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

Regulator of G-Protein Signaling 16 Is a Negative Modulator of Platelet Function and Thrombosis

© 2019 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley. Background: Members of the regulator of G-protein signaling (RGS) family inhibit G-protein coupled receptor signaling by modulating G-protein activity. In platelets, there are 3 different RGS isoforms that are expressed at the protein level, including RGS16. Recently, we have shown that CXCL12 regulates platelet function via RGS16. However, the role of RGS16 in platelet function and thrombus formation is poorly defined. Methods and Results: We used a genetic knockout mouse model approach to examine the role(s) of RGS16 in platelet activation by using a host of in vitro and in vivo assays. We observed that agonist-induced platelet aggregation, secretion, and integrin activation were much more pronounced in platelets from the RGS16 knockout (Rgs16−/−) mice relative to their wild type (Rgs16+/+) littermates. Furthermore, the Rgs16−/− mice had a markedly shortened bleeding time and were more susceptible to vascular injury–associated thrombus formation than the controls. Conclusions: These findings support a critical role for RGS16 in regulating hemostatic and thrombotic functions of platelets in mice. Hence, RGS16 represents a potential therapeutic target for modulating platelet function

Arhgef1 Plays a Vital Role in Platelet Function and Thrombogenesis

© 2019 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley. Background: Platelets are the cellular mediators of hemostasis and thrombosis, and their function is regulated by a number of molecular mediators, such as small GTPases. These small GTPases are themselves regulated by guanine nucleotide exchange factors such as Arhgefs, several of which are found in platelets, including the highly expressed Arhgef1. However, the role of Arhgef1 in platelets has not yet been investigated. Methods and Results: We employed mice with genetic deletion of Arhgef1 (ie, Arhgef1−/−) and investigated their platelet phenotype by employing a host of in vivo and in vitro platelet assays. Our results indicate that Arhgef1−/− mice had prolonged carotid artery occlusion and tail bleeding times. Moreover, platelets from these mice exhibited defective aggregation, dense and α granule secretion, αIIbβ3 integrin activation, clot retraction and spreading, in comparison to their wild-type littermates. Finally, we also found that the mechanism by which Arhgef1 regulates platelets is mediated in part by a defect in the activation of the RhoA–Rho-associated kinase axis, but not Rap1b. Conclusions: Our data demonstrate, for the first time, that Arhgef1 plays a critical role in platelet function, in vitro and in vivo

The G-protein βγ subunits regulate platelet function

© 2020 Elsevier Inc. Aims: G-protein coupled receptors (GPCRs) tightly regulate platelet function by interacting with various physiological agonists. An essential mediator of GPCR signaling is the G protein αβγ heterotrimers, in which the βγ subunits are central players in downstream signaling. Herein, we investigated the role of Gβγ subunits in platelet function, hemostasis and thrombogenesis. Methods: To achieve this goal, platelets from both mice and humans were employed in the context of a small molecule inhibitor of Gβγ, namely gallein. We used an aggregometer to examine aggregation and dense granules secretion. We also used flow cytometry for P-selectin and PAC1 to determine the impact of inhibiting Gβγ on α -granule secretion and αIIbβ3 activation. Clot retraction and the platelet spreading assay were used to examine Gβγ role in outside-in platelet signaling, whereas Western blot was employed to examine its role in Akt activation. Finally, we used the bleeding time assay and the FeCl3-induced carotid-artery injury thrombosis model to determine Gβγ contribution to in vivo platelet function. Results: We observed that gallein inhibits platelet aggregation and secretion in response to agonist stimulation, in both mouse and human platelets. Furthermore, gallein also exerted inhibitory effects on integrin αIIbβ3 activation, clot retraction, platelet spreading and Akt activation/phosphorylation. Finally, gallein\u27s inhibitory effects manifested in vivo, as documented by its ability to modulate physiological hemostasis and delay thrombus formation. Conclusion: Our findings demonstrate, for the first time, that Gβγ subunits directly regulate GPCR-dependent platelet function, in vitro and in vivo. Moreover, these data highlight Gβγ as a novel therapeutic target for managing thrombotic disorders

The JUUL E-Cigarette Elevates the Risk of Thrombosis and Potentiates Platelet Activation

