The lupus-susceptibility locus, Sle3, mediates enhanced resistance to bacterial infections

The genetic predisposition to many autoimmune diseases is inherited as a polygenic trait. It is conceivable that some of the causative alleles in these diseases became prevalent in the population by conferring a survival benefit against environmental assaults, such as infections. We used mice cogenic for genetic loci predisposing to systemic lupus erythomatosus to test the hypothesis that some of these genetic loci protect the host from bacterial infections. Mice with the Sle3 lupus-susceptibility locus on a wild-type background were found to have enhanced antibacterial responses in the context of pneumonia and intra-abdominal sepsis than wild-type animals. This was associated with markedly augmented accumulation of neutrophils in infected tissues, and was bone marrow transferable and dependent on the presence of neutrophils, but not lymphocytes. There was no difference in in vitro leukocyte killing of bacteria nor influx of phagocytes between lupus-susceptible and wild-type animals, but neutrophils from lupus-susceptible mice displayed markedly reduced rate of apoptosis, associated with altered expression of Bcl-2 family proteins, contributing to their greater accumulation. Importantly, deliberate inhibition of apoptosis in wild-type animals significantly boosted the accumulation of neutrophils at the site of infection and resulted in an enhanced antimicrobial response. These observations support the concept that some of the genetic loci that mediate autoimmunity may also confer augmented antimicrobial innate immunity.     

RpoS proteolysis is regulated by a mechanism that does not require the SprE (RssB) response regulator phosphorylation site

In Escherichia coli the response regulator SprE (RssB) facilitates degradation of the sigma factor RpoS by delivering it to the ClpXP protease. This process is regulated: RpoS is degraded in logarithmic phase but becomes stable upon carbon starvation, resulting in its accumulation. Because SprE contains a CheY domain with a conserved phosphorylation site (D58), the prevailing model posits that this control is mediated by phosphorylation. To test this model, we mutated the conserved response regulator phosphorylation site (D58A) of the chromosomal allele of sprE and monitored RpoS levels in response to carbon starvation. Though phosphorylation contributed to the SprE basal activity, we found that RpoS proteolysis was still regulated upon carbon starvation. Furthermore, our results indicate that phosphorylation of wild-type SprE occurs by a mechanism that is independent of acetyl phosphate.     

Mannose-binding lectin augments the uptake of lipid A, Staphylococcus aureus, and Escherichia coli by Kupffer cells through increased cell surface expression of scavenger receptor A

We investigated roles of scavenger receptor A (SR-A) and mannose-binding lectin (MBL) in the uptake of endotoxin and bacteria by Kupffer cells. When [3H]lipid A was injected into retro-orbital plexus of mice, significantly less accumulation of lipid A in the liver was observed in SR-A-deficient mice and wild-type mice coinjected with fucoidan or acetylated low-density lipoprotein, which are known ligands for SR-A. Isolated Kupffer cells were able to take up [3H]lipid A in a time-dependent manner. The amount of lipid A associated with nonadherent Kupffer cells derived from SR-A-deficient mice was reduced by approximately 80% when compared with wild-type cells, indicating an important role of SR-A in endotoxin uptake by Kupffer cells. The lipid A uptake by Kupffer cells was significantly enhanced in the presence of rMBL. Coincubation of fucoidan with [3H]lipid A significantly inhibited the basal and the MBL-stimulated uptake of lipid A by Kupffer cells. Preincubation of MBL with Kupffer cells also increased the uptake of lipid A. These results indicate that MBL augments the SR-A-mediated uptake of lipid A by Kupffer cells. Consistently, the exposure of MBL to Kupffer cells increased cell surface SR-A expression. The phagocytosis of Staphylococcus aureus and Escherichia coli by Kupffer cells was also enhanced by preincubation of MBL with the cells. In addition, MBL bound to lipid A, LPS, and S. aureus, and precipitated S. aureus. This study demonstrates important roles of SR-A and MBL in the uptake of endotoxin and bacteria by Kupffer cells.     

