A Small Non-Coding RNA Mediates Transcript Stability and Expression of Cytochrome bd Ubiquinol Oxidase Subunit I in Rickettsia conorii.

Small regulatory RNAs (sRNAs) are now widely recognized for their role in the post-transcriptional regulation of bacterial virulence and growth. We have previously demonstrated the biogenesis and differential expression of several sRNAs in Rickettsia conorii during interactions with the human host and arthropod vector, as well as the in vitro binding of Rickettsia conorii sRNA Rc_sR42 to bicistronic cytochrome bd ubiquinol oxidase subunits I and II (cydAB) mRNA. However, the mechanism of regulation and the effect of sRNA binding on the stability of the cydAB bicistronic transcript and the expression of the cydA and cydB genes are still unknown. In this study, we determined the expression dynamics of Rc_sR42 and its cognate target genes, cydA and cydB, in mouse lung and brain tissues during R. conorii infection in vivo and employed fluorescent and reporter assays to decode the role of sRNA in regulating cognate gene transcripts. Quantitative RT-PCR revealed significant changes in the expression of sRNA and its cognate target gene transcripts during R. conorii infection in vivo, and a greater abundance of these transcripts was observed in the lungs compared to brain tissue. Interestingly, while Rc_sR42 and cydA exhibited similar patterns of change in their expression, indicating the influence of sRNA on the mRNA target, the expression of cydB was independent of sRNA expression. Further, we constructed reporter plasmids of sRNA and cydAB bicistronic mRNA to decipher the role of sRNA on CydA and CydB expression. We observed increased expression of CydA in the presence of sRNA but detected no change in CydB expression in the presence or absence of sRNA. In sum, our results demonstrate that the binding of Rc_sR42 is required for the regulation of cydA but not cydB. Further studies on understanding the influence of this interaction on the mammalian host and tick vector during R. conorii infection are in progress.

Sensing the Messenger: Potential Roles of Cyclic-di-GMP in Rickettsial Pathogenesis.

Pathogenic bacteria causing human rickettsioses, transmitted in nature by arthropod vectors, primarily infect vascular endothelial cells lining the blood vessels, resulting in 'endothelial activation' and onset of innate immune responses. Nucleotide second messengers are long presumed to be the stimulators of type I interferons, of which bacterial cyclic-di-GMP (c-di-GMP) has been implicated in multiple signaling pathways governing communication with other bacteria and host cells, yet its importance in the context of rickettsial interactions with the host has not been investigated. Here, we report that all rickettsial genomes encode a putative diguanylate cyclase pleD, responsible for the synthesis of c-di-GMP. In silico analysis suggests that although the domain architecture of PleD is apparently well-conserved among different rickettsiae, the protein composition and sequences likely vary. Interestingly, cloning and sequencing of the pleD gene from virulent (Sheila Smith) and avirulent (Iowa) strains of R. rickettsii reveals a nonsynonymous substitution, resulting in an amino acid change (methionine to isoleucine) at position 236. Additionally, a previously reported 5-bp insertion in the genomic sequence coding for pleD (NCBI accession: NC_009882) was not present in the sequence of our cloned pleD from R. rickettsii strain Sheila Smith. In vitro infection of HMECs with R. rickettsii (Sheila Smith), but not R. rickettsii (Iowa), resulted in dynamic changes in the levels of pleD up to 24 h post-infection. These findings thus provide the first evidence for the potentially important role(s) of c-di-GMP in the determination of host-cell responses to pathogenic rickettsiae. Further studies into molecular mechanisms through which rickettsial c-di-GMP might regulate pathogen virulence and host responses should uncover the contributions of this versatile bacterial second messenger in disease pathogenesis and immunity to human rickettsioses.

Comparative transcriptomic analysis of Rickettsia conorii during in vitro infection of human and tick host cells.

