Microbial Pathogenesis 77 (2014) 42e52

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The role for TolA in enterohemorrhagic Escherichia coli pathogenesis and virulence gene transcription Jason K. Morgan*, Jose A. Ortiz, James T. Riordan University of South Florida, Department of Cell Biology, Microbiology and Molecular Biology, Tampa, FL 33620, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 June 2014 Received in revised form 14 October 2014 Accepted 21 October 2014 Available online 22 October 2014

Loss of the periplasm spanning protein TolA in Escherichia coli leads to activation of the Rcs phosphorelay, and is required for full virulence in Gram-negative pathogens such as Salmonella enterica and Dickeya dadantii. This study explores the role for TolA in the pathogenesis of enterohemorrhagic E. coli (EHEC) and the effect of its mutation on the transcription of key EHEC virulence genes controlled by Rcs phosphorelay, including the type III secretion system (T3SS) (espA and tir), the E. coli common pilus (ecpA), and motility (fliC). Promoter activity for T3SS regulator ler was substantially higher following inactivation of tolA, and corresponded with a similar elevation in espA and tir transcription. Likewise, ecpA transcription was increased in EHECDtolA. Conversely, and in-line with previous studies, inactivation of tolA resulted in complete loss of motility and decreased fliC transcription. For all genes examined, altered transcription observed for EHECDtolA was dependent on the outer-membrane lipoprotein RcsF. Despite elevated virulence gene transcription, in tolA deleted strains virulence of EHEC in the Galleria mellonella wax worm model was substantially attenuated in a manner at least partly dependent on RcsF, and adherence to cultured HT-29 colonic epithelial cells was markedly reduced. The results of this study broaden the role for TolA in EHEC pathogenesis, and suggest that significant outer-membrane perturbations are able to promote transcription of important EHEC adherence factors. © 2014 Elsevier Ltd. All rights reserved.

Keywords: EHEC TolA RcsF Adherence genes Outer-membrane integrity Rcs phosphorelay

1. Introduction Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a virulent pathogen associated with food-and water-borne outbreaks [1,2]. EHEC caused infections can lead to hemorrhagic colitis (bloody diarrhea) and the life-threatening hemolytic uremic syndrome (HUS) as a result of Shiga toxin (Stx) production [3,4]. Successful intestinal colonization by this pathogen is, in part, reliant upon a type III secretion system (T3SS) encoded within the locus of enterocyte effacement (LEE) pathogenicity island [5]. The T3SS translocon apparatus, composed of a filament structure (EspA) and a pore complex (EspD/EspB), facilitates intimate attachment to host intestinal epithelial cells through direct binding of intimin on the surface of the bacterium with the translocated intimin receptor (Tir) on the cell surface [6e8]. The LEE encodes all of the genes required for production of the T3SS and contains 5 major operons (LEE1eLEE5), the first of which encodes the positive LEE gene regulator, Ler [9e11]. The regulatory

* Corresponding author. University of South Florida, 4202 E. Fowler Ave., ISA 2015, Tampa, FL 33620, USA. E-mail address: [email protected] (J.K. Morgan). http://dx.doi.org/10.1016/j.micpath.2014.10.010 0882-4010/© 2014 Elsevier Ltd. All rights reserved.

factors which contribute to the control of the ler promoter have been extensively studied [12e23]. Likewise, numerous exogenous signals have been shown to modulate LEE gene transcription, including host derived ethanolamine, fucose, and epinephrine/ norepinephrine [24e27]. LEE gene transcription is further enhanced by conditions which mimic the host intestinal milieu such as low pH, osmolarity, availability of ammonium, and the presence of the bicarbonate ion [28e30]. One important positive regulator of the LEE in EHEC includes the Rcs phosphorelay response regulator RcsB which indirectly regulates LEE gene transcription through grvA [13], and is required for full bicarbonate mediated stimulation of the LEE [28,31]. The Rcs phosphorelay is composed of a sensor kinase (RcsC), a histidine phospho-transferase (RcsD), and a response regulator (RcsB) [32,33]. The sensor kinase RcsC is able to communicate membrane perturbations caused by osmotic shock, membrane damaging agents and growth on solid media, and is essential for normal biofilm formation [34]. RcsB binds to specific DNA sequences (RcsB box) at promoters to activate or repress transcription and, in its phosphorylated state, can bind as a homodimer or as a heterodimer with other regulatory proteins, such as RcsA [32]. In its activated state, the RcsB/RcsA heterodimer represses transcription of flagellar genes [35] and simultaneously activates genes involved

