The impact of bacterial morphology on virulence and transmission attributes of pathogens is poorly understood. The prevalent enteric pathogen Campylobacter jejuni displays a helical shape postulated as important for colonization and host interactions. However, this had not previously been demonstrated experimentally. C. jejuni is thus a good organism for exploring the role of factors modulating helical morphology on pathogenesis. We identified an uncharacterized gene, designated pgp1 (peptidoglycan peptidase 1), in a calcofluor white-based screen to explore cell envelope properties important for C. jejuni virulence and stress survival. Bioinformatics showed that Pgp1 is conserved primarily in curved and helical bacteria. Deletion of pgp1 resulted in a striking, rod-shaped morphology, making pgp1 the first C. jejuni gene shown to be involved in maintenance of C. jejuni cell shape. Pgp1 contributes to key pathogenic and cell envelope phenotypes. In comparison to wild type, the rod-shaped pgp1 mutant was deficient in chick colonization by over three orders of magnitude and elicited enhanced secretion of the chemokine IL-8 in epithelial cell infections. Both the pgp1 mutant and a pgp1 overexpressing strain – which similarly produced straight or kinked cells – exhibited biofilm and motility defects. Detailed peptidoglycan analyses via HPLC and mass spectrometry, as well as Pgp1 enzyme assays, confirmed Pgp1 as a novel peptidoglycan DL-carboxypeptidase cleaving monomeric tripeptides to dipeptides. Peptidoglycan from the pgp1 mutant activated the host cell receptor Nod1 to a greater extent than did that of wild type. This work provides the first link between a C. jejuni gene and morphology, peptidoglycan biosynthesis, and key host- and transmission-related characteristics.
Campylobacter jejuni is a helical, highly motile, Gram-negative ε-Proteobacterium and a prevalent zoonotic organism existing asymptomatically in the intestinal tract of birds and other animal species [1]–[3]. However, ingestion of as few as 500 bacteria can result in human disease [4]. C. jejuni is the leading cause of foodborne gastroenteritis in the developed world, causing an acute self-limiting infection of varying severity that can give rise to severe complications such as inflammatory bowel disease, reactive arthritis, and Guillain-Barré syndrome (GBS) [5]. C. jejuni lacks many of the frequently identified virulence factors encoded by other enteric pathogens such as pili, enterotoxins, and specialized secretion mechanisms [6], [7]. Genes affecting fundamental aspects of C. jejuni biology in hosts other than humans, such as stress survival, transmission, and asymptomatic colonization, also affect virulence in disease models. We found that C. jejuni strain 81-176 binds calcofluor white (CFW), a compound that reacts with β1–3 and β1–4 carbohydrate linkages and fluoresces under long wave UV light [8], [9]. The carbohydrate responsible for CFW reactivity in C. jejuni has not yet been identified, although it was previously shown not to be one of the well-characterized surface polysaccharides expressed by C. jejuni: the capsular polysaccharide, lipooligosaccharide (LOS), N-linked glycoproteins, or O-linked flagellar glycoproteins [10]. C. jejuni mutants with altered CFW reactivity can be readily identified in screens. All C. jejuni CFW hyper- or hyporeactive mutants characterized to date exhibit changes in pathogenesis, virulence, fundamental, and/or stress survival phenotypes ([10], [11]; E. Frirdich and E. C. Gaynor, unpublished). For instance, CFW hyper-reactive mutants overproduce biofilms, while hypo-reactive mutants are biofilm-defective. Other attributes associated with altered CFW reactivity have included defects in colonization, host cell interactions, cell envelope components, and stress survival. A CFW hypofluorescent mutant with a lesion in a novel peptidoglycan peptidase gene (pgp1) serves as the basis of this study. The helical shape of C. jejuni has long been postulated to be critical for pathogenic attributes such as the ability to burrow through the mucosal layer during infection of zoonotic and human hosts. However, genetic components involved in modulating C. jejuni morphology had not previously been identified. Morphology