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

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.

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An Ehrlichia chaffeensis Tandem Repeat Protein Interacts with Multiple Host Targets Involved in Cell Signaling, Transcriptional Regulation, and Vesicle Trafficking

Infection and Immunity ◽

10.1128/iai.00027-09 ◽

2009 ◽

Vol 77 (5) ◽

pp. 1734-1745 ◽

Cited By ~ 55

Author(s):

Abdul Wakeel ◽

Jeeba A. Kuriakose ◽

Jere W. McBride

Keyword(s):

Transcriptional Regulation ◽

Cell Signaling ◽

Host Cell ◽

Tandem Repeat ◽

Vesicle Trafficking ◽

Effector Proteins ◽

Polycomb Group ◽

Mononuclear Phagocytes ◽

Ehrlichia Chaffeensis ◽

Protein Tyrosine

ABSTRACT Ehrlichia chaffeensis is an obligately intracellular bacterium that exhibits tropism for mononuclear phagocytes forming cytoplasmic membrane-bound microcolonies called morulae. To survive and replicate within phagocytes, E. chaffeensis exploits the host cell by modulating a number of host cell processes, but the ehrlichial effector proteins involved are unknown. In this study, we determined that p47, a secreted, differentially expressed, tandem repeat (TR) protein, interacts with multiple host proteins associated with cell signaling, transcriptional regulation, and vesicle trafficking. Yeast two-hybrid analysis revealed that p47 interacts with polycomb group ring finger 5 (PCGF5) protein, Src protein tyrosine kinase FYN (FYN), protein tyrosine phosphatase non-receptor type 2 (PTPN2), and adenylate cyclase-associated protein 1 (CAP1). p47 interaction with these proteins was further confirmed by coimmunoprecipitation assays and colocalization in HeLa cells transfected with p47-green fluorescent fusion protein (AcGFP1-p47). Moreover, confocal microscopy demonstrated p47-expressing dense-cored (DC) ehrlichiae colocalized with PCGF5, FYN, PTPN2, and CAP1. An amino-terminally truncated form of p47 containing TRs interacted only with PCGF5 and not with FYN, PTPN2, and CAP1, indicating differences in p47 domains that are involved in these interactions. These results demonstrate that p47 is involved in a complex network of interactions involving numerous host cell proteins. Furthermore, this study provides a new insight into the molecular and functional distinction of DC ehrlichiae, as well as the effector proteins involved in facilitating ehrlichial survival in mononuclear phagocytes.

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Ehrlichia chaffeensis TRP120 Interacts with a Diverse Array of Eukaryotic Proteins Involved in Transcription, Signaling, and Cytoskeleton Organization

Infection and Immunity ◽

10.1128/iai.05608-11 ◽

2011 ◽

Vol 79 (11) ◽

pp. 4382-4391 ◽

Cited By ~ 38

Author(s):

Tian Luo ◽

Jeeba A. Kuriakose ◽

Bing Zhu ◽

Abdul Wakeel ◽

Jere W. McBride

Keyword(s):

Host Cell ◽

Molecular Interactions ◽

Tandem Repeat ◽

Ring Finger ◽

Polycomb Group ◽

Mononuclear Phagocytes ◽

Ehrlichia Chaffeensis ◽

Content Type ◽

Cytoskeleton Organization ◽

Cell Defense

ABSTRACTEhrlichia chaffeensisis an obligately intracellular bacterium that exhibits tropism for mononuclear phagocytes and survives by evading host cell defense mechanisms. Recently, molecular interactions betweenE. chaffeensis47-kDa tandem repeat (TR) protein (TRP47) and the eukaryotic host cell have been described. In this investigation, yeast (Saccharomyces cerevisiae) two-hybrid analysis demonstrated thatE. chaffeensis-secreted tandem repeat protein 120 (TRP120) interacts with a diverse group of host cell proteins associated with major biological processes, including transcription and regulation, cell signaling, protein trafficking, and actin cytoskeleton organization. Twelve target proteins with the highest frequency of interaction with TRP120 were confirmed by cotransformation in yeast. Host targets, including human immunoglobulin lambda locus (IGL), cytochromecoxidase subunit II (COX2), Golgi-associated gamma adaptin ear-containing ARF binding protein 1 (GGA1), polycomb group ring finger 5 (PCGF5), actin gamma 1 (ACTG1), and unc-13 homolog D (UNC13D;Caenorhabditis elegans), colocalized strongly with TRP120 in HeLa cells and withE. chaffeensisdense-cored morulae and areas adjacent to morulae in the host cytoplasm. The TR domain of TRP120 interacted only with PCGF5, indicating that distinct TRP120 domains contribute to specific host target interactions and that multiple domains are required to reconstitute TRP120 interactions with other host targets. Three previously defined molecular interactions between TRP47 and host proteins, PCGF5, IGLL1, and CAP1, were also associated with TRP120, demonstrating that molecular cross talk occurs betweenEhrlichiaTRPs and host targets. These findings further support the role of TRPs as effectors that reprogram the host cell.

