Pili Binding to Asialo-GM1 on Epithelial Cells Can Mediate Cytotoxicity or Bacterial Internalization by Pseudomonas aeruginosa

The interaction of Pseudomonas aeruginosa type IV pili and the glycosphingolipid asialo-GM1 (aGM1) can mediate bacterial adherence to epithelial cells, but the steps subsequent to this adherence have not been elucidated. To investigate the result of the interaction of pili and aGM1, we used polarized epithelial monolayers of Madin-Darby canine kidney (MDCK) cells in culture, which contained little detectable aGM1 on their apical surface but were able to incorporate exogenous aGM1. Compared to an untreated monolayer, P. aeruginosa PA103 displayed an eightfold increase in association with and fivefold more cytotoxicity toward MDCK cells pretreated with aGM1. Cytotoxicity of either carrier-treated or aGM1-treated monolayers required the type III secreted protein ExoU. Asialo-GM1 pretreatment of MDCK monolayers likewise augmented bacterial internalization of an isogenic invasive strain approximately fourfold. These increases were not seen in monolayers treated with GM1, the sialyated form of the glycolipid, and were inhibited by treatment with an antibody to aGM1. Also, the aGM1-mediated adhesion, cytotoxicity, and internalization required intact type IV pili since nonpiliated PA103 mutants were unaffected by aGM1 pretreatment of MDCK cells. These results demonstrate that epithelial cell injury and bacterial internalization can proceed from the same adhesin-receptor interaction, and they indicate that P. aeruginosa exoproducts solely determine the steps subsequent to adhesion.     

The Pseudomonas aeruginosa PhoP-PhoQ Two-Component Regulatory System Is Induced upon Interaction with Epithelial Cells and Controls Cytotoxicity and Inflammation

The adaptation of Pseudomonas aeruginosa to its environment, including the host, is tightly controlled by its network of regulatory systems. The two-component regulatory system PhoPQ has been shown to play a role in the virulence and polymyxin resistance of P. aeruginosa as well as several other Gram-negative species. Dysregulation of this system has been demonstrated in clinical isolates, yet how it affects virulence of P. aeruginosa is unknown. To investigate this, an assay was used whereby bacteria were cocultured with human bronchial epithelial cells. The interaction of wild-type (WT) bacteria that had adhered to epithelial cells led to a large upregulation of the expression of the oprH-phoP-phoQ operon and its target, the arn lipopolysaccharide (LPS) modification operon, in a PhoQ-dependent manner, compared to cells in the supernatant that had failed to adhere. Relative to the wild type, a phoQ mutant cocultured on epithelial cells produced less secreted protease and lipase and, like the phoQ mutant, piv, lipH, and lasB mutants demonstrated reduced cytotoxicity toward epithelial cells. Mutation in phoQ also resulted in alterations to lipid A and to increased inflammatory LPS. These data indicate that mutation of phoQ results in a phenotype that is similar to the less virulent but more inflammatory phenotype of clinical strains isolated from chronic-stage cystic fibrosis lung infections.     

Differential Sensitivity of Human Epithelial Cells to Pseudomonas aeruginosa Exoenzyme S

Exoenzyme S (ExoS) is an ADP-ribosyltransferase produced and directly translocated into eukaryotic cells by the opportunistic pathogen Pseudomonas aeruginosa. Model systems that allow bacterial translocation of ExoS have found ExoS to have multiple effects on eukaryotic cell function, affecting DNA synthesis, actin cytoskeletal structure, and cell matrix adherence. To understand mechanisms underlying differences observed in cell sensitivities to ExoS, we examined the effects of bacterially translocated ExoS on multiple human epithelial cell lines. Of the cell lines examined, confluent normal kidney (NK) epithelial cells were most resistant to ExoS, while tumor-derived cell lines were highly sensitive to ExoS. Analysis of the mechanisms of resistance indicated that cell association as well as an intrinsic resistance to morphological alterations were associated with increased resistance to ExoS. Conversely, increased sensitivity to ExoS appeared to be linked to epithelial cell growth, with tumor cells capable of undergoing non-contact-inhibited, anchorage-independent growth all being sensitive to ExoS, and NK cells becoming sensitive to ExoS when subconfluent and growing. Consistent with the possibility that growth-related, actin-based structures are involved in sensitivity to ExoS, scanning electron microscopy revealed cellular extensions from sensitive, growing cells to bacteria, which were not readily evident in resistant cells. In all studies, the severity of effects of ExoS on cell function directly correlated with the degree of Ras modification, indicating that sensitivity to ExoS in some manner related to the efficiency of ExoS translocation and its ADP-ribosylation of Ras. Our results suggest that factors expressed by growing epithelial cells are required for the bacterial contact-dependent translocation of ExoS; as normal epithelial cells differentiate into polarized confluent monolayers, expression of these factors is altered, and cells in turn become more resistant to the effects of ExoS.     

