scholarly journals Mutations in Ehrlichia chaffeensis Genes ECH_0660 and ECH_0665 Cause Transcriptional Changes in Response to Zinc or Iron Limitation

2021 ◽  
Author(s):  
Ascención Torres-Escobar ◽  
María D. Juárez-Rodríguez ◽  
Roman R. Ganta
Keyword(s):  
Iron Limitation ◽  
Ehrlichia Chaffeensis ◽  
Wild Type ◽  
Gene Encoding ◽  
Transcriptional Changes ◽  
Rapid Clearance ◽  
Human Monocytic Ehrlichiosis ◽  
Phage Proteins ◽  
Type Infection

Ehrlichia chaffeensis causes human monocytic ehrlichiosis by replicating within phagosomes of monocytes/macrophages. A function disruption mutation within the pathogen’s ECH_0660 gene encoding a phage head-to-tail connector protein resulted in the rapid clearance of the pathogen in vivo, while aiding to induce sufficient immunity in a host to protect against wild-type infection challenge. In this study, we describe the characterization of a cluster of seven genes spanning from ECH_0659 to ECH_0665, which contained four genes encoding bacterial phage proteins, including the ECH_0660 gene. Assessment of the promoter region upstream to the first gene of the seven genes (ECH_0659) in Escherichia coli demonstrated transcriptional enhancement under zinc and iron starvation. Further, transcription of the seven genes was significantly higher for E. chaffeensis having a mutation in the ECH_0660 gene compared to the wild-type pathogen under zinc and iron starving conditions. In contrast, transcription from the genes was mostly similar to wild-type or moderately downregulated for the ECH_0665 gene mutant with the function disruption. Recently, we reported that this mutation caused a minimal impact on the pathogen’s in vivo growth, as it persisted similar to wild-type. The current study is the first in describing how zinc and iron contribute to E. chaffeensis biology. Specifically, we demonstrated that the functional disruption in the gene encoding the predicted head-to-tail connector protein in E. chaffeensis results in the enhanced transcription of seven genes including those encoding phage proteins during zinc and iron limitation. IMPORTANCE Ehrlichia chaffeensis, a tick-transmitted bacterium, causes human monocytic ehrlichiosis by replicating within phagosomes of monocytes/macrophages. A function disruption mutation within the pathogen’s gene encoding a phage head-to-tail connector protein resulted in the rapid clearance of the pathogen in vivo, while aiding to induce sufficient immunity in a host to protect against wild-type infection challenge. In the current study, we investigated if the functional disruption in the predicted head-to-tail connector protein gene caused transcriptional changes resulting from metal ion limitations. This is the first study describing how zinc and iron may contribute to E. chaffeensis replication.

scholarly journals Attenuated Mutants of Ehrlichia chaffeensis Induce Protection against Wild-Type Infection Challenge in the Reservoir Host and in an Incidental Host

2015 ◽  
Vol 83 (7) ◽  
pp. 2827-2835 ◽  
Author(s):  
Arathy D. S. Nair ◽  
Chuanmin Cheng ◽  
Deborah C. Jaworski ◽  
Suhasini Ganta ◽  
Michael W. Sanderson ◽  
...  
Keyword(s):  
Reservoir Host ◽  
Ehrlichia Chaffeensis ◽  
Wild Type ◽  
Tissue Samples ◽  
Content Type ◽  
Vaccine Candidates ◽  
Rapid Clearance ◽  
Human Monocytic Ehrlichiosis ◽  
Insertion Mutations ◽  
Type Infection

