, West Grove, PA), and CTB(GM1)-FITC (Sigma) with treatment for 30 min. Following washing, cells were rendered permeable by treatment with CytoFix/CytoPerm (BD Biosciences) for 20 min before incubation with antibody to clathrin (clone X22; Affinity BioReagents) followed by aminomethylcuoumarin acetate (AMCA)-conjugated secondary antibody (Jackson, West Grove, PA). Cells were allowed to settle on glass slides and mounted with antifade aqueous mount medium (BioMedia Corp., CA). Deconvolution images were obtained using a DeltaVision Restoration microscope system (Applied Precision Inc., Issaquah, WA) and analyzed using DeltaVision SoftWorx (version 2.50) in cell sections of 0.2 µm. Quantification of fluorescent signals and their localization were accomplished for each 0.2-µm section in the linear range of the digital camera by object-based analysis algorithms using MetaMorph software (Universal Imaging Corporation, Downington, PA). Maximum projection views of cells are shown which combine all 0.2-µm images (z-stacks). A range of fluorescent signal exclusion thresholds was also applied in these analyses to validate the default value otherwise input by the software and as used for visualization.
Protein phosphotyrosine and coprecipitation.
Isolated B lymphocytes (1 x 106) were suspended in 50 µl of RPMI 1640 with 5% FCS and 10 mM HEPES. Cells were warmed to 37°C before stimulation with 1.2 µg/ml of goat F(ab')2 anti-mouse IgM. At the indicated times, 450 µl of lysis buffer was added (with substitution of Triton X-100 for NP-40). Antibodies to Igß and CD22, as well as Lyn, Syk, phospholipase C-2 (PLC-2), Shp-1, or Vav (10 µl) (Santa Cruz Biotechnology) were added to the lysates with 25 µl of protein A-Sepharose. The lysates were incubated for 4 h and washed three times in lysis buffer. Immune complexes were subjected to SDS-PAGE on a 5 to 15% gradient gel after elution in sample buffer. Gels were transferred to nitrocellulose, and tyrosine phosphorylation was detected by blotting with monoclonal antibody 4G10 (Santa Cruz Biotechnology). Blots were then stripped and reprobed with antibodies to Lyn, Syk, PLC-2, Shp-1, Ig, Igß, and CD22.
BrdU pulse-chase and flow cytometry. Mice were given drinking water ad libitum containing 0.8 mg bromodeoxyuridine (BrdU)/ml for 3 weeks (pulse) and returned to water lacking BrdU (chase) for 2 weeks. Animals were sacrificed at specified times, and lymphocytes were isolated and analyzed for BrdU incorporation. Single-cell suspensions from spleen, lymph node, or bone marrow were harvested and subjected to red blood cell lysis with NH4Cl. Cell surface binding of anti-CD4-phycoerythrin, CD8-allophycocyanin (APC), and B220-Cychrome antibodies (PharMingen) was carried out in 100 µl of fluorescence-activated cell sorter (FACS) buffer (2% FCS in PBS) with 1 x 106 cells on ice for 10 min. Cells were then resuspended in 0.5 ml of ice-cold PBS, and 1.2 ml ice-cold 95% ethanol was added dropwise under gentle mixing to fix the cells. After 30 min of incubation, cells were centrifuged and washed with 2 ml of PBS and then incubated in of 1 ml of PBS, 1% paraformaldehyde, and 0.01% Tween 20 for 30 min at 22°C. Cells were again centrifuged and resuspended in 1 ml of 0.15 M NaCl, 5 mM MgCl2, 10 µM HCl, and 50 Kunitz units/ml of DNase-1 for 10 min. at 22°C. After centrifugation, cells were washed with 2 ml of PBS, incubated in 10 µl of FITC-conjugated anti-BrdU antibody (Becton Dickinson) for 30 min, and then again sedimented, washed in 2 ml PBS, and finally resuspended in 0.5 ml FACS buffer for fluorescence-activated cell sorting analyses, performed on a FACScalibur flow cytometer using CellQuest software (Becton Dickinson). Flow cytometry using lectin conjugates was accomplished as described previously (16).
