scholarly journals Protein and DNA Biosynthesis Demonstrated in Host Cell-Free Phagosomes Containing Anaplasma phagocytophilum or Ehrlichia chaffeensis in Axenic Media

2021 ◽  
Vol 89 (4) ◽  
Author(s):  
Yuntao Zhang ◽  
Li Chen ◽  
Chandramouli Kondethimmanahalli ◽  
Huitao Liu ◽  
Roman R. Ganta
Keyword(s):  
Dna Synthesis ◽  
Host Cell ◽  
Anaplasma Phagocytophilum ◽  
Biochemical Characterization ◽  
Animal Health ◽  
Host Cells ◽  
Ehrlichia Chaffeensis ◽  
Axenic Medium ◽  
Content Type ◽  
Dna Biosynthesis

ABSTRACT Rickettsiae belong to the Anaplasmataceae family, which includes mostly tick-transmitted pathogens causing human, canine, and ruminant diseases. Biochemical characterization of the pathogens remains a major challenge because of their obligate parasitism. We investigated the use of an axenic medium for growth of two important pathogens—Anaplasma phagocytophilum and Ehrlichia chaffeensis—in host cell-free phagosomes. We recently reported that the axenic medium promotes protein and DNA biosynthesis in host cell-free replicating form of E. chaffeensis, although the bacterial replication is limited. We now tested the hypothesis that growth on axenic medium can be improved if host cell-free rickettsia-containing phagosomes are used. Purification of phagosomes from A. phagocytophilum- and E. chaffeensis-infected host cells was accomplished by density gradient centrifugation combined with magnet-assisted cell sorting. Protein and DNA synthesis was observed for both organisms in cell-free phagosomes with glucose-6-phosphate and/or ATP. The levels of protein and DNA synthesis were the highest for a medium pH of 7. The data demonstrate bacterial DNA and protein synthesis for the first time in host cell-free phagosomes for two rickettsial pathogens. The host cell support-free axenic growth of obligate pathogenic rickettsiae will be critical in advancing research goals in many important tick-borne diseases impacting human and animal health.

scholarly journals Ehrlichia chaffeensis TRP120 Binds a G+C-Rich Motif in Host Cell DNA and Exhibits Eukaryotic Transcriptional Activator Function

2011 ◽  
Vol 79 (11) ◽  
pp. 4370-4381 ◽  
Author(s):  
Bing Zhu ◽  
Jeeba A. Kuriakose ◽  
Tian Luo ◽  
Efren Ballesteros ◽  
Sharu Gupta ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Dna Binding ◽  
Host Cell ◽  
High Throughput ◽  
Tandem Repeat ◽  
Binding Sites ◽  
Host Gene ◽  
Host Cells ◽  
Ehrlichia Chaffeensis ◽  
Content Type

ABSTRACTEhrlichia chaffeensisis an obligately intracellular bacterium that modulates host cell gene transcription in the mononuclear phagocyte, but the host gene targets and mechanisms involved in transcriptional modulation are not well-defined. In this study, we identified a novel tandem repeat DNA-binding domain in theE. chaffeensis120-kDa tandem repeat protein (TRP120) that directly binds host cell DNA. TRP120 was observed by immunofluorescent microscopy in the nucleus ofE. chaffeensis-infected host cells and was detected in nuclear extracts by Western immunoblotting with TRP120-specific antibody. The TRP120 binding sites and associated host cell target genes were identified using high-throughput deep sequencing (Illumina) of immunoprecipitated DNA (chromatin immunoprecipitation and high-throughput DNA sequencing). Multiple em motif elicitation (MEME) analysis of the most highly enriched TRP120-bound sequences revealed a G+C-rich DNA motif, and recombinant TRP120 specifically bound synthetic oligonucleotides containing the motif. TRP120 target gene binding sites were mapped most frequently to intersecting regions (intron/exon; 49%) but were also identified in upstream regulatory regions (25%) and downstream locations (26%). Genes targeted by TRP120 were most frequently associated with transcriptional regulation, signal transduction, and apoptosis. TRP120 targeted inflammatory chemokine genes, CCL2, CCL20, and CXCL11, which were strongly upregulated duringE. chaffeensisinfection and were also upregulated by direct transfection with recombinant TRP120. This study reveals that TRP120 is a novel DNA-binding protein that is involved in a host gene transcriptional regulation strategy.