J.K. Morgan et al. / Microbial Pathogenesis 77 (2014) 42e52

in capsule synthesis during growth at low temperatures [36]. Signal transduction to RcsC and Rcs phosphorelay activation typically requires the action of an additional factor, RcsF, which is a ~14-kDa outer-membrane bound lipoprotein that orients towards the periplasm [37]. RcsF signaling has been demonstrated for mutations that affect the proper folding of periplasmic proteins, as well as those which disrupt the proper synthesis and localization of lipopolysaccharides and lipoproteins [38,39]. Activity is dependent on both the formation of nonconsecutive disulfides and an unstructured proline rich region to properly convey signals to the sensor kinase RcsC for Rcs phosphorelay activation [40,41]. Loss of tolA, encoding the periplasm spanning protein of the TolPal complex, has been shown to substantially activate the Rcs phosphorelay system through stimulation of the sensor kinase RcsC [42]. The TolA protein is part of a trans-envelope complex with TolQ and TolR, two additional inner-membrane bound proteins, which function together to interact with the outer-membrane Pal complex, forming an energized system with an essential role in maintaining outer-membrane integrity and cellular division [43e47]. The E. coli TolA periplasmic tandem repeat region is highly variable, and contributes to differences in the tolerance to agents which damage the outer-membrane, such as detergents [48,49]. Mutation of tolA leads to highly mucoid colonies and decreased adherence in E. coli strain MG1655, a phenotype that is dependent on RcsB [50]. Moreover, tolA deficient Salmonella typhimurium displays attenuated virulence in mouse infections, likely resulting from decreased LPS production and subsequently increased bile and serum susceptibility [51,52]. However, while Rcs activation in tolA deficient MG1655 decreased adherence [50], the effect of tolA deletion in EHEC pathogenesis and virulence gene regulation has not yet been explored, of particular significance due to the important role for Rcs in control of the LEE [13,53]. In this study the role for TolA in EHEC virulence was examined. Specifically, the effect of tolA inactivation on the transcriptional regulation of key adhesin and virulence genes was explored using qRT-PCR and promoter fusions. In addition, this study sought to determine the contribution of the outer-membrane lipoprotein RcsF to transcriptional responses mediated by loss of TolA in EHEC. 2. Methods 2.1. Bacterial strains and culture conditions The strains and plasmids used in this study are listed in Table 1. Strains were stocked at
80  C in glycerol diluted (15% v/v final) in Lysogeny Broth (LB), and were maintained in LB or on LB with 1.5% agar (LBA). Unless otherwise noted, overnight (18e20 h) cultures grown in LB (Fig. S1) were used to inoculate fresh LB to a final optical density 600 nm (OD600) of 0.05. Cultures were grown at 37  C in a rotary shaker (200 RPM) using a 1:10 media-to-flask volume. Antibiotics (SigmaeAldrich, St. Louis, MO) were added to cultures when required. 2.2. Genetic manipulations and plasmid construction Primers used for the genetic manipulation are provided in Table S1. For construction of isogenic deletion mutants, the l-Red assisted one-step inactivation method was used, adapted for enterohemorrhagic E. coli [54], and as described [53]. Confirmation of genetic constructs was performed using a combination of BccI (NEB) restriction mapping of PCR amplified products with primers flanking the target genomic regions compared with BccI restriction sites, and DNA sequencing (MWG Operon, Huntsville, AL). To complement tolA, a PCR product corresponding to the tolA ORF and 59-bp upstream of tolA (containing the native RBS) (nucleotide

43

Table 1 Strains and plasmids used in this study. Strain/ plasmid

Relevant characteristics

Strain name: DH5a Vector propagation, recA1 endA1 TW14359 WT 2006 outbreak, western U.S.A. EcRJM-1 TW14359DescN EcRJM-35 TW14359DgrlR::kan EcRJM-114 TW14359DecpA::kan EcRJM-118 TW14359DtolA EcRJM-119 TW14359DrcsF EcRJM-120 TW14359DrcsFDtolA::kan EcRJM-121 TW14359DhldE Plasmid name: pACYC177 Low copy cloning vector, AmpR KanR P15A pBAD-TOPO Ara inducible expression vector, AmpR pBR322 pRS551 lac fusion vector, AmpR KanR lacZþ ColE1 pMPM-A2 Low copy cloning vector, AmpR pMB1/f1 pRJM-2 pRS551 containing lerP905elacZ pCP20 Flp recombinase expression vector pKD4 Template plasmid for Kan cassette pKM208 Red-recombinase expression vector pRJM-34 pecpR, ecpR ORF and promoter in vector pMPM-A2 pRJM-19 pACYC177-tolA pRJM-57 pBAD-hldE pRJM-58 pBAD-rcsF

Reference

Nucleotide positiona

Lab stock [93] [53] [53] This This This This This

study study study study study

347685-348271 865752-866946 222559-223306 3994026-3995405

[94] Invitrogen [95] [55] [53] [96] [96] [96] This study

348346-349340

This study This study This study

865653-867022 3993929-3995481 222470-223383

a Nucleotide positions based on the published TW14359 (NC_013008) genome sequences (NCBI).

positions 865653-867022, GenBank Ref# NC_013008.1) was produced using primers TolA-59/XhoI and TolAþ1310/BamHI, and cloned into the XhoI/BamHI digested vector pACYC177, to create vector pRJM-19. To complement hldE, a PCR product corresponding to the hldE ORF and 76-bp upstream of hldE (containing the native RBS) (nucleotide positions 3993929-3995481, GenBank Ref# NC_013008.1) was produced using primers HldE-76 and HldEþ1,476, and inserted into pBAD-TOPO TA expression vector (Life Technologies), to create vector pRJM-57. To complement rcsF, a PCR product corresponding to the rcsF ORF and 77-bp upstream of rcsB (containing the native RBS) (nucleotide positions 222470223383, GenBank Ref# NC_013008.1) was produced using RcsF-77 and RcsFþ432, and inserted into pBAD-TOPO TA expression vector (Life Technologies), to create vector pRJM-58. The proper sequence and orientation of pBAD-TOPO vector inserts was determined through a combination of restriction mapping and DNA sequencing. Expression of ecpR was performed by cloning a fragment containing the entire ecpR open reading frame, and upstream promoter region, (nucleotide positions 348346-349340, GenBank Ref# NC_013008.1) into the BamHI/XhoI digested low copy vector pMPM-A2 [55] using primers EcpR-404/XhoI and EcpRþ637/BamHI, and validated as described above. 2.3. Construction of lacZ transcriptional promoter fusions and bgalactosidase assays The construction of the lerP905-lacZ reporter transcriptional fusion, pRJM-2, followed a previously described protocol using

