Serine protease autotransporters from Shigella flexneri and pathogenic Escherichia coli target a broad range of leukocyte glycoproteins

Fernando Ruiz-Pereza,b,1, Rezwanul Wahida, Christina S. Fahertya, Krishnan Kolappaswamyc, Liliana Rodrigueza, Araceli Santiagoa,b, Ebony Murphya, Alan Crossa, Marcelo B. Szteina, and James P. Nataroa,b

aCenter for Vaccine Development and cProgram of Comparative Medicine, Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201; and bDepartment of Pediatrics, University of Virginia School of Medicine, Charlottesville, VA 22908

Edited by Ralph R. Isberg, Tufts University School of Medicine, Boston, MA, and approved June 27, 2011 (received for review January 24, 2011) The serine protease autotransporters of Enterobacteriaceae (SPATEs) in mucosal colonization (11, 15, 16). However, the fact that not all are secreted by pathogenic Gram-negative bacteria through the producers of the class 2 SPATEs are mucosal pathogens suggested autotransporter pathway. We previously classified SPATE to us that cleavage of mucin-family substrates may provide an into two classes: cytotoxic (class 1) and noncytotoxic (class 2). Here, additional advantage to the pathogen (17, 18). we show that Pic, a class 2 SPATE produced by Shigella flex- A variety of leukocyte surface glycoproteins with vital roles in neri 2a, uropathogenic and enteroaggregative Escherichia coli numerous cellular functions are substituted with carbohydrates strains, targets a broad range of human leukocyte adhesion proteins. structurally similar to those found on human mucin glycoproteins Substrate specificity was restricted to glycoproteins rich in O-linked (19, 20). Here, we show that the substrates of mucin-active class 2 glycans, including CD43, CD44, CD45, CD93, CD162 (PSGL-1; P- SPATEs include glycoproteins located on the surface of nearly all glycoprotein ligand 1), and the surface-attached chemokine fractal- lineages of hematopoietic cells. These targets, including CD43, kine, all implicated in leukocyte trafficking, migration, and inflam- CD44, CD45, CD93, fractalkine, and PSGL-1 (P-selectin glyco- mation. N-terminal sequencing of proteolytic products revealed Pic protein ligand 1), may have diverse effects on the immune re- (protease involved in colonization) cleavage sites to occur before

sponse, including leukocyte , activation, migration, and MICROBIOLOGY Thr or Ser residues. The purified carbohydrate sLewis-X implied in signaling. Our data suggest broadly important mechanisms for inflammation and malignancy inhibited cleavage of PSGL-1 by Pic. these common virulence factors. Exposure of human leukocytes to purified Pic resulted in polymor- phonuclear cell activation, but impaired chemotaxis and transmigra- Results tion; Pic-treated T cells underwent programmed cell death. We also Mucinase Activity of S. flexneri and Potential Targets on Human Muc show that the Pic-related protease Tsh/Hbp, implicated in extrain- Proteins. We reported previously that Pic cleaves ovomucin and testinal infections, exhibited a spectrum of substrates similar to bovine submaxillarly mucin (BSM). After overnight incubation, those cleaved by Pic. In the guinea pig keratoconjunctivitis model, supernatants of S. flexneri 2a cause near complete degradation of fl a Shigella pic mutant induced greater in ammation than its parent the major mucin species (Fig. S1A). N-terminal sequencing of strain. We suggest that the class-2 SPATEs represent unique im- mucin breakdown products over time (Fig. S1B) revealed the mune-modulating bacterial virulence factors. first eight amino acids of the major breakdown fragments to be SETNAIIG; this motif corresponded to residues S588 and fol- glycoprotease | diarrhea lowing on the 462-kDa BSM glycoprotein (Bos Taurus, XP_ 002687422.1). BLAST analysis of the predicted Pic target re- mong the most important enteric pathogens worldwide are vealed 100% identity with sequences encoding the human Muc19 AShigella spp. and pathogenic Escherichia coli (1, 2), yet protein (XP_002344701.1), the 1,184-kDa apomucin protein vaccines for these agents are not available. One impediment to (NP_001106757.1), the Equinus submaxillary apomucin (XP_ such vaccine efforts is the fact that enteric pathogens modulate 001915445.1), and two more human hypothetical mucin proteins the host immune system to promote and prolong infection. Thus, (XP_002343203.1, XP_002347351.1). Interestingly, sequences understanding the impact of enteric pathogens on the human similar to SETNAIIG were found within a large number of other immune system is a high research priority. human Muc and Muc-related proteins, suggesting the possibi- fi We rst described a family of putative virulence factors call- lity that the Pic substrate profile may be broader than we ed the serine protease autotransporters of Enterobacteriaceae had supposed. (SPATEs), secreted by all pathogenic E. coli and Shigella spp. (3, 4). This family now numbers more than 20 proteases, with diverse Cleavage of Sialomucin (CD43) on Human Leukocytes by Pic-Producing functions. We have proposed that SPATEs can be divided phy- Pathogenic Strains. Among the mammalian Muc-related proteins logenetically into two distinct classes, designated 1 and 2 (5). Class are a large number of extracellular and membrane-associated 1 SPATEs are cytotoxic in vitro and induce mucosal damage on glycoproteins. To determine whether Pic cleaved such proteins, intestinal explants. Although the actions of class 1 SPATEs are not fully understood, several have been shown to enter eukaryotic cells – and to cleave cytoskeletal proteins (6 8). More enigmatic are the Author contributions: F.R.-P., C.S.F., M.B.S., and J.P.N. designed research; F.R.-P., R.W., class 2 SPATEs, which include (i) the thermostable hemagglutinin C.S.F., K.K., L.R., A.S., E.M., and A.C. performed research; A.C. and J.P.N. contributed (Tsh) from avian pathogenic E. coli, and its nearly identical ho- new reagents/analytic tools; F.R.-P., C.S.F., K.K., M.B.S., and J.P.N. analyzed data; and molog, hemoglobin-binding protein (Hbp) from human extra- F.R.-P. and J.P.N. wrote the paper. intestinal E. coli (9, 10); (ii) the Pic protease (protease involved The authors declare no conflict of interest. in colonization), found in Shigella flexneri, enteroaggregative This article is a PNAS Direct Submission. E. coli (EAEC), and uropathogenic E. coli (UPEC) (11–13); and Freely available online through the PNAS open access option. (iii) the EpeA (EHEC-plasmid encoded autotransporter) from 1To whom correspondence should be addressed. E-mail: [email protected]. enterohemorrhagic E. coli (14). Pic protease induces mucus re- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. lease, cleaves mucin, and confers a subtle competitive advantage 1073/pnas.1101006108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1101006108 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 8A by Pic on both PMNs and PBMCs, as ascertained by Western 25 E S 5000 immunoblot. Similar results were obtained using the supernatants c ic 3000