Pseudomonas aeruginosa Induces Type-III-Secretion-Mediated Apoptosis of Macrophages and Epithelial Cells

Pseudomonas aeruginosa is a gram-negative opportunistic pathogen that is cytotoxic towards a variety of eukaryotic cells. To investigate the effect of this bacterium on macrophages, we infected J774A.1 cells and primary bone-marrow-derived murine macrophages with the P. aeruginosa strain PA103 in vitro. PA103 caused type-III-secretion-dependent killing of macrophages within 2 h of infection. Only a portion of the killing required the putative cytotoxin ExoU. By three criteria, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assays, cytoplasmic nucleosome assays, and Hoechst staining, the ExoU-independent but type-III-secretion-dependent killing exhibited features of apoptosis. Extracellular bacteria were capable of inducing apoptosis, and some laboratory and clinical isolates of P. aeruginosa induced significantly higher levels of this form of cell death than others. Interestingly, HeLa cells but not Madin-Darby canine kidney cells were susceptible to type-III-secretion-mediated apoptosis under the conditions of these assays. These findings are consistent with a model in which the P. aeruginosa type III secretion system transports at least two factors that kill macrophages: ExoU, which causes necrosis, and a second, as yet unidentified, effector protein, which induces apoptosis. Such killing may contribute to the ability of this organism to persist and disseminate within infected patients.     

Relationship of Keratinocyte Growth Factor and Hepatocyte Growth Factor Levels in Rat Lung Lavage Fluid to Epithelial Cell Regeneration after Bleomycin

Keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) are known mitogens for normal alveolar Type 2 cells in vitro and in vivo. We wished to determine whether these two growth factors are involved in lung repair after epithelial cell necrosis by determining the levels of each factor in lung lavage fluid collected serially after bleomycin-induced injury, and how these values relate specifically to proliferation of bronchiolar and alveolar epithelial cells. Rats received an intratracheal injection of 1 unit bleomycin in 0.5 ml water and were killed at intervals up to 4 weeks with 1 muCi/g tritiated thymidine injected 1 hour before death. Early necrosis of bronchiolar epithelial (BR) cells and Type 1 alveolar epithelium was followed by an increase in inflammatory cell numbers and high protein levels in bronchoalveolar lavage (BAL) fluids. In addition, the levels of KGF and HGF, measured by enzyme-linked immunosorbent assay in BAL, increased as early as 3 days and peaked at 7-14 days, when KGF was measured at 160 pg/ml (n = 50) and HGF reached 460 pg/ml (n = 40). Both values dropped sharply after 2 weeks. Epithelial cell proliferation was quantitated as percentage of labeled cells in autoradiographs of methacrylate sections. Labeling of BR cells predominated in the first week and peaked at 7% at 3 days. Type 2 cell proliferation was delayed somewhat but occurred in 3 to 10 days with a peak of 7% labeled cells at 1 week. The results demonstrate that both HGF and KGF are present in the lung in greatly increased amounts soon after bleomycin-induced epithelial cell necrosis. These high levels are associated with both BR and alveolar epithelial cell proliferation.     

Macrophages and Epithelial Cells Respond Differently to the Pseudomonas aeruginosa Type III Secretion System

The multiple effects of Pseudomonas aeruginosa type III secretion have largely been attributed to variations in cytotoxin expression between strains. Here we show that the target cell type is also important. While lung epithelial cells showed significant changes in morphology but not viability when infected with P. aeruginosa, macrophages were efficiently killed by P. aeruginosa. Both responses were dependent on the type III secretion system.     