BACKGROUND: Pathogenic Rickettsia species belonging to the spotted fever group are arthropod-borne, obligate intracellular bacteria which exhibit preferential tropism for host microvascular endothelium in the mammalian hosts, resulting in disease manifestations attributed primarily to endothelial damage or dysfunction. Although rickettsiae are known to undergo evolution through genomic reduction, the mechanisms by which these pathogens regulate their transcriptome to ensure survival in tick vectors and maintenance by transovarial/transstadial transmission, in contrast to their ability to cause debilitating infections in human hosts remain unknown. In this study, we compare the expression profiles of rickettsial sRNAome/transcriptome and determine the transcriptional start sites (TSSs) of R. conorii transcripts during in vitro infection of human and tick host cells. RESULTS: We performed deep sequencing on total RNA from Amblyomma americanum AAE2 cells and human microvascular endothelial cells (HMECs) infected with R. conorii. Strand-specific RNA sequencing of R. conorii transcripts revealed the expression 32 small RNAs (Rc_sR's), which were preferentially expressed above the limit of detection during tick cell infection, and confirmed the expression of Rc_sR61, sR71, and sR74 by quantitative RT-PCR. Intriguingly, a total of 305 and 132 R. conorii coding genes were differentially upregulated (> 2-fold) in AAE2 cells and HMECs, respectively. Further, enrichment for primary transcripts by treatment with Terminator 5'-Phosphate-dependent Exonuclease resulted in the identification of 3903 and 2555 transcription start sites (TSSs), including 214 and 181 primary TSSs in R. conorii during the infection to tick and human host cells, respectively. Seventy-five coding genes exhibited different TSSs depending on the host environment. Finally, we also observed differential expression of 6S RNA during host-pathogen and vector-pathogen interactions in vitro, implicating an important role for this noncoding RNA in the regulation of rickettsial transcriptome depending on the supportive host niche. CONCLUSIONS: In sum, the findings of this study authenticate the presence of novel Rc_sR's in R. conorii, reveal the first evidence for differential expression of coding transcripts and utilization of alternate transcriptional start sites depending on the host niche, and implicate a role for 6S RNA in the regulation of coding transcriptome during tripartite host-pathogen-vector interactions.

Evolution, purification, and characterization of RC0497: a peptidoglycan amidase from the prototypical spotted fever species Rickettsia conorii.

Rickettsial species have independently lost several genes owing to reductive evolution while retaining those predominantly implicated in virulence, survival, and biosynthetic pathways. In this study, we have identified a previously uncharacterized Rickettsia conorii gene RC0497 as an N-acetylmuramoyl-L-alanine amidase constitutively expressed during infection of cultured human microvascular endothelial cells at the levels of both mRNA transcript and encoded protein. A homology-based search of rickettsial genomes reveals that RC0497 homologs, containing amidase_2 family and peptidoglycan binding domains, are highly conserved among the spotted fever group (SFG) rickettsiae. The recombinant RC0497 protein exhibits α-helix secondary structure, undergoes a conformational change in the presence of zinc, and exists as a dimer at higher concentrations. We have further ascertained the enzymatic activity of RC0497 via demonstration of its ability to hydrolyze Escherichia coli peptidoglycan. Confocal microscopy on E. coli expressing RC0497 and transmission immunoelectron microscopy of R. conorii revealed its localization predominantly to the cell wall, septal regions of replicating bacteria, and the membrane of vesicles pinching off the cell wall. In summary, we have identified and functionally characterized RC0497 as a peptidoglycan hydrolase unique to spotted fever rickettsiae, which may potentially serve as a novel moonlighting protein capable of performing multiple functions during host-pathogen interactions.

Activation of Mechanistic Target of Rapamycin (mTOR) in Human Endothelial Cells Infected with Pathogenic Spotted Fever Group Rickettsiae.