1000 0.08% pi P 0 S D No staining 10 0 10 1 10 2 10 3 10 4 fl

S 3500 B 42 042 ic B of wild-type S. exneri 2457T (Fig. 1 C and D). P 42 P P 15002500 99.89% 0 0 500 0 PBS 0 1 2 3 4 fl 10 10 10 10 10

3000 In addition to Pic, S. exneri 2457T secretes two other SPATE 2000

A Pic 1000 4.54% 0 10 0 10 1 10 2 10 3 10 4 proteins: SepA and SigA. Neither supernatants containing SepA 3500 PicS258A 2500

5001500 99.67% 0 fi B 10 0 10 1 10 2 10 3 10 4 nor puri ed SigA induced degradation of CD43, suggesting that

2457T Wt Cell number

500150025003500 10.25% 0 fi 10 0 10 1 10 2 10 3 10 4 the effect is speci c for Pic (Fig. 1 C and D). 2457T∆pic 5000

1000 3000 99.74% t c 0 fi igA 0 1 2 3 4 To con rm that Pic cleaves extracellular CD43 on intact leu- Dpi s 10 10 10 10 10 W T D 7T 7 7T A CD43-APC fl BS 45 45 45 ic g kocytes, we performed ow cytometry analyses of human PMNs P 2 2 2 P Si Lymphocytes 4000 fi 3000 or PBMCs treated with puri ed Pic, PicS258A, or supernatants of 2000 1000 C 0 0.00% No staining 10 0 10 1 10 2 10 3 10 4 1400 fl 1000 S. exneri. Staining with anti-CD16 (for PMNs), anti-CD3 (for 600 200 PBS 0 78.93% 10 0 10 1 10 2 10 3 10 4

3000 – t c 2000 lymphocytes), and anti-CD43 conjugated mAbs revealed that A 1000 Dpi Pic 0 0.05% 8 0 1 2 3 4 10 10 10 10 10

TW T 1600 S 7 7 S25 A S only CD43 was cleaved, becoming undetectable on the surfaces of c g PicS258A 400 800 1200 B 45 45 ic i B 0 73.91% P 2 2 P Pi S P 10 0 10 1 10 2 10 3 10 4 fi Cell number neutrophils and lymphocytes after treatment with puri ed Pic or

2457T Wt 1000 2000 3000 4000

0 0.17% D 10 0 10 1 10 2 10 3 10 4

1500 2000 Shigella supernatants, but CD43 was present on cells treated with 2457T∆pic 1000 500

0 77.11% 10 0 10 1 10 2 10 3 10 4 fi CD43-APC puri ed PicS258A (Fig. 1E).

Fig. 1. Cleavage of sialomucin (CD43) on human leukocytes by Pic. PMNs (A, Pic Cleaves a Broad Array of O-Glycosylated Mucin-Like Proteins. We C, and E) and PBMCs (B, D, and E) were isolated from healthy volunteers and also found that Pic cleaved other mucin type O-glycans involved in treated with log-phase supernatants from the following strains: EAEC 042, diverse functions of the immune system, including PSGL-1, CD44, Δ fi an isogenic EAEC042 pic mutant, or the protease de cient EAEC042- CD45, CD93, and fractalkine/CX3CL1 (Fig. 2A). None of the PicS258A strain; controls comprised 2 μM of purified Pic protein or PBS (A fi and B). Cells were alternatively treated with supernatants of S. flexneri puri ed proteins or bacterial supernatants cleaved recombinant 2457T, an isogenic Pic mutant, an isogenic SigA null mutant strain, or with intercellular adhesion molecule-1 (ICAM-1) (CD54) or lamp-1 2 μM of purified Pic, PicS258A, or SigA (C and D). Following 30-min incubation proteins (Fig. 2A). We treated human neutrophils and lympho- at 37 °C, samples were analyzed by SDS/PAGE followed by immunoblot (A–D) cytes with purified SPATE proteins and S. flexneri supernatants, or flow cytometry (E) using an Hrp- or APC-conjugated monoclonal antibody and analyzed the presence of the various targets by flow cytometry specific to the extracellular domain of human CD43. PBMC were gated by and confocal microscopy, using mAbs that recognize the extra- low side scatter, and PMNs were gated under high side scatter. Flow cyto- cellular domains of each molecule. All molecules that were sus- metry data are representative of at least four independent experiments. ceptible to cleavage in recombinant form were also degraded from the surfaces of both human neutrophils and lymphocytes (Fig. 2 B we first addressed CD43, also known as sialomucin, a major cell- and C). Shigella supernatants cleaved the extracellular domains of surface glycoprotein expressed on nearly all lineages of hemato- PSGL-1, CD45, CD93, and CD43 (Fig. S2), whereas CD3 and poietic cells. Human polymorphonuclear leukocytes (PMN) and CD16 were not affected (Fig. 2B). The heavily O-glycosylated protein CD93, which is present on neutrophils but not on lym- peripheral blood mononuclear cells (PBMC) isolated from blood phocytes, was also degraded by Pic (Fig. 2B). samples of healthy adult volunteers were treated with purified Pic or with the supernatants of EAEC strain 042; for comparison, Pic Recognizes O-, but Not N-Glycosylated Glycans. To ascertain the cells were treated with supernatants of a previously described relevance of carbohydrate substitution in Pic substrate recogni- isogenic null pic mutant, or with supernatants of 042PicS258A, tion, we used human recombinant CD44 and PSGL-1, which are which expresses Pic harboring a single amino acid mutation at the known to harbor both N- and O-linked oligosaccharides. We catalytic serine (Fig. 1 A and B). CD43 was completely degraded found that pretreatment of the glycans with neuraminidase, which