Defects in Type III Secretion Correlate with Internalization of Pseudomonas aeruginosa by Epithelial Cells

Previous characterization of Pseudomonas aeruginosa clinical isolates has demonstrated an inverse correlation between cytotoxicity and internalization by epithelial cells. To further investigate this relationship, we tested PA103, a cytotoxic P. aeruginosa strain, and 33 isogenic noncytotoxic transposon mutants for internalization by Madin-Darby canine kidney cells. The majority of the mutants were not internalized, demonstrating that an inverse correlation between cytotoxicity and bacterial uptake by epithelial cells is not absolute. Six of the noncytotoxic mutants, however, demonstrated measurable levels of internalization by standard aminoglycoside exclusion assays even though internalization of wild-type strain PA103 was not detectable. All six had evidence of protein secretion defects involving two proteins, a 40-kDa protein and a 32-kDa protein. These proteins, designated PepB (for Pseudomonas exoprotein B) and PepD, respectively, each had characteristics of type III transported proteins. In addition, nucleotide sequencing studies demonstrated that PepB and PepD are homologs of YopB and YopD, respectively, type III secreted proteins of Yersinia spp. necessary for the translocation of effector molecules into the cytoplasmic compartment of eukaryotic cells. Thus, while many mutations in PA103 result in loss of cytotoxicity without an appreciable increase in internalization, defects in transport of type III secretion proteins PepB and PepD correlate with both loss of cytotoxicity and gain of internalization. These results are consistent with type III secretion of an inhibitor of internalization that requires PepB and PepD for translocation into the host cell.     

Salmonella enterica Invasion of Polarized Epithelial Cells Is a Highly Cooperative Effort

The invasion of polarized epithelial cells by Salmonella enterica requires the cooperative activity of the Salmonella pathogenicity island 1 (SPI1)-encoded type III secretion system (T3SS) and the SPI4-encoded adhesin SiiE. The invasion of polarized cells is more efficient than that of nonpolarized cells, and we observed the formation of clusters of bacteria on infected cells. Here we demonstrate that the invasion of polarized cells is a highly cooperative activity. Using a novel live-cell imaging approach, we visualized the cooperative entry of multiple bacteria into ruffles induced on the apical surfaces of polarized cells. The induction of membrane ruffles by activity of Salmonella enables otherwise noninvasive mutant strains to enter polarized host cells. Bacterial motility and chemotaxis were of lower importance for cooperativity in polarized-cell invasion. We propose that cooperative invasion is a key factor for the very efficient entry into polarized cells and a factor contributing to epithelial damage and intestinal inflammation.     

Interruption of Multiple Cellular Processes in HT-29 Epithelial Cells by Pseudomonas aeruginosa Exoenzyme S

Exoenzyme S (ExoS), an ADP-ribosylating enzyme produced by the opportunistic pathogen Pseudomonas aeruginosa, is directly translocated into eukaryotic cells by bacterial contact. Within the cell, ExoS ADP-ribosylates the cell signaling protein Ras and causes inhibition of DNA synthesis and alterations in cytoskeletal structure. To further understand the interrelationship of the different cellular effects of ExoS, functional analyses were performed on HT-29 epithelial cells after exposure to ExoS-producing P. aeruginosa 388 and the non-ExoS-producing strain 388DeltaS. Two different mechanisms of morphological alteration were identified: (i) a more-transient and less-severe cell rounding caused by the non-ExoS-producing strain 388DeltaS and (ii) a more-severe, long-term cell rounding caused by ExoS-producing strain 388. Long-term effects of ExoS on cell morphology occurred in conjunction with ExoS-mediated inhibition of DNA synthesis and the ADP-ribosylation of Ras. ExoS was also found to cause alterations in HT-29 cell function, leading to the loss of cell adhesion and microvillus effacement. Nonadherent ExoS-treated cells remained viable but had a high proportion of modified Ras. While microvillus effacement was detected in both 388- and 388DeltaS-treated cells, effacement was more prevalent and rapid in cells exposed to strain 388. We conclude from these studies that ExoS can have multiple effects on epithelial cell function, with more severe cellular alterations associated with the enzymatic modification of Ras. The finding that ExoS had greater effects on cell growth and adherence than on cell viability suggests that ExoS may contribute to the P. aeruginosa infectious process by rendering cells nonfunctional.     

