Inside Epithelial Cells Research Articles

Evaluation of a tetracycline-inducible promoter in Staphylococcus aureus in vitro and in vivo and its application in demonstrating the role of sigB in microcolony formation.

An inducible promoter system provides a powerful tool for studying the genetic basis for virulence. A variety of inducible systems have been used in other organisms, including pXyl-xylR-inducible promoter, the pSpac-lacI system, and the arabinose-inducible P(BAD) promoter, but each of these systems has limitations in its application to Staphylococcus aureus. In this study, we demonstrated the efficacy of a tetracycline-inducible promoter system in inducing gene expression in S. aureus in vitro and inside epithelial cells as well as in an animal model of infection. Using the xyl/tetO promoter::gfp(uvr) fusion carried on a shuttle plasmid, we demonstrated that dose-dependent tetracycline induction, as measured by bacterial fluorescence, occurred in each of the above environments while basal activation under noninduced conditions remained low. To ascertain how the system can be used to elucidate the genetic basis of a pathogenic phenotype, we cloned the sigB gene downstream of the inducible promoter. Induction of SigB expression led to dose-dependent attachment of the tested strain to polystyrene microtiter wells. Additionally, bacterial microcolony formation, an event preceding mature biofilm formation, also increased with tetracycline induction of SigB.

Staphylococcus aureus RN6390 replicates and induces apoptosis in a pulmonary epithelial cell line.

Staphylococcus aureus frequently colonizes the airways of patients with compromised airway defenses (e.g., cystic fibrosis [CF] patients) for extended periods. Persistent and relapsing infections may be related to live S. aureus bacteria actively residing inside epithelial cells. In this study, we infected a respiratory epithelial cell line, which was derived from a CF patient, with S. aureus RN6390. Internalization of S. aureus was found to be time and dose dependent and could be blocked by cytochalasin D. Transmission electron microscopy revealed that internalized bacteria resided within endocytic vacuoles without any evidence of lysosomal fusion in a 24-h period. The results of internalization experiments and time-lapse fluorescence microscopy of epithelial cells infected with green fluorescent S. aureus indicate that, after an initial lag period of 7 to 9 h, intracellular bacteria began to replicate, with three to five divisions in a 24-h period, leading to apoptosis of infected cells. Induction of apoptosis required bacterial internalization and is associated with intracellular replication. The slow and gradual replication of S. aureus inside epithelial cells hints at the role of host factors or signals in bacterial growth and further suggests possible cross talk between host cells and S. aureus.

Immunologic, microscopic, and molecular evidence of Encephalitozoon intestinalis (Septata intestinalis) infection in mammals other than humans.

Encephalitozoon intestinalis (Septata intestinalis) is the second most prevalent microsporidian species infecting humans, but it has not been described in other animal species. This investigation examined 10 domestic animal stool samples (8 mammalian, 2 avian) containing spores detected by anti-Encephalitozoon monoclonal antibody immunofluorescence (FA). The presence of E. intestinalis but not Encephalitozoon hellem or Encephalitozoon cuniculi was confirmed in 6 of 8 mammalian stool samples by species-specific FA and polymerase chain reaction. Clusters of spores inside epithelial cells were observed in feces of five mammals (donkey, dog, pig, cow, and goat) using "quick-hot" Gram-chromotrope stain. None of the 10 samples reacted with anti-E. hellem or anti-E. cuniculi sera, nor were they amplified with species-specific primers for E. hellem and E. cuniculi. To our knowledge, this is the first identification of E. intestinalis in animals other than humans. The data shown herein suggest the possibility that E. intestinalis infection may be zoonotic in origin.

Detection of P-glycoprotein mediated MDR/MXR in Caranus maenas hepatopancreas by immuno-gold-silver labeling

Carcinus maenas were sampled in the German Bight for immunohistochemistry, histology and parallel chemical analysis of organochlorines. Incubations were performed with a monoclonal antibody against P-glycoprotein which mediates multixenobiotic resistance in order to investigate whether Carcinus maenas develop MXR in hepatopancreas. The data showed that MXR was highly expressed at the microvilli of epithelial cells while inside epithelial cells of the tubules MXR was restricted to a specific cell type, the transitional stage between fibrillar (F) cells and blister (B) cells. These cells were rich in lysosomes as was demonstrated by high acid phosphatase activity. In aging B cells, fusion events of the lysosomal compartment to one large vacuole were observed to be accompanied by a loss of MXR protein content. Subsequently, B cells were eliminated from the epithelium into the lumina where the cell debris was incorporated into the faeces. It is concluded that F/B cells appear to be specialized for accumulation of toxic compounds and their digestion and elimination.