B Neutrophils 500 A 600 DNA-DAPI Acn-FITC PSGL-1-PE CD43-APC All merged 1000 C pic 400 800 1200 D 1500 2500 Vehicle 300 99.36% pic Number Number

90.69% Number 97.81% Number 99.76% 200 Number 400 100 500 200 600

D 99.41% 0 0 0 0 0 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1234 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 500 PicS258A SigA Pic SepA 2457T Wt 2457T 042 042 Wt 042 PBS 2457T 400 600 300 1500 2500 Pic Number 14.91% Number Number 96.65% Number 2.26%

Number 9.14% 200 100 100 300 500 100 300 500

28.59% 500 0 0 0 0 0 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1234 10 10 10 10 10 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 Vehicle 500 PSGL-1 600 800 2000 600 400 99.22% Number 400 Number Cell number 93.81% Number 96.95% Number

S258A 99.34% Number 200 200 400 600 200

100 300 99.51% 01000 0 0 0 0 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 0 1234 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 600 600 2500 500 700 400 400 98.51%

CD45 300 Number

Number 96.20% Number SepA 91.74% Number PMN

99.06% Number 200 200 100 200 300 450

98.97% 500 1500 100 0 0 0 0 0 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1234 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 CD44 APC CD93 FITC CD45RO ECD CD162 PE CD16b PE Fractalkine Lymphocytes Pic 200 160 1400 100 120 160 120 1000 80

78.37% Number Number

100 Lym Number 99.83% Number 40 60 80 99.09% Vehicle Number 1.64% 69.28% 40 200 600 0 04080 0 0 020 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 0 10 1 10 2 10 3 10 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 1000 120 150 200 150 200 80 150 250 600 8.18% Number CD44 Number Number

13.02% Number 99.80% Number 0.66% 40

Pic 50 4.72% 50 100 050100 0200 0 0 0 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 0 10 1 10 2 10 3 10 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 140 1400 200

71.05% 100 150 Number Number 100 150 200 Number Number 100 Number S258A Cell number 99.67% 0.84% 67.90% 99.58% 50 200 600 1000 0 0 0 050 0 1 2 3 4 0 1 2 3 4 02060100 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 200 200 CD54 120 1500 150 150 250 100 PicS258A Number 74.79% Number SepA 100 Number Number 98.79% 1.11% 64.46% Number 99.72% 50 500 1000 0 050 04080 0 050 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 Lamp1 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 CD44 APC CD93 FITC CD45RO ECD CD162 PE CD3 APC-Cy7

Fig. 2. Cleavage of an O-glycosylated mucin-like protein family by Pic. (A) Five micrograms of glycosylated human recombinant proteins were incubated at 37 °C for 1 h with supernatants of S. flexneri 2457T, EAEC 042, the isogenic S. flexneriΔpic and EAEC042Δpic mutant strains, or with 2 μM of purified Pic, PicS258A, SepA, or SigA. Samples were analyzed by SDS/PAGE followed by immunoblot using monoclonal antibodies to the external domain of PSGL-1, CD45, CD44, and fractalkine. Control proteins included ICAM-1 (CD54) and LAMP-1. (B) Pic protein degrades the extracellular domain of O-glycosylated mucin-like proteins on human leukocytes. The 1 × 106 PMNs or PBMCs were isolated from human blood and incubated at 37 °C for 30 min with 2 μM of purified Pic, PicS258A, or SepA and analyzed by flow cytometry using monoclonal antibodies against the extracellular domains of CD44, CD45, PSGL-1, CD93, CD3, or CD16. Lymphocytes were gated by low side scatter using anti-CD3, but neutrophils were gated under high side scatter and anti-CD16 binding. Flow cytometry data are representative of at least four independent experiments. (C) To visualize degradation of mucin-like proteins, Pic-treated whole-blood leukocytes were stained for DNA, actin, CD43, and PSGL-1, followed by fluorescence microscopy. Arrows indicate PMNs and lymphocytes by virtue of nucleus and cytoplasm shape.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1101006108 Ruiz-Perez et al. Downloaded by guest on October 1, 2021 stained human neutrophils with FITC-conjugated anti-SLeX monoclonal antibody. As shown in Fig. S4, SLeX was found on A B Pic more than 90% of the untreated neutrophils, or on cells treated ated + Pic m. m.+ ntre -glyc -glyc eura eura 250 U N N N N Substrate Cleavage sequence with PicS258A or SepA. However, the cell population bearing the Mucin PKY337 - S338 ETN 100 CD44 RSS188 - T189 SGG SLeX carbohydrate was reduced by almost 35% following treat- CD44 CD44 PWI212 - T213 DST ment with purified Pic, compared with untreated cells (Fig. S4).

50 PSGL-1 EAL262 - S263 TEP

* 263 264 * PSGL-1 ALS - T EPS 25 Interestingly, increasing concentrations of synthetic SLeX car- CD45 NAI143 - S144 DVP μ 200 CD45 AVI212 - S213 TTT bohydrate (5 nM to 1.6 M) preincubated with Pic protein before CD162 100 Aliphac or small residue S/T with potenal treatment of the recombinant PSGL-1 protein engendered dose- O-linked sacharide 50 dependent inhibition of Pic protease activity on PSGL-1 (Fig. 4B). 25 Cleavage site We confirmed that the recombinant PSGL-1 used in the cleavage assays bear SLeX (Fig. 4C). Fig. 3. Proteolytic activity of Pic is dependent on O-, but not on N-glyco- sylation. (A) Five micrograms of recombinant CD44 or PSGL-1 proteins were Pic Impairs PMN Chemotaxis and Transmigration. We also assessed de-glycosylated with neuraminidase or N-glycosydase-F at 37 °C overnight; the effects of Pic on PMN function using in vitro chemotaxis deglycosylated proteins were then incubated with 2 μM of Pic at 37 °C for 1 h. assays. Calcein-labeled PMNs incubated with Pic, PicS258A, or Samples were analyzed by Western immunoblot using an anti-CD44 or anti- μ PSGL-1 antibodies. (B) Amino acid sequences flanking cleavage sites in four PBS vehicle control were applied to 3- M pore transwell mem- Pic substrates are shown. Asterisks indicate degraded products analyzed by branes and stimulated with IL-8 or fMLP (N-formyl-methionine- N-terminal sequencing. leucine-phenylalanine) as chemoattractants; after 4 h incubation, the neutrophils that had transmigrated toward the chemo- attractant were enumerated. We observed ∼20% translocation of removes both N- and O-linked oligosaccharides, prevented deg- untreated PMNs in this model (Fig. 5A), compared with only <2% radation of both glycoproteins, implicating carbohydrates in PMN migration when the PMNs were treated with Pic protease substrate recognition (Fig. 3A). In contrast, removing N-linked (Fig. 5A)(P < 0.01). Unexpectedly, the movement of PMNs in- saccharides with N-glycosidase-F did not protect CD44 or PSGL-1 cubated with PicS258A was significantly reduced compared with from degradation by Pic (Fig. 3A). Moreover, Pic was not able to buffer alone as negative control (P < 0.05) (Fig. 5A); denaturation degrade the heavily N-linked glycosylated proteins E-, P- or L- of PicS258A by heating completely abolished its effects (Fig. 5A).

