scholarly journals Genomic Properties and Temporal Analysis of the Interaction of an Invasive Escherichia albertii With Epithelial Cells

2020 ◽  
Vol 10 ◽  
Author(s):  
Fabiano T. Romão ◽  
Fernando H. Martins ◽  
Rodrigo T. Hernandes ◽  
Tadasuke Ooka ◽  
Fernanda F. Santos ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Temporal Analysis ◽  
Infection Process ◽  
Effector Proteins ◽  
Invasion Process ◽  
Etiologic Agents ◽  
Bacterial Interaction ◽  
Type Three Secretion System ◽  
Enterocyte Effacement

Diarrhea is one of the main causes of infant mortality worldwide, mainly in the developing world. Among the various etiologic agents, Escherichia albertii is emerging as an important human enteropathogen. E. albertii promote attaching and effacing (AE) lesions due to the presence of the locus of enterocyte effacement (LEE) that encodes a type three secretion system (T3SS), the afimbrial adhesin intimin and its translocated receptor, Tir, and several effector proteins. We previously showed that E. albertii strain 1551-2 invades several epithelial cell lineages by a process that is dependent on the intimin-Tir interaction. To understand the contribution of T3SS-dependent effectors present in E. albertii 1551-2 during the invasion process, we performed a genetic analysis of the LEE and non-LEE genes and evaluated the expression of the LEE operons in various stages of bacterial interaction with differentiated intestinal Caco-2 cells. The kinetics of the ability of the 1551-2 strain to colonize and form AE lesions was also investigated in epithelial HeLa cells. We showed that the LEE expression was constant during the early stages of infection but increased at least 4-fold during bacterial persistence in the intracellular compartment. An in silico analysis indicated the presence of a new tccP/espFU subtype, named tccP3. We found that the encoded protein colocalizes with Tir and polymerized F-actin during the infection process in vitro. Moreover, assays performed with Nck null cells demonstrated that the 1551-2 strain can trigger F-actin polymerization in an Nck-independent pathway, despite the fact that TccP3 is not required for this phenotype. Our study highlights the importance of the T3SS during the invasion process and for the maintenance of E. albertii 1551-2 inside the cells. In addition, this work may help to elucidate the versatility of the T3SS for AE pathogens, which are usually considered extracellular and rarely reach the intracellular environment.

scholarly journals Regulation of the Locus of Enterocyte Effacement in Attaching and Effacing Pathogens

2017 ◽  
Vol 200 (2) ◽  
Author(s):  
R. Christopher D. Furniss ◽  
Abigail Clements
Keyword(s):  
Secretion System ◽  
Effector Proteins ◽  
The Core ◽  
Genetic Feature ◽  
Gut Epithelium ◽  
Type Three Secretion System ◽  
Gut Mucosa ◽  
Enterocyte Effacement ◽  
Type Three Secretion ◽  
Complex Regulatory Network

ABSTRACTAttaching and effacing (AE) pathogens colonize the gut mucosa using a type three secretion system (T3SS) and a suite of effector proteins. The locus of enterocyte effacement (LEE) is the defining genetic feature of the AE pathogens, encoding the T3SS and the core effector proteins necessary for pathogenesis. Extensive research has revealed a complex regulatory network that senses and responds to a myriad of host- and microbiota-derived signals in the infected gut to control transcription of the LEE. These signals include microbiota-liberated sugars and metabolites in the gut lumen, molecular oxygen at the gut epithelium, and host hormones. Recent research has revealed that AE pathogens also recognize physical signals, such as attachment to the epithelium, and that the act of effector translocation remodels gene expression in infecting bacteria. In this review, we summarize our knowledge to date and present an integrated view of how chemical, geographical, and physical cues regulate the virulence program of AE pathogens during infection.

scholarly journals Differential Modulation by Ca2+ of Type III Secretion of Diffusely Adhering Enteropathogenic Escherichia coli

