Hemolysin Triggers Proteolysis of Host Proteins to Disrupt Cell Adhesion, Inﬂammatory, and Survival Pathways

Cell Host & Microbe Article

The UPEC Pore-Forming Toxin a-Hemolysin Triggers Proteolysis of Host Proteins to Disrupt Cell Adhesion, Inﬂammatory, and Survival Pathways

Bijaya K. Dhakal1 and Matthew A. Mulvey1,* 1Division of Microbiology and Immunology, Pathology Department, University of Utah School of Medicine, Salt Lake City, Utah, USA 84112-0565 *Correspondence: [email protected] DOI 10.1016/j.chom.2011.12.003

SUMMARY a rapid regenerative response in the bladder mucosa and underlying stromal cells (Mysorekar et al., 2009; Mysorekar et al., Uropathogenic Escherichia coli (UPEC), which are 2002; Shin et al., 2011). While the regeneration of bladder urothe- the leading cause of both acute and chronic urinary lial cells has been defined over the past few years with increasing tract infections, often secrete a labile pore-forming clarity, the host and bacterial factors that modulate bladder cell toxin known as a-hemolysin (HlyA). We show that exfoliation during the course of a urinary tract infection (UTI) stable insertion of HlyA into epithelial cell and macro- remain poorly understood. phage membranes triggers degradation of the cyto- In general, host cell exfoliation entails degradation of cell-cell and cell-matrix contacts, presumably requiring disassembly of skeletal scaffolding protein paxillin and other host focal adhesions and other functionally related structures. The regulatory proteins, as well as components of the manipulation of host cell exfoliation and turnover rates within k proinflammatory NF B signaling cascade. Proteol- mucosal barriers likely impacts the establishment, dissemina- ysis of these factors requires host serine proteases, tion, and persistence of diverse bacterial pathogens both within and paxillin degradation specifically involves the and outside of the urinary tract. For example, the dental path- serine protease mesotrypsin. The induced activation ogen Porphyromonas gingivalis secretes proteases that degrade of mesotrypsin by HlyA is preceded by redistribution focal adhesion-associated components like focal adhesion of mesotrypsin precursors from the cytosol into foci kinase (FAK) and the cytoskeletal scaffolding protein paxillin, along microtubules and within nuclei. HlyA intoxica- stimulating the exfoliation of oral keratinocytes and thereby tion also stimulated caspase activation, which creating a niche for bacterial expansion (Nakagawa et al., occurred independently of effects on host serine 2006). In contrast, in the intestinal tract where the continuous shedding and rapid turnover of epithelial cells is the norm, path- proteases. HlyA-induced proteolysis of host proteins ogens may benefit by hindering host cell exfoliation. Shigella likely allows UPEC to not only modulate epithelial flexneri and several other enteric pathogens accomplish this by cell functions, but also disable macrophages and stabilizing focal adhesions via secretion of a protein complex suppress inflammatory responses. (OspE) that interacts with integrin-linked kinase and consequently suppresses phosphorylation of FAK and paxillin (Kim et al., 2009). Likewise, Neisseria gonorrhoeae can attenuate INTRODUCTION normal host cell exfoliation within the vaginal mucosa by altering the composition of focal adhesions via engagement of specific The uroepithelial cells that comprise the stratified layers of the carcinoembryonic antigen-related cell adhesion molecule (CEA- bladder mucosa have an exceptionally slow turnover rate, but CAM) host receptors (Muenzner et al., 2010). Pathogens like will often exfoliate in response to infection with uropathogenic Haemophilus influenzae, Neisseria meningitidis, and Moraxella Escherichia coli (UPEC) (Bower et al., 2005). These bacteria catarrhalis may act similarly at other sites within the host. are able to bind and invade host cells, where they can replicate In light of these and related observations, we sought to to high levels, forming intracellular biofilm-like communities uncover possible mechanisms by which UPEC might disrupt (Anderson et al., 2003; Mulvey et al., 2001). Alternatively, inter- focal adhesion-associated components like paxillin and thereby nalized UPEC can enter a more quiescent state that likely facili- prime bladder epithelial cells for exfoliation. Our results indicate tates long-term bacterial persistence within the urinary tract that sublytic concentrations of the UPEC-associated pore-form- (Blango and Mulvey, 2010; Eto et al., 2006; Mulvey et al., ing toxin a-hemolysin (HlyA) can trigger rapid degradation of 2001). The exfoliation of uroepithelial cells can function to clear paxillin as well as a subset of other host proteins involved in adherent and intracellular bacteria, but may also promote the cell-cell and cell-matrix interactions. We show that HlyA also dissemination of UPEC, both within and outside of the host. induces proteolysis of key host cell signaling factors, including Consequently, the shedding of uroepithelial cells can be viewed those central to host inflammatory responses. HlyA-induced as a double-edged sword, potentially benefitting both host and proteolysis of these proteins is indirect, occurring in parallel pathogen. Exfoliation in response to infection or injury triggers with caspase activation via a pathway involving an atypical

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a 10 kDa N-terminal fragment of paxillin, followed by release of intact, full-length GFP (Figure 1B). GFP itself was not degraded in these assays. Additional work showed that lipopolysaccharide (LPS), the type 1 pilus-associated adhesin FimH, and host cell invasion by UPEC or other invasive pathogens were not sufﬁcient to trigger paxillin proteolysis within infected BECs (Figure S1). To gain further insight into what factor(s) elicit paxillin degradation in our infection assays, and to determine how widespread this phenomenon was, we utilized a panel of other distinct bacterial isolates to infect BECs. Of 20 UPEC isolates examined, 14 (60%) triggered proteolysis of paxillin (Figure 2A). In contrast, paxillin remained stable in BECs infected with any of several non-UPEC isolates tested. The non-E. coli uropatho- gens Staphylococcus saprophyticus, S. aureus, Enterococcus faecium, and Klebsiella pneumoniae also failed to induce paxillin degradation. With the exception of S. aureus, we noted that each strain that promoted loss of paxillin in these assays was also hemolytic on blood agar plates.