, nor -1 (MAC-1) integrin (Fig. S3). To addressed the potential effect of Pic on PMN transmigration MICROBIOLOGY To assess the Pic cleavage site, we treated recombinant PSGL-1, through endothelial cell monolayers, calcein-labeled PMNs were CD44, and CD45 with purified Pic and characterized the domi- treated with Pic and added to human lung microvascular endo- nant early degradation products by N-terminal sequencing (Fig. thelial cells (HBMVEC-L) monolayers cultured in transwells, and S3B). In all cases, Pic cleaved before a serine or threonine residue stimulated with IL-8 and fMLP as chemoatractants. Nearly 65% (Fig. 3B and Fig. S3C). Most of the putative cleavage sites oc- and 40% of untreated neutrophils transmigrated in the presence curred between Ser/Thr and aliphatic (I, V, L) or small amino of IL-8 and fMLP, respectively (Fig. 5A). In contrast, trans- acids (A, V, S, T). migration of cells treated with Pic protease in the presence of IL-8 or fMLP was ∼31% and 21%, respectively (both P < 0.01) (Fig. Inhibition of P-Selectin Binding to Human Leukocytes by Pic. To ad- 5A). We observed a 1.5-fold reduction of PMN transmigration dress the functional significance of Pic, we treated human neu- when the leukocytes were incubated with PicS258A compared trophils and lymphocytes with purified Pic, PicS258A, or PBS with untreated cells (P < 0.05) (Fig. 5A); this effect was abolished vehicle control for 30 min, followed by incubation with human P- by heat-treatment of the protease (Fig. 5A). Taken together, these selectin-IgG chimera. Binding of P-selectin chimera to leukocytes data suggest that interaction of Pic with O-glycoproteins directly was evaluated by flow cytometry using a FITC-conjugated anti- impairs PMN function. human Fc antibody. We observed the expected interaction be- tween P-selectin and untreated PMNs or PBMCs (Fig. 4A); Activation of the Oxidative Burst by Pic. The neutrophil however, cells treated with purified Pic exhibited diminished in- oxidative burst is also induced by stimulation of glycoproteins at teraction with P-selectin. Rolling interactions between leukocytes the plasma membrane (21). We found that LPS-free Pic at 2 μM and the are mediated by selectin–PSGL-1 in- (the minimal concentration required for depletion of more than teraction, and require branched Sialyl Lewis-X (SLeX)–O-glycan 90% of O-glycoproteins in a 1-h period) (Fig. S5) triggered the extensions on specific PSGL-1 amino acid residues. To evaluate respiratory burst in at least 10% to 15% of PMNs after 30-min whether SLeX saccharide was affected by Pic treatment, we incubation, and in more than 50% of cells after 60 min (Fig. 5B

Fig. 4. Inhibition of P-selectin binding to human leukocyte PMNs PMBCs SLeX (nM) 6 A B

× 600 by Pic. (A)1 10 human lymphocytes were treated with 2.08% 2000

Vehicle 1.45% SLeX 200 μ fi 1500 0 2 M puri ed Pic or PicS258A, or with PBS control for 30 min 0 a 100 50 5 Pic De-glyc PBS No P-Sel 0 1 2 3 4 0 1 2 3 4 25 followed for 30-min incubation with human P-selectin-IgG 10 10 10 10 10 10 10 10 10 10 500 600 82.08% 41.77% PSGL-1 chimera. Binding of P-selectin chimera to leukocytes was 300 Vehicle 200 0 fl 0 1 2 3 4 0 0 1 2 3 4 evaluated by ow cytometry using a FITC-conjugated anti 10 10 10 10 10 10 10 10 10 10 500 human Fc antibody. Untreated cells without incubation 300 35.89% 12.71% Pic SLeX Cell number 0300 with P-selectin chimera (No P-Sel) were also stained with 0100 a 200 PBS De-glyc Pic 400 800 0 1 2 3 4 0 1 2 3 4 1600 antihuman Fc antibody. (B) Inhibition of Pic protease ac- 10 10 10 10 10 10 10 10 10 10 400 800 82.16% tivity by SLeX carbohydrate was evaluated by Western blot PicS258A 400 37.37% PSGL-1 0 0200 μ μ 0 1 2 3 4 0 1 2 3 4 using 5 g of recombinant PSGL-1 and 2 M of Pic pre- 10 10 10 10 10 10 10 10 10 10 viously incubated with twofold increasing concentration of Anti-FC FITC-A Anti-FC FITC-A C 1 2 SLeX carbohydrate (5–1,600 nM). PSGL-1 or PSGL-1 degly- 1-PSGL-1 P-selectin binding cosylated by neuraminidase treatment (De-glyc) were in- 2-De-glyc. PSGL-1 a-SLeX cubated with Pic as controls. PBS, untreated PSGL-1; α-SLeX, PSGL-1 previously incubated with an anti-SLeX antibody before treatment with Pic. Western blot was developed with an anti PSGL-1 monoclonal antibody. (C) SLeX saccharide on PSGL-1 and deglycosylated PSGL-1 were analyzed by Western blot using a monoclonal antibody against SLeX.

