No abstract

Patients with acute myocardial infarction receive a P2Y receptor antagonist prior to reperfusion, a treatment that has reduced, but not eliminated, mortality, or heart failure. We tested whether the caspase-1 inhibitor VX-765 given at reperfusion (a requirement for clinical use) can provide sustained reduction of infarction and long-term preservation of ventricular function in a pre-clinical model of ischemia/reperfusion that had been treated with a P2Y receptor antagonist. To address, the hypothesis open-chest rats were subjected to 60-min left coronary artery branch occlusion/120-min reperfusion. Vehicle or inhibitors were administered intravenously immediately before reperfusion. With vehicle only, 60.3 ± 3.8% of the risk zone suffered infarction. Ticagrelor, a P2Y antagonist, and VX-765 decreased infarct size to 42.8 ± 3.3 and 29.2 ± 4.9%, respectively. Combining ticagrelor with VX-765 further decreased infarction to 17.5 ± 2.3%. Similar to recent clinical trials, combining ticagrelor and ischemic postconditioning did not result in additional cardioprotection. VX-765 plus another P2Y antagonist, cangrelor, also decreased infarction and preserved ventricular function when reperfusion was increased to 3 days. In addition, VX-765 reduced infarction in blood-free, isolated rat hearts indicating at least a portion of injurious caspase-1 activation originates in cardiac tissue. While the pro-drug VX-765 only protected isolated hearts when started prior to ischemia, its active derivative VRT-043198 provided the same amount of protection when started at reperfusion, indicating that even in blood-free hearts, caspase-1 appears to exert its injury only at reperfusion. Moreover, VX-765 decreased circulating IL-1β, prevented loss of cardiac glycolytic enzymes, preserved mitochondrial complex I activity, and decreased release of lactate dehydrogenase, a marker of pyroptosis. Our results are the first demonstration of a clinical-grade drug given at reperfusion providing additional, sustained infarct size reduction when added to a P2Y receptor antagonist.

SummarySalmonella enterica serovar Typhimurium periodically experiences acid stress in a variety of host and non-host environments. An encounter with non-lethal acid stress (pH > 4) induces an assortment of physiological changes, called the acid tolerance response (ATR), that helps the cell to tolerate extreme low pH (pH 3). These physiological changes differ in log phase and stationary phase cells and are controlled by different regulatory proteins. OmpR is an acidinduced response regulator critical to the stationary phase ATR but not to the log phase ATR. As OmpR also controls the expression of the acid-induced virulence operon ssrAB, acid shock induction of ompR was examined to gain insight into how Salmonella links virulence with survival at extreme acid pH. The results indicate that acid pH induces ompR from a promoter different from that used for basal expression. Transcription from this promoter is repressed by the histone-like protein H-NS and requires OmpR-P for induction. The classic sensor kinase EnvZ and acetyl phosphate collaborate to produce the optimum level of OmpR-P needed for autoinduction. Although OmpR-P is required for acid-induced expression of ompR in wild-type cells, OmpR is not needed for ompR transcription in the absence of H-NS. Thus, the role of OmpR-P in autoinduction is to help to counteract repression by H-NS. This evidence, combined with the finding that relaxing DNA supercoiling with novobiocin also increased ompR transcription, suggests that acid stress induces ompR by altering local Recently, a brute force screening method was used to identify several MudJ insertion mutants selectively defective in the stationary phase ATR (Bang et al., 2000). Although lacking the stationary phase ATR, these mutants exhibited normal log phase acid tolerance. Sequence analysis of the Mu junctions revealed that two of the insertions occurred within the regulatory gene ompR. The ompR product is part of a classic two-component signal transduction system in which EnvZ is the membranebound sensor kinase and OmpR is the response regulator (Mizuno and Mizushima, 1990). EnvZ appears to sense a variety of environmental signals, the most actively studied being osmolarity. Upon sensing a signal, EnvZ phosphorylates itself at histidine residue 243 (Igo and Silhavy, 1988;Roberts et al., 1994) and then transfers the phosphate to aspartate 55 of OmpR (Delgado et al., 1993). Phosphorylated OmpR then binds and activates target genes such as those encoding the outer membrane porins OmpC and OmpF.As one might predict, unphosphorylated OmpR is not sufficient to induce the stationary phase ATR, phosphorylation is critical. Mutants lacking two major OmpR phosphodonors, EnvZ and acetyl phosphate, are acid sensitive. However, this phenotype is suppressed by converting OmpR residue D55 (the phosphorylation site) to a glutamate. The D55E mutation mimics phosphorylation and results in a constitutively active OmpR protein that does not require phosphorylation to activate target genes. This result indicates that phosphoryla...