Calcium mobilization and protein phosphotyrosine analyses. Isolated B cells were measured for calcium flux following stimulation by Indo-1 loading followed by FACS analysis, as previously described (16). Radiolabeled thymidine incorporation was used to measure B-cell proliferation responses to goat F(ab')2 anti-mouse IgM (Jackson) as described previously (16).
Histology. Examinations of frozen embedded kidney, liver, or spleen (O.C.T. medium; Sakura Finetek, Torrance, CA) were performed on 5-µm sections fixed in formalin and rehydrated in PBS. Sections were incubated with anti-IgM-rhodamine or anti-IgG-FITC (Jackson ImmunoResearch Labs, West Grove, PA). Washed and stained sections were mounted in aqueous gel mount (Biomeda Corp., Foster City, CA) and observed by fluorescent microscopy at a magnification of x200 (Zeiss, Göttingen, Germany).
Lyn-deficient autoimmune disease. Antinuclear antibody (ANA) detection and serum antibody titers to double-stranded DNA (dsDNA), histones, Sm antigen, and total kidney protein were assayed as previously described (4).
Statistical analysis. Analyses of variance and Student's t test were used as described in the figure legends to compare the groups shown in each experiment. Results are expressed as means and standard errors of the mean unless otherwise indicated.
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RESULTS |
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ST6Gal-I promotes protein tyrosine phosphorylation in immune signaling.
Levels of protein expression and phosphotyrosine accumulation before and after IgM cross-linking were measured among immune signal transducers, including Ig/ß heterodimers, the Lyn and Syk tyrosine kinases, PLC-2, and the Vav guanine nucleotide exchange factor. These proteins were found at normal abundance among B cells lacking ST6Gal-I, while some migrated at slightly different molecular weights consistent with altered glycosylation. Measurements of tyrosine phosphorylation indicated, in contrast, that only 20 to 40% of normal levels were induced among positive signal transducers Ig/ß, Syk, and PLC-2 in ST6Gal-I deficiency (Fig. 2A). The Lyn tyrosine kinase, in comparison, which has roles in both positive and negative immune signaling, acquired 60% of normal phosphotyrosine induction, while no change in Vav phosphorylation was observed when corrected for protein level. These findings show that ST6Gal-I deficiency results in a selective and significant decrease in phosphotyrosine accumulation on key positive immune signal transduction mediators in response to B-cell activation.
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CD22 is required to accelerate IgM endocytosis in ST6Gal-I deficiency. The half-life of cell surface IgM was further investigated among mice lacking CD22 (25). CD22 deficiency alone moderately increased the half-life of cell surface IgM among naïve resting B cells compared with wild-type B cells, while the absence of both CD22 and ST6Gal-I reduced IgM endocytosis and restored IgM cell surface half-life to normal (Fig. 5A). Consistent with recent observations, the absence of CD22 further reduced IgM-clathrin colocalization in ST6Gal-I deficiency to levels observed among wild-type B cells (Fig. 5B) (6). In addition, CD45 half-life at the cell surface and colocalization with clathrin were unaffected by loss of CD22 and ST6Gal-I (Fig. 5C) (data not shown). These findings reveal that the elevated level of cell surface IgM-clathrin colocalization and the glycoprotein-selective increase in the rate of IgM endocytosis which are provoked by ST6Gal-I deficiency both require CD22. The loss of CD22 further elevated ST6Gal-I-deficient B-cell immune responses involving Ca2+ mobilization and cell proliferation, consistent with recent findings (6; data not shown). Thus, in ST6Gal-I deficiency, CD22 attenuates immune signal transduction and induces IgM BCR endocytosis coincident with increased IgM-CD22 colocalization and constitutive Shp-1 recruitment to CD22. Uncoupling Shp-1 recruitment to CD22 might therefore elevate BCR immune responses among ST6Gal-I-deficient B cells.