Food sensitizes C. elegans avoidance behaviours through acute dopamine signalling

Many behavioural states are modulated by food availability and nutritional status. Here, we report that in Caenorhabditis elegans, the presence of an external food source enhances avoidance responses to soluble repellents sensed by the polymodal ASH neurons. This enhancement requires dopamine signalling and is mimicked by exogenous dopamine. Food modulation is dependent on the mechanosensory cilia of the dopaminergic neurons, indicating that dopamine is released in response to sensation of bacteria. Activation of the dopamine neurons leads within seconds to a transient state of increased sensory acuity. In vivo imaging experiments indicate that this dopamine-dependent sensitization results in part from modality-specific increases in the magnitude and duration of gustatory responses in the ASH neurons. The D1-like dopamine receptor DOP-4 acts cell autonomously in ASH to mediate effects on response magnitude. Thus, dopamine functions as a direct signal of the presence of food to control context-dependent behavioural states.     

Bacterial SOS response: a food safety perspective

The SOS response is a conserved inducible pathway in bacteria that is involved in DNA repair and restart of stalled replication forks. Activation of the SOS response can result in stress resistance and mutagenesis. In food processing facilities and during food preservation, bacteria are exposed to stresses and stimuli that potentially activate the SOS response, resulting in resistant or adapted bacteria. This review places the bacterial SOS response in a food safety perspective by providing an overview of the known triggers of the SOS response mechanism and its impact on the survival of spoilage and pathogenic bacteria.     

Salmonella chemoreceptors McpB and McpC mediate a repellent response to L-cystine: a potential mechanism to avoid oxidative conditions

Chemoreceptors McpB and McpC in Salmonella enterica have been reported to promote chemotaxis in LB motility-plate assays. Of the chemicals tested as potential effectors of these receptors, the only response was towards L-cysteine and its oxidized form, L-cystine. Although enhanced radial migration in plates suggested positive chemotaxis to both amino acids, capillary assays failed to show an attractant response to either, in cells expressing only these two chemoreceptors. In vivo fluorescence resonance energy transfer (FRET) measurements of kinase activity revealed that in wild-type bacteria, cysteine and cystine are chemoeffectors of opposing sign, the reduced form being a chemoattractant and the oxidized form a repellent. The attractant response to cysteine was mediated primarily by Tsr, as reported earlier for Escherichia coli. The repellent response to cystine was mediated by McpB/C. Adaptive recovery upon cystine exposure required the methyl-transferase/-esterase pair, CheR/CheB, but restoration of kinase activity was never complete (i.e. imperfect adaptation). We provide a plausible explanation for the attractant-like responses to both cystine and cysteine in motility plates, and speculate that the opposing signs of response to this redox pair might afford Salmonella a mechanism to gauge and avoid oxidative environments.     

Experimental evolution of a novel pathway for glycerol dissimilation inEscherichia coli

Wild-type Escherichia coli utilizes glycerol aerobically through an inducible pathway mediated by an ATP-dependent kinase and a glycerol 3-phosphate dehydrogenase which is a flavoprotein. A mutant, strain ECL424, employing a novel pathway for glycerol utilization was isolated. The novel pathway is mediated by an NAD-linked dehydrogenase and a dihydroxyacetone specific enzyme II of the phosphoenolpyruvate phosphotransferase system. This study describes the selection from strain ECL424, a derivative which grows more rapidly on glycerol. The derivative, strain ECL428, produces twice the parental levels of both the dehydrogenase and the enzyme II during growth on glycerol. The function of the dehydrogenase in wild-type cells is unknown, although hydroxyacetone (acetol), 3-hydroxy-2-butanone (acetoin), and 1-amino-2-propanone are gratuitous inducers. The induction can be prevented by glucose whose effect can be cancelled by external cyclic AMP. The effects of hydroxyacetone, glucose, and cyclic AMP are attenuated in the two mutants in which the dehydrogenase is produced at high basal levels. The dihydroxyacetone specific enzyme II is inducible by the substrate in both wild-type and mutant strains and serves for growth on the triose.     

