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.     

Role of the Corneal Epithelial Basement Membrane in Ocular Defense against Pseudomonas aeruginosa

Pseudomonas aeruginosa can invade corneal epithelial cells and translocates multilayered corneal epithelia in vitro, but it does not penetrate the intact corneal epithelium in vivo. In healthy corneas, the epithelium is separated from the underlying stroma by a basement membrane containing extracellular matrix proteins and pores smaller than bacteria. Here we used in vivo and in vitro models to investigate the potential of the basement membrane to defend against P. aeruginosa. Transmission electron microscopy of infected mouse corneas in vivo showed penetration of the stroma by P. aeruginosa only where the basement membrane was visibly disrupted by scratch injury, suggesting that the intact basement membrane prevented penetration. This hypothesis was explored using an in vitro Matrigel Transwell model to mimic the corneal basement membrane. P. aeruginosa translocation of multilayered corneal epithelia grown on Matrigel was ∼100-fold lower than that of cells grown without Matrigel (P < 0.005, t test). Matrigel did not increase transepithelial resistance. Matrigel-grown cells blocked translocation by a P. aeruginosa protease mutant. Without cells, Matrigel also reduced traversal of P. aeruginosa and the protease mutant. Fluorescence microscopy revealed a relative accumulation of bacteria at the superficial epithelium of cells grown on Matrigel at 3 h compared to cells grown on uncoated filters. By 5 h, bacteria accumulated beneath the cells, suggesting direct trapping by the Matrigel. These findings suggest that the basement membrane helps defend the cornea against infection via physical barrier effects and influences on the epithelium and that these roles could be compromised by P. aeruginosa proteases.Pseudomonas aeruginosa is an important opportunistic pathogen, commonly affecting burn victims, individuals with cystic fibrosis, patients in hospital intensive care units, and contact lens wearers (9-11). In the absence of contact lens wear, the cornea is remarkably resistant to infection, with P. aeruginosa effectively colonizing this tissue only if it is injured or otherwise compromised (41). To initiate clinically significant corneal pathology, P. aeruginosa (and almost all other microbes) must first access the corneal stroma, which is normally protected by a multilayered epithelium and associated basement membrane (1).The corneal epithelial basement membrane is secreted by the overlying epithelia and is comprised of sheets of extracellular matrix constituents, including type IV collagen, heparan sulfate proteoglycan, and various glycoproteins (laminin, entactin, nidogen, and fibronectin) and growth factors that mediate cellular function (1). Quantitative imaging of the corneal epithelial basal membrane in the rhesus macaque has shown a complex cross-linking of fibers and proteins intermingled with pores ranging from 30 to 400 nm in size (2). As shown for other basement membranes in the body (17, 19, 33), the corneal basement membrane anchors epithelial cells and provides positional information for healing, tissue regeneration, and repair (44). In vitro, Matrigel forms an artificial basement membrane with pores ranging from 26 to 359 nm in size, is composed of laminin, collagen IV, heparan sulfate proteoglycans, entactin, nidogen, and naturally occurring growth factors, and closely resembles the natural corneal basement membrane (2, 26).It has been shown that P. aeruginosa isolates can translocate MDCK cell monolayers (6, 22) and that translocation and virulence were reduced by mutation of genes encoding multidrug resistance efflux systems (23). Purified elastase and exotoxin A from P. aeruginosa have each been shown to increase the permeability of MDCK cell monolayers (4), and purified elastase increases alveolar permeability in vivo (5). However, the role of the basement membrane in P. aeruginosa translocation has not been studied.We have previously shown that P. aeruginosa can translocate multilayered corneal epithelia in vitro and that human tear fluid reduced both translocation in vitro and virulence in vivo in murine models of corneal infection (28). While this and other studies have focused on the role of the tear film and corneal epithelium in defense against P. aeruginosa keratitis (14), little attention has been given to the basement membrane. Previous studies have reported that the epithelial basement membranes of other tissues can form a physical barrier to potential pathogens, including human papillomavirus, herpes simplex virus, and Rift Valley fever virus (25, 42, 43, 47). In this study, it was hypothesized that the corneal basement membrane forms a physical barrier to defend against the penetration of P. aeruginosa, since its pores are smaller than the size of bacteria, and that P. aeruginosa proteases can functionally overcome that defense. This hypothesis was examined correlatively using transmission electron microscopy of in vivo-infected mouse corneas and tested directly using a quantitative in vitro Matrigel-based model system to mimic the corneal epithelium and its associated basement membrane.     