Staphylococcus aureus Invasion of Bovine Mammary Epithelial Cells

Staphylococcus aureus is a frequent cause of mastitis in dairy cows. However, pathogenesis of the infection has not been completely defined. We report the invasion of two strains of S. aureus into a bovine mammary epithelial cell line and a bovine mammary epithelial cell primary culture. Invasion of S. aureus into bovine mammary cells was time-dependent. Transmission electron microscopy of bovine mammary cells invaded by S. aureus showed intracellular replication of the bacterium within membrane-bound vacuoles. Invasion was reduced significantly when bovine mammary epithelial cells were treated with inhibitors of F-actin microfilament polymerization but not when these cells were treated with inhibitors of microtubule formation. Results indicated that S. aureus is capable of invading and replicating inside bovine mammary epithelial cells. Data also suggested that S. aureus invasion of bovine mammary epithelial cells requires active participation of specific components of the cytoskeleton of the epithelial cell.

Invasion of Bovine Mammary Epithelial Cells by Streptococcus dysgalactiae

Two strains of Streptococcus dysgalactiae, isolated from cows with mastitis, were assayed for their ability to invade, multiply, and induce damage to a bovine mammary epithelial cell line, MAC-T. Invasion of S. dysgalactiae into MAC-T cells was time-dependent, and invasion was inhibited in a dosedependent manner by cytochalasin B and D, but not by colchicine. The invasion process did not appear to affect viability of mammary epithelial cells, but cellular damage was induced, as indicated by a time-dependent release of increasing amounts of lactate dehydrogenase. No net intracellular bacterial growth was observed, but S. dysgalactiae survived inside MAC-T cells. These results indicated that S. dysgalactiae invaded epithelial cells, induced cellular damage, and was capable of persisting inside bovine mammary epithelial cells.

Targeting of Salmonella typhimurium to vesicles containing lysosomal membrane glycoproteins bypasses compartments with mannose 6-phosphate receptors.

Salmonella typhimurium is an intracellular bacterial pathogen that remains enclosed in vacuoles (SCV) upon entry into the host cell. In this study we have examined the intracellular trafficking route of S. typhimurium within epithelial cells. Indirect immunofluorescence analysis showed that bacteria initiated fusion with lysosomal membrane glycoprotein (lgp)-containing compartments approximately 15 min after bacterial internalization. This process was completed approximately 75 min later and did not require microtubules. Cation-independent (CI)- or cation-dependent (CD)-mannose 6-phosphate receptors (M6PRs) were not observed at detectable levels in SCV. Lysosomal enzymes showed a different distribution in SCV: lysosomal-acid phosphatase (LAP) was incorporated into these vacuoles with the same kinetics as lgps, while cathepsin D was present in a low proportion (approximately 30%) of SCV. Uptake experiments with fluid endocytic tracers such as fluorescein-dextran sulphate (F-DX) or horseradish-peroxidase (HRP) showed that after 2 h of uptake, F-DX was present in approximately 75% of lgp-containing vesicles in uninfected cells, while only approximately 15% of SCV contained small amounts of the tracer during the same uptake period. SCV also showed only partial fusion with HRP-preloaded secondary lysosomes, with approximately 30% of SCV having detectable amounts of HRP at 6 h after infection. These results indicate that SCV show limited accessibility to fluid endocytic tracers and mature lysosomes, and are therefore functionally separated from the endocytic route. Moreover, the unusual intracellular trafficking route of S. typhimurium inside epithelial cells has allowed us to establish the existence of two different lgp-containing vesicles in Salmonella-infected cells: one population is separated from the endocytic route, fusogenic with incoming SCV and may arise from a secretory pathway, while the second involves the classical secondary or mature lysosomes.