Attributed to the tropism for host microvascular endothelium lining the blood vessels, vascular inflammation and dysfunction represent salient features of rickettsial pathogenesis, yet the details of fundamentally important pathogen interactions with host endothelial cells (ECs) as the primary targets of infection remain poorly appreciated. Mechanistic target of rapamycin (mTOR), a serine/threonine protein kinase of the phosphatidylinositol kinase-related kinase family, assembles into two functionally distinct complexes, namely mTORC1 (Raptor) and mTORC2 (Rictor), implicated in the determination of innate immune responses to intracellular pathogens via transcriptional regulation. In the present study, we investigated activation status of mTOR and its potential contributions to host EC responses during Rickettsia rickettsii and R. conorii infection. Protein lysates from infected ECs were analyzed for threonine 421/serine 424 phosphorylation of p70 S6 kinase (p70 S6K) and that of serine 2448 on mTOR itself as established markers of mTORC1 activation. For mTORC2, we assessed phosphorylation of protein kinase B (PKB or Akt) and protein kinase C (PKC), respectively, on serine 473 and serine 657. The results suggest increased phosphorylation of p70 S6K and mTOR during Rickettsia infection of ECs as early as 3 h and persisting for up to 24 h post-infection. The steady-state levels of phospho-Akt and phospho-PKC were also increased. Infection with pathogenic rickettsiae also resulted in the formation of microtubule-associated protein 1A/1B-light chain 3 (LC3-II) puncta and increased lipidation of LC3-II, a response significantly inhibited by introduction of siRNA targeting mTORC1 into ECs. These findings thus yield first evidence for the activation of both mTORC1 and mTORC2 during EC infection in vitro with Rickettsia species and suggest that early induction of autophagy in response to intracellular infection might be regulated by this important pathway known to function as a central integrator of cellular immunity and inflammation.

Rickettsia rickettsii Whole-Cell Antigens Offer Protection against Rocky Mountain Spotted Fever in the Canine Host.

Rocky Mountain spotted fever (RMSF) is a potentially fatal tick-borne disease in people and dogs. RMSF is reported in the United States and several countries in North, Central, and South America. The causative agent of this disease, Rickettsia rickettsii, is transmitted by several species of ticks, including Dermacentor andersoni, Rhipicephalus sanguineus, and Amblyomma americanum RMSF clinical signs generally include fever, headache, nausea, vomiting, muscle pain, lack of appetite, and rash. If untreated, it can quickly progress into a life-threatening illness in people and dogs, with high fatality rates ranging from 30 to 80%. While RMSF has been known for over a century, recent epidemiological data suggest that the numbers of documented cases and the fatality rates remain high in people, particularly during the last two decades in parts of North America. Currently, there are no vaccines available to prevent RMSF in either dogs or people. In this study, we investigated the efficacies of two experimental vaccines, a subunit vaccine containing two recombinant outer membrane proteins as recombinant antigens (RCA) and a whole-cell inactivated antigen vaccine (WCA), in conferring protection against virulent R. rickettsii infection challenge in a newly established canine model for RMSF. Dogs vaccinated with WCA were protected from RMSF, whereas those receiving RCA developed disease similar to that of nonvaccinated R. rickettsii-infected dogs. WCA also reduced the pathogen loads to nearly undetected levels in the blood, lungs, liver, spleen, and brain and induced bacterial antigen-specific immune responses. This study provides the first evidence of the protective ability of WCA against RMSF in dogs.

Expression Profiling of Long Noncoding RNA Splice Variants in Human Microvascular Endothelial Cells: Lipopolysaccharide Effects In Vitro.

Endothelial cell interactions with lipopolysaccharide (LPS) involve both activating and repressing signals resulting in pronounced alterations in their transcriptome and proteome. Noncoding RNAs are now appreciated as posttranscriptional and translational regulators of cellular signaling and responses, but their expression status and roles during endothelial interactions with LPS are not well understood. We report on the expression profile of long noncoding (lnc) RNAs of human microvascular endothelial cells in response to LPS. We have identified a total of 10,781 and 8310 lncRNA transcripts displaying either positive or negative regulation of expression, respectively, at 3 and 24 h posttreatment. A majority of LPS-induced lncRNAs are multiexonic and distributed across the genome as evidenced by their presence on all chromosomes. Present among these are a total of 44 lncRNAs with known regulatory functions, of which 41 multiexonic lncRNAs have multiple splice variants. We have further validated splice variant-specific expression of EGO (NONHSAT087634) and HOTAIRM1 (NONHSAT119666) at 3 h and significant upregulation of lnc-IL7R at 24 h. This study illustrates the genome-wide regulation of endothelial lncRNA splice variants in response to LPS and provides a foundation for further investigations of differentially expressed lncRNA transcripts in endothelial responses to LPS and pathophysiology of sepsis/septic shock.