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.     

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.     

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.     

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.     

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.     

Susceptibility of Epithelial Cells to Pseudomonas aeruginosa Invasion and Cytotoxicity Is Upregulated by Hepatocyte Growth Factor

Normal cell polarity protects epithelial cells against Pseudomonas aeruginosa invasion and cytotoxicity. Using epithelial cell clones with selective defects in sorting of membrane constituents, and using hepatocyte growth factor pretreatment, we found that polarized susceptibility to P. aeruginosa can be altered without disrupting tight junctions. The results also showed that cellular susceptibility factors for invasion and cytotoxicity are not the same, although both are localized to the basolateral cell surface in polarized epithelial cells.     

Examining the Role of Actin-Plasma Membrane Association in Pseudomonas aeruginosa Infection and Type III Secretion Translocation in Migratory T24 Epithelial Cells

The opportunistic pathogen Pseudomonas aeruginosa targets wounded epithelial barriers, but the cellular alteration that increases susceptibility to P. aeruginosa infection remains unclear. This study examined how cell migration contributes to the establishment of P. aeruginosa infections using (i) highly migratory T24 epithelial cells as a cell culture model, (ii) mutations in the type III secretion (T3S) effector ExoS to manipulate P. aeruginosa infection, and (iii) high-resolution immunofluorescent microscopy to monitor ExoS translocation. ExoS includes both GTPase-activating (GAP) and ADP-ribosyltransferase (ADPRT) activities, and P. aeruginosa cells expressing wild-type ExoS preferentially bound to the leading edge of T24 cells, where ExoS altered leading-edge architecture and actin anchoring in conjunction with interrupting T3S translocation. Inactivation of ExoS GAP activity allowed P. aeruginosa to be internalized and secrete ExoS within T24 cells, but as with wild-type ExoS, translocation was limited in association with disruption of actin anchoring. Inactivation of ExoS ADPRT activity resulted in significantly enhanced T3S translocation by P. aeruginosa cells that remained extracellular and in conjunction with maintenance of actin-plasma membrane association. Infection with P. aeruginosa expressing ExoS lacking both GAP and ADPRT activities resulted in the highest level of T3S translocation, and this occurred in conjunction with the entry and alignment of P. aeruginosa and ExoS along actin filaments. Collectively, in using ExoS mutants to modulate and visualize T3S translocation, we were able to (i) confirm effector secretion by internalized P. aeruginosa, (ii) differentiate the mechanisms underlying the effects of ExoS GAP and ADPRT activities on P. aeruginosa internalization and T3S translocation, (iii) confirm that ExoS ADPRT activity targeted a cellular substrate that interrupted T3S translocation, (iv) visualize the ability of P. aeruginosa and ExoS to align with actin filaments, and (v) demonstrate an association between actin anchoring at the leading edge of T24 cells and the establishment of P. aeruginosa infection. Our studies also highlight the contribution of ExoS to the opportunistic nature of P. aeruginosa infection through its ability to exert cytotoxic effects that interrupt T3S translocation and P. aeruginosa internalization, which in turn limit the P. aeruginosa infectious process.     

Interleukin-7 Produced by Intestinal Epithelial Cells in Response to Citrobacter rodentium Infection Plays a Major Role in Innate Immunity against This Pathogen

Interleukin-7 (IL-7) engages multiple mechanisms to overcome chronic viral infections, but the role of IL-7 in bacterial infections, especially enteric bacterial infections, remains unclear. Here we characterized the previously unexplored role of IL-7 in the innate immune response to the attaching and effacing bacterium Citrobacter rodentium. C. rodentium infection induced IL-7 production from intestinal epithelial cells (IECs). IL-7 production from IECs in response to C. rodentium was dependent on gamma interferon (IFN-γ)-producing NK1.1⁺ cells and IL-12. Treatment with anti-IL-7Rα antibody during C. rodentium infection resulted in a higher bacterial burden, enhanced intestinal damage, and greater weight loss and mortality than observed with the control IgG treatment. IEC-produced IL-7 was only essential for protective immunity against C. rodentium during the first 6 days after infection. An impaired bacterial clearance upon IL-7Rα blockade was associated with a significant decrease in macrophage accumulation and activation in the colon. Moreover, C. rodentium-induced expansion and activation of intestinal CD4⁺ lymphoid tissue inducer (LTi) cells was completely abrogated by IL-7Rα blockade. Collectively, these data demonstrate that IL-7 is produced by IECs in response to C. rodentium infection and plays a critical role in the protective immunity against this intestinal attaching and effacing bacterium.     