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J.K. Morgan et al. / Microbial Pathogenesis 77 (2014) 42e52

vector pRS551 [31]. b-galactosidase activity (Miller Units) was measured as previously described and compared between strains using the appropriate t-test or by Tukey's HSD following a significant F-test (n ¼ 3, a ¼ 0.05) (R ver. 3.1.0) [22,56]. To ensure that potential alterations in cell shape did not affect optical density measurements used in Miller assay unit calculations, wild-type TW14359, TW14359DtolA, TW14359DtolA ptolA optical density measurements were compared with the CFU counts at specified time points, and no significant difference was observed between strains at the specified OD600 values (Fig. S3). 2.4. RNA purification and qRT-PCR

centrifuged (5 min at 5000 g) and the bacterial pellet was resuspended in fresh DMEM without FBS. For each strain, 200 ml of culture was added to each well of a 6-well plate for cell infection. Inoculated plates were then centrifuged at 500 g for 5 min and incubated at 37  C with 5% CO2. Following 2 h of co-incubation, each well was washed 4 times using 1 ml of sterile PBS to remove non-adherent cells, and adherent cells were removed using 500 ml of PBS with 0.1% Triton X-100. Finally, cells were enumerated through serial dilution and plating on LBA agar. Each experiment was performed using at least 3 biological replicates. Stress survival assays were performed using either DMEM or PBS with 0.1% Triton X-100 to ensure that TW14359DtolA survival was not affected in either condition (Fig. S3).

Primers for qRT-PCR are provided in Table S1 in supplemental materials. RNA purification, cDNA synthesis, qRT-PCR cycling conditions and data analysis followed previously described protocols [57]. qRT-PCR was performed using a Realplex2 Mastercycler (Eppendorf). Cycle threshold (Ct) data were normalized to rrsH (16S rRNA gene) and normalized cycle threshold values (DCt) were transformed to arbitrary transcript expression levels using 2
DCt/ 10
6 as described [57,58]. Expression levels were compared using the appropriate t-test or by Tukey's HSD following a significant Ftest (n ¼ 3, a ¼ 0.05) (R ver. 3.1.0).

Assays for bacterial motility were performed as previously described [53]. Briefly, overnight LB cultures were diluted to fresh LB at an OD600 of 0.05 and 2 ml was inoculated onto LB plates containing 0.25% (W/V) agar [62]. Following inoculation, plates were incubated at 37  C for 8 h and motility was determined by measuring the lateral growth (mm) on the agar surface. Experiments were performed using 3 biological replicates.

2.5. Virulence assays using Galleria mellonella model of infection

3. Results

G. mellonella is an emerging model of bacterial infection, and has shown a positive correlation of virulence patterns in mice for major virulence determinants in gram-negative human pathogens [59e61]. Assays for virulence in G. mellonella were performed using 5th instar larvae (Georgia Crickets, Winder, GA). Upon arrival, larvae were stored on wood chips at 4  C, and were used within 2 weeks of receipt. Larvae between weighing between 0.2 and 0.25 g, free of melanization (dark spots indicating infection) and injury, were selected for use. One milliliter samples of bacteria grown to mid-exponential phase (OD600 of 0.5) were pelleted by centrifugation (5000 g) and washed twice with PBS with repeated centrifugation between washes, and finally re-suspended in 1 ml of PBS. G. mellonella larvae (n ¼ 10) were inoculated with 105 CFU of wild-type TW14359, mutant and complement derivatives of TW14359, diluted in PBS (10 ml total vol.) or diluent (control) by hemolymph injection via the 4th posterior proleg using a 26 gauge, 50 ul Hamilton syringe (Hamilton Company). Inoculated larvae were placed in sterile petri dishes and incubated at 37  C in the dark for 96 h. Virulence was quantified by calculating the percent survival for each trial at 24 h. In each assay, larvae were scored as dead if they did not respond to touch. Both un-injected and PBS only injected control larvae were included in each trial, and none were killed during the course of experimentation. As an additional control, stress survival assays were performed and showed that TW14359DtolA was unaffected by exposure to PBS (Fig. S3).

3.1. The effect of tolA deletion on ler promoter activity in EHEC

2.6. Mammalian tissue culture and adherence assays Maintenance and culture of HT-29 colonic intestinal cells was performed as previously described [31]. Briefly, HT-29 cells were grown to confluency in 6-well tissue culture plates at 37  C with 5% CO2. Prior to infection with bacterial strains, media was aspirated and each well was washed once with PBS (phosphate-buffered saline) and replaced with DMEM without FBS. For adherence experiments, bacterial cultures grown overnight in LB were diluted to and OD600 of 0.05 in fresh LB and grown to mid-logarithmic phase (OD600 of 0.5) and 1 ml of culture was removed to a sterile 1.5 ml tube. Tubes containing bacterial cultures were then briefly

2.7. Motility assays

Given the important role of Rcs phosphorelay in E. coli gene regulation [63], particularly in regulation of the LEE pathogenicity island through grvA [13], it was of interest to explore the effects of a tolA mutation LEE expression in EHEC. It was initially hypothesized that a knockout of tolA, resulting in strong Rcs phosphorelay activation, would have a positive effect on LEE gene transcription. Therefore, a ler promoter-lacZ fusion containing both mapped ler promoters was used to assay ler promoter activity. As expected, activity was significantly increased exponential growth (OD600 of 0.5) in TW14359DtolA compared to TW14359 (p < 0.001) (Fig. 1A). Further, while overall ler promoter activity decreased during lateexponential phase, in agreement with the current model of LEE gene regulation [64], the differential promoter activity between TW14359 and TW14359DtolA collapsed during later stages of growth (3e5 h) (Fig. 1A). Loss of TolA results in dramatic alterations to the outermembrane, specifically related to the formation of membrane vesicles, but also through substantially decreased O-antigen production and localization [65,66]. Decreased production of LPS could potentially alter the composition of the outer-membrane sufficient to stimulate stress sensing components of the Rcs phosphorelay, such as RcsF [37]. Thus, it was hypothesized that aberrant production or localization of lipopolysaccharide (LPS) could at least partially explain the dramatic increase in ler promoter activity observed for TW14359DtolA. In order to test this, an LPS deficient strain was created by deleting hldE (previously rfaE and waaE), encoding an enzyme required for ADP-heptose formation [67], resulting in a truncated LPS inner-core oligosaccharide. Indeed, promoter activity for ler was slightly but significantly increased in TW14359DhldE (1.23-fold; p ¼ 0.017), although it remained significantly lower than that of TW14359DtolA (2.11-fold; p ¼ 0.007) (Fig. 1B). During stationary phase, however, neither tolA or hldE mutants displayed ler promoter activity above that of wildtype TW14359, suggesting that deletion of tolA increases transcription of the LEE encoded regulator Ler, and that increased ler promoter activity is relegated to periods of exponential growth. Furthermore, while LPS potentially plays a role in ler promoter

