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The serine protease of pic mediates a dose-dependent mucolytic activity after binding to sugar constituents of the mucin substrate
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The serine protease of pic mediates a dose-dependent mucolytic activity after binding to sugar constituents of the mucin substrate






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The serine protease of pic mediates a dose-dependent mucolytic activity after binding to sugar constituents of the mucin substrate Document Transcript

  • 1. The serine protease motif of Pic mediates a dose-dependent mucolytic activity after binding to sugar constituents of the mucin substrate Javier Gutie´rrez-Jime´nez, Ivonne Arciniega, Fernando Navarro-Garcı´a* Department of Cell Biology, Centro de Investigacio´n y de Estudios Avanzados (CINVESTAV-IPN), Zacatenco, Ap. Postal 14-740, 07000 Me´xico, DF, Mexico a r t i c l e i n f o Article history: Received 23 January 2008 Received in revised form 14 April 2008 Accepted 18 April 2008 Available online 2 May 2008 Keywords: Pic Autotransporter protein Mucin Proteolytic activity Mucinase Lectin activity a b s t r a c t The pic gene is harbored on the chromosomes of three important pathogens: enteroaggregative Escherichia coli (EAEC), uropathogenic E. coli (UPEC), and Shigella flexneri. Since Pic is secreted into the intestinal lumen during EAEC infection, we sought to identify intestinal–mucosal substrates for Pic. Pic did not damage epithelial cells, cleave fodrin, or degrade host defense proteins embedded in the mucus layer (sIgA, lactoferrin and lysozyme). However, by using a solid–phase assay to evaluate the mucinolytic activity of EAEC Pic, we documented a specific, dose-dependent mucinolytic activity. A serine protease inhibitor and an enzymatically inactive variant of Pic were used to show that the Pic serine protease motif is required for mucinolytic activity. Pic binds mucin, and this binding was blocked in competition assays using monosaccharide constituents of the oligosaccharide side chains of mucin. Moreover, Pic mucinolytic activity decreased when sialic acid was removed from mucin. Thus, Pic is a mucinase with lectin-like activity that can be related to its reported hemagglutinin activity. Our results suggest that EAEC may secrete Pic into the intestinal lumen as a strategy for penetrating the gel-like mucus layer during EAEC colonization. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Enteroaggregative Escherichia coli (EAEC) is associated with persistent pediatric diarrhea and belong to diarrheagenic patho- types recognized in E. coli [1]. This pathotype is defined by its aggregating pattern of adherence to HEp-2 cells and shows two prominent pathogenic features: (i) formation of a thick mucus blanket on the intestinal mucosa and (ii) mucosal damage by cytotoxins [2,3]. Another recognized enteric bacterial pathogen is Shigella flexneri, an intracellular pathogen that causes bacillary dysentery by invasion and tissue damage of colonic epithelium. The stools are generally watery, purulent, bloody, and mucoid [4]. These enteric pathogens share the pic/set chromosomal locus, which in S. flexneri is found on the she pathogenicity island [5]. This locus encodes a 109 kDa protein named Pic (Protein involved in colonization), and a protein homologue named PicU has recently been identified in the CFT073 strain of uropathogenic E. coli (UPEC) [6]. Pic is one of two autotransporter proteins secreted by EAEC that belong to the serine protease autotransporter of Enterobacteriaceae (SPATE) family [7]. The full contribution of these proteins to pathogenesis remains elusive: no universal contribution has been suggested and different activities have been reported for various members of the SPATE family [8,9]. Unlike plasmid-encoded toxin (Pet), the other autotransporter secreted by EAEC, Pic has no enterotoxic or cytotoxic activities [8,10]. However, the cytotoxic effect of Pic has only been tested on HEp-2 cells with a single concentration of toxin [8]. Several biological roles for Pic have been reported, including hemaggluti- nation, degradation of complement protein, and mucinolytic activity against bovine submaxillary gland mucin (BSM) and mouse intestinal mucus [7]. PicU has also shown an in vitro mucinolytic activity against BSM [6]. However, another group was unable to identify a mucinase activity