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YopM is a leucine-rich repeat (LRR)-containing effector in several Yersinia species, including Yersinia pestis and Y. pseudotuberculosis. Different Yersinia strains encode distinct YopM isoforms with variable numbers of LRRs but conserved C-terminal tails. A 15-LRR isoform in Y. pseudotuberculosis YPIII was recently shown to bind and inhibit caspase-1 via a YLTD motif in LRR 10, and attenuation of YopM− YPIII was reversed in mice lacking caspase-1, indicating that caspase-1 inhibition is a major virulence function of YopMYPIII. To determine if other YopM proteins inhibit caspase-1, we utilized Y. pseudotuberculosis strains natively expressing a 21-LRR isoform lacking the YLTD motif (YopM32777) or ectopically expressing a Y. pestis 15-LRR version with a functional (YopMKIM) or inactivated (YopMKIM D271A) YLTD motif. Results of mouse and macrophage infections with these strains showed that YopM32777, YopMKIM, and YopMKIM D271A inhibit caspase-1 activation, indicating that the YLTD motif is dispensable for this activity. Analysis of YopMKIM deletion variants revealed that LRRs 6 to 15 and the C-terminal tail are required to inhibit caspase-1 activation. YopM32777, YopMKIM, and YopMKIM deletion variants were purified, and binding partners in macrophage lysates were identified. Caspase-1 bound to YopMKIM but not YopM32777. Additionally, YopMKIM bound IQGAP1 and the use of Iqgap1−/− macrophages revealed that this scaffolding protein is important for caspase-1 activation upon infection with YopM− Y. pseudotuberculosis. Thus, while multiple YopM isoforms inhibit caspase-1 activation, their variable LRR domains bind different host proteins to perform this function and the LRRs of YopMKIM target IQGAP1, a novel regulator of caspase-1, in macrophages.
IMPORTANCE Activation of caspase-1, mediated by macromolecular complexes termed inflammasomes, is important for innate immune defense against pathogens. Pathogens can, in turn, subvert caspase-1-dependent responses through the action of effector proteins. For example, the Yersinia effector YopM inhibits caspase-1 activation by arresting inflammasome formation. This caspase-1 inhibitory activity has been studied in a specific YopM isoform, and in this case, the protein was shown to act as a pseudosubstrate to bind and inhibit caspase-1. Different Yersinia strains encode distinct YopM isoforms, many of which lack the pseudosubstrate motif. We studied additional isoforms and found that these YopM proteins inhibit caspase-1 activation independently of a pseudosubstrate motif. We also identified IQGAP1 as a novel binding partner of the Yersinia pestis YopMKIM isoform and demonstrated that IQGAP1 is important for caspase-1 activation in macrophages infected with Yersinia. Thus, this study reveals new insights into inflammasome regulation during Yersinia infection.
Recognition of microbial pathogens by the innate immune system is a crucial component of host defense. Pattern recognition receptors recognize conserved features of microbes, termed pathogen-associated molecular patterns (PAMPs), and mobilize host defenses against both extracellular and intracellular pathogens (1). Detection of extracellular PAMPs by Toll-like receptors (TLRs) or of cytosolic PAMPs by nucleotide-binding oligomerization domain leucine-rich repeat (LRR) receptors (NLRs) initiates cellular events important for clearance of pathogens, such as expression of proinflammatory cytokines or inflammasome formation (2, 3).
PAMPs and other danger signals associated with pathogens that access the host cytosol trigger the formation and activation of the inflammasome, a multioligomeric complex that serves as a molecular platform for the recruitment and activation of proinflammatory caspase-1 (4). Formation of the inflammasome is coordinated by protein-protein interactions between individual NLRs and in some cases an adaptor protein, adaptor protein apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC). Some inflammasomes (e.g., the NLRP3 inflammasome) require distinct signals for priming and activation. Induction of transcription downstream of TLR stimulation by a PAMP (signal 1) leads to the synthesis of NLRP3 and prointerleukin-1β (pro-IL-1β). The NLRP3 inflammasome is subsequently assembled and activated by a wide variety of stimuli (signal 2), such as pore-forming toxins or extracellular ATP (5, 6). In contrast, the NLRC4 inflammasome does not require a priming step and is more selective for activating stimuli such as bacterial flagellin and components of bacterial type III secretion systems (T3SS) (7, 8). While the regulation and composition of different inflammasomes are variable, they all have the ability to activate caspase-1. Caspase-1 activation by the inflammasome induces a form of cell death termed pyroptosis (9). In addition, the proinflammatory cytokines IL-1β and IL-18, synthesized as inactive precursors, rely on the activity of caspase-1 for their maturation and secretion from the host cell (4). The caspase-1-dependent processes of pyroptosis and cytokine secretion act in concert to promote innate immune responses, enabling clearance of invading pathogens. Thus, evasion of innate immune responses dependent on caspase-1 is an important strategy of pathogenic bacteria.
Pathogenic Yersinia species (Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica) employ a contact-dependent T3SS for the delivery of Yersinia outer protein (Yop) effectors into the cytosol of infected host cells (10). Yop effectors function to modulate key host processes to promote Yersinia pathogenesis (11). Delivery of effector Yops requires the assembly of the T3SS injectisome on the bacterial surface and insertion of the YopB/D translocon into the plasma membrane of host cells. Consequences of membrane perturbation by the T3SS translocon are assembly of inflammasomes and activation of caspase-1 during the infection of macrophages by Yersinia (12, 13). However, Yop effectors counteract the activation of inflammasomes and caspase-1. Activation of caspase-1 by the T3SS translocon is mediated primarily by the NLRP3 inflammasome and is counteracted by the function of the effector YopK, which interacts with the translocon and prevents caspase-1 activation (14). Recently, a second effector, YopM, an LRR-containing protein that is required for Yersinia virulence (15, 16), has been shown to inhibit NLRP3 inflammasome formation and activation of caspase-1 (17).
YopM consists of an N-terminal secretion signal, followed by two α-helices that fold into an LRR region, which spans most of the protein (18). The YopM protein terminates in a short, unstructured but conserved C-terminal tail of 24 amino acids. Interestingly, YopM appears to be devoid of catalytic activity, and instead, this protein acts as a scaffold to bind multiple host proteins. YopM was found to bind two host kinases, protein kinase C-related kinase 2 (PRK2) and the 90-kDa ribosomal S6 kinase (RSK1), and increase their activity toward a heterologous substrate (19, 20). RSK1 binds to the C-terminal tail of YopM, and PRK2 binds to the LRR region (21, 22). How complexes of YopM/RSK/PRK contribute to Yersinia virulence remains unknown. The LRR region and the C-terminal tail are both required for the virulence function of YopM (21, 22). Additionally, the C-terminal tail is required for the formation of high-molecular-weight complexes by YopM in macrophages (21). Each strain of Yersinia encodes one isoform of YopM, but there are a number of different isoforms found in the genus (23), and depending on the specific isoform, the LRR region contains between 13 and 21 repeats and the LRRs can be variable in sequence.