Ruiz-Perez et al. PNAS Early Edition | 3of6 Downloaded by guest on October 1, 2021 Fig. 5. Impairment of PMN chemotaxis and transmigration through endothelial cell monolayers by Pic. (A) Chemotaxis: 3 × 105 calcein-AM labeled PMNs were treated with 2 μM of Pic, PicS258A, or PBS vehicle control in the upper chamber of Fbg-coated transwell inserts; 100 mM of IL-8 or 100 nM of fMLP were added to the lower chamber. Penetration of cells through the membrane was measured after 4 h fluorometrically. Transmigration: PMNs preincubated as above were applied to the upper chamber of inserts supporting HBMVEC-L, and transmigrated PMNs were enumerated as mentioned before. Data shown are means and SEM of at least three independent experiments performed in triplicate; dates were combined and analyzed by one-way ANOVA. The numbers of migrated cells were normalized to vehicle control with chemoattractant (**P < 0.01, *P < 0.05). (B) Activation of neutrophil oxidative burst by Pic: 1 × 106 human neutrophils were labeled with dihydrorhodamine-123 dye for 5 min, immediately treated with 2 μM of LPS-free purified Pic, PicS258A, heat-denatured PicS258A, or PBS vehicle control, and analyzed by flow cytometry for oxidative burst, indicated by FITC fluorescence at 30- and 60-min time points. For positive control, 100 ng/mL of PMA was used. The cell population was gated to distinguish vehicle-treated and PMA-treated controls. Flow cytometry data are representative of three independent experiments.

and Fig. S6). The protease mutant PicS258A activated the oxi- Effect of Pic in the Guinea Pig Keratoconjunctivitis Model. Our dative burst to the same degree as Pic (Fig. 5B and Fig. S6), in vitro studies of Pic protease suggested both pro- and antiin- whereas heat-treatment of PicS258A abolished this effect (Fig. 5B flammatory contributions to Shigella pathogenesis. To discern and Fig. S6), further ruling out an effect of contaminating heat- which of these effects prevailed in the setting of acute Shigella- fl stable LPS. induced in ammation, we used the widely used guinea pig kera- toconjunctivitis model (26), in which the evolution of the in- Pic Induces Apoptosis of Activated T Cells. Cross-linking of PSGL-1, flammatory response to Shigella infection can be observed over CD43, CD44, CD45, and CD99 on activated T cells induces ap- time. Compared with wild-type S. flexneri 2a strain 2457T, the pic optosis (22–25). To determine whether Pic binding or cleavage of protease mutant induced a greater inflammatory response over fi O-glycans on activated T cells induces apoptosis, we isolated the rst 2 d of the infection (Fig. S8); after this, time differences in fl lymphocytes from healthy volunteers and activated the cells with the in ammation response were less apparent. We ruled out the ConA, then treated the cells with Pic; cells were stained with possibility of incidental mutations in 2457Tpic by reconstructing annexin V or propidium iodide (PI), to detect apoptosis or ne- the native pic using allelic exchange. We simultaneously ruled out any effect of SetAB enterotoxin encoded in the com- crosis, respectively. More than 50% of cells treated with Pic un- plementary strand of the pic gene by creating stop codons in the derwent apoptosis in these experiments, which was comparable to setAB gene without affecting the Pic amino acid sequence (see SI the levels induced by cross-linking of CD3 and CD28 with specific Materials and Methods). S. flexneri 2457T harboring the recon- mAbs (Fig. 6). Cells treated with PicS258A exhibited reduced structed pic/setAB gene induced inflammation over the first 2 d levels of apoptosis compared with those treated with active Pic; that was similar to the wild-type and significantly less than that however, the proportion of apoptotic cells was approximately induced by the pic mutant (Fig. S8)(P < 0.05). twofold higher than that observed in untreated cells (Fig. 6). Necrosis was apparent in all treatments, but exclusively after onset Other Class 2 SPATEs Induce Effects Similar to Pic. To determine if of apoptosis (Annexin-V/PI double staining). Apoptosis phe- other members of the class 2 SPATEs cleave targets similar to nomenon was also confirmed by detecting fragmented DNA in those recognized by Pic, we treated human leukocytes with the Tunel assays (Fig. S7). purified Tsh protein of avian pathogenic E. coli, which differs in

Lymphocyte gate Isotype control Vehicle 4 4 10 10

3 3 0.80% 6.55% 10 10 2 2 PI PI PI PI 10 10 SSC 1 1 74.70% 1000 2000 3000 4000 10 10 R1: 55.21% 0.00% 17.95% 99.86% 0 0 0 10 0 1000 2000 3000 4000 1010 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 FSC AnnexinV FITC AnnexinV FITC Pic PicS258A Anti- CD3/CD28 4 4 4 Necrosis 10 10 10 3 3 3 0.22% 13.71% Fig. 6. Pic triggers cell death of activated human T cells. Ac- 10 10

0.32% 6.63% 10 0.11% 6.37% tivated human T lymphocytes on day 5 after stimulation with 2 2 2 PI PI PI μ μ fi 10 10

10 ConA (2 g/mL) were incubated with 2 M of puri ed Pic, PI PI PI 1 1 1 PicS258A, or control anti-CD3/CD28 antibodies overnight. Fol- 35.01% 59.19% 10

10 18.73% 58.51% 33.86% 10 67.34% lowing double staining with annexin-V FITC and PI, cells were 35.01% 58.51% 0 0 0 fl 10

