Pathways involved in the synergistic activation of macrophages by lipoteichoic acid and hemoglobin.

Lipoteichoic acid (LTA) is a Gram-positive cell surface molecule that is found in both a cell-bound form and cell-free form in the host during an infection. Hemoglobin (Hb) can synergize with LTA, a TLR2 ligand, to potently activate macrophage innate immune responses in a TLR2- and TLR4-dependent way. At low levels of LTA, the presence of Hb can result in a 200-fold increase in the secretion of IL-6 following macrophage activation. Six hours after activation, the macrophage genes that are most highly up-regulated by LTA plus Hb activation compared to LTA alone are cytokines, chemokines, receptors and interferon-regulated genes. Several of these genes exhibit a unique TLR4-dependent increase in mRNA levels that continued to rise more than eight hours after stimulation. This prolonged increase in mRNA levels could be the result of an extended period of NF-κB nuclear localization and the concurrent absence of the NF-κB inhibitor, IκBα, after stimulation with LTA plus Hb. Dynasore inhibition experiments indicate that an endocytosis-dependent pathway is required for the TLR4-dependent up-regulation of IL-6 secretion following activation with LTA plus Hb. In addition, interferon-ß mRNA is present after activation with LTA plus Hb, suggesting that the TRIF/TRAM-dependent pathway may be involved. Hb alone can elicit the TLR4-dependent secretion of TNF-α from macrophages, so it may be the TLR4 ligand. Hb also led to secretion of high mobility group box 1 protein (HMGB1), which synergized with LTA to increase secretion of IL-6. The activation of both the TLR2 and TLR4 pathways by LTA plus Hb leads to an enhanced innate immune response.

Inactivation of DltA modulates virulence factor expression in Streptococcus pyogenes.

BACKGROUND: D-alanylated lipoteichoic acid is a virtually ubiquitous component of gram-positive cell walls. Mutations in the dltABCD operon of numerous species exhibit pleiotropic effects, including reduced virulence, which has been attributed to increased binding of cationic antimicrobial peptides to the more negatively charged cell surface. In this study, we have further investigated the effects that mutating dltA has on virulence factor expression in Streptococcus pyogenes. METHODOLOGY/PRINCIPAL FINDINGS: Isogenic Delta dltA mutants had previously been created in two distinct M1T1 isolates of S. pyogenes. Immunoblots, flow cytometry, and immunofluorescence were used to quantitate M protein levels in these strains, as well as to assess their ability to bind complement. Bacteria were tested for their ability to interact with human PMN and to grow in whole human blood. Message levels for emm, sic, and various regulatory elements were assessed by quantitative RT-PCR. Cell walls of Delta dltA mutants contained much less M protein than cell walls of parent strains and this correlated with reduced levels of emm transcripts, increased deposition of complement, increased association of bacteria with polymorphonuclear leukocytes, and reduced bacterial growth in whole human blood. Transcription of at least one other gene of the mga regulon, sic, which encodes a protein that inactivates antimicrobial peptides, was also dramatically reduced in Delta dltA mutants. Concomitantly, ccpA and rofA were unaffected, while rgg and arcA were up-regulated. CONCLUSIONS/SIGNIFICANCE: This study has identified a novel mechanism for the reduced virulence of dltA mutants of Streptococcus pyogenes in which gene regulatory networks somehow sense and respond to the loss of DltA and lack of D-alanine esterification of lipoteichoic acid. The mechanism remains to be determined, but the data indicate that the status of D-alanine-lipoteichoic acid can significantly influence the expression of at least some streptococcal virulence factors and provide further impetus to targeting the dlt operon of gram-positive pathogens in the search for novel antimicrobial compounds.

Lipoteichoic acid synergizes with glycosphingolipids to potently stimulate secretion of interleukin-6 from human blood cells.

In the present study, we found that lipoteichoic acid (LTA) synergizes with glycosphingolipids to stimulate human blood cells to secrete cytokines. We employed globoside, kerasin, and lactosylceramide as representative neutral glycosphingolipids and mixed gangliosides GM(2) and GM(3) as representative acidic glycosphingolipids. LTA and the glycosphingolipids enhanced cytokine secretion by human whole blood, peripheral blood mononuclear cells, and purified monocytes in a dose-dependent manner. The level of synergy ranged up to approximately 10-fold greater than the additive stimulation caused by LTA and glycosphingolipid alone. The greatest synergy was observed with GM(3). We also found that LTA synergizes with the synthetic bacterial lipopeptide mimic Pam3CysK4. In contrast, the glycosphingolipids suppressed the stimulation caused by Pam3CysK4. The stimulation of human cells requires the simultaneous presence of LTA and the glycosphingolipids and probably requires their physical interactions, as shown by dot blotting and nondenaturing polyacrylamide gel electrophoresis experiments. We hypothesize that the enhanced stimulation is due to heterooligomers that form between LTA and glycosphingolipids at the subcritical micelle concentrations used in these experiments. Previous studies showed that LTA also synergizes with hemoglobin. The data taken together suggest that LTA may be a pathogen-associated molecular pattern, although its full activity requires the presence of a synergistic partner(s).