LaRock and Cookson presented evidence that YopM proteins containing 15 LRRs encoded by Y. pseudotuberculosis strain YPIII and Y. pestis strain CO92 bind directly to caspase-1 to inhibit its activity (17). These two isoforms of YopM, as exemplified by YopMYPIII, contain a consensus caspase-1 cleavage motif (Tyr-Leu-Thr-Asp or YLTD) present in the 10th LRR and appear to mimic pseudosubstrate caspase-1 inhibitors (17). YopMYPIII was shown to inhibit capsase-1 activity in vitro, to bind to the 10-kDa subunit of active caspase-1 in lysates of macrophages, and to inhibit the recruitment of caspase-1 to macromolecular complexes of NLRP3 (NLRP3 foci) in YPIII-infected macrophages (17). In contrast, a YopMYPIII variant in which the fourth position of the YLTD sequence was changed to A (D271A) was defective in all of these activities (17). LaRock and Cookson proposed that YopMYPIII uses the YLTD sequence to bind directly to caspase-1 to inhibit its activity and sequester it, resulting in the formation of arrested or "pre-NLRP3 inflammasomes" (17). Importantly, the virulence defect of a YPIII yopM mutant was reversed in mice lacking caspase-1, indicating that YopM functions to inhibit capase-1-directed immune functions in vivo (17).
The findings of LaRock and Cookson (17) provide the first molecular understanding of how a YopM protein can inhibit host innate immune responses, yet they raise a number of important questions. For example, do YopM isoforms that lack the YLTD sequence (of which there are many among different Yersinia species and strains) also inhibit the activation of caspase-1? Is the C-terminal RSK1-binding tail of YopM, which is essential for its virulence function, also required to inhibit caspase-1? Does the LRR domain of YopM target other host proteins in addition to caspase-1 to inhibit the function of the NLRP3 inflammasome?
Here, we studied two distinct YopM isoforms, YopM32777 from Y. pseudotuberculosis 32777 and YopMKIM from Y. pestis KIM. The former contains 21 LRRs and lacks the YLTD sequence, while the latter contains 15 LRRs and the YLTD sequence in LRR 10. We demonstrate that both YopM isoforms inhibit the activation of caspase-1 in mice or macrophages infected with Y. pseudotuberculosis. In addition, using deletion variants of YopMKIM, we provide evidence that both LRRs 6 to 15 and the C-terminal tail are necessary to inhibit the activation of caspase-1. Analysis of a YopMKIM D271A variant showed that the YLTD sequence is dispensable for the inhibition of caspase-1 activation in this isoform. We also identify the large scaffolding protein IQGAP1 (IQ motif-containing GTPase-activating protein 1) (24) as a novel interacting partner of the YopMKIM LRR domain and show that IQGAP1 is required for caspase-1 activation in macrophages infected with a Y. pseudotuberculosis yopM mutant. These results suggest that distinct YopM isoforms target different host proteins to inhibit caspase-1 activation and show that IQGAP1 is required for the activation of caspase-1 in macrophages infected with Yersinia.
Sequence comparison of Y. pestis KIM and Y. pseudotuberculosis 32777 YopM proteins.Two well-studied YopM isoforms, YopMKIM and YopM32777, contain 15 and 21 LRRs, respectively. Here, we have aligned the YopM32777 and YopMKIM sequences by using ClustalW (see Fig. S1 in the supplemental material). This analysis indicates that the N terminus and the C-terminal tail are conserved in YopMKIM and YopM32777 and the first two and last eight LRRs are well aligned (see Fig. S1). YopM32777 appears to have additional repeats within the region corresponding to LRRs 3 to 7 of YopMKIM. To account for these additional LRRs in YopM32777, they are numbered as subrepeats of LRRs 3, 5, and 7 of YopMKIM (see Fig. S1). The primary-structure alignment of YopMKIM and YopM32777 by this convention is shown in Fig. 1. The YLTD motif in LRR 10 of YopMKIM is absent from the YopM32777 isoform. Instead, YopM32777 has the sequence DLTD (Fig. 1; see Fig. S1).
Figure S1
Copyright © 2014 Chung et al.
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A Y. pseudotuberculosis 32777 yopM mutant is fully virulent in mice lacking caspase-1.Y. pseudotuberculosis yopM mutants are significantly attenuated in intravenous mouse models of infection (21, 22). LaRock and Cookson showed that the virulence defect of a YPIII yopM mutant was rescued in C57BL/6 mice lacking caspase-1 (17). We used Caspase-1−/− C57BL/6 mice, which have been determined to be functionally Caspase-11−/− as well (25) (here referred to as Caspase-1/11−/− mice), to investigate if the virulence of a 32777 yopM mutant would be restored in the absence of caspase-1. Caspase-1/11−/− mice were infected with equivalent numbers of CFU of 32777 or 32777ΔyopM (Table 1), and the time to death was monitored. As expected, 32777 was virulent in Caspase-1/11−/− mice following intravenous infection (Fig. 2, solid line). We previously showed that the 32777ΔyopM mutant is attenuated in C57BL/6 mice by intravenous challenge (22), a phenotype that was confirmed in independent experiments (data not shown). A reversal of the virulence defect of the 32777ΔyopM mutant was observed in Caspase-1/11−/− mice, as all of the mice succumbed to infection (Fig. 2, dotted line). The data are consistent with those of LaRock and Cookson (17) and suggest that caspase-1-directed immune responses in vivo are responsible for the attenuation of Y. pseudotuberculosis yopM mutants. Moreover, these data point to inhibition of caspase-1 activation as a conserved function shared by distinct YopM isoforms. However, because the mice used are Caspase-1/11−/−, the possibility cannot be ruled out that caspase-11-directed responses in vivo contribute to the attenuation of Y. pseudotuberculosis yopM mutants.
YopM32777 inhibits caspase-1 activation in macrophages infected with Y. pseudotuberculosis.To obtain more direct evidence that YopM32777 inhibits the activation of caspase-1, we infected lipopolysaccharide (LPS)-primed, bone marrow-derived macrophages (BMDMs) from C57BL/6 mice with 32777 or 32777ΔyopM and measured the activation of caspase-1. Caspase-1 activation was inhibited in C57BL/6 BMDMs infected with 32777 compared to that in 32777ΔyopM, as shown by Western blotting of macrophage lysates for cleaved (10-kDa) caspase-1 (Fig. 3A, compare lanes 1 and 5). Infection with a 32777ΔyopK mutant (Table 1) was used as a positive control for caspase-1 cleavage (Fig. 3A, lane 9). Additionally, unlike 32777, the 32777ΔyopM strain failed to inhibit IL-1β secretion (Fig. 3B) or pyroptosis (Fig. 3C), as determined by enzyme-linked immunosorbent assay (ELISA) or lactate dehydrogenase (LDH) release, respectively. Activation of caspase-1 by 32777ΔyopM required a functional T3SS, as caspase-1 was not activated when BMDMs were infected with a 32777ΔyopMyopB mutant (Table 1; see Fig. S2A in the supplemental material), as measured by Western blotting for cleaved caspase-1 (see Fig. S2B) and ELISA for secreted IL-1β (see Fig. S2C). Because caspase-11 can contribute to NLRP3-dependent activation of caspase-1 in response to cytosolic LPS (25, 26), we examined the role of caspase-11 in the response of BMDMs to infection with the 32777ΔyopM mutant. BMDMs from Caspase-1−/−, Caspase-11−/−, or Caspase-1/11−/− mice were infected and analyzed as described above. We observed similar amounts of cleaved caspase-1 in C57BL/6 and Caspase-11−/− BMDMs infected with 32777ΔyopM or 32777ΔyopK (Fig. 3A, compare lanes 5 and 8 and lanes 9 and 12). Likewise, the absence of caspase-11 did not significantly decrease the levels of secreted IL-1β in BMDMs infected with 32777ΔyopM (Fig. 3B). Interestingly, pyroptosis was partially caspase-11 dependent in BMDMs infected with the yopM mutant (Fig. 3C). As expected, IL-1β secretion (Fig. 3B) and pyroptosis (Fig. 3C) were at background levels in Caspase-1−/− and Caspase-1/11−/− BMDMs infected with 32777ΔyopM. The data show that YopM32777 inhibits caspase-1 activation, demonstrating that this activity is not restricted to the 15 LRR isoforms that contain a YLTD motif. In addition, the data show that caspase-11 is not required for the activation of caspase-1 in macrophages infected with a Y. pseudotuberculosis yopK or yopM mutant but does contribute to pyroptosis in the latter case.