associated with PicU or the EAEC homologue [11]. Thus, the biological role of Pic in these pathogens is unknown. However, the possibility of a mucinolytic activity for Pic suggests an effective strategy for penetrating the intestinal mucus layer and/or obtaining a carbon source for the metabolic process. In this work, we further studied this possibility. We tested different intestinal mucosa substrates for Pic and using a solid– phase assay to evaluate mucinolytic activity of Pic, we found a specific, dose-dependent mucinolytic activity for EAEC Pic. This activity depended on its serine protease motif and on Pic binding to monosaccharide constituents of the oligosaccharide side chains of mucin. * Corresponding author. Tel.: þ52 5 5747 3990; fax: þ52 5 5747 3393. E-mail address: fnavarro@cell.cinvestav.mx (F. Navarro-Garcı´a). Contents lists available at ScienceDirect Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath 0882-4010/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2008.04.006 Microbial Pathogenesis 45 (2008) 115–123
  • 2. 2. Results 2.1. Interaction of Pic with epithelial cells Some SPATE members (i.e. Pet, EspC, Sat, and EspP) display cytotoxic activities against mammalian cells [3,8,9,12]. To determine whether Pic is cytotoxic to intestinal epithelial cells, HT29-C1 cells were incubated with various doses of Pic for up to 12 h. Potential cytotoxic effects were then detected by Giemsa staining to visualize the morphology of intoxicated cells. As a neg- ative control, cells were treated with concentrated supernatants from non-toxigenic HB101 (pACYC184). As a positive control, cells were treated for 4 h with culture supernatant containing Pet pro- tein at 37 mg/ml (355 nM). Cells in the negative control exhibited a normal cell morphology (Fig. 1A), whereas cell rounding and detachment from the glass substratum was observed for cells in the positive control (Fig. 1B). HT29-C1 cells treated with culture supernatant containing 364 nM Pic exhibited normal cell mor- phology, indicating that Pic did not cause a cytotoxic effect (Fig.1C). Since cytotoxicity could be dose dependent, HT29-C1 cells were incubated with 546, 728, 910, 1366 and 1821 nM of Pic for 12 h at 37 C (Fig. 1D-F). None of these doses produced a cytotoxic effect. Pic was also unable to produce a cytotoxic effect in another epi- thelial cell line, HEp-2 cells (data not shown). Confocal microscopy was used to further study possible interactions between Pic and epithelial cells. After incubation with 910 nM Pic, HEp-2 cells were immunostained with anti-Pic anti- bodies and rhodamine-labeled phalloidin to visualize the actin cytoskeleton. HEp-2 cells were treated with 355 nM Pet protein as a positive control in parallel experiments. Consistent with previous results [3,13], Pet-treated cells showed cytoskeleton contraction (Fig. 1G), loss of actin stress fibers (Fig. 1G), and an intracellular distribution of Pet (Fig. 1H-I). In contrast, a higher concentration of Pic (910 nM) was unable to produce any of the Pet-related effects: Pic did not damage the actin cytoskeleton (Fig. 1J) and was not present in HEp-2 cells (Fig. 1K). We were also unable to detect Pic binding to the cell membrane (Fig. 1L). Identical results were obtained with an even higher 1.8 mM concentration of Pic (data not shown). Fodrin cleavage is involved with the cytotoxic effects elicited by Pet and EspC. To explore whether Pic is able to cleave fodrin, we performed in vitro degradation experiments using a recombinant, 109 kDa GST–fodrin [14]. Previous work has shown that GST–fodrin is a substrate of Pet and EspC [12,15]. GST–fodrin was exposed to Pic at several times (0, 30 and 180 min) and then resolved by SDS- PAGE. At time zero, fodrin and Pic were detected as a band of 109 kDa: both proteins have the same molecular weights and run at the same position in SDS-PAGE gels (Fig. 2A). After 30 and 180 min of incubation, no breakdown products of GST–fodrin were detected (Fig. 2A). The band from time zero which corresponds to GST– fodrin and Pic was identical to the bands from samples taken after 30 and 180 min of incubation. The same result, indicating no Pic- mediated cleavage of GST–fodrin, was obtained after a 6 h incubation (data not shown). In contrast, Pet and EspC were able to cleave GST–fodrin in 60 min to generate different breakdown products on SDS-PAGE (Fig. 2A). Pet and EspC were also able to bind fodrin as determined by overlay analyses, whereas Pic was unable to bind fodrin in the same assay (Fig. 2B). All these data suggest that Pic, unlike Pet and EspC [3,12], is unable to interact with and damage epithelial cells of the intestinal mucosa. 