Atypical Roles for Campylobacter jejuni Amino Acid ATP Binding ...

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May 9, 2008 - Ann E. Lin,1 Kirsten Krastel,2 Rhonda I. Hobb,3 Stuart A. Thompson,3 ... shock, the paqQ mutant was more susceptible than the wild type to the ...

INFECTION AND IMMUNITY, Nov. 2009, p. 4912–4924 0019-9567/09/$12.00 doi:10.1128/IAI.00571-08 Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Vol. 77, No. 11

Atypical Roles for Campylobacter jejuni Amino Acid ATP Binding Cassette Transporter Components PaqP and PaqQ in Bacterial Stress Tolerance and Pathogen-Host Cell Dynamics䌤 Ann E. Lin,1 Kirsten Krastel,2 Rhonda I. Hobb,3 Stuart A. Thompson,3 Dennis G. Cvitkovitch,2 and Erin C. Gaynor1* Department of Microbiology and Immunology, Life Sciences Centre, The University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia, V6T 1Z3,1 and Dental Research Institute, University of Toronto, 124 Edward St., Toronto, Ontario, M5G 1G6,2 Canada, and Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia 309123 Received 9 May 2008/Returned for modification 7 July 2008/Accepted 19 August 2009

Campylobacter jejuni is a human pathogen causing severe diarrheal disease; however, our understanding of the survival of C. jejuni during disease and transmission remains limited. Amino acid ATP binding cassette (AA-ABC) transporters in C. jejuni have been proposed as important pathogenesis factors. We have investigated a novel AA-ABC transporter system, encoded by cj0467 to cj0469, by generating targeted deletions of cj0467 (the membrane transport component) and cj0469 (the ATPase component) in C. jejuni 81-176. The analyses described here have led us to designate these genes paqP and paqQ, respectively (pathogenesisassociated glutamine [q] ABC transporter permease [P] and ATPase [Q]). We found that loss of either component resulted in amino acid uptake defects, most notably diminished glutamine uptake. Altered resistance to a series of environmental and in vivo stresses was also observed: both mutants were hyperresistant to aerobic and organic peroxide stress, and while the ⌬paqP mutant was also hyperresistant to heat and osmotic shock, the ⌬paqQ mutant was more susceptible than the wild type to the latter two stresses. The ⌬paqP and ⌬paqQ mutants also displayed a surprising but statistically significant increase in recovery from macrophages and epithelial cells in short-term intracellular survival assays. Annexin V, 4ⴕ,6-diamidino-2-phenylindole (DAPI), and Western blot analyses revealed that macrophages infected with the ⌬paqP or ⌬paqQ mutant exhibited transient but significant decreases in cell death and extracellular signal-regulated kinase–mitogenactivated protein kinase activation compared to levels in wild-type-infected cells. The ⌬paqP mutant was not defective in either short-term or longer-term mouse colonization, consistent with its increased stress survival and diminished host cell damage phenotypes. Collectively, these results demonstrate a unique correlation of an AA-ABC transporter with bacterial stress tolerances and host cell responses to pathogen infection. estly thermophilic organism, C. jejuni survives optimally under a 12% CO2, 5% O2 atmosphere at 37 to 42°C (24, 61) and is sensitive to a spectrum of conditions, including aerobic, oxidative, heat, and osmotic stresses, as well as nutrient limitation (2, 24, 43). Many of these conditions are frequently encountered both inside and outside its natural animal hosts. C. jejuni harbors several stress response proteins that allow the bacterium to traverse the transmission and colonization environments; many of these have also been characterized as virulence factors (3, 10, 25, 55, 66). Although C. jejuni is primarily an extracellular pathogen, host cell invasion is an important virulence property (69, 86, 96). C. jejuni is also capable of surviving intracellularly both in intestinal epithelial cells and in immune cells such as monocytes and macrophages (18, 32, 52), which has been hypothesized to be an important virulence attribute for persistent infection and immune system evasion (32, 41, 96). Successful intracellular survival likewise includes overcoming physiological stresses, such as oxidative environments generated by the human gut and by host epithelial or immune cells. Another feature of C. jejuni pathogenesis is the stimulation of host cell signal transduction events to trigger an inflammatory response and host cell death (9, 32, 35, 49, 78, 90). The mitogen-activated protein kinase (MAPK) family plays an im-

Campylobacter jejuni, a highly prevalent, invasive human pathogen, is responsible for 5 to 15% of all diarrheal disease worldwide (1, 96). Infection with this gram-negative bacterium causes acute gastroenteritis, predominantly in the duodenum, ileum, and colon. In severe cases, C. jejuni can also lead to postinfectious complications such as Guillain-Barre´ syndrome, the primary cause of acute ascending bilateral paralysis (96). Despite its prevalence, our current knowledge of the virulence mechanisms of C. jejuni and its survival throughout pathogenesis to cause disease in the human gastrointestinal tract is significantly limited compared to that for other enteric pathogens, such as Escherichia coli and Salmonella spp. C. jejuni must overcome a series of physiological stresses throughout the infectious process. This includes surviving transmission environments, such as water reservoirs, farms, and food-processing plants, and in vivo environments, such as the intestinal tracts of poultry, the human gut, and intestinal epithelial cells. As a capnophilic, microaerophilic, and mod* Corresponding author. Mailing address: The University of British Columbia, Department of Microbiology and Immunology, Life Sciences Centre, 2350 Health Sciences Mall, Vancouver, British Columbia, V6T 1Z3, Canada. Phone: (604) 822-2710. Fax: (604) 822-6041. E-mail: [email protected] 䌤 Published ahead of print on 24 August 2009. 4912

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portant role in mediating signal transduction and is activated by a range of environmental stimuli (73). There are three major MAPK families in mammals, extracellular signal-regulated kinases 1 and 2 (Erk1/2), c-Jun NH2-terminal kinase (Jnk), and the p38 kinases, all of which lead to gene expression changes important for regulating diverse cellular activities (23, 74). C. jejuni infection of INT407 human epithelial cells and of Caco-2 and T84 human intestinal epithelial cells has been shown to trigger Erk1/2, Jnk, and p38 kinase phosphorylation (15, 35, 49). In other bacteria, pathogen-induced Erk activation has been shown to be important for the induction of macrophage apoptosis (22, 83, 95), and lipopolysaccharide (LPS) has been determined to be responsible for MAPK stimulation in monocytes and macrophages (26, 68, 85). Several lines of investigation have focused on the effects of C. jejuni infection on apoptotic and proinflammatory responses in epithelial cells (9, 15, 49, 90), and C. jejuni-induced apoptosis and cytokine release have been observed in human 28SC monocytes and THP-1 macrophages (32, 37). However, the mechanisms underlying C. jejuni-induced host cell death and inflammatory response activation in macrophages still require further examination. Several studies have identified ATP-binding cassette (ABC) transporters as important not only for bacterial physiology but also for pathogenesis and host cell infection (12, 48, 65, 82). ABC transporters, typically situated in the bacterial cytoplasmic membrane, have been shown to dictate various physiological processes by importing nutrients or metabolites such as amino acids, short peptides, and organic or inorganic ions (72). ABC transporters comprise three functional subunits: two alpha-helical integral transmembrane proteins (permeases), which channel the substrate across the bacterial membrane, and a periplasmic or cytoplasmic ATP binding protein (ATPase), which binds and hydrolyzes ATP to actively transfer the substrate from the periplasm into the cytoplasm. A separate substrate-binding domain is often coupled to the system, although it is frequently encoded by a gene located outside of the permease- and ATPase-encoding operon (17). The permease subunit is required for ABC transporter functionality, and dysfunctional ATPase domains likewise frequently result in inactivation of substrate transport (16, 56, 57). Components of amino acid ABC (AA-ABC) transporter systems identified previously in C. jejuni include the glutamate/aspartate substrate binding protein Peb1 (48, 53), the cysteine binding protein CjaA (54), and the SdaA and SdaC serine transporters (87). The periplasmic solute binding protein Peb1 and the cysteine binding protein CjaA have also been proposed previously to function as surface antigens and adhesins important for host cell colonization and pathogenesis (48, 54). Although AA-ABC transporters have been shown to be important for host cell colonization in vitro and in vivo, they have not previously been implicated in C. jejuni stress modulation or host cell death and signal transduction events. In a previous study, we found that transcription of the putative C. jejuni AA-ABC transporter system encoded by cj0467 to cj0469 was induced during in vitro infection of INT407 cells (25), suggesting a role for these genes in pathogenesis. To test this and to explore the biological function(s) of this system, two independent mutants were constructed, one lacking a functional integral transmembrane protein (⌬paqP/cj0467 per-