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Ehrlichia chaffeensis TRP32 Is a Nucleomodulin That Directly Regulates Expression of Host Genes Governing Differentiation and Proliferation

Infection and Immunity ◽

10.1128/iai.00657-16 ◽

2016 ◽

Vol 84 (11) ◽

pp. 3182-3194 ◽

Cited By ~ 12

Author(s):

Tierra R. Farris ◽

Paige S. Dunphy ◽

Bing Zhu ◽

Clayton E. Kibler ◽

Jere W. McBride

Keyword(s):

Dna Binding ◽

Host Cell ◽

Gene Transcription ◽

Noncoding Rna ◽

Target Genes ◽

Immune Cell ◽

Luciferase Reporter ◽

Ehrlichia Chaffeensis ◽

Content Type

Ehrlichia chaffeensis is an obligately intracellular bacterium that reprograms the mononuclear phagocyte through diverse effector-host interactions to modulate numerous host cell processes, including transcription. In a previous study, we reported that E. chaffeensis TRP32, a type 1 secreted effector, interacts with multiple host nucleus-associated proteins and also autoactivates reporter gene expression in yeast. In this study, we demonstrate that TRP32 is a nucleomodulin that binds host DNA and alters host gene transcription. TRP32 enters the host cell nucleus via a noncanonical translocation mechanism that involves phosphorylation of Y179 located in a C-terminal trityrosine motif. Both genistein and mutation of Y179 inhibited TRP32 nuclear entry. An electromobility shift assay (EMSA) demonstrated TRP32 host DNA binding via its tandem repeat domain. TRP32 DNA-binding and motif preference were further confirmed by supershift assays, as well as competition and mutant probe analyses. Using chromatin immunoprecipitation with next-generation sequencing (ChIP-seq), we determined that TRP32 binds a G-rich motif primarily within ±500 bp of the gene transcription start site. An ontology analysis identified genes involved in processes such as immune cell differentiation, chromatin remodeling, and RNA transcription and processing as primary TRP32 targets. TRP32-bound genes ( n = 1,223) were distributed on all chromosomes and included several global regulators of proliferation and inflammation such as those encoding FOS, JUN, AKT3, and NRAS and noncoding RNA genes microRNA 21 (miRNA 21) and miRNA 142. TRP32 target genes were differentially regulated during infection, the majority of which were repressed, and direct repression/activation of these genes by TRP32 was confirmed in vitro with a cellular luciferase reporter assay.

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Simultaneous Ribosome Profiling of Human Host Cells Infected with Toxoplasma gondii

mSphere ◽

10.1128/msphere.00292-19 ◽

2019 ◽

Vol 4 (3) ◽

Cited By ~ 6

Author(s):

Michael J. Holmes ◽

Premal Shah ◽

Ronald C. Wek ◽

William J. Sullivan

Keyword(s):

