Background: Three T6SSs are present in P. aeruginosa. H1-T6SS secretes bacteriolytic toxins.Results: H2-T6SS is regulated by quorum sensing and Fur and modulates internalization in epithelial cells through PI3K-Akt host pathway activation.Conclusion: H2-T6SS plays a role in virulence.Significance: In contrast to the anti-prokaryotic H1-T6SS, H2-T6SS targets human cells. Those T6SSs can carry out different functions important in establishing infection.

Pseudomonas aeruginosa , an important opportunistic pathogen of man, exploits numerous factors for initial attachment to the host, an event required to establish bacterial infection. In this paper, we rigorously explore the role of two major bacterial adhesins, type IV pili (Tfp) and flagella, in bacterial adherence to distinct host receptors at the apical (AP) and basolateral (BL) surfaces of polarized lung epithelial cells and induction of subsequent host signaling and pathogenic events. Using an isogenic mutant of P. aeruginosa that lacks flagella or utilizing beads coated with purified Tfp, we establish that Tfp are necessary and sufficient for maximal binding to host N-glycans at the AP surface of polarized epithelium. In contrast, experiments utilizing a P. aeruginosa isogenic mutant that lacks Tfp or using beads coated with purified flagella demonstrate that flagella are necessary and sufficient for maximal binding to heparan sulfate (HS) chains of heparan sulfate proteoglycans (HSPGs) at the BL surface of polarized epithelium. Using two different cell-free systems, we demonstrate that Tfp-coated beads show highest binding affinity to complex N-glycan chains coated onto plastic plates and preferentially aggregate with beads coated with N-glycans, but not with single sugars or HS. In contrast, flagella-coated beads bind to or aggregate preferentially with HS or HSPGs, but demonstrate little binding to N-glycans. We further show that Tfp-mediated binding to host N-glycans results in activation of phosphatidylinositol 3-kinase (PI3K)/Akt pathway and bacterial entry at the AP surface. At the BL surface, flagella-mediated binding to HS activates the epidermal growth factor receptor (EGFR), adaptor protein Shc, and PI3K/Akt, and induces bacterial entry. Remarkably, flagella-coated beads alone can activate EGFR and Shc. Together, this work provides new insights into the intricate interactions between P. aeruginosa and lung epithelium that may be potentially useful in the development of novel treatments for P. aeruginosa infections.

The adhesion force and specificity in the first experimental evidence for cell–cell recognition in the animal kingdom were assigned to marine sponge cell surface proteoglycans. However, the question whether the specificity resided in a protein or carbohydrate moiety could not yet be resolved. Here, the strength and species specificity of cell–cell recognition could be assigned to a direct carbohydrate–carbohydrate interaction. Atomic force microscopy measurements revealed equally strong adhesion forces between glycan molecules (190–310 piconewtons) as between proteins in antibody–antigen interactions (244 piconewtons). Quantitative measurements of adhesion forces between glycans from identical species versus glycans from different species confirmed the species specificity of the interaction. Glycan-coated beads aggregated according to their species of origin, i.e., the same way as live sponge cells did. Live cells also demonstrated species selective binding to glycans coated on surfaces. These findings confirm for the first time the existence of relatively strong and species-specific recognition between surface glycans, a process that may have significant implications in cellular recognition.

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Pseudomonas aeruginosa, an important opportunistic pathogen of humans, exploits epithelial damage to establish infection. We have rigorously explored the role of N-glycoproteins and heparan sulfate proteoglycans (HSPGs) in P. aeruginosa-mediated attachment and subsequent downstream events at the apical (AP) and basolateral (BL) surfaces of polarized epithelium. We demonstrate that the N-glycan chains at the AP surface are necessary and sufficient for binding, invasion, and cytotoxicity to kidney (MDCK) and airway (Calu-3) cells grown at various states of polarization on Transwell filters. Upregulation of N-glycosylation enhanced binding, whereas pharmacologic inhibition of N-glycosylation or infection of MDCK cells defective in N-glycosylation resulted in decreased binding. In contrast, at the BL surface, the HS moiety of HSPGs mediated P. aeruginosa binding, cytotoxicity, and invasion. In incompletely polarized epithelium, HSPG abundance was increased at the AP surface, explaining its increased susceptibility to P. aeruginosa colonization and damage. Using MDCK cells grown as three-dimensional cysts as a model for epithelial organs, we show that P. aeruginosa specifically colocalized with HS-rich areas at the BL membrane but with complex N-glycans at the AP surface. Finally, P. aeruginosa bound to HS chains and N-glycans coated on plastic surfaces, showing the highest binding affinity toward isolated HS chains. Together, these findings demonstrate that P. aeruginosa recognizes distinct receptors on the AP and BL surfaces of polarized epithelium. Changes in the composition of N-glycan chains and/or in the distribution of HSPGs may explain the enhanced susceptibility of damaged epithelium to P. aeruginosa.Ninety-five percent of all infectious agents enter through mucosal surfaces of the gastrointestinal, genitourinary, and respiratory tracts (reviewed in reference 35). These mucosal surfaces are usually lined by a single layer of epithelial cells, which serves as the primary barrier against the entry of most infectious agents and can be considered a primary component of the innate immune system. Epithelial cells form highly polarized cell layers with apical (AP) and basolateral (BL) surfaces that exhibit distinct protein, lipid, and glycoconjugate compositions. Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen of humans that exploits injured mucosa to cause acute and chronic infections with high morbidity and mortality (reviewed in references 26 and 31). In the setting of epithelial injury and immunocompromise, this Gram-negative pathogen causes serious infections in patients with extensive burns, corneal trauma, or catheter-related bladder injury or in those on ventilators. In addition, P. aeruginosa chronically colonizes the lungs of patients with cystic fibrosis (CF) (4), leading to severe pulmonary damage and death. Despite aggressive antibiotic therapy, the fatality rate for many P. aeruginosa infections is 40%, and new approaches to treatment are even more critical now that antibiotic resistance...

