scholarly journals Klebsiella pneumoniae Translocates across the Intestinal Epithelium via Rho GTPase- and Phosphatidylinositol 3-Kinase/Akt-Dependent Cell Invasion

2014 ◽  
Vol 83 (2) ◽  
pp. 769-779 ◽  
Author(s):  
Chun-Ru Hsu ◽  
Yi-Jiun Pan ◽  
Ju-Yun Liu ◽  
Chun-Tang Chen ◽  
Tzu-Lung Lin ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Klebsiella Pneumoniae ◽  
Cell Invasion ◽  
Intestinal Epithelium ◽  
Intestinal Barrier ◽  
Dependent Manner ◽  
Phosphatidylinositol 3 Kinase ◽  
Content Type ◽  
Junction Protein ◽  
Phosphatidylinositol 3

Klebsiella pneumoniaeis an important pathogen that causes hospital-acquired septicemia and is associated with the recent emergence of community-acquired pyogenic liver abscess (PLA). Clinical typing suggests thatK. pneumoniaeinfections originate from the gastrointestinal reservoir. However, the underlying mechanism remains unknown. Here, we have sought to determine howK. pneumoniaepenetrates the intestinal barrier. We identified that bacteremia and PLA clinical isolates adhered to and invaded intestinal epithelial cells. Internalization ofK. pneumoniaein three different human colonic cell lines was visualized by confocal microscopy and three-dimensional (3D) imaging. Using a Transwell system, we demonstrated that theseK. pneumoniaeisolates translocated across a polarized Caco-2 monolayer. No disruptions of transepithelial electrical resistance and altered distribution of tight junction protein ZO-1 or occludin were observed. Therefore,K. pneumoniaeappeared to penetrate the intestinal epithelium via a transcellular pathway. Using specific inhibitors, we characterized the host signaling pathways involved. Inhibition by cytochalasin D and nocodazole suggested that actin and microtubule cytoskeleton were both important forK. pneumoniaeinvasion. A Rho inhibitor, ML141, LY294002, and an Akt1/2 inhibitor diminishedK. pneumoniaeinvasion in a dose-dependent manner, indicating that Rho family GTPases and phosphatidylinositol 3-kinase (PI3K)/Akt signaling were required. By a mouse model of gastrointestinal colonization,in vivoinvasion ofK. pneumoniaeinto colonic epithelial cells was demonstrated. Our results present evidence to describe a possible mechanism of gastrointestinal translocation forK. pneumoniae. Cell invasion by manipulating host machinery provides a pathway for gut-colonizedK. pneumoniaecells to penetrate the intestinal barrier and access extraintestinal locations to cause disease.

scholarly journals Klebsiella pneumoniae Reduces SUMOylation To Limit Host Defense Responses

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Joana Sá-Pessoa ◽  
Kornelia Przybyszewska ◽  
Filipe Nuno Vasconcelos ◽  
Amy Dumigan ◽  
Christian G. Frank ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Klebsiella Pneumoniae ◽  
Glycogen Synthase ◽  
Defense Responses ◽  
Multidrug Resistant ◽  
Lung Epithelial Cells ◽  
Type I ◽  
Dependent Manner ◽  
Content Type ◽  
Medical Needs

ABSTRACT Klebsiella pneumoniae is an important cause of multidrug-resistant infections worldwide. Understanding the virulence mechanisms of K. pneumoniae is a priority and timely to design new therapeutics. Here, we demonstrate that K. pneumoniae limits the SUMOylation of host proteins in epithelial cells and macrophages (mouse and human) to subvert cell innate immunity. Mechanistically, in lung epithelial cells, Klebsiella increases the levels of the deSUMOylase SENP2 in the cytosol by affecting its K48 ubiquitylation and its subsequent degradation by the ubiquitin proteasome. This is dependent on Klebsiella preventing the NEDDylation of the Cullin-1 subunit of the ubiquitin ligase complex E3-SCF-βTrCP by exploiting the CSN5 deNEDDylase. Klebsiella induces the expression of CSN5 in an epidermal growth factor receptor (EGFR)-phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT)-extracellular signal-regulated kinase (ERK)-glycogen synthase kinase 3 beta (GSK3β) signaling pathway-dependent manner. In macrophages, Toll-like receptor 4 (TLR4)-TRAM-TRIF-induced type I interferon (IFN) via IFN receptor 1 (IFNAR1)-controlled signaling mediates Klebsiella-triggered decrease in the levels of SUMOylation via let-7 microRNAs (miRNAs). Our results revealed the crucial role played by Klebsiella polysaccharides, the capsule, and the lipopolysaccharide (LPS) O-polysaccharide, to decrease the levels of SUMO-conjugated proteins in epithelial cells and macrophages. A Klebsiella-induced decrease in SUMOylation promotes infection by limiting the activation of inflammatory responses and increasing intracellular survival in macrophages. IMPORTANCE Klebsiella pneumoniae has been singled out as an urgent threat to human health due to the increasing isolation of strains resistant to “last-line” antimicrobials, narrowing the treatment options against Klebsiella infections. Unfortunately, at present, we cannot identify candidate compounds in late-stage development for treatment of multidrug-resistant Klebsiella infections; this pathogen is exemplary of the mismatch between unmet medical needs and the current antimicrobial research and development pipeline. Furthermore, there is still limited evidence on K. pneumoniae pathogenesis at the molecular and cellular levels in the context of the interactions between bacterial pathogens and their hosts. In this research, we have uncovered a sophisticated strategy employed by Klebsiella to subvert the activation of immune defenses by controlling the modification of proteins. Our research may open opportunities to develop new therapeutics based on counteracting this Klebsiella-controlled immune evasion strategy.

