Experimental Eye Research 145 (2016) 278e288
Contents lists available at ScienceDirect
Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
Research article
Glycan involvement in the adhesion of Pseudomonas aeruginosa to tears Liisa Kautto a, b, Terry Nguyen-Khuong a, b, Arun Everest-Dass a, b, Andrea Leong c, Zhenjun Zhao c, Mark D.P. Willcox c, Nicolle H. Packer a, b, *, Robyn Peterson a, b a b c
Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia Biomolecular Frontiers Research Centre, Macquarie University, Sydney, Australia School of Optometry and Vision Science, University of New South Wales, Sydney, Australia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 April 2015 Received in revised form 21 January 2016 Accepted in revised form 21 January 2016 Available online 3 February 2016
The human eye is constantly bathed by tears, which protect the ocular surface via a variety of mechanisms. The O-linked glycans of tear mucins have long been considered to play a role in binding to pathogens and facilitating their removal in the tear flow. Other conjugated glycans in tears could similarly contribute to pathogen binding and removal but have received less attention. In the work presented here we assessed the contribution of glycan moieties, in particular the protein attached Nglycans, presented by the broad complement of tear proteins to the adhesion of the opportunistic pathogen Pseudomonas aeruginosa, a leading cause of microbial keratitis and ulceration of the cornea. Our adhesion assay involved immobilising the macromolecular components of tears into the wells of a polyvinyl difluoride (PVDF) microtitre filter plate and probing the binding of fluorescently labelled bacteria. Three P. aeruginosa strains were studied: a cytotoxic strain (6206) and an invasive strain (6294) from eye infections, and an invasive strain (320) from a urinary tract infection (UTI). The ocular isolates adhered two to three times more to human tears than to human saliva or porcine gastric mucin, suggesting ocular niche-specific adaptation. Support for the role of the N-glycans carried by human tear proteins in the binding and removal of P. aeruginosa from the eye was shown by: 1) pre-incubation of the bacteria with free component sugars, galactose, mannose, fucose and sialyl lactose (or combination thereof) inhibiting adhesion of all the P. aeruginosa strains to the immobilised tear proteins, with the greatest inhibition of binding of the ocular cytotoxic 6206 and least for the invasive 6294 strain; 2) preincubation of the bacteria with N-glycans released from the commercially available human milk lactoferrin, an abundant protein that carries N-linked glycans in tears, inhibiting the adhesion to tears of the ocular bacteria by up to 70%, which was significantly more binding inhibition than by the same amount of intact human lactoferrin or by the plant-derived N-glycans released from the rice recombinant lactoferrin; 3) pre-incubation of the bacteria with N-linked glycans released from human tear proteins inhibiting the adhesion of the ocular P. aeruginosa strains to immobilised tear proteins; 4) inhibition by the N-glycans from lactoferrin of the ability of an ocular strain of P. aeruginosa to invade corneal epithelial cells; 5) removal of terminal sialic acid and fucose moieties from the tear glycoproteins with a2-3,6,8 neuraminidase (sialidase) and a1-2,3,4 fucosidase resulting in a reduction in binding of the UTI P. aeruginosa isolate, but not the adhesion of the ocular cytotoxic (6206) or invasive (6294) isolates. Glycosidase activity was validated by mass spectrometry. In all cases, the magnitude of inhibition of bacterial adhesion by the N-glycans was consistently greater for the cytotoxic ocular strain than for the invasive ocular strain. Ocular P. aeruginosa isolates seems to exhibit different adhesion mechanism than previously known PAI and PAII lectin adhesion. The work may contribute towards the development of glycan-focused therapies to prevent P. aeruginosa infection of the eye. Crown Copyright © 2016 Published by Elsevier Ltd. All rights reserved.
Keywords: Pseudomonas aeruginosa Tears Glycosylation N-linked glycans Lactoferrin
1. Introduction * Corresponding author. Balaclava Road, Macquarie University, 2109, NSW, Australia. E-mail address: [email protected] (N.H. Packer). http://dx.doi.org/10.1016/j.exer.2016.01.013 0014-4835/Crown Copyright © 2016 Published by Elsevier Ltd. All rights reserved.
