Successful opportunistic pathogens, of which P. aeruginosa is a prime example, exploit specific niches in the host in order to facilitate attachment, colonization, damage, and dissemination. Our work was inspired by the fact that long carbohydrate chains of various glycoconjugates, including HSPGs and N-glycoproteins, could potentially serve as bacterial receptors since they are prominent cell surface-exposed structures at the mucosal epithelium. We extensively characterized the distribution of HSPGs and the structure of N-glycan chains in cultured epithelial cells grown at various states of polarization. Since P. aeruginosa requires preexisting epithelial damage and the loss of at least some degree of polarity in order to cause disease, these in vitro epithelial cell culture systems recapitulate important aspects of human infections and serve as a useful model to further dissect mechanisms of disease. Using comprehensive and multifaceted approaches, we demonstrated that complex N-glycan chains are necessary and sufficient to mediate P. aeruginosa binding at the AP surface, whereas the HS moieties of HSPGs mediate binding at the BL surface of the polarized epithelium. During epithelial injury and dedifferentiation, HSPG presentation at the AP surface is increased, explaining at least in part the predilection of this important pathogen for such injured tissues. Changes in the composition of N-glycan chains and/or in the polarized segregation of HSPGs could contribute to the pathogenesis of acute and chronic diseases.
We first focused on N-glycosylation, as increased or altered expression of N-glycans could result in enhanced susceptibility to P. aeruginosa
infections in the setting of acute or chronic injury. Using chemical and enzymatic inhibitors, we showed that N-glycans are important contributors to P. aeruginosa
binding, entry, and damage at the AP surface of airway and kidney cells grown at various states of polarity as confluent 2D monolayers. ConAr
MDCK cells, which are defective in N-glycosylation, were particularly informative in identifying the role of N-glycans in P. aeruginosa
binding and subsequent internalization and host injury. Although the specific defect in these cells is not known, the fact that N-glycosylation could be restored by growing these cells in the presence of excess Man or Glc suggested that they are defective in the activities of PMM and PMI enzymes (46
). When P. aeruginosa
was added to the AP surface of ConAr
MDCK cells, decreased binding, entry, and cytotoxicity were observed compared to those in wt cells, whereas no difference was observed at the BL surface. While it has previously been suggested that ConAr
MDCK cells form more highly polarized monolayers, which leads to resistance to P. aeruginosa
), we found no evidence for altered polarity in these cells under our experimental conditions. Importantly, when ConAr
MDCK cells were grown in the presence of Man or Glc, P. aeruginosa
binding, entry, and cytotoxicity were restored to wild-type levels at the AP surface. We could also enhance the binding and subsequent damage at the AP surface of wt MDCK and airway epithelial cells grown in the presence of excess Man or Glc. We demonstrated increased colocalization of bacteria with more complex N-glycan patches on the AP membrane of highly organized 3D cysts grown in the presence of Man to upregulate N-glycosylation. Finally, P. aeruginosa
preferentially bound in vitro
to a mixture of complex N-glycans over an individual sugar. Together, these results suggest that N-glycan chains of one or more N-glycosylated proteins at the AP surface serve as important receptors for AP binding of P. aeruginosa
. The N-glycosylated molecule is unlikely to be the previously identified receptors CD95 (Fas receptor), integrin, and fibronectin, as these molecules are preferentially expressed at the BL surface (50
). Likewise, it is unlikely to be CFTR, since similar results were obtained with Calu-3 cells, which express high levels of CFTR at the AP surface, and wt MDCK cells, which express very little CFTR (18
). Future studies will be aimed at identifying this important molecule. In summary, N-glycans and particular enzymes in the N-glycosylation pathway could be potential targets for novel therapeutic approaches in treatment of P. aeruginosa
infections. Most importantly, simple sugars could be used to either modify the course of the disease or competitively inhibit bacterial binding and subsequent infection, a line of treatment currently being investigated.
While these results identified N-glycan chains as AP receptors, they did not reveal the identity of the BL receptor or the receptor that is upregulated in incompletely polarized cells. We therefore examined whether HSPGs, which are abundantly expressed on the BL surface (3
), can serve as specific BL receptors for P. aeruginosa
. We demonstrated that HSPGs are upregulated in incompletely polarized cells and lose their polarized distribution. Competitive inhibition, enzymatic removal, or desulfation of HS chains decreased P. aeruginosa
binding, entry, and cytotoxicity at the BL surface of well-polarized cells and at both surfaces in incompletely polarized cells. We corroborated these results by showing that P. aeruginosa
binds to HS in vitro
, with the highest affinity among all tested compounds, and that it preferentially binds to HS-rich patches on the BL surface of 3D cysts.
If N-glycans can function as binding receptors and are found on both the AP and BL surfaces, why do they not contribute to P. aeruginosa
binding to the BL surface? First, the identity of the protein core that is N-glycosylated may also contribute to binding specificity and the BL N-glycoproteins may not function as binding receptors or may not be readily accessible. In addition, or alternatively, the binding affinity for HSPGs may be higher than that for N-glycan chains (as suggested by our in vitro
data). While our results demonstrate that AP N-glycans can function as receptors, in healthy hosts with well-polarized intact mucosal barriers P. aeruginosa
does not cause disease. This observation likely reflects the importance of local defense mechanisms, such as a functional innate immune system. However, upregulation of AP HSPGs, particularly in the context of altered local innate immune responses, may offer an explanation for the increased binding and subsequent damage seen when P. aeruginosa
infects incompletely polarized but intact monolayers, such as might be seen during regenerating epithelium. In more severe injury, when the monolayer is disrupted, increased access to HSPGs on the BL surface may also contribute to enhanced susceptibility to infection. Interestingly, it has been shown that inhibition of shedding of the HSPG syndecan-1 or degradation of the shed HS chains attenuates mouse lung infection (40
). These results suggest an additional possible role for secreted HSPGs in mediating microbial virulence. They also indirectly suggest that P. aeruginosa
binds to HS chains on syndecan-1, thus supporting the work presented here.
