scholarly journals Expression of the EspB Protein of EnteropathogenicEscherichia coli within HeLa Cells Affects Stress Fibers and Cellular Morphology

1999 ◽  
Vol 67 (1) ◽  
pp. 120-125 ◽  
Author(s):  
Kathleen A. Taylor ◽  
Paul W. Luther ◽  
Michael S. Donnenberg
Keyword(s):  
Host Cell ◽  
Hela Cells ◽  
Type Iii Secretion ◽  
Cell Clone ◽  
Host Cells ◽  
Stress Fibers ◽  
Cell Cytoplasm ◽  
Eukaryotic Promoter ◽  
Host Cell Cytoplasm ◽  
Attached Bacteria

ABSTRACT The EspB protein of enteropathogenic Escherichia coli(EPEC) is essential for the signaling events that lead to the accumulation of actin beneath intimately attached bacteria, a process that is known as the attaching and effacing effect. EspB is targeted to the host cell cytoplasm by a type III secretion apparatus. To determine the effect of intracellular EspB on the host cell cytoskeleton, we transfected HeLa cells with a plasmid containing the espBgene under the control of an inducible eukaryotic promoter. A HeLa cell clone that expressed espB mRNA and EspB protein after induction was selected for further study. The expression of EspB in these cells caused a dramatic change in cell morphology and a marked reduction in actin stress fibers. Cells expressing EspB were significantly impaired in their ability to support invasion by EPEC andSalmonella typhimurium. However, the expression of EspB within host cells could not compensate for the lack of EspB expression by an espB mutant strain of EPEC to restore attaching and effacing activity. These studies suggest that EspB is a cytoskeletal toxin that is translocated to the host cell cytoplasm, where it causes a redistribution of actin.

scholarly journals The EspB Protein of EnteropathogenicEscherichia coli Is Targeted to the Cytoplasm of Infected HeLa Cells

1998 ◽  
Vol 66 (11) ◽  
pp. 5501-5507 ◽  
Author(s):  
Kathleen A. Taylor ◽  
Colin B. O’Connell ◽  
Paul W. Luther ◽  
Michael S. Donnenberg
Keyword(s):  
Host Cell ◽  
Hela Cells ◽  
Type Iii Secretion ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Cell Cytoplasm ◽  
Type Iii ◽  
Host Cell Cytoplasm ◽  
Infected Cells

ABSTRACT The EspB protein of enteropathogenic Escherichia coli(EPEC) is exported via a type III secretion apparatus. EspB is critical for signaling the host cell and for the development of the attaching and effacing lesion characteristic of EPEC infection. We used cellular fractionation and confocal laser scanning microscopy to determine the cellular location of EspB during infection of HeLa cells. Both methods indicated that EspB is targeted to the cytoplasm of infected cells. Using mutants, we found that EspB targeting to the host cell cytoplasm requires the type III secretion apparatus and the secreted proteins EspA and EspD, but not intimin. These results provide insights into the function of the type III secretion apparatus of EPEC and the functions of the Esp proteins.

scholarly journals Eukaryotic Cell Permeabilisation to Identify New Putative Chlamydial Type III Secretion System Effectors Secreted within Host Cell Cytoplasm

2020 ◽  
Vol 8 (3) ◽  
pp. 361 ◽  
Author(s):  
Carole Kebbi-Beghdadi ◽  
Ludovic Pilloux ◽  
Virginie Martin ◽  
Gilbert Greub
Keyword(s):  
Host Cell ◽  
Type Iii Secretion ◽  
Plasma Membranes ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Cell Cytoplasm ◽  
Type Iii ◽  
Host Cell Cytoplasm ◽  
Adverse Pregnancy