J.K. Morgan et al. / Microbial Pathogenesis 77 (2014) 42e52

Fig. 1. ler promoter activity following loss of TolA. (A) b-galactosidase activity in Miller units of lerP905-lacZ measured for LB cultures during lag, exponential, and lateexponential growth phases (from 1 to 5 h) for TW14359 (filled) and TW14359DtolA (empty). Dashed lines denote OD600 measurements at the specified time-points for TW14359 (triangle) and TW14359DtolA (circle). Asterisks denote significance by a Student's t-test (***, p < 0.001; n ¼ 3). (B) b-galactosidase activity in Miller units of lerP905-lacZ for exponential (OD600 ¼ 0.5) and stationary (OD600 ¼ 3.5) phase cultures in TW14359 (black), TW14359DhldE (gray), and TW14359DtolA (empty). For both panels, asterisks denote significance by a Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001; n ¼ 3) and error bars represent the standard deviation.

stimulation, these results suggest that the promoter activation observed for TW14359DtolA is largely independent. 3.2. Role for the outer-membrane component lipoprotein RcsF in DtolA mediated LEE and ECP regulation Indicative of Rcs phosphorelay activation, TolA deficient E. coli colonies are highly mucoid owing to activation of Rcs regulated capsular polysaccharide (cps) genes [42]. In this study, RcsF was investigated in-lieu of the response regulator RcsB in part to minimize the pleiotropic effects of an rcsB deletion [63], but also because the mechanistic basis behind Rcs stimulation in a TolA deficient strain has not yet been fully characterized. As predicted, mutation of rcsF in TW14359DtolA substantially reduced the mucoidy colony phenotype observed on solid agar (data not shown). Furthermore, since deletion of tolA activates the Rcs phosphorelay, which has been shown to increase LEE gene transcription and, it was hypothesized that increased ler promoter

45

activity in TW14359DtolA was at least partly dependent RcsF. To test this, activity was determined from cultures grown to midexponential phase (OD600 ¼ 0.5) in LB. As previously observed, ler promoter activity in TW14359DtolA was significantly higher than wild-type TW14359 (2.8-fold; p < 0.05), while activity in TW14359DrcsF was only slightly reduced compared with wild-type TW14359 (p ¼ 0.053) (Fig. 2A). As predicted, ler promoter activity in TW14359DtolADrcsF was significantly lower than TW14359DtolA (p < 0.05), although activity remained slightly but significantly higher than that of wild type TW14359 (p < 0.05) (Fig. 2A). Based on the known role for Ler in positively regulating the LEE [9,68], it was predicted that increased ler promoter activity would ultimately lead to higher levels of LEE gene transcription. Therefore, qRT-PCR was used to determine expression of key LEE genes espA and tir, encoding the translocon filament protein and the translocated intimin receptor, respectively. As expected, significantly increased transcription was observed for both espA (3.7-fold; p ¼ 0.024) and tir (2.35-fold; p ¼ 0.013) in TW14359DtolA compared with wild type TW14359 (Fig. 2B). Transcript levels for espA and tir were slightly but significantly lower than wild-type TW14359 in the tolA complement and rcsF mutant strains (Fig. 2B). Conversely, since the RcsB directly represses motility [35], the major flagellin subunit gene (fliC) was included as a control and, as was expected, transcript levels were significantly decreased in TW14359DtolA compared with wild type TW14359 (12-fold; p ¼ 0.012) (Fig. 2B). Taken together, these findings support the hypothesis that the increased LEE expression phenotype of TW14359DtolA is at least partially dependent on the outermembrane lipoprotein RcsF. It was also of interest to determine the effect of a tolA mutation on the expression of another Rcs phosphorelay regulated adhesin, the E. coli common pilus (ECP). In neonatal meningitic E. coli (NMEC), the ecp operon is directly regulated by RcsB in conjunction with the ecp encoded regulator, EcpR [24]. However, the requirement for RcsF in ecp regulation has not yet been described. It was thus predicted that a tolA mutation would similarly increase transcription of ecpA, encoding the major ECP pilin subunit. Indeed, ecpA transcript levels in TW14359DtolA were significantly higher than wild-type TW14359 (6-fold; p ¼ 0.004), and deletion of rcsF in TW14359 and TW14359DtolA completely abrogated ecpA upregulation (Fig. 2B). These results indicate deletion of tolA, which has been shown to cause perturbations in the cell envelope [65], is able to increase transcription of genes involved in adherence, such as those involved in type III secretion and in ECP production. Loss of TolA resulted in significantly higher levels of both LEE and ecp gene transcription compared with wild-type TW14359. As this was dependent on RcsF, it was of interest to recapitulate transcriptional changes following overexpression of rcsF under the control of the pBAD promoter in TW14359DrcsF. Based on the role for RcsB in positive regulation of the LEE and ecp operons [13,53,69], it was predicted that overexpression of rcsF would mirror the transcriptional changes in these operons observed for TW14359DtolA. Indeed, transcription of espA (LEE) and ecpA (ECP) was significantly higher (19.4-fold; p ¼ 0.001 and 13.2-fold; p ¼ 0.017, respectively) in TW14359DrcsF overexpressing rcsF compared with wild-type TW14359, in-line with the preceding experiments (Fig. 3). And, as expected, overexpression of rcsF in TW14359DrcsF significantly decreased transcription of fliC compared with wild-type TW14359 (451-fold; p < 0.001), congruent with the role for Rcs phosphorelay in repression of motility [62]. Finally, whereas transcription of ecpA and fliC was not significantly altered in TW14359DrcsF compared with wildtype TW14359, loss of rcsF did slightly but significantly increase transcription of ecpA compared with wild-type TW14359 (2.2fold; p ¼ 0.017). In-contrast with these observations,