scholarly journals Anaplasma phagocytophilum Outer Membrane Protein A Interacts with Sialylated Glycoproteins To Promote Infection of Mammalian Host Cells

2012 ◽  
Vol 80 (11) ◽  
pp. 3748-3760 ◽  
Author(s):  
Nore Ojogun ◽  
Amandeep Kahlon ◽  
Stephanie A. Ragland ◽  
Matthew J. Troese ◽  
Juliana E. Mastronunzio ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Anaplasma Phagocytophilum ◽  
Protein A ◽  
Host Cells ◽  
Mammalian Host ◽  
Content Type ◽  
Outer Membrane Protein A ◽  
Sialylated Glycoproteins

ABSTRACTAnaplasma phagocytophilumis the tick-transmitted obligate intracellular bacterium that causes human granulocytic anaplasmosis (HGA).A. phagocytophilumbinding to sialyl Lewis x (sLex) and other sialylated glycans that decorate P selectin glycoprotein 1 (PSGL-1) and other glycoproteins is critical for infection of mammalian host cells. Here, we demonstrate the importance ofA. phagocytophilumouter membrane protein A (OmpA) APH_0338 in infection of mammalian host cells. OmpA is transcriptionally induced during transmission feeding ofA. phagocytophilum-infected ticks on mice and is upregulated during invasion of HL-60 cells. OmpA is presented on the pathogen's surface. Sera from HGA patients and experimentally infected mice recognize recombinant OmpA. Pretreatment ofA. phagocytophilumorganisms with OmpA antiserum reduces their abilities to infect HL-60 cells. The OmpA N-terminal region is predicted to contain the protein's extracellular domain. GlutathioneS-transferase (GST)-tagged versions of OmpA and OmpA amino acids 19 to 74 (OmpA19-74) but not OmpA75-205bind to, and competitively inhibitA. phagocytophiluminfection of, host cells. Pretreatment of host cells with sialidase or trypsin reduces or nearly eliminates, respectively, GST-OmpA adhesion. Therefore, OmpA interacts with sialylated glycoproteins. This study identifies the firstA. phagocytophilumadhesin-receptor pair and delineates the region of OmpA that is critical for infection.

scholarly journals The Helicobacter pylori Autotransporter ImaA Tempers the Bacterium's Interaction with α5β1 Integrin

2016 ◽  
Vol 85 (1) ◽  
Author(s):  
William E. Sause ◽  
Daniela Keilberg ◽  
Soufiane Aboulhouda ◽  
Karen M. Ottemann
Keyword(s):  
Helicobacter Pylori ◽  
Host Cell ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Wild Type ◽  
Content Type ◽  
Type Iv ◽  
H Pylori ◽  
Α5β1 Integrin ◽  
Cell Interface

ABSTRACT The human pathogen Helicobacter pylori uses the host receptor α5β1 integrin to trigger inflammation in host cells via its cag pathogenicity island (cag PAI) type IV secretion system (T4SS). Here, we report that the H. pylori ImaA protein (HP0289) decreases the action of the cag PAI T4SS via tempering the bacterium's interaction with α5β1 integrin. Previously, imaA-null mutants were found to induce an elevated inflammatory response that was dependent on the cag PAI T4SS; here we extend those findings to show that the elevated response is independent of the CagA effector protein. To understand how ImaA could be affecting cag PAI T4SS activity at the host cell interface, we utilized the Phyre structural threading program and found that ImaA has a region with remote homology to bacterial integrin-binding proteins. This region was required for ImaA function. Unexpectedly, we observed that imaA mutants bound higher levels of α5β1 integrin than wild-type H. pylori, an outcome that required the predicted integrin-binding homology region of ImaA. Lastly, we report that ImaA directly affected the amount of host cell β1 integrin but not other cellular integrins. Our results thus suggest a model in which H. pylori employs ImaA to regulate interactions between integrin and the T4SS and thus alter the host inflammatory strength.

scholarly journals Toxoplasma gondii Development of Its Replicative Niche: in Its Host Cell and Beyond