Figure S2
Copyright © 2014 Chung et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Differential binding of caspase-1 by YopMKIM and YopM32777.LaRock and Cookson showed that purified YopMYPIII bound to cleaved caspase-1 in lysates derived from BMDMs infected with the YPIII yopM mutant (17). Because YopMKIM and YopM32777 both inhibited the activation of caspase-1, we wanted to determine if both proteins bind to cleaved caspase-1. To investigate the binding of YopMKIM and YopM32777 to caspase-1, these proteins were purified as fusions to glutathione S-transferase (GST) and incubated with lysates of BMDMs treated with LPS and ATP to activate caspase-1. Western blotting was used to detect the binding of caspase-1 or RSK1 to the GST-YopM fusion proteins or GST alone as a control. As shown in Fig. 4A, GST-YopMKIM, but not GST, bound to procaspase-1 (50 kDa), as well as RSK1 (compare lanes 6 and 3). Weak binding of cleaved caspase-1 to GST-YopMKIM was also detected (Fig. 4A, lane 6). Interestingly, we did not detect binding of procaspase-1 or cleaved caspase-1 to GST-YopM32777 (Fig. 4A, lane 9). These data suggest that sequence differences account for the differential binding of YopMKIM and YopM32777 to caspase-1. We previously constructed a series of deletion variants of YopMKIM missing different numbers of LRRs or the C terminus (Fig. 1) (22). We used GST fusions of two of the YopMKIM deletion variants (Δ6-15 and Δ12-C) to delineate the regions of the protein required for binding to procaspase-1 and cleaved caspase-1. As shown in Fig. 4B, GST-YopMKIM (lane 6) and the Δ12-C variant (lane 12) bound to both procaspase-1 and cleaved caspase-1, while the Δ6-15 variant (lane 9) failed to interact with either protein. As expected, the Δ12-C variant failed to bind RSK1, while the Δ6-15 variant retained this activity (Fig. 4B, compare lanes 12 and 9). Thus, binding to both procaspase-1 and cleaved caspase-1 requires LRRs 6 to 11 of YopMKIM. Taken together, these data demonstrate differential binding of caspase-1 by YopMKIM and YopM32777 in vitro, even though both proteins inhibit the activation of caspase-1 in vivo. The use of a different isoform (YopMYPIII) or a different stimulus (infection) to activate caspase-1 in macrophages could explain why LaRock and Cookson failed to detect binding of procaspase-1 in their experiments (17).
LRRs 6 to 15 and the C terminus of YopMKIM are necessary for inhibition of activation of caspase-1.To investigate which domains of YopMKIM are required to inhibit the activation of caspase-1 in macrophages, the Δ6-15, Δ12-C, and ΔC deletion variants (Fig. 1) (22) and the wild-type protein were expressed in 32777ΔyopM under the control of the native YopM promoter (Table 1). A Yop secretion assay confirmed that the expression and stability of the YopMKIM deletion variants were similar to those of the wild-type protein (Fig. 5A). These strains, as well as the 32777 and 32777ΔyopM controls, were subsequently used to infect C57BL/6 BMDMs to assess whether the LRR region of the protein is sufficient to inhibit the activation of caspase-1. As expected, 32777ΔyopM was defective for inhibition of IL-1β secretion in BMDMs and this phenotype was complemented by the expression of YopMKIM (Fig. 5B). BMDMs infected with the strain expressing the Δ6-15 variant secreted an amount of IL-1β similar to that secreted by those infected with 32777ΔyopM (Fig. 5B). Interestingly, strains expressing either the Δ12-C or the ΔC variant failed to inhibit IL-1β secretion (Fig. 5B and C), suggesting that the C terminus of YopMKIM is important for inhibition of caspase-1 activation. Taken together, the data reveal that both the region comprising LRRs 6 to 15 and the C-terminal tail of YopMKIM are required for inhibition of the activation of caspase-1.
A YLTD motif in YopMKIM is not required to inhibit the activation of caspase-1.Mutation of the aspartic acid residue in the YLTD motif in YopMYPIII to an alanine (D271A) abrogated its ability to bind to and inhibit the activation of caspase-1 (17). We introduced the identical mutation into the gene encoding YopMKIM to assess if the YLTD motif is required for the Y. pestis protein to inhibit the activation of caspase-1. Figure 6A shows that the YopMKIM D271A protein is produced by 32777ΔyopM at the same levels as the wild-type protein in a Yop secretion assay (compare lanes 2 and 3). Surprisingly, when we infected C57BL/6 BMDMs with 32777ΔyopM expressing the YopMKIM D271A variant, both caspase-1 cleavage (Fig. 6B, lane 5) and IL-1β secretion (Fig. 6C) were inhibited, arguing that the YLTD motif in YopMKIM is not required to inhibit the activation of caspase-1.
YopMKIM interacts with IQGAP1.To determine if novel host proteins bind to YopMKIM, pulldown assays with LPS-primed C57BL/6 BMDM lysates and GST, GST-YopMKIM, or a GST-YopMKIM Δ6-15 or Δ12-C deletion variant were carried out. Bound proteins were, eluted, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and visualized by silver staining. As shown in Fig. 7A, an ~200-kDa protein bound to GST-YopMKIM (lane 2) and the Δ12-C variant (lane 5) but not to GST (lane 1) or the Δ6-15 variant (lane 4). The ~200-kDa protein was not detected when the pulldown was done with GST-YopMKIM in lysis buffer alone (Fig. 7A, lane 3), suggesting that it was specific to the macrophage lysate. The region of the gel containing the ~200-kDa protein was excised and subjected to mass spectrometry analysis, and the corresponding protein was identified as the murine scaffolding protein IQGAP1 (24). Interestingly, IQGAP1 has previously been identified as a target of the type III effector proteins Ibe of enteropathogenic Escherichia coli (27) and SseI of Salmonella enterica serovar Typhimurium (28). In these cases, IQGAP1 was important for pedestal formation in epithelial cells by E. coli (27) or inhibition of macrophage and dendritic cell migration by S. Typhimurium (28). To verify the results obtained by mass spectrometry, samples from pulldown experiments were analyzed by Western blotting with an antibody against IQGAP1. As shown in Fig. 7B, IQGAP1 bound specifically to GST-YopMKIM (lane 5) and the Δ12-C variant (lane 11). These data indicate that YopMKIM interacts with IQGAP1 and that LRRs 6 to 11 are required for this interaction. Similar experiments performed with GST-YopM32777 showed that IQGAP1 does not bind to this isoform (data not shown). It remains to be determined whether the interaction of IQGAP1 and YopMKIM is direct or mediated by a cofactor.