Susceptibility to Plasmodium yoelii Preerythrocytic Infection in BALB/c Substrains Is Determined at the Point of Hepatocyte Invasion

After transmission by Anopheles mosquitoes, Plasmodium sporozoites travel to the liver, infect hepatocytes, and rapidly develop as intrahepatocytic liver stages (LS). Rodent models of malaria exhibit large differences in the magnitude of liver infection, both between parasite species and between strains of mice. This has been mainly attributed to differences in innate immune responses and parasite infectivity. Here, we report that BALB/cByJ mice are more susceptible to Plasmodium yoelii preerythrocytic infection than BALB/cJ mice. This difference occurs at the level of early hepatocyte infection, but expression levels of reported host factors that are involved in infection do not correlate with susceptibility. Interestingly, BALB/cByJ hepatocytes are more frequently polyploid; thus, their susceptibility converges on the previously observed preference of sporozoites to infect polyploid hepatocytes. Gene expression analysis demonstrates hepatocyte-specific differences in mRNA abundance for numerous genes between BALB/cByJ and BALB/cJ mice, some of which encode hepatocyte surface molecules. These data suggest that a yet-unknown receptor for sporozoite infection, present at elevated levels on BALB/cByJ hepatocytes and also polyploid hepatocytes, might facilitate Plasmodium liver infection.     

Pseudomonas aeruginosa Invasion and Cytotoxicity Are Independent Events, Both of Which Involve Protein Tyrosine Kinase Activity

Pseudomonas aeruginosa clinical isolates exhibit invasive or cytotoxic phenotypes. Cytotoxic strains acquire some of the characteristics of invasive strains when a regulatory gene, exsA, that controls the expression of several extracellular proteins, is inactivated. exsA mutants are not cytotoxic and can be detected within epithelial cells by gentamicin survival assays. The purpose of this study was to determine whether epithelial cell invasion precedes and/or is essential for cytotoxicity. This was tested by measuring invasion (gentamicin survival) and cytotoxicity (trypan blue staining) of PA103 mutants deficient in specific exsA-regulated proteins and by testing the effect of drugs that inhibit invasion for their effect on cytotoxicity. A transposon mutant in the exsA-regulated extracellular factor exoU was neither cytotoxic nor invasive. Furthermore, several of the drugs that inhibited invasion did not prevent cytotoxicity. These results show that invasion and cytotoxicity are mutually exclusive events, inversely regulated by an exsA-encoded invasion inhibitor(s). Both involve host cell protein tyrosine kinase (PTK) activity, but they differ in that invasion requires Src family tyrosine kinases and calcium-calmodulin activity. PTK inhibitor drugs such as genistein may have therapeutic potential through their ability to block both invasive and cytotoxicity pathways via an action on the host cell.     

Invasion of Human Oral Epithelial Cells by Prevotella intermedia

Invasion of oral epithelial cells by pathogenic oral bacteria may represent an important virulence factor in the progression of periodontal disease. Here we report that a clinical isolate of Prevotella intermedia, strain 17, was found to invade a human oral epithelial cell line (KB), whereas P. intermedia 27, another clinical isolate, and P. intermedia 25611, the type strain, were not found to invade the cell line. Invasion was quantified by the recovery of viable bacteria following a standard antibiotic protection assay and observed by electron microscopy. Cytochalasin D, cycloheximide, monodansylcadaverine, and low temperature (4°C) inhibited the internalization of P. intermedia 17. Antibodies raised against P. intermedia type C fimbriae and against whole cells inhibited invasion, but the anti-type-C-fimbria antibody inhibited invasion to a greater extent than the anti-whole-cell antibody. This work provides evidence that at least one strain of P. intermedia can invade an oral epithelial cell line and that the type C fimbriae and a cytoskeletal rearrangement are required for this invasion.     