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mease mutant) and the other lacking a functional ATP binding protein (⌬paqQ/cj0469 ATPase mutant). Extensive analyses of both mutant strains demonstrated that this ABC transporter system primarily transports glutamine, as well as other amino acids at a more moderate level, and that the loss of function of this system results in increased recovery of bacteria from both epithelial cells and macrophages in a short-term intracellular survival assay. Although initially surprising, this observation was found to be consistent with an overall increased resistance of the mutants to aerobic and organic peroxide stresses, as well as with a transient decrease in host cell death and Erk activation in macrophages. Differential effects of the two mutations on other stress responses were also observed, suggesting potentially distinct roles for the different components in certain bacterial functions. Together, these data suggest that this system helps modulate both expected and unexpected physiological properties of C. jejuni and demonstrate for the first time that a bacterial AA-ABC transporter is involved in bacterial stress survival and in host cell damage and signal transduction events following infection. MATERIALS AND METHODS Bacterial strains, cells, media, and growth conditions. Wild-type (WT) Campylobacter jejuni 81-176 and mutants were cultured in Mueller-Hinton (MH) broth (Oxoid Ltd., Hampshire, England) or agar supplemented with 10 ␮g/ml of vancomycin and 5 ␮g/ml of trimethoprim (MHTV) in a microaerophilic and capnophilic (C. jejuni growth) environment at 37°C. C. jejuni growth environments were generated using Oxoid CampyGen gas packs in an enclosed container or a trigas incubator with 6% O2 and 12% CO2. C. jejuni 81-176 ⌬paqP and ⌬paqQ mutants were cultured under the same conditions with the addition of 50 ␮g/ml of kanamycin. For overnight broth cultures, freshly growing plates of bacteria were inoculated into MH broth to a starting optical density at 600 nm (OD600) of approximately 0.004. The flasks were placed in an enclosed jar under 6% O2–12% CO2 and grown at 37°C with shaking at 200 rpm. All E. coli DH5␣ strains were grown in Luria-Bertani agar or broth at 37°C. RAW 264.7 and INT407 cells were cultured at 37°C in humidified air with 5% CO2. RAW 264.7 cells were maintained in Dulbecco’s modified Eagle medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (Gibco), while INT407 cells were maintained in minimal essential medium (Gibco) supplemented with 10% FBS. Construction of C. jejuni ⌬paqP (cj0467) and ⌬paqQ (cj0469) mutants. To construct the ⌬paqP (cj0467) and ⌬paqQ (cj0469) mutant strains in C. jejuni 81-176, the target genes were amplified by PCR from C. jejuni chromosomal DNA prepared using a DNA extraction kit (Promega, Nepean, Ontario, Canada), with primers paqPF1 (5⬘-TCTAGAGAAGATGGAGAAATTTTG-3⬘) and paqPR1 (5⬘-TCTAGAACACCACAAAAAGCCAT-3⬘), as well as paqQF1 (5⬘TCTAGATCCTTGCAGAGTATTC-3⬘) and paqQR1 (5⬘-TCTAGATACCAA CTGAGCTAAACC-3⬘), yielding 1.3-kb and 1.2-kb fragments, respectively, with XbaI sites at the flanking regions. The PCR products were purified (Qiagen, Mississauga, Ontario, Canada) and cloned into the pGEM-T vector (Promega); these constructs were designated pGEM-paqP and pGEM-paqQ. Restriction digestion was performed, using the enzyme HindIII for paqP and MscI with BamHI for paqQ, to remove approximately 400 bp from paqP and 290 bp from paqQ. A nonpolar aphA-3 cassette encoding kanamycin resistance (Kanr) was digested from the pUC18K2 plasmid (51) by using the HindIII or MscI and BamHI enzymes and was ligated to the digested pGEM-paqP and pGEM-paqQ vectors, creating suicide vectors carrying the ⌬paqP::aphA-3 and ⌬paqQ::aphA-3 deletion constructs. Following selection and amplification in E. coli DH5␣, the mutagenic plasmids were purified from DH5␣ using a Qiagen Midi-prep kit and were transformed into WT C. jejuni 81-176 by natural transformation or by electroporation as previously described (11). Because pGEM-T is a suicide vector in C. jejuni, colonies recovered from MHTV-kanamycin plates should represent stable chromosomal integrants resulting from double-crossover homologous recombination. Genomic DNA from several mutant clones was prepared using the Wizard genomic DNA kit (Promega). The resulting mutants were designated the ⌬paqP and ⌬paqQ mutants. Insertional inactivation of the paqP