Enzymatic digestion increases permeability of the outer blood-retinal barrier for high-molecular-weight substances

The purpose of the study was to investigate whether lysosomal enzymes can participate in damaging the outer blood-retinal barrier and to examine the role of glycosaminoglycans in maintaining the barrier function for high-molecular-weight substances. The ciliary artery was cannulated in freshly enucleated pig eyes. Perfusion was performed with buffer (controls), with heparinase (substrate: heparan sulfate), or with lysosomal enzymes freshly prepared from pig retinal pigment epithelium at 36 degrees C, followed by perfusion with the tracer native ferritin (NF) or the marker cationized ferritin (CF). The eyes were examined by electron microscopy. In controls treated with buffer alone, NF was found in high concentration in the lumina of the choroidal capillaries; however, little NF was found in Bruch's membrane (BsM). The tracer did not penetrate to any extent beyond BsM. In eyes digested with heparinase or lysosomal enzymes, significantly higher numbers of tracer molecules were found in BsM. Furthermore, NF penetrated BsM and was apparent in the subretinal space and also inside retinal pigment epithelial cells, probably due to pinocytosis. The results indicate that heparan sulfate proteoglycan is important for the maintenance of the outer blood-retinal barrier and that lysosomal proteases may participate in damaging this barrier, causing increased permeability to high-molecular-weight substances.

Effect of endocytosis inhibitors on alveolar clearance of albumin, immunoglobulin G, and SP-A in rabbits

Protein in the alveolar space may be cleared by endocytosis and degradation inside alveolar epithelial cells, by transcytosis across the alveolar epithelium, or by restricted diffusion through the epithelium. The relative contributions of these three pathways to clearance of large quantities of protein from the air spaces is not known. This study investigated the effects of monensin and nocodazole, agents which inhibit endocytosis in cell culture, on alveolar epithelial protein transport in anesthetized rabbits. There was evidence that monensin and nocodazole inhibited endocytosis by the alveolar epithelium in vivo. Nocodazole increased the number of vesicles in the alveolar epithelium and capillary endothelium. Monensin increased vesicle density in the endothelium. These results suggested that the inhibitors disrupted microtubules or interrupted cellular membrane traffic in the lung. Both inhibitors decreased lung parenchymal uptake of immunoreactive human albumin from the air spaces. Monensin and nocodazole inhibited albumin uptake in cultured alveolar type II cells. Monensin increased the amount of 125I-labeled surfactant protein A associated with the lungs, compared with the quantity remaining in the air space 2 h after instillation. Although the drugs decreased alveolar epithelial protein uptake, they did not decrease alveolar clearance of 125I-labeled immunoglobulin G or 131I-labeled albumin in anesthetized rabbits. Thus monensin- and nocodazole-sensitive protein-uptake pathways do not account for most alveolar protein clearance when the distal air spaces are filled with a protein solution.

Intracellular replication is essential for the virulence of Salmonella typhimurium.

Salmonella typhimurium is a facultative intracellular parasite, capable of penetrating, surviving, and multiplying within diverse eukaryotic cell types, including epithelial and phagocytic cells. We have been studying intracellular replication of S. typhimurium and found that it is essential in the pathogenesis of this bacterium. A total of 45,000 independent mini-Mu MudJ transposon mutants in S. typhimurium SL1344 were screened in Madin-Darby canine kidney (MDCK) epithelial cells with a beta-lactam, cefotaxime, to enrich for mutants defective for intracellular replication. Ten different auxotrophic (purine, pyrimidine, purine/methionine, and valine/isoleucine) and three prototrophic replication-defective mutants (Rep-) were identified. All Rep- mutants showed no differences in aerobic and anaerobic growth patterns, motility, serum sensitivity, mouse macrophage survival, iron uptake, and phosphate requirements. All Rep- mutants were unable to multiply inside MDCK, HeLa, and Caco-2 epithelial cells. When required nutrients for various auxotrophs were supplemented, auxotrophs then replicated inside MDCK cells. Although the parental strain multiplies in large vacuoles inside MDCK cells that distort the host cells, MDCK cells infected with the Rep- mutants appeared relatively normal and few bacteria were seen inside vacuoles. The purine auxotrophs and the three prototrophic Rep- mutants were highly attenuated in mice, and oral and intraperitoneal LD50 levels were 3 to 4 orders of magnitude higher than the wild type level. The three prototrophs were invasive and persisted in the murine organs such as livers and spleens for at least 3 weeks. Therefore, these prototrophic genes are needed for intracellular replication and are essential to the virulence of S. typhimurium.