2014 ◽  
Vol 13 (8) ◽  
pp. 965-976 ◽  
Author(s):  
Ira J. Blader ◽  
Anita A. Koshy
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Host Cells ◽  
Content Type ◽  
Obligate Intracellular ◽  
Cellular Processes ◽  
High Prevalence ◽  
Life Threatening ◽  
Cell Processes ◽  
Cellular Pathways

ABSTRACTIntracellular pathogens can replicate efficiently only after they manipulate and modify their host cells to create an environment conducive to replication. While diverse cellular pathways are targeted by different pathogens, metabolism, membrane and cytoskeletal architecture formation, and cell death are the three primary cellular processes that are modified by infections.Toxoplasma gondiiis an obligate intracellular protozoan that infects ∼30% of the world's population and causes severe and life-threatening disease in developing fetuses, in immune-comprised patients, and in certain otherwise healthy individuals who are primarily found in South America. The high prevalence ofToxoplasmain humans is in large part a result of its ability to modulate these three host cell processes. Here, we highlight recent work defining the mechanisms by whichToxoplasmainteracts with these processes. In addition, we hypothesize why some processes are modified not only in the infected host cell but also in neighboring uninfected cells.

scholarly journals Fluorescence-Reported Allelic Exchange Mutagenesis-Mediated Gene Deletion Indicates a Requirement for Chlamydia trachomatis Tarp during In Vivo Infectivity and Reveals a Specific Role for the C Terminus during Cellular Invasion

2020 ◽  
Vol 88 (5) ◽  
Author(s):  
Susmita Ghosh ◽  
Elizabeth A. Ruelke ◽  
Joshua C. Ferrell ◽  
Maria D. Bodero ◽  
Kenneth A. Fields ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Host Cell ◽  
Host Cells ◽  
Actin Binding ◽  
Wild Type ◽  
Content Type ◽  
Allelic Exchange ◽  
Binding Domains

ABSTRACT The translocated actin recruiting phosphoprotein (Tarp) is a multidomain type III secreted effector used by Chlamydia trachomatis. In aggregate, existing data suggest a role of this effector in initiating new infections. As new genetic tools began to emerge to study chlamydial genes in vivo, we speculated as to what degree Tarp function contributes to Chlamydia’s ability to parasitize mammalian host cells. To address this question, we generated a complete tarP deletion mutant using the fluorescence-reported allelic exchange mutagenesis (FRAEM) technique and complemented the mutant in trans with wild-type tarP or mutant tarP alleles engineered to harbor in-frame domain deletions. We provide evidence for the significant role of Tarp in C. trachomatis invasion of host cells. Complementation studies indicate that the C-terminal filamentous actin (F-actin)-binding domains are responsible for Tarp-mediated invasion efficiency. Wild-type C. trachomatis entry into HeLa cells resulted in host cell shape changes, whereas the tarP mutant did not. Finally, using a novel cis complementation approach, C. trachomatis lacking tarP demonstrated significant attenuation in a murine genital tract infection model. Together, these data provide definitive genetic evidence for the critical role of the Tarp F-actin-binding domains in host cell invasion and for the Tarp effector as a bona fide C. trachomatis virulence factor.

scholarly journals Core N-Glycan Structures Are Critical for the Pathogenicity of Cryptococcus neoformans by Modulating Host Cell Death

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Eun Jung Thak ◽  
Su-Bin Lee ◽  
Shengjie Xu-Vanpala ◽  
Dong-Jik Lee ◽  
Seung-Yeon Chung ◽  
...  
Keyword(s):  
Cell Death ◽  
Fungal Infection ◽  
Host Cell ◽  
Host Cells ◽  
Glycan Structure ◽  
Macrophage Cell ◽  
Content Type ◽  
Host Cell Death ◽  
Intact Core