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and paqQ genes via insertion of the Kanr cassette was verified by PCR and sequencing analysis as well as by Southern blot analysis. The ⌬paqP mutant was complemented by natural transformation of plasmid pRRC-paqP, which was generated by inserting the paqP gene into the pRRC vector (39) using XbaI sites introduced during the initial amplification. Natural transformation was performed as described above, and colonies were isolated from MHTV plates supplemented with kanamycin and chloramphenicol. The resultant strain was designated the ⌬pacPc strain. RNA extraction and RT-PCR analysis. C. jejuni RNA was isolated as previously described (25). Reverse transcription of the purified RNA was performed using SuperScript II mix and random primers (Invitrogen, Burlington, Ontario, Canada), followed by purification using the Qiagen PCR purification kit. The purified cDNA products were PCR amplified for nssR by using primers nssRF1 (5⬘AGAACTTTTATCTAGTGTAGG-3⬘) and nssRR1 (5⬘-CGTCCTTAAATC TAATGC-3⬘) and for tuf by using primers tufF1 (5⬘-GCGTGGTATTACTATT GCTAC-3⬘) and tufR1 (5⬘-TCGAAGTCAGTGTGTGGAG-3⬘). RNA was confirmed as DNA free by reverse transcriptase PCR (RT-PCR) minus the RT enzyme. Southern blot analysis. Genomic DNA was isolated using a DNA isolation kit as described above. For each Southern blot, 100 ng of DNA from each strain was digested with EcoRV, which cuts in the middle of the Kanr cassette but does not cut either paqP or paqQ. DNA was separated on a 0.75% agarose gel and was directly blotted onto polyvinylidene difluoride membranes by using 0.25 N NaOH and 0.75 M NaCl as the transfer solution, according to the manufacturer’s instructions, with DIG High Prime DNA labeling and detection starter kit II (Roche Applied Science, Mannheim, Germany). All probes were nonradioactively labeled using this kit. The paqP fragment was obtained by digesting the pGEM-paqP plasmid with XbaI, while the paqQ fragment was obtained by digesting the pGEM-paqQ plasmid with XbaI and MscI. The resulting fragments were denatured by heating at 99°C for 10 min and were placed on ice. Hexanucleotides, digoxigenin, and Klenow fragment were added to the denatured fragment and incubated overnight at 37°C as described in the DIG DNA labeling kit instructions. The blot was hybridized with these probes and was visualized using enhanced chemiluminescence (Perkin-Elmer, Waltham, MA). Insertion of the Kanr cassette into paqP and paqQ should result in the generation of two smaller probe-reactive fragments (due to EcoRV digestion of the Kanr cassette) rather than the larger WT-sized fragment. In the case of the ⌬paqQ mutant, only one smaller fragment is readily visible at the exposure shown. Amino acid transport assay. C. jejuni cells were grown for 15 h to the midlog-growth phase in 15 ml of MH broth. Cultures were harvested by centrifugation, washed twice in M9 minimal medium (90.2 mM Na2HPO4, 22.0 mM KH2PO4, 8.56 mM NaCl, 18.7 mM NH4Cl, 2 mM MgSO4, 22.2 mM glucose, 10 ␮M CaCl2 [pH 7.4]), and resuspended in the same medium to an approximate OD600 of 1.0. The resuspension was kept on ice for no longer than 4 h. A 150-␮l aliquot of the cell suspension was added to 1.5 ml of M9 minimal medium containing 0.5% (vol/vol) lactic acid (Sigma-Aldrich, Oakville, Ontario, Canada) and was allowed to equilibrate by incubation at 37°C for 3 min. The assay was then initiated by the addition of 5 ␮M 14C-labeled amino acid (6.9 to 9.36 GBq mmol⫺1) (Perkin-Elmer). Samples (0.1 ml) were withdrawn at the indicated time points, collected by vacuum filtration through 0.22-␮m-pore-size membrane filters (Millipore Corp., Billerica, MA), and washed twice with M9 minimal medium. Sample filters were then immersed in Filter-Count scintillation cocktail (Fisher Scientific, Ontario, Canada) and counted in a Beckman Coulter scintillation counter. Transporter activity was expressed as nanomoles of amino acid transported per milligram of dry cells per minute. Gentamicin protection assay for adherence, invasion, and intracellular survival. INT407 cells (a human epithelial cell line) or RAW 264.7 cells (a murine macrophage cell line) were seeded into 24-well tissue culture plates at semiconfluence (⬃5 ⫻ 105 cells/ml) and allowed to grow for 20 h prior to infection. Mid-log-phase WT C. jejuni and mutants from overnight shaking cultures were added to prewarmed minimal essential medium or Dulbecco’s modified Eagle medium, which was used to infect the cells at a multiplicity of infection (MOI) of ⬃200 for 3 h. The cells were then washed three times with phosphate-buffered saline (PBS) to remove any unbound bacteria. To assay adherence, 1 ml of 1% Triton X-100 in PBS was added to some of the wells for 5 min to disrupt the cells; the samples were then plated onto MH agar and grown for 48 h at 37°C in a C. jejuni growth environment as described above. To assay invasion, fresh medium supplemented with 10% FBS and 150 ␮g/ml of gentamicin was added, and the cells were incubated at 37°C for 2 h before being washed and treated with Triton X-100 as described above. To assay short-term intracellular survival, fresh medium supplemented with 10% FBS containing 10 ␮g/ml gentamicin was added, and the cells were incubated for an additional 4 h before being washed and treated with Triton X-100; this is also referred to as the 9-h-postinfection time

INFECT. IMMUN. point. To assay long-term intracellular survival, cells were left in 10 ␮g/ml gentamicin for an additional 19 h postinvasion (or 24 h after initial infection) before being subjected to washes and Triton X-100 treatment. The susceptibility of each strain to gentamicin was tested by determining the MIC using an Etest strip (AB Biodisk). In vivo colonization using a mouse model. BALB/cByJ mice from Jackson Laboratories (Bar Harbor, ME) were housed at the animal care center at the Medical College of Georgia, with seven mice per experimental group. Each mouse was infected with 5 ⫻ 109 CFU of WT or ⌬paqP C. jejuni bacteria via oral gavage as previously described (60). C. jejuni bacteria shed in fecal pellets from each mouse at 7, 14, 19, 28, and 35 days postinfection were homogenized and enumerated on MH agar containing 5% (vol/vol) sheep’s blood, 20 ␮g/ml cefoperazone, 10 ␮g/ml vancomycin, and 2 ␮g/ml amphotericin B. The level of detection was 1 ⫻ 102 CFU/g of fecal pellet. All animal treatments were carried out in accordance with NIH guidelines for the care and use of laboratory animals, using procedures approved by the Medical College of Georgia Institutional Care and Use Committee. Oxidative, aerotolerance, heat, and osmotic stress assays. To assay oxidative stress, C. jejuni bacteria from overnight cultures in MH broth were inoculated into fresh MH broth to an OD600 of ⬃ 0.6; 1 ml of these cultures was subsequently added to each well of a 24-well plate. tert-Butyl hydroperoxide (t-BOOH; Sigma-Aldrich) and hydrogen peroxide (H2O2; Sigma-Aldrich) were made in MH medium at various concentrations. A 1-ml volume of the oxidative agent was added to C. jejuni cultures and incubated for 30 min at 37°C under C. jejuni growth conditions before cultures were harvested for CFU enumeration. Aerotolerance was examined by diluting C. jejuni bacteria, grown to mid-log phase in MH broth to an initial OD600 of ⬃0.004, in fresh MH broth and growing the bacteria in a shaking culture at 200 rpm and 37°C under atmospheric conditions. To assess limited CO2 stress and heat stress, mid-log-phase bacteria were serially diluted, spotted onto MH agar plates, and incubated at 37°C in a 5% CO2 incubator or at 45°C under C. jejuni growth conditions. Osmotic stress was examined by spotting the dilutions onto MH agar plates supplemented with 0.17 M NaCl and incubating at 37°C in a C. jejuni growth environment. Cellular death and viability detection. (i) Annexin V staining. RAW 264.7 cells were plated at semiconfluence on coverslips in 24-well plates and were cultured for 24 h before infection with equal CFU of C. jejuni, followed by gentamicin treatment as described above. At the end of a 9-h infection (3 h of infection, 2 h of gentamicin treatment, and an additional 4 h of intracellular survival as described above), cells were washed three times with PBS and stained with annexin V fluorescein and propidium iodide according to the manufacturer’s instructions (Roche Applied Sciences). Annexin V labeling was visualized using a Nikon eclipse TE2000 microscope (Nikon Instruments Inc., Mississauga, Ontario, Canada) fitted with appropriate filter sets for detecting fluorescence. (ii) DAPI staining and immunofluorescence. Following infection, RAW 264.7 cells were washed and fixed as described by Guttman et al. (28). Cellular nuclei were labeled with 4⬘,6-diamidino-2-phenylindole (DAPI) and were mounted using Vectashield (Vector Labs, Burlington, Ontario, Canada). Relative cell viability was fluorescently quantified by counting DAPI-stained cell nuclei in multiple randomly selected fields in a manner similar to that previously described by Barnes et al. (7). Cell lysate preparation and Western blotting. RAW 264.7 cells were grown on 150-mm-diameter tissue culture dishes and infected at an MOI of ⬃200 for 9 h. Cells were washed three times with PBS containing 1 mM CaCl2 and 1 mM MgCl2, followed by treatment with radioimmunoprecipitation assay lysis buffer (150 mM NaCl, 50 mM Tris [pH 7.4], 5 mM EDTA, 1% Nonidet P-40, 1% deoxycholic acid, 10% sodium dodecyl sulfate) for 10 min on ice. Western blotting was performed according to the method of Guttman et al. (29). Briefly, equal amounts of total proteins were loaded and separated on 10% sodium dodecyl sulfate-polyacrylamide gels and were then transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Mississauga, Ontario, Canada). Membranes were blocked with 4% skim milk and washed in Tris-buffered saline with 0.1% Tween 20 three times, for 5 min each time. A primary mouse antiphospho-Erk1/2 antibody (Cell Signaling Technology, Beverly, MA) was used at a dilution of 1:2,000, and a rabbit anti-Erk antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was used at a 1:2,000 dilution (0.1 ␮g/ml). Rabbit anti-phosphoJnk (Thr183/Tyr185) (Cell Signaling Technology) and rabbit anti-Jnk1 (Santa Cruz Biotechnology) antibodies were used at a 1:1,000 dilution. After the membrane was washed, a horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology) was used at 1:5,000 for labeling. Signals were detected by enhanced chemiluminescence (Perkin-Elmer).