Chlamydia trachomatis and Waddlia chondrophila are strict intracellular bacteria belonging to the Chlamydiales order. C. trachomatis is the most frequent bacterial cause of genital and ocular infections whereas W. chondrophila is an opportunistic pathogen associated with adverse pregnancy outcomes and respiratory infections. Being strictly intracellular, these bacteria are engaged in a complex interplay with their hosts to modulate their environment and create optimal conditions for completing their life cycle. For this purpose, they possess several secretion pathways and, in particular, a Type III Secretion System (T3SS) devoted to the delivery of effector proteins in the host cell cytosol. Identifying these effectors is a crucial step in understanding the molecular basis of bacterial pathogenesis. Following incubation of infected cells with perfringolysin O, a pore-forming toxin that binds cholesterol present in plasma membranes, we analysed by mass spectrometry the protein content of the host cell cytoplasm. We identified 13 putative effectors secreted by C. trachomatis and 19 secreted by W. chondrophila. Using Y. enterocolitica as a heterologous expression and secretion system, we confirmed that four of these identified proteins are secreted by the T3SS. Two W. chondrophila T3SS effectors (hypothetical proteins Wcw_0499 and Wcw_1706) were further characterised and demonstrated to be early/mid-cycle effectors. In addition, Wcw_1706 is associated with a tetratricopeptide domain-containing protein homologous to C. trachomatis class II chaperone. Furthermore, we identified a novel C. trachomatis effector, CT460 that localises in the eukaryotic nucleus when ectopically expressed in 293 T cells.

Toxoplasma gondii reorganizes the host cell architecture during spontaneous cyst formation in vitro

Parasitology ◽  
2017 ◽  
Vol 145 (8) ◽  
pp. 1027-1038 ◽  
Author(s):  
T. C. Paredes-Santos ◽  
E. S. Martins-Duarte ◽  
W. de Souza ◽  
M. Attias ◽  
R. C. Vommaro
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Cyst Wall ◽  
Cyst Formation ◽  
Acute Stage ◽  
Host Cells ◽  
Cell Cytoplasm ◽  
Chronic Stage ◽  
Host Cell Cytoplasm

AbstractToxoplasma gondii is an intracellular protozoan parasite that causes toxoplasmosis, a prevalent infection related to abortion, ocular diseases and encephalitis in immuno-compromised individuals. In the untreatable (and life-long) chronic stage of toxoplasmosis, parasitophorous vacuoles (PVs, containing T. gondii tachyzoites) transform into tissue cysts, containing slow-dividing bradyzoite forms. While acute-stage infection with tachyzoites involves global rearrangement of the host cell cytoplasm, focused on favouring tachyzoite replication, the cytoplasmic architecture of cells infected with cysts had not been described. Here, we characterized (by fluorescence and electron microscopy) the redistribution of host cell structures around T. gondii cysts, using a T. gondii strain (EGS) with high rates of spontaneous cystogenesis in vitro. Microtubules and intermediate filaments (but not actin microfilaments) formed a ‘cage’ around the cyst, and treatment with taxol (to inhibit microtubule dynamics) favoured cystogenesis. Mitochondria, which appeared adhered to the PV membrane, were less closely associated with the cyst wall. Endoplasmic reticulum (ER) profiles were intimately associated with folds in the cyst wall membrane. However, the Golgi complex was not preferentially localized relative to the cyst, and treatment with tunicamycin or brefeldin A (to disrupt Golgi or ER function, respectively) had no significant effect on cystogenesis. Lysosomes accumulated around cysts, while early and late endosomes were more evenly distributed in the cytoplasm. The endocytosis tracer HRP (but not BSA or transferrin) reached bradyzoites after uptake by infected host cells. These results suggest that T. gondii cysts reorganize the host cell cytoplasm, which may fulfil specific requirements of the chronic stage of infection.

scholarly journals The Hydrophilic Translocator for Vibrio parahaemolyticus, T3SS2, Is Also Translocated

2012 ◽  
Vol 80 (8) ◽  
pp. 2940-2947 ◽  
Author(s):  
Xiaohui Zhou ◽  
Jennifer M. Ritchie ◽  
Hirotaka Hiyoshi ◽  
Tetsuya Iida ◽  
Brigid M. Davis ◽  
...  
Keyword(s):  
Host Cell ◽  
Vibrio Parahaemolyticus ◽  
Diarrheal Disease ◽  
Host Cells ◽  
Cell Cytoplasm ◽  
Host Cell Membrane ◽  
Membrane Pore ◽  
Content Type ◽  
Bacterial Proteins ◽  
Host Cell Cytoplasm