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J.K. Morgan et al. / Microbial Pathogenesis 77 (2014) 42e52

Fig. 2. The role for RcsF in DtolA mediated virulence gene regulation. (A) b-galactosidase activity in Miller units of lerP905-lacZ measured for LB cultures grown to mid-log phase (OD600 ¼ 0.5). Plots with different lower-case letters differ significantly by Tukey's HSD test following a significant F-test (p < 0.05, n ¼ 3), and error bars represent the standard deviation. (B) Plots for transcript levels determined by qRT-PCR of tir, espA, fliC, and ecpA (as indicated below each box) for wild type TW14359 (black), TW14359DtolA (white), TW14359DtolA ptolA (gray), TW14359DrcsF (blue), and TW14359DrcsFDtolA (dark red). Samples were taken from exponential phase (OD600 ¼ 0.5) LB cultures. Asterisks denote significance from wild-type TW14359 determined by a Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001; n ¼ 3), and error bars represent the standard deviation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

overexpression of rcsF in either wild-type TW14359 or TW14359DtolA had no significant effect on ecpA transcription. Furthermore, rcsF overexpression in TW14359DtolA abrogated increased espA transcription (p ¼ 0.011). Collectively, these findings support a significant role for RcsF in the altered adhesin gene expression phenotype of TW14359DtolA.

3.3. tolA deletion mitigates EHEC adherence to cultured epithelial cells and virulence in the G. mellonella model As increased transcription of LEE and ECP was observed in TW14359DtolA, it was hypothesized that adherence to cultured HT29 colonic epithelial cells would be enhanced in this background

Fig. 3. Effect of RcsF overexpression on espA, fliC, and ecpA transcription in EHEC. (A) Plots for transcript levels determined by qRT-PCR of espA, fliC, and ecpA (as indicated below each box) for wild type TW14359 (black), TW14359DrcsF (white), and TW14359DrcsF pBAD-rcsF (gray) or (B) TW14359 (black), TW14359DtolA (white), TW14359 pBAD-rcsF (gray), and TW14359DtolA pBAD-rcsF (blue). Samples were taken from exponential phase (OD600 ¼ 0.5) LB cultures supplemented with 0.1% L-arabinose. Asterisks denote significance from wild-type TW14359 determined by a Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001; n ¼ 3), and error bars represent the standard deviation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

J.K. Morgan et al. / Microbial Pathogenesis 77 (2014) 42e52

when compared to wild type TW14359. This was not however observed to be the case, as TW14359DtolA adherence was significantly reduced compared to wild-type TW14359 after 2 h of coincubation with HT-29 cells (p < 0.001) (Fig. 4) and complementation of tolA restored near wild-type levels of adherence. While adherence remained significantly reduced in TW14359DrcsFDtolA compared with wild-type TW14359 (p < 0.001), adherence was further reduced when compared to TW14359DtolA (p ¼ 0.024) (Fig. 4). These findings show that, despite increased LEE and ECP gene expression in TW14359DtolA, adherence to colonic epithelial cells is significantly reduced in this background, and that reduced adherence is enhanced in the absence of rcsF. Given the effects of tolA deletion on LEE and ECP gene expression, and on adherence to intestinal cells, it was of interest to determine the contribution of tolA to EHEC virulence. As G. mellonella has been shown to be an appropriate invertebrate virulence model for type III secretion in enteropathogenic E. coli (EPEC) [60], the effect of tolA deletion in EHEC O157:H7 on G. mellonella survival was determined at 24 h post-injection. Larvae were injected with 1  105 CFU of each strain, the concentration of which represents the LD50 as determined by injecting larvae with 10-fold dilutions (from 102 to 106 CFU) of wild-type strain TW14359. In the silkworm model, a separate insect model for bacterial pathogenesis [70], LPS production was shown to play a significant role in the virulence of EHEC [71]. As tolA mutants are predicted to be defective in O-antigen polymerization and localization to the outer membrane [72], it was hypothesized that decreased EHEC virulence observed in G. mellonella would similarly result from a deficiency in LPS production. In agreement with the latter study [70], the LPS deficient strain TW14359DhldE was significantly attenuated for virulence (83.3% survival; p ¼ 0.022) compared with wild-type TW14359 (40% survival), and complementation of hldE restored wild-type levels of virulence (33.3% survival) (Fig. 5A).

