Crosstalk between Virulence Loci: Regulation of Salmonella enterica Pathogenicity Island 1 (SPI-1) by Products of the std Fimbrial Operon

Invasion of intestinal epithelial cells is a critical step in Salmonella infection and requires the expression of genes located in Salmonella pathogenicity island 1 (SPI-1). A key factor for SPI-1 expression is DNA adenine (Dam) methylation, which activates synthesis of the SPI-1 transcriptional activator HilD. Dam-dependent regulation of hilD is postranscriptional (and therefore indirect), indicating the involvement of unknown cell functions under Dam methylation control. A genetic screen has identified the std fimbrial operon as the missing link between Dam methylation and SPI-1. We show that all genes in the std operon are part of a single transcriptional unit, and describe three previously uncharacterized ORFs (renamed stdD, stdE, and stdF). We present evidence that two such loci (stdE and stdF) are involved in Dam-dependent control of Salmonella SPI-1: in a Dam− background, deletion of stdE or stdF suppresses SPI-1 repression; in a Dam+ background, constitutive expression of StdE and/or StdF represses SPI-1. Repression of SPI-1 by products of std operon explains the invasion defect of Salmonella Dam− mutants, which constitutively express the std operon. Dam-dependent repression of std in the ileum may be required to permit invasion, as indicated by two observations: constitutive expression of StdE and StdF reduces invasion of epithelial cells in vitro (1,000 fold) and attenuates Salmonella virulence in the mouse model (>60 fold). In turn, crosstalk between std and SPI-1 may play a role in intestinal infections by preventing expression of SPI-1 in the caecum, an intestinal compartment in which the std operon is known to be expressed

Gene Transcription and Splicing of T-Type Channels Are Evolutionarily-Conserved Strategies for Regulating Channel Expression and Gating

T-type calcium channels operate within tightly regulated biophysical constraints for supporting rhythmic firing in the brain, heart and secretory organs of invertebrates and vertebrates. The snail T-type gene, LCav3 from Lymnaea stagnalis, possesses alternative, tandem donor splice sites enabling a choice of a large exon 8b (201 aa) or a short exon 25c (9 aa) in cytoplasmic linkers, similar to mammalian homologs. Inclusion of optional 25c exons in the III–IV linker of T-type channels speeds up kinetics and causes hyperpolarizing shifts in both activation and steady-state inactivation of macroscopic currents. The abundant variant lacking exon 25c is the workhorse of embryonic Cav3 channels, whose high density and right-shifted activation and availability curves are expected to increase pace-making and allow the channels to contribute more significantly to cellular excitation in prenatal tissue. Presence of brain-enriched, optional exon 8b conserved with mammalian Cav3.1 and encompassing the proximal half of the I–II linker, imparts a ∼50% reduction in total and surface-expressed LCav3 channel protein, which accounts for reduced whole-cell calcium currents of +8b variants in HEK cells. Evolutionarily conserved optional exons in cytoplasmic linkers of Cav3 channels regulate expression (exon 8b) and a battery of biophysical properties (exon 25c) for tuning specialized firing patterns in different tissues and throughout development

Acid-evoked Ca2+ signalling in rat sensory neurones: effects of anoxia and aglycaemia

Ischaemia excites sensory neurones (generating pain) and promotes calcitonin gene-related peptide release from nerve endings. Acidosis is thought to play a key role in mediating excitation via the activation of proton-sensitive cation channels. In this study, we investigated the effects of acidosis upon Ca2+ signalling in sensory neurones from rat dorsal root ganglia. Both hypercapnic (pHo 6.8) and metabolic-hypercapnic (pHo 6.2) acidosis caused a biphasic increase in cytosolic calcium concentration ([Ca2+] i ). This comprised a brief Ca2+ transient (half-time approximately 30 s) caused by Ca2+ influx followed by a sustained rise in [Ca2+] i due to Ca2+ release from caffeine and cyclopiazonic acid-sensitive internal stores. Acid-evoked Ca2+ influx was unaffected by voltage-gated Ca2+-channel inhibition with nickel and acid sensing ion channel (ASIC) inhibition with amiloride but was blocked by inhibition of transient receptor potential vanilloid receptors (TRPV1) with (E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b][1,4] dioxin-6-yl)acrylamide (AMG 9810; 1 microM) and N-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl) tetrahydropryazine-1(2H)-carbox-amide (BCTC; 1 microM). Combining acidosis with anoxia and aglycaemia increased the amplitude of both phases of Ca2+ elevation and prolonged the Ca2+ transient. The Ca2+ transient evoked by combined acidosis, aglycaemia and anoxia was also substantially blocked by AMG 9810 and BCTC and, to a lesser extent, by amiloride. In summary, the principle mechanisms mediating increase in [Ca2+] i in response to acidosis are a brief Ca2+ influx through TRPV1 followed by sustained Ca2+ release from internal stores. These effects are potentiated by anoxia and aglycaemia, conditions also prevalent in ischaemia. The effects of anoxia and aglycaemia are suggested to be largely due to the inhibition of Ca2+-clearance mechanisms and possible increase in the role of ASICs