Sponges were the earliest multicellular organisms to evolve through the development of cell recognition and adhesion processes mediated by cell surface proteoglycans. Information on sponges has an extra added value because, as a group, they are the oldest Metazoans alive and contribute more to our understanding of life on earth than knowledge of other animal groups. Although the proteoglycans are emerging as key players in various physiological and pathophysiological cellular events, little is known about the carbohydrate moiety of the proteoglycan molecule. Until recently there was no evidence provided for the existence of specific and biologically significant carbohydrate-carbohydrate interaction. We show here that the interaction between single oligosaccharides of surface proteoglycans is relatively strong (in the 200-300 piconewtons range) and in the same range as other relevant biological interactions, like those between antibodies and antigens. This carbohydrate-carbohydrate recognition is highly species-specific and perfectly mimics specific cell-cell recognition. Both the strength and the species-specificity of the carbohydrate-carbohydrate interaction are guaranteed by polyvalency, by compositional and architectural differences between carbohydrates, and by the arrangement of the carbohydrate chain in a three-dimensional context. Ca(2+)-ions are essential and probably provide coordinating forces. Our findings confirm the existence and character of species-specific carbohydrate-carbohydrate recognition fundamental to cell recognition and adhesion events.

The Cambrian explosion of life was a relatively short period approximately 540 Ma that marked a generalized acceleration in the evolution of most animal phyla, but the trigger of this key biological event remains elusive. Sponges are the oldest extant Precambrian metazoan phylum and thus a valid model to study factors that could have unleashed the rise of multicellular animals. One such factor is the advent of self-/non-self-recognition systems, which would be evolutionarily beneficial to organisms to prevent germ-cell parasitism or the introduction of deleterious mutations resulting from fusion with genetically different individuals. However, the molecules responsible for allorecognition probably evolved gradually before the Cambrian period, and some other (external) factor remains to be identified as the missing triggering event. Sponge cells associate through calcium-dependent, multivalent carbohydrate-carbohydrate interactions of the g200 glycan found on extracellular proteoglycans. Single molecule force spectroscopy analysis of g200-g200 binding indicates that calcium affects the lifetime (+Ca/-Ca: 680 s/3 s) and bond reaction length (+Ca/-Ca: 3.47 A/2.27 A). Calculation of mean g200 dissociation times in low and high calcium within the theoretical framework of a cooperative binding model indicates the nonlinear and divergent characteristics leading to either disaggregated cells or stable multicellular assemblies, respectively. This fundamental phenomenon can explain a switch from weak to strong adhesion between primitive metazoan cells caused by the well-documented rise in ocean calcium levels at the end of Precambrian time. We propose that stronger cell adhesion allowed the integrity of genetically uniform animals composed only of "self" cells, facilitating genetic constitutions to remain within the metazoan individual and be passed down inheritance lines. The Cambrian explosion might have been triggered by the coincidence in time of primitive animals endowed with self-/non-self-recognition and of a surge in seawater calcium that increased the binding forces between their calcium-dependent cell adhesion molecules.

Treatment of acute and chronic pulmonary infections caused by opportunistic pathogen Pseudomonas aeruginosa is limited by the increasing frequency of multidrug bacterial resistance. Here, we describe a novel adjunctive therapy in which administration of a mix of simple sugars-mannose, fucose, and galactose-inhibits bacterial attachment, limits lung damage, and potentiates conventional antibiotic therapy. The sugar mixture inhibits adhesion of nonmucoid and mucoid P. aeruginosa strains to bronchial epithelial cells in vitro. In a murine model of acute pneumonia, treatment with the sugar mixture alone diminishes lung damage, bacterial dissemination to the subpleural alveoli, and neutrophil- and IL-8-driven inflammatory responses. Remarkably, the sugars act synergistically with anti-Pseudomonas antibiotics, including β-lactams and quinolones, to further reduce bacterial lung colonization and damage. To probe the mechanism, we examined the effects of sugars in the presence or absence of antibiotics during growth in liquid culture and in an ex vivo infection model utilizing freshly dissected mouse tracheas and lungs. We demonstrate that the sugar mixture induces rapid but reversible formation of bacterial clusters that exhibited enhanced susceptibility to antibiotics compared with individual bacteria. Our findings reveal that sugar inhalation, an inexpensive and safe therapeutic, could be used in combination with conventional antibiotic therapy to more effectively treat P. aeruginosa lung infections.