10 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 1010 0 10 1 10 2 10 3 10 4 analyzed by ow cytometry to determine the percentage of AnnexinV FITC AnnexinV FITC AnnexinV FITC dead cells. Without gating, 10% of 10,000 total events were displayed. Flow cytometry data are representative of at least Apoptosis three independent experiments.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1101006108 Ruiz-Perez et al. Downloaded by guest on October 1, 2021 only two residues from Hbp, found in human invasive E. coli. Tsh matory response signature. The recognition of mucin by Pic via its degraded all leukocyte O-glycans tested, including PSGL-1, glycan substitutions has been previously suggested (40). As in that CD43, CD93, and fractalkine (Fig. 7). prior report, we found that protease-deficient Pic retains some functions, presumably via noncatalytic substrate binding. Discussion We were surprised that Pic induced PMN activation and pro- Pic efficiently cleaves leukocyte surface glycoproteins involved in grammed T-cell death. The effects on PMN activation and T-cell diverse human immune functions. In this article, we show that apoptosis could result from cross-linking of PSGL-1, CD43, Pic substrates include a remarkable array of O-linked glycans CD44, CD45, or CD99 on leukocytes; such cross-linking by spe- modified with SLeX. cific antibodies or their natural ligands is known to result in ac- One of these targets, CD43, is among the most abundant mu- tivation of the oxidative bust and apoptosis (22–25, 41). Beside the cin-like leukocyte surface glycoproteins, and is expressed on O-glycoproteins tested in this study, CD34, CD68, CD99, CD164, nearly all lineages of hematopoietic cells, including cells of the and the recently discovered Tim (T-cell Ig-mucin domain) cluster, bone marrow, the thymus, and peripheral lymphoid tissues (27). a family of proteins harboring mucin-like domains, represent In its native form, CD43 prevents leukocytes from adhering to potential targets for Pic and Pic-related proteases. neighboring cells, thereby permitting migration in response to Cleavage of the mucin-like glycoproteins involved in leukocyte chemokine attraction. Upon processing, the protein plays an trafficking prevent chemoattraction and migration of leukocytes, opposite role: facilitating binding of the leukocyte to neighboring and may also inhibit their antibacterial functions once arrived at cells (28, 29). Szabady et al. reported recently that the StcE pro- the nidus of infection. In another scenario, premature nonspecific tease of enterohemorrhagic E. coli inhibits PMN chemoattraction activation of PMNs may enhance inflammation, including aggra- and function by cleavage of CD43 (30). Our data suggest that vated tissue damage and increased recruitment of leukocytes to leukocyte CD43 may also be affected by the pathogens S. flexneri, the area. Moreover, apoptosis of T cells by Pic would prolong an EAEC, UPEC, avian pathogenic E. coli, and human invasive E. aberrant and misdirected inflammatory response, given the cen- coli, thereby underscoring the pathogenic importance of this tral role of T-cell–generated IFN-γ in sterilization of Shigella in- phenomenon among diverse systems (Fig. S9). fection (42). In the Guinea pig keratoconjuctivitis model, Pic Pic’s other targets could be at least as important as CD43. Pic produced an antiinflammatory effect. Should this effect prevail in cleaves PSGL-1, involved in leukocyte trafficking and tethering the setting of human shigellosis, it could retard induction of innate to endothelial selectins (reviewed in ref. 31); the multifunc- and adaptive responses that would be expected to control bacte-

tional cell-surface glycoprotein CD44, controlling rial multiplication. Further understanding of the role of Pic in MICROBIOLOGY through cell–cell and cell–matrix interactions (32, 33); the CD45 shigellosis will require more detailed investigations in multiple receptor-like protein tyrosine phosphatase, involved in cell ad- models, perhaps including volunteer studies. hesion, migration, modulation of cytokine production and sig- Pic is expressed almost exclusively by S. flexneri 2a, which is naling (34, 35); the CD93 glycoprotein, promoting migration, consistently the dominant Shigella serotype worldwide; Pic may of antibody and complement opsonized particles account for this epidemiologic dominance. Interestingly, Pic and (36, 37); and fractalkine/CX3CL1, a membrane-bound chemo- Tsh are also associated with E. coli strains that cause extra- kine that functions not only as a chemoattractant but also as an intestinal infections (43), supporting our hypothesis that their adhesion molecule induced on activated endothelial cells by pro- roles involve targeting of general immune functions operating at fl in ammatory cytokines (38, 39). Cleavage of these diverse sub- diverse niches (Fig. S9). Further research in our laboratories will strates could result in dramatic paralysis of the leukocyte- explore these potential roles and their clinical implications. We mediated response at a local level. recognize, in addition, that the broad functionality of Pic and ’ fi The mechanism of Pic s substrate speci city is noteworthy. related proteases presents therapeutic opportunities against a SLeX is a tetrasaccharide carbohydrate that is usually attached to large number of inflammatory disorders. O-glycans, such as PSGL-1, but which is also found on other gly- cans, including mucin, CD43, and CD44. This molecule has a vital Materials and Methods role in cell-to-cell recognition processes and is a determinant of Detailed experimental procedures are given in SI Materials and Methods. both E- and P-selectins. Binding to this particular glycan group Purification of SPATE proteins was previously described (5). Transmigra- provides important functional specificity for Pic, which has ap- tion, chemotaxis and oxidative burst assays were performed according to parently exploited the host’s use of SLeX as a common inflam- described protocols (44–46). Apoptosis induced by Pic on activated T lym-

PMNs

A 150 87.87% B 5000 6000 500 100 5000 4000 400 Number 4000 3000

98.56%% 300 50 3000 90.69% 87.87% Number Number 99.78%

Vehicle 2000 200 2000 Number 0 1000 1000 0 0 0 1 2 3 4 0 1 2 3 4 10 100 0 10 1 10 2 10 3 10 4 0 1 2 3 4

10 10 10 10 10 10 10 10 10 10 0 10 10 10 10 10 CD162 PE-A CD43 APC-A CD93 FITC-A CD16 APC-Alexa700-A 150

87.23% 150 25 75 50 6000 450 5000 400 5000 100 350 4000 4000 Number Tsh 300 87.23% 3000 250 3000 25.54% 50 200 Number 15.39% Number 6.51% 2000 2000 150 Number 0 100 1000 1000

10 50 0 10 1 10 2 10 3 10 4 0 0 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 0 100 101 102 103 104 CD93 FITC-A CD162 PE-A CD43 APC-A CD16 APC-Alexa700-A 140 78.77% 120 6000 5000 400 100 5000 ll number 350 000 er 0 4 b 8 4000 Num e

60 78. 77% 3000 Tsh/Inh 250 300 3000 Fig. 7. Tsh degrades the extracellular domain of O-glyco- 40 200 C 99. 28% 87. 44% Number Number 2000 96. 55% 20 2000 150 0 1000 100 1000 0 0 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 50 0 10 1 10 2 10 3 10 4 100 101 102 103 104 0 CD162 PE-A CD43 APC-A CD93 FITC-A CD16 APC-Alexa700-A sylated mucin-like proteins on human leukocytes. (A)1× 250 86.54% 450 3000 200 5000

