Stimulation of soluble guanylate cyclase exerts antiinflammatory actions in the liver through a VASP/NF-κB/NLRP3 inflammasome circuit

Soluble guanylate cyclase (sGC) catalyzes the conversion of guanosine triphosphate into cyclic guanosine-3',5'-monophosphate, a key second messenger in cell signaling and tissue homeostasis. It was recently demonstrated that sGC stimulation is associated with a marked antiinflammatory effect in the liver of mice with experimental nonalcoholic steatohepatitis (NASH). Here, we investigated the mechanisms underlying the antiinflammatory effect of the sGC stimulator praliciguat (PRL) in the liver. Therapeutic administration of PRL exerted antiinflammatory and antifibrotic actions in mice with choline-deficient l-amino acid-defined high-fat diet-induced NASH. The PRL antiinflammatory effect was associated with lower F4/80- and CX3CR1-positive macrophage infiltration into the liver in parallel with lower Ly6CHigh- and higher Ly6CLow-expressing monocytes in peripheral circulation. The PRL antiinflammatory effect was also associated with suppression of hepatic levels of interleukin (IL)-1β, NLPR3 (NACHT, LRR, and PYD domain-containing protein 3), ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain), and active cleaved-caspase-1, which are components of the NLRP3 inflammasome. In Kupffer cells challenged with the classical inflammasome model of lipopolysaccharide plus adenosine triphosphate, PRL inhibited the priming (expression of Il1b and Nlrp3) and blocked the release of mature IL-1β. Mechanistically, PRL induced the protein kinase G (PKG)-mediated phosphorylation of the VASP (vasodilator-stimulated phosphoprotein) Ser239 residue which, in turn, reduced nuclear factor-κB (NF-κB) activity and Il1b and Nlrp3 gene transcription. PRL also reduced active cleaved-caspase-1 levels independent of pannexin-1 activity. These data indicate that sGC stimulation with PRL exerts antiinflammatory actions in the liver through mechanisms related to a PKG/VASP/NF-κB/NLRP3 inflammasome circuit

Klebsiella pneumoniae Multiresistance Plasmid pMET1: Similarity with the Yersinia pestis Plasmid pCRY and Integrative Conjugative Elements

Dissemination of antimicrobial resistance genes has become an important public health and biodefense threat. Plasmids are important contributors to the rapid acquisition of antibiotic resistance by pathogenic bacteria.The nucleotide sequence of the Klebsiella pneumoniae multiresistance plasmid pMET1 comprises 41,723 bp and includes Tn1331.2, a transposon that carries the bla(TEM-1) gene and a perfect duplication of a 3-kbp region including the aac(6')-Ib, aadA1, and bla(OXA-9) genes. The replication region of pMET1 has been identified. Replication is independent of DNA polymerase I, and the replication region is highly related to that of the cryptic Yersinia pestis 91001 plasmid pCRY. The potential partition region has the general organization known as the parFG locus. The self-transmissible pMET1 plasmid includes a type IV secretion system consisting of proteins that make up the mating pair formation complex (Mpf) and the DNA transfer (Dtr) system. The Mpf is highly related to those in the plasmid pCRY, the mobilizable high-pathogenicity island from E. coli ECOR31 (HPI(ECOR31)), which has been proposed to be an integrative conjugative element (ICE) progenitor of high-pathogenicity islands in other Enterobacteriaceae including Yersinia species, and ICE(Kp1), an ICE found in a K. pneumoniae strain causing primary liver abscess. The Dtr MobB and MobC proteins are highly related to those of pCRY, but the endonuclease is related to that of plasmid pK245 and has no significant homology with the protein of similar function in pCRY. The region upstream of mobB includes the putative oriT and shares 90% identity with the same region in the HPI(ECOR31).The comparative analyses of pMET1 with pCRY, HPI(ECOR31), and ICE(Kp1 )show a very active rate of genetic exchanges between Enterobacteriaceae including Yersinia species, which represents a high public health and biodefense threat due to transfer of multiple resistance genes to pathogenic Yersinia strains

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Expression of Soluble Guanylate Cyclase in Rat and Human Hepatocytes

Soluble Guanylate Cyclase (sGC) is a key enzyme in the nitric oxide (NO) signaling pathway. sGC binds NO to produce cyclic guanosine-3’,5’-monophosphate (cGMP). The NO-sGC-cGMP pathway is directly involved in a number of physiological functions including smooth muscle vasodilation. Deficiencies in this system, such as reduced NO tone, can result in cardiovascular dysfunction. Stimulators of sGC have been developed to synergize with and enhance NO signaling, creating potential therapies for a number of diseases. Currently, it is known that sGC is expressed in tissues such as the brain, kidney, lung, and liver. However, as an intracellular enzyme, sGC expression in specific cell types within these tissues remains to be explored. This study aims to uncover the expression of sGC in rat and human hepatocyte cells, the major cell type in the liver responsible for metabolizing and detoxifying the body. To begin, the expression of sGC subunits α1 and β1 was assessed by immunohistochemistry (IHC) staining on a paraffin fixed rat liver slice. The results uncovered the localization of sGC in stellate, endothelial and hepatocytes surrounding the vasculature in a rat liver. To confirm positive expression of sGC in hepatocytes, branched DNA (bDNA) probes for sGC were used to confirm expression of sGC in isolated, cryopreserved rat and human hepatocyte cells. Finally, the functionality of the sGC enzyme was shown in vitro by stimulating both rat and human hepatocytes in the presence of an sGC stimulator with and without the NO-donor (DETA). Target engagement by the sGC stimulator was determined through the quantitation of cGMP

