阪崎杆菌侵袭人脑微血管内皮细胞对细胞骨架的影响

目的观察阪崎杆菌侵袭人脑血管内皮细胞后,细胞骨架中微丝和微管的变化。方法用荧光染色技术观察阪崎杆菌侵袭人脑微血管内皮细胞后,细胞骨架中的微丝和微管的变化,观察微丝和微管的抑制剂对阪崎杆菌侵袭人脑微血管内皮细胞的影响。结果阪崎杆菌侵袭人脑微血管内皮细胞后,细胞骨架结构发生明显的改变,微丝和微管部分断裂,模糊,散在分布,微丝抑制剂(细胞松弛素D)和微管抑制剂(秋水仙素)可以抑制阪崎杆菌侵袭人脑微血管内皮细胞。结论阪崎杆菌侵袭人脑血管内皮细胞破坏细胞骨架结构,微丝和微管抑制剂抑制阪崎杆菌侵袭。     

Disassembly of F-actin cytoskeleton after interaction of Bacillus cereus with fully differentiated human intestinal Caco-2 cells

In the present study, the role of direct procaryote-eucaryote interactions in the virulence of Bacillus cereus was investigated. As a model of human enterocytes, differentiated Caco-2 cells were used. Infection of fully differentiated Caco-2 cells with B. cereus in the exponential phase of growth, in order to minimize the concentration of spores or sporulating microorganisms, shows that a strain-dependent cytopathic effect develops. Interestingly, addition of 3-h-old cultures of some strains resulted in complete detachment of the cultured cells after a 3-h infection whereas no such effect was found after a 3-h infection with 16-h-old cultures. Infection of enterocyte-like cells with B. cereus leads to disruption of the F-actin network and necrosis. Even though the effect of secreted factors cannot be ruled out, direct eucaryote-procaryote interaction seems to be necessary. In addition, we observed that some B. cereus strains were able to be internalized in Caco-2 cells. Our findings add a new insight into the mechanisms of virulence of B. cereus in the context of intestinal infection.     

Outer membrane protein A expression in Enterobacter sakazakii is required to induce microtubule condensation in human brain microvascular endothelial cells for invasion

Enterobacter sakazakii (ES) causes neonatal meningitis and necrotizing enterocolitis with case-fatality rates among infected infants ranging from 40 to 80%. Very little is known about the mechanisms by which these organisms cause disease. Here, we demonstrate that ES invades human brain microvascular endothelial cells (HBMEC) with higher frequency when compared with epithelial cells and endothelial cells from different origins. The entry of ES into HBMEC requires the expression of outer membrane protein A (OmpA), as the OmpA-deletion mutant was sevenfold less invasive than the wild type ES and the bacterium does not multiply inside HBMEC. Anti-OmpA antibodies generated against the OmpA of Escherichia coli K1, which also recognize the OmpA of ES, did not prevent the invasion of ES in HBMEC. ES invasion depends on microtubule condensation in HBMEC and is independent of actin filament reorganization. Both PI3-kinase and PKC-alpha were activated during ES entry into HBMEC between 15min and 30min of infection. Concomitantly, overexpression of dominant negative forms of PI3-kinase and PKC-alpha significantly inhibited the invasion of ES into HBMEC. In summary, ES invasion of HBMEC is dependent on the expression of OmpA similar to that of E. coli K1; however, the epitopes involved in the interaction with HBMEC appears to be different.     

