scholarly journals Human Granulocytic Ehrlichiosis Agent and Ehrlichia chaffeensis Reside in Different Cytoplasmic Compartments in HL-60 Cells

1999 ◽  
Vol 67 (3) ◽  
pp. 1368-1378 ◽  
Author(s):  
Jason Mott ◽  
Roy E. Barnewall ◽  
Yasuko Rikihisa
Keyword(s):  
Membrane Protein ◽  
Transferrin Receptor ◽  
Brefeldin A ◽  
Ehrlichia Chaffeensis ◽  
Inclusion Type ◽  
Granulocytic Ehrlichiosis ◽  
Histocompatibility Complex ◽  
Human Granulocytic Ehrlichiosis ◽  
Late Endosomes ◽  
Immunofluorescence Labeling

ABSTRACT The human granulocytic ehrlichiosis (HGE) agent resides and multiplies exclusively in cytoplasmic vacuoles of granulocytes. Double immunofluorescence labeling was used to characterize the nature of the HGE agent replicative inclusions and to compare them with inclusions containing the human monocytic ehrlichia, Ehrlichia chaffeensis, in HL-60 cells. Although both Ehrlichiaspp. can coinfect HL-60 cells, they resided in separate inclusions. Inclusions of both Ehrlichia spp. were not labeled with either anti-lysosome-associated membrane protein 1 or anti-CD63. Accumulation of myeloperoxidase-positive granules were seen around HGE agent inclusions but not around E. chaffeensis inclusions. 3-(2,4-Dinitroanilino)-3′-amino-N-methyldipropylamine and acridine orange were not localized to either inclusion type. Vacuolar-type H+-ATPase was not colocalized with HGE agent inclusions but was weakly colocalized with E. chaffeensisinclusions. E. chaffeensis inclusions were labeled with the transferrin receptor, early endosomal antigen 1, and rab5, but HGE agent inclusions were not. Some HGE agent and E. chaffeensis inclusions colocalized with major histocompatibility complex class I and II antigens. These two inclusions were not labeled for annexins I, II, IV, and VI; α-adaptin; clathrin heavy chain; or β-coatomer protein. Vesicle-associated membrane protein 2 colocalized to both inclusions. The cation-independent mannose 6-phosphate receptor was not colocalized with either inclusion type. Endogenously synthesized sphingomyelin, from C6-NBD-ceramide, was not incorporated into either inclusion type. Brefeldin A did not affect the growth of either Ehrlichia sp. in HL-60 cells. These results suggest that the HGE agent resides in inclusions which are neither early nor late endosomes and does not fuse with lysosomes or Golgi-derived vesicles, while E. chaffeensis resides in an early endosomal compartment which accumulates the transferrin receptor.

scholarly journals Ehrlichia chaffeensis and E. sennetsu, but Not the Human Granulocytic Ehrlichiosis Agent, Colocalize with Transferrin Receptor and Up-Regulate Transferrin Receptor mRNA by Activating Iron-Responsive Protein 1

1999 ◽  
Vol 67 (5) ◽  
pp. 2258-2265 ◽  
Author(s):  
Roy E. Barnewall ◽  
Norio Ohashi ◽  
Yasuko Rikihisa
Keyword(s):  
Cell Line ◽  
Transferrin Receptor ◽  
Leukemia Cell ◽  
Binding Activity ◽  
Leukemia Cell Line ◽  
Acute Monocytic Leukemia ◽  
Ehrlichia Chaffeensis ◽  
Mrna Levels ◽  
Granulocytic Ehrlichiosis ◽  
Human Granulocytic Ehrlichiosis