SummaryThe stationary phase expression of many conserved, adaptive bacterial proteins is dependent on RpoS, a second vegetative sigma factor. The regulation of RpoS itself, however, is complex and not fully understood, particularly at the level of transcription. In this report, we show that the observed hydrogen peroxide sensitivity of a mutant defective in expression of barA, a bacterial virulence factor, can be explained by a reduction in catalase activity, an RpoS-controlled function. Levels of katE mRNA, encoding the major catalase of Escherichia coli, were much lower in the barA mutant, suggesting that BarA is required for the expression of this RpoSregulated gene. Expression of another RpoS-regulated gene, osmY, was also found to be severely reduced in the barA mutant. Employing Western analyses with anti-RpoS antisera and Northern analyses using probes specific for rpoS, we found that BarA is required for the exponential phase induction of RpoS itself. Operon lacZ fusion expression studies and Northern analyses indicate that BarA itself is maximally expressed in early exponential phase cultures immediately preceding the transcriptional induction of RpoS. Results of primer extension studies indicate that exponential phase expression from the rpoSp1 promoter is reduced by more than 85% in a barA mutant but could be efficiently complemented by a plasmid-borne copy of barA in trans. These results suggest that regulatory signals that are operant in exponentially growing cultures play an important role in effecting stationary phase gene expression.

The obligate intracytoplasmic pathogen Rickettsia prowazekii relies on the transport of many essential compounds from the cytoplasm of the eukaryotic host cell in lieu of de novo synthesis, an evolutionary outcome undoubtedly linked to obligatory growth in this metabolite-replete niche. The paradigm for the study of rickettsial transport systems is the ATP/ADP translocase Tlc1, which exchanges bacterial ADP for host cell ATP as a source of energy, rather than as a source of adenylate. Interestingly, the R. prowazekii genome encodes four open reading frames that are highly homologous to the well-characterized ATP/ADP translocase Tlc1. Therefore, by annotation, the R. prowazekii genome encodes a total of five ATP/ADP translocases: Tlc1, Tlc2, Tlc3, Tlc4, and Tlc5. We have confirmed by quantitative reverse transcriptase PCR that mRNAs corresponding to all five tlc homologues are expressed in R. prowazekii growing in L-929 cells and have shown their heterologous protein expression in Escherichia coli, suggesting that none of the tlc genes are pseudogenes in the process of evolutionary meltdown. However, we demonstrate by heterologous expression in E. coli that only Tlc1 functions as an ATP/ADP transporter. A survey of nucleotides and nucleosides has determined that Tlc4 transports CTP, UTP, and GDP. Intriguingly, although GTP was not transported by Tlc4, it was an inhibitor of CTP and UTP uptake and demonstrated a K i similar to that of GDP. In addition, we demonstrate that Tlc5 transports GTP and GDP. We postulate that Tlc4 and Tlc5 serve the primary function of maintaining intracellular pools of nucleotides for rickettsial nucleic acid biosynthesis and do not provide the cell with nucleoside triphosphates as an energy source, as is the case for Tlc1. Although heterologous expression of Tlc2 and Tlc3 was observed in E. coli, we were unable to identify substrates for these proteins.Rickettsia prowazekii is the etiological agent of epidemic typhus in humans and is designated as a select agent based on its potential use as an agent of bioterrorism. R. prowazekii is an obligate intracellular bacterium and grows only within the cytoplasm of a eukaryotic host cell, unbounded by a host-derived membrane vesicle. As a consequence of growth in this nutrient-rich niche, R. prowazekii has evolved several unique transport systems specific for large, charged metabolites, such as UDP-glucose, S-adenosylmethionine, ATP, NAD, UMP, GMP, and AMP (11,12,45,52,54,55). The ability to transport substrates that are present in the host cell cytosol and rarely available in the extracellular milieu has likely contributed to the evolution of the small-sized rickettsial genome (834 open reading frames [ORFs]); many of the de novo biosynthetic pathways characteristic of free-living bacteria are no longer present in rickettsiae (8). The ATP/ADP translocase Tlc1 is the most thoroughly characterized of these transport systems (1-5, 13, 14, 16, 18, 27, 36, 50, 53, 56). Tlc1 is an obligateexchange transport system that catalyzes the equilibration of...