PI3K-dependent host cell actin rearrangements are required for Cronobacter sakazakii invasion of human brain microvascular endothelial cells

Cronobacter sakazakii (C. sakazakii) is an opportunistic pathogen that can cause neonatal sepsis and meningitis. The mechanism involved in the pathogenesis of C. sakazakii meningitis remains largely unknown. Previous studies indicated that bacterial invasion of brain microvascular endothelial cells is required for penetration into the central nervous system. In this study, we found that C. sakazakii invasion of human brain microvascular endothelial cells (HBMEC) was significantly inhibited by cytochalasin D, a disrupting agent of actin microfilaments. Disassembly of actin stress fibers and cortical actin fibers was observed in HBMEC infected with C. sakazakii. C. sakazakii infection leads to increased Akt phosphorylation in HBMEC, which was blocked by treatment with PI3K inhibitors. Meanwhile, PI3K and Akt inhibitors significantly inhibited C. sakazakii invasion of HBMEC. Our further results illustrated that the C. sakazakii-induced Akt activation and C. sakazakii invasion were attenuated in HBMEC transfected with dominant-negative PI3K (Δp110). More importantly, the actin filaments rearrangements in HBMEC induced by C. sakazakii were effectively blocked by PI3K inhibitors treatment and transfection with Δp110. Taken together, our findings demonstrated that PI3K-mediated actin rearrangements are required for C. sakazakii invasion of HBMEC.     

Persistent infection of human intestinal Caco-2 cell line by Coxsackieviruses B

Coxsackieviruses B (CV-B, Picornaviridae family, genus Enterovirus) are characterized by their ability to cause cytopathic effects in tissue culture. These viruses are considered highly cytolytic, but can establish persistence/latency in susceptible cells, indicating that a regulatory mechanism may exist to shut off viral protein synthesis and replication under certain situations. The persistence of coxsackieviral B RNA is of great interest because of its implication in the pathogenesis of several chronic human diseases. However, a few studies have dealt with the persistence of these viruses at the intestinal level. The aim of this study is to test the capacity of the six CV-B serotypes to establish persistent infection in human intestinal Caco-2 cell line. Ten CV-B isolates, including CV-B3 prototype strain (Nancy) and a recombinant isolate (B3-B4), were tested. Six CV-B isolates were found to establish persistent infections in Caco-2 cell line. Persistent replication was proved by the detection of viral RNA from cell cultures, VP1 capsid protein detection by immunofluorescence (IF) staining, and the release of infectious particles up to two months and a half after infection without any obvious cytolysis. In addition, our results suggest that the establishment of a persistent infection is serotype-independent.     

Contribution of Listeria monocytogenes RecA to acid and bile survival and invasion of human intestinal Caco-2 cells

The food-borne pathogen Listeria monocytogenes is able to colonize the human gastro-intestinal tract and subsequently cross the intestinal barrier. Thus, for L. monocytogenes to become virulent, it must survive the low pH of the stomach, high bile concentrations in the small intestine, and invade the epithelial cells. In this study, we show that RecA, which is an important factor in DNA repair and the activator of the SOS response, contributes to the resistance against acid and bile and to the ability of L. monocytogenes to adhere and invade human intestine epithelial cells. Activation of recA was shown with a promoter reporter after exposure to low pH and high bile concentrations and during adhesion and invasion of Caco-2 intestinal epithelial cells. Furthermore, an in-frame recA deletion mutant showed reduced survival after exposure to low pH and high bile concentrations. This mutant also showed a deficiency in adhesion and invasion of Caco-2 cells. These results suggest that RecA may contribute to the colonization of the human gastro-intestinal tract and crossing of the intestinal barrier.     

Activation of transepithelial ion transport by secretin in human intestinal Caco-2 cells