IQGAP1 is important for caspase-1 activation in macrophages infected with Y. pseudotuberculosis.The interaction between YopMKIM and the scaffolding protein IQGAP1 suggested to us that the latter protein might be important for the activation of caspase-1 in macrophages infected with Yersinia. We assessed the role of IQGAP1 in the activation of caspase-1 by infecting LPS-primed BMDMs from wild-type 129 mice or 129 Iqgap1−/− mice with Y. pseudotuberculosis and measuring caspase-1 cleavage and IL-1β secretion. There was less cleavage of caspase-1 in Iqgap1−/− BMDMs, than in BMDMs from 129 wild-type controls when they were infected with 32777ΔyopM (Fig. 8A, compare lanes 8 and 3). Additionally, there was significantly less secretion of IL-1β in Iqgap1−/− BMDMs than in the wild-type controls when they were infected with 32777ΔyopM (Fig. 8B). We noted that secretion of IL-1β by Iqgap1−/− BMDMs infected with 32777ΔyopM was not completely abolished but rather severely decreased (Fig. 8B), indicating that IQGAP1 is important but not essential for activation of caspase-1 under these conditions. Interestingly, in response to extracellular ATP, a canonical activator of the NLRP3 inflammasome, there was no noticeable difference in the amount of cleaved caspase-1 (Fig. 8A, compare lanes 5 and 10) or secreted IL-1β (Fig. 8B) between 129 and Iqgap1−/− BMDMs. Failure to secrete IL-1β was not due to a defect of YopB/D translocon insertion in Iqgap1−/− BMDMs, as there was no noticeable difference in YopM translocation between 129 and Iqgap1−/− BMDMs (Fig. 8C, compare lanes 1 and 4 and lanes 3 and 6). Additionally, there was no detectable difference between 129 and Iqgap1−/− BMDMs in the kinetics of their cell rounding response to effectors that disrupt the actin cytoskeleton (data not shown), indicating that IQGAP1 is not required for translocation. These data suggest that IQGAP1 is specifically important for activation of the NLRP3 inflammasome in macrophages during Yersinia infection but dispensable for other NLRP3-dependent stimuli that lead to caspase-1 activation. To determine if IQGAP1 is required for the formation of macromolecular complexes of NLRP3 or a later step in inflammasome assembly, we used immunofluorescence microscopy to detect NLRP3 focus formation in 129 or Iqgap1−/− BMDMs infected with Y. pseudotuberculosis. NLRP3 foci were not detected in uninfected 129 BMDMs but were detected in these cells infected with 32777 (see Fig. S3 in the supplemental material). In addition, NLRP3 foci formed in 129 and Iqgap1−/− BMDMs infected with 32777ΔyopM (see Fig. S3), suggesting that IQGAP1 functions downstream of NLRP3 activation. These results are consistent with the idea that YopM inhibits the recruitment of procaspase-1 to NLRP3 preinflammasomes (17) through targeting of IQGAP1.
Figure S3
Copyright © 2014 Chung et al.
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Summary.We have extended the results of LaRock and Cookson (17) by showing that (i) distinct YopM isoforms inhibit the activation of caspase-1, (ii) the LRR region and C-terminal tail are both required for this activity, and (iii) YopMKIM binds to IQGAP1, which we have identified as a novel regulator of inflammasome function in macrophages. A model of the activation of the NLRP3 inflammasome in macrophages in response to Yersinia infection and mechanisms of caspase-1 inhibition by YopM is presented in Fig. 9. We speculate that translocon insertion and effector translocation generate distinct signals that sequentially stimulate the formation of NLRP3 preinflammasomes (signal 2) and recruitment of caspase-1 to NLRP3 foci (signal 3) to complete inflammasome formation (Fig. 9). Translocon insertion could generate signal 2 by causing ion fluxes or other types of plasma membrane perturbation, while the entry of effectors, or PAMPs, into the cytosol may generate signal 3 (Fig. 9). Our data show that the C-terminal tail and LRRs 6 to 15 of YopMKIM are required to inhibit caspase-1 activation (mechanism A in Fig. 9). Preliminary results of translocation assays indicate that the Δ6-15 and Δ12-C variants exhibit reduced translocation and/or stability in infected macrophages, compared to the ΔC variant and YopMKIM (data not shown). Therefore, the Δ6-15 and Δ12-C variants may be defective for inhibition of caspase-1 activation, in part because of reduced translocation or stability. The requirement for the C terminus, which is necessary for virulence in vivo (21, 22), may reflect its role in the multimerization of YopM (Fig. 9) (21). It will be important to determine if RSK1, by promoting YopM multimerization or exhibiting kinase activity, has an important role in the inhibition of caspase-1 cleavage. We suggest that LRRs 6 to 11 in YopMKIM are essential for inhibition of caspase-1 activation because they target the protein to IQGAP1 (Fig. 9). It is unclear how IQGAP1 functions in this novel capacity to activate caspase-1, but as shown in Fig. 9, it is possible that it is important to transmit signal 3 for the recruitment of procaspase-1 to NLRP3 preinflammasomes. Using immunofluorescence microscopy we were unable to detect the localization of IQGAP1 to NLRP3 foci in macrophages infected with yopM mutant Y. pseudotuberculosis (data not shown). IQGAP1 is a ubiquitously expressed scaffolding protein that plays a role in diverse cellular processes (29, 30), and although it does not appear to localize to NLRP3 foci, it does interact with multiple host proteins, including Rho GTPases and kinases (29), that could regulate inflammasome assembly. IQGAP1 could also control caspase-1 cleavage by regulating actin organization or transcription, as these processes are known to be important for activation of the NLRP3 inflammasome. It remains to be determined if and how YopMKIM inhibits IQGAP1 function. Preliminary experiments show that steady-state levels of IQGAP1 are unaffected by the presence of YopMKIM in macrophages infected with Y. pseudotuberculosis. A final issue that remains to be addressed is the role of the YLTD motif (17), which appears to be required for YopMYPIII to inhibit procaspase-1 recruitment to NLRP3 foci, activation of caspase-1 in macrophages, and caspase-1 activity in vitro (mechanism B, Fig. 9), but is dispensable for YopM32777 and YopMKIM to inhibit the activation of caspase-1 in macrophages. YopMYPIII and YopMKIM are 99.5% identical at the amino acid level (17), making it unlikely that amino acid differences between these isoforms could explain the different results obtained. It seems more likely that in 15-LRR YopM isoforms that contain a YLTD motif, the sequence naturally plays a cryptic role in the inhibition of caspase-1, and the specific in vitro experimental conditions used by LaRock and Cookson uncovered an unusually important role for this motif. A Y. pseudotuberculosis strain expressing a 15-LRR isoform (YopMIP2666) in which repeats 10 to 11 were deleted was fully virulent in a mouse model of infection (21), arguing that the YLTD motif is dispensable for caspase-1 inhibition in vivo. A better understanding of how different YopM isoforms inhibit the activation of caspase-1 in macrophages is likely to lead to new insights into how inflammasomes are regulated and promote host protection against microbial infections.
