Kathleen H. Cox

Saccharomyces cerevisiae GATA sequences function as TATA elements during nitrogen catabolite repression and when Gln3p is excluded from the nucleus by overproduction of Ure2p

Saccharomyces cerevisiae selectively uses good nitrogen sources (glutamine) in preference to poor ones (proline) by repressing GATA factor-dependent transcription of the genes needed to transport and catabolize poor nitrogen sources, a physiological process designated nitrogen catabolite repression (NCR). We show that some NCR-sensitive genes (CAN1,DAL5, DUR1,2, and DUR3) produce two transcripts of slightly different sizes. Synthesis of the shorter transcript is NCR-sensitive and that of the longer transcript is not. The longer transcript also predominates in gln3Δ mutants irrespective of the nitrogen source provided. We demonstrate that the longer mRNA species arises through the use of an alternative transcription start site generated by Gln3p-binding sites (GATAAs) being able to act as surrogate TATA elements. The ability of GATAAs to serve as surrogate TATAs, i.e. when synthesis of the shorter, NCR-sensitive transcripts are inhibited, correlates with sequestration of enhanced green fluorescent protein (EGFP)-Gln3p in the cytoplasm in a way that is indistinguishable from that seen with EGFP-Ure2p. However, when the shorter, NCR-sensitive DAL5transcript predominates, EGFP-Gln3p is nuclear. These data suggest that the mechanism underlying NCR involves the cytoplasmic association of Ure2p with Gln3p, an interaction that prevents Gln3p from reaching it is binding sites upstream of NCR-sensitive genes.

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 NH3 or excess nitrogen ⊣ Mks1p ⊣ Ure2p ⊣ Gln3p → DAL5, and rapamycin or limiting nitrogen ⊣ Torp → Tap42 ⊣ Mks1p → Rtg1/3p → CIT2, respectively. In current models, Mks1p positively regulates both Gln3p (and DAL5 expression) and Rtg1/3p (and CIT2expression). 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 orGAP1. (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 CIT2expression 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.

Genome-wide transcriptional analysis in S. cerevisiae by mini-array membrane hybridization

Access to the powerful micro‐array analytical methods used for genome‐wide transcriptional analysis has so far been restricted by the high cost and/or lack of availability of the sophisticated instrumentation and materials needed to perform it. Mini‐array membrane hybridization provides a less expensive alternative. The reliability of this technique, however, is not well documented and its reported use has, up to this point, been very limited. Our objective was to test whether or not mini‐array membrane hybridization would reliably identify genes whose expression was controlled by a specific set of genetic and/or physiological signals. Our results demonstrate that mini‐array hybridization can correctly identify genes whose expression is known to be controlled by the GATA‐factor regulatory network in S. cerevisiae and in addition can reliably identify genes not previously reported to be associated with this nitrogen control system. Copyright

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 ΔdltA 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 ΔdltA 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 ΔdltA 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.

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.

Variant AE2 Anion Exchanger Transcripts Accumulate in Multiple Cell Types in the Chicken Gastric Epithelium

Molecular analyses have resulted in the isolation of two chicken stomach AE2 anion exchanger cDNAs, AE2-1 and AE2-2. The 4.3-kilobase (kb) AE2-1 cDNA contains an open reading frame that encodes a predicted polypeptide of 135 kDa that is homologous to AE2 anion exchangers from other species. The partial 1.7-kb AE2-2 cDNA, which differs from the AE2-1 cDNA in two regions, would be predicted to encode an AE2 polypeptide with an alternative N-terminal cytoplasmic tail. Examination of the distribution of these variant transcripts has revealed that AE2 transcripts ranging in size from 4.4 to 7.3 kb accumulate in various adult tissues. However, in the stomach, the unique sequence at the 5′-end of AE2-1 is preferentially associated with transcripts that range in size from 4.5 to 4.9 kb, while the unique sequence at the 5′-end of AE2-2 is preferentially associated with the 7.3-kb AE2 RNA species. In situ hybridization analyses have further revealed that AE2 transcripts accumulate to very high levels within the acid-secreting epithelial cells of the profound gland in the stomach and, to a lesser extent, within the mucus-secreting cells of the superficial gland that line the stomach lumen. This result suggests that AE2 anion exchangers are involved in the regulation of intracellular pH in each of these gastric epithelial cell types.

Enhancement of Macrophage Stimulation by Lipoteichoic Acid and the Costimulant Hemoglobin Is Dependent on Toll-Like Receptors 2 and 4

ABSTRACT 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.

Lipoteichoic Acid Synergizes with Glycosphingolipids To Potently Stimulate Secretion of Interleukin-6 from Human Blood Cells

ABSTRACT 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 GM2 and GM3 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 ∼10-fold greater than the additive stimulation caused by LTA and glycosphingolipid alone. The greatest synergy was observed with GM3. 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).

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-β 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.