Secretin stimulates bicarbonate secretion from pancreatic duct cells, but what influence secretin exerts on intestinal tissues remains to be clarified. The aim of this study is to examine effects of secretin on ion transport in intestinal epithelial Caco-2 cells. We mounted monolayers of Caco-2 cells grown on permeable supports for 21-28 d in a Ussing chamber and measured short-circuit currents (I(sc)). Addition of secretin (5-100 nM) to the basolateral solution dose-dependently induced biphasic increases of I(sc) (transient and sustained phase). Dibutyryl cyclic AMP (200 microM), forskolin (10 microM), and 3-isobutyl-1-methylxanthine (IBMX, 1 mM) also induced I(sc) responses similar to the administration of secretin. Addition of 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 100 microM) or benzamil (100 microM) to the apical solution markedly reduced the secretin-induced I(sc) increase in the transient phase. A selective antagonist of cAMP-dependent protein kinase (PKA), N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89, 1 microM), and a membrane permeable Ca(2+) chelator, 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA/AM, 10 microM) reduced the secretin-induced I(sc). Basolateral addition of 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, 1 mM) suppressed the sustained phase I(sc) increase. Secretin also induced alkalinization of the apical solution (DeltapH, 0.053 +/- 0.013). The alkalinization did not occur when DIDS (1 mM) was added to the basolateral solution or Na(+) was removed from the solutions. Taken together, our observations suggest: (1) secretin stimulates a benzamil-sensitive Na(+) influx and an NPPB-sensitive Cl(-) efflux across the apical membrane through PKA-dependent and Ca(2+)-sensitive pathways; and (2) secretin also induces alkalinization of the apical solution through the activation of a DIDS-sensitive Na(+)-HCO(3)(-) cotransport in the basolateral membrane of Caco-2 cells.     

Rotavirus induces apoptosis in fully differentiated human intestinal Caco-2 cells

Rotaviruses, which are the main cause of viral gastroenteritis in young children, induce structural and functional damages in infected mature enterocytes of the small intestine. To investigate a relationship between rotavirus infection and cell death by apoptosis, we used the human intestinal Caco-2 cell line. We demonstrated by several methods including TUNEL and ELISA detection of cytoplasmic histone-associated DNA fragments that the infection of fully differentiated Caco-2 cells by the RRV rotavirus strain induces apoptosis. Rotavirus infection leads to the loss of mitochondrial membrane potential and the release of cytochrome C from mitochondria. We showed that rotavirus-induced apoptosis was dependent of the multiplicity of infection and increased with time from 4 h to 24 h of infection. Flow cytometric analysis showed that DNA fragmentation occurs in productively infected cells, suggesting that rotavirus induces apoptosis by a direct mechanism. We also demonstrated that non-replicative RRV particles are not sufficient to induce apoptosis and viral gene expression seems required. Intracellular calcium plays a role in RRV-induced apoptosis because treatment with an intracellular calcium ion chelator (BAPTA-AM) partially inhibited apoptosis.     

Pseudomonas aeruginosaentry into Caco-2 cells is enhanced in repairing wounded monolayers

Human respiratory cells participating in the repair of epithelial wounds have been shown to be highly susceptible toPseudomonas aeruginosaadherence. To ascertain whether such susceptibility is a common feature of different repairing epithelial cells, Caco-2 cell monolayers were chemically injured, reincubated for 48 h to partially repair and exposed to bacteria. Cells edging the wounds that spread and migrate to re-establish cell confluence were called ‘repairing cells’ while cells far from the wounds were called ‘non-repairing cells’. By light microscopy, bacteria were seen to adhere to and to enter into both repairing and non-repairing cells. The percentage of intracellular bacteria in repairing cells was significantly higher than in non-repairing cells. The higher susceptibility of repairing monolayers to bacterial entry was confirmed by the gentamicin exclusion assay.P. aeruginosaentry into Caco-2 cells was greatly enhanced in non-injured confluent monolayers treated with EDTA to disrupt intercellular junctions. As tight junction disfunctions have been described during the wound repair process, we speculate that exposure of basolateral receptors to bacterial ligands may account for the enhancement ofP. aeruginosainternalization by repairing monolayers.     

Cronobacter spp. (previously Enterobacter sakazakii) invade and translocate across both cultured human intestinal epithelial cells and human brain microvascular endothelial cells