Liver Anti-Fibrosis Therapy with Mesenchymal Stem Cells Secreting Hepatocyte Growth Factor

Abstract

The objective of this study is to investigate the anti-fibrotic effect of combined mesencymal stem cells (MSCs) and gene therapy on liver fibrosis. When transfected by the complex with a plasmid DNA of hepatocyte growth factor (HGF) and the spermine-introduced pullulan of gene carrier, MSCs secreted HGF protein over 1 week. The HGF secreted from transfected MSC had the biological activity to promote the albumin production of hepatocytes. After intravenous injection, the HGF-secreting MSCs (HGF-MSC) accumulated in the liver. The injection of HGF-MSC decreased the fibrosis area in a rat model of liver fibrosis to a significantly great extent compared with that of original MSC. In the in vitro experiment, the higher number of HGF-transfected MSCs was migrated by stromal cell-derived factor (SDF)-1α more strongly than the original MSC. Considering the promotion of SDF-1α secretion in the liver fibrosis, it is possible that, when transplanted, genetically-engineered MSCs are accumulated in the liver due to their higher response to SDF-1α. It is concluded that the intravenous injection of genetically-engineered MSCs is a promising therapy for liver fibrosis.     

IL-18 Receptor Expression on Epithelial Cells is Upregulated by TNF Alpha

IL-18 is a multifunctional cytokine that augments both innate and acquired immunity and potentiates Th1 and Th2 reactions. We studied the expression of IL-18 receptor (IL-18R) on renal and respiratory epithelial cell lines. Both cell lines upregulated IL-18R mRNA and IL-18R membrane expression in response to TNF alpha and other proinflammatory cytokines. The function of IL-18R was confirmed by induction of IL-8 release from epithelial cells in response to recombinant IL-18. Epithelial cells may represent an important target for IL-18, mainly under inflammatory conditions associated with TNF alpha release.     

Adherence of Burkholderia pseudomallei Cells to Cultured Human Epithelial Cell Lines Is Regulated by Growth Temperature

We have investigated the adherence of Burkholderia pseudomallei, cultured under a number of different conditions, to six human epithelial cell lines. While several complex medium compositions had relatively little effect on adherence, growth at 30 degrees C was found to significantly increase adherence to all cell lines relative to that of cultures grown at 37 degrees C (P < 0.001).     

Vascular Permeability Factor of Pseudomonas aeruginosa

The production of vascular permeability factor (PF) by certain strains of Pseudomonas aeruginosa has been demonstrated in rabbits injected intradermally with culture filtrates followed by intravenous injection with Pontamine Sky Blue 6BX. The dose-response curve was found to be rectilinear when lesion diameters, within the range of 10 to 20 mm, were plotted against log dose. Thus, PF in test filtrates can be measured with reasonable accuracy by the concomitant testing of a reference PF. In contrast to the titers of PF obtained with Vibrio cholerae cultures, those with strains of P. aeruginosa were rather low. Thus far, PF has been demonstrated only in shallow still cultures of P. aeruginosa and not in shake cultures. A variety of commercial media were tested for the production of PF, but none was satisfactory. A synthetic medium that gave more reproducible and higher yields of PF was developed. Cultivation at 30 C generally gave higher yields of PF than at 37 C. PF was destroyed by heating at 60 C for 30 min or by digesting with trypsin or Pronase. Strains producing larger amounts of PF appeared to have greater virulence when inoculated onto the surface of burns in mice than those yielding little or no PF.     

Adherence to and Penetration of Human Intestinal Caco-2 Epithelial Cell Monolayers by Pseudomonas aeruginosa

Clinical isolates of Pseudomonas aeruginosa from blood adhered to and penetrated intestinal Caco-2 cell monolayers to a greater degree than did isolates from sputum, with a concomitant drastic decrease in transepithelial electrical resistance. PAO-PR1, an avirulent exotoxin A mutant of PAO1, did not cause a decrease in the resistance. The Caco-2 monolayer system may be useful for the evaluation of certain P. aeruginosa virulence factor activities.