© The Author(s) 2020. Background: Smoking is the main preventable cause of death in the United States and worldwide and is associated with serious cardiovascular health consequences, including thrombotic diseases. Recently, electronic cigarettes (e-cigarettes) and, in particular JUUL, have attained wide popularity among smokers, nonsmokers, pregnant females, and even the youth, which is alarming. Interestingly, there is/are no information/studies regarding the effect of JUUL on cardiovascular diseases, specifically in the context of modulation of platelet activation. Thus, it is important to discern the cardiovascular disease health risks associated with JUUL. Methods and Results: We used a passive e-vape vapor inhalation system where C57BL/6J mice (10-12 weeks old) were exposed to JUUL e-cigarette vape. Menthol flavored JUUL pods containing 5% nicotine by weight were used as the e-liquid. Mice were exposed to a total of 70 puffs daily for 2 weeks; 3-second puff duration, and 25-second puff interval. The effects of JUUL relative to clean air were analyzed, on mouse platelet function in vitro (eg, aggregation) and in vivo (eg, FeCl3-induced carotid artery injury thrombosis model). Our results indicate that short-term exposure to JUUL e-cigarette causes hyperactivation of platelets and shortens the thrombus occlusion as well as hemostasis/bleeding times, relative to clean air (medians of 14 vs. 200 seconds, P \u3c.01 and 35 vs. 295 seconds, P \u3c.001, respectively). Conclusion: Our findings document—for the first time—that short-term exposure to the JUUL e-cigarette increases the risk of thrombotic events, in part by modulating platelet function, such as aggregation and secretion, in mice

Short-term exposure to waterpipe/hookah smoke triggers a hyperactive platelet activation state and increases the risk of thrombogenesis

© 2020 American Heart Association, Inc. OBJECTIVE: Cardiovascular disease is a major public health problem. Among cardiovascular disease’s risk factors, tobacco smoking is considered the single most preventable cause of death, with thrombosis being the main mechanism of cardiovascular disease mortality in smokers. While tobacco smoking has been on the decline, the use of waterpipes/hookah has been rising, mainly due to the perception that they are less harmful than regular cigarettes. Strikingly, there are few studies on the negative effects of waterpipes on the cardiovascular system, and none regarding their direct contribution to thrombus formation. APPROACH AND RESULTS: We used a waterpipe whole-body exposure protocol that mimics real-life human exposure scenarios and investigated its effects, relative to clean air, on platelet function, hemostasis, and thrombogenesis. We found that waterpipe smoke (WPS)–exposed mice exhibited both shortened thrombus occlusion and bleeding times. Further, our results show that platelets from WPS-exposed mice are hyperactive, with enhanced agonist-induced aggregation, dense and αgranule secretion, αIIbβ3 integrin activation, phosphatidylserine expression, and platelet spreading, when compared with clean air–exposed platelets. Finally, at the molecular level, it was found that Akt (protein kinase B) and ERK (extracellular signal-regulated kinases) phosphorylation are enhanced in the WPS and in nicotine-treated platelets. CONCLUSIONS: Our findings demonstrate that WPS exposure directly modulates hemostasis and increases the risk of thrombosis and that this is mediated, in part, via a state of platelet hyperactivity. The negative health impact of WPS/hookah, therefore, should not be underestimated. Moreover, this study should also help in raising public awareness of the toxic effects of waterpipe/hookah

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

Genetic map of pMET1 and comparison to plasmid pCRY and chromosomal elements.

<p>A. The genetic maps of pMET1 and pCRY are compared showing the homologous regions. The arrows indicate genes locations and orientation. Genes with different functions are shown with different colors and if the genes in the different structures shown are homologus they are represented with the same colors. Yellow: mobilization; green: replication and partition; red: antibiotic resistance; purple: virB/pilX-like; blue: transposition; grey: unknown. Since pCRY is smaller than pMET1, to represent it in circular form a dotted line was added to fill the gap. Solid line represents non-homologous DNA. B. Comparison of a pMET1 region with chromosomal HPIs or ICEs is shown using a linearized version of the plasmid. The HPIs shown are those from <i>E. coli</i> ECOR31 (HPI_ECOR31) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001800#pone.0001800-Schubert1" target="_blank">[43]</a>, <i>K. pneumoniae</i> NTUH-K2044 (ICE_Kp1) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001800#pone.0001800-Lin1" target="_blank">[44]</a>, and <i>Y. pestis</i> KIM (HPI_Yp)<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001800#pone.0001800-Schubert1" target="_blank">[43]</a>. The diagram shows the HP core regions, which are not at scale and are represented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001800#pone.0001800-Schubert1" target="_blank">[43]</a>, and the RB-HPIs. The sequence described in this manuscript has been deposited in GenBank, accession number is EU383016.</p

Replication region of pMET1.

<p>A. The bar shows a genetic map of the pMET1 replication region and the GC content plot generated using a window size of 100 bp on top. Recombinant clones were obtained by inserting the indicated fragments into pCR2.1 or ligated to the pUC4K <i>aph</i> cassette. The ability to be maintained in <i>E. coli</i> C2110 (a <i>polA</i> mutant) of the recombinant plasmids made using pCR2.1 as vector is indicated to the right by a + or − sign. The ability to generate kanamycin resistant colonies in <i>E. coli</i> TOP10 of the indicated fragments when ligated to the <i>aph</i> cassette from pUC4K is also represented by a + or − sign. B. BLASTP comparison of the amino acid sequences of the putative RepA proteins from pMET1 and pCRY.</p