ABSTRACT Cryptococcus neoformans is a human-pathogenic fungal pathogen that causes life-threatening meningoencephalitis in immunocompromised individuals. To investigate the roles of N-glycan core structure in cryptococcal pathogenicity, we constructed mutant strains of C. neoformans with defects in the assembly of lipid-linked N-glycans in the luminal side of the endoplasmic reticulum (ER). Deletion of ALG3 (alg3Δ), which encodes dolichyl-phosphate-mannose (Dol-P-Man)-dependent α-1,3-mannosyltransferase, resulted in the production of truncated neutral N-glycans carrying five mannose residues as a major species. Despite moderate or nondetectable defects in virulence-associated phenotypes in vitro, the alg3Δ mutant was avirulent in a mouse model of systemic cryptococcosis. Notably, the mutant did not show defects in early stages of host cell interaction during infection, including attachment to lung epithelial cells, opsonic/nonopsonic phagocytosis, and manipulation of phagosome acidification. However, the ability to drive macrophage cell death was greatly decreased in this mutant, without loss of cell wall remodeling capacity. Furthermore, deletion of ALG9 and ALG12, encoding Dol-P-Man-dependent α-1,2-mannosyltransferases and α-1,6-mannosyltransferases, generating truncated core N-glycans with six and seven mannose residues, respectively, also displayed remarkably reduced macrophage cell death and in vivo virulence. However, secretion levels of interleukin-1β (IL-1β) were not reduced in the bone marrow-derived dendritic cells obtained from Asc- and Gsdmd-deficient mice infected with the alg3Δ mutant strain, excluding the possibility that pyroptosis is a main host cell death pathway dependent on intact core N-glycans. Our results demonstrated N-glycan structures as a critical feature in modulating death of host cells, which is exploited by as a strategy for host cell escape for dissemination of C. neoformans. IMPORTANCE We previously reported that the outer mannose chains of N-glycans are dispensable for the virulence of C. neoformans, which is in stark contrast to findings for the other human-pathogenic yeast, Candida albicans. Here, we present evidence that an intact core N-glycan structure is required for C. neoformans pathogenicity by systematically analyzing alg3Δ, alg9Δ, and alg12Δ strains that have defects in lipid-linked N-glycan assembly and in in vivo virulence. The alg null mutants producing truncated core N-glycans were defective in inducing host cell death after phagocytosis, which is triggered as a mechanism of pulmonary escape and dissemination of C. neoformans, thus becoming inactive in causing fatal infection. The results clearly demonstrated the critical features of the N-glycan structure in mediating the interaction with host cells during fungal infection. The delineation of the roles of protein glycosylation in fungal pathogenesis not only provides insight into the glycan-based fungal infection mechanism but also will aid in the development of novel antifungal agents.

scholarly journals EtpE Binding to DNase X Induces Ehrlichial Entry via CD147 and hnRNP-K Recruitment, Followed by Mobilization of N-WASP and Actin

mBio ◽  
2015 ◽  
Vol 6 (6) ◽  
Author(s):  
Dipu Mohan Kumar ◽  
Mingqun Lin ◽  
Qingming Xiong ◽  
Mathew James Webber ◽  
Comert Kural ◽  
...  
Keyword(s):  
Cell Surface ◽  
Host Cell ◽  
Actin Polymerization ◽  
Ehrlichia Chaffeensis ◽  
Content Type ◽  
Hnrnp K ◽  
Obligate Intracellular ◽  
Human Proteins ◽  
Coated Bead ◽  
Human Monocytic Ehrlichiosis