MicroRNA Signature of Human Microvascular Endothelium Infected with Rickettsia rickettsii.

MicroRNAs (miRNAs) mediate gene silencing by destabilization and/or translational repression of target mRNA. Infection of human microvascular endothelial cells as primary targets of Rickettsiarickettsii, the etiologic agent of Rocky Mountain spotted fever, triggers host responses appertaining to alterations in cellular gene expression. Microarray-based profiling of endothelial cells infected with R.rickettsii for 3 or 24 h revealed differential expression of 33 miRNAs, of which miRNAs129-5p, 200a-3p, 297, 200b-3p, and 595 were identified as the top five up-regulated miRNAs (5 to 20-fold, p ≤ 0.01) and miRNAs 301b-3p, 548a-3p, and 377-3p were down-regulated (2 to 3-fold, p ≤ 0.01). Changes in the expression of selected miRNAs were confirmed by q-RT-PCR in both in vitro and in vivo models of infection. As potential targets, expression of genes encoding NOTCH1, SMAD2, SMAD3, RIN2, SOD1, and SOD2 was either positively or negatively regulated. Using a miRNA-specific mimic or inhibitor, NOTCH1 was determined to be a target of miRNA 200a-3p in R. rickettsii-infected human dermal microvascular endothelial cells (HMECs). Predictive interactome mapping suggested the potential for miRNA-mediated modulation of regulatory gene networks underlying important host cell signaling pathways. This first demonstration of altered endothelial miRNA expression provides new insights into regulatory elements governing mechanisms of host responses and pathogenesis during human rickettsial infections.

Bortezomib Effects on Human Microvascular Endothelium in vitro.

Cellular oxidative stress in the endothelium of blood vessels leads to several pathophysiological sequelae, including vascular damage and dysfunction, inflammation and atherosclerosis. Heme oxygenase-1 (HO-1) provides protection against oxidative stress-induced cell death and plays a crucial role in the regulation of cyclooxygenase-2 (COX-2) in endothelial cells. In the present study, we have investigated the effects of bortezomib, a clinically used proteasome inhibitor, on the regulation of HO-1 and COX-2 in cultured human microvascular endothelial cells (HMECs). Bortezomib treatment of HMECS induced dose- and time-dependent expression of HO-1 and COX-2 mRNA and protein, and triggered nuclear translocation of nuclear factor erythroid 2-related transcription factor (Nrf2). These findings suggest that HO-1/COX-2-mediated induction of antioxidant mechanisms via Nrf2 activation may contribute to the cytoprotective effects of bortezomib in microvascular endothelium.

Bacterial small RNAs in the Genus Rickettsia.

BACKGROUND: Rickettsia species are obligate intracellular Gram-negative pathogenic bacteria and the etiologic agents of diseases such as Rocky Mountain spotted fever (RMSF), Mediterranean spotted fever, epidemic typhus, and murine typhus. Genome sequencing revealed that R. prowazekii has ~25 % non-coding DNA, the majority of which is thought to be either "junk DNA" or pseudogenes resulting from genomic reduction. These characteristics also define other Rickettsia genomes. Bacterial small RNAs, whose biogenesis is predominantly attributed to either the intergenic regions (trans-acting) or to the antisense strand of an open reading frame (cis-acting), are now appreciated to be among the most important post-transcriptional regulators of bacterial virulence and growth. We hypothesize that intergenic regions in rickettsial species encode for small, non-coding RNAs (sRNAs) involved in the regulation of its transcriptome, leading to altered virulence and adaptation depending on the host niche. RESULTS: We employed a combination of bioinformatics and in vitro approaches to explore the presence of sRNAs in a number of species within Genus Rickettsia. Using the sRNA Identification Protocol using High-throughput Technology (SIPHT) web interface, we predicted over 1,700 small RNAs present in the intergenic regions of 16 different strains representing 13 rickettsial species. We further characterized novel sRNAs from typhus (R. prowazekii and R. typhi) and spotted fever (R. rickettsii and R. conorii) groups for their promoters and Rho-independent terminators using Bacterial Promoter Prediction Program (BPROM) and TransTermHP prediction algorithms, respectively. Strong σ70 promoters were predicted upstream of all novel small RNAs, indicating the potential for transcriptional activity. Next, we infected human microvascular endothelial cells (HMECs) with R. prowazekii for 3 h and 24 h and performed Next Generation Sequencing to experimentally validate the expression of 26 sRNA candidates predicted in R. prowazekii. Reverse transcriptase PCR was also used to further verify the expression of six putative novel sRNA candidates in R. prowazekii. CONCLUSIONS: Our results yield clear evidence for the expression of novel R. prowazekii sRNA candidates during infection of HMECs. This is the first description of novel small RNAs for a highly pathogenic species of Rickettsia, which should lead to new insights into rickettsial virulence and adaptation mechanisms.