is maintained in most bacteria by the peptidoglycan (PG) layer [12]. PG is composed of glycan chains consisting of β1–4 linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues that are cross-linked by short peptides. PG synthesis is a highly regulated process [13], [14] taking place at the bacterial inner membrane, where lipid II precursors are polymerized by glycosyltransferase and DD-transpeptidase reactions. In addition to the synthetic enzymes, PG hydrolases are required to cleave bonds in PG or PG fragments for insertion of nascent PG into the mature layer, regulation of cell wall growth, cell separation, PG turnover and recycling, cell lysis, and the release of PG fragments in host-pathogen interactions [15]. Almost every glycosidic and amide bond in PG can be cleaved by one or more specific PG hydrolases, and this redundancy makes it difficult to assign a specific function to a hydrolase [15], [16]. It has been speculated that murein synthases and hydrolases are part of a multienzyme complex for PG assembly [16], and several interactions between PG enzymes have been reported [14]. Campylobacter PG contains meso-diaminopimelic acid (meso-Dap) in its peptide side chains and is modified by O-acetylation [17], [18], but the detailed muropeptide composition had not previously been elucidated. Bioinformatic analyses identified 3 putative PG synthases in C. jejuni: the penicillin-binding proteins (PBPs) PBP1A (cjj81176_0536), PBP (cjj81176_0550), and PBP2 (cjj81176_0680), but there are no predicted homologs of low-molecular weight PBPs such as DD-endo- or carboxypeptidases. C. jejuni does have homologs of the Csd1 and CcmA endopeptidases and of the Csd3/HdpA endo/carboxypeptidase discovered recently in Helicobacter pylori [19], [20]. Deletion of each of these genes in H. pylori resulted in curved-rod morphologies with reduced levels of PG cross-linking [19], but preliminary evidence indicates that these enzymes may have a different function in C. jejuni (E. Frirdich and E. C. Gaynor, unpublished). Here, we report the first C. jejuni gene, pgp1, to be identified with a role in helical morphology. Structural PG analyses of C. jejuni wild-type 81-176 and Δpgp1 mutant strains showed striking alterations in the mutant, with enzyme assays confirming Pgp1 as a novel DL-carboxypeptidase. Furthermore, using the Δpgp1 mutant with a straight morphology, the importance of C. jejuni helical shape and PG in pathogenesis attributes was examined; we identified roles in motility, biofilm formation, chick colonization, and stimulation of host cell proinflammatory mediators Nod1 and IL-8.
As noted, screening for C. jejuni hypofluorescent mutants was previously shown to identify genes involved in pathogenesis-related phenotypes [10]. However, as the Tn7 transposon (Tn) used previously for mutant library construction was reproducibly found to have inserted in intergenic regions (M. K. McLennan and E. C. Gaynor, unpublished), a new random Tn library was generated as part of this study, using the highly efficient mariner system of in vitro Tn mutagenesis developed for C. jejuni [21]. The mariner library was plated onto CFW-containing plates and, from approximately 10,000 colonies screened, 400 hypofluorescent mutants were isolated. Of the Tn insertions mapped, 8 were in distinct regions of the gene cjj81176_1344 (Figure 1; the CFW hypofluorescent phenotype is described below). The 1344 gene was named pgp1 (peptidoglycan peptidase 1) to describe its function and identification as the first C. jejuni PG peptidase to be characterized. The pgp1 gene product is highly conserved in mainly helical and vibrioid bacteria, primarily within the ε- and δ-Proteobacteria but also in a few extremophiles outside the ε-Proteobacteria (Table S1). The H. pylori homolog is described by Sycuro et al. [22]. Pgp1 was annotated as a putative periplasmic protein. However, bioinformatics using conserved domain searches and the threading program PHYRE identified an N-terminal signal peptide, a zinc binding site, and metallocarboxypeptidase catalytic residues and folds. Pgp1 contains a conserved domain at its N-terminus similar to that of the M14 family of metallocarboxypeptidases. To explore functional consequences of loss of Pgp1 function in