Gene Expression ◽

Toxoplasma Gondii ◽

Host Cell ◽

Ribosome Profiling ◽

Host Gene ◽

Host Cells ◽

Host Gene Expression ◽

Content Type ◽

Quiescent Cells ◽

Study Gene Expression

ABSTRACT Toxoplasma gondii is a ubiquitous obligate intracellular parasite that infects the nucleated cells of warm-blooded animals. From within the parasitophorous vacuole in which they reside, Toxoplasma tachyzoites secrete an arsenal of effector proteins that can reprogram host gene expression to facilitate parasite survival and replication. Gaining a better understanding of how host gene expression is altered upon infection is central for understanding parasite strategies for host invasion and for developing new parasite therapies. Here, we applied ribosome profiling coupled with mRNA measurements to concurrently study gene expression in the parasite and in host human foreskin fibroblasts. By examining the parasite transcriptome and translatome, we identified potential upstream open reading frames that may permit the stress-induced preferential translation of parasite mRNAs. We also determined that tachyzoites reduce host death-associated pathways and increase survival, proliferation, and motility in both quiescent and proliferative host cell models of infection. Additionally, proliferative cells alter their gene expression in ways that are consistent with massive transcriptional rewiring, while quiescent cells were best characterized by reentry into the cell cycle. We also identified a translational control regimen consistent with mechanistic target of rapamycin (mTOR) activation in quiescent cells and, to a lesser degree, in proliferative cells. This study illustrates the utility of the method for dissection of gene expression programs simultaneously in the parasite and host. IMPORTANCE Toxoplasma gondii is a single-celled parasite that has infected up to one-third of the world’s population. Significant overhauls in gene expression in both the parasite and the host cell accompany parasite invasion, and a better understanding of these changes may lead to the development of new therapeutic agents. In this study, we employed ribosome profiling to determine the changes that occur at the levels of transcription and translation in both the parasite and the infected host cell at the same time. We discovered features of Toxoplasma mRNAs that suggest a means for controlling parasite gene expression under stressful conditions. We also show that differences in host gene expression occur depending on whether they are confluent or not. Our findings demonstrate the feasibility of using ribosomal profiling to interrogate the host-parasite dynamic under a variety of conditions.

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Ehrlichia chaffeensis TRP75 Interacts with Host Cell Targets Involved in Homeostasis, Cytoskeleton Organization, and Apoptosis Regulation To Promote Infection

mSphere ◽

10.1128/msphere.00147-18 ◽

2018 ◽

Vol 3 (2) ◽

Cited By ~ 2

Author(s):

Tian Luo ◽

Shubhajit Mitra ◽

Jere W. McBride

Keyword(s):

Host Cell ◽

Tandem Repeat ◽

Effector Proteins ◽

Intracellular Bacterium ◽

Ehrlichia Chaffeensis ◽

Host Proteins ◽

Content Type ◽

Target Proteins ◽

Repeat Protein

ABSTRACT Ehrlichia chaffeensis is an obligately intracellular bacterium that exhibits tropism for mononuclear phagocytes. The mechanisms involved in E. chaffeensis infection of the host cell and evasion of host defenses are not fully defined, but a subset of type 1 secreted tandem repeat protein (TRP) effectors play important roles. Recently, we determined molecular interactions of TRP120, TRP47, and TRP32 with the eukaryotic host cell. In this investigation, we used yeast two-hybrid analysis to reveal that another E. chaffeensis tandem repeat protein, TRP75, interacts with a diverse group of human proteins associated with organismal and tissue homeostasis, multiple metabolic processes and regulation, response to reactive oxygen species, signal transduction, and protein modifications. Thirteen identified host target proteins associated with actin cytoskeleton reorganization or apoptosis were examined in detail and confirmed to interact with TRP75 at different levels as determined by coimmunoprecipitation assays. These protein interactions were visualized by immunofluorescence confocal microscopy during infection and colocalized with Ehrlichia morulae with different intensities. Moreover, small interfering RNAs (siRNAs) ( n = 86) were used to knock down identified TRP75-interacting host proteins separately, and their influence on ehrlichial infection was investigated by real-time quantitative PCR (qPCR). Knockdown of 74/86 (86%) TRP75 target proteins had a significant negative effect on ehrlichial infection. The results of this study further support the idea of a role of Ehrlichia TRPs as effectors that interact with a complex array of host proteins to promote ehrlichial infection. IMPORTANCE Human monocytic ehrlichiosis (HME) is caused by an obligatory intracellular bacterium, E. chaffeensis , and is one of the most prevalent, life-threatening emerging infectious zoonoses in the United States. The mechanisms through which E. chaffeensis invades and establishes an intracellular niche are not well understood but are dependent on secreted ehrlichial effector proteins. The significance of this study is in addressing how intracellular pathogens, particularly those with small genomes such as Ehrlichia , exploit a limited number of secreted effector proteins such as tandem repeat proteins (TRPs) to manipulate complex eukaryotes and to regulate host cell processes through molecular pathogen-host interplay. The results of our studies highlight the broader role of ehrlichial TRPs in promoting infection and help define the mechanisms through which obligately intracellular bacteria modulate host cell function for survival.