Ehrlichia chaffeensis, a tick-borne rickettsial organism, causes the disease human monocytic ehrlichiosis. The pathogen also causes disease in several other vertebrates, including dogs and deer. In this study, we assessed two clonally purifiedE. chaffeensismutants with insertions within the genes Ech_0379 and Ech_0660 as vaccine candidates in deer and dogs. Infection with the Ech_0379 mutant and challenge with wild-typeE. chaffeensis1 month following inoculation with the mutant resulted in the reduced presence of the organism in blood compared to the presence of wild-type infection in both deer and dogs. The Ech_0660 mutant infection resulted in its rapid clearance from the bloodstream. The wild-type infection challenge following Ech_0660 mutant inoculation also caused the pathogen's clearance from blood and tissue samples as assessed at the end of the study. The Ech_0379 mutant-infected and -challenged animals also remained positive for the organism in tissue samples in deer but not in dogs. This is the first study that documents that insertion mutations inE. chaffeensisthat cause attenuated growth confer protection against wild-type infection challenge. This study is important in developing vaccines to protect animals and people againstEhrlichiaspecies infections.

scholarly journals Ehrlichia chaffeensis and Its Invasin EtpE Block Reactive Oxygen Species Generation by Macrophages in a DNase X-Dependent Manner

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
Omid Teymournejad ◽  
Mingqun Lin ◽  
Yasuko Rikihisa
Keyword(s):  
Host Cell ◽  
Cell Receptor ◽  
Ros Generation ◽  
Ehrlichia Chaffeensis ◽  
Biological Processes ◽  
Wild Type ◽  
New Approach ◽  
Host Cell Receptor ◽  
Life Threatening ◽  
Human Monocytic Ehrlichiosis

ABSTRACT The obligatory intracellular pathogen Ehrlichia chaffeensis lacks most genes that confer resistance to oxidative stress but can block reactive oxygen species (ROS) generation by host monocytes-macrophages. Bacterial and host molecules responsible for this inhibition have not been identified. To infect host cells, Ehrlichia uses the C terminus of its surface invasin, entry-triggering protein of Ehrlichia (EtpE; EtpE-C), which directly binds the mammalian cell surface receptor glycosylphosphatidylinositol-anchored protein DNase X. We investigated whether EtpE-C binding to DNase X blocks ROS production by mouse bone marrow-derived macrophages (BMDMs). On the basis of a luminol-dependent chemiluminescence assay, E. chaffeensis inhibited phorbol myristate acetate (PMA)-induced ROS generation by BMDMs from wild-type, but not DNase X−/−, mice. EtpE-C is critical for inhibition, as recombinant EtpE-C (rEtpE-C)-coated latex beads, but not recombinant N-terminal EtpE-coated or uncoated beads, inhibited PMA-induced ROS generation by BMDMs from wild-type mice. DNase X is required for this inhibition, as none of these beads inhibited PMA-induced ROS generation by BMDMs from DNase X−/− mice. Previous studies showed that E. chaffeensis does not block ROS generation in neutrophils, a cell type that is a potent ROS generator but is not infected by E. chaffeensis. Human and mouse peripheral blood neutrophils did not express DNase X. Our findings point to a unique survival mechanism of ROS-sensitive obligate intramonocytic bacteria that involves invasin EtpE binding to DNase X on the host cell surface. This is the first report of bacterial invasin having such a subversive activity on ROS generation. IMPORTANCE Ehrlichia chaffeensis preferentially infects monocytes-macrophages and causes a life-threatening emerging tick-transmitted infectious disease called human monocytic ehrlichiosis. Ehrlichial infection, and hence the disease, depends on the ability of this bacterium to avoid or overcome powerful microbicidal mechanisms of host monocytes-macrophages, one of which is the generation of ROS. Our findings reveal that an ehrlichial surface invasin, EtpE, not only triggers bacterial entry but also blocks ROS generation by host macrophages through its host cell receptor, DNase X. As ROS sensitivity is an Achilles’ heel of this group of pathogens, understanding the mechanism by which E. chaffeensis rapidly blocks ROS generation suggests a new approach for developing effective anti-infective measures. The discovery of a ROS-blocking pathway is also important, as modulation of ROS generation is important in a variety of ailments and biological processes. IMPORTANCE Ehrlichia chaffeensis preferentially infects monocytes-macrophages and causes a life-threatening emerging tick-transmitted infectious disease called human monocytic ehrlichiosis. Ehrlichial infection, and hence the disease, depends on the ability of this bacterium to avoid or overcome powerful microbicidal mechanisms of host monocytes-macrophages, one of which is the generation of ROS. Our findings reveal that an ehrlichial surface invasin, EtpE, not only triggers bacterial entry but also blocks ROS generation by host macrophages through its host cell receptor, DNase X. As ROS sensitivity is an Achilles’ heel of this group of pathogens, understanding the mechanism by which E. chaffeensis rapidly blocks ROS generation suggests a new approach for developing effective anti-infective measures. The discovery of a ROS-blocking pathway is also important, as modulation of ROS generation is important in a variety of ailments and biological processes.