47

Interestingly, virulence was significantly attenuated in TW14359DtolA (83.3% survival; p < 0.001) at 24 h post-injection compared with wild-type TW14359 (40% survival) (Fig. 5A). And, although the virulence of TW14359DrcsFDtolA was restored to near wild-type levels (50% survival), deletion of rcsF alone was observed to significantly increased virulence (13.3% survival; p ¼ 0.001) compared with wild-type TW14359 (Fig. 5A), suggesting that the restoration of virulence observed in TW14359DrcsFDtolA is the result of an overall increase in virulence following the loss of RcsF. This is supported by the observation that TW14359DrcsFDtolA remained significantly less virulent than TW14359DrcsF (p ¼ 0.037). Since loss of rcsF mitigates the increased ecp and LEE gene expression phenotype, it was of interest to determine their role for EHEC virulence in G. mellonella. To explore this, ECP and T3SS deficient and overexpressing strains were assayed. Interestingly, neither deletion of ecpA nor overexpression of the ecp encoded transcriptional activator, EcpR, resulted in significant changes in larval survival. In contrast, loss of type III secretion (TW14359DescN) or its overexpression (TW14359DgrlR::kan) [53] did slightly, but not significantly, alter larval survival compared with wild-type TW14359 (Fig. 5B). Collectively, these findings demonstrate the requirement of TolA for virulence in G. mellonella, and suggest that the decreased virulence phenotype of TW14359DtolA is at least in-part due to both decreased LPS production and to Rcs phosphorelay activation. 3.4. Loss of tolA abrogates motility in EHEC Activation of the Rcs phosphorelay is known to repress motility through the promoter upstream of flhDC [35]; although RcsB has been shown to be required for motility repression following loss of tolA, the specific role for RcsF in this phenotype has not been explored. Therefore, motility was assessed for wild type and mutant derivatives, as determined by lateral growth on motility agar. As was expected, TW14359DtolA displayed a complete loss of motility, and complementation of tolA on a low-copy vector restored motility to that of wild-type TW14359 (Fig. 6). Whereas motility was partially restored in TW14359DrcsFDtolA, motility remained significantly lower than that of wild-type TW14359 (2.8fold; p < 0.001). Deletion of rcsF alone did not significantly affect motility compared with wild-type TW14359, indicating that the partial restoration of motility observed in TW14359DrcsFDtolA was not simply due to a loss of RcsF. 4. Discussion

Fig. 4. Loss of TolA reduces adherence of EHEC to cultured HT-29 colonic epithelial cells. Adherence to HT-29 colonic epithelial cells plotted as CFU/ml recovered after 2 h of co-culture at 37  C with 5% CO2. Each plot represents the average of 3 independent experiments. Asterisks denote significance by a Student's t-test (*, p < 0.05; ***, p < 0.001; n ¼ 3) and error bars represent the standard deviation.

The results of this study have shown that mutation of tolA in EHEC strain TW14359 leads to the upregulation of important adherence genes, including LEE encoded espA and tir, and the E. coli common pilus (ECP) major fimbrial subunit gene, ecpA. Inexplicably, this increase in LEE and ECP expression was not correlated with increased pathogenic potential, tolA null strains being characterized by reduced adherence to HT-29 intestinal cells and virulence attenuation in G. mellonella. The Tol-Pal system serves an integral role in the maintenance of outer-membrane integrity and cell division [44] and, as previously reported, mutation of tolA leads to increased Rcs phosphorelay activation [42]. In this study, ler promoter activity was significantly increased following mutation of tolA, and was further shown to be at least partly independent of alterations in LPS, specifically following deletion of hldE. It is hypothesized that increased ler promoter activity in TW14359DtolA is communicated through the RcsB-GrvA pathway for LEE gene regulation [13]. This is supported by the observation that full ler promoter stimulation in TW14359DtolA is

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Fig. 5. The role for TolA in EHEC virulence using Galleria mellonella. (Panels A and B) Percent survival of G. mellonella larvae following injection of 10 larvae with 105 CFU of wild-type TW14359 or its derivatives at 24 h post injection. Each plot represents the average of 3 independent experiments. For all plots, error bars represent the standard error (SEM). Asterisks denote significance by a Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001; n ¼ 3).

dependent on RcsF. Moreover, transcript levels for the LEE encoded espA and tir were both elevated in TW14359DtolA, and their transcriptional activation was similarly dependent on RcsF, suggesting that potential alterations in membrane integrity resulting from tolA deletion are LEE activating in a manner requiring the Rcs phosphorelay sensor RcsF. These findings are consistent with previous reports of the Rcs component genes in positively regulating the LEE in EHEC [13,53].

While the mechanistic basis of RcsF signal transduction to the sensor kinase RcsC has not yet been determined, it has been shown that both perturbations to the outer-membrane and sensing of mislocalization of outer-membrane lipoproteins stimulate RcsF signaling to RcsC [38,39]. Thus, activation of the LEE in TW14359DtolA may be due to RcsF sensing of pleiotropic cellular effects following loss of TolA. Indeed, alterations in LPS localization appears to increase ler promoter activity, albeit at a low level.

Fig. 6. TolA and RcsF dependent repression of motility. (left) Motility as measured by lateral growth on representative motility plates for wild-type TW14359 and mutant derivative strains. (right) Measurements of motility (lateral growth in mm) following 8 h of growth on motility agar. Asterisks denote significance by a Student's t-test (*, p < 0.05; n ¼ 3) compared with wild-type TW14359, and error bars represent the standard deviation.