The diacylated lipopeptide FSL-1 enhances phagocytosis of bacteria by macrophages through a Toll-like receptor 2-mediated signalling pathway

A significant amount of evidence has been accumulated to show that Toll-like receptors (TLRs) function as sensors for microbial invasion. However, little is known about how signalling triggered by TLRs leads to the phagocytosis of pathogens. This study was designed to determine whether stimulation of TLR2 mainly with the lipopeptide FSL-1 plays a role in the phagocytosis of pathogens by macrophages. FSL-1 enhanced the phagocytosis of Escherichia coli to a markedly greater extent than it did that of Staphylococcus aureus, but did not enhance the phagocytosis of latex beads. FSL-1 stimulation resulted in enhanced phagocytosis of bacteria by macrophages from TLR2(+/+) mice but not by those from TLR2(-/-) mice. Chinese hamster ovary cells stably expressing TLR2 failed to phagocytose these bacteria, but the cells expressing CD14 did. FSL-1 induced upregulation of the expression of phagocytic receptors, including MSR1, CD36, DC-SIGN and Dectin-1 in THP-1 cells. Human embryonic kidney 293 cells transfected with DC-SIGN and MSR1 phagocytosed these bacteria. These results suggest that the FSL-1-induced enhancement of phagocytosis of bacteria by macrophages may be explained partly by the upregulation of scavenger receptors and the C-type lectins through TLR2-mediated signalling pathways, and that TLR2 by itself does not function as a phagocytic receptor.     

Regulation of the yeast pheromone response pathway by G protein subunits.

The yeast GPA1, STE4, and STE18 genes encode proteins homologous to the respective alpha, beta and gamma subunits of the mammalian G protein complex which appears to mediate the response to mating pheromones. Overexpression of the STE4 protein by the galactose-inducible GAL1 promoter caused activation of the pheromone response pathway which resulted in cell-cycle arrest in late G1 phase and induction of the FUS1 gene expression, thereby suppressing the sterility of the receptor-less mutant delta ste2. Disruption of STE18, in turn, suppressed activation of the pheromone response induced by overexpression of STE4, suggesting that the STE18 product is required for the STE4 action. However, overexpression of both the STE4 and STE18 proteins did not generate a stronger pheromone response than overexpression of STE4 in the presence of wild-type levels of STE18. These results suggest that the beta subunit is the limiting component for the pheromone response and support the idea that beta and gamma subunits act as a positive regulator. Furthermore, overexpression of GPA1 prevented cell-cycle arrest but not FUS1 induction mediated by overexpression of STE4. This implies that the alpha subunit acts as a negative regulator presumably through interacting with beta and gamma subunits in the mating pheromone signaling pathway.     

A survival pathway for Caenorhabditis elegans with a blocked unfolded protein response

The unfolded protein response (UPR) counteracts stress caused by unprocessed ER client proteins. A genome-wide survey showed impaired induction of many UPR target genes in xbp-1 mutant Caenorhabditis elegans that are unable to signal in the highly conserved IRE1-dependent UPR pathway. However a family of genes, abu (activated in blocked UPR), was induced to higher levels in ER-stressed xbp-1 mutant animals than in ER-stressed wild-type animals. RNA-mediated interference (RNAi) inactivation of a representative abu family member, abu-1 (AC3.3), activated the ER stress marker hsp-4::gfp in otherwise normal animals and killed 50% of ER-stressed ire-1 and xbp-1 mutant animals. Abu-1(RNAi) also enhanced the effect of inactivation of sel-1, an ER-associated protein degradation gene. The nine abu genes encode highly related type I transmembrane proteins whose lumenal domains have sequence similarity to a mammalian cell surface scavenger receptor of endothelial cells that binds chemically modified extracellular proteins and directs their lysosomal degradation. Our findings that ABU-1 is an intracellular protein located within the endomembrane system that is induced by ER stress in xbp-1 mutant animals suggest that ABU proteins may interact with abnormal ER client proteins and this function may be particularly important in animals with an impaired UPR.     