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

Infection and Immunity ◽

10.1128/iai.00638-20 ◽

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.

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Novel Transcriptional Regulons for Autotrophic Cycle Genes in Crenarchaeota

Journal of Bacteriology ◽

10.1128/jb.00249-15 ◽

2015 ◽

Vol 197 (14) ◽

pp. 2383-2391 ◽

Cited By ~ 7

Author(s):

Semen A. Leyn ◽

Irina A. Rodionova ◽

Xiaoqing Li ◽

Dmitry A. Rodionov

Keyword(s):

Carbon Dioxide ◽

Transcriptional Regulation ◽

Comparative Genomics ◽

Dna Binding ◽

Transcriptional Control ◽

Binding Assays ◽

Promoter Regions ◽

Content Type ◽

Genome Context

ABSTRACTAutotrophic microorganisms are able to utilize carbon dioxide as their only carbon source, or, alternatively, many of them can grow heterotrophically on organics. Different variants of autotrophic pathways have been identified in various lineages of the phylumCrenarchaeota. Aerobic members of the orderSulfolobalesutilize the hydroxypropionate-hydroxybutyrate cycle (HHC) to fix inorganic carbon, whereas anaerobicThermoprotealesuse the dicarboxylate-hydroxybutyrate cycle (DHC). Knowledge of transcriptional regulation of autotrophic pathways inArchaeais limited. We applied a comparative genomics approach to predict novel autotrophic regulons in theCrenarchaeota. We report identification of two novel DNA motifs associated with the autotrophic pathway genes in theSulfolobales(HHC box) andThermoproteales(DHC box). Based on genome context evidence, the HHC box regulon was attributed to a novel transcription factor from the TrmB family named HhcR. Orthologs of HhcR are present in allSulfolobalesgenomes but were not found in other lineages. A predicted HHC box regulatory motif was confirmed byin vitrobinding assays with the recombinant HhcR protein fromMetallosphaera yellowstonensis. For the DHC box regulon, we assigned a different potential regulator, named DhcR, which is restricted to the orderThermoproteales. DhcR inThermoproteus neutrophilus(Tneu_0751) was previously identified as a DNA-binding protein with high affinity for the promoter regions of two autotrophic operons. The global HhcR and DhcR regulons reconstructed by comparative genomics were reconciled with available omics data inMetallosphaeraandThermoproteusspp. The identified regulons constitute two novel mechanisms for transcriptional control of autotrophic pathways in theCrenarchaeota.IMPORTANCELittle is known about transcriptional regulation of carbon dioxide fixation pathways inArchaea. We previously applied the comparative genomics approach for reconstruction of DtxR family regulons in diverse lineages ofArchaea. Here, we utilize similar computational approaches to identify novel regulatory motifs for genes that are autotrophically induced in microorganisms from two lineages ofCrenarchaeotaand to reconstruct the respective regulons. The predicted novel regulons in archaeal genomes control the majority of autotrophic pathway genes and also other carbon and energy metabolism genes. The HhcR regulon was experimentally validated by DNA-binding assays inMetallosphaeraspp. Novel regulons described for the first time in this work provide a basis for understanding the mechanisms of transcriptional regulation of autotrophic pathways inArchaea.

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

Infection and Immunity ◽

10.1128/iai.00450-16 ◽

2016 ◽

Vol 85 (1) ◽

Cited By ~ 5

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.

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

Eukaryotic Cell ◽

10.1128/ec.00081-14 ◽

2014 ◽

Vol 13 (8) ◽

pp. 965-976 ◽

Cited By ~ 32

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.

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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

Infection and Immunity ◽

10.1128/iai.00841-19 ◽

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.

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