As a gram-negative opportunistic pathogen, Pseudomonas aeruginosa can cause a wide range of infections in vulnerable
L. Kautto et al. / Experimental Eye Research 145 (2016) 278e288
individuals, including manifestations in the lungs (Venkatakrishnan et al., 2013), intestines (Ohara and Itoh, 2003), urinary tract (Chiarini et al., 1987) and the skin (Wu et al., 2011). Infection of the eye by P. aeruginosa is a leading cause of bacterial keratitis, which can subsequently lead to corneal perforation and blindness unless treated effectively (Willcox, 2007, 2012). Factors that can increase susceptibility of the eye to P. aeruginosa infection include trauma to the cornea, the presence of pre-existing ocular disease, extended contact lens wear, immunosuppression and presumed immune-incompetency in premature infants (Kreger, 1983; Yildiz et al., 2012). An important initial factor in P. aeruginosa infection is the ability of the bacterium to adhere to the glycans (sugars) on host epithelial cells. The surface lectins and adhesins of P. aeruginosa have been well characterised in the context of respiratory infection (reviewed in Venkatakrishnan et al., 2013). The most well-known lectins are PA-IL and PA-IIL with a preferred binding affinity for galactose and fucose/mannose, respectively (Chen et al., 1998; Sabin et al., 2006; Imberty et al., 2004). In addition, PA-IIL and P. aeruginosa flagellum proteins favour adhesion to glycan configurations characteristic of Lewis blood group antigens that contain terminal fucose epitopes (Scharfman et al., 2001; Marotte et al., 2007). These glycan moieties can serve as binding receptors on human epithelial cell surfaces and are also found in the secretory fluids that bathe them, which in turn provide competitive decoys to enable pathogen removal (Stanley and Cummings, 2009; Everest-Dass et al., 2012; Peterson et al., 2013). The ocular surface, being constantly exposed to environmental pathogens, is particularly vulnerable to infection but is protected by a variety of mechanisms conveyed by the tears that constantly bathe the eye. Tear fluid consists of a thin outer lipid layer, and an inner aqueous layer that merges with mucin at the glycocalyx of the corneal epithelial cells (Peters and Colby, 2012). Several proteins in the tear fluid have antimicrobial activities, including secretory immunoglobulin A (sIgA; Willcox and Lan, 1999), lysozyme (Ridley, 1928), and the iron-binding lactoferrin (Flanagan and Willcox, 2009). However, the ability of tears to protect against P. aeruginosa is not directly dependent on antibacterial effects since this particular bacterium can grow readily in tear fluid (Fleiszig et al., 2003). This implies the involvement of other factors, such as adhesion and removal of the pathogen via the glycan moieties of the secreted glycoproteins in tears. To date, investigation of glycan involvement in P. aeruginosa adhesion to tears has focused predominantly on the ability of the tear mucins to bind to P. aeruginosa, a mechanism attributed to the abundant O-linked glycan chains, which attach to the oxygen atom of serine or threonine residues on the mucin proteins (Fleiszig et al., 1994, 2003bib_Fleiszig_et_al_2003; Aristoteli and Willcox, 2001; Aristoteli and Willcox, 2003; Guzman-Aranguez and Argueso, 2010; Argüeso, 2013). This is reflective of similar research on P. aeruginosa in the context of respiratory infection, where mucus is in abundance and of pathological concern (reviewed in Venkatakrishnan et al., 2013). However, the secreted mucins (MUC5AC and MUC 7) represent only a small fraction of the total protein content of tear fluid (Argüeso et al., 2002), and evidence has emerged that P. aeruginosa also binds to other tear proteins (McNamara et al., 2005). Prime candidates for such adhesion are tear glycoproteins that carry N-linked glycans, which are attached to the nitrogen atom of asparagine residues on the protein backbone (Stanley et al., 2009). At least 43 N-linked glycoproteins have been identified in tears including lactoferrin (constituting up to 30% of total tear protein; Flanagan and Willcox, 2009), secretory immunoglobulin A (sIgA), Prolactin Inducible Protein (PIP), Deleted in Malignant Brain Tumours 1 (DMBT1) and clusterin (Zhou et al., 2009), all of which
279
have been associated with pathogen adhesion and removal in other human secretory fluids (Barboza et al., 2012; Schroten et al., 1998; Schenkels et al., 1997; Ahn et al., 2008; Ligtenberg et al., 2010; Sabatte et al., 2011). N-linked glycans from human basal tears have been recently characterised, demonstrating a variety of complex structures with potential binding epitopes (NguyenKhuong et al., 2014). Nonetheless, the contribution of N-linked glycans in tears to pathogen adhesion and removal has received little attention. Tear fluid also contains a small amount of glycolipid (