scholarly journals Oxidative stress increases megalin expression in the renal proximal tubules during the normoalbuminuric stage of diabetes mellitus

2018 ◽  
Vol 314 (3) ◽  
pp. F462-F470 ◽  
Author(s):  
Yoshifumi Kurosaki ◽  
Akemi Imoto ◽  
Fumitaka Kawakami ◽  
Masanori Yokoba ◽  
Tsuneo Takenaka ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Diabetes Mellitus ◽  
Hydrogen Peroxide ◽  
High Glucose ◽  
Renal Cortex ◽  
Dependent Manner ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphorylated Akt ◽  
Stress Dependent ◽  
Phosphatidylinositol 3

Megalin, an endocytic receptor expressed in proximal tubule cells, plays a critical role in renal tubular protein reabsorption and is associated with the albuminuria observed in diabetic nephropathy. We have previously reported increased oxidant production in the renal cortex during the normoalbuminuric stage of diabetes mellitus (DM); however, the relationship between oxidative stress and renal megalin expression during the normoalbuminuric stage of DM remains unclear. In the present study, we evaluated whether oxidative stress affects megalin expression in the normoalbuminuric stage of DM in a streptozotocin-induced diabetic rat model and in immortalized human proximal tubular cells (HK-2). We demonstrated that increased expression of renal megalin accompanies oxidative stress during the early stage of DM, before albuminuria development. Telmisartan treatment prevented the diabetes-induced elevation in megalin level, possibly through an oxidative stress-dependent mechanism. In HK-2 cells, hydrogen peroxide significantly increased megalin levels in a dose- and time-dependent manner; however, the elevation in megalin expression was decreased following prolonged exposure to severe oxidative stress induced by 0.4 mmol/l hydrogen peroxide. High-glucose treatment also significantly increased megalin expression in HK-2 cells. Concurrent administration of the antioxidant N-acetyl-cysteine blocked the effects of high glucose on megalin expression. Furthermore, the hydrogen peroxide-induced increase in megalin expression was blocked by treatment with phosphatidylinositol 3-kinase and Akt inhibitors. Increase of phosphorylated Akt expression was also seen in the renal cortex of diabetic rats. Taken together, our results indicate that mild oxidative stress increases renal megalin expression through the phosphatidylinositol 3-kinase-Akt pathway in the normoalbuminuric stage of DM.

scholarly journals A Micro-engineered Human Colon Intestine-Chip Platform to Study Leaky Barrier

2020 ◽  
Author(s):  
Athanasia Apostolou ◽  
Rohit A. Panchakshari ◽  
Antara Banerjee ◽  
Dimitris V. Manatakis ◽  
Maria D. Paraskevopoulou ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Intestinal Epithelium ◽  
Unmet Need ◽  
Intestinal Barrier ◽  
Human Colon ◽  
Epithelial Barrier ◽  
Host Immune System ◽  
Intestinal Epithelial ◽  
Barrier Disruption ◽  
Interleukin 22