ABSTRACTObligate intracellular bacteria, such asEhrlichia chaffeensis, perish unless they can enter eukaryotic cells.E. chaffeensisis the etiological agent of human monocytic ehrlichiosis, an emerging infectious disease. To infect cells,Ehrlichiauses theCterminus of the outer membrane invasinentry-triggeringprotein (EtpE) ofEhrlichia(EtpE-C), which directly binds the mammalian cell surface glycosylphosphatidyl inositol-anchored protein, DNase X. How this binding drivesEhrlichiaentry is unknown. Here, using affinity pulldown of host cell lysates with recombinant EtpE-C (rEtpE-C), we identified two new human proteins that interact with EtpE-C: CD147 and heterogeneous nuclear ribonucleoprotein K (hnRNP-K). The interaction of CD147 with rEtpE-C was validated by far-Western blotting and coimmunoprecipitation of native EtpE with endogenous CD147. CD147 was ubiquitous on the cell surface and also present around foci of rEtpE-C-coated-bead entry. Functional neutralization of surface-exposed CD147 with a specific antibody inhibitedEhrlichiainternalization and infection but not binding. Downregulation of CD147 by short hairpin RNA (shRNA) impairedE. chaffeensisinfection. Functional ablation of cytoplasmic hnRNP-K by a nanoscale intracellular antibody markedly attenuated bacterial entry and infection but not binding. EtpE-C also interacted with neuronal Wiskott-Aldrich syndrome protein (N-WASP), which is activated by hnRNP-K. Wiskostatin, which inhibits N-WASP activation, and cytochalasin D, which inhibits actin polymerization, inhibitedEhrlichiaentry. Upon incubation with host cell lysate, EtpE-C but not an EtpE N-terminal fragment stimulatedin vitroactin polymerization in an N-WASP- and DNase X-dependent manner. Time-lapse video images revealed N-WASP recruitment at EtpE-C-coated bead entry foci. Thus, EtpE-C binding to DNase X drivesEhrlichiaentry by engaging CD147 and hnRNP-K and activating N-WASP-dependent actin polymerization.IMPORTANCEEhrlichia chaffeensis, an obligate intracellular bacterium, causes a blood-borne disease called human monocytic ehrlichiosis, one of the most prevalent life-threatening emerging tick-transmitted infectious diseases in the United States. The survival ofEhrlichiabacteria, and hence, their ability to cause disease, depends on their specific mode of entry into eukaryotic host cells. Understanding the mechanism by whichE. chaffeensisenters cells will create new opportunities for developing effective therapies to prevent bacterial entry and disease in humans. Our findings reveal a novel cellular signaling pathway triggered by an ehrlichial surface protein called EtpE to induce its infectious entry. The results are also important from the viewpoint of human cell physiology because three EtpE-interacting human proteins, DNase X, CD147, and hnRNP-K, are hitherto unknown partners that drive the uptake of small particles, including bacteria, into human cells.

scholarly journals Chlamydia-Infected Cells Continue To Undergo Mitosis and Resist Induction of Apoptosis

2004 ◽  
Vol 72 (1) ◽  
pp. 451-460 ◽  
Author(s):  
Whitney Greene ◽  
Yangming Xiao ◽  
Yanqing Huang ◽  
Grant McClarty ◽  
Guangming Zhong
Keyword(s):  
Dna Synthesis ◽  
Host Cell ◽  
Chlamydial Infection ◽  
Host Cells ◽  
Infected Host ◽  
Apoptosis Induction ◽  
Infected Cells ◽  
Induction Of Apoptosis

ABSTRACT Both anti- and proapoptotic activities have been reported to occur during chlamydial infection. To reconcile the apparent controversy, we compared host cell apoptotic responses to infection with 17 different chlamydial serovars and strains. None of the serovars caused any biologically significant apoptosis in the infected host cells. Host cells in chlamydia-infected cultures can continue to undergo DNA synthesis and mitosis. Chlamydia-infected cells are resistant to apoptosis induction, although the extent of the antiapoptotic ability varied between serovars. These observations have demonstrated that an anti- but not proapoptotic activity is the prevailing event in chlamydia-infected cultures.

scholarly journals The Sinorhizobium (Ensifer) fredii HH103 Nodulation Outer Protein NopI Is a Determinant for Efficient Nodulation of Soybean and Cowpea Plants

2016 ◽  
Vol 83 (5) ◽  
Author(s):  
Irene Jiménez-Guerrero ◽  
Francisco Pérez-Montaño ◽  
Carlos Medina ◽  
Francisco Javier Ollero ◽  
Francisco Javier López-Baena
Keyword(s):  
Adenylate Cyclase ◽  
Host Cell ◽  
Host Range ◽  
Pathogenic Bacteria ◽  
Defense Responses ◽  
Host Cells ◽  
Broad Host Range ◽  
Sinorhizobium Fredii ◽  
Symbiotic Efficiency ◽  
Content Type