2.2. The serine protease motif of Pic is involved in the dose-dependent mucinolytic activity High molecular weight molecules such as mucin (>2 Â 106 Da) are not resolved by SDS-PAGE because they are unable to enter the gel. Thus, as an alternative method to analyze the interaction of Pic with mucin, we employed a colorimetric technique [16] and stan- dardized the degradation assay by using commercial BSM and Proteinase K (PK). BSM was coupled to biotin, adjusted to different concentrations (0.1, 0.2, 0.4 and 0.8 mg/ml), adsorbed to microtiter plates, and exposed to 1 or 10 mg of PK per well. BSM-coated wells were incubated with PBS as a negative control. The assay showed that BSM was efficiently degraded by 1 mg of PK since this protease treatment dramatically reduced the mucin-dependent absorbance of the treated wells (Fig. 3A). Reduction of the mucin-dependent absorbance was related to PK concentration, as 10 mg of PK reduced absorbance values more than 1 mg of PK (Fig. 3A). To evaluate the mucinolytic activity of Pic, we used commercial hog stomach mucin (HSM) as well as BSM. Both substrates were coupled to biotin and adsorbed to microtiter plates as mentioned above. Degradation assays showed that Pic at 1 or 10 mg per well was able to degrade HSM in a dose-dependent fashion (Fig. 3B). Thus, 1 mg of Pic degraded around 32% of the HSM sample, while 10 mg of Pic degraded around 56% of the HSM. Similar results were obtained when BSM was used as the substrate (data not shown). These data indicate that Pic is able to degrade mucin in a quanti- tative, dose-dependent manner. Since the serine protease motif harbored by SPATE proteins is crucial for their biological activities [9], we analyzed the role of this motif on the mucinolytic activity of Pic. Pic was preincubated for 30 min with the serine protease inhibitor phenylmethylsulphonyl fluoride (PMSF) and then added at 1 or 10 mg per well to HSM- coated plates. Absorbance values obtained with PMSF-treated Pic were similar to those obtained when HSM was incubated with PBS (Fig. 3C). This demonstrated that PMSF-treated Pic was unable to degrade HSM at either 1 or 10 mg of Pic per well. To further assess the role of the serine protease motif on the mucinolytic activity of Pic, site-directed mutagenesis was used to convert its predicted catalytic serine residue (S258) to isoleucine. This mutated protein, Pic S258I, was used at 1 or 10 mg per well in the HSM degradation assay. As with PMSF-treated Pic, absorbance values obtained with Pic S258I were similar to those values obtained when HSM was incubated with PBS (Fig. 3D). These data demonstrate that the Pic serine protease motif is crucial to cleave mucin, since the muci- nolytic activity is abolished by blocking this motif with an inhibitor or by a mutation. 2.3. Interaction of Pic with proteins immersed in the mucus layer We analyzed whether Pic displays proteolytic activity against proteins embedded in the intestinal mucus such as lysozyme, lactoferrin and secretory IgA (sIgA). Each potential substrate was incubated with Pic for 0, 0.5, 3, or 4 h before degradation products were analyzed by SDS-PAGE. Pic and lysozyme were resolved at time zero as full-length proteins of 109 and 14 kDa, respectively. Lysozyme was also resolved as a full-length protein without deg- radation products after 0.5, 3, and 4 h of incubation, indicating that Pic is unable to cleave lysozyme in vitro (Fig. 4A). When apolactoferrin was incubated with Pic, SDS-PAGE analysis showed at time zero two bands of 109 and 80 kDa for Pic and apolacto- ferrin, respectively. Both protein bands were preserved without degradation products after 0.5, 3, and 4 h of incubation, demon- strating that Pic was also unable to cleave apolactoferrin (Fig. 4B). Since sIgA is a heteromeric protein, its interaction with Pic was analyzed by SDS-PAGE under reducing and non-reducing condi- tions. In both cases, Pic and sIgA proteins were incubated for 1, 4 and 8 h as previously described [17]. Under non-reducing condi- tions (no 2-b-mercaptoethanol and heating), SDS-PAGE showed Pic as a full-length protein band of 109 kDa while sIgA was observed as a high molecular weight protein (around 400 kDa) which hardly entered into the separating gel. This profile was J. Gutie´rrez-Jime´nez et al. / Microbial Pathogenesis 45 (2008) 115–123116
