Bacteria use a variety of secreted virulence factors to manipulate host cells, thereby causing significant morbidity and mortality. We report a mechanism for the long-distance delivery of multiple bacterial virulence factors, simultaneously and directly into the host cell cytoplasm, thus obviating the need for direct interaction of the pathogen with the host cell to cause cytotoxicity. We show that outer membrane–derived vesicles (OMV) secreted by the opportunistic human pathogen Pseudomonas aeruginosa deliver multiple virulence factors, including β-lactamase, alkaline phosphatase, hemolytic phospholipase C, and Cif, directly into the host cytoplasm via fusion of OMV with lipid rafts in the host plasma membrane. These virulence factors enter the cytoplasm of the host cell via N-WASP–mediated actin trafficking, where they rapidly distribute to specific subcellular locations to affect host cell biology. We propose that secreted virulence factors are not released individually as naked proteins into the surrounding milieu where they may randomly contact the surface of the host cell, but instead bacterial derived OMV deliver multiple virulence factors simultaneously and directly into the host cell cytoplasm in a coordinated manner. Gram-negative pathogens are responsible for 2 million annual hospital-acquired infections, adding tremendously to U.S. healthcare costs. Pseudomonas aeruginosa, an opportunistic human pathogen, is commonly associated with nosocomial infections, particularly ventilator-associated infections and pseudomonal pneumonia in immunocompromised patients with cystic fibrosis, chronic obstructive pulmonary disease, ventilator-associated pneumonia, community-acquired pneumonia, and bronchiectasis. We have identified the mechanism for a secretion system that Gram-negative bacteria use to strategically deliver toxins to the host to promote bacterial virulence and host colonization, a pathway that we hope to target to develop new therapies to treat P. aeruginosa infections. Our findings have significant implications for the study of Gram-negative bacterial pathogenesis. We propose that secreted virulence factors are not released individually as naked proteins into the surrounding milieu where they may randomly contact the surface of the host cell, but instead bacterial-derived outer membrane vesicles (OMV) deliver multiple virulence factors simultaneously and directly into the host cell cytoplasm in a coordinated manner. This long-distance bacterial communication to the host via OMV is reminiscent of the delivery of signaling proteins and miRNA between eukaryotic cells via exosomes, and may represent a general protein secretion strategy used by both pathogen and host.

Enhanced antibiotic resistance of Pseudomonas aeruginosa in the cystic fibrosis (CF) lung is thought to be due to the formation of biofilms. However, there is no information on the antibiotic resistance of P. aeruginosa biofilms grown on human airway epithelial cells or on the effects of airway cells on biofilm formation by P. aeruginosa. Thus we developed a coculture model and report that airway cells increase the resistance of P. aeruginosa to tobramycin (Tb) by >25-fold compared with P. aeruginosa grown on abiotic surfaces. Therefore, the concentration of Tb required to kill P. aeruginosa biofilms on airway cells is 10-fold higher than the concentration achievable in the lungs of CF patients. In addition, CF airway cells expressing ΔF508-CFTR significantly enhanced P. aeruginosa biofilm formation, and ΔF508 rescue with wild-type CFTR reduced biofilm formation. Iron (Fe) content of the airway in CF is elevated, and Fe is known to enhance P. aeruginosa growth. Thus we investigated whether enhanced biofilm formation on ΔF508-CFTR cells was due to increased Fe release by airway cells. We found that airway cells expressing ΔF508-CFTR released more Fe than cells rescued with WT-CFTR. Moreover, Fe chelation reduced biofilm formation on airway cells, whereas Fe supplementation enhanced biofilm formation on airway cells expressing WT-CFTR. These data demonstrate that human airway epithelial cells promote the formation of P. aeruginosa biofilms with a dramatically increased antibiotic resistance. The ΔF508-CFTR mutation enhances biofilm formation, in part, by increasing Fe release into the apical medium.

Angiotensin IV (Ang IV) exerts profound effects on memory and learning, a phenomenon ascribed to its binding to a specific AT₄ receptor. However the AT₄ receptor has recently been identified as the insulin‐regulated aminopeptidase (IRAP). In this study, we demonstrate that AT₄ receptor ligands, including Ang IV, Nle¹‐Ang IV, divalinal‐Ang IV, and the structurally unrelated LVV‐hemorphin‐7, are all potent inhibitors of IRAP catalytic activity, as assessed by cleavage of leu‐β‐naphthylamide by recombinant human IRAP. Both Ang IV and divalinal–Ang IV display competitive kinetics, indicating that AT₄ ligands mediate their effects by binding to the catalytic site of IRAP. The AT₄ ligands also displaced [¹²⁵I]‐Nle¹‐Ang IV or [¹²⁵I]‐divalinal1‐Ang IV from IRAP‐HEK293T membranes with high affinity, which was up to 200‐fold greater than in the catalytic assay; this difference was not consistent among the peptides, and could not be ascribed to ligand degradation. Although some AT₄ ligands were subject to minor cleavage by HEK293T membranes, none were substrates for IRAP. Of a range of peptides tested, only vasopressin, oxytocin, and met‐enkephalin were rapidly cleaved by IRAP. We propose that the physiological effects of AT₄ ligands result, in part, from inhibition of IRAP cleavage of neuropeptides involved in memory processing.