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DISCUSSION |
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/ß, Syk, and PLC-2 upon IgM stimulation. Similar effects of ST6Gal-I deficiency were observed among MD4 transgenic B-cell populations expressing antigen receptors to hen egg lysozyme but which had never been exposed to antigen. These findings imply that diminished B-cell activation responses conferred by ST6Gal-I deficiency are not developmental phenotypes acquired by altered antigen receptor signaling during B-cell ontogeny, but rather reflect a role of ST6Gal-I protein glycosylation in promoting mature B-cell immune function.
ST6Gal-I- and CD22-dependent regulation of IgM signaling and endocytosis.
Attenuation and extinction of B-cell immune signaling have been associated with IgM internalization in fused lipid raft-clathrin domains, interactions of IgM BCRs with CD22 and Shp-1, and disruption of IgM BCR interaction with Ig/ß heterodimers (7, 9, 11, 30, 41, 43, 46). The B-cell phenotype induced by ST6Gal-I deficiency is reminiscent of these findings. Increased IgM BCR endocytosis was associated with elevated IgM-clathrin colocalization among GM1-positive membrane domains. Absence of ST6Gal-I increased the level of IgM-CD22 colocalization, which was further observed among clathrin domains. In addition, no change in Ig/ß expression at the B-cell surface was detected on ST6Gal-I-deficient B cells, implying the disruption of IgM-Ig/ß complexes prior to IgM endocytosis (data not shown). While CD22 may be excluded from lipid rafts, we concur with a recent report (6) that the majority of CD22 normally resides among clathrin-rich domains. Nevertheless, almost half of colocalized IgM-CD22 is outside of clathrin-rich domains in ST6Gal-I deficiency, implying that CD22 associates with the BCR and dampens immune responsiveness prior to arrival in clathrin-rich domains and subsequent endocytosis. Interestingly, a 40% reduction of cell surface IgM levels was invariably observed on B cells lacking ST6Gal-I, CD22, and Lyn, either singly or in combination (16; data not shown). Therefore, the level of cell surface IgM BCR expression does not correlate with the magnitude of immune responses to anti-IgM stimulation and implicates instead altered BCR colocalization and trafficking as responsible for dampened immune signaling in ST6Gal-I deficiency. The mechanism of CD22-dependent trafficking of cell surface IgM is not resolved; however, CD22 Siglec ligand binding may be involved in this process.
ST6Gal-I sialylation in CD22 Siglec-dependent and -independent binding. The physiologic role of CD22 Siglec binding has been difficult to perceive. As CD22 is the only molecule detected on the B-cell surface with Siglec binding activity for counter-receptors produced by ST6Gal-I (5, 19), it would be expected that CD22 mutations specifically disabling Siglec binding would recapitulate phenotypes observed in ST6Gal-I deficiency. CD22 Siglec mutations targeted to the mouse germ line and studied among primary B cells have indeed produced phenotypes similar to those observed in ST6Gal-I deficiency, including attenuated BCR-induced proliferation (32). In contrast, an exogenous approach to disrupt CD22 Siglec ligands using glycan ligand mimetics increased antigen receptor-stimulated Ca2+ mobilization when added to established B-cell lines, suggesting a reduction of CD22 Siglec-dependent association with IgM complexes (19). Moreover, study of a B-cell line derived from CD22-deficient mice revealed augmented Ca2+ mobilization upon expression of mutant CD22 molecules disabled in Siglec binding (18). Such findings appear difficult to reconcile. From the earliest discordances observed among CD22 mutant mice, it seems likely that mouse strain differences may contribute to alterations in the outcome involving CD22 function in BCR-induced proliferation (25, 27, 28, 32, 38). In addition, the use of primary B cells may be relevant, as a study using the Daudi B-cell line reported no increase in CD22 endocytosis in the absence of CD22 Siglec ligands (50).