Degradation of Paxillin Is Initiated by HlyA Hemolytic activity associated with UPEC isolates is generally attributable to the pore-forming exotoxin a-hemolysin (HlyA), a prototypical member of the RTX (repeats-in-toxin) protein family encoded within the hlyCABD operon (Cavalieri et al., 1984; Linhartova´ et al., 2010; Marrs et al., 2005). We found that targeted deletion of hlyA in UTI89 rendered this strain nonhemo- Figure 1. Paxillin Is Degraded in UPEC-Infected Bladder cells lytic and unable to stimulate paxillin degradation in BECs (Fig- (A) Lysates from uninfected (UI) BECs and BECs that were infected with UTI89 ure 2B). Complementation of the hlyA mutant with a plasmid for the indicated time periods were probed by western blot using antibodies (pSF4000) encoding the complete hly operon restored the specific for paxillin. Staining for b-actin levels in each sample served as a loading control. wild-type phenotypes. In many UPEC isolates, including UTI89, (B) Lysates from BECs transfected with the plasmids pEGFP or pEGFP-paxillin the hly operon is genetically linked to a locus encoding another and then infected with UTI89 for 0, 2, or 4 hr were analyzed by western blot toxin known as cytotoxic necrotizing factor-1 (CNF1) (Marrs using antibodies specific for GFP (top), paxillin (middle), or actin (bottom). See et al., 2005). Both HlyA and CNF1 have the potential to influence also Figure S1. host cell death and tissue turnover rates within the bladder (Doye et al., 2002; Mills et al., 2000; Smith et al., 2008), but in our assays neither deletion of the cnf1 gene nor expression of recombinant host serine protease known as mesotrypsin. Finally, we demon- CNF1 affected the ability of UTI89 to stimulate paxillin degrada- strate that HlyA can similarly affect macrophages, highlighting tion (Figure 2C). unexpected roles for HlyA as a modulator of both phagocyte The hly operon also encodes the acyltransferase HlyC, which and epithelial cell functionality and survival. is responsible for acylating two conserved lysine residues within maturing HlyA toxin molecules. In the absence of acylation medi- RESULTS ated by HlyC, HlyA is secreted and interacts with host cell membranes, but fails to oligomerize to form functional pores UPEC Triggers Proteolysis of Paxillin (Herlax et al., 2009; Stanley et al., 1998). Infection of BECs with Paxillin is a 68 kDa multidomain scaffold protein that can modu- WAM582, a K-12 strain that carries the complete wild-type hly late the dynamics of cytoskeletal rearrangements and host cell operon, promoted rapid degradation of paxillin similar to hly- adhesion via coordinated interactions with numerous structural positive UPEC isolates (Figure 2D). In contrast, paxillin remained and regulatory proteins (Deakin and Turner, 2008). The stability stable in BECs following infection with the isogenic mutant of paxillin in human bladder epithelial cells (BECs) infected with WAM783, which encodes the hly operon minus hlyC. Of note, the reference UPEC isolate UTI89 was monitored by western both WAM783 and WAM582 express similar amounts of HlyA blot analysis. Infection of BECs with UTI89 resulted in decreased as determined by western blot (data not shown). Complementa- levels of paxillin within about 1.5 hr, and by 4 hr the protein tion of WAM783 with a plasmid encoding HlyC made this strain became undetectable (Figure 1A). UTI89-mediated degradation (WAM783/pHlyC) hemolytic and able to induce paxillin degrada- of paxillin was confirmed in BECs made to express enhanced tion (Figure 2D). Crude, bacteria-free preparations of acylated green fluorescent protein (eGFP) fused to the N terminus of pax- HlyA recovered from WAM783/pHlyC culture supernatants also illin. In these transfected cells, degradation of GFP-paxillin in triggered paxillin degradation, while nonacylated HlyA prepara- response to infection with UTI89 was observed to occur in a step- tions isolated from WAM783 cultures had no effect on the wise fashion, resulting first in the release of GFP fused to stability of paxillin (Figure 2E).

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Figure 2. HlyA Triggers Paxillin Degradation in BECs along with the Disruption of Host Actin and Microtubule Networks (A) Western blots show levels of paxillin and actin in 5637 BECs following 4 hr infections with UPEC isolates, non-UPEC E. coli strains, or non-E. coli uropathogenic bacteria. Uninfected (UI) control cells were treated with M9 medium without bacteria. Bacterial strains were scored as hemolytic (+) based on their ability to lyse erythrocytes on blood agar plates. (B–D) Western blots show paxillin and actin levels present in BECs at the indicated time points after infection with wild-type UTI89, UTI89DhlyA, UTI89DhlyA/ pSF4000, UTI89Dcnf1, UTI89Dcnf1/pHLK102, WAM783 (+hlyABD DhlyC), or WAM582 (+hlyCABD).