400 PBS 6 2500 150 350 4000 Number 2000 300 10 PMNs and PBMCs were isolated from human blood and 100 86.54% 3000

SepA 250 1500 99.64% 91.74% Number Number 200 82.07% 50 2000 1000 150 0 Number 500 1000 100 0 0 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 50 0 10 1 10 2 10 3 10 4 100 101 102 103 104 0 μ fi CD162 PE-A CD43 APC-A CD93 FITC-A CD16 APC-Alexa700-A incubated at 37 °C for 30 min. with 1 M of puri ed Tsh or CD43-APC CD93-PE Pic CD162-PE CD16-Alexa 700 Tsh previously inactivated with AEBSF (serine protease in- PBMCs hibitor). SepA was used as a protein control for digestion. 350 160 100 100 140 300 80 80 0 2 60 60 100 1 80 200 250 SigA fl 60 40 40 VhilVehicle 57. 12% 150 Number Number 60. 72% 164%1.64% 88. 72% Samples were analyzed by ow cytometry using mono- Number 40 100 Number 20 20 20 50 10 0 10 1 10 2 10 3 10 4 0 0 0 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 CD93 FITC-A 10 0 0 10 1 10 2 10 3 10 CD162 PE-A CD43 APC-A CD3 APC-Cy7-A clonal antibodies against the extracellular domain of PSGL- 400 200 350 350 350 300 300 300 150 250 250 250 200 200 200 Tsh 100 1, CD43, CD93, CD3, and CD16. PMNs and PBMCs were

150 SepA 150

150 3.38% 0.66%

Number Number 87.25% 9.36% 50 Number 100 Number 100 100 50

50 10 0 10 1 10 2 10 3 10 4 50 0 CD93 FITC-A 0 0 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 0 10 1 10 2 10 3 10 CD162 PE-A CD43 APC-A CD3 APC-Cy7-A gated as in Fig 2B. Flow cytometry results are representa- 120 350 120 200 300 100 100 80 250 150 80 200 Tsh/Inh 60 tive of at least three independent experiments. (B) Human 60 100 Cell number 52.21% 150 40

Number Number Tsh 40 54.60% 0.62% 84.69% Number 100 50 20 20 Number 50 0 1 2 3 4

0 0 10 10 10 10 10 0 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 0 10 0 10 1 10 2 10 3 10 CD93 FITC-A μ fi CD162 PE-A CD43 APC-A CD3 APC-Cy7-A recombinant fractalkine was treated with 2 M of puri ed 400 400 300 200 350 350 250 300 300 150 250 200 Pic, SepA, Tsh, or SigA for 1 h at 37 °C and analyzed by 250

SepA 200 150 200

77.14% 100 150 Number Number 0.83% 90.00% 150

100 79.11%

Number Fractalkine 50 100 100 Number 50 50

10 0 10 1 10 2 10 3 10 4 50 0 0 0 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 CD93 FITC-A 10 0 0 10 1 10 2 10 3 10 Western blot using a monoclonal antibody against human CD162 PE-A CD43 APC-A CD3 APC-Cy7-A CD162-PE CD43-APC CD93-PE CD3-APC-Cy7 fractalkine.

Ruiz-Perez et al. PNAS Early Edition | 5of6 Downloaded by guest on October 1, 2021 phocytes was determined according to the protocol recently published Subjects committee. Animal experiments were conducted according to the (41). The keratoconjunctivitis model is described in SI Materials and Methods protocol 0210016, approved by the University of Maryland at Baltimore- and ref. 26. International Animal Care and Use Committee.

Ethics Statement. Blood from healthy donators were obtained under in- ACKNOWLEDGMENTS. We thank Regina Harley and Jill Harper for technical formed consent under protocol HP-00042957, approved by the Institutional assistance. This work was supported by United States Public Health Service Review Board of the University of Maryland School of Medicine Human Grants AI-33096 and AI-43615 (to J.P.N.).