The mechanism of Cronobacter pathogenesis in neonatal meningitis and potential virulence factors (aside from host cell invasion ability) remain largely unknown. To ascertain whether Cronobacter can invade and transcytose across intestinal epithelial cells, enter into the blood stream and then transcytose across the blood-brain-barrier, we have utilized human intestinal INT407 and Caco-2 cells and brain microvascular endothelial cell (HBMEC) monolayers on Transwell filters as experimental model systems. Our data indicate a wide range of heterogeneity with respect to invasion efficiency among twenty-three Cronobacter isolates screened. For selected isolates, we observed significant levels of transcytosis for Cronobacter sakazakii across tight monolayers of both Caco-2 and HBMEC, mimicking in vivo ability to cross the intestine as well as the blood brain barrier, and at a frequency equivalent to that of a control meningitis-causing Escherichia coli K1 strain. Finally, EM analysis demonstrated intracellular Cronobacter bacteria within host vacuoles in HBMEC, as well as transcytosed bacteria at the basolateral surface. These data reveal that certain Cronobacter isolates can invade and translocate across both cultured human intestinal epithelial cells and HBMEC, thus demonstrating a potential path for neonatal infections of the central nervous system (CNS) following oral ingestion.     

Regulation of apical transporter of L-DOPA in human intestinal Caco-2 cells

The present study examined the nature of the apical inward L-3,4-dihydroxyphenylalanine (L-DOPA) transporter in human intestinal epithelial Caco-2 cells, and whether protein kinases modulate the activity of this transporter. The apical inward transfer of L-DOPA was promoted through an energy-dependent and sodium-insensitive transporter (Km=33 microM; Vmax=2932 pmol/mg protein/6 min). This transporter was insensitive to N-(methylamino)-isobutyric acid, but competitively inhibited by 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BCH; IC50=83 microM). The organic cation inhibitor decynium 24 failed to affect the accumulation of L-DOPA, whereas the organic anion inhibitor 4,4'-diisothiocynatostilbene-2,2'-disulphonic acid (DIDS) competitively inhibited L-DOPA uptake (IC50=83 microM). However, the apical-to-basal and basal-to-apical transepithelial transport and the cell accumulation of [3H]-PAH was close to that of [14C]-sorbitol and insensitive to DIDS (300 microM). Modulators of protein kinase A (PKA) [cyclic adenosine monophosphate (cAMP), forskolin, H-89 and cholera toxin], protein kinase G (PKG) [cyclic guanosine monophosphate (GMP), zaprinast, LY 83583 and sodium nitroprusside] and protein kinase C (PKC) (phorbol 12,13-dibutirate and chelerythrine) failed to affect the accumulation of L-DOPA. The Ca2+/calmodulin inhibitors calmidazolium and trifluoperazine inhibited L-DOPA uptake (IC50s of 53 and 252 microM, respectively), but the rise of intracellular Ca2+ by A23187 (1 microM) and thapsigargin (1 microM) played no role on L-DOPA uptake. It is concluded that Caco-2 cells take up L-DOPA over the apical cell border through the sodium-independent and pH-sensitive L-type amino acid transporter.     

beta2-Adrenoceptor-mediated suppression of human intestinal mast cell functions is caused by disruption of filamentous actin dynamics

Previous studies indicated potent inhibitory effects of beta2-adrenoceptor (beta2AR) activation on the immunological mediator release of mast cells (MC). Here, we studied effects of beta2AR agonists on human MC mediator release, and in particular on MC proliferation, adhesion, and migration. MC were isolated from human intestinal mucosa, purified, and cultured in the presence of stem cell factor (SCF). beta2AR activation by epinephrine, norepinephrine, and salbutamol suppressed the IgE receptor-dependent release of histamine, lipid mediators, and TNF-alpha, and inhibited SCF-dependent MC proliferation and migration. Moreover, beta2-adrenergic stimulation interfered with MC adhesion to fibronectin and human endothelial cells. Using fluorescent phallacidin, we found that beta2AR activation reduced the amount of filamentous actin (F-actin) within minutes, whereas MC stimulation by either IgE receptor cross-linking or SCF caused F-actin accumulation. Interestingly, this activation-induced F-actin increase was abolished by previous beta2-adrenergic stimulation. Finally, we demonstrated that disruption of the F-actin cytoskeleton by latrunculin B mimicked the effects of beta2AR agonists on MC adhesion and migration. Our results argue for an important role of F-actin interference in beta2AR-mediated MC inhibition. Furthermore, the data support the concept of neuroimmune interactions regulating intestinal MC distribution, density, and functionality in vivo.     