Enhancement of macrophage stimulation by lipoteichoic acid and the costimulant hemoglobin is dependent on Toll-like receptors 2 and 4.

Macrophage stimulation by lipoteichoic acid (LTA) and hemoglobin (Hb) requires Toll-like receptors 2 and 4 (TLR2 and -4). There are two distinct temporal phases of interleukin-6 (IL-6) production. The first results in a slight enhancement of IL-6 secretion in response to LTA plus Hb compared to that with LTA alone and is TLR4 independent. The second requires TLR4 and accounts for most of the additional stimulation seen with LTA plus Hb.

Monocyte and macrophage activation by lipoteichoic Acid is independent of alanine and is potentiated by hemoglobin.

Lipoteichoic acids (LTAs) are Gram-positive bacterial cell wall components that elicit mononuclear cell cytokine secretion. Cytokine-stimulating activity is thought to be dependent on retaining a high level of ester-linked D-alanine residues along the polyglycerol phosphate backbone. However, Streptococcus pyogenes LTA essentially devoid of D-alanine caused human and mouse cells to secrete as much IL-6 as LTA with a much higher D-alanine content. Furthermore, hemoglobin (Hb) markedly potentiates the stimulatory effect of various LTAs on mouse macrophages or human blood cells, regardless of their d-alanine content. LTA and Hb appear to form a molecular complex, based on the ability of each to affect the other's migration on native acrylamide gels, their comigration on these gels, and the ability of LTA to alter the absorption spectra of Hb. Because S. pyogenes is known to release LTA and secrete at least two potent hemolytic toxins, LTA-Hb interactions could occur during streptococcal infections and might result in a profound alteration of the local inflammatory response.

Actin cytoskeleton is required for nuclear accumulation of Gln3 in response to nitrogen limitation but not rapamycin treatment in Saccharomyces cerevisiae.

Saccharomyces cerevisiae selectively utilizes good nitrogen sources in preference to poor ones by down-regulating transcription of genes encoding proteins that transport and degrade poor nitrogen sources when excess nitrogen is available. This regulation is designated nitrogen catabolite repression (NCR). When cells are transferred from a good to a poor nitrogen source (glutamine to proline) or treated with rapamycin, an inhibitor of the protein kinases Tor1/2, Gln3 (NCR-sensitive transcription activator) moves from the cytoplasm into the nucleus. Gln3 re-accumulates in the cytoplasm when cells are returned to a good nitrogen source. However, Gln3 is not uniformly distributed in the cytoplasm. Such non-uniform distribution could result from a variety of interactions including association with a cytoplasmic vesicular system or components of the cytoskeleton. We used latrunculin, a drug that disrupts the actin cytoskeleton by inhibiting actin polymerization, to determine whether the actin cytoskeleton participates in intracellular Gln3 movement. Latrunculin-treatment prevents nuclear accumulation of Gln3 and NCR-sensitive transcription in cells transferred from ammonia to proline medium but does not prevent its accumulation in the cytoplasm of cells transferred from proline to glutamine medium. In contrast, rapamycin-induced nuclear accumulation of Gln3 is not demonstrably affected by latrunculin treatment. These data indicate the actin cytoskeleton is required for nuclear localization of Gln3 in response to limiting nitrogen but not rapamycin-treatment. Therefore, the actin cytoskeleton either participates in the response of Gln3 intracellular localization to nitrogen limitation before Tor1/2, or Tor1/2 inhibition only mimics the outcome of nitrogen limitation rather than directly regulating it.

Cytoplasmic compartmentation of Gln3 during nitrogen catabolite repression and the mechanism of its nuclear localization during carbon starvation in Saccharomyces cerevisiae.