Downregulation of VRK1 by p53 in response to DNA damage is mediated by the autophagic pathway

Human VRK1 induces a stabilization and accumulation of p53 by specific phosphorylation in Thr18. This p53 accumulation is reversed by its downregulation mediated by Hdm2, requiring a dephosphorylated p53 and therefore also needs the removal of VRK1 as stabilizer. This process requires export of VRK1 to the cytosol and is inhibited by leptomycin B. We have identified that downregulation of VRK1 protein levels requires DRAM expression, a p53-induced gene. DRAM is located in the endosomal-lysosomal compartment. Induction of DNA damage by UV, IR, etoposide and doxorubicin stabilizes p53 and induces DRAM expression, followed by VRK1 downregulation and a reduction in p53 Thr18 phosphorylation. DRAM expression is induced by wild-type p53, but not by common human p53 mutants, R175H, R248W and R273H. Overexpression of DRAM induces VRK1 downregulation and the opposite effect was observed by its knockdown. LC3 and p62 were also downregulated, like VRK1, in response to UV-induced DNA damage. The implication of the autophagic pathway was confirmed by its requirement for Beclin1. We propose a model with a double regulatory loop in response to DNA damage, the accumulated p53 is removed by induction of Hdm2 and degradation in the proteasome, and the p53-stabilizer VRK1 is eliminated by the induction of DRAM that leads to its lysosomal degradation in the autophagic pathway, and thus permitting p53 degradation by Hdm2. This VRK1 downregulation is necessary to modulate the block in cell cycle progression induced by p53 as part of its DNA damage response.     

Fine balance in the regulation of DnaB helicase by DnaC protein in replication in Escherichia coli.

The DnaC protein of Escherichia coli is essential for replication in vivo and in vitro. In the initiation of replication of a minichromosome at its origin, DnaC delivers the DnaB helicase from a DnaB.DnaC complex to the future replication fork and then departs. However, if an excess of DnaC was present in subsequent steps, it severely inhibited replication by slowing the DnaB helicase at the replication fork. When DnaB was present at a level equimolar with the excess DnaC, the inhibition was relieved, implying that the ratio of DnaC to DnaB is critical for achieving optimal replication activity and avoiding inhibition by DnaC. In vivo, overproduction of DnaC slowed cell growth. This slowing was alleviated by overproducing DnaB at the same time. E. coli strains with a dnaCts gene defective in chromosomal initiation were complemented by the wild-type gene in trans. On the other hand, strains with an elongation-defective dnaCts gene were not complemented by the wild-type dnaC gene. The dominance of the mutant protein suggests that it remains tightly complexed with DnaB at the replication fork, inhibiting elongation even in the presence of the wild-type DnaC.     

Region-specific enhancement of basal extracellular and cocaine-evoked dopamine levels following constitutive deletion of the Serotonin(1B) receptor

The behavioral effects of cocaine are enhanced following constitutive deletion of the serotonin(1B) receptor. The neural substrates mediating the enhanced response to cocaine are unknown. The present studies determined whether basal dopamine dynamics or cocaine-evoked dopamine levels are altered in projection areas of mesostriatal or mesoaccumbens dopamine neurons following serotonin(1B) receptor deletion. Male wild-type and serotonin(1B) knockout mice were implanted with microdialysis guide cannulas aimed at the dorsal striatum or nucleus accumbens. The zero net flux method of quantitative microdialysis was used to quantify basal extracellular dopamine concentrations (DA(ext)) and the extraction fraction of dopamine (E(d)), which provides an index of dopamine uptake. Conventional microdialysis techniques were used to quantify cocaine (0, 5.0, and 20.0 mg/kg)-evoked dopamine overflow. Basal DA(ext) and E(d) did not differ in striatum of wild-type and knockout mice. Similarly, cocaine-stimulated dopamine overflow did not differ between genotype. The basal E(d) did not differ in the nucleus accumbens of wild-type and knockout mice. However, DA(ext) was significantly elevated in the nucleus accumbens of knockout mice. Cocaine-evoked dopamine overflow (nM) was also enhanced in the nucleus accumbens of knockout mice. However, the cocaine-induced increase in dopamine levels, relative to basal values, did not differ between genotype. These data demonstrate that deletion of the serotonin(1B) receptor is associated with increases in basal DA(ext) in the nucleus accumbens. This increase is not associated with an alteration in E(d), suggesting increased basal dopamine release in these animals. It is hypothesized that these alterations in presynaptic neuronal activity are a compensatory response to constitutive deletion of the serotonin(1B) receptor and may contribute to the enhanced behavioral effects of psychostimulants observed in knockout mice.     