Contents lists available at ScienceDirect
Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
Research article
Glycan involvement in the adhesion of Pseudomonas aeruginosa to tears Liisa Kautto a, b, Terry Nguyen-Khuong a, b, Arun Everest-Dass a, b, Andrea Leong c, Zhenjun Zhao c, Mark D.P. Willcox c, Nicolle H. Packer a, b, *, Robyn Peterson a, b a b c
Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia Biomolecular Frontiers Research Centre, Macquarie University, Sydney, Australia School of Optometry and Vision Science, University of New South Wales, Sydney, Australia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 April 2015 Received in revised form 21 January 2016 Accepted in revised form 21 January 2016 Available online 3 February 2016
The human eye is constantly bathed by tears, which protect the ocular surface via a variety of mechanisms. The O-linked glycans of tear mucins have long been considered to play a role in binding to pathogens and facilitating their removal in the tear flow. Other conjugated glycans in tears could similarly contribute to pathogen binding and removal but have received less attention. In the work presented here we assessed the contribution of glycan moieties, in particular the protein attached Nglycans, presented by the broad complement of tear proteins to the adhesion of the opportunistic pathogen Pseudomonas aeruginosa, a leading cause of microbial keratitis and ulceration of the cornea. Our adhesion assay involved immobilising the macromolecular components of tears into the wells of a polyvinyl difluoride (PVDF) microtitre filter plate and probing the binding of fluorescently labelled bacteria. Three P. aeruginosa strains were studied: a cytotoxic strain (6206) and an invasive strain (6294) from eye infections, and an invasive strain (320) from a urinary tract infection (UTI). The ocular isolates adhered two to three times more to human tears than to human saliva or porcine gastric mucin, suggesting ocular niche-specific adaptation. Support for the role of the N-glycans carried by human tear proteins in the binding and removal of P. aeruginosa from the eye was shown by: 1) pre-incubation of the bacteria with free component sugars, galactose, mannose, fucose and sialyl lactose (or combination thereof) inhibiting adhesion of all the P. aeruginosa strains to the immobilised tear proteins, with the greatest inhibition of binding of the ocular cytotoxic 6206 and least for the invasive 6294 strain; 2) preincubation of the bacteria with N-glycans released from the commercially available human milk lactoferrin, an abundant protein that carries N-linked glycans in tears, inhibiting the adhesion to tears of the ocular bacteria by up to 70%, which was significantly more binding inhibition than by the same amount of intact human lactoferrin or by the plant-derived N-glycans released from the rice recombinant lactoferrin; 3) pre-incubation of the bacteria with N-linked glycans released from human tear proteins inhibiting the adhesion of the ocular P. aeruginosa strains to immobilised tear proteins; 4) inhibition by the N-glycans from lactoferrin of the ability of an ocular strain of P. aeruginosa to invade corneal epithelial cells; 5) removal of terminal sialic acid and fucose moieties from the tear glycoproteins with a2-3,6,8 neuraminidase (sialidase) and a1-2,3,4 fucosidase resulting in a reduction in binding of the UTI P. aeruginosa isolate, but not the adhesion of the ocular cytotoxic (6206) or invasive (6294) isolates. Glycosidase activity was validated by mass spectrometry. In all cases, the magnitude of inhibition of bacterial adhesion by the N-glycans was consistently greater for the cytotoxic ocular strain than for the invasive ocular strain. Ocular P. aeruginosa isolates seems to exhibit different adhesion mechanism than previously known PAI and PAII lectin adhesion. The work may contribute towards the development of glycan-focused therapies to prevent P. aeruginosa infection of the eye. Crown Copyright © 2016 Published by Elsevier Ltd. All rights reserved.
Keywords: Pseudomonas aeruginosa Tears Glycosylation N-linked glycans Lactoferrin
1. Introduction * Corresponding author. Balaclava Road, Macquarie University, 2109, NSW, Australia. E-mail address: [email protected] (N.H. Packer). http://dx.doi.org/10.1016/j.exer.2016.01.013 0014-4835/Crown Copyright © 2016 Published by Elsevier Ltd. All rights reserved.