ABSTRACT Ehrlichia chaffeensis and E. sennetsu are genetically divergent obligatory intracellular bacteria of human monocytes and macrophages, and the human granulocytic ehrlichiosis (HGE) agent is an obligatory intracellular bacterium of granulocytes. Infection with both E. chaffeensis and E. sennetsu, but not HGE agent, in the acute monocytic leukemia cell line THP-1 almost completely inhibited by treatment with deferoxamine, a cell-permeable iron chelator. Transferrin receptors (TfRs) accumulated on both E. chaffeensis and E. sennetsu, but not HGE agent, inclusions in THP-1 cells or the cells of the promyelocytic leukemia cell line HL-60. Reverse transcription-PCR showed an increase in the level of TfR mRNA 6 h postinfection which peaked at 24 h postinfection with bothE. chaffeensis and E. sennetsu infection in THP-1 or HL-60 cells. In contrast, HGE agent in THP-1 or HL-60 cells induced no increase in TfR mRNA levels. Heat treatment of E. chaffeensis or the addition of monodansylcadaverine, a transglutaminase inhibitor, 3 h prior to infection inhibited the up-regulation of TfR mRNA. The addition of oxytetracycline 6 h after E. chaffeensis infection caused a decrease in TfR mRNA which returned to the basal level by 24 h postinfection. These results indicate that both internalization and continuous proliferation of ehrlichial organisms or the production of ehrlichial proteins are required for the up-regulation of TfR mRNA. Results of electrophoretic mobility shift assays showed that both E. chaffeensis and E. sennetsu infection increased the binding activity of iron-responsive protein 1 (IRP-1) to the iron-responsive element at 6 h postinfection and remained elevated at 24 h postinfection. However, HGE agent infection had no effect on IRP-1 binding activity. This result suggests that activation of IRP-1 and subsequent stabilization of TfR mRNA comprise the mechanism of TfR mRNA up-regulation by E. chaffeensis and E. sennetsu infection.

scholarly journals Cloning and Expression of the 44-Kilodalton Major Outer Membrane Protein Gene of the Human Granulocytic Ehrlichiosis Agent and Application of the Recombinant Protein to Serodiagnosis

1998 ◽  
Vol 36 (6) ◽  
pp. 1666-1673 ◽  
Author(s):  
N. Zhi ◽  
N. Ohashi ◽  
Y. Rikihisa ◽  
H. W. Horowitz ◽  
G. P. Wormser ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Immunoblot Analysis ◽  
Major Outer Membrane Protein ◽  
Cloning And Expression ◽  
Western Immunoblot ◽  
Granulocytic Ehrlichiosis ◽  
Human Granulocytic Ehrlichiosis ◽  
Western Immunoblot Analysis

A 44-kDa major outer membrane protein of the human granulocytic ehrlichiosis (HGE) agent is an immunodominant antigen in human infection. A gene encoding this protein was cloned and sequenced. Southern blot results revealed the existence of multigenes homologous to the P44 gene in the genome of the HGE agent. The recombinant 44-kDa protein (rP44) was expressed by using expression vector pET30a. The reactivity of the affinity-purified rP44 was evaluated by Western immunoblot analysis and dot blot immunoassay. Western immunoblot analysis showed that mouse anti-rP44 serum reacted with 44- to 42-kDa proteins in six different HGE agent strains tested except strain 2, in which three proteins of 42, 40, and 38 kDa were recognized. Eleven HGE patient serum samples, a horse anti-HGE serum, and a horse anti-Ehrlichia equi serum recognized the rP44 protein. This suggests that rP44 is an HGE-E. equi group-specific antigen. Neither human anti-Ehrlichia chaffeensis serum nor rabbit anti-Borrelia burgdorferi serum reacted with rP44. Sera from two patients coinfected with the HGE agent and B. burgdorferi reacted positively with rP44 and the HGE agent. Sera from 20 HGE patients with indirect fluorescent-antibody (IFA) titers ranging from 1:20 to 1:2,560 gave distinct positive reactions in a dot immunoblot assay. There was a positive correlation between the color densities of the dot reactions and the IFA titers when greater than 50 ng of recombinant antigen per dot was used. The use of the affinity-purified rP44 protein as antigen would provide a more specific, consistent, and simpler serodiagnosis for HGE than the use of whole infected cells or purified HGE agents.

scholarly journals Effects of Anaplasma phagocytophila on NADPH Oxidase Components in Human Neutrophils and HL-60 Cells