C. jejuni, a non-polar pgp1 targeted deletion strain was constructed (designated Δpgp1 above the gene cluster in Figure 1). To verify lack of polar effects on the downstream gene, an insertional mutation was also created in 1343. The Δ1343 mutant exhibited wild-type phenotypes for shape, motility, biofilm formation and CFW reactivity (data not shown; these phenotypes for Δpgp1 are described below). The level of 1343 mRNA was also determined to be identical in Δpgp1 and in the wild-type strains by semi-quantitative RT-PCR (data not shown) The pgp1 gene is located in the middle of a putative operon, and the location of the pgp1 promoter is unclear from sequence analysis (Figure 1). Therefore, complementation was first attempted by expressing the pgp1 gene from the cat promoter of the vector pRRC (CmR) [23] integrated into an rRNA spacer region of Δpgp1. This construct contained a similar region of pgp1 as shown for pEF20 in Figure 1 (pEF20 will be described further below) and did not complement Δpgp1 (data not shown). However, integration of this same construct into the C. jejuni wild type strain also resulted in morphological and phenotypic alterations (described below), suggesting that pgp1 copy number was likely important for complementation, and that the level of pgp1 expression from the cat promoter was higher than optimal. To generate a complementing strain expressing pgp1 from its native promoter, increasing amounts of the region upstream of pgp1 were cloned with pgp1 into pRRC. These regions were cloned in the opposite direction as cat (to avoid overexpression from the cat promoter) and integrated into an rRNA spacer region of Δpgp1. These constructs yielded varying degrees of complementation, with the pEF35R (Figure 1) construct complementing nearly all Δpgp1 phenotypes to wild-type levels. The complemented strain was verified by PCR and designated Δpgp1c. Since the pgp1 gene product is highly conserved in helical and vibrioid bacteria, the morphology of the Δpgp1 mutant was examined. The Δpgp1 mutant displayed a striking change compared to wild type and had lost the characteristic helical shape (Figure 2A), adopting a straight morphology (Figure 2B). Complementation restored the helical shape in approximately 95% of the cells (Figure 2C). Consistent with the phenotype of the mariner insertion mutants, the Δpgp1 mutant was hyporeactive to CFW, with wild-type reactivity restored by complementation (Figure 2D). The Δpgp1 mutant displayed no obvious flagellar structural defects (Figure 2B), but did exhibit a slight motility defect after incubation for 20 h on 0. 4% agar plates, producing halos that were, on average, about 82. 5% of wild type (Figure 2E). Motility was restored to wild-type levels in the complemented strain. Biofilm levels, as assessed by a crystal violet assay, were approximately 1. 2-, 1. 5- and 1. 6-fold lower in the Δpgp1 mutant compared to wild type at days 1,2, and 3, respectively. Biofilm production was partially restored in Δpgp1c (Figure 2F). No differences between wild type and Δpgp1 were observed for growth, stress survival, capsule and LOS migration on acrylamide gels, membrane protein composition, and sensitivity to antimicrobial compounds (Table S2). To assess the effects of pgp1 overexpression, pgp1 was expressed from either the cat or aphA-3 promoter in pRRC (data not shown) or pRRK (pEF20 in Figure 1), respectively, and integrated at an rRNA spacer region of wild-type C. jejuni. Both pRRC and pRRK derivatives had the same effects on wild type, so only the results for pEF20 are shown. The pgp1 overexpressing strain displayed an altered cell shape in approximately 50% of the population, producing kinked and straight cells among the helical cells (Figure 3A). This strain also exhibited reduced motility and a defect in biofilm formation (Figure 3B & C). Reverse transcriptase-quantitative PCR (RT-qPCR) confirmed that the levels of pgp1 mRNA were 5. 1-fold higher in the overexpressing strain than in wild type. In addition, there was a 1. 2-fold increase in pgp1 mRNA levels in the complementing strain Δpgp1c in comparison to wild type, which may explain the partial complementation of some phenotypes. Expression of pgp1 in E. coli had no effect on cell shape (data not shown).