scholarly journals Endothelial cell-specific reduction of heparan sulfate suppresses glioma growth in mice

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Takamasa Kinoshita ◽  
Hiroyuki Tomita ◽  
Hideshi Okada ◽  
Ayumi Niwa ◽  
Fuminori Hyodo ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Heparan Sulfate ◽  
Vascular Endothelium ◽  
Wild Type ◽  
Brain Capillaries ◽  
Gene Encoding ◽  
Glioma Growth ◽  
Underlying Mechanisms ◽  
The Brain

Abstract Purpose Heparan sulfate (HS) is one of the factors that has been suggested to be associated with angiogenesis and invasion of glioblastoma (GBM), an aggressive and fast-growing brain tumor. However, it remains unclear how HS of endothelial cells is involved in angiogenesis in glioblastoma and its prognosis. Thus, we investigated the effect of endothelial cell HS on GBM development. Methods We generated endothelial cell-specific knockout of Ext1, a gene encoding a glycosyltransferase and essential for HS synthesis, and murine GL261 glioblastoma cells were orthotopically transplanted. Two weeks after transplantation, we examined the tumor progression and underlying mechanisms. Results The endothelial cell-specific Ext1 knockout (Ext1CKO) mice exhibited reduced HS expression specifically in the vascular endothelium of the brain capillaries compared with the control wild-type (WT) mice. GBM growth was significantly suppressed in Ext1CKO mice compared with that in WT mice. After GBM transplantation, the survival rate was significantly higher in Ext1CKO mice than in WT mice. We investigated how the effect of fibroblast growth factor 2 (FGF2), which is known as an angiogenesis-promoting factor, differs between Ext1CKO and WT mice by using an in vivo Matrigel assay and demonstrated that endothelial cell-specific HS reduction attenuated the effect of FGF2 on angiogenesis. Conclusions HS reduction in the vascular endothelium of the brain suppressed GBM growth and neovascularization in mice.

scholarly journals Overproduction of SecA Suppresses the Export Defect Caused by a Mutation in the Gene Encoding the Escherichia coli Export Chaperone SecB

1999 ◽  
Vol 181 (10) ◽  
pp. 3010-3017 ◽  
Author(s):  
Heather A. Cook ◽  
Carol A. Kumamoto
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane Proteins ◽  
Protein A ◽  
In Vivo Studies ◽  
Wild Type ◽  
Gene Encoding ◽  
Precursor Proteins ◽  
Additional Support ◽  
Insight Into

ABSTRACT SecB is a cytosolic protein required for rapid and efficient export of particular periplasmic and outer membrane proteins inEscherichia coli. SecB promotes export by stabilizing newly synthesized precursor proteins in a nonnative conformation and by targeting the precursors to the inner membrane. Biochemical studies suggest that SecB facilitates precursor targeting by binding to the SecA protein, a component of the membrane-embedded translocation apparatus. To gain more insight into the functional interaction of SecB and SecA, in vivo, mutations in the secA locus that compensate for the export defect caused by the secBmissense mutation secBL75Q were isolated. Two suppressors were isolated, both of which led to the overproduction of wild-type SecA protein. In vivo studies demonstrated that the SecBL75Q mutant protein releases precursor proteins at a lower rate than does wild-type SecB. Increasing the level of SecA protein in the cell was found to reverse this slow-release defect, indicating that overproduction of SecA stimulates the turnover of SecBL75Q-precursor complexes. These findings lend additional support to the proposed pathway for precursor targeting in which SecB promotes targeting to the translocation apparatus by binding to the SecA protein.

