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Serine Protease Autotransporters from Shigella Flexneri and Pathogenic

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Serine Protease Autotransporters from Shigella Flexneri and Pathogenic 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 proteins 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 protein 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-selectin 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 apoptosis, activation, migration, and 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., MICROBIOLOGY 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 | August 2, 2011 | vol. 108 | no. 31 | 12881–12886 Downloaded by guest on October 2, 2021 8A by Pic on both PMNs and PBMCs, as ascertained by Western 25 E Neutrophils 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.
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