scholarly journals Identification of the Secretion and Translocation Domain of the Enteropathogenic and Enterohemorrhagic Escherichia coli Effector Cif, Using TEM-1 β-Lactamase as a New Fluorescence-Based Reporter

2004 ◽  
Vol 186 (16) ◽  
pp. 5486-5495 ◽  
Author(s):  
Xavier Charpentier ◽  
Eric Oswald
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Host Cell ◽  
Effector Proteins ◽  
Host Cells ◽  
Reporter System ◽  
Type Iii ◽  
Cycle Arrest ◽  
Lambdoid Prophage

ABSTRACT Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) strains are human and animal pathogens that inject effector proteins into host cells via a type III secretion system (TTSS). Cif is an effector protein which induces host cell cycle arrest and reorganization of the actin cytoskeleton. Cif is encoded by a lambdoid prophage present in most of the EPEC and EHEC strains. In this study, we analyzed the domain that targets Cif to the TTSS by using a new reporter system based on a translational fusion of the effector proteins with mature TEM-1 β-lactamase. Translocation was detected directly in living host cells by using the fluorescent β-lactamase substrate CCF2/AM. We show that the first 16 amino acids (aa) of Cif were necessary and sufficient to mediate translocation into the host cells. Similarly, the first 20 aa of the effector proteins Map, EspF, and Tir, which are encoded in the same region as the TTSS, mediated secretion and translocation in a type III-dependent but chaperone-independent manner. A truncated form of Cif lacking its first 20 aa was no longer secreted and translocated, but fusion with the first 20 aa of Tir, Map, or EspF restored both secretion and translocation. In addition, the chimeric proteins were fully able to trigger host cell cycle arrest and stress fiber formation. In conclusion, our results demonstrate that Cif is composed of a C-terminal effector domain and an exchangeable N-terminal translocation signal and that the TEM-1 reporter system is a convenient tool for the study of the translocation of toxins or effector proteins into host cells.

scholarly journals Distribution, Functional Expression, and Genetic Organization of Cif, a Phage-Encoded Type III-Secreted Effector from Enteropathogenic and Enterohemorrhagic Escherichia coli

2007 ◽  
Vol 190 (1) ◽  
pp. 275-285 ◽  
Author(s):  
Estelle Loukiadis ◽  
Rika Nobe ◽  
Sylvia Herold ◽  
Clara Tramuta ◽  
Yoshitoshi Ogura ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Functional Expression ◽  
Effector Proteins ◽  
Enterohemorrhagic Escherichia Coli ◽  
Host Cells ◽  
E Coli ◽  
Type Iii ◽  
Related Phenotype ◽  
Enterocyte Effacement ◽  
Lambdoid Prophage

ABSTRACT Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) inject effector proteins into host cells via a type III secretion system encoded by the locus of enterocyte effacement (LEE). One of these effectors is Cif, encoded outside the LEE by a lambdoid prophage. In this study, we demonstrated that the Cif-encoding prophage of EPEC strain E22 is inducible and produces infectious phage particles. We investigated the distribution and functional expression of Cif in 5,049 E. coli strains of human, animal, and environmental origins. A total of 115 E. coli isolates from diverse origins and geographic locations carried cif. The presence of cif was tightly associated with the LEE, since all the cif-positive isolates were positive for the LEE. These results suggested that the Cif-encoding prophages have been widely disseminated within the natural population of E. coli but positively selected within the population of LEE-positive strains. Nonetheless, 66% of cif-positive E. coli strains did not induce a typical Cif-related phenotype in eukaryotic cells due to frameshift mutations or insertion of an IS element in the cif gene. The passenger region of the prophages carrying cif was highly variable and showed various combinations of IS elements and genes coding for other effectors such as nleB, nleC, nleH, nleG, espJ, and nleA/espI (some of which were also truncated). This diversity and the presence of nonfunctional effectors should be taken into account to assess EPEC and EHEC pathogenicity and tropism.

scholarly journals Eimeria bovis infections induce G1 cell cycle arrest and a senescence-like phenotype in endothelial host cells

Parasitology ◽  
2020 ◽  
pp. 1-13
Author(s):  
Zahady D. Velásquez ◽  
Sara López-Osorio ◽  
Daniel Waiger ◽  
Carolina Manosalva ◽  
Learta Pervizaj-Oruqaj ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Endothelial Cells ◽  
Cell Cycle Arrest ◽  
Host Cell ◽  
Cyclin B1 ◽  
Host Cells ◽  
Phenotypic Change ◽  
Cyclin E1 ◽  
Cycle Arrest ◽  
Eimeria Bovis