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FIG. 1. Generation of nonpolar, single-insert ⌬paqP and ⌬paqQ disruption strains. (A) Genomic organization of the cj0467, cj0468, and cj0469 genes, encoding a putative AA-ABC transporter system. The aphA-3 cassette, encoding kanamycin resistance, was used to create insertiondeletions in the cj0467 (paqP) and cj0469 (paqQ) genes by double crossover, generating the ⌬paqP and ⌬paqQ mutant strains. (B) Southern blotting of EcoRV-digested genomic DNA using paqP and paqQ probes confirms that the target gene in each mutant was disrupted. (C) RT-PCR was performed to assay the transcription of genes upstream of paqP (nssR) and downstream of paqQ (tuf) in the deletion strains.

RESULTS Construction of targeted, nonpolar paqP and paqQ disruption strains. The putative AA-ABC transporter system encoded by cj0467 to cj0469 is highly conserved and occurs in the same genomic context among C. jejuni strains, as described in CampyDB (http://www.xbase.bham.ac.uk/campydb). Given this conservation, together with our previous study describing the upregulation of these genes during cell infections (25), we will refer to the cj0467-to-cj0469 gene numbers annotated for strain NCTC11168, although our studies were performed using the invasive strain 81-176 (in 81-176, this locus is annotated as CJJ81176_0492 to CJJ81176_0494). cj0467 and cj0468 are predicted to encode integral membrane proteins, while cj0469 is predicted to encode an ATP binding protein (CampyDB). This system exhibits significant similarity to glutamine ABC transporter components: NCBI BLAST searches and searches of the Archaeal and Bacterial ABC transporter database (http: //www-abcdb.biotoul.fr/) revealed that Cj0467 and Cj0468 are ⬃43% identical to the glutamine ABC transporter permease GlnP, and that Cj0469 is ⬃60% identical to GlnQ, in Streptococcus pneumoniae, Pseudomonas spp., and Helicobacter spp. Cj0467 and Cj0469 also exhibit homology to two uncharacterized C. jejuni proteins that were previously annotated as GlnP (Cj0940c) and GlnQ (Cj0902) based on slightly higher initial BLAST search homologies (37.5% identity to E. coli GlnP for Cj0940c versus 33% for Cj0467; 56.2% identity to Bacillus stearothermophilus GlnQ for Cj0902 versus 55% for Cj0469) (34, 62, 63). To avoid confusion with the previous annotation, and because our data (described below) indicate that Cj0467 and Cj0469 participate both in glutamine transport and in other important biological processes, we have designated these proteins PaqP and PaqQ, respectively (for pathogenesis-associated glutamine [q] ABC transporter permease [P] and ATPase [Q]). To investigate the role of this system in C. jejuni, paqP and paqQ were individually disrupted using a nonpolar Kanr cassette (Fig. 1A). PCR, sequencing, and Southern blot analyses confirmed that the resultant ⌬paqP and ⌬paqQ mutants harbored disruptions in the appropriate genes (Fig. 1B; also data

not shown). To verify that Kanr insertion into both paqP and paqQ was nonpolar, RT-PCR was used to confirm that nssR and tuf were transcribed in both mutant strains (Fig. 1C) and that paqQ was transcribed in the ⌬paqP strain (data not shown). WT and mutant strains exhibited similar growth characteristics in MH broth and on MH agar at both 37°C and 42°C (data not shown). The C. jejuni ⌬paqP and ⌬paqQ mutants are defective in L-glutamine uptake and moderately defective in the uptake of other amino acids. To investigate the functionality of PaqP and PaqQ, transport assays were performed using different radioactively labeled amino acids. Based on the strong homology to GlnP and GlnQ described above, together with a previous report suggesting that this AA-ABC system may participate in cysteine transport (54), the WT and the two mutants were grown to mid-log phase and assayed for rates of uptake of 14 14 14 L-[ C]glutamine, L-[ C]glutamate, L-[ C]aspartate, and 14 L-[ C]cysteine. Both mutants exhibited significant defects in glutamine uptake: at 1 min, glutamine transport levels of both the ⌬paqP (⬃1.97 nmol/min/mg [dry weight]) and the ⌬paqQ (⬃1.66 nmol/min/mg [dry weight]) mutant were ⬍50% that of the WT strain (⬃4.12 nmol/min/mg [dry weight]) (Fig. 2A). An intermediate defect was observed for glutamate uptake (Fig. 2B), while more-moderate decreases in cysteine and aspartate uptake were observed (Fig. 2C and D). This suggests that this AA-ABC transporter system has a greater affinity for glutamine transport but also transports other amino acids to a lesser extent. The complemented ⌬paqPc strain partially restored glutamine uptake, as a two-tailed t test did not show a statistically significant difference between the WT and ⌬paqPc strains (Fig. 2E). Enhanced recovery of ⌬paqP and ⌬paqQ mutants from RAW 264.7 macrophages and INT407 epithelial cells in a short-term intracellular survival assay. In a previous microarray analysis, we observed increases in levels of cj0467 to cj0469 mRNAs in C. jejuni during INT407 cell infections (25). Thus, we were interested in exploring whether this system might influence the interaction of C. jejuni with host cells. RAW

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FIG. 2. The ⌬paqP and ⌬paqQ mutants exhibit reduced levels of uptake of glutamine and other amino acids relative to those of the WT. (A through D) WT C. jejuni 81-176 (squares and solid line) and the ⌬paqP (circles and dashed line) and ⌬paqQ (triangles and dotted line) mutants were grown with shaking in MH broth for 15 h to early-log phase (⬃0.3 OD600 unit/ml) and then harvested and assayed for high-affinity transport of L-[14C]glutamine (A), L-[14C]glutamate (B), L-[14C]cysteine (C), or L-[14C]aspartate (D) at a final concentration of 5 ␮M. Samples were taken at the time points shown. (E) The ⌬paqP and ⌬paqQ mutants both showed statistically significant differences in glutamine uptake from the WT, while the ⌬paqP-complemented (⌬paqPc) strain did not. Rates of uptake were determined from three separate biological replicate cultures, with duplicate samples harvested for each strain at each time point. Asterisks indicate statistical significance (P ⬍ 0.05) by a two-tailed t test. Note the differences in the scales of the y axes, reflecting the levels of amino acid uptake observed.