Inhibition of Aminoglycoside 6′-N-Acetyltransferase Type Ib-Mediated Amikacin Resistance by Antisense Oligodeoxynucleotides

Amikacin has been very useful in the treatment of infections caused by multiresistant bacteria because it is refractory to the actions of most modifying enzymes. However, the spread of AAC(6′)-I-type acetyltransferases, enzymes capable of catalyzing inactivation of amikacin, has rendered this antibiotic all but useless in some parts of the world. The aminoglycoside 6′-N-acetyltransferase type Ib, which is coded for by the aac(6′)-Ib gene, mediates resistance to amikacin and other aminoglycosides. RNase H mapping and computer prediction of the secondary structure led to the identification of five regions accessible for interaction with antisense oligodeoxynucleotides in the aac(6′)-Ib mRNA. Oligodeoxynucleotides targeting these regions could bind to native mRNA with different efficiencies and mediated RNase H digestion. Selected oligodeoxynucleotides inhibited AAC(6′)-Ib synthesis in cell-free coupled transcription-translation assays. After their introduction into an Escherichia coli strain harboring aac(6′)-Ib by electroporation, some of these oligodeoxynucleotides decreased the level of resistance to amikacin. Our results indicate that use of antisense compounds could be a viable strategy to preserve the efficacies of existing antibiotics to which bacteria are becoming increasingly resistant

Complete Nucleotide Sequence of Klebsiella pneumoniae Multiresistance Plasmid pJHCMW1

The multiresistance plasmid pJHCMW1, harbored by a clinical Klebsiella pneumoniae strain isolated from a neonate with meningitis, was sequenced. A circular sequence of 11,354 bp was generated, of which 7,993 bp make up Tn1331, a transposon including the antibiotic resistance genes aac(6′)-Ib, aadA1, bla(OXA-9), and bla(TEM-1). The gene aac(6′)-Ib is included in a gene cassette, and both aadA1 and bla(OXA-9) are included in a single-gene cassette that may have arisen as a consequence of a recombination event involving two integrons. The pJHCMW1 plasmid replicates through a ColE1-like RNA-regulated mechanism, includes a functional oriT, and two loci with similarity to XerCD site-specific recombination target sites involved in plasmid stabilization by the resolution of multimers. One of these two loci, mwr, is active and has been the subject of previous studies, and the other, dxs, is not functional but binds the recombinase XerD with low affinity. Two additional open reading frames were identified, one with low similarity to two hypothetical membrane proteins from Mycobacterium tuberculosis and Mycobacterium leprae and the other with low similarity to psiB, a gene encoding a function that facilitates the establishment of the transferring plasmid in the recipient bacterial cell during the process of conjugation

External Guide Sequences Targeting the aac(6′)-Ib mRNA Induce Inhibition of Amikacin Resistance▿

The dissemination of AAC(6′)-I-type acetyltransferases have rendered amikacin and other aminoglycosides all but useless in some parts of the world. Antisense technologies could be an alternative to extend the life of these antibiotics. External guide sequences are short antisense oligoribonucleotides that induce RNase P-mediated cleavage of a target RNA by forming a precursor tRNA-like complex. Thirteen-nucleotide external guide sequences complementary to locations within five regions accessible for interaction with antisense oligonucleotides in the mRNA that encodes AAC(6′)-Ib were analyzed. While small variations in the location targeted by different external guide sequences resulted in big changes in efficiency of binding to native aac(6′)-Ib mRNA, most of them induced high levels of RNase P-mediated cleavage in vitro. Recombinant plasmids coding for selected external guide sequences were introduced into Escherichia coli harboring aac(6′)-Ib, and the transformant strains were tested to determine their resistance to amikacin. The two external guide sequences that showed the strongest binding efficiency to the mRNA in vitro, EGSC3 and EGSA2, interfered with expression of the resistance phenotype at different degrees. Growth curve experiments showed that E. coli cells harboring a plasmid coding for EGSC3, the external guide sequence with the highest mRNA binding affinity in vitro, did not grow for at least 300 min in the presence of 15 μg of amikacin/ml. EGSA2, which had a lower mRNA-binding affinity in vitro than EGSC3, inhibited the expression of amikacin resistance at a lesser level; growth of E. coli harboring a plasmid coding for EGSA2, in the presence of 15 μg of amikacin/ml was undetectable for 200 min but reached an optical density at 600 nm of 0.5 after 5 h of incubation. Our results indicate that the use of external guide sequences could be a viable strategy to preserve the efficacy of amikacin

Genetic structures located upstream of <i>parF</i> and <i>parG.</i>

<p>A. The direct repeats within the pMET1 putative <i>parH</i>-like locus are shown in red. The diagram also shows the −35 and −10 sequences, as well as the inverted repeats (arrows). The inverted repeat within the putative <i>parH</i> locus is shown in blue. The beginning of the ParF amino acid sequence including the deviant Walker motif A and motif A' are shown. B. Logo plot <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001800#pone.0001800-Crooks1" target="_blank">[60]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001800#pone.0001800-Schneider1" target="_blank">[61]</a> of a multiple alignment of the direct repeats shown in red.</p