Atrial natriuretic factor stimulates exocrine pancreatic secretion in the rat through NPR-C receptors

Increasing evidence supports the role of atrial natriuretic factor (ANF) in the modulation of gastrointestinal physiology. The effect of ANF on exocrine pancreatic secretion and the possible receptors and pathways involved were studied in vivo. Anesthetized rats were prepared with pancreatic duct cannulation, pyloric ligation, and bile diversion into the duodenum. ANF dose-dependently increased pancreatic secretion of fluid and proteins and enhanced secretin and CCK-evoked response. ANF decreased chloride secretion and increased the pH of the pancreatic juice. Neither cholinergic nor adrenergic blockade affected ANF-stimulated pancreatic secretion. Furthermore, ANF response was not mediated by the release of nitric oxide. ANF-evoked protein secretion was not inhibited by truncal vagotomy, atropine, or Nomega-nitro-l-arginine methyl ester administration. The selective natriuretic peptide receptor-C (NPR-C) receptor agonist cANP-(4-23) mimicked ANF response in a dose-dependent fashion. When the intracellular signaling coupled to NPR-C receptors was investigated in isolated pancreatic acini, results showed that ANF did not modify basal or forskolin-evoked cAMP formation, but it dose-dependently enhanced phosphoinositide hydrolysis, which was blocked by the selective PLC inhibitor U-73122. ANF stimulated exocrine pancreatic secretion in the rat, and its effect was not mediated by nitric oxide or parasympathetic or sympathetic activity. Furthermore, CCK and secretin appear not to be involved in ANF response. Present findings support that ANF exerts a stimulatory effect on pancreatic exocrine secretion mediated by NPR-C receptors coupled to the phosphoinositide pathway.     

An aerobic recA-, umuC-dependent pathway of spontaneous base-pair substitution mutagenesis in Escherichia coli

Antimutator alleles indentify genes whose normal products are involved in spontaneous mutagenesis pathways. Mutant alleles of the recA and umuC genes of Escherichia coli, whose wild-type alleles are components of the inducible SOS response, were shown to cause a decrease in the level of spontaneous mutagenesis. Using a series of chromosomal mutant trp alleles, which detect point mutations, as a reversion assay, it was shown that the reduction in mutagenesis is limited to base-pair substitutions. Within the limited number of sites than could be examined, transversions at AT sites were the favored substitutions. Frameshift mutagenesis was slightly enhanced by a mutant recA allele and unchanged by a mutant umuC allele. The wild-type recA and umuC genes are involved in the same mutagenic base-pair substitution pathway, designated "SOS-dependent spontaneous mutagenesis" (SDSM), since a recAumuC strain showed the same degree and specificity of antimutator activity as either single mutant strain. The SDSM pathway is active only in the presence of oxygen, since wild-type, recA, and umuC strains all show the same levels of reduced spontaneous mutagenesis anaerobically. The SDSM pathway can function in starving/stationary cells and may, or may not, be operative in actively dividing cultures. We suggest that, in wild-type cells, SDSM results from basal levels of SOS activity during DNA synthesis. Mutations may result from synthesis past cryptic DNA lesions (targeted mutagenesis) and/or from mispairings during synthesis with a normal DNA template (untargeted mutagenesis). Since it occurs in chromosomal genes of wild-type cells, SDSM may be biologically significant for isolates of natural enteric bacterial populations where extended starvation is often a common mode of existence.