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen chronically infecting the lungs of patients with chronic obstructive pulmonary disease (COPD), pneumonia, cystic fibrosis (CF), and bronchiectasis. Cif (PA2934), a bacterial toxin secreted in outer membrane vesicles (OMV) by P. aeruginosa, reduces CFTR-mediated chloride secretion by human airway epithelial cells, a key driving force for mucociliary clearance. The aim of this study was to investigate the mechanism whereby Cif reduces CFTR-mediated chloride secretion. Cif redirected endocytosed CFTR from recycling endosomes to lysosomes by stabilizing an inhibitory effect of G3BP1 on the deubiquitinating enzyme (DUB), USP10, thereby reducing USP10-mediated deubiquitination of CFTR and increasing the degradation of CFTR in lysosomes. This is the first example of a bacterial toxin that regulates the activity of a host DUB. These data suggest that the ability of P. aeruginosa to chronically infect the lungs of patients with COPD, pneumonia, CF, and bronchiectasis is due in part to the secretion of OMV containing Cif, which inhibits CFTR-mediated chloride secretion and thereby reduces the mucociliary clearance of pathogens. In this manuscript, we present a detailed mechanistic study of how a secreted P. aeruginosa virulence factor disrupts mucociliary clearance in the lung by inactivating a host cell deubiquitinating enzyme (USP10), thereby facilitating the degradation of the CFTR secretory chloride channel, which reduces mucociliary clearance by human airway epithelial cells. To our knowledge, this is the first report of a bacterial toxin that alters host innate immune defense by hijacking host proteins involved in the ubiquitin proteolytic system. P. aeruginosa is recognized as the most common bacterial pathogen in ventilator-associated pneumonia and is commonly isolated from community-acquired pneumonia patients. While P. aeruginosa can cause devastating acute infections, many studies have documented that P. aeruginosa can also generate chronic infections in immunocompromised individuals, including cystic fibrosis, chronic obstructive pulmonary disease and bronchiectasis patients. In these patients, the ineffective host immune response to the bacterial colonization is thought to play a large role in deteriorating lung function and ultimately the death of the patient. Our findings have significant implications to the study of P. aeruginosa infections in the lung, particularly the ability of P. aeruginosa to disrupt critical host innate immune response mechanisms.

We previously reported that Pseudomonas aeruginosa PA14 secretes a protein that can reduce the apical membrane expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Here we report that we have used a proteomic approach to identify this secreted protein as PA2394, and we have named the gene cif, for CFTR inhibitory factor. We demonstrate that Cif is a secreted protein and is found associated with outer membrane-derived vesicles. Expression of Cif in Escherichia coli and purification of the C-terminal six-His-tagged Cif protein showed that Cif is necessary and sufficient to mediate the reduction in apical membrane expression of CFTR and a concomitant reduction in CFTR-mediated Cl⁻ ion secretion. Cif demonstrates epoxide hydrolase activity in vitro and requires a highly conserved histidine residue identified in α/β hydrolase family enzymes to catalyze this reaction. Mutating this histidine residue also abolishes the ability of Cif to reduce apical membrane CFTR expression. Finally, we demonstrate that the cif gene is expressed in the cystic fibrosis (CF) lung and that nonmucoid isolates of P. aeruginosa show greater expression of the gene than do mucoid isolates. We propose a model in which the Cif-mediated decrease in apical membrane expression of CFTR by environmental isolates of P. aeruginosa facilitates the colonization of the CF lung by this microbe.

Approximately one‐quarter of people over the age of 65 are estimated to suffer some form of cognitive impairment, underscoring the need for effec tive cognitive‐enhancing agents. Insulin‐regulated ami nopeptidase (IRAP) is potentially an innovative tar get for the development of cognitive enhancers, as its peptide inhibitors exhibit memory‐enhancing effects in both normal and memory‐impaired rodents. Using a homology model of the catalytic domain of IRAP and virtual screening, we have identified a class of nonpeptide, small‐molecule inhibitors of IRAP. Structure‐based computational development of an initial “hit” resulted in the identification of two divergent families of compounds. Subsequent medicinal chemistry performed on the highest affinity compound produced inhibitors with nanomolar affinities ( K _i 20‐700 nM) for IRAP. In vivo efficacy of one of these inhibitors was demonstrated in rats with an acute dose (1 nmol in 1 μl) administered into the lateral ventricles, improving performance in both spatial working and recognition memory paradigms. We have identified a family of specific IRAP inhibi tors that is biologically active which will be useful both in understanding the physiological role of IRAP and potentially in the development of clinically useful cogni tive enhancers. Notably, this study also provides unequiv ocal proof of principal that inhibition of IRAP results in memory enhancement.— Albiston, A. L., Morton, C. J., Ng, H. L., Pham, V., Yeatman, H. R., Ye, S., Ruani, N., Fernando, R. N., De Bundel, D., Ascher, D. B., Men delsohn, F. A. O., Parker, M. W., Chai, S. Y. Identification and characterization of a new cognitive enhancer based on inhibition of insulin‐regulated aminopeptidase. FASEB J. 22, 4209–4217 (2008)