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Cumulatively, these results indicate that the insertion of acyl- HlyA-Dependent Activation of Caspases and Host Serine ated HlyA into host membranes can trigger paxillin degradation, Proteases but do not rule out possible involvement of additional bacterial HlyA is not known to have any inherent proteolytic activity, sug- factors common to both K-12 and UPEC isolates. This process gesting that it may stimulate paxillin degradation by activation of did not require lytic concentrations of HlyA, as BECs intoxicated one or more host proteases. Using pharmacological inhibitors, with HlyA in these experiments remained for the most part intact we tested the possible involvement of several of the better- and viable as determined by trypan blue exclusion assays. known proteolytic pathways in eukaryotic cells. Our results indi- However, bacterial strains expressing wild-type HlyA did cause cated that the proteosome, calpains, and lysosomal proteases substantial rounding and eventual lifting of the BECs (Figures like cathepsins are likely not critical for paxillin degradation in 2F, S2A, and S2B), coincident with massive disruption of host response to HlyA (Figures S4A–S4C). microtubule and actin networks (Figure 2G). We also examined possible involvement of caspases, a family of conserved aspartate-specific cysteine proteases (caspases, Effects of HlyA Membrane Insertion and Downstream CASP1–12) that have central roles in the initiation and execution Signaling Events of apoptosis (Chowdhury et al., 2008). Using fluorochrome- In addition to acylation by HlyC, the lytic activity of HlyA is also labeled inhibitor of caspases (FLICA) reagents (Bedner et al., dependent upon extracellular Ca2+ (Bauer and Welch, 1996; Os- 2000), we found that wild-type UTI89, but not the isogenic hlyA tolaza and Gon˜ i, 1995). This cation binds C-terminal GGXGXD mutant, stimulates rapid activation of the executioner caspases repeats within HlyA and promotes proper folding and insertion CASP3 and CASP7 in BECs (Figure 3A). The addition of suramin, of the toxin into the host cell plasma membrane where it likely which interferes with the effects of HlyA on BECs (see Figure S3), becomes concentrated and oligomerizes within cholesterol- prevented caspase activation by UTI89 (Figure 3B). These data rich detergent resistant microdomains (Baka´ s et al., 1998; Herlax indicate that HlyA expression by UPEC activates caspases et al., 2009; Ludwig et al., 1988). In our assays, depletion of within BECs, in agreement with previous studies showing that cholesterol (using methyl b-cyclodextran, MbCD) or chelation HlyA can promote apoptosis in a variety of host cell types of extracellular Ca2+ (using EGTA) prevented the stable associa- (Chen et al., 2006; Jonas et al., 1993; Mobley et al., 1990; Russo tion of HlyA with BECs (Figure S3G), completely inhibiting both et al., 2005). HlyA-induced caspase activation in our assays the cytotoxic effects of HlyA and paxillin degradation within correlated with the degradation of paxillin, suggesting a func- UTI89-infected BECs (Figures 2H, 2I, and S3H and data not tional link between these events. However, the treatment of shown). UTI89-infected BECs with the CASP3/7-specific inhibitor PFTs like HlyA can stimulate intracellular Ca2+ fluxes, the Z-DEVD-FMK, the pan-caspase inhibitor Z-VAD-FMK, or the release of cellular K+ ions, and activation of mitogen-activated CASP1/4 inhibitor Z-YVAD-FMK failed to prevent loss of paxillin protein (MAP) kinases (Bhakdi et al., 1986; Koschinski et al., downstream of HlyA intoxication (Figure 3C). Thus, while HlyA 2006; Porta et al., 2011). However, neither intracellular Ca2+ stimulates caspase activation within infected BECs, these prote- fluxes nor K+ efflux affected the loss of paxillin within UTI89-in- ases are not integral to paxillin degradation in our experiments. fected BECs, and inhibition of MAP kinase signaling caused at Caspases often act in concert with other proteolytic enzymes, best only a modest delay in paxillin degradation (Figures 2I and including serine proteases (Moffitt et al., 2007). These enzymes S2C–S2F). In addition, two other PFTs (a-toxin from S. aureus make up more than one third of all known proteases and func- and aerolysin from Aeromonas hydrophila) with the ability to alter tion in wide-ranging biological processes (Page and Di Cera, cellular cation fluxes and MAP kinase signaling failed to trigger 2008). A role for serine proteases as mediators of paxillin degra- the proteolysis of paxillin (Figure S2G). We also ruled out the dation in response to HlyA was assessed using the chymo- involvement of host purinergic P2X receptors and pannexin1 trypsin-like serine protease inhibitor tosyl-L-phenylalanine- channels, which were recently proposed to amplify the hemolytic chloromethyl ketone (TPCK) and the trypsin-like serine protease effects of HlyA (Figure S3)(Skals et al., 2009). Interestingly, three inhibitor tosyl-L-lysine-chloromethyl ketone (TLCK). TPCK had P2X antagonists—suramin, Evans Blue, and Brilliant Blue G—did no notable effect on paxillin stability in UTI89-infected BECs effectively block HlyA-induced paxillin degradation in our (data not shown), whereas TLCK completely inhibited HlyA- assays, but appeared to do so by nonspecifically inhibiting the induced paxillin proteolysis (Figure 3D). Of note, TLCK did not stable association of HlyA with BECs, similarly to MbCD and interfere with the stable association of HlyA with BECs (Fig- EGTA (Figure S3G). Thus, while our data refute the involvement ure S3G), and also had no effect on either HlyA-induced round- of P2X receptors and pannexin1 as necessary facilitators of ing of target host cells (Figure S3H) or HlyA-dependent activa- HlyA-induced paxillin degradation, they substantiate the idea tion of caspases (Figure 3B). The latter observation, and the that loss of paxillin within infected BECs requires the stable inability of caspase inhibitors to block paxillin degradation in insertion of HlyA into target host membranes. our assays, indicates that serine proteases are activated in

(E) Western blots show paxillin and actin levels in 5637 BECs following 0 to 4 hr incubations with preparations of crude, bacteria-free nonacylated HlyA (HlyA783)or wild-type HlyA (HlyA783/pHlyC). (F) Phase contrast microscopy of BECs infected with UTI89 or UTI89DhlyA for the indicated times. Scale bar, 50 mm. (G) Confocal images of BECs infected for 4 hr with UTI89 or UTI89DhlyA and then stained to visualize microtubules or F-actin. Scale bar, 20 mm. (H and I) BECs were infected with UTI89 for the indicated times in the presence of MbCD (5 mM), EGTA (4 mM), the intracellular Ca2+ chelator BAPTA-AM (10 mM), or vehicle alone (DMSO, control). Paxillin and actin levels were assessed by western blots. See also Figure S2 and Table S1.