Missense mutations in the gene encoding prothrombin corresponding to Arg596 cause antithrombin resistance and thrombomodulin resistance

2016 ◽  
Vol 116 (12) ◽  
pp. 1022-1031 ◽  
Author(s):  
Yuki Takagi ◽  
Moe Murata ◽  
Toshihiro Kozuka ◽  
Yukiko Nakata ◽  
Ryo Hasebe ◽  
...  
Keyword(s):  
Surface Plasmon Resonance ◽  
Residual Activity ◽  
Base Substitution ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Missense Mutations ◽  
Wild Type ◽  
Gene Encoding ◽  
Single Base

SummaryAntithrombin (AT) and thrombomodulin (TM) play important roles in the process of natural anticoagulation in vivo. Recently, we reported that the prothrombin Yukuhashi mutation (p.Arg596Leu) was associated with AT and TM resistance-related thrombophilia. To assess the AT and TM resistances associated with other missense mutations by single base substitution in the Arg596 codon, we generated recombinant variants (596Gln, 596Trp, 596Gly, and 596Pro) and investigated the effects on AT and TM anticoagulant functions. All variants except 596Pro were secreted in amounts comparable to that of the wild-type but exhibited variable procoagulant activities. After a 30-minute inactivation by AT, the relative residual activity of wild-type thrombin decreased to 15 ± 4.0%, in contrast to values of all variants were maintained at above 80%. The thrombin–AT complex formation, as determined by enzyme-linked immunosorbent assay, was reduced with all tested variants in the presence and absence of heparin. In the presence of soluble TM (sTM), the relative fibrinogen clotting activity of wild-type thrombin decreased to 16 ± 0.12%, whereas that of tested variants was 37%–56%. In a surface plasmon resonance assay, missense Arg596 mutations reduced thrombin–TM affinity to an extent similar to the reduction of fibrinogen clotting inhibition. In the presence of sTM or cultured endothelial-like cells, APC generation was enhanced differently by variant thrombins in a thrombin–TM affinity- dependent manner. These data indicate that prothrombin Arg596 missense mutations lead to AT and TM resistance in the variant thrombins and suggest that prothrombin Arg596 is important for AT- and TM- mediated anticoagulation.

scholarly journals Neisseria meningitidis Polynucleotide Phosphorylase Affects Aggregation, Adhesion, and Virulence

2016 ◽  
Vol 84 (5) ◽  
pp. 1501-1513 ◽  
Author(s):  
Jakob Engman ◽  
Aurel Negrea ◽  
Sara Sigurlásdóttir ◽  
Miriam Geörg ◽  
Jens Eriksson ◽  
...  
Keyword(s):  
Neisseria Meningitidis ◽  
Posttranslational Modifications ◽  
Human Cells ◽  
Bacterial Attachment ◽  
Polynucleotide Phosphorylase ◽  
Gene Encoding ◽  
Content Type ◽  
Type Iv ◽  
Transcriptional Changes

Neisseria meningitidisautoaggregation is an important step during attachment to human cells. Aggregation is mediated by type IV pili and can be modulated by accessory pilus proteins, such as PilX, and posttranslational modifications of the major pilus subunit PilE. The mechanisms underlying the regulation of aggregation remain poorly characterized. Polynucleotide phosphorylase (PNPase) is a 3′–5′ exonuclease that is involved in RNA turnover and the regulation of small RNAs. In this study, we biochemically confirm that NMC0710 is theN. meningitidisPNPase, and we characterize its role inN. meningitidispathogenesis. We show that deletion of the gene encoding PNPase leads to hyperaggregation and increased adhesion to epithelial cells. The aggregation induced was found to be dependent on pili and to be mediated by excessive pilus bundling. PNPase expression was induced following bacterial attachment to human cells. Deletion of PNPase led to global transcriptional changes and the differential regulation of 469 genes. We also demonstrate that PNPase is required for full virulence in anin vivomodel ofN. meningitidisinfection. The present study shows that PNPase negatively affects aggregation, adhesion, and virulence inN. meningitidis.