Endothelial Mechanistic Target of Rapamycin Activation with Different Strains of R. rickettsii: Possible Role in Rickettsial Pathogenesis.

Rickettsia rickettsii is an obligate intracellular pathogen that primarily targets endothelial cells (ECs), leading to vascular inflammation and dysfunction. Mechanistic target of rapamycin (mTOR) regulates several cellular processes that directly affect host immune responses to bacterial pathogens. Here, we infected ECs with two R. rickettsii strains, avirulent (Iowa) and highly virulent Sheila Smith (SS) to identify differences in the kinetics and/or intensity of mTOR activation to establish a correlation between mTOR response and bacterial virulence. Endothelial mTOR activation with the highly virulent SS strain was significantly higher than with the avirulent Iowa strain. Similarly, there was increased LC3-II lipidation with the virulent SS strain compared with the avirulent Iowa strain of R. rickettsii. mTOR inhibitors rapamycin and Torin2 significantly increased bacterial growth and replication in the ECs, as evidenced by a more than six-fold increase in rickettsia copy numbers at 48 h post-infection. Further, the knockdown of mTOR with Raptor and Rictor siRNA resulted in a higher rickettsial copy number and the altered expression of the pro-inflammatory cytokines interleukin (IL)-1α, IL-6, and IL-8. These results are the first to reveal that endothelial mTOR activation and the early induction of autophagy might be governed by bacterial virulence and have established the mTOR pathway as an important regulator of endothelial inflammation, host immunity, and microbial replication.

UAP56 is an important mediator of angiotensin II/platelet derived growth factor induced vascular smooth muscle cell DNA synthesis and proliferation.

Angiotensin (Ang) II and platelet-derived growth factor (PDGF) are important mediators of pathologic vascular smooth muscle cell (VSMC) proliferation. Identifying downstream mediators of Ang II and PDGF signaling may provide insights for therapies to improve vascular proliferative diseases. We have previously demonstrated that breakpoint cluster region (Bcr) is an important mediator of Ang II/PDGF signaling in VSMC. We have recently reported that the DExD/H box protein UAP56 is an interacting partner of Bcr in regulating VSMC DNA synthesis. We hypothesized that UAP56 itself is an important regulator of VSMC proliferation. In this report we demonstrate that knockdown of UAP56 inhibits Ang II/PDGF induced VSMC DNA synthesis and proliferation, and inhibits E2F transcriptional activity. In addition, we demonstrate that UAP56 is present in the vessel wall of low-flow carotid arteries. These findings suggest that UAP56 is a regulator of VSMC proliferation and identify UAP56 as a target for preventing vascular proliferative disease.

UAP56 is a novel interacting partner of Bcr in regulating vascular smooth muscle cell DNA synthesis.

Bcr is a serine/threonine kinase that is a critical regulator of vascular smooth muscle cell inflammation and proliferation. We have previously demonstrated that Bcr acts in part via phosphorylation and inhibition of PPARγ. We have identified the RNA helicase UAP56 as another substrate of Bcr. In this report we demonstrate that knockdown of UAP56 blocks Bcr induced DNA synthesis in vascular smooth muscle cells (VSMC). We also found that over expression of Bcr increased the expression of cyclin E and decreased the expression of p27. Knockdown of UAP56 reversed the effect of Bcr on cyclin E and p27 expression. Furthermore, we found that Bcr binds to UAP56 and demonstrate that binding of UAP56 to Bcr is critical for Bcr induced DNA synthesis in VSMC. Our data identify UAP56 as an important binding partner of Bcr and a novel target for inhibiting vascular smooth muscle cell proliferation.