Interleukin-8 Production by Human Airway Epithelial Cells in Response to Pseudomonas aeruginosa Clinical Isolates Expressing Type a or Type b Flagellins

Pseudomonas aeruginosa lung infection is a major cause of morbidity and mortality worldwide. P. aeruginosa flagellin, the main structural protein of the flagellar filament, is a virulence factor with proinflammatory activity on respiratory epithelial cells. P. aeruginosa bacteria express one of two isoforms of flagellin (type a or b) that differ in their primary amino acid sequences as well as in posttranslational glycosylation. In this study, the distribution of type a and b flagellins among 3 P. aeruginosa laboratory strains and 14 clinical isolates (1 ulcerative keratitis, 3 cystic fibrosis, and 10 acute pneumonia isolates) was determined, and their abilities to stimulate interleukin-8 (IL-8) production by human airway epithelial cells was compared. By comparison with the PAK (type a) and PAO1 (type b) prototype laboratory strains, 10/14 (71.4%) of clinical isolates expressed type a and 4/14 (28.6%) expressed type b flagellins. Among four cell lines surveyed, BEAS-2B cells were found to give the greatest difference between constitutive and flagellin-stimulated IL-8 production. All 17 flagellins stimulated IL-8 production by BEAS-2B cells (range, 700 to 4,000 pg/ml). However, no discernible differences in IL-8 production were evident when comparing type a versus type b flagellins or flagellins from laboratory versus clinical strains or among the clinical strains.Pseudomonas aeruginosa is a Gram-negative, aerobic, rod-shaped bacterium with a unipolar flagellum. P. aeruginosa is a clinically important opportunistic human pathogen, and its respiratory tract infections are a leading cause of morbidity and mortality in patients with cystic fibrosis, ventilator-associated pneumonia, and compromised immune systems (6). Hospital-acquired pneumonia constitutes the second leading type of nosocomial infection, and P. aeruginosa is the most commonly isolated bacterium from these cases (36). P. aeruginosa lung colonization in cystic fibrosis patients induces a neutrophil-dominated airway inflammatory response that, if untreated, ultimately leads to lung failure and death (41). P. aeruginosa also causes severe eye and urinary tract infections in immunocompromised patients, particularly those with HIV, and in individuals with severe burn wounds (42). Despite antibiotic treatment, mortality rates as high as 40% may occur in acute infections, and multidrug-resistant isolates are increasingly reported (11).Respiratory epithelial cells play a crucial role in the inflammatory response during P. aeruginosa infection (33). Airway epithelial cells produce cytokines and chemokines that initiate and amplify host innate and adaptive immune responses following bacterial colonization. For example, epithelial cells exposed to P. aeruginosa produce interleukin-8 (IL-8), the major chemokine associated with neutrophil extravasation from the vasculature into the lumen of the airways (17). IL-8 and neutrophils are present in increased amounts in the lungs of patients with P. aeruginosa infections (8). A diverse array of P. aeruginosa gene products stimulate IL-8 production by respiratory epithelial cells, including flagellin and pilin, the primary structural proteins of bacterial flagella and pili respectively (9).In addition to its ability to stimulate a proinflammatory host response, P. aeruginosa flagellin also constitutes a bacterial virulence factor. Multiple studies have demonstrated a role for P. aeruginosa flagella in the pathogenesis of experimental and clinical diseases (16, 22, 25). Using a burned-mouse model, nonflagellated P. aeruginosa strains expressing a mutant flagellin gene showed a significant decrease in virulence that was restored when flagellin expression was reinstated (29). Pulmonary infection of mice with P. aeruginosa devoid of flagella also resulted in reduced airway colonization and decreased mortality compared with those in mice infected with flagellated bacteria (12). Because flagella are one of the most immunostimulatory products of P. aeruginosa, it should be possible to modulate airway inflammation and reduce mortality using flagellin-based therapeutics without predisposing the host to invasive bacterial infection. However, to develop such therapies, the immunostimulatory bacterial component, as well as the epithelial cell responses that are activated, requires thorough characterization.Flagellins isolated from laboratory reference strains of P. aeruginosa have been classified as type a or b based upon molecular mass and reactivity with specific antisera (28). The type a flagellins have more variable molecular masses (45 to 52 kDa), whereas the type b proteins show an invariant size of about 53 kDa (1, 4). The discrepancy in sizes between type a and b flagellins results from differences in their primary amino acid sequences as well as in posttranslational glycosylation (4, 39, 43-45). The P. aeruginosa flagellar typing system was developed based upon the analysis of defined laboratory strains, and to our knowledge, clinical isolates, particularly from acute bacterial pneumonia patients, have not been extensively characterized in this manner. Therefore, the present study was undertaken to assess the distribution of type a and b flagellins among a panel of P. aeruginosa clinical isolates and to compare the abilities of the two protein isoforms to stimulate a proinflammatory response by respiratory epithelial cells.     