scholarly journals The conserved carboxy-terminal domain of Saccharomyces cerevisiae TFIID is sufficient to support normal cell growth

1991 ◽  
Vol 11 (10) ◽  
pp. 4809-4821
Author(s):  
D Poon ◽  
S Schroeder ◽  
C K Wang ◽  
T Yamamoto ◽  
M Horikoshi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Growth ◽  
Transcription Initiation ◽  
Mutant Form ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Gene Encoding ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal

We have examined the structure-function relationships of TFIID through in vivo complementation tests. A yeast strain was constructed which lacked the chromosomal copy of SPT15, the gene encoding TFIID, and was therefore dependent on a functional plasmid-borne wild-type copy of this gene for viability. By using the plasmid shuffle technique, the plasmid-borne wild-type TFIID gene was replaced with a family of plasmids containing a series of systematically mutated TFIID genes. These various forms of TFIID were expressed from three different promoter contexts of different strengths, and the ability of each mutant form of TFIID to complement our chromosomal TFIID null allele was assessed. We found that the first 61 amino acid residues of TFIID are totally dispensable for vegetative cell growth, since yeast strains containing this deleted form of TFIID grow at wild-type rates. Amino-terminally deleted TFIID was further shown to be able to function normally in vivo by virtue of its ability both to promote accurate transcription initiation from a large number of different genes and to interact efficiently with the Gal4 protein to activate transcription of GAL1 with essentially wild-type kinetics. Any deletion removing sequences from within the conserved carboxy-terminal region of S. cerevisiae TFIID was lethal. Further, the exact sequence of the conserved carboxy-terminal portion of the molecule is critical for function, since of several heterologous TFIID homologs tested, only the highly related Schizosaccharomyces pombe gene could complement our S. cerevisiae TFIID null mutant. Taken together, these data indicate that all important functional domains of TFIID appear to lie in its carboxy-terminal 179 amino acid residues. The significance of these findings regarding TFIID function are discussed.

NADPH Cytochrome P-450 Oxidoreductase and Susceptibility to Ketoconazole

1998 ◽  
Vol 42 (7) ◽  
pp. 1756-1761 ◽  
Author(s):  
K. Venkateswarlu ◽  
Diane E. Kelly ◽  
Nigel J. Manning ◽  
Steven L. Kelly
Keyword(s):  
Ergosterol Biosynthesis ◽  
Squalene Epoxidase ◽  
Wild Type ◽  
Gene Encoding ◽  
Nadph Cytochrome ◽  
Cytochrome P 450 ◽  
Different Levels ◽  
Donor System

ABSTRACT The phenotype of a strain of Saccharomyces cerevisiaecontaining a disruption of the gene encoding NADPH cytochrome P-450 oxidoreductase (CPR) was quantified biochemically and microbiologically, as were those of various transformants of this strain after expression of native CPR, cytochrome P-45051 (CYP51), and a fusion protein of CYP51-CPR (FUS). Only a 4-fold decrease in ergosterol biosynthesis was observed for the cpr strain, but ketoconazole sensitivity increased 200-fold, indicating hypersensitivity to the alternative electron donor system incpr strains. Both phenotypes could be reversed in transformants expressing the CPR and FUS, indicating the availability of the CPR in FUS as well as the expressed native CPR for monoxygenase-associated reactions. The complementation of function was observed both in vitro and in vivo for the monoxygenases squalene epoxidase, CYP51, and CYP61 in the ergosterol biosynthesis pathway with which CPR is coupled. Overexpression of CYP51 and FUS produced different levels of ketoconazole resistance in wild-type cells, indicating that the availability of CPR may limit the potential of overproduction of CYP51 as a mechanism of resistance to azole antifungal agents.

scholarly journals IRF-8 extinguishes neutrophil production and promotes dendritic cell lineage commitment in both myeloid and lymphoid mouse progenitors