Bacterial strains and plasmids.A description of the Y. pseudotuberculosis strains used in this study is provided in Table 1. A frame-shift mutation was introduced into yopK in 32777 as previously described (31), generating 32777ΔyopK. An in-frame deletion of yopB was constructed in the 32777ΔyopM background as previously described (31), generating 32777ΔyopMyopB. Vectors encoding YopMKIM and YopMKIM deletion variants under the control of the native YopM promoter were generated in pMMB67EH and used to transform 32777ΔyopM as previously described (22). The pYopMKIM Δ12-C vector was reconstructed in this study by the same approach and was analyzed independently of the other deletion variants, as shown in Fig. 5C. The vector pYopMKIM D271A was constructed as follows. The D271A codon change was introduced into pYopMKIM with the QuikChange site-directed mutagenesis kit (Agilent) and primers described in reference 17. The plasmids constructed in this study were verified by sequencing. 32777 and 32777ΔyopM were transformed with a plasmid expressing green fluorescent protein (GFP) by mating with E. coli s17-1λpir harboring p67GFP3.1 as previously described (32). For infection of BMDMs, Y. pseudotuberculosis strains were grown overnight in Luria broth (LB) at 28°C. The following day, cultures were diluted 1:40 in LB containing 20 mM MgCl2 and 20 mM sodium oxalate, grown at 28°C for 1 h, and then shifted to 37°C for 2 h. For analysis of secreted Yops, yersiniae were diluted and grown as described above, except at 28°C for 2 h, followed by a temperature shift to 37°C for 4 h.
Mouse infections.Y. pseudotuberculosis cultures were grown overnight in LB at 28°C, washed twice with phosphate-buffered saline (PBS), and suspended to approximately 1.5 × 104 CFU/ml. A volume of 100 µl from this suspension was delivered by tail vein injection into Caspase-1/11−/− mice (33) (a gift from Adrianus van der Velden, Stony Brook University). Time to death was monitored for 21 days, at which point the remaining mice were euthanized. All mice were handled according to the guidelines for the humane care and use of experimental animals, and the procedures used were approved by the Stony Brook University Institutional Animal Care and Use Committee.
Bone marrow isolation and culture conditions.Bone marrow was isolated from femur exudates of 6- to 8-week-old female C57BL/6 wild-type (Jackson Laboratories), Caspase-1−/− (34), Caspase-11−/− (35), or Caspase-1/11−/− (36) mice or 129 wild-type or Iqgap1−/− (37) mice as previously described (38). At 18 h prior to infection, BMDMs were seeded into tissue culture plates in Dulbecco's modified Eagle medium (Invitrogen) supplemented with 10% fetal bovine serum (HyClone), 15% L-cell-conditioned medium, 1 mM sodium pyruvate, 2 mM glutamate, and 100 ng/ml E. coli LPS (Sigma). BMDMs were used to seed six-well plates at a density of 0.8 × 106 cells/well for all of the experiments except those in Fig. 3, where 48-well plates were seeded at 2.0 × 105 cells/well.
Macrophage infection or stimulation conditions.LPS-primed BMDMs were left uninfected or infected with Y. pseudotuberculosis grown under the conditions described above at a multiplicity of infection (MOI) of 30. Tissue culture plates were centrifuged for 5 min at 95 × g to facilitate the contact of yersiniae with BMDMs. Plates containing BMDMs were subsequently incubated at 37°C with 5% CO2. At 90 min postinfection, cell supernatants were collected and analyzed for secreted IL-1β and LDH release while cell lysates were harvested for Western blotting of host proteins. As a positive control for activation of caspase-1, BMDMs were primed with 100 ng/ml E. coli LPS for 4 h and subsequently given 2.5 mM adenosine triphosphate (ATP) for 1 h.
Western blotting of macrophage cell lysates.BMDMs were washed twice with cold Hanks balanced salt solution to remove residual phenol red and lysed in lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl [pH 8.0], protease inhibitor cocktail [Complete, Mini, EDTA-free; Roche]). Proteins were resolved by SDS-PAGE with NuPAGE Novex 4 to 12% Bis-Tris gels (Invitrogen) unless indicated otherwise, transferred onto a polyvinylidene difluoride (PVDF) membrane, and probed with rabbit polyclonal anti-caspase-1 (Santa Cruz Biotechnologies), rabbit polyclonal anti-RSK1 (Abcam), and mouse monoclonal anti-IQGAP1 (BD Biosciences) antibodies. Horseradish peroxidase (HRP)-conjugated anti-rabbit (Cell Signaling) and anti-mouse (Jackson ImmunoResearch) antibodies were used as secondary reagents. To control for loading, Western blots were stripped and reprobed with anti-β-actin antibody conjugated to HRP (Sigma-Aldrich). Signals in Western blot assays were detected with Amersham ECL Prime Western blotting detection reagent (GE Healthcare).
ELISA.Levels of secreted IL-1β in supernatants collected from BMDMs were analyzed with a commercially available ELISA kit (R&D Biosystems) according to the manufacturer's instructions.
Cytotoxicity assay.The LDH released into supernatants of BMDMs was quantified with the LDH Cytotoxicity Assay kit (Clontech) according to the manufacturer's instructions.
Analysis of Yop secretion.Following growth of Yersinia cultures as described above, bacteria were removed by centrifugation and trichloroacetic acid (10%, wt/vol) was added to the supernatants to precipitate and collect secreted Yops as previously described (22). Samples were subsequently processed for Western blotting with mouse monoclonal antibodies (6-1CE or 2A3.3A8.1A2) that recognize epitopes in the N-terminal region of YopM (a gift from Susan Straley, University of Kentucky), mouse monoclonal anti-YopB antibodies (76.15, 190.11, or 328.1 [unpublished data]), or mouse monoclonal anti-YopE antibodies (149.27 or 202.19 [unpublished data]).
Purification of GST-YopM proteins.E. coli strains harboring pGEX-2T, pGEX-2T YopMKIM, pGEX-2T YopM32777, pDEST15 YopMKIMΔ6-15, or pDEST15 YopMKIMΔ12-C (22) were grown in LB at 37°C to an optical density (OD) at 600 nm of 0.3 to 0.6. Isopropyl-β-d-thiogalactopyranoside (IPTG) was then added to a final concentration of 0.1 mM, and the cultures were grown for an additional 4 h. Bacteria were collected by centrifugation and lysed with Lysonase and Bugbuster reagents (Novagen) according to the manufacturer's instructions. Supernatants containing bacterial proteins were added to GST-Bind Resin (Novagen) and incubated for 30 min at 4°C on a rotating shaker. Samples were washed, and bound GST and GST-fused proteins were eluted according to the manufacturer's instructions. Purified proteins were dialyzed against PBS and used for GST pulldown assays or stored at −80°C.
GST pulldown assay.Ten micrograms of purified GST or GST-fused protein was added to 20 µl of GST-Bind Resin (Novagen) and incubated at 4°C for 30 min. Beads were then washed three times with 1× GST Bind/Wash Buffer (Novagen). Uninfected, LPS-primed or LPS- and ATP-treated BMDMs were incubated in lysis buffer as described above, and the resulting lysates were added to the beads and incubated for 2 h at 4°C on a rotating shaker. Resin-containing bound proteins were washed four times in lysis buffer and boiled in sample buffer, and the eluted proteins were separated by SDS-PAGE. Proteins were transferred to PVDF membranes, and Western blotting was performed as described above.
Silver staining and mass spectrometry.Samples from GST pulldown assays were resolved by SDS-PAGE. Gels were subsequently stained with the SilverQuest Stain kit (Invitrogen) according to the manufacturer's instructions. Specific bands were excised from the gel, fragmented by trypsin digestion, and subjected to analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Yop translocation assays.Yop translocation assays were performed as previously described (39). Briefly, infected BMDMs were incubated in lysis buffer as described above and the soluble fraction of the lysate was collected and analyzed for the presence of YopM by Western blotting as described above.