  • 3. similar after 1, 4, and 8 h of incubation, indicating no degradation of sIgA by Pic (Fig. 4C). Under reducing conditions, SDS-PAGE showed Pic as a single band of 109 kDa and sIgA as three bands representing the secretory component (70 kDa), the heavy chains (52 kDa), and the light chains (23 kDa). None of these sIgA bands were degraded by Pic at any of the incubation times tested (Fig. 4D). All these data indicate that Pic is unable to cleave pro- teins embedded in the mucus layer such as lysozyme, lactoferrin and sIgA. Furthermore, the data demonstrate that there is substrate specificity for the serine protease motif of Pic. 2.4. Role of monosaccharide components of mucin in the interaction with Pic The ability of Pic to bind to bovine mucin was examined in a solid- phase assay using mucin-coated microtiter wells [18]. GST-coated Fig. 1. Pic does not cause cytotoxicity and is not internalized into cells. (A–F) Pic does not induce cytotoxic effects. HT29-C1 cells were incubated with different doses of Pic, 355 nM Pet, or left without treatment. After incubation, HT29-C1 cells were fixed, permeabilized and stained with Giemsa. Untreated cells (A). Cells treated with Pet as a positive control (B). Cells treated with purified Pic at 364 (C), 728 (D), 1366 (E) and 1821 nM (F). (G–L) Pic is not internalized into epithelial cells. HEp-2 cells were incubated with Pic (910 nM), Pet (355 nM) proteins or left without treatment. After incubation, the cells were fixed, permeabilized, and stained with rhodamine–phalloidin (red; G,J) and anti-Pic (K) or anti-Pet (H) antibodies followed by a secondary fluorescein-labeled goat anti-rabbit antibody (green). Slides were observed under confocal microscopy. (G–I) Pet-treated cells. (J–L) Pic-treated cells. Merged images are shown in panels I and L. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) J. Gutie´rrez-Jime´nez et al. / Microbial Pathogenesis 45 (2008) 115–123 117
  • 4. microtiter wells were used as a negative control. The binding of Pic to bovine mucin was higher than that to GST, as revealed by anti-Pic or anti-GST antibodies, respectively (Fig. 5A). To determine if any of the individual sugar components of mucin were important for Pic binding, the solid-phase assay was repeated with three mono- saccharide constituents of the oligosaccharide side chains of mucin: N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), and N-acetylneuraminic acid (sialic acid, NANA). Pic bound to all the three Fig. 2. Pic does not interact with fodrin. (A) Degradation assay. Purified GST–fodrin (1 mg) was incubated with 1 mg Pic, Pet or EspC proteins at different times and then separated by SDS-PAGE. The degradation products are indicated with a closed circle for Pet and with an asterisk for EspC. (B) Overlay assay. Pic (4 mg), Pet (4 mg), or EspC (4 mg) were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membrane containing these proteins was incubated with GST–fodrin (5 mg/ml) and the affinity reaction was revealed by using anti-GST antibodies and AP-labeled anti-mouse antibodies. Fig. 3. Pic mucinolytic activity is a dose- and serine protease motif-dependent event. Microplate wells were coated with biotin-labeled mucin, and then treated for 4 h at 37 C with 1 or 10 mg per well of either Proteinase K used as positive control (A), Pic (B), Pic pretreated for 30 min with the serine protease inhibitor (PMSF) (C), or the Pic S258I serine protease motif mutant (D). After incubation, the undigested labeled mucin was detected with streptavidin–peroxidase. Enzymatic activity was determined with different concentrations of HSM by plotting absorbance readings on a graph generated from a standard curve of labeled mucin in PBS. J. Gutie´rrez-Jime´nez et al. / Microbial Pathogenesis 45 (2008) 115–123118
