Chromatography on mannose agarose indicating an in vivo interaction of both

Chromatography on mannose agarose indicating an in vivo interaction of both proteins. The same method applied to a lecBdeficient mutant of P. Eliglustat site aeruginosa did not result in isolation of OprF. Moreover, OprF could be isolated from the outer membrane fraction by His-tagged LecB immobilized on Ni-NTA agarose and could also be detected by affinity binding to peroxidase labelled LecB. Apparently, co-purification of OprF depended on specific binding to LecB which could be abrogated by subsequent washing of the column with the LecB-specific sugar fucose. Efficient in vitro binding of peroxidase labelled LecB to OprF blotted onto PVDF membranes after SDS-PAGE clearly suggested that LecB recognized OprF. So far, we failed to obtain any experimental evidence for glycosylation of OprF. Hence, the mechanism of the interaction between LecB and OprF remains unknown and requires further investigation. Carbohydrate blood group antigens present on the surface of erythrocytes 22948146 can bind to LecB and thereby cause hemagglutination. We have observed that a P. aeruginosa lecB deficient strain showed a significantly decreased hemagglutination activity as compared to the corresponding wild-type strain (Fig. 4). Interestingly, a P. aeruginosa oprF deletion mutant showed the same decrease in hemagglutination activity which could not be increased by expression of lecB from a plasmid. This result also strongly suggests an interaction of LecB with OprF on the cell surface of P. aeruginosa. Interactions 25837696 of lectins with cell surface proteins of pathogenic bacteria have been reported before [55]. Lectins located at the tip of pili or agella including PapG and GafD of uropathogenic E. coli are referred to as adhesins, as they play a role in adherence to epithelial cells [56]. In an earlier report, we demonstrated that LecB is an important factor in the development of biofilms by P. aeruginosa [23]. Furthermore, it was suggested that both LecB and OprF contribute to bacterial adherence to A549 epithelial cells [30,54]. As P. aeruginosa is toxic to epithelial cells [57], promotion of adherence might manifest as increased cytotoxicity and consequent lung epithelial destruction. Therefore, it is tempting to speculate that LecB and OprF together may mediate adhesion of P. aeruginosa to receptors located on cells of either the same or of different species, thus enabling the colonization of host tissues as well as the formation of mono- or multispecies biofilms. Previously, it was reported that interferon gamma binds to OprF, resulting in the expression of another quorum-sensing dependent virulence determinant, the lectin LecA of P. aeruginosa. Interestingly, a fucosyl-residue is required for recognition of human interferon gamma by the receptor [58] suggesting that the fucose-specific LecB may act as an adaptor to mediate recognition of this cytokine by OprF on the bacterial surface. Thus, it would be interesting to test whether this regulatory effect on lecA expression through sensing of interferon gamma is still functional in a lecB-negative P. aeruginosa mutant.Lectin LecB Interacts with Porin OprFFigure 4. Hemagglutination of rabbit red blood cells after incubation with P. aeruginosa. Erythrocytes were treated with papain and Lcysteine and then incubated with either PBS buffer in the MedChemExpress LED 209 absence and presence of 20 mM fucose or PBS-buffer containing P. aeruginosa PAO1 wildtype and mutants DlecB and DoprF. The positive control additionally contained purified LecB protein (concentr.Chromatography on mannose agarose indicating an in vivo interaction of both proteins. The same method applied to a lecBdeficient mutant of P. aeruginosa did not result in isolation of OprF. Moreover, OprF could be isolated from the outer membrane fraction by His-tagged LecB immobilized on Ni-NTA agarose and could also be detected by affinity binding to peroxidase labelled LecB. Apparently, co-purification of OprF depended on specific binding to LecB which could be abrogated by subsequent washing of the column with the LecB-specific sugar fucose. Efficient in vitro binding of peroxidase labelled LecB to OprF blotted onto PVDF membranes after SDS-PAGE clearly suggested that LecB recognized OprF. So far, we failed to obtain any experimental evidence for glycosylation of OprF. Hence, the mechanism of the interaction between LecB and OprF remains unknown and requires further investigation. Carbohydrate blood group antigens present on the surface of erythrocytes 22948146 can bind to LecB and thereby cause hemagglutination. We have observed that a P. aeruginosa lecB deficient strain showed a significantly decreased hemagglutination activity as compared to the corresponding wild-type strain (Fig. 4). Interestingly, a P. aeruginosa oprF deletion mutant showed the same decrease in hemagglutination activity which could not be increased by expression of lecB from a plasmid. This result also strongly suggests an interaction of LecB with OprF on the cell surface of P. aeruginosa. Interactions 25837696 of lectins with cell surface proteins of pathogenic bacteria have been reported before [55]. Lectins located at the tip of pili or agella including PapG and GafD of uropathogenic E. coli are referred to as adhesins, as they play a role in adherence to epithelial cells [56]. In an earlier report, we demonstrated that LecB is an important factor in the development of biofilms by P. aeruginosa [23]. Furthermore, it was suggested that both LecB and OprF contribute to bacterial adherence to A549 epithelial cells [30,54]. As P. aeruginosa is toxic to epithelial cells [57], promotion of adherence might manifest as increased cytotoxicity and consequent lung epithelial destruction. Therefore, it is tempting to speculate that LecB and OprF together may mediate adhesion of P. aeruginosa to receptors located on cells of either the same or of different species, thus enabling the colonization of host tissues as well as the formation of mono- or multispecies biofilms. Previously, it was reported that interferon gamma binds to OprF, resulting in the expression of another quorum-sensing dependent virulence determinant, the lectin LecA of P. aeruginosa. Interestingly, a fucosyl-residue is required for recognition of human interferon gamma by the receptor [58] suggesting that the fucose-specific LecB may act as an adaptor to mediate recognition of this cytokine by OprF on the bacterial surface. Thus, it would be interesting to test whether this regulatory effect on lecA expression through sensing of interferon gamma is still functional in a lecB-negative P. aeruginosa mutant.Lectin LecB Interacts with Porin OprFFigure 4. Hemagglutination of rabbit red blood cells after incubation with P. aeruginosa. Erythrocytes were treated with papain and Lcysteine and then incubated with either PBS buffer in the absence and presence of 20 mM fucose or PBS-buffer containing P. aeruginosa PAO1 wildtype and mutants DlecB and DoprF. The positive control additionally contained purified LecB protein (concentr.

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