J.K. Morgan et al. / Microbial Pathogenesis 77 (2014) 42e52

Although tolA deletion results in activation of the Rcs phosphorelay, resulting in positive stimulation of the LEE, the extracytoplasmic stress sensing Cpx phosphorelay has also been reported to be active in tolA deficient E. coli [50]. This is of particular significance as, at least in enteropathogenic E. coli (EPEC), activation of the Cpx twocomponent system substantially represses LEE gene transcription [73]. Therefore, if the Cpx phosphorelay similarly represses LEE gene transcription in EHEC, one might expect LEE gene transcription to be reduced in TW14359DtolA. However, as LEE gene activity is significantly higher in TW14359DtolA compared with wild-type TW14359, activation by the Rcs phosphorelay appears to be dominant to any potential Cpx mediated LEE gene repression, at least in the conditions examined. It remains possible that additional sensory regulators may contribute to the observed LEE gene expression phenotype in TW14359DtolA, and future work is aimed at identifying the role for other factors in the regulatory phenotype. The E. coli common pilus (ECP) plays an integral role in adherence to epithelial cells and biofilm formation for EHEC [74] and, in neonatal meningitic E. coli (NMEC), RcsB is required for transcriptional activation of the ecp operon [69]. The first gene of the ecp operon, ecpR, encodes a positive regulator of ecp transcription, and both EcpR and RcsB bind upstream of ecpR to stimulate ECP production [75,76]. However, to-date, activation of ecp transcription by mutations which stimulate the Rcs phosphorelay had not yet been reported. Transcription of the ecp encoded fimbrillin gene, ecpA, was thus explored in TW14359DtolA. Loss of tolA significantly increased ecpA transcription, and was similarly dependent on RcsF for activation. Furthermore, RcsF overproduction in TW14359DrcsF increased ecp transcription to a higher level than that observed in TW14359DtolA. Binding of both non-phosphorylated and phosphorylated variants of RcsB to the ecpR promoter have been previously shown in NMEC [69], however the role for RcsB in ecp regulation in EHEC had not yet been clearly established. A previous study suggested that the RcsB binding site was dispensable for ecpR promoter activity in EPEC [75], whose promoter contains 99% nucleotide homology with that of EHEC. However, since Rcs stimulation significantly increased ecpA transcription, it is likely that RcsB has a similar regulatory role to that observed in NMEC for EHEC ecp transcriptional activation. Taken together, these findings show that ecp and LEE gene transcriptional activation is simultaneously observed in TW14359DtolA, and following overexpression of rcsF in TW14359DrcsF. While overexpression of rcsF In TW14359DrcsF resulted in predictably increased ecpA and espA transcription, similarly overexpressed rcsF in TW14359 or TW14359DtolA did not mirror those changes. Specifically, whereas ecpA was un-affected in both backgrounds, overproduction of rcsF in TW14359DtolA completely abrogated increased transcription compared with wild-type TW14359. While the specific mechanism behind this disparity was not explored in this study, it is possible that overexpression of rcsF in strains with intrinsic Rcs stimulation (such as TW14359DtolA), or possessing an intact copy of rcsF, yield Rcs phosphorelay stimulation higher than that observed for TW14359DrcsF overexpressing rcsF. Such hyper-stimulation may lead to differential transcription of Rcs regulated of genes which exert a negative effect on LEE gene transcription. For example, RcsB positively regulates transcription of the sRNA activator of RpoS translation, rprA [77,78], which is of significance since RpoS has been shown to negatively regulate LEE gene transcription [79,80]. However, future work is required to further refine the role of RcsF in LEE gene stimulation. Mutation of tolA resulted in completely abrogated motility, and in concomitantly repressed transcription of the major flagellar subunit encoding gene, fliC. This observation is markedly consistent with previous studies on tolA deletion in E. coli strain MG1655 and

49

on the role for RcsB in regulating motility in EHEC [35,50]. In EHEC, however, the LEE encoded activator GrlA is similarly able to repress motility in a manner dependent on RcsB [31,81], and the grlRA operon is positively regulated by Ler [82]. Therefore, it is possible that motility repression in TW14359DtolA is modulated by a synergistic combination of direct repression by phosphorylated RcsB and by increased levels of GrlA following LEE gene activation. Future work is required to determine the specific role for GrlA in motility repression, if any, in TW14359DtolA. Additionally, while motility was partially restored in TW14359DrcsFDtolA, overall motility was still reduced compared with wild-type TW14359. This finding is intriguing as fliC transcript levels were observed at wildtype levels following deletion of rcsF in TW14359DtolA. Since TolA has an important role in normal cell division, reduced motility may be a function of alterations in cell shape caused by disruption of normal cellular division in TW14359DtolA, as has been previously reported [44]. However, another study induced a filamentous cell morphology in E. coli using antibiotics, yet reported normal levels of chemotaxis and flagellation for bacteria up to 50 times the length of normal cells [83], suggesting that elongation of the cell in TW14359DtolA may not be directly responsible for reduced motility. Interestingly, a recent report in MG1655 demonstrated an important role for the Tol-Pal complex in directing the correct polar localization of chemoreceptors [84]. Therefore, it is possible that, although Rcs mediated motility repression of fliC is alleviated in TW14359DrcsFDtolA, the function or assembly of flagella is negatively affected by loss of TolA. Based on the previous findings for increased transcription of LEE and ecp encoded adhesins in TW14359DtolA, it was predicted that adherence to cultured epithelial cells would be enhanced. However, adherence to HT-29 colonic epithelial cells was significantly reduced in TW14359DtolA when compared with wild-type TW14359. This finding is particularly salient as it reveals that, while T3SS and ECP encoding genes are upregulated in TW14359DtolA, the TolA protein is required for wild-type levels of adherence. Although it remains a possibility that TolA has a role in biogenesis or function of the T3SS, the assembly of chaperoneusher pili (such as ECP) is predicted to occur independent of TolA [85]. Moreover, while decreased LPS localization is predicted for TolA deficient strains [72], previous reports have demonstrated that intimate adherence patterns of O-antigen deficient EHEC are not affected [86]. Indeed, loss of O-antigen has been reported to increase overall adherence to cultured epithelial cells for EHEC [87]. Thus, one potential explanation for the observed decreased adherence in TW14359DtolA is that, although the LEE is upregulated in TW14359DtolA, secretion of LEE and non-LEE encoded proteins may be negatively affected by loss of TolA. While it is possible that decreased adherence in TW14359DtolA results from the Rcs-mediated repression of one or more additional adhesions, such as curli [50,88], the observation that deletion of rcsF exaggerates the overall adherence defect of TW14359DtolA suggests that it is mostly independent of the Rcs phosphorelay. Alternatively, since increased ler promoter activity was relegated to exponential growth, the narrow window of increased LEE gene transcription may not be sufficient to positively modulate adherence through enhanced T3SS or ECP production. The results of this study also showed that mutation of tolA results in significantly attenuated virulence in the G. mellonella model for pathogenesis. In EPEC, a type III secretion deficient strain displayed markedly reduced virulence in the same model [60]; however, our work has demonstrated that deletion of tolA, which increases LEE gene transcription, significantly reduces virulence in G. mellonella. Indeed, loss of ECP production or type III secretion did not significantly affect virulence. As the insect immune response utilizes both humoral and cell based responses, including the production of