Interactions of Yersinia enterocolitica with polarized human intestinal Caco-2 cells

The in vitro interactions of Yersinia enterocolitica, Salmonella typhimurium and Escherichia coli with polarized human colonie carcinoma (Caco-2) cells are described. Invasion of a confluent Caco-2 cell monolayer by Yersinia and Salmonella took place within 4 h after contact, which was in marked contrast to E. coli which did not invade Caco-2 cells. Cytoplasmic extrusions developed on the apical membrane and indicated the site of entrance of bacteria into the Caco-2 cells. Intracellular Yersinia and Salmonella were surrounded by a vacuolar membrane. Single as well as multiple bacteria were enclosed within a single vacuole. At 6 h after contact some of the intracellular yersiniae were found free in the cytoplasm. Furthermore, morphological signs of degeneration of Caco-2 cells such as vacuolization and autophagy were observed. Caco-2 cells infected with Salmonella also showed degenerative changes but the salmonellae resided within membranebound vacuoles in contrast to Yersinia. These observations are in contrast to those described for the invasion of other cell lines (not derived from intestinal epithelium) by Yersinia and may reflect more closely the interactions between Yersinia and the intestinal epithelium during gastrointestinal infection.     

Cell association and invasion of Caco-2 cells by Campylobacter jejuni.

Adherence and invasion studies were conducted in monolayers of Caco-2 cells. Three-day-old monolayers were inoculated with Campylobacter jejuni 81-176 at a bacterium/cell ratio of 1,000:1. Saturation studies demonstrated time- and dose-dependent saturation curves for C. jejuni cell association and invasion into Caco-2 cells. Electron microscopy revealed intracellular C. jejuni located within membrane-bound vacuoles. Cell association and invasion were inhibited by 0.3 and 0.5 M concentrations of various sugars, including D-glucose, D-mannose, and D-fucose. However, there was no inhibition with the corresponding L-sugars, indicating physiological specificity. The inhibition of cell association with phloridzin was less pronounced. There was no inhibition of bacterial entry with monodansylcadaverine or g-strophanthin, indicating that it was unlikely that coated-pit formation is important in the invasion of C. jejuni into Caco-2 cells. Furthermore, there was no inhibition with cytochalasin D, vincristine, or vinblastine. Inhibition of cell association was demonstrated at 4 degrees C. Significantly decreased cell association and invasion were seen in potassium-depleted cells. Treatment of cells with bromelain also caused reduction in the number of C. jejuni binding to cells. A nonmotile aflagellate variant of C. jejuni also showed reduced invasion. The results of this study are consistent with energy-dependent invasion mechanisms. The results do not support an endocytic method of invasion for C. jejuni into Caco-2 cells.     

Efflux properties of basolateral peptide transporter in human intestinal cell line Caco-2

Small peptides and some pharmacologically active compounds are absorbed from the small intestine by the apical H⁺-coupled peptide transporter 1 (PEPT1) and the basolateral peptide transporter. Here we investigated the efflux properties of the basolateral peptide transporter in Caco-2 cells using two strategies, efflux measurements and a kinetic analysis of transepithelial transport of glycylsarcosine (Gly-Sar). [¹⁴C]Gly-Sar efflux through the basolateral membrane was not affected significantly by the external pH. Both approaches revealed that the basolateral peptide transporter was saturable in the efflux direction, and that the affinity was lower than that in the influx direction. For two peptide-like drugs, there was no difference in substrate recognition by the basolateral peptide transporter between the two sides of the membrane. Using the kinetic parameters of PEPT1 and the basolateral peptide transporter, a computational model of Gly-Sar transport in Caco-2 cells was constructed. The simulation fitted the experimental data well. Our findings suggested that substrate affinity of the basolateral peptide transporter is apparently asymmetric, but pH-dependence and substrate specificity are symmetric for the two directions of transport. The behaviour of Gly-Sar in Caco-2 cells could be predicted by a mathematical model describing the peptide transporters.     