  • 5. monosaccharides, showing a higher affinity for GalNAc than for GlcNAc or sialic acid. However, Pic had a higher affinity for mucin than for any of the individual monosaccharides (Fig. 5A). To further analyze the interaction of Pic with these mono- saccharides, GlcNAc, GalNAc and sialic acid were used in inhibition experiments. Pretreating Pic with GlcNAc reduced Pic binding to bovine mucin by 70% compared to the untreated Pic control (Fig. 5B), indicating that GlcNAc is a mucin constituent recognized by Pic. GalNAc decreased Pic binding to mucin by 34% compared to the untreated control. However, when Pic was pretreated with sialic acid, the percentage of Pic binding to mucin increased by 35% compared to the untreated control (Fig. 5B). This suggested that sialic acid may act as a bridge between Pic and mucin, thereby enhancing Pic binding. Interestingly, when Pic was preincubated with mucin and then added to monosaccharide-coated wells, mucin was able to block Pic binding to all three sugars with equal and essentially complete efficiency (around 97% inhibition as compared with the untreated Pic) (Fig. 5C). Our data suggested that Pic binding to the sugar residues of mucin could be important for Pic-mediated degradation of the protein component of mucin. To test this hypothesis, we used neuraminidase to remove sialic acid from BSM. A colorimetric assay [19] demonstrated that the amount of sialic acid in com- mercial BSM decreased from 0.602 mM to 0.419 mM after treat- ment with 0.06 U/ml of neuraminidase. Both untreated and neuraminidase-treated mucins were coupled to biotin for use in the solid-phase degradation assay. Pic (9 nM) degraded both mucins at concentrations of 0.1, 0.2, 0.4 and 0.8 mg mucin/ml (Fig. 5D). However, we observed a significant decrease of Pic activity against neuraminidase-treated mucin in comparison with untreated mucin (Fig. 5D). The maximum difference was observed at 0.2 mg/ml of mucin, where Pic degraded 50% of the untreated mucin versus 17% of the neuraminidase-treated mucin (Fig. 5D). These data indicate that the presence of sugars on mucin constitutes an important factor for Pic binding and its mucinolytic activity. Fig. 4. Pic is unable to cleave lysozyme, apolactoferrin or sIgA. Purified Pic protein (1 mg) was incubated with 1 mg lysozyme, apolactoferrin, or sIgA at different times and then separated by SDS-PAGE at 12%, 10%, and 8%, respectively. (A) Pic–lysozyme interaction for 0.5, 3, or 4 h. (B) Pic–lactoferrin interaction for 0.5, 3, or 4 h. (C-D) Pic–sIgA interaction for 1, 4, or 8 h; gels (8% and 12%) were run under non-reducing (C) and reducing (D) conditions. J. Gutie´rrez-Jime´nez et al. / Microbial Pathogenesis 45 (2008) 115–123 119
  • 6. 3. Discussion Epidemiological studies have shown that the pic gene is associ- ated with diarrheogenic strains of EAEC [20,21]. Since the Pic protein is secreted into the intestinal lumen during EAEC infection, the aim of this work was to identify intestinal mucosa substrates for Pic. Here, we show that Pic does not directly damage epithelial cells. Pic does not cleave the actin-binding protein fodrin, and it does not cleave host defense proteins such as sIgA, lactoferrin, or lysozyme that are embedded in the mucus layer. Instead, Pic binds and cleaves mucin. Pic binds to the monosaccharide constituents of the mucin oligo- saccharide side chains, and its serine protease motif is required for mucin cleavage. Secretion of a thick mucus layer over the intestinal epithelium is a hallmark of EAEC infection; Pic mucinolytic activity could explain why the thick mucus does not inhibit EAEC infection. Dutta et al. previously showed that 20 mg/ml of Pic is not cyto- toxic to HEp-2 cells [8]. Here, toxin dose response experiments performed after 4–12 h of intoxication conclusively demonstrated that Pic is not cytotoxic to HT29-C1 cells, an intestinal epithelial cell line. Confocal microscopy further showed that, at even highest dose and incubation time used, Pic (i) does not bind to the plasma membrane of epithelial cells; (ii) is not internalized into epithelial cells; and (iii) does not produce cytoskeletal damage in epithelial cells. Unlike the SPATEs Pet and EspC, Pic did not bind and cleave fodrin. This observation supports the data reported by Dutta et al. [8] but is contrary to the data reported by Parham et al. [6]. The discrepancies may be attributed to different sources of fodrin/ spectrin: we used a consistent source of recombinant aII spectrin, whereas the other authors used purified erythrocyte spectrin which can vary in substrate quality and thus produce unclear results. Together, our data suggest that Pic is unable to interact with and damage the epithelial cells of the intestinal mucosa. Pic has been reported to be a serine protease with mucinase activity that is involved with multiple aspects of EAEC pathogenesis [7]. We decided to further explore its capacity to degrade host defense proteins that are embedded