Blood ◽  
2012 ◽  
Vol 119 (9) ◽  
pp. 2003-2012 ◽  
Author(s):  
Amy M. Becker ◽  
Drew G. Michael ◽  
Ansuman T. Satpathy ◽  
Roger Sciammas ◽  
Harinder Singh ◽  
...  
Keyword(s):  
Cell Lineage ◽  
Wild Type ◽  
Multipotent Progenitors ◽  
Dc Potential ◽  
Lymphoid Progenitors ◽  
Cellular Context ◽  
Transcriptional Changes ◽  
Microarray Studies ◽  
Myeloid Progenitors

Abstract While most blood lineages are assumed to mature through a single cellular and developmental route downstream of HSCs, dendritic cells (DCs) can be derived from both myeloid and lymphoid progenitors in vivo. To determine how distinct progenitors can generate similar downstream lineages, we examined the transcriptional changes that accompany loss of in vivo myeloid potential as common myeloid progenitors differentiate into common DC progenitors (CDPs), and as lymphoid-primed multipotent progenitors (LMPPs) differentiate into all lymphoid progenitors (ALPs). Microarray studies revealed that IFN regulatory factor 8 (IRF-8) expression increased during each of these transitions. Competitive reconstitutions using Irf8−/− BM demonstrated cell-intrinsic defects in the formation of CDPs and all splenic DC subsets. Irf8−/− common myeloid progenitors and, unexpectedly, Irf8−/− ALPs produced more neutrophils in vivo than their wild-type counterparts at the expense of DCs. Retroviral expression of IRF-8 in multiple progenitors led to reduced neutrophil production and increased numbers of DCs, even in the granulocyte-macrophage progenitor (GMP), which does not normally possess conventional DC potential. These data suggest that IRF-8 represses a neutrophil module of development and promotes convergent DC development from multiple lymphoid and myeloid progenitors autonomously of cellular context.

scholarly journals Identification and Characterization of the Bacillus thuringiensis phaZ Gene, Encoding New Intracellular Poly-3-Hydroxybutyrate Depolymerase

2006 ◽  
Vol 188 (21) ◽  
pp. 7592-7599 ◽  
Author(s):  
Chi-Ling Tseng ◽  
Hui-Ju Chen ◽  
Gwo-Chyuan Shaw
Keyword(s):  
Bacillus Thuringiensis ◽  
Wild Type ◽  
Gene Encoding ◽  
Significant Similarity ◽  
Phb Depolymerase ◽  
New Type ◽  
Identification And Characterization

ABSTRACTA gene that codes for a novel intracellular poly-3-hydroxybutyrate (PHB) depolymerase has now been identified in the genome ofBacillus thuringiensissubsp.israelensisATCC 35646. This gene, previously annotated as a hypothetical 3-oxoadipate enol-lactonase (PcaD) gene and now designatedphaZ, encodes a protein that shows no significant similarity with any known PHB depolymerase. Purified His-tagged PhaZ could efficiently degrade trypsin-activated native PHB granules as well as artificial amorphous PHB granules and release 3-hydroxybutyrate monomer as a hydrolytic product, but it could not hydrolyze denatured semicrystalline PHB. In contrast, purified His-tagged PcaD ofPseudomonas putidawas unable to degrade trypsin-activated native PHB granules and artificial amorphous PHB granules. TheB. thuringiensisPhaZ was inactive againstp-nitrophenylpalmitate, tributyrin, and triolein. Sonication supernatants of the wild-typeB. thuringiensiscells exhibited a PHB-hydrolyzing activity in vitro, whereas those prepared from aphaZmutant lost this activity. ThephaZmutant showed a higher PHB content than the wild type at late stationary phase of growth in a nutrient-rich medium, indicating that this PhaZ can function as a PHB depolymerase in vivo. PhaZ contains a lipase box-like sequence (G-W-S102-M-G) but lacks a signal peptide. A purified His-tagged S102A variant had lost the PHB-hydrolyzing activity. Taken together, these results indicate thatB. thuringiensisharbors a new type of intracellular PHB depolymerase.

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