264.7 murine macrophages and INT407 human epithelial cells were infected with WT, ⌬paqP, and ⌬paqQ strains and assayed for adherence, invasion, and intracellular survival using a gentamicin protection assay. No significant differences in cell adherence or invasion were observed between the mutants and the WT strain (Fig. 3). However, the recovery of intracellular ⌬paqP and ⌬paqQ mutants at 9 h postinfection (short-term intracellular survival) was approximately 20-fold higher than that of the WT in RAW 264.7 cells (Fig. 3A) and approximately 10-fold higher than that of the WT in INT407 cells (Fig. 3B). Human intestinal Caco-2 epithelial cells were also tested, and similar results were observed (data not shown). This re-

producible phenotype is statistically significant but transient; no differences in intracellular recovery between WT and mutant strains were observed 24 h postinfection (data not shown). WT and mutant strains were equally susceptible to gentamicin by Etest strip analyses (MIC, 1.5 ␮g/ml), suggesting that the elevated short-term intracellular recovery of the mutants was not due to differences in gentamicin resistance. No significant differences in colonization in vivo were observed between mutant and WT C. jejuni strains using a mouse infection model. To explore a potential in vivo role for this system, we infected BALB/cByJ mice with WT and ⌬paqP mutant strains using a previously established mouse coloniza-

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FIG. 3. The ⌬paqP and ⌬paqQ mutants exhibit statistically significant increases in recovery from macrophages and epithelial cells at a short-term intracellular survival time point. The WT (squares), ⌬paqP (circles), and ⌬paqQ (triangles) strains were grown overnight in MH broth with shaking to log phase. A murine macrophage cell line, RAW 264.7 (A), and a human epithelial cell line, INT407 (B), were infected with bacteria at an MOI of ⬃200. After 3 h, the cells were washed with PBS, and some of the wells were harvested for enumeration of CFU of adherent bacteria (3-h-postinfection time point). The remaining wells were treated with 150 ␮g/ml of gentamicin for an additional 2 h to kill extracellular bacteria and washed, and some of the wells were harvested for enumeration of CFU of invaded bacteria (5-h-postinfection time point). Fresh medium with 10 ␮g/ml gentamicin was added to the remaining wells, and after an additional 4 h, intracellular (IC) bacteria were recovered and plated for enumeration of CFU (9-h-postinfection time point). All experiments included triplicate infections for each strain at each time point. Asterisks indicate statistical significance (P ⬍ 0.05) by a two-tailed t test. This result is representative of three separate repeats.

tion protocol (60, 64). Colonization was monitored for 7 to 35 days. Levels of colonization with the C. jejuni ⌬paqP mutant did not differ significantly from those with the WT at any given time point (Fig. 4). It should be noted that although WT C. jejuni colonized the mouse intestinal tract at a significant and high level up to 28 days, this and other tractable animal models do not consistently trigger inflammation, and thus only colonization, not virulence, can be assayed (13, 31). The ⌬paqP and ⌬paqQ C. jejuni mutants exhibit increased resistance to limited CO2, aerobic, and organic peroxide stresses. C. jejuni must overcome a multitude of environmental stresses in both extracellular and intracellular environments (52). As a capnophilic and microaerophilic organism, C. jejuni requires elevated levels of CO2 for normal growth and is sensitive to atmospheric levels of O2. To examine whether the

FIG. 4. The ⌬paqP mutant is not defective in mouse colonization in vivo. WT C. jejuni (solid circles) and the ⌬paqP mutant (open circles) colonized BALB/cByJ mice at similar levels from day 7 to day 35 postinfection. The dashed horizontal line indicates the level of detection, which was 1 ⫻ 102 CFU/g fecal pellet.

growth of the ⌬paqP or ⌬paqQ mutant is affected under suboptimal gas conditions, mid-log-phase bacteria were serially diluted, spotted onto MH agar, and allowed to grow in a 5% CO2 environment (versus an ideal environment of 12% CO2). Under these conditions, both the ⌬paqP and the ⌬paqQ mutant survived better than the WT strain (Fig. 5A). To evaluate aerobic sensitivity specifically, C. jejuni WT and mutant strains were grown in broth culture under standard C. jejuni conditions (see Materials and Methods), shifted to normal aerobic atmospheric conditions, and assayed for survival after 4 to 6 h by enumeration of CFU. Consistent with the 5% CO2 growth observations, both mutants exhibited sustained survival under aerobic conditions relative to that of the WT strain (Fig. 5B). O2- and CO2-related stress responses are often closely associated with other oxidative stress responses in C. jejuni (2, 6). We thus investigated whether loss of this AA-ABC transporter system in C. jejuni might also influence responses to reactive oxygen species (ROS). WT C. jejuni and the ⌬paqP and ⌬paqQ mutants were treated for 30 min with either hydrogen peroxide (H2O2), an inorganic peroxide generated by macrophages as a defense mechanism to eradicate intramacrophage pathogens (2), or t-BOOH, an organic peroxide also generated by host cells (6). While neither mutant exhibited altered sensitivity to H2O2 (Fig. 5C), the ⌬paqP mutant exhibited higher t-BOOH resistance than the WT at a statistically significant level (Fig. 5D). Although the ⌬paqQ mutant did not exhibit a statistically significant difference from the WT, experimental repeats consistently showed that the ⌬paqQ mutant exhibited higher tBOOH tolerance than the WT. Together, our data indicate that this AA-ABC system impacts the tolerance of low CO2 levels, aerobic stress, and at least one ROS condition in C. jejuni. Tolerance to heat and osmotic stress is differentially altered in the ⌬paqP and ⌬paqQ mutants. To examine if the ⌬paqP and ⌬paqQ mutants exhibit other stress tolerance alterations, the mutants were also subjected to heat and osmotic stress assays. To test heat tolerance, mid-log-phase bacteria were diluted to an OD600 of ⬃0.2 in MH broth, and serial dilutions

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FIG. 5. The ⌬paqP and ⌬paqQ mutants exhibit enhanced growth in a 5% CO2 environment, increased resistance to aerobic stress, and modestly increased resistance to t-BOOH but not hydrogen peroxide (H2O2) stress. (A and B) C. jejuni strains were grown under standard C. jejuni growth conditions overnight, serially diluted, plated onto MH agar, and either incubated in a 5% CO2 incubator overnight (A) or shifted to a shaking culture under atmospheric conditions for 4 h and 6 h (B). (C and D) Log-phase starting cultures of WT (squares), ⌬paqP (circles), and ⌬paqQ (triangles) strains were treated with either H2O2 (C) or t-BOOH (D) at various concentrations and were incubated microaerobically at 37°C for 30 min before being harvested for CFU enumeration. All samples were taken in triplicate at each concentration. The asterisk indicates statistical significance (P ⬍ 0.05) by a two-tailed t test.