in the mucus layer. Pic and the IgA1 protease autotransporter of Neisseria spp. both possess a con- sensus serine protease motif [22]. However, Pic was unable to cleave sIgA, an important immunoglobulin in the mucosal immu- nity against adherent pathogens [9]. Pic was also unable to cleave lactoferrin, a molecule with inhibitory effects on biofilm development, bacterial adhesion, and colonization [23]. Further- more, lactoferrin is a serine protease which has been reported to proteolytically cleave and inactivate three autotransporter coloni- zation factors: the IgA protease precursor protein; Hap, the non- pilus adhesin [24]; and Aae from A. actinomycetemcomitans [25]. Finally, Pic was unable to cleave lysozyme, an antimicrobial protein which specifically attacks and destroys peptidoglycan [26]. Fig. 5. Interaction of Pic with mucin. (A) Binding of Pic to mucin and monosaccharide constituents of mucin. Wells were coated with mucin, exposed to the indicated mono- saccharides or GST, and then blocked with BSA. The wells were subsequently incubated with increasing concentrations of Pic for 1 h at 37 C. Pic or GST binding was detected by using anti-Pic or anti-GST antibodies followed of a secondary antibody labeled with peroxidase. (B) Effect of monosaccharides on the binding of Pic to mucin. Pic was preincubated with the indicated sugars for 30 min at 37 C and then added to mucin-coated wells. Pic binding was detected as indicated in panel A. (C) Effect of mucin on the binding of Pic to the monosaccharide constituents of mucin. Pic was preincubated with mucin for 30 min at 37 C and then added to wells coated with the indicated sugars. Pic binding was detected as indicated in the panel A. (D) Effect of removing sialic acid from mucin on the Pic mucinolytic activity. Untreated and neuraminidase-treated mucins were coupled to biotin. The degradation assay on solid phase was performed using Pic at 1 mg per well as indicated in Fig. 3 legend. J. Gutie´rrez-Jime´nez et al. / Microbial Pathogenesis 45 (2008) 115–123120
  • 7. Studies of Pic as a mucinase have been complicated by the technologies used to demonstrate its mucinolytic activity. In the first study on Pic, SDS-PAGE analysis was used to monitor Pic mucinolytic activity against crude mouse mucus [7]. As the crude mucus was probably unable to enter the separating gel, the resulting data was unclear. The use of Sephacryl S-400 column chromatography of untreated crude mouse mucus versus mucus treated with Pic and measuring the optical density of PAS staining fractions was even more complicated [7]. Another methodology used by various authors [6–8] detects zones of mucin lysis on BSM in agarose plates after treatment with Pic (24 h at 37 C) and staining with amido black. Unfortunately, this is a rudimentary technique with no quantitative measurement after long incubations. It also produces inconsistent results. For example, with this methodology Pic showed mucinolytic activity against BSM and mouse crude mucin but did not show mucinolytic activity against HSM [8]. Heimer et al. [11], in contrast, were unable to identify a mucinase activity associated with Pic from either EAEC of UPEC. Here, we have shown that Pic is indeed a mucinase with mucino- lytic activity against both BSM and HSM. Use of the microplate assay with immobilized biotin-labeled mucin as substrate and a 4 h 37 C treatment with Pic allowed us to (i) clearly demonstrate the mucinolytic activity of Pic; (ii) establish that this effect is a dose- dependent event (from substrate or enzyme); and (iii) quantify Pic activity (i.e. percentages of mucin degradation at different Pic concentrations). We also demonstrated with the microplate assay that Pic mucinolytic activity depends on its serine protease motif since the use of a serine protease inhibitor (i.e. PMSF) or a serine protease motif mutant (Pic S258I) completely blocked mucinolytic activity against either BSM or HSM. The microplate assay used here could also resolve a controversy regarding the possible mucinolytic activity of the SPATE Tsh [11,27]. Collectively, our data indicate that Pic is functionally similar to EpeA, a SPATE from enterohemorrhagic E. coli which has mucinolytic activity but is not associated with a cytopathic effect on epithelial cells [28]. Mucin, the principal glycoprotein of mucous secretion, plays an important role in protection against microbial invasion because of its heavy glycosylation