The identities of CD22 binding partners depend upon the methodologies employed. IgM-CD22 interaction has been reported in ranges from 0.5% to 18% by coprecipitation and protein cross-linking (21, 30, 50). Those methods can detect closely adjacent and even direct interactions but may also disrupt selective in situ binding and can rely upon strict intermolecular distances. Quantitative fluorescent deconvolution microscopy cannot achieve this degree of spatial resolution; however, this technique can analyze native and intact B-cell surfaces. Our findings by this method are consistent with recent studies (6) that report a substantial although minor fraction of total cell surface IgM (
30%) is typically colocalized with CD22 in wild-type unstimulated peripheral B cells. A significant basal level of IgM-CD22 colocalization is consistent with the normal role of CD22 expression in modulating peripheral naïve B-cell activation thresholds. It is not clear, however, whether IgM-CD22 colocalization on B cells expressing ST6Gal-I involves a subset of total cell surface glycoproteins lacking CD22 Siglec ligands due to the phenomenon of microheterogeneity of glycan linkage formation in the Golgi apparatus. It remains possible that increased colocalization of IgM with CD22 in ST6Gal-I deficiency results from the absence of CD22 Siglec binding.
Many proteins are glycosylated by ST6Gal-I in B cells, and all would appear to be CD22 Siglec ligands by lectin coprecipitation from homogenized cell extracts (16; data not shown). However, CD22 interactions are likely more selective in situ. On the resting intact B-cell surface, CD22 Siglec binding was recently shown to predominantly involve cis ligands resulting in homotypic multimeric CD22 complexes (15). Our results by quantitative fluorescent deconvolution microscopy of entire intact as well as naïve B-cell surfaces appear consistent with those findings. We observed more dispersed CD22 expression at the cell surface in ST6Gal-I deficiency as well as a reduced fraction of total cell surface CD22 colocalized with IgM. These findings suggest that reduced homotypic CD22 binding occurs in the absence of Siglec ligands concurrent with increased heterotypic CD22 interactions among other cell surface molecules, including the IgM BCR, possibly by enabling CD22 Siglec-independent binding contributed by the conserved extracellular non-Siglec Ig-like domains. This may explain the significant amount of IgM cross-linking to CD22 observed on intact cell surfaces that lack Siglec ligands (50).
Control of Shp-1 recruitment to CD22 by ST6Gal-I protein glycosylation. The attenuation of BCR signaling by CD22 is attributed to Shp-1 recruitment following activation (7, 9, 11, 43). In wild-type B cells, a low level of Shp-1 coprecipitation with CD22 is observed prior to IgM cross-linking, with maximal Shp-1 recruitment occurring after several minutes of BCR stimulation. In contrast, ST6Gal-I deficiency induced maximal Shp-1 recruitment to CD22 prior to IgM stimulation and the resultant increase in phosphotyrosine on CD22. Nevertheless, constitutive Shp-1 recruitment to CD22 in ST6Gal-I deficiency may not simply reflect the absence of CD22 Siglec ligands. No similar increase in Shp-1 recruitment to CD22 was reported among B cells bearing mutant CD22 molecules with either deleted or likely inactive Siglec binding domains (32). It is possible that the absence of CD22 sialylation by ST6Gal-I alters CD22 conformation in promoting Shp-1 recruitment when CD22 is colocalized with IgM BCRs. Or perhaps ST6Gal-I deficiency increases access of Shp-1 to CD22 by altering glycoprotein trafficking and residence among membrane microdomains. It is widely accepted that Shp-1 recruitment to CD22 reflects binding of Shp-1 src homology 2 (SH2) domain sequences to phosphotyrosine within CD22 ITIM domains. Remarkably, the level of phosphotyrosine detected on CD22 in ST6Gal-I deficiency was reduced to negligible levels in the further absence of Lyn, and yet the constitutively high level of Shp-1 recruitment to CD22 remained in the absence of immune stimulation. Shp-1 recruitment was not sustained following BCR stimulation of ST6Gal-I-deficient B cells, revealing that Lyn function increases the half-life of CD22-Shp-1 complexes following BCR activation. While the mechanism of CD22-Shp-1 complex formation in ST6Gal-I deficiency remains to be established, it is possible that detection of phosphotyrosine by antibody binding is thwarted by ST6Gal-I deficiency or a mechanism exists by which CD22 and Shp-1 complexes can form independent of CD22 tyrosine phosphorylation.