were spotted onto MH agar and grown at 45°C, a mild heat stress condition for C. jejuni (10, 43, 61, 66). As a control, cultures were harvested and plated at 37°C (Fig. 6A). While the ⌬paqP mutant exhibited an increase in heat stress resistance over that of the WT, the ⌬paqQ mutant was more sensitive to heat stress than the WT (Fig. 6B). Similar results were obtained when the strains were subjected to growth under osmotic stress using MH agar with 0.17 M NaCl: the ⌬paqP mutant exhibited higher resistance to osmotic stress than the WT, while the ⌬paqQ mutant was more sensitive to NaCl (Fig. 6C). Macrophages infected with the C. jejuni ⌬paqP and ⌬paqQ mutants exhibit a transient reduction in cell death and Erk activation compared to those of macrophages infected with WT bacteria. The observations described above suggested that the enhanced intracellular recovery of the ⌬paqP and ⌬paqQ mutants can be attributed at least partly to increased survival via enhanced or altered bacterial stress tolerance. Since host cell death results in detachment of the cells from the tissue culture surface and a resultant loss of C. jejuni inside those cells from the intracellular survival assay counts, we hypothesized that the observed increase in the intracellular recovery of the mutants might also reflect increased host cell survival. To investigate this, we separately monitored levels of induced host cell death and cell viability using annexin V Fluor and DAPI staining, followed by visualization using fluorescence microscopy. At 9 h postinfection, annexin V staining showed that, in agreement with previous findings in THP-1 human macrophages (78), cell death was induced in WT C. jejuni-infected RAW 264.7 macrophages (Fig. 7A and B). However, we ob-

served a statistically significant reduction in the level of cell death in macrophages infected with the ⌬paqP or ⌬paqQ mutant compared to that in WT-infected macrophages (Fig. 7A and B). DAPI staining, followed by enumeration of cell nuclei on the coverslip, confirmed that a significantly lower number of viable cells remained on the coverslip when RAW 264.7 cells were infected with the WT than when they were infected with the ⌬paqP or ⌬paqQ mutant (Fig. 7C). Finally, to investigate whether PaqP or PaqQ might affect host cell signal transduction pathways, we examined the phosphorylation of several MAPKs known or suspected to be induced upon C. jejuni infection (15, 49, 90). At 9 h postinfection, Erk phosphorylation levels in RAW 264.7 macrophages infected with either the ⌬paqP or the ⌬paqQ mutant were markedly diminished relative to those in WT-infected cells (Fig. 7D). In contrast, Jnk and p38 phosphorylation levels were similar in macrophages infected with WT and mutant strains (Fig. 7D; also data not shown). Levels of phospho-Erk in INT407 or Caco-2 epithelial cells infected with either the ⌬paqP or the ⌬paqQ mutant were also similar to those observed for WT-infected cells (data not shown). Consistent with the cell infection data presented earlier, no differences in host cell death or phospho-Erk levels between WT- and mutantinfected cells were observed at other time points postinfection. DISCUSSION C. jejuni must survive diverse conditions encountered in the external environment, in the intestinal tract, and inside epithe-

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FIG. 6. The ⌬paqP mutant is more resistant, and the ⌬paqQ mutant is more susceptible, to heat and osmotic stresses than WT C. jejuni. Log-phase cultures were serially diluted (10-fold from an initial OD600 of ⬃0.2), plated, and incubated for 2 days at 37°C (A) and 45°C (B) on MH agar to assess the response to heat stress, or at 37°C on MH agar supplemented with 0.17 M NaCl (C) to assess the response to osmotic stress. Duplicate samples were assayed for each condition. Shown are results of a representative experiment with several repeats yielding similar results.

lial and immune system cells. External pressures challenging C. jejuni include heat, osmotic, oxidative, aerobic, and nutrient limitation stresses. Growing evidence also indicates that C. jejuni can survive intracellularly within host enterocytes and macrophages (18, 89, 96), suggesting that C. jejuni must also tolerate intracellular stresses for sustained survival. Recent studies have provided some insight into external C. jejuni stress survival strategies; however, the mechanisms underlying the ability of C. jejuni to circumvent certain stresses, such as aerobic, high osmolarity, and high temperature environments, remain less well elucidated (40, 81). Furthermore, the specific factors promoting intracellular bacterial survival and triggering host cell signal transduction pathways remain largely unknown. In this study, we have investigated the biological and pathogenesis-related roles of a putative C. jejuni AA-ABC transporter originally annotated as cj0467 to cj0469. Both the integral membrane protein (permease) and ATP binding protein (ATPase) components are frequently required to produce a functional AA-ABC transporter (59); nonetheless, to ensure a comprehensive analysis of the system’s functions, we generated independent nonpolar mutations in both the Cj0467 (PaqP) permease and the Cj0469 (PaqQ) ATPase. Bioinformatics analyses predicted this AA-ABC system to be a glutamine

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ABC transporter with high homology to glutamine ABC transporters in Helicobacter, Streptococcus, and Pseudomonas spp.; a previous study also suggested that this system may be a putative cysteine ABC transporter coupled to CjaA, a cysteine substrate binding protein (54). Our amino acid transport assays indicate that the transporter does not exhibit specificity exclusively for a single amino acid, since both the ⌬paqP and ⌬paqQ mutants were defective in the uptake of glutamine, glutamate, cysteine, and aspartate. Nevertheless, glutamine appeared to be a preferred substrate, since both mutants exhibited moresevere decreases in glutamine uptake than in the uptake of the other amino acids. Some residual glutamine transport was observed in the ⌬paqP and ⌬paqQ mutants, and preliminary glutamine uptake competition assays performed on the ⌬paqP and ⌬paqQ mutants indicate that that there is likely a redundant mechanism for glutamine transport in C. jejuni (unpublished observations). This is supported by the previous annotation of cj0940c and cj0902 as glnP and glnQ, which, like paqP and paqQ, are present in all sequenced strains of C. jejuni (34, 62, 63). Our previously published microarray analysis, identifying increased levels of cj0467 to cj0469 mRNA during INT407 cell infection (25), suggested a potential role for this AA-ABC transporter in C. jejuni pathogenesis. Infection assays using INT407 cells and RAW 264.7 macrophages revealed no significant differences in adherence, invasion, or long-term intracellular survival at 24 h between the ⌬paqP and ⌬paqQ mutants and the WT. Mouse colonization levels were likewise similar for C. jejuni WT- and mutant-infected animals, at both earlier and later time points. Surprisingly, however, a statistically significant increase in the recovery of bacteria in the short-term intracellular survival assay was observed for the ⌬paqP and ⌬paqQ mutants in both INT407 and RAW 264.7 infected cells. This is in contrast to previous studies of the C. jejuni Peb1 aspartate/glutamate binding protein mutant (48, 64) and of a group B Streptococcus glutamine ABC transporter ⌬glnQ mutant (82), where significant infection defects were observed both in vitro and in vivo. Our findings also deviate from observations for several other genes identified in the microarray cluster mentioned above as upregulated during cell infection: both ⌬spoT and pVir mutants show diminished levels of invasion (4, 5, 25), and the ⌬spoT mutant is also defective in intracellular survival (25). However, like the ⌬paqP and ⌬paqQ mutants, a ⌬cprS (cj1226c; identified in the same cluster) sensor kinase mutant exhibited a modest increase in intracellular survival (80); whether CprS participates in the regulation of paqP and paqQ remains to be determined. One initial hypothesis to explain the enhanced intracellular survival of the ⌬paqP and ⌬paqQ mutants was that they may be more resilient than the WT to stresses occurring in an intracellular environment. As a defense mechanism, host epithelial cells and macrophages produce oxygen derivatives and ROS to generate a highly oxidative environment (6, 67, 75). Consistent with this hypothesis, the ⌬paqP mutant exhibited a statistically significant increase in resistance to t-BOOH organic peroxide stress, while the ⌬paqQ mutant exhibited a similar and reproducible, albeit more moderate, phenotype. It was recently shown that a gamma glutamyl transpeptidase (GGT)-deficient C. jejuni mutant (⌬ggt) exhibited increased invasion efficiency concomitant with increased H2O2 resistance (7). GGT is in-