1. Kaper JB, Nataro JP, Mobley HL (2004) Pathogenic Escherichia coli. Nat Rev Microbiol 23. Bazil V, Brandt J, Tsukamoto A, Hoffman R (1995) Apoptosis of human hematopoietic 2:123–140. progenitor cells induced by crosslinking of surface CD43, the major sialoglycoprotein 2. O’Ryan M, Prado V, Pickering LK (2005) A millennium update on pediatric diarrheal of leukocytes. Blood 86:502–511. illness in the developing world. Semin Pediatr Infect Dis 16:125–136. 24. Bernard G, et al. (1997) Apoptosis of immature thymocytes mediated by E2/CD99. 3. Henderson IR, Navarro-Garcia F, Nataro JP (1998) The great escape: Structure and J Immunol 158:2543–2550. function of the autotransporter proteins. Trends Microbiol 6:370–378. 25. Klaus SJ, Sidorenko SP, Clark EA (1996) CD45 ligation induces programmed cell death 4. Henderson IR, Nataro JP (2001) Virulence functions of autotransporter proteins. Infect in T and B lymphocytes. J Immunol 156:2743–2753. Immun 69:1231–1243. 26. Sereny B (1957) Experimental keratoconjunctivitis shigellosa. Acta Microbiol Acad Sci 5. Dutta PR, Cappello R, Navarro-García F, Nataro JP (2002) Functional comparison of Hung 4:367–376. serine protease autotransporters of enterobacteriaceae. Infect Immun 70:7105–7113. 27. Rosenstein Y, Santana A, Pedraza-Alva G (1999) CD43, a molecule with multiple 6. Al-Hasani K, Navarro-Garcia F, Huerta J, Sakellaris H, Adler B (2009) The immunogenic functions. Immunol Res 20:89–99. SigA enterotoxin of Shigella flexneri 2a binds to HEp-2 cells and induces fodrin re- 28. Manjunath N, Johnson RS, Staunton DE, Pasqualini R, Ardman B (1993) Targeted distribution in intoxicated epithelial cells. PLoS ONE 4:e8223. disruption of CD43 gene enhances T lymphocyte adhesion. J Immunol 151:1528–1534. 7. Canizalez-Roman A, Navarro-García F (2003) Fodrin CaM-binding domain cleavage by 29. Seveau S, Keller H, Maxfield FR, Piller F, Halbwachs-Mecarelli L (2000) Neutrophil Pet from enteroaggregative Escherichia coli leads to actin cytoskeletal disruption. Mol polarity and locomotion are associated with surface redistribution of leukosialin Microbiol 48:947–958. (CD43), an antiadhesive membrane molecule. Blood 95:2462–2470. 8. Navarro-García F, Canizalez-Roman A, Luna J, Sears C, Nataro JP (2001) Plasmid- 30. Szabady RL, Lokuta MA, Walters KB, Huttenlocher A, Welch RA (2009) Modulation of encoded toxin of enteroaggregative Escherichia coli is internalized by epithelial cells. neutrophil function by a secreted mucinase of Escherichia coli O157:H7. PLoS Pathog Infect Immun 69:1053–1060. 5:e1000320. 9. Otto BR, van Dooren SJ, Nuijens JH, Luirink J, Oudega B (1998) Characterization of 31. Carlow DA, et al. (2009) PSGL-1 function in immunity and steady state homeostasis. a hemoglobin protease secreted by the pathogenic Escherichia coli strain EB1. J Exp Immunol Rev 230:75–96. Med 188:1091–1103. 32. Lesley J, Hyman R, Kincade PW (1993) CD44 and its interaction with extracellular 10. Provence DL, Curtiss R, 3rd (1994) Isolation and characterization of a gene involved in matrix. Adv Immunol 54:271–335. hemagglutination by an avian pathogenic Escherichia coli strain. Infect Immun 62: 33. Jalkanen S, Jalkanen M (1992) Lymphocyte CD44 binds the COOH-terminal heparin- 1369–1380. binding domain of fibronectin. J Cell Biol 116:817–825. 11. Henderson IR, Czeczulin J, Eslava C, Noriega F, Nataro JP (1999) Characterization of 34. Harvath L, Balke JA, Christiansen NP, Russell AA, Skubitz KM (1991) Selected anti- pic, a secreted protease of Shigella flexneri and enteroaggregative Escherichia coli. bodies to leukocyte common antigen (CD45) inhibit human neutrophil chemotaxis. Infect Immun 67:5587–5596. J Immunol 146:949–957. 12. Parham NJ, et al. (2004) PicU, a second serine protease autotransporter of ur- 35. Lai JC, Wlodarska M, Liu DJ, Abraham N, Johnson P (2010) CD45 regulates migration, opathogenic Escherichia coli. FEMS Microbiol Lett 230:73–83. proliferation, and progression of double negative 1 thymocytes. J Immunol 185: 13. Rajakumar K, Sasakawa C, Adler B (1997) Use of a novel approach, termed island 2059–2070. probing, identifies the Shigella flexneri she pathogenicity island which encodes 36. Steinberger P, et al. (2002) Identification of human CD93 as the phagocytic C1q re- a homolog of the immunoglobulin A protease-like family of proteins. Infect Immun ceptor (C1qRp) by expression cloning. J Leukoc Biol 71:133–140. 65:4606–4614. 37. Bohlson SS, Zhang M, Ortiz CE, Tenner AJ (2005) CD93 interacts with the PDZ domain- 14. Leyton DL, Sloan J, Hill RE, Doughty S, Hartland EL (2003) Transfer region of pO113 containing adaptor protein GIPC: Implications in the modulation of phagocytosis. from enterohemorrhagic Escherichia coli: Similarity with R64 and identification of J Leukoc Biol 77:80–89. a novel plasmid-encoded autotransporter, EpeA. Infect Immun 71:6307–6319. 38. Bazan JF, et al. (1997) A new class of membrane-bound chemokine with a CX3C motif. 15. Harrington SM, et al. (2009) The Pic protease of enteroaggregative Escherichia coli Nature 385:640–644. promotes intestinal colonization and growth in the presence of mucin. Infect Immun 39. Fong AM, et al. (1998) Fractalkine and CX3CR1 mediate a novel mechanism of leu- 77:2465–2473. kocyte capture, firm adhesion, and activation under physiologic flow. J Exp Med 188: 16. Navarro-Garcia F, et al. (2010) Pic, an autotransporter protein secreted by different 1413–1419. pathogens in the Enterobacteriaceae family, is a potent mucus secretagogue. Infect 40. Gutiérrez-Jiménez J, Arciniega I, Navarro-García F (2008) The serine protease motif of Immun 78:4101–4109. Pic mediates a dose-dependent mucolytic activity after binding to sugar constituents 17. Restieri C, Garriss G, Locas MC, Dozois CM (2007) Autotransporter-encoding se- of the mucin substrate. Microb Pathog 45:115–123. quences are phylogenetically distributed among Escherichia coli clinical isolates and 41. Chen SC, et al. (2004) Cross-linking of P-selectin glycoprotein ligand-1 induces death reference strains. Appl Environ Microbiol 73:1553–1562. of activated T cells. Blood 104:3233–3242. 18. Parham NJ, et al. (2005) Distribution of the serine protease autotransporters of the 42. Le-Barillec K, et al. (2005) Roles for T and NK cells in the innate immune response to Enterobacteriaceae among extraintestinal clinical isolates of Escherichia coli. J Clin Shigella flexneri. J Immunol 175:1735–1740. Microbiol 43:4076–4082. 43. Heimer SR, Rasko DA, Lockatell CV, Johnson DE, Mobley HL (2004) Autotransporter 19. Ohtsubo K, Marth JD (2006) Glycosylation in cellular mechanisms of health and dis- pic and tsh are associated with Escherichia coli strains that cause acute pyelo- ease. Cell 126:855–867. nephritis and are expressed during urinary tract infection. Infect Immun 72:593–597. 20. Marth JD, Grewal PK (2008) Mammalian glycosylation in immunity. Nat Rev Immunol 44. Yamamoto H, Sedgwick JB, Vrtis RF, Busse WW (2000) The effect of transendothelial 8:874–887. migration on eosinophil function. Am J Respir Cell Mol Biol 23:379–388. 21. Lund-Johansen F, et al. (1993) Activation of human and granulocytes by 45. Lokuta MA, Huttenlocher A (2005) TNF-alpha promotes a stop signal that inhibits monoclonal antibodies to glycosylphosphatidylinositol-anchored antigens. Eur J Im- neutrophil polarization and migration via a p38 MAPK pathway. J Leukoc Biol 78: munol 23:2782–2791. 210–219. 22. Artus C, et al. (2006) CD44 ligation induces caspase-independent cell death via a novel 46. Smith JA, Weidemann MJ (1993) Further characterization of the neutrophil oxidative calpain/AIF pathway in human erythroleukemia cells. Oncogene 25:5741–5751. burst by flow cytometry. J Immunol Methods 162:261–268.

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