Pseudomonas aeruginosa Evasion of Phagocytosis Is Mediated by Loss of Swimming Motility and Is Independent of Flagellum Expression

Pseudomonas aeruginosa is a pathogenic Gram-negative bacterium that causes severe opportunistic infections in immunocompromised individuals; in particular, severity of infection with P. aeruginosa positively correlates with poor prognosis in cystic fibrosis (CF) patients. Establishment of chronic infection by this pathogen is associated with downregulation of flagellar expression and of other genes that regulate P. aeruginosa motility. The current paradigm is that loss of flagellar expression enables immune evasion by the bacteria due to loss of engagement by phagocytic receptors that recognize flagellar components and loss of immune activation through flagellin-mediated Toll-like receptor (TLR) signaling. In this work, we employ bacterial and mammalian genetic approaches to demonstrate that loss of motility, not the loss of the flagellum per se, is the critical factor in the development of resistance to phagocytosis by P. aeruginosa. We demonstrate that isogenic P. aeruginosa mutants deficient in flagellar function, but retaining an intact flagellum, are highly resistant to phagocytosis by both murine and human phagocytic cells at levels comparable to those of flagellum-deficient mutants. Furthermore, we show that loss of MyD88 signaling in murine phagocytes does not recapitulate the phagocytic deficit observed for either flagellum-deficient or motility-deficient P. aeruginosa mutants. Our data demonstrate that loss of bacterial motility confers a dramatic resistance to phagocytosis that is independent of both flagellar expression and TLR signaling. These findings provide an explanation for the well-documented observation of nonmotility in clinical P. aeruginosa isolates and for how this phenotype confers upon the bacteria an advantage in the context of immune evasion.Pseudomonas aeruginosa is an opportunistic Gram-negative bacterial pathogen that causes severe infections in immunocompromised patients and in the pulmonary compartment of patients suffering from cystic fibrosis (CF) (13, 14). In CF patients, disease severity is positively correlated with colonization by P. aeruginosa and the establishment of chronic infection. As part of the colonization process, the bacteria undergo a number of genetic changes that assist in their ability to survive in the mammalian host and to evade detection and clearance by the immune system (9, 21). One such change that has been phenotypically characterized for P. aeruginosa is loss of flagellar motility (12, 17). Furthermore, the loss of flagellar gene expression and motility function is associated with increased bacterial burdens and increased disease severity in CF patients (12, 17). While downregulation of flagellar expression has been inferred to confer a survival advantage on P. aeruginosa once it colonizes the host by evasion of both phagocytic receptors and TLR5-driven inflammatory signaling, the exact contribution of flagellum downregulation with respect to successful immune evasion is unclear (5, 17, 18).Nonopsonic phagocytosis of P. aeruginosa by murine and human macrophages has previously been reported to require the expression of a flagellum, and the interpretation of these results concluded that the flagellum is a necessary ligand for triggering phagocytic internalization of the bacteria (18). Furthermore, flagellar expression is reported to be critical for inducing inflammation during P. aeruginosa infection, and loss of flagellar gene expression results in impaired inflammatory responses and attenuated bacterial clearance (5). Here, we provide data that challenge the current paradigm that the flagellum functions as a primary phagocytic ligand for P. aeruginosa ingestion by immune cells with the formal demonstration that motility, rather than loss of flagellar expression, confers the advantage toward P. aeruginosa evasion of phagocytosis.In these studies, we use P. aeruginosa motility-defective mutants to assess the role of bacterial motility in regard to phagocytic recognition by innate immune cells. When present in an aqueous environment, P. aeruginosa can swim via rotation of a single polar, monotrichous flagellum (27); there is currently no evidence that P. aeruginosa bacteria produce lateral flagella or alter their cell morphology as a direct function of motility. For the purposes of this report, motility refers to flagellum-based bacterial movement in an aqueous environment unless specifically indicated. Of note, P. aeruginosa is also capable of a flagellum-independent type of motility termed twitching in which type IV pilus filaments that extend from the cell body adhere to a surface and then retract, thus propelling the bacterium forward (23). Bacterial flagellar motility occurs through a motor complex that provides energy for rotational torque of a helical filament of repeating flagellin subunits that act as a propeller. The rotor, a multimer complex composed of FliG, FliM, and FliN, acts as a molecular switch and determines clockwise or counterclockwise rotation (27). In P. aeruginosa, the stator complex, which provides a stationary housing for the rotor, is composed of at least four partially redundant integral membrane proteins, MotAB and MotCD. Deletion of all four stators allows for flagellar assembly, but the structure cannot rotate and so the mutant is nonmotile (swimming and swarming defective) (27).Previous reports have concluded that an intact flagellum is required for phagocytic recognition of P. aeruginosa (18). However, here we demonstrate that the phagocytic resistance exhibited by swimming motility-defective bacteria is not due to loss of flagellum-mediated activation of immune cells, since bacteria expressing a nonfunctional flagellum exhibit phagocytic resistance comparable to that of flagellum-deficient bacteria and loss of MyD88 signaling in phagocytic cells does not recapitulate this defect for phagocytosis. Rather, with the use of a variety of in vitro, ex vivo, and in vivo infection models, we show that loss of P. aeruginosa motility dramatically alters immune responses to these bacteria compared to those for motile isogenic bacterial strains and that it is the loss of flagellum-mediated motility, but not flagellum expression itself, that results in dramatic bacterial resistance to phagocytosis by murine and human phagocytes. These studies provide an explanation for the clinical observation that P. aeruginosa isolates obtained from CF hosts often exhibit a nonmotile phenotype and explain how this phenotype can confer a survival advantage for bacteria that modulate or lose their motility during an active infection.     