ABSTRACT The type III secretion system (T3SS) is a specialized secretion apparatus that is commonly used by many plant and animal pathogenic bacteria to deliver proteins, termed effectors, to the interior of the host cells. These effectors suppress host defenses and interfere with signal transduction pathways to promote infection. Some rhizobial strains possess a functional T3SS, which is involved in the suppression of host defense responses, host range determination, and symbiotic efficiency. The analysis of the genome of the broad-host-range rhizobial strain Sinorhizobium fredii HH103 identified eight genes that code for putative T3SS effectors. Three of these effectors, NopL, NopP, and NopI, are Rhizobium specific. In this work, we demonstrate that NopI, whose amino acid sequence shows a certain similarity with NopP, is secreted through the S. fredii HH103 T3SS in response to flavonoids. We also determined that NopL can be considered an effector since it is directly secreted to the interior of the host cell as demonstrated by adenylate cyclase assays. Finally, the symbiotic phenotype of single, double, and triple nopI, nopL, and nopP mutants in soybean and cowpea was assayed, showing that NopI plays an important role in determining the number of nodules formed in both legumes and that the absence of both NopL and NopP is highly detrimental for symbiosis. IMPORTANCE The paper is focused on three Rhizobium-specific T3SS effectors of Sinorhizobium fredii HH103, NopL, NopP, and NopI. We demonstrate that S. fredii HH103 is able to secrete through the T3SS in response to flavonoids the nodulation outer protein NopI. Additionally, we determined that NopL can be considered an effector since it is secreted to the interior of the host cell as demonstrated by adenylate cyclase assays. Finally, nodulation assays of soybean and cowpea indicated that NopI is important for the determination of the number of nodules formed and that the absence of both NopL and NopP negatively affected nodulation.

scholarly journals Divergent Evolution of Legionella RCC1 Repeat Effectors Defines the Range of Ran GTPase Cycle Targets

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
A. Leoni Swart ◽  
Bernhard Steiner ◽  
Laura Gomez-Valero ◽  
Sabina Schütz ◽  
Mandy Hannemann ◽  
...  
Keyword(s):  
Host Cell ◽  
Legionella Pneumophila ◽  
Small Gtpase ◽  
Effector Proteins ◽  
Host Cells ◽  
Divergent Evolution ◽  
Ran Gtpase ◽  
Content Type ◽  
Gtpase Cycle ◽  
Gtpase Ran

ABSTRACT Legionella pneumophila governs its interactions with host cells by secreting >300 different “effector” proteins. Some of these effectors contain eukaryotic domains such as the RCC1 (regulator of chromosome condensation 1) repeats promoting the activation of the small GTPase Ran. In this report, we reveal a conserved pattern of L. pneumophila RCC1 repeat genes, which are distributed in two main clusters of strains. Accordingly, strain Philadelphia-1 contains two RCC1 genes implicated in bacterial virulence, legG1 (Legionella eukaryotic gene 1), and ppgA, while strain Paris contains only one, pieG. The RCC1 repeat effectors localize to different cellular compartments and bind distinct components of the Ran GTPase cycle, including Ran modulators and the small GTPase itself, and yet they all promote the activation of Ran. The pieG gene spans the corresponding open reading frames of legG1 and a separate adjacent upstream gene, lpg1975. legG1 and lpg1975 are fused upon addition of a single nucleotide to encode a protein that adopts the binding specificity of PieG. Thus, a point mutation in pieG splits the gene, altering the effector target. These results indicate that divergent evolution of RCC1 repeat effectors defines the Ran GTPase cycle targets and that modulation of different components of the cycle might fine-tune Ran activation during Legionella infection. IMPORTANCE Legionella pneumophila is a ubiquitous environmental bacterium which, upon inhalation, causes a life-threatening pneumonia termed Legionnaires’ disease. The opportunistic pathogen grows in amoebae and macrophages by employing a “type IV” secretion system, which secretes more than 300 different “effector” proteins into the host cell, where they subvert pivotal processes. The function of many of these effector proteins is unknown, and their evolution has not been studied. L. pneumophila RCC1 repeat effectors target the small GTPase Ran, a molecular switch implicated in different cellular processes such as nucleocytoplasmic transport and microtubule cytoskeleton dynamics. We provide evidence that one or more RCC1 repeat genes are distributed in two main clusters of L. pneumophila strains and have divergently evolved to target different components of the Ran GTPase activation cycle at different subcellular sites. Thus, L. pneumophila employs a sophisticated strategy to subvert host cell Ran GTPase during infection.

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