Abstract Apicomplexan parasites are well-known to modulate their host cells at diverse functional levels. As such, apicomplexan-induced alteration of host cellular cell cycle was described and appeared dependent on both, parasite species and host cell type. As a striking evidence of species-specific reactions, we here show that Eimeria bovis drives primary bovine umbilical vein endothelial cells (BUVECs) into a senescence-like phenotype during merogony I. In line with senescence characteristics, E. bovis induces a phenotypic change in host cell nuclei being characterized by nucleolar fusion and heterochromatin-enriched peripheries. By fibrillarin staining we confirm nucleoli sizes to be increased and their number per nucleus to be reduced in E. bovis-infected BUVECs. Additionally, nuclei of E. bovis-infected BUVECs showed enhanced signals for HH3K9me2 as heterochromatin marker thereby indicating an infection-induced change in heterochromatin transition. Furthermore, E. bovis-infected BUVECs show an enhanced β-galactosidase activity, which is a well-known marker of senescence. Referring to cell cycle progression, protein abundance profiles in E. bovis-infected endothelial cells revealed an up-regulation of cyclin E1 thereby indicating a cell cycle arrest at G1/S transition, signifying a senescence key feature. Similarly, abundance of G2 phase-specific cyclin B1 was found to be downregulated at the late phase of macromeront formation. Overall, these data indicate that the slow proliferative intracellular parasite E. bovis drives its host endothelial cells in a senescence-like status. So far, it remains to be elucidated whether this phenomenon indeed reflects an intentionally induced mechanism to profit from host cell-derived energy and metabolites present in a non-dividing cellular status.

scholarly journals Eimeria tenella ROP kinase EtROP1 induces G0/G1 cell cycle arrest and inhibits host cell apoptosis

2019 ◽  
Vol 21 (7) ◽  
Author(s):  
Mamadou Amadou Diallo ◽  
Alix Sausset ◽  
Audrey Gnahoui‐David ◽  
Adeline Ribeiro E Silva ◽  
Aurélien Brionne ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Host Cell ◽  
Cell Apoptosis ◽  
Eimeria Tenella ◽  
Cycle Arrest ◽  
Host Cell Apoptosis ◽  
G1 Cell Cycle Arrest

scholarly journals The Stringent Response and Cell Cycle Arrest in Escherichia coli

PLoS Genetics ◽  
2008 ◽  
Vol 4 (12) ◽  
pp. e1000300 ◽  
Author(s):  
Daniel J. Ferullo ◽  
Susan T. Lovett
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Stringent Response ◽  
Cycle Arrest

scholarly journals Human Immunodeficiency Virus Type 1 Vpr-Dependent Cell Cycle Arrest through a Mitogen-Activated Protein Kinase Signal Transduction Pathway

2005 ◽  
Vol 79 (17) ◽  
pp. 11366-11381 ◽  
Author(s):  
Naoto Yoshizuka ◽  
Yuko Yoshizuka-Chadani ◽  
Vyjayanthi Krishnan ◽  
Steven L. Zeichner
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Human Immunodeficiency Virus ◽  
Cell Cycle Arrest ◽  
Host Cell ◽  
Human Immunodeficiency Virus Type ◽  
Erk Pathway ◽  
Cycle Arrest ◽  
Immunodeficiency Virus

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Vpr protein has important functions in advancing HIV pathogenesis via several effects on the host cell. Vpr mediates nuclear import of the preintegration complex, induces host cell apoptosis, and inhibits cell cycle progression at G2, which increases HIV gene expression. Some of Vpr's activities have been well described, but some functions, such as cell cycle arrest, are not yet completely characterized, although components of the ATR DNA damage repair pathway and the Cdc25C and Cdc2 cell cycle control mechanisms clearly play important roles. We investigated the mechanisms underlying Vpr-mediated cell cycle arrest by examining global cellular gene expression profiles in cell lines that inducibly express wild-type and mutant Vpr proteins. We found that Vpr expression is associated with the down-regulation of genes in the MEK2-ERK pathway and with decreased phosphorylation of the MEK2 effector protein ERK. Exogenous provision of excess MEK2 reverses the cell cycle arrest associated with Vpr, confirming the involvement of the MEK2-ERK pathway in Vpr-mediated cell cycle arrest. Vpr therefore appears to arrest the cell cycle at G2/M through two different mechanisms, the ATR mechanism and a newly described MEK2 mechanism. This redundancy suggests that Vpr-mediated cell cycle arrest is important for HIV replication and pathogenesis. Our findings additionally reinforce the idea that HIV can optimize the host cell environment for viral replication.

scholarly journals Dynamics of the Type III Secretion System Activity of Enteropathogenic Escherichia coli

mBio ◽  
2013 ◽  
Vol 4 (4) ◽  
Author(s):  
Erez Mills ◽  
Kobi Baruch ◽  
Gili Aviv ◽  
Mor Nitzan ◽  
Ilan Rosenshine
Keyword(s):  
Escherichia Coli ◽  
Type Iii Secretion ◽  
Public Health Problem ◽  
Effector Proteins ◽  
Host Cells ◽  
Global Public Health ◽  
Secretion Systems ◽  
Comprehensive Description ◽  
Content Type ◽  
Type Iii