and its ability to form a gel. However, enteric pathogens have developed various strategies (motility, adhesion, and proteolytic cleavage of mucin) for penetrating this protective gel-like layer [29]. Many enteric pathogens are able to bind mucin as an initial event towards colonization [30], but few pathogens express proteins that exhibit features of both a lectin and a pro- tease. A soluble Zn-metalloprotease, hemagglutinin/protease (HapA) [31], exhibits both of these features and proteolytically degrades several physiologically important host proteins including mucin [32]. Here, we show that Pic is a mucinase that displays a lectin-like activity by binding to monosaccharide constituents of the oligosaccharide side chains of mucin. This dual function might be related to other activities attributed to Pic, such as its ability to inactivate complement and its role in hemagglutination. Our collective data indicate that Pic, which is secreted by three important pathogens (EAEC, UPEC, and S. flexneri), is a mucinase with a lectin-like activity. The lectin-like activity of Pic can be related to its previously reported hemagglutinin activity which is also found in its homologue Tsh from avian pathogenic E. coli [7,33]. Finally, this work suggests an important role for Pic in penetrating the protective gel-like mucus layer during EAEC (as well as UPEC and S. flexneri) colonization. 4. Material and methods 4.1. Strains and plasmids As previously described [7], the minimal Pic clone HB101(pPic1) was used to obtain Pic protein. Supernatant proteins from the non- toxigenic HB101 (pACYC184) were used as a negative control. The strains were maintained on L-agar or L-broth containing 10 mg/ml tetracycline. Clone 18531, representing bp 2531–4689 of human aII spectrin, was cloned into the inducible bacterial expression vector pGEX-3X. The strains were maintained on L-agar or L-broth con- taining 100 mg/ml ampicillin. Clone 18531 was kindly provided by Stabatch and Morrow [14]. 4.2. Antibodies and recombinant protein preparation Broth cultures from HB101(pPic) were incubated overnight at 37 C and then centrifuged at 7000 Â g for 15 min. The culture supernatant was filtered throughout 0.22 mm cellulose acetate membrane filters (Corning, Cambridge, MA), concentrated 100-fold in an ultrafree centrifugal filter device with a 100 kDa cut-off (Millipore, Bedford, MA), filter-sterilized again and stored at À20 C for up to 3 months. Rabbit anti-Pic polyclonal antibodies were elicited by excising proteins from polyacrylamide gels and injecting the gel slices subcutaneously into rabbits in two doses, 2 weeks apart. The antibody responses and specificity were determined by immuno- blotting and the gamma fractions from the antisera were obtained. GST–fodrin was prepared as previously described [14]. Briefly, overnight bacterial culture from clone 18531 expressed in BL21 was induced with IPTG. The supernatant of the lysate was affinity absorbed on glutathione–Sepharose beads at 4 C and washed with 1 mM DTT. The bound peptide was eluted during 10 min at room temperature in the same buffer containing PBS, 1 mM DTT and 10 mM reduced glutathione. 4.3. Culture and tissue culture assays HEp-2 cells were propagated in humidified 5% CO2–95% air at 37 C in Dulbecco’s modified Eagle’s medium (DMEM) supple- mented with 5% fetal bovine serum (Hyclone), 1% non-essential amino acids, 5 mM L-glutamine, penicillin (100 units/ml), and streptomycin (100 mg/ml). For experimental use, subconfluent HEp- 2 cells were resuspended with EDTA–trypsin, plated into eight-well LabTek slides (VWR), and allowed to grow to 70% confluence. The HT29-C1 clone, obtained from Daniel Louvard (Institut Pasteur, Paris, France), was grown in DMEM supplemented with 10% fetal bovine serum, 44 mM sodium bicarbonate, 10 mg of human trans- ferrin (Sigma, St. Louis, Mo.), 50 IU of streptomycin, and 50 mg/ml of penicillin. HT29-C1 cells were grown at 37 C in humidified 10% CO2–90% air; medium was changed 6 days/week. For experimental use, HT29-C1 cells were prepared in eight-well LabTek slides and grown to 70% confluence (about 3 days). For all experiments, Pic was diluted directly into tissue culture medium (without antibiotics or serum) and added to the target cells at a final volume of 250 ml per well (eight-well LabTek slides). Following the specified incubation times in humidified atmosphere of 5% CO2–95% air at 37 C, the medium was aspirated, cells