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antimicrobial peptides (AMPs) and macrophage-like hemocytes, respectively [89], bacterial susceptibility to host immune defenses and/or phagocytosis may be reduced as a result of decreased LPS production in TW14359DtolA [72]. In support of this, production of LPS has an integral role in EHEC virulence using the silkworm model of virulence [71]. Since the LPS deficient strain TW14359DhldE was similarly attenuated following injection, it is likely that the virulence defect observed for TW14359DtolA is at least in-part due to decreased LPS production. Our results also showed that virulence was increased following loss of RcsF either alone or in TW14359DtolA. One possible explanation for this observation is that surface antigen availability may be partially masked by Rcs regulated capsule synthesis, which is in-contrast with a recent report in Klebsiella pneumoniae using the G. mellonella model wherein the authors demonstrate a positive role for capsule synthesis in virulence [90]. Future studies are aimed at further delineating the specific role for the Rcs phosphorelay and capsule synthesis in EHEC virulence using the G. mellonella model. To conclude, this study has shown that mutation of tolA in EHEC results in significantly higher transcription of RcsB regulated EHEC adhesins and reduced transcription of motility genes, and that both were dependent on the outer-membrane lipoprotein RcsF. Ultimately, however, TolA was required for maximal adherence to cultured colonic epithelial cells and virulence in the G. mellonella model for pathogenesis. Increased transcription of key EHEC adherence and colonization factors, such as ECP and the T3SS, may be triggered by sensing significant perturbations to the outermembrane by host factors present in the lumen or intestinal mucosa, such as antimicrobial peptides [91,92], to promote colonization. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.micpath.2014.10.010. References [1] J.P. Nataro, J.B. Kaper, Diarrheagenic Escherichia coli, Clin. Microbiol. Rev. 11 (1998) 142e201. [2] J.M. Rangel, P.H. Sparling, C. Crowe, P.M. Griffin, D.L. Swerdlow, Epidemiology of Escherichia coli O157:H7 outbreaks, United States, 1982e2002, Emerg. Infect. Dis. 11 (2005) 603e609. [3] H. Karch, P.I. Tarr, M. Bielaszewska, Enterohaemorrhagic Escherichia coli in human medicine, Int. J. Med. Microbiol. 295 (2005) 405e418. [4] K.A. Eaton, D.I. Friedman, G.J. Francis, J.S. Tyler, V.B. Young, J. Haeger, et al., Pathogenesis of renal disease due to enterohemorrhagic Escherichia coli in germ-free mice, Infect. Immun. 76 (2008) 3054e3063. [5] N.T. Perna, G.F. Mayhew, G. Posfai, S. Elliott, M.S. Donnenberg, J.B. Kaper, et al., Molecular evolution of a pathogenicity island from enterohemorrhagic Escherichia coli O157:H7, Infect. Immun. 66 (1998) 3810e3817. [6] J.B. Kaper, J.P. Nataro, H.L. Mobley, Pathogenic Escherichia coli, Nat. Rev. Microbiol. 2 (2004) 123e140. [7] R. DeVinney, M. Stein, D. Reinscheid, A. Abe, S. Ruschkowski, B.B. Finlay, Enterohemorrhagic Escherichia coli O157:H7 produces Tir, which is translocated to the host cell membrane but is not tyrosine phosphorylated, Infect. Immun. 67 (1999) 2389e2398. [8] B. Kenny, R. DeVinney, M. Stein, D.J. Reinscheid, E.A. Frey, B.B. Finlay, Enteropathogenic E. coli (EPEC) transfers its receptor for intimate adherence into mammalian cells, Cell 91 (1997) 511e520. [9] J.L. Mellies, S.J. Elliott, V. Sperandio, M.S. Donnenberg, J.B. Kaper, The Per regulon of enteropathogenic Escherichia coli: identification of a regulatory cascade and a novel transcriptional activator, the locus of enterocyte effacement (LEE)-encoded regulator (Ler), Mol. Microbiol. 33 (1999) 296e306. [10] V. Sperandio, J.L. Mellies, R.M. Delahay, G. Frankel, J.A. Crawford, W. Nguyen, et al., Activation of enteropathogenic Escherichia coli (EPEC) LEE2 and LEE3 operons by Ler, Mol. Microbiol. 38 (2000) 781e793. [11] S.J. Elliott, V. Sperandio, J.A. Giron, S. Shin, J.L. Mellies, L. Wainwright, et al., The locus of enterocyte effacement (LEE)-encoded regulator controls expression of both LEE- and non-LEE-encoded virulence factors in enteropathogenic and enterohemorrhagic Escherichia coli, Infect. Immun. 68 (2000) 6115e6126. [12] V.K. Sharma, R.L. Zuerner, Role of hha and ler in transcriptional regulation of the esp operon of enterohemorrhagic Escherichia coli O157:H7, J. Bacteriol. 186 (2004) 7290e7301.

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