Apical organelle discharge by Cryptosporidium parvum is temperature, cytoskeleton, and intracellular calcium dependent and required for host cell invasion

The apical organelles in apicomplexan parasites are characteristic secretory vesicles containing complex mixtures of molecules. While apical organelle discharge has been demonstrated to be involved in the cellular invasion of some apicomplexan parasites, including Toxoplasma gondii and Plasmodium spp., the mechanisms of apical organelle discharge by Cryptosporidium parvum sporozoites and its role in host cell invasion are unclear. Here we show that the discharge of C. parvum apical organelles occurs in a temperature-dependent fashion. The inhibition of parasite actin and tubulin polymerization by cytochalasin D and colchicines, respectively, inhibited parasite apical organelle discharge. Chelation of the parasite's intracellular calcium also inhibited apical organelle discharge, and this process was partially reversed by raising the intracellular calcium concentration by use of the ionophore A23187. The inhibition of parasite cytoskeleton polymerization by cytochalasin D and colchicine and the depletion of intracellular calcium also decreased the gliding motility of C. parvum sporozoites. Importantly, the inhibition of apical organelle discharge by C. parvum sporozoites blocked parasite invasion of, but not attachment to, host cells (i.e., cultured human cholangiocytes). Moreover, the translocation of a parasite protein, CP2, to the host cell membrane at the region of the host cell-parasite interface was detected; an antibody to CP2 decreased the C. parvum invasion of cholangiocytes. These data demonstrate that the discharge of C. parvum sporozoite apical organelle contents occurs and that it is temperature, intracellular calcium, and cytoskeleton dependent and required for host cell invasion, confirming that apical organelles play a central role in C. parvum entry into host cells.     

Surfactants enhance the tight-junction permeability of food allergens in human intestinal epithelial Caco-2 cells

BACKGROUND: Food additives are responsible for certain allergic types of symptoms. Here Caco-2 cell monolayers were used as a model of the intestinal epithelium for the study of the effect of a food grade surfactant. We determined whether or not the presence of a surfactant enhances the transportation of food allergens across human intestinal epithelial Caco-2 cells. METHODS: This study investigated sucrose monoester fatty acids, which are a food grade surfactant. As an in vitro model of human epithelial cells, Caco-2 cells were grown in monolayers and exposed to different doses of the surfactant in conjunction with ovomucoid, a major egg white allergen. The integrity of the monolayer was assessed by measuring transepithelial electrical resistance (TEER). The permeability of tight junctions and transport of the antigen were studied. RESULTS: TEER correlated with the permeability of tight junctions. TEER significantly decreased upon exposure to a surfactant, indicating an increase in ovomucoid permeability without degradation. The surfactant induced shortening in microvilli, actin disbandment and structural separation of tight junctions. The results indicate that food grade surfactants can increase the paracellular uptake of food allergens.     

Campylobacter jejuni motility and invasion of Caco-2 cells.

We investigated the influence of motility on Campylobacter jejuni binding and invasion of Caco-2 cells. C. jejuni was motile in soft agar at basic (pH 8.5) and neutral pH values representative of the intestinal environment. However, C. jejuni was immobilized at pH 5.0. The inability of C. jejuni to swarm on soft agar at pH 5.0 was not related to flagellar depolymerization or loss of viability. In tissue culture medium, C. jejuni displayed typical periods of straight swimming punctuated by tumbling behavior. This behavior was altered when the viscosity of the medium was adjusted to mimic the viscosity of intestinal mucus. C. jejuni showed longer periods of straight swimming with significantly increased velocity followed by pauses instead of tumbles. The binding and invasion of C. jejuni in Caco-2 cells also increased significantly in high-viscosity growth medium. We speculate that the swimming behavior of C. jejuni in a viscous environment may be an important factor in the interaction of these organisms with host epithelial cells. The pH, which affects C. jejuni motility, may also influence the tropism of these organisms.