Regulation of inducible heme oxygenase and cyclooxygenase isozymes in a mouse model of spotted fever group rickettsiosis.

Vascular endothelial cells (ECs) lining the blood vessels are the preferred primary targets of pathogenic Rickettsia species in the host. In response to oxidative stress triggered by infection, ECs launch defense mechanisms such as expression of heme oxygenase-1 (HO-1). Previous evidence from an established animal model of Rocky Mountain spotted fever also suggests selective modulation of anti-oxidant enzyme activities in the target host tissues. In this study, we have examined the expression profiles of HO-1 and COX-2 in different tissues during Rickettsia conorii infection of susceptible C3H/HeN mice. RNA hybridization with murine HO-1 and COX-2-specific complementary DNA probes revealed increased HO-1 expression in the liver and brain of mice infected with three different doses of R. conorii ranging from 2.25×10(3) to 2.25×10(5) pfu, relatively non-remarkable changes in the lungs, and a trend for down-regulation in the spleen. The most prominent HO-1 response was evident in the liver with ∼4-fold increase on day 4 post-infection, followed by a decline on day 7. HO-1 expression in the brain, however, peaked with significantly higher levels on day 7. Following infection with both sub-lethal as well as lethal doses of infection, the transcript encoding COX-2 also displayed a pattern of increased expression in the liver and brain. Although immunohistochemical staining revealed increased abundance of HO-1 protein in the liver of infected mice, adjoining serial sections did not exhibit positive staining for COX-2 in infected tissues. The levels of monocyte chemoattractant protein-1 (MCP-1) and keratinocyte-derived cytokine (KC) were significantly higher in the sera of infected mice and corresponded with the onset and severity of the disease. Treatment of infected animals with anti-oxidants α-lipoic acid and N-acetylcysteine and HO inhibitor stannous protoporphyrin (SnPPIX) showed only selective beneficial effects on HO-1 and COX-2 expression in the liver and spleen and serum levels of KC and MCP-1. R. conorii infection of susceptible mice, therefore, results in selective regulation of the expression of HO-1 and COX-2 in a manner dependent on the target host tissue's cellular environment and the propensity of infection with rickettsiae.

Rickettsia conorii infection stimulates the expression of ISG15 and ISG15 protease UBP43 in human microvascular endothelial cells.

Rickettsia conorii, an obligate intracellular bacterium and the causative agent of Mediterranean spotted fever, preferentially infects microvascular endothelial cells of the mammalian hosts leading to onset of innate immune responses, characterized by the activation of intracellular signaling mechanisms, release of pro-inflammatory cytokines and chemokines, and killing of intracellular rickettsiae. Our recent studies have shown that interferon (IFN)-ß, a cytokine traditionally considered to be involved in antiviral immunity, plays an important role in the autocrine/paracrine regulation of host defense mechanisms and control of R. conorii growth in the host endothelial cells. Here, we show that R. conorii infection induces the expression of ISG15 (an interferon-stimulated gene coding a protein of 17kD) and UBP43 (an ISG15-specific protease) at the levels of mRNA and protein and report the evidence of ISGylation of as yet unidentified target proteins in cultured human microvascular endothelium. Infection-induced expression of ISG15 and UBP43 requires intracellular replication of rickettsiae and production of IFN-ß, because treatment with tetracycline and presence of an antibody capable of neutralizing IFN-ß activity resulted in near complete attenuation of both responses. Inhibition of R. conorii-induced ISG15 by RNA interference results in significant increase in the extent of rickettsial replication, whereas UBP43 knockdown yields a reciprocal inhibitory effect. In tandem, these results demonstrate the stimulation of interferon-ß-mediated innate immune mechanisms capable of perturbing the growth and replication of pathogenic rickettsiae and provide first evidence for ISG15-mediated post-translational modification of host cellular proteins during infection with an intracellular bacterium.