Regulated intracellular localization of Gln3, the transcriptional activator responsible for nitrogen catabolite repression (NCR)-sensitive transcription, permits Saccharomyces cerevisiae to utilize good nitrogen sources (e.g. glutamine and ammonia) in preference to poor ones (e.g. proline). During nitrogen starvation or growth in medium containing a poor nitrogen source, Gln3 is nuclear and NCR-sensitive transcription is high. However, when cells are grown in excess nitrogen, Gln3 is localized to the cytoplasm with a concomitant decrease in gene expression. Treating cells with the Tor protein inhibitor, rapamycin, mimics nitrogen starvation. Recently, carbon starvation has been reported to cause nuclear localization of Gln3 and increased NCR-sensitive transcription. Here we show that nuclear localization of Gln3 during carbon starvation derives from its indirect effects on nitrogen metabolism, i.e. Gln3 does not move into the nucleus of carbon-starved cells if glutamine rather than ammonia is provided as the nitrogen source. In addition, these studies have clearly shown Gln3 is not uniformly distributed in the cytoplasm, but rather localizes to punctate or tubular structures. Analysis of these images by deconvolution microscopy suggests that Gln3 is concentrated in or associated with a highly structured system in the cytosol, one that is possibly vesicular in nature. This finding may impact significantly on how we view (i) the mechanism by which Tor regulates the intracellular localization of Gln3 and (ii) how proteins move into and out of the nucleus.

Gln3 phosphorylation and intracellular localization in nutrient limitation and starvation differ from those generated by rapamycin inhibition of Tor1/2 in Saccharomyces cerevisiae.

The ability of the cell to sense environmental conditions and alter gene expression in response to them is critical to its survival. In Saccharomyces cerevisiae, the Tor1/2 serine/threonine kinases are global regulators situated at the top of a signal cascade reported to receive and transmit nutritional signals associated with the nitrogen supply of the cell. At the other end of that cascade is Gln3, one of two transcriptional activators responsible for most nitrogen catabolic gene expression. When nitrogen is in excess, Tor1/2 are active, and Gln3 is phosphorylated and localizes to the cytoplasm. If Tor1/2 are inhibited by rapamycin or mutation, Gln3 becomes dephosphorylated, accumulates in the nucleus, and mediates nitrogen catabolite repression (NCR)-sensitive transcription. The observations that Gln3 also accumulates in the nuclei of cells provided with poor nitrogen sources or during nitrogen starvation has led to the conclusion that Tor1/2 control intracellular Gln3 localization and NCR-sensitive transcription by regulating Gln3 phosphorylation/dephosphorylation. To test this model, we compared Gln3 phosphorylation states and intracellular localizations under a variety of physiological conditions known to elicit different levels of NCR-sensitive transcription. Our data indicate that: (i) observable Gln3 phosphorylation levels do not correlate in a consistent way with the quality or quantity of the nitrogen source provided, the intracellular localization of Gln3, or the capacity to support NCR-sensitive transcription. (ii) Gln3-Myc(13) is hyperphosphorylated during nitrogen and carbon starvation, but this uniform response does not correlate with Gln3 intracellular localization. (iii) Gln3-Myc(13) dephosphorylation and nuclear localization correlate with one another at early but not late times after rapamycin treatment. These data suggest that rapamycin treatment and growth with poor nitrogen sources bring about nuclear accumulation of Gln3 but likely do so by different mechanisms or by a common mechanism involving molecules other than Gln3 and/or other than the levels of Gln3-Myc(13) phosphorylation thus far detected by others and ourselves.

Mks1p is required for negative regulation of retrograde gene expression in Saccharomyces cerevisiae but does not affect nitrogen catabolite repression-sensitive gene expression.

The Tor1/2p signal transduction pathway regulates nitrogen catabolite repression (NCR)-sensitive (GAP1, GAT1, DAL5) and retrograde (CIT2, DLD3, IDH1/2) gene expression by controlling intracellular localization of the transcription activators, Gln3p and Gat1p, and Rtg1p and Rtg3p, respectively. The accepted pathway for this regulation is NH(3) or excess nitrogen dash, vertical Mks1p dash, vertical Ure2p dash, vertical Gln3p --> DAL5, and rapamycin or limiting nitrogen dash, vertical Torp --> Tap42 dash, vertical Mks1p --> Rtg1/3p --> CIT2, respectively. In current models, Mks1p positively regulates both Gln3p (and DAL5 expression) and Rtg1/3p (and CIT2 expression). Here, in contrast, we show the following. (i) Mks1p is a strong negative regulator of CIT2 expression and does not effect NCR-sensitive expression of DAL5 or GAP1. (ii) Retrograde carbon and NCR-sensitive nitrogen metabolism are not linked by the quality of the nitrogen source, i.e. its ability to elicit NCR, but by the product of its catabolism, i.e. glutamate or ammonia. (iii) In some instances, we can dissociate rapamycin-induced CIT2 expression from Mks1p function, i.e. rapamycin does not suppress Mks1p-mediated down-regulation of CIT2 expression. These findings suggest that currently accepted models of Tor1/2p signal transduction pathway regulation require revision.