2003 ◽  
Vol 71 (4) ◽  
pp. 1725-1732 ◽  
Author(s):  
Tina Ide ◽  
Silke Michgehl ◽  
Sabine Knappstein ◽  
Gerhard Heusipp ◽  
M. Alexander Schmidt
Keyword(s):  
Escherichia Coli ◽  
Type Iii Secretion ◽  
Effector Proteins ◽  
Host Cells ◽  
Chromosomal Locus ◽  
Type Iii ◽  
Calcium Chelation ◽  
Host Plasma Membrane ◽  
Enterocyte Effacement

ABSTRACT Enteropathogenic Escherichia coli (EPEC) strains are a common cause of persistent diarrhea among infants, primarily in developing countries. The pathogenicity of EPEC is associated with the expression and secretion of bacterial proteins encoded by the chromosomal locus of enterocyte effacement (LEE). The LEE-encoded type III-secreted proteins EspA, EspB, and EspD are part of a molecular syringe, which is used by EPEC to translocate effector proteins directly into the cytoplasm of host cells. The type III-secreted translocated intimin receptor (Tir) protein is thought to be delivered by an Esp-dependent mechanism into the host cell, and this is followed by insertion into the host plasma membrane, where the protein serves as the receptor for intimin, an afimbrial bacterial adhesin. Type III secretion is subject to environmental regulation, and secretion can be induced in vitro by growing bacteria in cell culture medium. In this study we found that Ca2+ is involved in the regulation of type III secretion both in classical locally adherent EPEC and in atypical diffusely adherent EPEC. Interestingly, we observed contrasting secretion of Esp proteins and Tir in response to Ca2+. While the secretion of Tir is clearly enhanced and the protein is integrated into HeLa membranes under calcium chelation conditions, Esp secretion is strongly reduced under these conditions. These data suggest that under Ca2+-depleted conditions Tir might be secreted into the medium and integrated into host membranes by an Esp-independent mechanism, without the need for a functional type III translocation machinery.

scholarly journals The bulk of unpolymerized actin in Xenopus egg extracts is ATP-bound.

1995 ◽  
Vol 6 (2) ◽  
pp. 227-236 ◽  
Author(s):  
J Rosenblatt ◽  
P Peluso ◽  
T J Mitchison
Keyword(s):  
Actin Polymerization ◽  
Critical Concentration ◽  
Large Fraction ◽  
Nucleotide Exchange ◽  
Chromatography Analysis ◽  
Thymosin Beta 4 ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Non-muscle cells contain 15-500 microM actin, a large fraction of which is unpolymerized. Thus, the concentration of unpolymerized actin is well above the critical concentration for polymerization in vitro (0.2 microM). This fraction of actin could be prevented from polymerization by being ADP bound (therefore less favored to polymerize) or by being ATP bound and sequestered by a protein such as thymosin beta 4, or both. We isolated the unpolymerized actin from Xenopus egg extracts using immobilized DNase 1 and assayed the bound nucleotide. High-pressure liquid chromatography analysis showed that the bulk of soluble actin is ATP bound. Analysis of actin-bound nucleotide exchange rates suggested the existence of two pools of unpolymerized actin, one of which exchanges nucleotide relatively rapidly and another that apparently does not exchange. Native gel electrophoresis of Xenopus egg extracts demonstrated that most of the soluble actin exists in complexes with other proteins, one of which might be thymosin beta 4. These results are consistent with actin polymerization being controlled by the sequestration and release of ATP-bound actin, and argue against nucleotide exchange playing a major role in regulating actin polymerization.

scholarly journals Rho GTPases as Key Molecular Players within Intestinal Mucosa and GI Diseases

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 66
Author(s):  
Rashmita Pradhan ◽  
Phuong A. Ngo ◽  
Luz d. C. Martínez-Sánchez ◽  
Markus F. Neurath ◽  
Rocío López-Posadas
Keyword(s):  
Intestinal Mucosa ◽  
Rho Gtpases ◽  
Current Knowledge ◽  
Cell Types ◽  
Effector Proteins ◽  
Special Focus ◽  
Specific Cell ◽  
Rho Proteins