Modification of Ras in Eukaryotic Cells by Pseudomonas aeruginosa Exoenzyme S

Genetic and functional data suggest that Pseudomonas aeruginosa exoenzyme S (ExoS), an ADP-ribosyltransferase, is translocated into eukaryotic cells by a bacterial type III secretory mechanism activated by contact between bacteria and host cells. Although purified ExoS is not toxic to eukaryotic cells, ExoS-producing bacteria cause reduced proliferation and viability, possibly mediated by bacterially translocated ExoS. To investigate the activity of translocated ExoS, we examined in vivo modification of Ras, a preferred in vitro substrate. The ExoS-producing strain P. aeruginosa 388 and an isogenic mutant strain, 388ΔexoS, which fails to produce ExoS, were cocultured with HT29 colon carcinoma cells. Ras was found to be ADP-ribosylated during coculture with 388 but not with 388ΔexoS, and Ras modification by 388 corresponded with reduction in HT29 cell DNA synthesis. Active translocation by bacteria was found to be required, since exogenous ExoS, alone or in the presence of 388ΔexoS, was unable to modify intracellular Ras. Other ExoS-producing strains caused modification of Ras, indicating that this is not a strain-specific event. ADP-ribosylation of Rap1, an additional Ras family substrate for ExoS in vitro, was not detectable in vivo under conditions sufficient for Ras modification, suggesting possible ExoS substrate preference among Ras-related proteins. These results confirm that intracellular Ras is modified by bacterially translocated ExoS and that the inhibition of target cell proliferation correlates with the efficiency of Ras modification.     

Dectin-1 is Inducible and Plays an Essential Role for Mycobacteria-Induced Innate Immune Responses in Airway Epithelial Cells

Introduction  

Airway epithelial cells are the first cells to be challenged upon contact with mycobacteria. In response, they express pattern-recognition receptors that play fundamental roles as sentinels and mediators of pulmonary innate immunity. The c-type lectin Dectin-1 is expressed predominantly on the surface of myeloid lineage cells. In this study, we examined the induction, regulation, and functions of Dectin-1 in pulmonary epithelial cells.     