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Figure 3. Activation of Caspases and TLCK-Sensitive Serine Proteases by HlyA (A) Caspase-3/7 activation over time in 5637 BECs infected with UTI89 or UTI89DhlyA, as determined by fluorometry of FLICA-treated samples. Data are expressed as the mean ± SEM of three independent experiments carried out in duplicate or triplicate. *p % 0.0001, as determined by Student’s t test. (B) Effects of suramin (1 mM) and TLCK (100 mM) on caspase-3/7 activation in BECs infected for 4 hr with UTI89, as quantified by fluorometry of FLICA-treated samples. Control UTI89-infected samples were treated with vehicle alone. Data are expressed as the mean ± SEM of four independent experiments performed in triplicate. *p % 0.0001, versus control values, as determined by Student’s t test. (C) Western blots showing levels of paxillin and actin in BECs infected with UTI89 for the indicated times in the presence of 100 mM of the CASP3/7-specific inhibitor Z-DEVD-FMK, the pan-caspase inhibitor Z-VAD-FMK, the CASP1/4 inhibitor Z-YVAD-FMK, or the negative control Z-FA-FMK. (D) Paxillin and actin levels in BECs infected with UTI89 for the indicated times in the presence or absence of TLCK (100 mM), as determined by western blots. (E) Phase contrast (left) and corresponding fluorescent images (right) of BECs infected with UTI89 or UTI89DhlyA for 4 hr in the presence of the fluorescent probe FSLCK (5 mM). Scale bar, 20 mm. (F) Fluorometric quantification of the levels of FSLCK-labeled, active serine proteases over time in BECs infected with UTI89 or UTI89DhlyA. Data are expressed as the mean ± SEM of three independent experiments carried out in triplicate. *p % 0.0001, as determined by Student’s t test. (G) Immunoblots probed using anti-FITC antibody to detect FSLCK-tagged proteins recovered from uninfected BECs, BECs infected with UTI89DhlyA, or BECs infected with UTI89 ± 1 mM suramin. (H) Levels of activated FSLCK-tagged serine proteases present in the bladder mucosa of mice 5 hr after infection with UTI89 or UTI89DhlyA were quantified by fluorometry. Data are expressed as the mean ± SEM from at least five mice. *p = 0.0017, as determined by Student’s t test. (I) Activated serine proteases present in the bladder mucosa of uninfected mice or mice infected with UTI89 or UTI89DhlyA for 5 hr were detected, following treatment with FSLCK, by western blots probed with anti-FITC antibody. Further analysis by mass spectroscopy identified the 23 kDa band (arrow) as mesotrypsin. See also Figure S3.

response to HlyA intoxication in parallel with, but independent FSLCK-tagged protein bands were detected in lysates from of, caspases. BECs following a 3 hr infection with wild-type UTI89 (Figure 3G). Carboxyfluorescein-spacer-leucine chloromethyl ketone The number and intensities of the bands were markedly reduced (FSLCK) covalently binds and fluorescently tags activated serine in lysates from BECs infected with the hlyA mutant, and nearly proteases (Grabarek and Darzynkiewicz, 2002). Using FSLCK as absent in lysates from uninfected BECs or BECs that were in- a probe, we observed by fluorescent microscopy high levels of fected with UTI89 in the presence of suramin. In total, these activated serine proteases in UTI89-infected BECs, versus rela- results indicate that HlyA can trigger the robust activation of tively low levels in host cells infected with the isogenic hlyA TLCK-sensitive serine proteases within BECs, one or more of mutant (Figure 3E). Fluorometric quantitation of BECs probed which mediate paxillin degradation. with FSLCK confirmed that wild-type UTI89 caused significant activation of serine proteases within 2 hr of infection, while the Mesotrypsin Promotes Paxillin Degradation hlyA mutant elicited almost no changes above background Downstream of HlyA (Figure 3F). By western blot analysis, using antibody specific To examine the ability of HlyA to influence activation of serine for the carboxyfluorescein fluorochrome within FSLCK, multiple proteases in vivo within the bladder, we utilized FSLCK in