2002 ◽  
Vol 70 (3) ◽  
pp. 1359-1366 ◽  
Author(s):  
Jason Mott ◽  
Yasuko Rikihisa ◽  
Shohko Tsunawaki
Keyword(s):  
Nadph Oxidase ◽  
Human Peripheral Blood ◽  
Human Neutrophils ◽  
Double Immunofluorescence ◽  
Granulocytic Ehrlichiosis ◽  
Protein Levels ◽  
Cytoplasmic Vacuoles ◽  
Human Granulocytic Ehrlichiosis ◽  
Blood Neutrophils ◽  
Immunofluorescence Labeling

ABSTRACT The human granulocytic ehrlichiosis agent, Anaplasma phagocytophila, resides and multiplies exclusively in cytoplasmic vacuoles of granulocytes. A. phagocytophila rapidly inhibits the superoxide anion (O2 −) generation by human neutrophils in response to various stimuli. To determine the inhibitory mechanism, the influence of A. phagocytophila on protein levels and localization of components of the NADPH oxidase were examined. A. phagocytophila decreased levels of p22 phox , but not gp91 phox , p47 phox , p67 phox , or P40 phox reactive with each component-specific antibody in human peripheral blood neutrophils and HL-60 cells. Double immunofluorescence labeling revealed that p47 phox , p67 phox , Rac2, and p22 phox did not colocalize with A. phagocytophila inclusions in neutrophils or HL-60 cells, and p22 phox levels were also reduced. A. phagocytophila did not prevent either membrane translocation of cytoplasmic p47 phox and p67 phox or phosphorylation of p47 phox upon stimulation by phorbol myristate acetate. The inhibitory signals for O2 − generation was independent of several signals required for A. phagocytophila internalization. These results suggest that rapid alteration in p22 phox induced by binding of A. phagocytophila to neutrophils is involved in the inhibition of O2 − generation. Absence of colocalization of NADPH oxidase components with the inclusion further protects A. phagocytophila from oxidative damage.

scholarly journals Vamp-7 Mediates Vesicular Transport from Endosomes to Lysosomes

1999 ◽  
Vol 146 (4) ◽  
pp. 765-776 ◽  
Author(s):  
Raj J. Advani ◽  
Bin Yang ◽  
Rytis Prekeris ◽  
Kelly C. Lee ◽  
Judith Klumperman ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Membrane Trafficking ◽  
Polyclonal Antibodies ◽  
Vesicular Transport ◽  
Brefeldin A ◽  
Immunohistochemical Analysis ◽  
Endocytic Pathway ◽  
Permeabilized Cells ◽  
Transport Vesicles ◽  
Late Endosomes

A more complete picture of the molecules that are critical for the organization of membrane compartments is beginning to emerge through the characterization of proteins in the vesicle-associated membrane protein (also called synaptobrevin) family of membrane trafficking proteins. To better understand the mechanisms of membrane trafficking within the endocytic pathway, we generated a series of monoclonal and polyclonal antibodies against the cytoplasmic domain of vesicle-associated membrane protein 7 (VAMP-7). The antibodies recognize a 25-kD membrane-associated protein in multiple tissues and cell lines. Immunohistochemical analysis reveals colocalization with a marker of late endosomes and lysosomes, lysosome-associated membrane protein 1 (LAMP-1), but not with other membrane markers, including p115 and transferrin receptor. Treatment with nocodozole or brefeldin A does not disrupt the colocalization of VAMP-7 and LAMP-1. Immunoelectron microscopy analysis shows that VAMP-7 is most concentrated in the trans-Golgi network region of the cell as well as late endosomes and transport vesicles that do not contain the mannose-6 phosphate receptor. In streptolysin- O–permeabilized cells, antibodies against VAMP-7 inhibit the breakdown of epidermal growth factor but not the recycling of transferrin. These data are consistent with a role for VAMP-7 in the vesicular transport of proteins from the early endosome to the lysosome.

scholarly journals Human ehrlichiosis

2006 ◽  
Vol 63 (4) ◽  
pp. 403-408 ◽  
Author(s):  
Milomir Djokic ◽  
Petar Curcic ◽  
Darko Nozic ◽  
Branislav Lako ◽  
Vesna Begovic ◽  
...  
Keyword(s):  
Bacterial Species ◽  
The United States ◽  
Amblyomma Americanum ◽  
Ehrlichia Chaffeensis ◽  
Granulocytic Ehrlichiosis ◽  
Human Granulocytic Ehrlichiosis ◽  
General Weakness ◽  
Infected Cells ◽  
Human Monocytic Ehrlichiosis ◽  
Human Ehrlichiosis