Rho proteins operate as key regulators of the cytoskeleton, cell morphology and trafficking. Acting as molecular switches, the function of Rho GTPases is determined by guanosine triphosphate (GTP)/guanosine diphosphate (GDP) exchange and their lipidation via prenylation, allowing their binding to cellular membranes and the interaction with downstream effector proteins in close proximity to the membrane. A plethora of in vitro studies demonstrate the indispensable function of Rho proteins for cytoskeleton dynamics within different cell types. However, only in the last decades we have got access to genetically modified mouse models to decipher the intricate regulation between members of the Rho family within specific cell types in the complex in vivo situation. Translationally, alterations of the expression and/or function of Rho GTPases have been associated with several pathological conditions, such as inflammation and cancer. In the context of the GI tract, the continuous crosstalk between the host and the intestinal microbiota requires a tight regulation of the complex interaction between cellular components within the intestinal tissue. Recent studies demonstrate that Rho GTPases play important roles for the maintenance of tissue homeostasis in the gut. We will summarize the current knowledge on Rho protein function within individual cell types in the intestinal mucosa in vivo, with special focus on intestinal epithelial cells and T cells.

scholarly journals Ras-mediated activation of the TORC2–PKB pathway is critical for chemotaxis

2010 ◽  
Vol 190 (2) ◽  
pp. 233-245 ◽  
Author(s):  
Huaqing Cai ◽  
Satarupa Das ◽  
Yoichiro Kamimura ◽  
Yu Long ◽  
Carole A. Parent ◽  
...  
Keyword(s):  
Cell Migration ◽  
Actin Polymerization ◽  
Time Course ◽  
Specific Inhibitor ◽  
Ras Proteins ◽  
Extracellular Signaling ◽  
Upstream Regulator ◽  
Pkb Phosphorylation ◽  
G Protein Coupled

In chemotactic cells, G protein–coupled receptors activate Ras proteins, but it is unclear how Ras-associated pathways link extracellular signaling to cell migration. We show that, in Dictyostelium discoideum, activated forms of RasC prolong the time course of TORC2 (target of rapamycin [Tor] complex 2)-mediated activation of a myristoylated protein kinase B (PKB; PKBR1) and the phosphorylation of PKB substrates, independently of phosphatidylinositol-(3,4,5)-trisphosphate. Paralleling these changes, the kinetics of chemoattractant-induced adenylyl cyclase activation and actin polymerization are extended, pseudopodial activity is increased and mislocalized, and chemotaxis is impaired. The effects of activated RasC are suppressed by deletion of the TORC2 subunit PiaA. In vitro RasCQ62L-dependent PKB phosphorylation can be rapidly initiated by the addition of a PiaA-associated immunocomplex to membranes of TORC2-deficient cells and blocked by TOR-specific inhibitor PP242. Furthermore, TORC2 binds specifically to the activated form of RasC. These results demonstrate that RasC is an upstream regulator of TORC2 and that the TORC2–PKB signaling mediates effects of activated Ras proteins on the cytoskeleton and cell migration.

scholarly journals Tethering DNA Damage Checkpoint Mediator Proteins Topoisomerase IIβ-binding Protein 1 (TopBP1) and Claspin to DNA Activates Ataxia-Telangiectasia Mutated and RAD3-related (ATR) Phosphorylation of Checkpoint Kinase 1 (Chk1)

2011 ◽  
Vol 286 (22) ◽  
pp. 19229-19236 ◽  
Author(s):  
Laura A. Lindsey-Boltz ◽  
Aziz Sancar
Keyword(s):  
Dna Damage ◽  
Ataxia Telangiectasia ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Effector Proteins ◽  
Ataxia Telangiectasia Mutated ◽  
Checkpoint Kinase ◽  
Atr Kinase