ABSTRACTThe intestinal epithelial barrier supports the symbiotic relationship between the microbiota colonizing the intestinal epithelium and the host immune system to maintain homeostasis. Leaky barrier is increasingly recognized as part of the pathogenesis of a number of chronic conditions in addition to inflammatory and infectious diseases. As our understanding on the regulation of the barrier remains limited, effective therapeutic targeting for the compromised barrier is still an unmet need. Here we combined advancements on the organoids and Organ-on-Chip technologies to establish a micro-engineered Colon Intestine-Chip for studying development and regulation of the human intestinal barrier. Our data demonstrate the significance of the endothelium in co-culture with the epithelial cells within a tissue-relevant microenvironment for the establishment of a tight epithelial barrier of polarized cells. Pathway analysis of the RNA sequencing (RNA-Seq), revealed significant upregulation of mechanisms relevant to the maturation of the intestinal epithelium in organoid-derived epithelial cells in co-culture with endothelium as compared to organoids maintained in suspension. We provide evidence that the Colon Intestine-Chip platform responds to interferon gamma (IFNγ), a prototype cytokine utilized to model inflammation-induced barrier disruption, by induction of apoptosis and reorganization of the apical junctional complexes as shown with other systems. We also describe the mechanism of action of interleukin 22 (IL-22) on mature, organoid-derived intestinal epithelial cells that is consistent with barrier disruption. Overall we propose the Colon Intestine-Chip as a promising human organoid-derived platform to decipher mechanisms driving the development of leaky gut in patients and enable their translation for this unmet medical need.

scholarly journals Both Src-Dependent and -Independent Mechanisms Mediate Phosphatidylinositol 3-Kinase Regulation of Colony-Stimulating Factor 1-Activated Mitogen-Activated Protein Kinases in Myeloid Progenitors

2000 ◽  
Vol 20 (18) ◽  
pp. 6779-6798 ◽  
Author(s):  
Angel W.-M. Lee ◽  
David J. States
Keyword(s):  
Kinase Inhibitors ◽  
Pi3 Kinase ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Colony Stimulating Factor ◽  
Phosphatidylinositol 3 Kinase ◽  
Stimulating Factor ◽  
In Cells ◽  
Phosphatidylinositol 3 ◽  
Myeloid Progenitors

ABSTRACT Colony-stimulating factor 1 (CSF-1) supports the proliferation, survival, and differentiation of bone marrow-derived cells of the monocytic lineage. In the myeloid progenitor 32D cell line expressing CSF-1 receptor (CSF-1R), CSF-1 activation of the extracellular signal-regulated kinase (ERK) pathway is both Ras and phosphatidylinositol 3-kinase (PI3-kinase) dependent. PI3-kinase inhibition did not influence events leading to Ras activation. Using the activity of the PI3-kinase effector, Akt, as readout, studies with dominant-negative and oncogenic Ras failed to place PI3-kinase downstream of Ras. Thus, PI3-kinase appears to act in parallel to Ras. PI3-kinase inhibitors enhanced CSF-1-stimulated A-Raf and c-Raf-1 activities, and dominant-negative A-Raf but not dominant-negative c-Raf-1 reduced CSF-1-provoked ERK activation, suggesting that A-Raf mediates a part of the stimulatory signal from Ras to MEK/ERK, acting in parallel to PI3-kinase. Unexpectedly, a CSF-1R lacking the PI3-kinase binding site (ΔKI) remained capable of activating MEK/ERK in a PI3-kinase-dependent manner. To determine if Src family kinases (SFKs) are involved, we demonstrated that CSF-1 activated Fyn and Lyn in cells expressing wild-type (WT) or ΔKI receptors. Moreover, CSF-1-induced Akt activity in cells expressing ΔKI is SFK dependent since Akt activation was prevented by pharmacological or genetic inhibition of SFK activity. The docking protein Gab2 may link SFK to PI3-kinase. CSF-1 induced Gab2 tyrosyl phosphorylation and association with PI3-kinase in cells expressing WT or ΔKI receptors. However, only in ΔKI cells are these events prevented by PP1. Thus in myeloid progenitors, CSF-1 can activate the PI3-kinase/Akt pathway by at least two mechanisms, one involving direct receptor binding and one involving SFKs.

scholarly journals Wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase, induces accumulation of DNA double-strand breaks

2020 ◽  
Vol 61 (2) ◽  
pp. 171-176 ◽  
Author(s):  
Makoto Ihara ◽  
Kazuko Shichijo ◽  
Satoshi Takeshita ◽  
Takashi Kudo
Keyword(s):  
Dna Damage ◽  
Specific Inhibitor ◽  
Double Strand Breaks ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
Phosphatidylinositol 3 Kinase ◽  
Strand Breaks ◽  
Γ Ray ◽  
Scid Cells ◽  
Phosphatidylinositol 3