Role of lipopolysaccharide in virulence of Pseudomonas aeruginosa

The role of lipopolysaccharide (LPS) in the virulence of Pseudomonas aeruginosa was studied. The virulence of several P. aeruginosa strains for burned mice was found to be directly related to the dispersion of LPS into either the phenol or the water phase after extraction. Virulence decreased as the proportion of LPS recovered from the phenol phase increased. No similar correlation was observed when several other strain characteristics were investigated. This phenomenon was studied in greater detail by using the "smooth"-specific phage E79 to select mutants altered in LPS structure. One such mutant, PA220-R2, was extensively characterized. LPS isolated from PA220-R2 was found to be completely deficient in high-molecular-weight polysaccharide material. This alteration rendered the strain serum sensitive and dramatically changed the reaction with O-specific typing sera and sensitivity to typing phages. However, motility, toxin A and elastase production, and 22 metabolic functions remained unchanged. PA220-R2 was found to be comparatively nonvirulent, with a 50% lethal dose more than 1,000-fold higher than that of its parent for burned mice. This was due to the inability of PA220-R2 to establish an infection in burned skin.     

ExsB Is Required for Correct Assembly of the Pseudomonas aeruginosa Type III Secretion Apparatus in the Bacterial Membrane and Full Virulence In Vivo

Pseudomonas aeruginosa is responsible for high-morbidity infections of cystic fibrosis patients and is a major agent of nosocomial infections. One of its most potent virulence factors is a type III secretion system (T3SS) that injects toxins directly into the host cell cytoplasm. ExsB, a lipoprotein localized in the bacterial outer membrane, is one of the components of this machinery, of which the function remained elusive until now. The localization of the exsB gene within the exsCEBA regulatory gene operon suggested an implication in the T3SS regulation, while its similarity with yscW from Yersinia spp. argued in favor of a role in machinery assembly. The present work shows that ExsB is necessary for full in vivo virulence of P. aeruginosa. Furthermore, the requirement of ExsB for optimal T3SS assembly and activity is demonstrated using eukaryotic cell infection and in vitro assays. In particular, ExsB promotes the assembly of the T3SS secretin in the bacterial outer membrane, highlighting the molecular role of ExsB as a pilotin. This involvement in the regulation of the T3S apparatus assembly may explain the localization of the ExsB-encoding gene within the regulatory gene operon.     

Secretory Component Production by Polarized Epithelial Cells from the Human Female Reproductive Tract

At mucosal surfaces, the polymeric Ig receptor (pIgR) is responsible for transporting polymeric IgA across epithelial cells. The purpose of this study was to determine whether normal epithelial cells from the female reproductive tract form tight junctions and produce secretory component, the external domain of the pIgR. Uterine, cervical and vaginal tissues from women at different stages of the menstrual cycle and following menopause were used to prepare purified epithelial cell sheets, which were cultured in cell chambers. Transepithelial resistance was measured and the media from apical and basolateral compartments assayed for secretory component. Secretory component produced by uterine epithelial cells accumulated preferentially in apical compartment and correlated with increased transepithelial resistance. Seeding as epithelial sheets at 1×10⁶ cells/cm² of matrix coated cell chambers was required for growth. Epithelial cells from endo-cervix and ecto-cervix, but not the vagina, also showed preferential production and release of secretory component into the apical chamber. In conclusion, normal epithelial cells from the human female reproductive tract grow to confluence, become polarized and produce secretory component. Our results suggest that uterine and cervical epithelial cells play a key regulatory role in the control of IgA transcytosis from tissue into secretions.     

Virulence and the Role of Iron in Pseudomonas aeruginosa Infection

The virulence of Pseudomonas aeruginosa can be enhanced by passage in mice or rabbits. Enhanced virulence has some specificity for the host in which the passage is done. Experimental infection in the peritoneal cavity of cannulated rabbits has shown that the injection of iron compounds can lead to a rapid and fatal growth of an otherwise nonlethal dose of bacteria. In vitro the unsaturated iron-binding proteins present in the peritoneal fluid can halve the growth rate of P. aeruginosa. The restricted rate of growth is restored to normal if the iron-binding proteins are saturated with iron. Exactly the same results are achieved with purified transferrin. Both fatal and nonfatal infections with P. aeruginosa cause a sharp fall in the percentage of saturation with Fe of the plasma and peritoneal fluid. In both normal and infected animals the peritoneal fluid is invariably less saturated than the plasma. Specific antiserum not only protects against death but also against the fall in iron saturation of the plasma and peritoneal fluid. In both fatal and nonfatal infections a high proportion of viable bacteria are unphagocytized in the peritoneal cavity.