ABSTRACT Type III secretion systems (TTSSs) are employed by pathogens to translocate host cells with effector proteins, which are crucial for virulence. The dynamics of effector translocation, behavior of the translocating bacteria, translocation temporal order, and relative amounts of each of the translocated effectors are all poorly characterized. To address these issues, we developed a microscopy-based assay that tracks effector translocation. We used this assay alongside a previously described real-time population-based translocation assay, focusing mainly on enteropathogenic Escherichia coli (EPEC) and partly comparing it to Salmonella. We found that the two pathogens exhibit different translocation behaviors: in EPEC, a subpopulation that formed microcolonies carried out most of the translocation activity, while Salmonella executed protein translocation as planktonic bacteria. We also noted variability in host cell susceptibility, with some cells highly resistant to translocation. We next extended the study to determine the translocation dynamics of twenty EPEC effectors and found that all exhibited distinct levels of translocation efficiency. Further, we mapped the global effects of key TTSS-related components on TTSS activity. Our results provide a comprehensive description of the dynamics of the TTSS activity of EPEC and new insights into the mechanisms that control the dynamics. IMPORTANCE EPEC and the closely related enterohemorrhagic Escherichia coli (EHEC) represent a global public health problem. New strategies to combat EPEC and EHEC infections are needed, and development of such strategies requires better understanding of their virulence machinery. The TTSS is a critical virulence mechanism employed by these pathogens, and by others, including Salmonella. In this study, we aimed at elucidating new aspects of TTSS function. The results obtained provide a comprehensive description of the dynamics of TTSS activity of EPEC and new insights into the mechanisms that control these changes. This knowledge sets the stage for further analysis of the system and may accelerate the development of new ways to treat EPEC and EHEC infections. Further, the newly described microscopy-based assay can be readily adapted to study the dynamics of TTSS activity in other pathogens.

scholarly journals Multiple RNA virus matrix proteins interact with SLD5 to manipulate host cell cycle

2021 ◽  
Vol 102 (12) ◽  
Author(s):  
Li Zhu ◽  
Xinyu Li ◽  
Henan Xu ◽  
Lifeng Fu ◽  
George Fu Gao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Host Cell ◽  
Cell Cycle Progression ◽  
Matrix Protein ◽  
Rna Viruses ◽  
Rna Virus ◽  
M Protein ◽  
Cycle Progression ◽  
Cycle Arrest

The matrix protein of many enveloped RNA viruses regulates multiple stages of viral life cycle and has the characteristics of nucleocytoplasmic shuttling. We have previously demonstrated that matrix protein 1 (M1) of an RNA virus, influenza virus, blocks host cell cycle progression by interacting with SLD5, a member of the GINS complex, which is required for normal cell cycle progression. In this study, we found that M protein of several other RNA viruses, including VSV, SeV and HIV, interacted with SLD5. Furthermore, VSV/SeV infection and M protein of VSV/SeV/HIV induced cell cycle arrest at G0/G1 phase. Importantly, overexpression of SLD5 partially rescued the cell cycle arrest by VSV/SeV infection and VSV M protein. In addition, SLD5 suppressed VSV replication in vitro and in vivo, and enhanced type Ⅰ interferon signalling. Taken together, our results suggest that targeting SLD5 by M protein might be a common strategy used by multiple enveloped RNA viruses to block host cell cycle. Our findings provide new mechanistic insights for virus to manipulate cell cycle progression by hijacking host replication factor SLD5 during infection.

scholarly journals Apoptosis and Necrosis on T47D Cells Induced by Shiga-Like Toxin from Local Isolates of Escherichia coli O157:H7

2019 ◽  
Author(s):  
I Wayan - Suardana ◽  
I Gusti Ngurah - Sudisma ◽  
Komang Januartha Putra Pinatih ◽  
Dyah Ayu Widiasih
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Cancer Therapy ◽  
Cell Cycle Arrest ◽  
Annexin V ◽  
Local Strain ◽  
T47d Cells ◽  
E Coli ◽  
Cycle Arrest ◽  
Apoptosis And Necrosis