were washed twice with PBS and processed by means of the following methods: (i) Giemsa staining. Cells were fixed with 70% methanol and stained with 10% Giemsa (Sigma). Slides were analyzed at a 100Â magnification by standard bright field light microscopy [3]. (ii) Immunostaining. Cells were fixed with 2% formalin–PBS, washed, permeabilized by adding 0.1% Triton X-100 in PBS, and stained with 0.05 mg/ml of tetramethyl rhodamine isothiocyanate (TRITC) phalloidin and with a rabbit anti-Pic polyclonal antibody as previously described [12], followed by an anti-rabbit fluorescein- labeled antibody. Slides were mounted on Gelvatol, covered with glass coverslips, and examined under a Leica TCS SP2 confocal microscope. J. Gutie´rrez-Jime´nez et al. / Microbial Pathogenesis 45 (2008) 115–123 121
  • 8. 4.4. Overlay assay Overlay assays were performed as previously reported [15]. Briefly, about 2 mg of protein (Pet, Pic or EspC) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Individual BSA-blocked strips were then incubated for 1 h with 5 mg/ml GST–fodrin. Strips were incubated with anti-GST mouse polyclonal antiserum (1:1000) for 1 h, followed of a goat anti-mouse AP- conjugated antibody (1:500) for 1 h. Binding was detected using 1-Step NBT/BCIP substrate. 4.5. Degradation assay Two micrograms of GST–fodrin, or lactoferrin, lysozyme and secretory IgA (Sigma) were mixed with an equal volume of 2Â digestion buffer containing Pic or Pet. Reactions were carried out at 30 C at several times and stopped by the addition of 4 Â SDS sample buffer. All samples were analyzed by SDS-PAGE; the gels were stained with Coomassie blue [15]. 4.6. Site-directed mutagenesis Site-directed mutagenesis was performed using the Quik- ChangeÔ Kit from Stratagene as specified by the manufacturer. The template used for construction of the site-directed mutant (pPicS258I) was pPic1, as we had previously done to produce Pet S260I [3]. The primers used for this purpose were 50-GAGCCCCTGGGGATATTGGTTCTCCTTTGT-30 and 50-ACAAAGGA- GAACCAATATCCCCCAGGGGCTC-30. The primers encompassed res- idues 1743–1772 of the pic sequence (AF097644) but encoded a T instead of a G at nucleotide 1758, thereby substituting an isoleucine for the serine at residue 258. Following mutagenesis, the S258I mutation was confirmed by double-strand sequencing of the area encompassing the serine protease active site. 4.7. Detection and quantification of mucinolytic activity The presence of mucinolytic activity was quantified in a micro- plate assay using immobilized biotin-labeled mucin as substrate [16]. Briefly, microplate wells were coated with biotin-labeled mucin, and concentrated supernatants containing Pic were added and incubated for 4 h at 37 C in sodium acetate buffer. The un- digested labeled mucin was detected with streptavidin–peroxidase. Enzymatic activity was determined by reporting absorbance read- ings on a plot obtained with a standard curve of labeled mucin. Activity was expressed as the percentage of decrease in absorbance compared with that from control wells devoid of enzyme. 4.8. Pic binding assays 4.8.1. Immobilized mucin and monosaccharide assays A modified solid-phase mucin binding assay was performed with BSM or monosaccharides (GlcNAc, GalNAc, and NANA from Sigma) as previously described [18]. Briefly, the bovine mucin (in HEPES) and 50 mM monosaccharides (in 1 M NaOH) were added to microtiter plate wells. Pic was added to the wells at various con- centrations. Reaction was detected by using a polyclonal anti-Pic antibody (1:700), followed by incubation with a secondary anti- body (HRP-coupled anti-rabbit). The color was developed with o-phenylene-diamine, the reaction was stopped with 2 N H2SO4, and the color was read at 490 nm. 4.8.2. Inhibition of Pic binding to mucin or monosaccharides Three monosaccharide constituents of the oligosaccharide side chains of mucin, N-acetylglucosamine (GlcNAc), N- acetylgalactosamine (GalNAc) and N-acetylneuraminic acid (sialic acid, NANA), were used in the solid-phase assay. Microtiter plate wells were coated with bovine mucin as described above. Solutions of GlcNAc and GalNAc (100 mM) in PBS or sialic acid (50 mM) in 10 mM Tris–HCl were mixed with Pic (2.8 nM). Pic in PBS served as the control. Samples from Pic-sugar suspension were added to mucin-coated wells. The mucin adherence assay was continued as described above. 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