Background. Human ehrlichiosis is a newly recognized disease. It is a tick-borne disease caused by several bacterial species of the genhus Erlichia. These are small gram-negative pleomorphic cocci, that are obligatory intracellular bacteria. Tick Ixodes is the principle vector in Europe, and Amblyomma americanum in the United States. Bacterial organisms replicate in a tick, and are transmited from infected cells in a vector to the blood cells of animals or humans. Human ehrlichiosis is a name for a group of diseases caused by different species of Ehrlichia. One of them is the disease named human monocytic ehrlichiosis, caused by Ehrlichia chaffeensis, and the other is a human granulocytic ehrlichiosis caused by Anaplasma phagocytophilia. Case report. We reported a 23-year-old patient admitted for the clinical treatment with the symptoms of high febrility (above 40 ?C), headache, vomiting, general weakness and exhaustion, but without data on a tick bite. The patient was treated with trimetoprim-sulfamethoxazole for a week when Ehrlichia chaffeensis was confirmed by the immunofluoroscence test, and the therapy contimed with doxacyclin. Conclusion. Human ehrlichiosis is also present in our country, so this disease should be considered everyday, especially in infectology practice.

Incidence and Prevalence of Infection with Human Granulocytic Ehrlichiosis Agent in Germany. A Prospective Study in Young Healthy Subjects

Infection ◽  
2001 ◽  
Vol 29 (5) ◽  
pp. 271-273 ◽  
Author(s):  
R. Woessner ◽  
B.C. Gaertner ◽  
M.T. Grauer ◽  
K. Weber ◽  
N. Mueller-Lantzsch ◽  
...  
Keyword(s):  
Prospective Study ◽  
Healthy Subjects ◽  
Granulocytic Ehrlichiosis ◽  
Human Granulocytic Ehrlichiosis ◽  
A Prospective Study

scholarly journals Relationship between invariant chain expression and major histocompatibility complex class II transport into early and late endocytic compartments.

1993 ◽  
Vol 177 (3) ◽  
pp. 583-596 ◽  
Author(s):  
P Romagnoli ◽  
C Layet ◽  
J Yewdell ◽  
O Bakke ◽  
R N Germain
Keyword(s):  
Major Histocompatibility Complex ◽  
Mhc Class Ii ◽  
Class Ii ◽  
Endocytic Pathway ◽  
Invariant Chain ◽  
Major Histocompatibility ◽  
Histocompatibility Complex ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Endocytic Compartments

Invariant chain (Ii), which associates with major histocompatibility complex (MHC) class II molecules in the endoplasmic reticulum, contains a targeting signal for transport to intracellular vesicles in the endocytic pathway. The characteristics of the target vesicles and the relationship between Ii structure and class II localization in distinct endosomal subcompartments have not been well defined. We demonstrate here that in transiently transfected COS cells expressing high levels of the p31 or p41 forms of Ii, uncleaved Ii is transported to and accumulates in transferrin-accessible (early) endosomes. Coexpressed MHC class II is also found in this same compartment. These early endosomes show altered morphology and a slower rate of content movement to later parts of the endocytic pathway. At more moderate levels of Ii expression, or after removal of a highly conserved region in the cytoplasmic tail of Ii, coexpressed class II molecules are found primarily in vesicles with the characteristics of late endosomes/prelysosomes. The Ii chains in these late endocytic vesicles have undergone proteolytic cleavage in the lumenal region postulated to control MHC class II peptide binding. These data indicate that the association of class II with Ii results in initial movement to early endosomes. At high levels of Ii expression, egress to later endocytic compartments is delayed and class II-Ii complexes accumulate together with endocytosed material. At lower levels of Ii expression, class II-Ii complexes are found primarily in late endosomes/prelysosomes. These data provide evidence that the route of class II transport to the site of antigen processing and loading involves movement through early endosomes to late endosomes/prelysosomes. Our results also reveal an unexpected ability of intact Ii to modify the structure and function of the early endosomal compartment, which may play a role in regulating this processing pathway.

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