Abstract Background Apoptosis and cell cycle arrest induction are targeted in the strategy of cancer therapy. Furthermore, bacterial toxins such as Shiga-like toxin producing Escherichia coli have been suggested to be used as a novel therapeutic agent against tumor malignancies, either as independent anti-neoplastic agents, or in combination treatment with chemo or radiotherapy. The aim of study was to investigate the potency of Shiga-like toxin originating from local strains of E. coli O157:H7 which was known less toxic than ATCC 43894 as a new cancer therapy. Methods As many as 10 culture cells T47D cell line were subjected by crude extract Shiga-like toxin originating from five local isolates of E. coli O157:H7 with each codes KL-48(2), SM-25(1), SM-7(1), DS-21(4), and one isolate ATCC 43894 as a control with IC50 doses, respectively. The treatment was observed for 24 h, with two replications. An FITC-Annexin V and PI assay was used to observe apoptosis and necrosis effect, and simultaneously with cell cycle analysis using propidium iodide (PI) staining. Results The study shown that T47D cells treated with Shiga-like toxin from local strain KL-48 (2) show the lowest viable cell, followed by SM7(1), ATCC 43894, SM-25(1), DS-21(4) in contrary with the control cells with each percentages at 15.20, 16.36, 22.17, 22.64, 33.86, and 94.36%, respectively. The results were also confirmed by the induction of the cell cycle arrest in phase G0-G1 as inactive phase, i.e. 66.41, 63.37, 61.52, 55.36 and 47.28% for T47D cells treated with toxins of KL-48(2), ATCC 43894, SM 25(1), SM 7(1), and DS 21(4), respectively. Conclusions These results show tendency deleterious effect of Shiga-like toxin from local isolates on T47D cells, so It is concluded that they have potency as a good anticancer drug against Gb3-expressing breast cancer.

scholarly journals Legionella pneumophila translocated translation inhibitors are required for bacterial-induced host cell cycle arrest

2019 ◽  
Vol 116 (8) ◽  
pp. 3221-3228 ◽  
Author(s):  
Asaf Sol ◽  
Erion Lipo ◽  
Dennise A. de Jesús-Díaz ◽  
Connor Murphy ◽  
Mildred Devereux ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Cell Cycle Arrest ◽  
Host Cell ◽  
Legionella Pneumophila ◽  
S Phase ◽  
Phase Cell ◽  
Cycle Arrest ◽  
Bacterial Replication ◽  
Cell Cycle Machinery

The cell cycle machinery controls diverse cellular pathways and is tightly regulated. Misregulation of cell division plays a central role in the pathogenesis of many disease processes. Various microbial pathogens interfere with the cell cycle machinery to promote host cell colonization. Although cell cycle modulation is a common theme among pathogens, the role this interference plays in promoting diseases is unclear. Previously, we demonstrated that the G1 and G2/M phases of the host cell cycle are permissive for Legionella pneumophila replication, whereas S phase provides a toxic environment for bacterial replication. In this study, we show that L. pneumophila avoids host S phase by blocking host DNA synthesis and preventing cell cycle progression into S phase. Cell cycle arrest upon Legionella contact is dependent on the Icm/Dot secretion system. In particular, we found that cell cycle arrest is dependent on the intact enzymatic activity of translocated substrates that inhibits host translation. Moreover, we show that, early in infection, the presence of these translation inhibitors is crucial to induce the degradation of the master regulator cyclin D1. Our results demonstrate that the bacterial effectors that inhibit translation are associated with preventing entry of host cells into a phase associated with restriction of L. pneumophila. Furthermore, control of cyclin D1 may be a common strategy used by intracellular pathogens to manipulate the host cell cycle and promote bacterial replication.

scholarly journals The First 45 Amino Acids of SopA Are Necessary for InvB Binding and SPI-1 Secretion

2006 ◽  
Vol 188 (7) ◽  
pp. 2411-2420 ◽  
Author(s):  
Wendy Higashide ◽  
Daoguo Zhou
Keyword(s):  
Type Iii Secretion ◽  
Catalytic Domain ◽  
Effector Proteins ◽  
Host Cells ◽  
Amino Acid Residues ◽  
Secretion Systems ◽  
Reporter System ◽  
Type Iii ◽  
Serovar Typhimurium ◽  
Type Iii Protein Secretion

ABSTRACT Salmonella enterica serovar Typhimurium encodes two type III secretion systems (TTSSs) within pathogenicity island 1 (SPI-1) and island 2 (SPI-2). These type III protein secretion and translocation systems transport a panel of bacterial effector proteins across both the bacterial and the host cell membranes to promote bacterial entry and subsequent survival inside host cells. Effector proteins contain secretion and translocation signals that are often located at their N termini. We have developed a ruffling-based translocation reporter system that uses the secretion- and translocation-deficient catalytic domain of SopE, SopE78-240, as a reporter. Using this assay, we determined that the N-terminal 45 amino acid residues of Salmonella SopA are necessary and sufficient for directing its secretion and translocation through the SPI-1 TTSS. SopA1-45, but not SopA1-44, is also able to bind to its chaperone, InvB, indicating that SPI-1 type III secretion and translocation of SopA require its chaperone.

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