Bcr kinase activation by angiotensin II inhibits peroxisome-proliferator-activated receptor gamma transcriptional activity in vascular smooth muscle cells.

Bcr is a serine/threonine kinase activated by platelet-derived growth factor that is highly expressed in the neointima after vascular injury. Here, we demonstrate that Bcr is an important mediator of angiotensin (Ang) II and platelet-derived growth factor-mediated inflammatory responses in vascular smooth muscle cells (VSMCs). Among transcription factors that might regulate Ang II-mediated inflammatory responses we found that ligand-mediated peroxisome proliferator-activated receptor (PPAR)gamma transcriptional activity was significantly decreased by Ang II. Ang II increased Bcr expression and kinase activity. Overexpression of Bcr significantly inhibited PPARgamma activity. In contrast, knockdown of Bcr using Bcr small interfering RNA and a dominant-negative form of Bcr (DN-Bcr) reversed Ang II-mediated inhibition of PPARgamma activity significantly, suggesting the critical role of Bcr in Ang II-mediated inhibition of PPARgamma activity. Point-mutation and in vitro kinase analyses showed that PPARgamma was phosphorylated by Bcr at serine 82. Overexpression of wild-type Bcr kinase did not inhibit ligand-mediated PPARgamma1 S82A mutant transcriptional activity, indicating that Bcr regulates PPARgamma activity via S82 phosphorylation. DN-Bcr and Bcr small interfering RNA inhibited Ang II-mediated nuclear factor kappaB activation in VSMCs. DN-PPARgamma reversed DN-Bcr-mediated inhibition of nuclear factor kappaB activation, suggesting that PPARgamma is downstream from Bcr. Intimal proliferation in low-flow carotid arteries was decreased in Bcr knockout mice compared with wild-type mice, suggesting the critical role of Bcr kinase in VSMC proliferation in vivo, at least in part, via regulating PPARgamma/nuclear factor kappaB transcriptional activity.

MicroRNA-424 regulates the expression of CX3CL1 (fractalkine) in human microvascular endothelial cells during Rickettsia rickettsii infection.

Cytokines and chemokines trigger complex intracellular signaling through specific receptors to mediate immune cell recruitment and activation at the sites of infection. CX3CL1 (Fractalkine), a membrane-bound chemokine also capable of facilitating intercellular interactions as an adhesion molecule, contributes to host immune responses by virtue of its chemoattractant functions. Published studies have documented increased CX3CL1 expression in target tissues in a murine model of spotted fever rickettsiosis temporally corresponding to infiltration of macrophages and recovery from infection. Because pathogenic rickettsiae primarily target vascular endothelium in the mammalian hosts, we have now determined CX3CL1 mRNA and protein expression in cultured human microvascular endothelial cells (HMECs) infected in vitro with Rickettsia rickettsii. Our findings reveal 15.5 ± 4.0-fold and 12.3 ± 2.3-fold increase in Cx3cl1 mRNA expression at 3 h and 24 h post-infection, coinciding with higher steady-state levels of the corresponding protein in comparison to uninfected HMECs. Since CX3CL1 is a validated target of microRNA (miR)-424-5p (miR-424) and our earlier findings demonstrated robust down-regulation of miR-424 in R. rickettsii-infected HMECs, we further explored the possibility of regulation of CX3CL1 expression during rickettsial infection by miR-424. As expected, R. rickettsii infection resulted in 87 ± 5% reduction in miR-424 expression in host HMECs. Interestingly, a miR-424 mimic downregulated R. rickettsii-induced expression of CX3CL1, whereas an inhibitor of miR-424 yielded a converse up-regulatory effect, suggesting miR-424-mediated regulation of CX3CL1 during infection. Together, these findings provide the first evidence for the roles of a host microRNA in the regulation of an important bifunctional chemokine governing innate immune responses to pathogenic rickettsiae.

UAP56 is an important regulator of protein synthesis and growth in cardiomyocytes.