Flagella and type IV pili are the predominant P. aeruginosa
adhesins. Studies are under way to determine their binding specificities toward N-glycans and HSPGs and whether they play different roles in adhesion at the AP versus BL surface. Previous studies have suggested that glycosphingolipids may serve as AP receptors for type IV pili (10
), although these findings remain controversial (13
). Flagellar components have been shown to bind to Lewis X derivatives that are found on secreted mucins (48
). Moreover, Toll-like receptor 5, predominantly found on the AP surface, has been found to bind flagellin (24
). Finally, there can be flagellum- and pilus-independent bacterial binding to the cell surface, since two different lectins have been described for P. aeruginosa
, specifically binding to either Fuc or Gal, sugars present in both N- and O-glycan chains (8
There is a great deal of potential to use heparin or heparin-like structures as drugs to treat a wide range of disorders, including respiratory diseases (12
). Although heparin has been in use as an anticoagulant for over 70 years, heparan sulfate-like structures are attracting considerable interest as a source of new therapeutics, since it has been proposed that the main purpose of heparin is in a defensive mechanism at sites of tissue injury against invading bacteria and other foreign materials (36
). HSPGs are attractive as therapeutic targets since a wide range of viral and bacterial pathogens are known to bind to HS chains (2
), and our work further shows that the sulfate moieties of HSPGs function as P. aeruginosa
-binding receptors. Drugs that disrupt HSPG-pathogen interactions may be an effective strategy for preventing or treating acute P. aeruginosa
infections, particularly as inhaled therapy for pneumonia.
An important recent advance in understanding epithelial cell biology is the ability to grow epithelial cells as 3D cysts in a collagen-Matrigel matrix, which recapitulates the development and structural organization of tubular structures such as the lung alveolus. (5
). While there are a few reports of studies of the interactions of pathogens with epithelial cells grown as aggregates (7
), to the best of our knowledge our study is the first that examines the interaction of bacteria with epithelial cells grown as organized 3D cysts. This approach required that we overcome several technical challenges. First, the cysts had to be treated briefly with collagenase in order to expose their surface. Second, bacterial binding had to be quantified by direct confocal microscopy, as bacteria also bind avidly to the collagen-Matrigel matrix. Third, during the 7 days it takes for mature cysts to form, the cells become highly polarized, and bacterial binding is limited. In addition, N-glycans and HSPGs are expressed at high levels in a highly polarized manner, with uniform distributions on the AP and BL sides, respectively. Therefore, we either exposed the BL surface of BL-side-out cysts to heparinase III (to correlate binding to HS-rich domains) or utilized ConAr
MDCK cells (to correlate binding to Man-rich domains). Altogether, cysts provide a promising avenue for further study of host-pathogen interactions in vitro
in a biologically relevant context, particularly as they relate to microbes that infect tubular or luminal structures. Future directions will include observing these events by time-lapse confocal microscopy to delineate the temporal and spatial events that occur during bacterial attachment, internalization, and host damage.
While our work examined epithelial cells in various states of polarization as a model for acute or chronic epithelial injury, upregulation of N-glycosylation and/or altered expression or localization of HSPGs may be specifically relevant to the chronic P. aeruginosa
infections that result in end-stage lung disease in patients with CF. CFTR itself is a highly glycosylated protein, and there are published data that suggest that the sugar composition of membrane glycoproteins and secreted mucins is altered in CF, though the exact mechanism remains unknown (45
). We are currently testing whether N-glycosylation or HSPGs are upregulated in lung tissues from patients with CF.
Another possible way in which upregulation of HSPG expression could affect both chronic and acute P. aeruginosa
infections is through enhancing the binding and dimerization of growth factors and their receptors as well as by stabilizing these receptors. Of particular relevance are the epidermal growth factor (EGF) and its receptor, EGFR. EGFR activation is important in wound repair. In addition, EGFR has been shown to be hyperphosphorylated in CF patients (6
). HSPGs also bind and stabilize the activity of interleukin-8 (IL-8), which may play a role in IL-8-driven hyperinflammatory responses in CF (16
). Thus, upregulation of HSPGs during acute and chronic epithelial injury may amplify P. aeruginosa
-induced changes in growth factor signaling. In preliminary studies, we have found that P. aeruginosa
activates EGFR in an HS-dependent manner.
The studies presented here on the pathogenesis of P. aeruginosa infections provide key insights into how changes in the presentation of AP and BL molecules contribute to acute and chronic P. aeruginosa infections. The use of cultured epithelial cells grown as 3D cysts provides a new and powerful in vitro tool that has the potential to offer better insights into understanding the complex mechanism(s) involved in the establishment of the infection and in subsequent organ damage. Our data show an important role for HS chains and N-glycans in mediating microbial adherence, entry, and cytotoxicity and provide a basis for designing therapeutic strategies based on these interactions. The use of combined therapeutic strategies that target N-glycosylation, HSPG synthesis, or polarized segregation of these molecules may provide powerful new therapeutic approaches to correct the abnormal epithelial architecture that occurs in the setting of chronic lung disease, including CF, in order to improve clinical outcomes. In summary, our work establishes fertile ground for better understanding the connection between specific physiological alterations in various acute and chronic disorders and the predilection to infections by P. aeruginosa.