Abstract Wortmannin, a fungal metabolite, is a specific inhibitor of the phosphatidylinositol 3-kinase (PI3K) family, which includes double-stranded DNA dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated kinase (ATM). We investigated the effects of wortmannin on DNA damage in DNA-PK-deficient cells obtained from severe combined immunodeficient mice (SCID cells). Survival of wortmannin-treated cells decreased in a concentration-dependent manner. After treatment with 50 μM wortmannin, survival decreased to 60% of that of untreated cells. We observed that treatment with 20 and 50 μM wortmannin induced DNA damage equivalent to that by 0.37 and 0.69 Gy, respectively, of γ-ray radiation. The accumulation of DNA double-strand breaks (DSBs) in wortmannin-treated SCID cells was assessed using pulsed-field gel electrophoresis. The maximal accumulation was observed 4 h after treatment. Moreover, the presence of DSBs was confirmed by the ability of nuclear extracts from γ-ray-irradiated SCID cells to produce in vitro phosphorylation of histone H2AX. These results suggest that wortmannin induces cellular toxicity by accumulation of spontaneous DSBs through inhibition of ATM.

scholarly journals Clostridium perfringens Delta-Toxin Damages the Mouse Small Intestine

Toxins ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 232 ◽  
Author(s):  
Soshi Seike ◽  
Masaya Takehara ◽  
Keiko Kobayashi ◽  
Masahiro Nagahama
Keyword(s):  
Epithelial Cells ◽  
Clostridium Perfringens ◽  
Intestinal Epithelial Cells ◽  
Cell Damage ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Fluid Accumulation ◽  
Ileal Loop ◽  
Junction Protein ◽  
E Cadherin

Clostridium perfringens strains B and C cause fatal intestinal diseases in animals. The secreted pore-forming toxin delta-toxin is one of the virulence factors of the strains, but the mechanism of intestinal pathogenesis is unclear. Here, we investigated the effects of delta-toxin on the mouse ileal loop. Delta-toxin caused fluid accumulation and intestinal permeability to fluorescein isothiocyanate (FITC)-dextran in the mouse ileal loop in a dose- and time-dependent manner. Treatment with delta-toxin induced significant histological damage and shortening of villi. Delta-toxin activates a disintegrin and metalloprotease (ADAM) 10, leading to the cleavage of E-cadherin, the epithelial adherens junction protein, in human intestinal epithelial Caco-2 cells. In this study, E-cadherin immunostaining in mouse intestinal epithelial cells was almost undetectable 1 h after toxin treatment. ADAM10 inhibitor (GI254023X) blocked the toxin-induced fluid accumulation and E-cadherin loss in the mouse ileal loop. Delta-toxin stimulated the shedding of intestinal epithelial cells. The shedding cells showed the accumulation of E-cadherin in intracellular vesicles and the increased expression of active caspase-3. Our findings demonstrate that delta-toxin causes intestinal epithelial cell damage through the loss of E-cadherin cleaved by ADAM10.

scholarly journals The mRNA-binding protein IGF2BP1 maintains intestinal barrier function by up-regulating occludin expression

2020 ◽  
Vol 295 (25) ◽  
pp. 8602-8612
Author(s):  
Vikash Singh ◽  
Chethana P. Gowda ◽  
Vishal Singh ◽  
Ashwinkumar S. Ganapathy ◽  
Dipti M. Karamchandani ◽  
...  
Keyword(s):  
Intestinal Epithelium ◽  
Barrier Function ◽  
Binding Protein ◽  
Intestinal Barrier ◽  
Intestinal Barrier Function ◽  
Intestinal Epithelial ◽  
Adult Mice ◽  
Junction Protein ◽  
Mrna Binding Protein ◽  
Mrna Binding

Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is an mRNA-binding protein that has an oncofetal pattern of expression. It is also expressed in intestinal tissue, suggesting that it has a possible role in intestinal homeostasis. To investigate this possibility, here we generated Villin CreERT2:Igf2bp1flox/flox mice, which enabled induction of an IGF2BP1 knockout specifically in intestinal epithelial cells (IECs) of adult mice. Using gut barrier and epithelial permeability assays and several biochemical approaches, we found that IGF2BP1 ablation in the adult intestinal epithelium causes mild active colitis and mild-to-moderate active enteritis. Moreover, the IGF2BP1 deletion aggravated dextran sodium sulfate–induced colitis. We also found that IGF2BP1 removal compromises barrier function of the intestinal epithelium, resulting from altered protein expression at tight junctions. Mechanistically, IGF2BP1 interacted with the mRNA of the tight-junction protein occludin (Ocln), stabilizing Ocln mRNA and inducing expression of occludin in IECs. Furthermore, ectopic occludin expression in IGF2BP1-knockdown cells restored barrier function. We conclude that IGF2BP1-dependent regulation of occludin expression is an important mechanism in intestinal barrier function maintenance and in the prevention of colitis.

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