Endogenous control of ST6Gal-I function. The immune modulatory capability of ST6Gal-I may be normally regulated among B cells, perhaps as indicated by unmasking of CD22 Siglec binding following immune stimulation. Although no change in ST6Gal-I RNA levels occurs subsequent to BCR activation of wild-type B cells (data not shown), multiple changes in B-cell surface glycan linkages occur which mimic those observed on ST6Gal-I-deficient B cells bearing unmasked CD22 (10, 36) (see Fig. S3 in the supplemental material). Decreased ST6Gal-I function may result from the induction of glycosyltransferases that compete for glycoprotein substrates, by the expression or activation of endogenous sialidases, or perhaps from proteolysis in the stem region abolishing glycosyltransferase substrate access in the Golgi apparatus by releasing the catalytic domain as a secreted fragment. Except in the instance of a rapid desialylation at the cell surface, it appears unlikely that these mechanisms are involved during the early postactivation responses that result in rapid Shp-1 recruitment to CD22 and IgM endocytosis. Interestingly, the normal induction of BCR endocytosis in response to IgM stimulation among wild-type B cells, which also results in a cell surface IgM half-life of less than 2 h, was not affected by the loss of CD22 or Lyn (data not shown). The similar decrease of IgM BCR cell surface half-life in ST6Gal-I deficiency, which in contrast is CD22 dependent and occurs in the absence of IgM stimulation, likely represents a distinct pathway to endocytosis that typically employs ST6Gal-I and CD22 function to restrain IgM BCR internalization in promoting naive mature B-cell immune function.
Posttranslational modifications contributing to intracellular immune signal transduction pathways include kinase and phosphatase enzymes that regulate protein phosphorylation. Less evident has been whether protein glycosylation, confined to extracellular compartments and topologically separated from protein phosphorylation, may participate in the formation of these intracellular signals. We have observed humoral immune deficits in the absence of protein glycosylation by ST6Gal-I that result in diminished tyrosine phosphorylation of proteins associated with the BCR complex, in findings that reflect both cell-type-specific and glycoprotein-selective roles for ST6Gal-I. Such focused purpose is increasingly evident among the discerned functions of mammalian glycosyltransferases, as further exemplified by the specificity of ST8Sia-II and -IV in neural NCAM function, FT-8 in lung epithelial transforming growth factor ß receptor signaling, and GnT-4a in pancreatic ß-cell glucose transporter expression (1, 26, 47, 48). Diminished B-cell immune responses observed in ST6Gal-I deficiency reflect constitutive disruption of a CD22-dependent mechanism that controls glycoprotein-selective cell surface modulation of IgM trafficking and colocalization with CD22-Shp-1 complexes among unstimulated mature B cells coincident with enhancing the rate of IgM endocytosis. Therefore, ST6Gal-I operates with CD22 in an immune regulatory pathway that converges with clathrin-dependent IgM endocytosis in establishing thresholds for naïve mature peripheral BCR activation. Diminishing ST6Gal-I function spares B-cell viability yet has a therapeutic effect in the context of the severe systemic lupus erythematosus phenotype due to Lyn deficiency and possibly other autoimmune disease syndromes arising from abnormal and hyperimmune B-cell activity.
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ACKNOWLEDGMENTS |
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J.D.M. is a founder of Abaron Biosciences, Inc., a company that is developing drugs related to the research described in the manuscript. The University of California—San Diego is also an equity holder. The terms of this arrangement have been reviewed and approved by the University of California—San Diego, in accordance with its conflict of interest policies.
This research was funded by NIH grants HL57345 (J.D.M.), AI050143 (J.C.P.), and GM25042 (B.E.C.). J.D.M. is supported as an Investigator of the Howard Hughes Medical Institute.
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FOOTNOTES |
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Supplemental material for this article may be found at http://mcb.asm.org/.
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