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FIG. 7. Cells infected with the ⌬paqP or ⌬paqQ mutant exhibit lower levels of cell death and Erk activation than WT infected cells. (A) C. jejuni-infected RAW 264.7 cells were stained with annexin V fluorescein to visualize dying cells at 9 h postinfection in a gentamicin protection assay as described in Materials and Methods. Frames shown are one representative view of several different frames. UI, uninfected; DIC, differential interference contrast. Bar, 5 ␮M. (B) The level of annexin V-stained cells as a percentage of the total number of cells at ⫻10 magnification was determined by counting several random frames with ⬎100 cells/frame (reflected in error bars). Asterisks denote statistically significant differences between WT and mutant strains (P ⬍ 0.05). (C) C. jejuni-infected RAW 264.7 cells were stained with DAPI, and cell viability was assessed by counting cell nuclei per random field. Results for cells infected with the WT or the ⌬paqP or ⌬paqQ mutant are shown. Error bars reflect numbers of DAPI counts from multiple random fields. (D) At 9 h postinfection, phospho-Erk was induced in RAW 264.7 macrophages infected with the WT but not in those infected with the ⌬paqP or ⌬paqQ mutant. Erk 1/2 phosphorylation (42 and 44 kDa) was detected by Western blotting using an anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibody. The membrane was stripped and reprobed with an anti-Erk antibody to assess total Erk levels as a control. Jnk phosphorylation (46 kDa and 54 kDa) was detected by Western blotting using an anti-phospho-Sapk/Jnk (Thr183/Tyr185) antibody. The membrane was stripped and reprobed with an anti-Jnk1 antibody to assess total Jnk levels as a control.

volved in glutathione and glutamine catabolism in both Helicobacter pylori and C. jejuni (33, 77), and glutathione has previously been implicated in resistance to oxidant-mediated killing in eukaryotes (76, 84). Although a connection between our observations and GGT is plausible, several lines of evidence suggest that our AA-ABC transporter system affects stress responses independently of GGT: our mutants were not hyperresistant to H2O2 (Fig. 6); a ⌬ggt mutant was previously found to be defective in mouse colonization (34); and there was no apparent alteration of GGT activity in our mutant strains (unpublished observations). In C. jejuni, organic peroxides, such as t-BOOH, are degraded by an alkyl hydroperoxide reductase, AhpC (42). It remains to be determined whether

PaqP and PaqQ are involved in this organic peroxide degradation pathway or whether they participate in t-BOOH stress tolerance by a novel mechanism. Another unexpected finding was the enhanced ability of the ⌬paqP and ⌬paqQ mutants to grow in a suboptimal CO2 environment and to survive better than the WT during aerobic stress. The ability to move between aerobic, microaerobic/ capnophilic, and anaerobic environments is an important pathogenesis and transmission property of C. jejuni for which the molecular mechanisms are poorly understood. C. jejuni relies on amino acids as primary carbon sources, with complete catabolism predicted only for aspartate, asparagine, glutamate, glutamine, serine, and proline. Amino acids such as asparagine

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and glutamine are likewise predicted to provide key sources of nitrogen via deamination reactions to aspartate and glutamate. During respiration, C. jejuni can utilize both O2 and a number of non-O2 terminal electron acceptors (i.e., nitrate and nitrite); the latter are critical for growth under low-O2 conditions (40). One interpretation of our data is that diminished glutamine uptake, such as that occurring in the ⌬paqP and ⌬paqQ mutants, may lead to lower levels of available nitrogen, thereby resulting in a shift to aerobic respiration. No anaerobic survival defects for ⌬paqP or ⌬paqQ mutants have been observed (unpublished observations); however, it is interesting that paqP and paqQ occur directly downstream of, and are likely cotranscribed with, nssR, encoding a nitrosative stress response regulator (19). Despite the dissimilar phenotypes of ⌬paqP and ⌬paqQ mutants versus the ⌬nssR mutant, it will be interesting in the future to explore the relevance of nitrogen assimilation and PaqP/Q to anaerobic/aerobic survival, as well as potential connections between PaqP/Q and NssR. Previous studies of C. jejuni suggested that cross talk exists between aerobic and heat or osmotic stress responses (3, 10, 47, 66, 70, 79). We thus hypothesized that ⌬paqP and ⌬paqQ mutants might also exhibit increased resistance to heat and salt treatments. As predicted, the ⌬paqP permease mutant tolerated heat and osmotic stresses better than the WT; however, the ⌬paqQ ATPase mutant was more sensitive than the WT to both heat and osmotic stresses. Thus, PaqP and PaqQ appear to exhibit differential functions in thermoregulation and osmoregulation. No similar observations have been reported previously; thus, no literature is currently available to help account for this phenomenon. However, several hypotheses may explain why loss of a permease or ATPase could have different consequences for specific aspects of bacterial physiology. First, as soluble proteins, ATPases harbor a level of promiscuity (30, 71, 92, 93). Thus, an ATPase may couple with other ABC transporter systems in the absence of a functional permease, while a system that has lost ATPase function but retains functional permease proteins could be partially rescued by another ATP binding protein. This is consistent with the high degree of homology (52.9% identity) observed between PaqQ and Cj0902/GlnQ. Alternatively, the absence of an ATPase may result in reduced ATP consumption, causing a general disruption in energy balance. Another interpretation is that the PaqP permease and PaqQ ATPase may exert distinct effects on the gating of small solutes or metabolites important for balancing the membrane electropotential gradient, which has been associated with osmoregulation and shock resistance in E. coli (8). Extensive future studies will be required to distinguish between these possibilities. In addition to characterizing physiological differences between the C. jejuni mutants, we also investigated if this AAABC transporter system might affect host cell viability. As noted, this would also be expected to affect the CFU counts in the intracellular survival assay, since dead cells (and bacteria inside them) lift from the wells and would be washed away prior to CFU enumeration. Interestingly, mutant-infected macrophages, but not mutant-infected INT407 cells, exhibited approximately two- to fourfold-higher viability and lower levels of annexin V staining than WT-infected cells in a short-term intracellular survival assay. Thus, the additive effect of increased stress resistance and diminished host cell death may