The ataxia-telangiectasia mutated and RAD3-related (ATR) kinase initiates DNA damage signaling pathways in human cells after DNA damage such as that induced upon exposure to ultraviolet light by phosphorylating many effector proteins including the checkpoint kinase Chk1. The conventional view of ATR activation involves a universal signal consisting of genomic regions of replication protein A-covered single-stranded DNA. However, there are some indications that the ATR-mediated checkpoint can be activated by other mechanisms. Here, using the well defined Escherichia coli lac repressor/operator system, we have found that directly tethering the ATR activator topoisomerase IIβ-binding protein 1 (TopBP1) to DNA is sufficient to induce ATR phosphorylation of Chk1 in an in vitro system as well as in vivo in mammalian cells. In addition, we find synergistic activation of ATR phosphorylation of Chk1 when the mediator protein Claspin is also tethered to the DNA with TopBP1. Together, these findings indicate that crowding of checkpoint mediator proteins on DNA is sufficient to activate the ATR kinase.

scholarly journals Rho-Proteins and Downstream Pathways as Potential Targets in Sepsis and Septic Shock: What Have We Learned from Basic Research

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1844
Author(s):  
Maria Luísa da Silveira Hahmeyer ◽  
José Eduardo da Silva-Santos
Keyword(s):  
Septic Shock ◽  
Actin Polymerization ◽  
Current Knowledge ◽  
Cecal Ligation ◽  
Experimental Models ◽  
Rho Proteins ◽  
Growing Cell ◽  
Sepsis And Septic Shock

Sepsis and septic shock are associated with acute and sustained impairment in the function of the cardiovascular system, kidneys, lungs, liver, and brain, among others. Despite the significant advances in prevention and treatment, sepsis and septic shock sepsis remain global health problems with elevated mortality rates. Rho proteins can interact with a considerable number of targets, directly affecting cellular contractility, actin filament assembly and growing, cell motility and migration, cytoskeleton rearrangement, and actin polymerization, physiological functions that are intensively impaired during inflammatory conditions, such as the one that occurs in sepsis. In the last few decades, Rho proteins and their downstream pathways have been investigated in sepsis-associated experimental models. The most frequently used experimental design included the exposure to bacterial lipopolysaccharide (LPS), in both in vitro and in vivo approaches, but experiments using the cecal ligation and puncture (CLP) model of sepsis have also been performed. The findings described in this review indicate that Rho proteins, mainly RhoA and Rac1, are associated with the development of crucial sepsis-associated dysfunction in different systems and cells, including the endothelium, vessels, and heart. Notably, the data found in the literature suggest that either the inhibition or activation of Rho proteins and associated pathways might be desirable in sepsis and septic shock, accordingly with the cellular system evaluated. This review included the main findings, relevance, and limitations of the current knowledge connecting Rho proteins and sepsis-associated experimental models.

SOX9 is required for kidney fibrosis and activates NAV3 to drive renal myofibroblast function

2021 ◽  
Vol 14 (672) ◽  
pp. eabb4282 ◽  
Author(s):  
Sayyid Raza ◽  
Elliot Jokl ◽  
James Pritchett ◽  
Katherine Martin ◽  
Kim Su ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Actin Polymerization ◽  
Kidney Diseases ◽  
Kidney Injury ◽  
Pharmacological Intervention ◽  
Kidney Fibrosis ◽  
Genome Wide ◽  
Vitro Model ◽  
Sustained Activation

Renal fibrosis is a common end point for kidney injury and many chronic kidney diseases. Fibrogenesis depends on the sustained activation of myofibroblasts, which deposit the extracellular matrix that causes progressive scarring and organ failure. Here, we showed that the transcription factor SOX9 was associated with kidney fibrosis in humans and required for experimentally induced kidney fibrosis in mice. From genome-wide analysis, we identified Neuron navigator 3 (NAV3) as acting downstream of SOX9 in kidney fibrosis. NAV3 increased in abundance and colocalized with SOX9 after renal injury in mice, and both SOX9 and NAV3 were present in diseased human kidneys. In an in vitro model of renal pericyte transdifferentiation into myofibroblasts, we demonstrated that NAV3 was required for multiple aspects of fibrogenesis, including actin polymerization linked to cell migration and sustained activation of the mechanosensitive transcription factor YAP1. In summary, our work identifies a SOX9-NAV3-YAP1 axis involved in the progression of kidney fibrosis and points to NAV3 as a potential target for pharmacological intervention.

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