ABSTRACTThe pathogenesis of the diarrheal disease caused byVibrio parahaemolyticus, a leading cause of seafood-associated enteritis worldwide, is dependent upon a type III secretion system, T3SS2. This apparatus enables the pathogen to inject bacterial proteins (effectors) into the cytosol of host cells and thereby modulate host processes. T3SS effector proteins transit into the host cell via a membrane pore (translocon) typically formed by 3 bacterial proteins. We have identified the third translocon protein for T3SS2: VopW, which was previously classified as an effector protein for a homologous T3SS inV. cholerae. VopW is a hydrophilic translocon protein; like other such proteins, it is not inserted into the host cell membrane but is required for insertion of the two hydrophobic translocators, VopB2 and VopD2, that constitute the membrane channel. VopW is not required for secretion of T3SS2 effectors into the bacterial culture medium; however, it is essential for transfer of these proteins into the host cell cytoplasm. Consequently, deletion ofvopWabrogates the virulence ofV. parahaemolyticusin several animal models of diarrheal disease. Unlike previously described hydrophilic translocators, VopW is itself translocated into the host cell cytoplasm, raising the possibility that it functions as both a translocator and an effector.

scholarly journals Chlamydia trachomatis secretion of hypothetical protein CT622 into host cell cytoplasm via a secretion pathway that can be inhibited by the type III secretion system inhibitor compound 1

Microbiology ◽  
2011 ◽  
Vol 157 (4) ◽  
pp. 1134-1144 ◽  
Author(s):  
Siqi Gong ◽  
Lei Lei ◽  
Xiaotong Chang ◽  
Robert Belland ◽  
Guangming Zhong
Keyword(s):  
Host Cell ◽  
Type Iii Secretion ◽  
Fusion Proteins ◽  
Hypothetical Protein ◽  
Secretion System ◽  
Cell Cytoplasm ◽  
Host Cell Cytoplasm ◽  
Cell Cytosol ◽  
H Protein ◽  
Host Cell Cytosol

Using antibodies raised with C. trachomatis fusion proteins, we localized a hypothetical protein encoded by the ORF ct622 in the cytoplasm of C. trachomatis-infected mammalian cells. The detection was specific since the antibody labelling of CT622 protein was removed by preabsorption with CT622 but not other fusion proteins. We similarly confirmed that CT621, a known secretion protein encoded by a hypothetical ORF downstream of ct622, was secreted into host cell cytosol. Proteins CT622 and CT621 displayed a similar secretion pattern, with both intra-inclusion and host cell cytosol localization, that was distinct from that of CPAF (chlamydial protease/proteasome-like activity factor). However, the expression and secretion kinetics differed significantly between CT622 and CT621: CT622 mRNA was detected at 2 h, protein at 6 h and secretion of protein into host cell cytoplasm at 36 h post-infection, while CT621 mRNA was detected at 8 h, protein at 16 h and secretion at 24 h. The secretion of both CT622 and CT621 was blocked by N′-(3,5-dibromo-2-hydroxybenzylidene)-4-nitrobenzohydrazide (compound 1), an inhibitor known to target the type III secretion system of bacteria. These results suggest that CT621 and CT622 may fulfil different functions during chlamydial intracellular growth. Further characterization of these proteins may generate important information for understanding chlamydial pathogenesis.

scholarly journals Identification of a Family of Effectors Secreted by the Type III Secretion System That Are Conserved in Pathogenic Chlamydiae

2010 ◽  
Vol 79 (2) ◽  
pp. 571-580 ◽  
Author(s):  
Sandra Muschiol ◽  
Gaelle Boncompain ◽  
François Vromman ◽  
Pierre Dehoux ◽  
Staffan Normark ◽  
...  
Keyword(s):  
Host Cell ◽  
Type Iii Secretion ◽  
Effector Proteins ◽  
Main Process ◽  
Cell Cytoplasm ◽  
Type Iii ◽  
Amino Terminal ◽  
Cell Functions ◽  
Host Cell Cytoplasm ◽  
Infectious Cycle