As a gram-negative opportunistic pathogen, Pseudomonas aeruginosa can cause a wide range of infections in vulnerable
L. Kautto et al. / Experimental Eye Research 145 (2016) 278e288
individuals, including manifestations in the lungs (Venkatakrishnan et al., 2013), intestines (Ohara and Itoh, 2003), urinary tract (Chiarini et al., 1987) and the skin (Wu et al., 2011). Infection of the eye by P. aeruginosa is a leading cause of bacterial keratitis, which can subsequently lead to corneal perforation and blindness unless treated effectively (Willcox, 2007, 2012). Factors that can increase susceptibility of the eye to P. aeruginosa infection include trauma to the cornea, the presence of pre-existing ocular disease, extended contact lens wear, immunosuppression and presumed immune-incompetency in premature infants (Kreger, 1983; Yildiz et al., 2012). An important initial factor in P. aeruginosa infection is the ability of the bacterium to adhere to the glycans (sugars) on host epithelial cells. The surface lectins and adhesins of P. aeruginosa have been well characterised in the context of respiratory infection (reviewed in Venkatakrishnan et al., 2013). The most well-known lectins are PA-IL and PA-IIL with a preferred binding affinity for galactose and fucose/mannose, respectively (Chen et al., 1998; Sabin et al., 2006; Imberty et al., 2004). In addition, PA-IIL and P. aeruginosa flagellum proteins favour adhesion to glycan configurations characteristic of Lewis blood group antigens that contain terminal fucose epitopes (Scharfman et al., 2001; Marotte et al., 2007). These glycan moieties can serve as binding receptors on human epithelial cell surfaces and are also found in the secretory fluids that bathe them, which in turn provide competitive decoys to enable pathogen removal (Stanley and Cummings, 2009; Everest-Dass et al., 2012; Peterson et al., 2013). The ocular surface, being constantly exposed to environmental pathogens, is particularly vulnerable to infection but is protected by a variety of mechanisms conveyed by the tears that constantly bathe the eye. Tear fluid consists of a thin outer lipid layer, and an inner aqueous layer that merges with mucin at the glycocalyx of the corneal epithelial cells (Peters and Colby, 2012). Several proteins in the tear fluid have antimicrobial activities, including secretory immunoglobulin A (sIgA; Willcox and Lan, 1999), lysozyme (Ridley, 1928), and the iron-binding lactoferrin (Flanagan and Willcox, 2009). However, the ability of tears to protect against P. aeruginosa is not directly dependent on antibacterial effects since this particular bacterium can grow readily in tear fluid (Fleiszig et al., 2003). This implies the involvement of other factors, such as adhesion and removal of the pathogen via the glycan moieties of the secreted glycoproteins in tears. To date, investigation of glycan involvement in P. aeruginosa adhesion to tears has focused predominantly on the ability of the tear mucins to bind to P. aeruginosa, a mechanism attributed to the abundant O-linked glycan chains, which attach to the oxygen atom of serine or threonine residues on the mucin proteins (Fleiszig et al., 1994, 2003bib_Fleiszig_et_al_2003; Aristoteli and Willcox, 2001; Aristoteli and Willcox, 2003; Guzman-Aranguez and Argueso, 2010; Argüeso, 2013). This is reflective of similar research on P. aeruginosa in the context of respiratory infection, where mucus is in abundance and of pathological concern (reviewed in Venkatakrishnan et al., 2013). However, the secreted mucins (MUC5AC and MUC 7) represent only a small fraction of the total protein content of tear fluid (Argüeso et al., 2002), and evidence has emerged that P. aeruginosa also binds to other tear proteins (McNamara et al., 2005). Prime candidates for such adhesion are tear glycoproteins that carry N-linked glycans, which are attached to the nitrogen atom of asparagine residues on the protein backbone (Stanley et al., 2009). At least 43 N-linked glycoproteins have been identified in tears including lactoferrin (constituting up to 30% of total tear protein; Flanagan and Willcox, 2009), secretory immunoglobulin A (sIgA), Prolactin Inducible Protein (PIP), Deleted in Malignant Brain Tumours 1 (DMBT1) and clusterin (Zhou et al., 2009), all of which
279
have been associated with pathogen adhesion and removal in other human secretory fluids (Barboza et al., 2012; Schroten et al., 1998; Schenkels et al., 1997; Ahn et al., 2008; Ligtenberg et al., 2010; Sabatte et al., 2011). N-linked glycans from human basal tears have been recently characterised, demonstrating a variety of complex structures with potential binding epitopes (NguyenKhuong et al., 2014). Nonetheless, the contribution of N-linked glycans in tears to pathogen adhesion and removal has received little attention. Tear fluid also contains a small amount of glycolipid (