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conjunction with a well-established mouse UTI model. By fluor- paxillin degradation. To examine this possibility, lysates from ometry, significantly higher levels of activated serine proteases BECs that were infected with wild-type UTI89 in the presence were detected within the bladder mucosa of mice following or absence of TLCK, or with UTI89DhlyA, were assayed by infection with wild-type UTI89 versus the hlyA mutant (Figure 3H). western blot analyses using antibodies specific for a panel Western blots of bladder mucosa lysates from both infected and of 24 host proteins. Nearly half of these were degraded in uninfected mice revealed only two FSLCK-tagged protein bands a TLCK-sensitive fashion within 4 hr of infection with wild-type with apparent molecular weights of about 23 and 70 kDa (Fig- UTI89, but not the hlyA mutant (Figures 5A and 5B). Although ure 3I). Similarly sized FSLCK-tagged protein bands were also these assays were too restricted to discern any definitive pattern detected, among many others, in UTI89-infected BECs in our in the types of host proteins that are degraded in response to cell culture-based assays (arrowheads, Figure 3G). FSLCK HlyA intoxication, they do demonstrate the ability of HlyA to labeling of the 23 kDa protein band was consistently more stimulate the loss of many proteins with key roles in mediating intense following infection of either cultured BECs or mouse host cell adherence and signal transduction. These include the bladders with wild-type UTI89 relative to UTI89DhlyA-infected paxillin homolog Hic-5, the docking proteins HEF-1 and p130- samples and uninfected controls. CAS, and the adherens junction component and signaling factor Using an immunoprecipitation approach coupled with mass b-catenin. spectroscopy, we identified the 23 kDa FSLCK-tagged protein In addition to its role as a mediator of cell adhesion, b-catenin in lysates from UTI89-infected mouse bladders as a TLCK-sensi- also functions in developmental and oncogenic cascades within tive host serine protease known as mesotrypsin. This enzyme is the Wnt signaling pathway and as a negative regulator of the generated via processing of three zymogens, known as trypsin- proinflammatory transcription factor NFkB(Deng et al., 2002). ogens 3, 4, and 5, that are produced as splice variants of the Interestingly, the TLCK-sensitive degradation of b-catenin in gene PRSS3 (Nakanishi et al., 2010). These trypsinogen isoforms UTI89-infected BECs occurs in step with loss of the prototypical differ only in their N-terminal region, which is cleaved at inhibitor of NFkB signaling, IkBa (Figure 5B). Usually, IkBa is conserved DDDDK-I sequences by the type II transmembrane ubiquitinated and subsequently degraded by the proteasome serine protease enteropeptidase to produce active mesotrypsin in response to proinflammatory signals (Rahman and McFadden, (Light and Janska, 1989). The mesotrypsin enzyme is highly 2011). However, the rapid loss of IkBa in our assays required similar to trypsins 1 and 2, major pancreatic digestive proteases HlyA and one or more TLCK-sensitive protease, indicating a derived from the zymogenic precursors trypsinogens 1 and 2, proteasome-independent mechanism for IkBa degradation in respectively (Nakanishi et al., 2010). However, relative to trypsins response to HlyA intoxication. Coincident with loss of IkBa and 1 and 2, mesotrypsin has distinct substrate specificity and b-catenin, we also observed HlyA-dependent and TLCK-sensi- a higher level of resistance to many natural, endogenous tive degradation of the NFkB subunit RelA (also known as p65) protease inhibitors (Nakanishi et al., 2010; Sahin-To´ th, 2005). (Figure 5B). This correlated with significantly reduced expression Among trypsinogens 1 through 5, only trypsinogen 4 tran- of the NFkB-regulated cytokine IL-6 by HlyA-intoxicated BECs in scripts were detected by RT-PCR in the human BECs used in response to added lipopolysaccharide (LPS, Figure 5C). In total, our cell culture-based assays (Figure 4A). Transcription of the these data indicate that HlyA can indirectly disrupt various activating enzyme enteropeptidase in these host cells was also aspects of host cell signaling, including key components of the observed. Using siRNA to silence expression of trypsinogen 4 NFkB pathway, via effects on serine proteases. (PRSS3, Figure 4B), we were able to significantly delay the degradation of paxillin in UTI89-infected BECs (Figure 4C). HlyA-Induced Proteolysis of Macrophage Proteins These data indicate that the active form of trypsinogen 4, meso- We next asked if the effects of HlyA on the stability of select host trypsin, is at least partially responsible for loss of paxillin in proteins could be observed in host cell types other than BECs. HlyA-intoxicated BECs. Interestingly, we found that trypsinogen Using both the RAW264.7 macrophage cell line and mouse peri- 4 undergoes radical redistribution in response to HlyA, as in- toneal macrophages, we found that wild-type UTI89, but not the ferred from using immunofluorescence microscopy with anti- hlyA mutant, triggered the degradation of paxillin as well as IkBa body directed against the enteropeptidase cleavage site DDDDK and RelA (Figures 6A–6C). Labeling of activated serine proteases (anti-ECS antibody, Figure 4D). Use of the anti-ECS antibody with the FSLCK reagent was also more intense in macrophages serves as a reasonable surrogate for direct immunodetection infected with wild-type UTI89 versus UTI89DhlyA (Figure 6D). of trypsinogen 4, as none of the other known trypsinogens Thus, the capacity of HlyA to activate serine proteases and appear to be expressed by the BECs used in our assays (see promote the loss of select host proteins is not limited to BECs. Figure 4A) (Nakanishi et al., 2010). In the presence of HlyA, trypsinogen 4 moves from a diffuse, primarily cytosolic localization to DISCUSSION a more reticulated and punctate distribution at the cell periphery, along microtubules, and within and around nuclei (Figures 4D In recent years, sublytic concentrations of several bacterial PFTs and S4D). This process is initiated within 1 hr of infection with have been shown to modulate important aspects of host cell wild-type UTI89, preceding the loss of paxillin. physiology, including MAP kinase and Akt signaling, inflamma- some activation, plasma membrane repair systems, histone TLCK-Sensitive, HlyA-Dependent Proteolysis methylation, the unfolded protein response, and mitochondrial of Multiple Host Proteins function (Aroian and van der Goot, 2007; Bischof et al., 2008; Induced activation of mesotrypsin and, perhaps, other TLCK- Gonzalez et al., 2011; Hamon et al., 2007; Kao et al., 2011; Los sensitive proteases by HlyA likely has effects extending beyond et al., 2011; Porta et al., 2011; Stavru et al., 2011; Wiles et al.,

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Figure 4. HlyA-Induced Paxillin Degradation Is Facilitated by Mesotrypsin (A) 5637 BECs express enteropeptidase and trypsinogen 4, but not other trypsinogens, as determined by RT-PCR. (B) Semiquantitative RT-PCR shows levels of PRSS3 (encoding trypsinogen 4) and GAPDH transcripts in BECs 72 hr after transfection with either control nonspecific siRNA or PRSS3-specific siRNA. (C) Western blots show levels of paxillin and actin present in lysates recovered from BECs treated with control, nonspecific siRNA or PRSS3-specific siRNA and infected with UTI89 for the indicated times. (D) Confocal micrographs of uninfected BECs or BECs that were infected with UTI89 or UTI89DhlyA for the indicated times and then stained to visualize trypsinogens (using ECS antibody, green), LAMP1 (red), and nuclei (blue). The boxed area in each micrograph is enlarged and placed directly below the corresponding image to show details. Scale bars, 20 mm (first and third rows) and 5 mm (second and fourth rows). See also Figure S4.