Fluorescence microscopy.BMDMs were seeded onto glass coverslips pretreated with ethanol and acetone and sterilized to remove contaminants. BMDMs were left uninfected or infected as described above, except that the tissue culture medium was supplemented with 0.5 mM IPTG to induce GFP expression. BMDMs were washed twice with PBS, fixed with 2.5% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with bovine serum albumin as previously described (40). BMDMs were incubated with a primary rabbit anti-NLRP3 polyclonal antibody (Santa Cruz Biotechnologies). Binding of the primary antibody was visualized by the addition of Alexa Fluor 594-conjugated anti-rabbit antibody (Invitrogen), and DNA was stained by the addition of 4,6′-diamidino-2-phenylindole (DAPI) dilactate. Coverslips were mounted onto glass slides with ProLong Gold antifade reagent (Invitrogen) and imaged with an Axiovert S100 (Zeiss) with a 32× objective. Images were taken with a SPOT camera (Diagnostic Instruments) and processed with Adobe Photoshop CS 5.1.
Statistical analysis.Experimental data analyzed for significance with GraphPad Prism 6.0 were from at least three independent experiments. Probability (P) values for IL-1β and LDH experiments were calculated by one-way analysis of variance (ANOVA) or grouped two-way ANOVA with Turkey's multiple-comparison posttest. P values from mouse survival experiments were calculated by log rank test. P values of <0.05 were considered significant.
We thank Joe McPhee, Susan Straley, Jason Tam, Adrianus van der Velden, Veena Sangkhae, and Ian Hitchcock for providing reagents; Galina Romanov for preparation of macrophage cultures and strain constructions; and Patricio Mena for help with mouse infections.
This research was supported by awards from the NIH to J.B.B. (R01AI099222 and U54AI057158-Lipkin) or I.E.B. (R01AI103062 and R21AI109267), Research Scholar Grant RSG-09-033-01-CSM from the American Cancer Society to V.A.S., and shared equipment grant S10RR025072 to T.K.
- Received 29 May 2014
- Accepted 5 June 2014
- Published 1 July 2014
- Copyright © 2014 Chung et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.


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This new decal
sheet from Canuck
Model Products
provides markings for 2 aircraft including D.H. Vampire - AB+3
Standard Service Markings. This sheet provides AB+3 markings and stencils for D.H. Vampire's in RCAF service.
The instruction sheet is well laid out and easy to follow. The single water slide decal sheet is of the highest quality and very clearly printed.
Reference Sources:
NOTE: Due to the limited amount of colour reference materials, the light blue of the 410Sqn stripes is a best educated guess. The colour was selected based on the few colour photos available as well as the known standard RCAF paints available at the time and in use on other aircraft. The modeller may wish to mask and paint a different shade of blue to their preference.
The Vampire has always been a favourite aircraft of mine due to it's very unique design. This decal sheet captures two much loved squadrons including Cougar Squadron with it's leaping red cougar artwork and 442 City of Vancouver Squadron with its big red bat face with intimidating fangs. It's good to see RCAF Vampire markings and the Cougar Squadron bird would be an excellent companion to other Cougar Squadron models..
This decal sheet is also available in 1/72.
I would like to thank Canuck Model Products for providing me with this product sample.
Steve Bamford
Photos and text © by Steve Bamford

subset: commoncrawl


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Hey there ...Next weak we will hit the 9800 and then up to 11650 .Good luck . ... (Read More)15 hours ago· Reply
will the upcoming corporate reporting seasons affects the crypto prices? ... (Read More)20 hours ago· 1 · Reply
Hello everyone! I want to make my own tokens, I found on internet a platform to do it named mintme, have you ever hearded about it? can you please give me some advices to do it in the most efficent way? Thank you All! ... (Read More)Jul 13, 2020 16:04 GMT· Reply
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Wednesday is a Min Pin/Whippet mix. She's is about 2 years old and weighs 25lbs. This sweetie is very mellow and just wants to be near you. If you are looking for someone to talk to after a long day who will adore you and listen to your problems, look no further. She will looking at you lovingly in exchange for some rubbing and pets. Wednesday currently lives in a foster home in Texas. If you choose to adopt her then Rescue Road trips will transport her north. The adoption fee includes transport!
Fairfield County
Danbury, Connecticut
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Looking for something to put you in a good mood or to get your creative juices flowing? I don't know about you but checking out unique crafty videos and simply admiring artworks sure is satisfying! Below are some of the coolest contemporary artists you'd want to know more about and their inspiring works.
#1 Cj Hendry
Catherine Jenna "Cj" Hendry is a contemporary artist known for her hyper-realistic drawings. If you think those are merely paint splatter works, look closely. She has also collaborated with renowned designer Christian Louboutin for Art Basel Hong Kong.
View this post on Instagram
Was scrolling through my phone and deleting old shit and found a heap of old pics from 2017. These are some photos from the Louboutin collaboration in Hong Kong, and such great memories from my old studio in Tribeca. Geeee time flies what the heck. Also those shoes people are holding, are crayon Louboutin heels we got moulded where the viewers were encouraged to draw on the walls because why wouldn't you want to draw on the walls!!
#2 Callen Schaub
Abstract artist Callen Schaub is known for his unconventional techniques and live performances of spin and pendulum paintings. He was diagnosed with dyslexia when he was a child and has since used painting and drawing as a medium for self-expression. He has worked with brands such as Bombay Sapphire and YSL, among others.
#3 Leonardo Frigo
London-based artist and violinist Leonardo Frigo creates intricate illustrations using his fountain pen and handmade ink. As his website reads, "Leonardo creates this unique art form with the premise that his instruments can not only be listened to while played or merely visually appreciated in their original form, but read like storybooks."
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In #Crete, Minos' daughter #Ariadne fell madly in love with #theseus and helped him navigate the labyrinth. In most accounts she gave him a ball of thread, allowing him to retrace his path. Theseus killed the #Minotaur with the sword of Aegeus and led the other Athenians back out of the #labyrinth. #handpainted #handmade #skate #skateboard #ink#blackink#inking#tatoo#greekmithology #grecia#mitologia#bespoke#christmas #gift#artauction #arte #auction
#4 Haruya Abe
Japanese potter Haruya Abe is known for his meticulously patterned ceramics. Using a technique known as yokoku, patterns are carved into a mold and then pressed onto the tableware shaped on the potter's wheel while pieces of clay are removed to create detailed patterns on vases and decorative bowls.
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Thank you @instagram Please take a look. ・ #repost @instagram ・・・ The only thing more mesmerizing than this honeycomb pattern is seeing Japanese potter Abe Haruya (@abe_haruya) create it, scrape by scrape. ・ 今日は人間ドッグで、初バリウムでした。 下剤のお陰でお腹ピーピーだし、気持ち悪いし。 そんなこんなで @instagram にて紹介していただきました。 IGTVご覧いただけたら嬉しいです。 ・ #健康第一#健康診断で体調不良 #なんだなかぁ
#5 Maayan Zilberman
Maayan Zilberman is the founder of Sweet Saba, a New-York based candy line of "grown-up" candies that emulate real objects. As her website reads, "these poignant handmade sculptures offer a multi-sensory experience through memory, unexpected flavor, and a sense of humor."
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Earth Day. One of the big reasons I stepped away from designing/manufacturing clothes, and why I moved toward a creative practice that is both ephemeral and minimal in ingredients and footprint, is so the focus could be on it bringing momentary joy, laughter, wonder… without it collecting dust or ultimately sitting in a trash pit. Obviously I'm by no means saving the planet by making sugar sculptures that dissolve, but if we all think a bit more about what we're making and where it ends up, maybe we can help our planet even just a bit. We can all do something. ❤🍭❤ #earthday
#6 Diana Beltran Herrera
Colombian artist and designer Diana Beltran Herrera is known for her whimsical, lifelike sculptures of birds, insects, fish, and plants using ordinary materials like wire, cardboard, plastic, and paper.