ABSTRACTChlamydiae are Gram-negative, obligate intracellular pathogens that replicate within a membrane-bounded compartment termed an inclusion. Throughout their development, they actively modify the eukaryotic environment. The type III secretion (TTS) system is the main process by which the bacteria translocate effector proteins into the inclusion membrane and the host cell cytoplasm. Here we describe a family of type III secreted effectors that are present in all pathogenic chlamydiae and absent in the environment-related species. It is defined by a common domain of unknown function, DUF582, that is present in four or five proteins in eachChlamydiaceaespecies. We show that the amino-terminal extremity of DUF582 proteins functions as a TTS signal. DUF582 proteins fromC. trachomatisCT620, CT621, and CT711 are expressed at the middle and late phases of the infectious cycle. Immunolocalization further revealed that CT620 and CT621 are secreted into the host cell cytoplasm, as well as within the lumen of the inclusion, where they do not associate with bacterial markers. Finally, we show that DUF582 proteins are present in nuclei of infected cells, suggesting that members of the DUF582 family of effector proteins may target nuclear cell functions. The expansion of this family of proteins in pathogenic chlamydiae and their conservation among the different species suggest that they play important roles in the infectious cycle.

scholarly journals Fusion of Chlamydia trachomatis-Containing Inclusions Is Inhibited at Low Temperatures and Requires Bacterial Protein Synthesis

1998 ◽  
Vol 66 (11) ◽  
pp. 5364-5371 ◽  
Author(s):  
Christiaan Van Ooij ◽  
Ellen Homola ◽  
Eleanor Kincaid ◽  
Joanne Engel
Keyword(s):  
Protein Synthesis ◽  
Chlamydia Trachomatis ◽  
Host Cell ◽  
Host Cells ◽  
Bacterial Protein ◽  
Cell Cytoplasm ◽  
Mammalian Cell Lines ◽  
Host Cell Cytoplasm ◽  
Early Endosomes ◽  
Bacterial Protein Synthesis

ABSTRACT The human pathogen Chlamydia trachomatis is an obligate intracellular bacterium with a unique developmental cycle. Within the host cell cytoplasm, it resides within a membrane-bound compartment, the inclusion. A distinguishing characteristic of the C. trachomatis life cycle is the fusion of the chlamydia-containing inclusions with each other in the host cell cytoplasm. We report that fusion of inclusions does not occur at 32°C in multiple mammalian cell lines and with three different serovars of C. trachomatis. The inhibition of fusion was inclusion specific; the fusion with sphingolipid-containing secretory vesicles and the interaction with early endosomes were unaffected by incubation at 32°C. The inhibition of fusion of the inclusions was not primarily the result of delayed maturation of the inclusion, as infectious progeny was produced in host cells incubated at 32°C, and the unfused inclusions remained competent to fuse up to 48 h postinfection. The ability to reverse the inhibition of fusion by shifting the infected cells from 32 to 37°C allowed the measurement of the rate and the time of fusion of the inclusions after entry of the bacteria. Most significantly, we demonstrate that fusion of inclusions with each other requires bacterial protein synthesis and that the required bacterial protein(s) is present, but inactive or not secreted, at 32°C.

scholarly journals α-Helices in the Type III Secretion Effectors: A Prevalent Feature with Versatile Roles

2021 ◽  
Vol 22 (11) ◽  
pp. 5412
Author(s):  
Anastasia D Gazi ◽  
Michael Kokkinidis ◽  
Vasiliki E Fadouloglou
Keyword(s):  
Host Cell ◽  
Type Iii Secretion ◽  
Defense Mechanisms ◽  
Cell Envelope ◽  
Active Form ◽  
Main Function ◽  
Secretion Systems ◽  
Cell Cytoplasm ◽  
Type Iii ◽  
Host Cell Cytoplasm

Type III Secretion Systems (T3SSs) are multicomponent nanomachines located at the cell envelope of Gram-negative bacteria. Their main function is to transport bacterial proteins either extracellularly or directly into the eukaryotic host cell cytoplasm. Type III Secretion effectors (T3SEs), latest to be secreted T3S substrates, are destined to act at the eukaryotic host cell cytoplasm and occasionally at the nucleus, hijacking cellular processes through mimicking eukaryotic proteins. A broad range of functions is attributed to T3SEs, ranging from the manipulation of the host cell’s metabolism for the benefit of the bacterium to bypassing the host’s defense mechanisms. To perform this broad range of manipulations, T3SEs have evolved numerous novel folds that are compatible with some basic requirements: they should be able to easily unfold, pass through the narrow T3SS channel, and refold to an active form when on the other side. In this review, the various folds of T3SEs are presented with the emphasis placed on the functional and structural importance of α-helices and helical domains.

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