2008). The effects of PFTs on target host cells are often attribut- Degradation of paxillin in response to HlyA intoxication is due able to toxin-induced fluctuations in intracellular K+ and/or Ca2+ at least in part to activation of mesotrypsin. This protease has levels. In our efforts to identify UPEC-associated factors that recently been shown to regulate the differentiation and desqua- influence BEC detachment, using paxillin degradation as a mation of keratinocytes within the outermost layer of the skin readout, we found that the PFT HlyA can trigger activation of epidermis (Nakanishi et al., 2010). We suggest that mesotrypsin TLCK-sensitive serine proteases independent of K+ and Ca2+ might serve a similar function within the bladder mucosa. Unlike fluxes. This process required stable interaction between HlyA its close relatives, trypsins 1 and 2, mesotypsin is more widely and the host cells, likely involving oligomerization and integration expressed and is resistant to many naturally occurring trypsin of the acylated, Ca2+-bound toxin into cholesterol-rich microdo- inhibitors (Nakanishi et al., 2010; Sahin-To´ th, 2005). In some mains within the host plasma membrane. Two other bacterial cases, mesotrypsin can also degrade endogenous protease PFTs, a-toxin and aerolysin, had no effect on the stability of inhibitors, meaning it has the capacity to regulate the function- paxillin, even though they, like HlyA, can stimulate K+ and Ca2+ ality of other proteolytic enzymes (Sahin-To´ th, 2005). In our fluxes (Krause et al., 1998; Seeger et al., 1984). These results assays, silencing the expression of PRSS3 significantly delayed indicate that the capacity of HlyA to promote loss of paxillin is HlyA-induced degradation of paxillin in BECs. Eventual loss not shared by all PFTs. of paxillin in these assays likely results from our inability to

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Figure 5. Widespread Effects of HlyA on Host Protein Stability and NFkB Signaling (A–B) Levels of the host proteins indicated were assessed by western blot using lysates from 5637 BECs infected for 0 to 4 hr with UTI89DhlyA or wild-type UTI89 ± TLCK (100 mM). (C) 5637 BECs were infected with UTI89DhlyA or wild-type UTI89 in the presence of LPS (5 mg/ml) or incubated with LPS alone, and 4 hr later IL-6 levels in the cell culture supernatants were measured by ELISA. Data are expressed as the mean ± SEM of results from at least three independent experiments performed in triplicate; p values were determined by Student’s t test. completely extinguish expression of PRSS3 and/or the involve- location of bacteria (Troeger et al., 2007). HlyA can also atten- ment of other host, or perhaps even bacterial, trypsin-like serine uate the viability, chemotaxis, and effector functions of host proteases. The proteolysis of paxillin and other host proteins in phagocytes like neutrophils and macrophages (Cavalieri and response to HlyA intoxication correlated with the redistribution Snyder, 1982a, 1982b; Gadeberg et al., 1989; Russo et al., of mesotrypsin precursors from the cytosol to punctate struc- 2005). tures situated primarily along microtubules, both at the cell Results presented here suggest molecular processes by periphery and around and within host nuclei. The means by which sublytic levels of HlyA can modulate the functionality which HlyA triggers these positional changes and actually and viability of diverse host cell types. The ability of HlyA to elicit signals mesotrypsin activation within BECs require further degradation of paxillin as well as other cytoskeletal regulatory investigation. and structural components may not only promote bladder cell UPEC strains that express HlyA cause more extensive tissue exfoliation, but can also act to cripple phagocytes. Coordinated damage within the urinary tract, correlating with more severe activation of both serine proteases and caspases in response to clinical outcomes (Johnson, 1991; Marrs et al., 2005; Smith HlyA intoxication represents an additional mechanism by which et al., 2008). In both murine and tissue culture model systems, this PFT can compromise host cell viability. The capacity of HlyA hlyA-positive isolates stimulate more rapid and extensive shed- to stimulate degradation of b-catenin, IkBa, and the NFkB ding of BECs than isogenic hlyA-negative mutants (see Figures subunit RelA may enable UPEC and related strains to promote S2A and S2B) (Smith et al., 2006; Smith et al., 2008). HlyA is host cell death while also attenuating host inﬂammatory also an important virulence factor associated with other closely responses. The TLCK-sensitive nature of these degradative related E. coli strains that can cause pneumonia, peritonitis, processes suggests a role for HlyA-activated serine proteases and septicemia (May et al., 2000; O’Hanley et al., 1991; Russo like mesotrypsin in the regulation of NFkB signaling. These et al., 2005; Welch et al., 1981; Wiles et al., 2009). In the gut, results add to the growing list of mechanisms used by microbial HlyA mediates the formation of focal leaks within colonic pathogens to disturb NFkB-dependent proinﬂammatory path- epithelial cells and can thereby promote the paracellular trans- ways (Rahman and McFadden, 2011).

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Figure 6. HlyA Triggers the Activation of Serine Proteases and Degradation of Host Proteins within Macrophages (A–B) Western blots showing levels of paxillin and actin in either RAW264.7 or mouse peritoneal macrophages following infection with either UTI89 or UTI89DhlyA for the indicated times. (C) Western blots showing levels of IkBa, RelA, and actin in RAW264.7 cells following infection with UTI89 or UTI89DhlyA for the times indicated. (D) Phase contrast (left) and corresponding ﬂuorescent images (right) of peritoneal macrophages infected with UTI89 or UTI89DhlyA for 3 hr in presence FSLCK (5 mM) to label activated serine proteases. Scale bar, 50 mm.