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A full size image of my dwarf kingfisher. #king #bird #paradise #malaysia #dwarf #kingfisher #asia #southeastasia #nature #wild #free #exotic #paradisebirds #birdsofparadise #paper #cut #papercut #inspiration #beautiful #artdaily #art #paperart #paperartist #paperbird #paperbirds #handmade #papercraft #papercutartist #paperlicious #papersculpture #paperbirds
Cover image from @maayan.zilberman


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Home buying
Why didn't anyone tell me buying a house sucks! I'm so freaking stressed out right now. Working with a lender, being told all we need is our down payment money at closing (seller is covering closing costs). Then suddenly today I get the loan application… more »
100 Miles of Nowhere plan
Jason and I will be riding 33 laps of a 3.33 mile section of the Jordan River Parkway. We will be based in Germania Park, off of 53rd South. We'd love to have you come and join us for a few laps, or more. There is a playground at Germania if your kids… more »
In the Market
Michael and I have started looking for a house...it is tiring to say the least. We found one we liked and went to make an offer only to find that someone else had already made an offer. We discussed bidding higher, I wanted to go for it, and then got… more »
Zombies are Real!
The government told me so! So if you need to get prepared for the coming Zombie Apocalypse, check the CDC site for some helpful preparedness tips. more »
Last night's post might have given some of you the wrong idea. I have a fantastic husband. He wonderful all the time...he even stopped playing last night and finished converting all our wedding photographs to JPGs for me (a process that he actually… more »
Portal 2 Widow
Sunday night Portal 2 came out. For those of you who are unfamiliar with this, it is a computer game (also on PS3 and Xbox 360). Granted, having played the first Portal, I can tell you that it is a fun game, and I can see how someone can play it for… more »
Season 6
100 Miles of ... Nowhere?
I just signed up for a "bike race." The Fat Cyclist's 100 Miles of Nowhere. What is it? It's a bike race, kinda. It's a solo event, kinda. It's for charity, definitely. The first 100 Miles of Nowhere was the Fat Cyclist and some buddies, riding their… more »


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You can't go wrong with this anniversary card for anyone celebrating an anniversary in your life. Whether it's a friend, a sibling, a parent, or a child, this anniversary card is perfect for all anniversaries! The heart-shaped rose is beautiful, and the sentiment is personal enough to be heartfelt but generic enough that this card could be for anyone!
This anniversary card is always a favorite, and it's easy to see why. The breathtaking roses and heart-shaped petals capture your attention, but the real beauty is in the message. Our hearts just melt every time we read, "May all your days be spent together".
Pink and red cut-out hearts make this one zesty anniversary card! Do you like a minimalist look that still spells heartfelt love? This anniversary greeting card is for you! Send it to a couple you know who is celebrating their anniversary. Wish your friends or family a happy anniversary with warm wishes to enjoy their special day. Isn't love fantastic? Tell them so! Send an anniversary card today and spread the love.
This gem of a Thank You card is the perfect balance of simple and fun. The abstract painting of the heart and butterflies is stunning, while the simplicity of the "thank you" message allows you to personalize this card however you'd like. This makes it suitable for any occasion, and, let's be honest– everyone's bound to love this gorgeous card!
How many times do you thank people every day? Even if you are thankful for something small, by sending a thank you card will raise the spirits of a person who did something nice for you.
Want to make someone smile while also saying thanks? This card is a the perfect pick! A happy face emoji will get the message out that you're grateful for their birthday wishes. And the words are sure to remind them what a big smile they left on your face that day. Send this greeting and they're sure to be grinning just as wide!
If a friend, coworker, or family member is feeling under the weather, why not send them a card to cheer them up! Send the Sunny Thoughts Get Well Soon Card to let them know you're rooting for their full recovery. Even if they're very sick or injured, this sunshine yellow card is sure to put a smile on their sweet face.
Tulips were once seen as a symbol of charity during the Victorian Era, but they are also a symbol of love and good wishes. Send this Get Well card to tell a sick loved one you hope they feel better. The bright colors and "Get well soon" message will lift their spirits and help them get over their sickness sooner! Send it today!
If one of your friends or family members has caught a cold or is feeling under the weather, send them a cute Get Well card to help them feel better! The message on this Get Well card will tell them you care and the sight of a dog with a hot water bottle will have them laughing at the silly picture and feeling better in no time!
Do you have a friend, coworker, or family member who loves dogs? Send them this Thinking of You card! This card shows a sweet Chihuahua as it sleeps on an open book, waiting for its owner and friend to return. Black fancy cursive font says, "You are in my thoughts." This card is the perfect message to send to the loved ones on your mind.
This funny bear is on his way to anyone you chose to send this card to. He has his head in the clouds and has whoever you send this "thinking of you" card to on his mind. He is flying around in his trusty plane, while wearing his flying cap and scarf to keep him warm. It will not take him long before he makes a safe landing in the mailbox of your choosing. As soon as they open it up, they will have this fuzzy guy tell them just how much you have been thinking of them.
Sending this card is the perfect way to let someone know that, "Hey, I am thinking of you often." It will also show them how much you can't wait to see them very soon. It displays a beautiful leaf border, surrounding your nice thoughts for someone. Use this Nature Leaves card the next time you are thinking of someone near and dear to you.
When a loved one passes away, it's often difficult to find the right words to express how you feel. This encouraging card is a gentle reminder that you are thinking of them and wishing them comfort and solace. Your hope for them is that all the wonderful memories they shared can bring a sense of lasting peace.
During a dark time in someone's life, it's important to let them know that brighter days are ahead, and that each one gets easier as long as they hold onto the precious memories of a loved one. Beautiful, elegant white flowers, symbolizing purity, adorn this touching card, which will provide comfort and support when they need it most.
A soothing gathering of purple flowers frames this beautiful sympathy card, which offers comforting words to console them during this difficult moment. Whether it's a family member, friend or co-worker, the message is one of kindness and compassion, which will stay with them for a long time to come.
A sea of stars frame the inspirational words in this graduation card, letting someone special know that they continue to shine bright in your eyes, especially with their latest "amazing accomplishment!" This time can be very exciting and emotional for any graduate, but knowing they have your support and encouragement will provide comfort as they see what exciting adventures lie ahead."
They've earned their degree and have gotten that much closer to pursing their dreams. This card will remind a special college graduate that they should be proud of all they've accomplished so far. It's no easy task and they've come out on top. Whatever they do next in life, they should know that they are a true success and this is a moment that deserves to be remembered.
In life, there are moments that we will always remember. College graduation is one of them. As the special graduate in your life takes that important next step in life, let them know that it's their time to shine and whatever journey they go on from here, it's up to them to create their own success story. After all, they've earned it.
Few events in our lives are as monumental as purchasing a new house. It's a huge investment and can be very exciting. Know someone who just bought a new home? Congratulate them in style by sending the Tiny Model Home card. The tiny model home is shown next to a hand holding a set of keys while the message wishes comfort and coziness!
Having a space of your own, some privacy and comfort, it's a good feeling! Maybe one of the best in the world. Especially when you have someone (or someones) you love to share it with. Send the Your Own Space Congratulations on New House card to wish the new homeowners well! The photo card shows a young couple hugging among their boxes of belongings. Very sweet!