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account for the ⬃2-fold-higher levels of recovered mutant versus WT bacteria in macrophages compared to INT407 cells, while in INT407 cells, increased stress resistance of the mutants may be the primary contributing factor. The transient but consistent macrophage death finding adds to previous reports that have also drawn tangible connections between host cell cytotoxicity and specific C. jejuni virulence factors, although the literature differs considerably depending on the C. jejuni strain and host cell type used. For instance, one study reported that apoptosis of 28SC monocytes induced by infection with C. jejuni strain 81-176 was dependent on cytolethal distending toxin (CDT) (32), a DNase-like molecule that causes host cell cycle arrest (45, 91). However, another study reported that apoptosis of THP-1 monocytes by C. jejuni strain F38011 was independent of CDT and lipooligosaccharide (in contrast to LPS observations for other pathogens [27, 58]) and instead involved secreted C. jejuni Cia proteins (44, 78). A third study reported that T84 enterocytes underwent oncosis in response to C. jejuni infection, a response that was dependent on the C. jejuni strain used and on the FlaAB flagellins but was independent of CDT (38). We have not observed any obvious differences in secreted protein profiles between our WT strain and our ⌬paqP and ⌬paqQ mutant strains (unpublished observations); nonetheless, it will be interesting to explore, in future work, potential connections between our findings and those of the studies described above. To explore mechanisms underlying the effect of this AAABC transporter system on macrophage death, we also investigated whether several host cell MAPK proteins essential for eukaryotic growth and survival (73, 74) exhibited altered phosphorylation profiles in WT- compared to mutant-infected cells. C. jejuni is known to activate these signaling pathways, which in turn lead to significant downstream effects, including cytokine production and host cell damage (15, 49, 90). Interestingly, deletion of paqP or paqQ in C. jejuni resulted in a transient abrogation of Erk activation in RAW 264.7 macrophage infections (Fig. 7D). This appeared to be specific both for Erk (versus Jnk and p38) and for macrophages (versus epithelial cells). Our observation that this system participates in Erk activation during macrophage infection was unexpected, and it is interesting to hypothesize that this may at least partially contribute to the observed PaqP- and PaqQ-induced macrophage death that we observed. Erk has been shown to impact apoptosis, although its precise role is controversial: some studies report that phospho-Erk is important for cell proliferation, survival, and anti-apoptosis (36, 46, 50), while other studies implicate Erk activation as proapoptotic in a number of different pathways in various cell types (22, 88, 94, 97). Activation of Erk and proinflammatory cytokines is often stimulated by virulence determinants such as LPS via toll-like receptor (TLR) signaling (26, 85). Future work will be required to determine if this is the case during C. jejuni infection; to date, however, no connections have been made between C. jejuni and host cell TLR4 activation, and as noted above, at least one study suggested that C. jejuni-induced macrophage apoptosis may be independent of lipooligosaccharide (78). Interestingly, glutamine supplementation has been shown to reduce apoptosis of human intestinal epithelial cells in a manner that may interface with the Erk activation pathway (20, 21, 46), and

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glutamine depletion was found to elicit apoptosis in epithelial cells and immune cells such as T cells and neutrophils (14). One working hypothesis to explain the increased host cell death observed for WT-infected versus ⌬paqP or ⌬paqQ mutant-infected cells is that intracellular C. jejuni may hijack glutamine from host cells and that this AA-ABC transporter system participates in this process. Preliminary experiments exploring glutamine supplementation during cell infections have not yet identified an effect on C. jejuni-induced macrophage apoptosis (unpublished observations). However, this hypothesis is consistent with our previous observations that C. jejuni requires the stringent response (spoT) and polyphosphate kinase 1 (ppk1) for survival both in low-nutrient environments and in an intracellular environment (11, 25), as well as with recent work suggesting that C. jejuni resides intracellularly in an anaerobic, and likely nutrient-poor, vacuole (89). In summary, we have provided evidence that the AA-ABC transporter system components PaqP and PaqQ participate in several key aspects of C. jejuni physiology and pathogenesis, some of which are novel not only for C. jejuni but for other bacteria as well. This work also reflects an emerging theme connecting basic metabolic processes with stress survival and virulence-associated properties in bacterial pathogenesis research. ACKNOWLEDGMENTS We thank Michael Gold for kindly providing the anti-Erk, anti-Jnk, and anti-p38 antibodies. We also thank members of the Gaynor lab, particularly Sarah Svensson and Emilisa Frirdich, for helpful discussions. This study was supported by Canadian Institutes of Health Research (CIHR) operating grant MOP-68981 and a Burroughs Wellcome Fund Career Development Award in the Biomedical Sciences to E.C.G. E.C.G. is supported by a Canada Research Chair award and a Michael Smith Foundation for Health Research (MSFHR) Scholar Award. A.E.L. is a recipient of a CIHR-Canada Graduate Student Master’s award and an MSFHR Junior Graduate Scholarship. S.A.T. and R.I.H. are supported by U.S. National Institutes of Health grants AI055715, AI058284, and AI061026. REFERENCES 1. Allos, B. M. 2001. Campylobacter jejuni infections: update on emerging issues and trends. Clin. Infect. Dis. 32:1201–1206. 2. Alter, T., and K. Scherer. 2006. Stress response of Campylobacter spp. and its role in food processing. J. Vet. Med. B 53:351–357. 3. Andersen, M. T., L. Brondsted, B. M. Pearson, F. Mulholland, M. Parker, C. Pin, J. M. Wells, and H. Ingmer. 2005. Diverse roles for HspR in Campylobacter jejuni revealed by the proteome, transcriptome and phenotypic characterization of an hspR mutant. Microbiology 151:905–915. 4. Bacon, D. J., R. A. Alm, D. H. Burr, L. Hu, D. J. Kopecko, C. P. Ewing, T. J. Trust, and P. Guerry. 2000. Involvement of a plasmid in virulence of Campylobacter jejuni 81-176. Infect. Immun. 68:4384–4390. 5. Bacon, D. J., R. A. Alm, L. Hu, T. E. Hickey, C. P. Ewing, R. A. Batchelor, T. J. Trust, and P. Guerry. 2002. DNA sequence and mutational analyses of the pVir plasmid of Campylobacter jejuni 81-176. Infect. Immun. 70:6242– 6250. 6. Baillon, M. L., A. H. van Vliet, J. M. Ketley, C. Constantinidou, and C. W. Penn. 1999. An iron-regulated alkyl hydroperoxide reductase (AhpC) confers aerotolerance and oxidative stress resistance to the microaerophilic pathogen Campylobacter jejuni. J. Bacteriol. 181:4798–4804. 7. Barnes, I. H., M. C. Bagnall, D. D. Browning, S. A. Thompson, G. Manning, and D. G. Newell. 2007. Gamma-glutamyl transpeptidase has a role in the persistent colonization of the avian gut by Campylobacter jejuni. Microb. Pathog. 43:198–207. 8. Berger, E. A., and L. A. Heppel. 1974. Different mechanisms of energy coupling for the shock-sensitive and shock-resistant amino acid permeases of Escherichia coli. J. Biol. Chem. 249:7747–7755. 9. Borrmann, E., A. Berndt, I. Hanel, and H. Kohler. 2007. Campylobacterinduced interleukin-8 responses in human intestinal epithelial cells and primary intestinal chick cells. Vet. Microbiol. 124:115–124.

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