Logan, UT) at 37 C in the presence of 5% CO2. Peritoneal and RAW264.7 macrophages were used as described in Supplemental Experimental Procedures. Bacterial strains employed in this study are listed in Table S1. All E. coli strains were grown static in M9 minimal medium at 37C for 48 hr prior to use (Wiles et al., 2008). S. ﬂexneri (ATCC 12022), S. enterica ser. Typhimurium (SL1344), S. saprophyticus, S. aureus, E. faecium, and K. pneumoniae were grown with shaking in Luria-Bertani (LB) broth (Difco) overnight. Bacteria were diluted in M9 medium (or LB broth for the non-E. coli isolates) to

OD600 0.5 prior to use. Antibodies, puriﬁed toxins, and other reagents used are noted in Supplemental Information.

Infection Assays, Western Blots, and Gene Expression Analysis Confluent BEC monolayers grown in 6-well plates were serum starved overnight in 1 ml of RPMI prior to infection or drug treatments. When appropriate, BECs were treated with the indicated compounds or vehicle alone for 1 hr prior to infection with UPEC. Host cells were infected with 100 ml of diluted bacterial cultures, corresponding to a multiplicity of infection of about 25. Fresh drugs, vehicle, or other compounds were added at this time and maintained for the duration of each experiment as needed. Sur- Our observations that HlyA disrupts NFkB signaling and cyto- amin was also added fresh again at 2 hr after infection to maintain its effective- kine expression complement previous findings showing that ness. An aliquot of M9 medium was added as a control to the uninfected HlyA can inactivate Akt, another key regulator of host cell survival samples. Plates were centrifuged at 600 3 g for 5 min to facilitate and synchro- and inflammation (Wiles et al., 2008). Together, these data nize bacterial contact with the host cells. At the specified times, BECs were washed with PBS2+ (PBS supplemented Ca2+ and Mg2+) prior to lysis in explain in large part the capacity of some UPEC isolates to RIPA buffer supplemented with 13 complete protease inhibitors (Roche suppress host inflammatory responses, as reported by others Applied Science) and 1 mM phenylmethanesulfonylfluoride (Sigma-Aldrich). (Billips et al., 2007; Hilbert et al., 2008; Hunstad et al., 2005; Equivalent protein amounts (typically 50 mg of each sample, as determined Loughman and Hunstad, 2011). Interestingly, in some of these by BCA assays; Pierce) were resolved by SDS-PAGE, transferred to Immobi- earlier studies mutations that alter the ability of UPEC to dampen lon PVDF-FL membrane (Millipore), and processed for western blot analysis as host cytokine expression levels were mapped to genes that previously described (Eto et al., 2007; Wiles et al., 2008). Gene cloning, silencing, semiquantitative RT-PCR, and the isolation of crude HlyA prepara- affect the bacterial cell envelope and, potentially, the secretion tions were carried out as detailed in Supplemental Experimental Procedures. of toxins like HlyA. In conclusion, our data reveal an unexpected process by which UPEC and related pathogens can manipulate Detection and Quantification of Activated Caspases and Serine host survival and inflammatory pathways via HlyA-dependent Proteases activation of host proteolytic cascades. Confluent, serum-starved BEC monolayers in 24-well plates were treated with the FLICA reagents FAM-DEVD-FMK (13), SR-DEVD-FMK (13), or FSLCK (5 mM, ImmunoChemistry Technologies; Bloomington, MN) and then infected EXPERIMENTAL PROCEDURES with UTI89 or UTI89ΔhlyA ± suramin (1 mM) or TLCK (100 mM) as indicated. At the specified times, BECs were washed 33 with PBS2+ and lysed in TTD buffer Host Cells and Bacterial Strains containing 0.25% Triton X-100, 10 mM Tris (pH 7.5), and 1 mM dithiothreitol. Human bladder epithelial cells, designated 5637 cells (ATCC HTB-9), were ob- Fluorescence measurements were obtained using a LS-5 Fluorescence spec- tained from the American Type Culture Collection and maintained in RPMI trophotometer (Perkin-Elmer; Waltham, MA), and readings were normalized to 1640 supplemented with heat-inactivated 10% fetal bovine serum (Hyclone, the total protein content in each sample, as determined by BCA assays.

66 Cell Host & Microbe 11, 58–69, January 19, 2012 ª2012 Elsevier Inc. Cell Host & Microbe HlyA-Induced Degradation of Host Proteins