Here you go. Take these keys, they belong to you now. That's a powerful moment. We mean of course the one where you are handed a set of House Keys for your new home. Wish a friend "congratulations!" with this lovely heartfelt card. In soft pastel colors, with a house-shaped keyring, it's sure to put a big smile on that new homeowner's face.
How cute is this little cartoon pug dog card? With his paws thrown out wide in either direction—you can just feel his good energy and high spirits! He radiates love from the bottom of his little puggin' heart. Tell someone "Hello!" and wish them a great day with the Doggone Best Day Funny Saying Hi Card. Perfect for dog people, animal lovers, or anyone who needs a quick pick-me-up!
Let someone bright, sunny, and special know they're on your mind today. How? By sending them the charming Hello Sunburst Saying Hi Card of course! Everyone likes to know they are being thought of fondly, and what person doesn't love a fun card?! Spread a warm smile on someone's face with this sunshine yellow card and its boisterously blue greeting of "hello."
They say an elephant never forgets, well now neither do you! Forget to say hello to a dear friend or coworker that is! Send the adorable Pink Elephants Saying Hi Card to wish a special someone in your life a fond hello and a wonderful day! The pink elephant looks calm, perhaps even a bit sleepy, it's sure to put a smile on the face of anyone who sees it.
Is your love as sweet as honey? Then celebrate your relationship with this funny love card! The silly message and design will make your sweetheart laugh and the gift of a card will make their day much happier and better! Your love is special and exciting, so make the most of your relationship by sending this funny love card to your partner!
Rings, chocolates, teddy bears, bracelets... All of these are great gifts to receive from the person you love most, but the only thing you really need is your sweetheart. To show your love how much you adore them, send this funny love card to celebrate your relationship and special connection. The sweet message will warm your partner's heart and show how much you care for them.
Kisses are the best and sweetest way to show someone how much you love them! To show your sweetheart just how much you care about them, send this love card! The design and message will make their heart race, while also showing them that you are thinking of them. Celebrate the love you have built with your partner by sending this love card today!
The day has finally come, someone you know is retiring, and the best part is that they have been a dedicated penny-saver their entire life! All their long years of work and dedication are about to pay off big time. This card represents the grand occasion perfectly. It features two older adults standing on top of coins that are piled high! It's their retirement! Send them this Penny-Saving Pays Off Happy Retirement card to mark this momentous occasion.
A couple relaxes in their lounge chairs, sipping a few drinks on a stunning, empty beach. The sun sets magnificently before them, and it looks incredible. Looking at this card makes you want to let out a big sigh of relief! Send this Relax By the Ocean card to congratulate someone on making it to their retirement. Time for them to kick back and enjoy some well-deserved time off!
Someone's retirement is fast approaching! Send that lucky soul the Next Exit Happy Retirement Card so they don't miss their turn. They've worked long and hard to reach this exciting point in their lives, so don't let it slip by uncelebrated. Let them know you hope they have lots of exciting trips planned now that they've got so much free time!


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Two men are facing drug charges after police found them with thousands of dollars worth of meth and heroin in Jamestown.
Authorities found the drugs and cash when they were called to a home on Sprague Street.
Richard Brown and Timothy Blakeslee were arrested and charged with possession of a controlled substance.


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This is the first report of a Papaya ringspot virus (PRSV) isolate from the northeastern region of India. The nucleotide sequence identity of PRSV-Meghalaya was in the range of 72.6 to 82.5% with other Indian PRSV isolates, and the highest identity of 84.4% was with a French isolate. Population genetic analysis indicated positive selection.
Papaya ringspot virus (PRSV) belongs to the family Potyviridae and the genus Potyvirus, with a positive-sense single-stranded RNA genome of ∼10,349 nucleotides (nt) in size, encompassing a single open reading frame (ORF) (1–5). PRSV is classified as either PRSV-P or PRSV-W type, depending on whether it infects papaya or cucurbits, respectively (6–8). PRSV is hypothesized to have originated in India ∼2,250 years ago, spread to East Asia ∼600 years ago, and reached the Americas ∼200 years ago (9). Although ∼5 complete PRSV sequences have been published from India (10, 11), none of them are from the northeastern region (NER) of India.
In 2015, leaf samples from a PRSV-infected papaya plant were collected from Umiam (erstwhile Barapani) in the state of Meghalaya, located in the NER of India. Total RNA extraction and cDNA synthesis were done as described by Patil et al. (12). Later, the cDNA was used for PCR amplification with potyvirus universal primers (13) and degenerate primers from this study. Cloning and sequencing of the coat protein (CP) and helper component proteinase (HcPro) regions indicated it to be a distinct sequence. Later, degenerate primers were designed at conserved regions of PRSV, and the entire PRSV-Meghalaya genome was real-time PCR (RT-PCR) amplified as 5 segments of ∼1.8 to 2.5 kb in size. These amplicons were cloned and sequenced as described by Patil et al. (12). The assembly of the contigs resulted in a complete genome size of 10,343 nt for the PRSV-Meghalaya isolate.
PRSV-Meghalaya was subjected to phylogenetic analysis, along with 21 full-length sequences of PRSV isolates from all over the world (12, 14, 15). PRSV-Meghalaya clustered with isolates from France, Mexico, Brazil, and the United States, in addition to all the other Indian PRSV isolates. It showed the highest nucleotide sequence identity of 84.4% with PRSV-France-E2, while the lowest sequence identity (70.6%) was with PRSV-South Korea (16), whereas the corresponding amino acid sequence identities were 89.5% with PRSV-France-E2 and 86.7% with PRSV-South Korea. PRSV-Meghalaya showed 82.5%, 81.7%, 77%, and 72.6% homology with nucleotide sequences of the four Indian isolates of PRSV (10, 11), namely, Pune, Delhi, Hyderabad, and Rajasthan, respectively.
To identify the most variable region of PRSV-Meghalaya, SimPlot was drawn for the sequences of 21 PRSV isolates (17). The genomic region of nt 241 to 1821 corresponding to P1 and nt 8841 to 9501 corresponding to the overlapping region of NIb-CP were the most variable. Recombination breakpoints were identified at 3 sites of PRSV-Meghalaya, namely nt 905 to 1134, nt 8844 to 9996, and nt 9205 to 10182, when analyzed using the RDP4 program (18). The degree of selective constraints at the amino acid level was estimated with MEGA 5.05 by analyzing separately the rate of nonsynonymous (dN) and synonymous (dS) substitutions with the Pamilo-Bianchi-Li method (19), and DnaSP 5.10 was used to evaluate the importance of natural selection (20). The proportion of nucleotide substitution between the nonsynonymous and synonymous sites (dN/ds) was 1.8, indicating a positive selection for the amino acid sequence conservation in PRSV-Meghalaya (21). This is the first report of a full-length sequence of a PRSV isolate from the NER of India, indicating its independent evolution.
Accession number(s).The complete genome sequence of PRSV isolate Meghalaya has been deposited in NCBI GenBank with the accession number MF356497.
We acknowledge the financial support from ICAR's Network Project on Transgenics in Crops (NPTC) and ICAR-NRCPB. R.M. acknowledges a postdoctoral fellowship from DST-SERB.
B.L.P. conceived the study idea, guided the entire work, analyzed the data, and wrote the manuscript. R.M. helped in cloning the PRSV sequences and in bioinformatics analysis.
- Received 13 April 2018.
- Accepted 17 April 2018.
- Published 31 May 2018.
- Copyright © 2018 Mishra and Patil.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.


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