UAP56, an ATP dependent RNA helicase that also has ATPase activity, is a DExD/H box protein that is phylogenetically grouped with the eukaryotic initiation factor eIF4A, the prototypical member of the DExD/H box family of helicases. UAP56, also known as BAT1, is an essential RNA splicing factor required for spliceosome assembly and mRNA export but its role in protein synthesis is not known. Here we demonstrate that UAP56 regulates protein synthesis and growth in cardiomyocytes. We found that wild-type (WT) UAP56 increased serum induced protein synthesis in HeLa cells. UAP56 mutants lacking ATPase and/or helicase activity inhibited protein synthesis compared with WT UAP56, suggesting that the ATPase and RNA helicase activity of UAP56 is important for protein synthesis. UAP56 siRNA inhibited phenylephrine (PE) induced protein synthesis in cardiomyocytes and inhibited PE induced cardiomyocyte hypertrophy. Our data demonstrate that UAP56 is an important regulator of protein synthesis and plays an important role in the regulation of cardiomyocyte growth.

Recent research milestones in the pathogenesis of human rickettsioses and opportunities ahead.

Infections caused by pathogenic Rickettsia species continue to scourge human health across the globe. From the point of entry at the site of transmission by arthropod vectors, hematogenous dissemination of rickettsiae occurs to diverse host tissues leading to 'rickettsial vasculitis' as the salient feature of pathogenesis. This perspective article accentuates recent breakthrough developments in the context of host-pathogen-vector interactions during rickettsial infections. The subtopics include potential exploitation of circulating macrophages for spread, identification of new entry mechanisms and regulators of actin-based motility, appreciation of metabolites acquired from and effectors delivered into the host, importance of the toxin-antitoxin module in host-cell interactions, effects of the vector microbiome on rickettsial transmission, and niche-specific riboregulation and adaptation. Further research on these aspects will advance our understanding of the biology of rickettsiae as intracellular pathogens and should enable design and development of new approaches to counter rickettsioses in humans and other hosts.

The VE-cadherin binding domain of fibrinogen induces endothelial barrier permeability and enhances transendothelial migration of malignant breast epithelial cells.

Fibrin deposition and exudation of plasma fibrinogen (Fg) have long been recognized as hallmarks of inflammation, cardiovascular disease and neoplasia. The Fg-beta(15-42) domain binds to the endothelial cell adhesion molecule, VE-cadherin, promoting endothelial cell proliferation, angiogenesis and leukocyte diapedesis. Furthermore, spontaneous blood-borne and lymphatic metastasis of some types of tumor emboli requires plasma fibrin(ogen); however, the molecular mechanisms by which this occurs are poorly understood. We sought to determine whether Fg-beta(15-42) and VE-cadherin binding interactions promote endothelial barrier permeability and breast cancer cell transendothelial migration (TEM) using transwell insert culture systems. Synthetic peptides containing/missing residues beta(15-17) critical for Fg-beta(15-42) binding to VE-cadherin, and antibodies that bind to Fg-beta(15-21) (T2G1) and VE-cadherin (BV9) were used to induce or inhibit Fg-mediated permeability and TEM. Fg induced dose-dependent permeability of human umbilical vein and microvascular endothelial but not epithelial cell barriers. Maximal Fg-induced endothelial permeability required Fg-beta(15-42) and VE-cadherin-binding interactions involving Fg-beta(15-17). Fg-induced TEM of malignant MDA-MB-231 and MCF-7 breast cancer cells also required Fg-beta(15-42) and VE-cadherin binding; however, such TEM was independent of E-cadherin or estrogen receptor expression. In contrast, Fg did not induce TEM of nonmalignant MCF-10A breast epithelial cells. Fg-induced endothelial permeability was retained in the presence of MDA-MB-231 but inhibited in the presence of MCF-10A cells. It is intriguing to speculate that loss of Fg-beta(15-42) binding by premalignant breast epithelial cells serves as a molecular switch to induce a highly aggressive, metastatic breast cancer phenotype. Hence, Fg-beta(15-42) represents a potential molecular target for therapeutic intervention of breast cancer metastasis.