Alternatively, equal amounts of protein from the FSLCK-treated samples were Bedner, E., Smolewski, P., Amstad, P., and Darzynkiewicz, Z. (2000). resolved by SDS-PAGE and probed by western blot analysis using anti-FITC Activation of caspases measured in situ by binding of fluorochrome-labeled and anti-actin antibodies. inhibitors of caspases (FLICA): correlation with DNA fragmentation. Exp. Cell To assay activation of serine proteases in vivo within the bladder mucosa, Res. 259, 308–313. adult female CBA/J mice (Jackson Laboratories; Bar Harbor, ME) were inocu- Bhakdi, S., Mackman, N., Nicaud, J.M., and Holland, I.B. (1986). Escherichia 8 Δ lated via transurethral catheterization with 10 cfu of UTI89 or UTI89 hlyA as coli hemolysin may damage target cell membranes by generating transmem- described (Mulvey et al., 1998). Mice were sacrificed at 5 hr postinoculation brane pores. Infect. Immun. 52, 63–69. and bladders were aseptically removed, bisected, splayed, and pinned down lumenal side up in mammalian Ringer solution (pH 7.4) containing Billips, B.K., Forrestal, S.G., Rycyk, M.T., Johnson, J.R., Klumpp, D.J., and 5 mM FSLCK. After a 20 min incubation at 37C, the splayed bladders were Schaeffer, A.J. (2007). Modulation of host innate immune response in the washed five times with Ringer solution, and the mucosal layer of each bladder bladder by uropathogenic Escherichia coli. Infect. Immun. 75, 5353–5360. was gently peeled off and lysed in TTD lysis buffer. FSLCK-tagged serine Bischof, L.J., Kao, C.Y., Los, F.C., Gonzalez, M.R., Shen, Z., Briggs, S.P., van proteases in each sample were quantified by fluorometry or visualized by der Goot, F.G., and Aroian, R.V. (2008). Activation of the unfolded protein western blot analysis as described above. response is required for defenses against bacterial pore-forming toxin in vivo. To identify the 23 kDa active serine protease present within the bladders of PLoS Pathog. 4, e1000176. UTI89-infected mice, mucosal layers isolated from four infected, FSLCK- Blango, M.G., and Mulvey, M.A. (2010). Persistence of uropathogenic treated bladders were pooled and lysed in RIPA buffer. FSLCK-tagged Escherichia coli in the face of multiple antibiotics. Antimicrob. Agents proteins were then immunoprecipitated using rabbit anti-FITC antibody immo- Chemother. 54, 1855–1863. bilized on Protein A Sepharose 4B beads, resolved by SDS-PAGE, and stained using GelCode Blue (Thermo Fisher Scientific). The 23 kDa band was then Bower, J.M., Eto, D.S., and Mulvey, M.A. (2005). Covert operations of uropa- excised, subjected to in-gel trypsin digestion and identified using liquid chro- thogenic Escherichia coli within the urinary tract. Traffic 6, 18–31. matography/mass spectrometry at the University of Utah core facility. Cavalieri, S.J., and Snyder, I.S. (1982a). Effect of Escherichia coli alpha-hemo- All mouse experiments were performed with approval from the local Institu- lysin on human peripheral leukocyte function in vitro. Infect. Immun. 37, tional Animal Care and Use Committee. 966–974. Cavalieri, S.J., and Snyder, I.S. (1982b). Effect of Escherichia coli alpha-hemo- Microscopy lysin on human peripheral leukocyte viability in vitro. Infect. Immun. 36, Phase contrast and fluorescence microscopic images of live BECs and macro- 455–461. phages were acquired using an Olympus CK40 microscope equipped with Cavalieri, S.J., Bohach, G.A., and Snyder, I.S. (1984). Escherichia coli alpha- 103 (Olympus A10PL NA 0.25) and 403 (Olympus CDPlan40FPL NA 0.55) hemolysin: characteristics and probable role in pathogenicity. Microbiol. objectives and a Canon PowerShot A640 camera. For confocal microscopy, Rev. 48, 326–343. BECs grown on 12 mm-diameter glass coverslips were fixed, stained, and imaged using an Olympus FV1000 microscope as previously described Chen, M., Tofighi, R., Bao, W., Aspevall, O., Jahnukainen, T., Gustafsson, L.E., (Dhakal and Mulvey, 2009). Ceccatelli, S., and Celsi, G. (2006). Carbon monoxide prevents apoptosis induced by uropathogenic Escherichia coli toxins. Pediatr. Nephrol. 21, 382–389. SUPPLEMENTAL INFORMATION Chowdhury, I., Tharakan, B., and Bhat, G.K. (2008). Caspases - an update. Supplemental Information includes four figures, one table, Supplemental Comp. Biochem. Physiol. B Biochem. Mol. Biol. 151, 10–27. Experimental Procedures, and Supplemental References and can be found Deakin, N.O., and Turner, C.E. (2008). Paxillin comes of age. J. Cell Sci. 121, with this article online at doi:10.1016/j.chom.2011.12.003. 2435–2444. Deng, J., Miller, S.A., Wang, H.Y., Xia, W., Wen, Y., Zhou, B.P., Li, Y., Lin, S.Y., ACKNOWLEDGMENTS and Hung, M.C. (2002). beta-catenin interacts with and inhibits NF-kappa B in human colon and breast cancer. Cancer Cell 2, 323–334. We thank the Fluorescence Microscopy and Mass Spectrometry and Proteo- mics Cores at the University of Utah for help with these studies. We are also Dhakal, B.K., and Mulvey, M.A. (2009). Uropathogenic Escherichia coli invades grateful to R.A. Welch, D.A. Low, T.M. Hooton, A.D. O’Brien, U. Sonnenborn, host cells via an HDAC6-modulated microtubule-dependent pathway. J. Biol. M.G. Caparon, M.A. Fisher, and J.S. Parkinson for supplying strains and other Chem. 284, 446–454. reagents. This work was supported by National Institutes of Health grants Doye, A., Mettouchi, A., Bossis, G., Cle´ ment, R., Buisson-Touati, C., Flatau, AI095647, DK068585, AI088086, and AI090369. G., Gagnoux, L., Piechaczyk, M., Boquet, P., and Lemichez, E. (2002). CNF1 exploits the ubiquitin-proteasome machinery to restrict Rho GTPase activation Received: July 6, 2011 for bacterial host cell invasion. Cell 111, 553–564. Revised: October 5, 2011 Eto, D.S., Sundsbak, J.L., and Mulvey, M.A. (2006). Actin-gated intracellular Accepted: December 2, 2011 growth and resurgence of uropathogenic Escherichia coli. Cell. Microbiol. 8, Published: January 18, 2012 704–717.

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