Bacterial cell shape programs and their impact on biology are becoming increasingly well-characterized for model organisms like E. coli and Caulobacter crescentus [30], [31]. However, studies linking bacterial shape and pathogenesis are in their infancy. This is the first report to address the morphogenesis program of the prevalent foodborne pathogen C. jejuni. Furthermore, apart from three annotated uncharacterized PBPs, no other C. jejuni PG-modifying enzymes have been identified. This is in spite of the fact that, as our study demonstrates, C. jejuni produces numerous muropeptide species indicative of additional unidentified and potentially novel PG hydrolases. Our identification and characterization of pgp1 provides the first link between cell shape and PG in C. jejuni. Pgp1 is conserved primarily in bacteria with curvature (Table S1). Although functions have not yet been ascribed for other putative shape-related proteins in C. jejuni, it is of note that helical ε- and δ-proteobacteria also typically contain the elongase and cytoskeletal components MreB, MreC, and RodA, but not MreD or RodZ. As such, unique PG and morphogenesis programs are likely to be identified both within and among these organisms, which in turn will lend new insight into their biology and pathogenesis. Enzyme assays demonstrated that Pgp1 is a metal-dependent DL-carboxypeptidase cleaving monomeric disaccharide tripeptides to disaccharide dipeptides. This is consistent with Pgp1 harboring an N-terminal M14 metallocarboxypeptidase domain present in two other PG dipeptidyl peptidases, E. coli MpaA and Bacillus sphaericus ENP1, which cleave tri- and tetrapeptides to dipeptides [32]–[35]. This also correlates with the reduction in dipeptides and increase in tripeptides in Δpgp1 PG. Decreased tetrapeptides in Δpgp1 may be an indirect consequence of the increased level of tripeptides affecting activity of DD-carboxypeptidases, or an indirect effect on other PG hydrolases via disruption of a putative enzyme complex. Efforts to identify Pgp1 binding partners that would support the latter hypothesis are underway. Our muropeptide analysis of C. jejuni 81-176 wild-type PG also revealed a muropeptide composition expected for a Gram-negative species, with several interesting differences from the related helical organism H. pylori [19], [36] and the rod-shaped E. coli [14], [24] (Table S5). For instance, unlike H. pylori, C. jejuni contains very low levels of pentapeptides, suggesting high DD-carboxypeptidase activity. C. jejuni PG also has a higher level of cross-linking than either other species, and a relatively short average glycan chain length. With pgp1 being the first C. jejuni gene directly linked with helical morphology, it allowed for the first direct comparison between a helical wild type strain and an isogenic targeted rod-shaped mutant in critical aspects of C. jejuni pathogenesis and survival. The modest motility defect of the rod-shaped Δpgp1 mutant may in part account for the modest biofilm defect [37]–[39]. Further corroborating the pgp1-biofilm connection are our observations that pgp1 is up-regulated in the hyperbiofilm-forming ΔcprS strain (S. Svensson and E. C. Gaynor, unpublished) and that Δpgp1 is hyporeactive to CFW [10]; the latter observation in turn continues to support a link between C. jejuni CFW reactivity and key biological and pathogenic processes. Isolated PG, which has a β1–4 disaccharide backbone, binds to CFW (E. Frirdich and E. C. Gaynor, unpublished), suggesting that changes in PG architecture in Δpgp1 may account for decreased CFW reactivity. However, as PG isolated from both wild type and mutant strains bound CFW equally well, a related explanation is that PG alterations affect the accessibility of CFW to binding sites on periplasmic PG molecules. The Δpgp1 mutant also allowed us to explore roles for shape and PG composition on host interactions. Surprisingly, the Δpgp1 mutant was not defective for host cell attachment or invasion in vitro and even displayed a slight increase in short-term intracellular survival. Increasing the viscosity of the media did not affect the ability of the mutant to invade epithelial cells (Figure S2). It is of note that although attachment and invasion to host cells increased for some C. jejuni strains in higher viscosity medium [28], this was not the case for our highly invasive wild-type strain 81-176, suggesting varying invasion programs among different C. jejuni strains. Intracellular C. jejuni was previously shown to induce host epithelial cell inflammatory responses via activation of the cytoplasmic Nod receptors [40], [41], which recognize distinct PG muropeptide molecules. The minimal molecule recognized by hNod2 is muramyl dipeptide (MDP), a structure common to PG from both Gram-negative and Gram–positive organisms [42]. hNod1 and mNod1 recognize DAP-containing muropeptides restricted to Gram-negative organisms. hNod1 exhibits a preference for tripeptides and mNod1 for tetrapeptides; however mNod1 also, to a lesser degree, recognizes DAP-containing tripeptides ([43]; J. Lee and S. Girardin, unpublished). Luciferase assays are a sensitive method to probe the capacity of a PG preparation to trigger Nod1 or Nod2 and provide complementary information to PG compositional data by HPLC and MS analyses. Using these assays, only mNod1 exhibited a statistically significant difference in activation by Δpgp1 PG compared to wild type. It is possible that C. jejuni modifies its PG in a manner that affects Nod signaling, as modifications of the PG backbone have been shown to affect how PG fragments are sensed by host cell receptors [44]. This poses an interesting question for future work. Our data also indicate that wild-type PG can activate hNod2, consistent with findings by Al-Sayeqh et al. (2010) who also showed NF-κB activation in HEK293T cells transfected with Nod2 and infected with live C. jejuni. While Zilbauer et al. (2007) did not observe C. jejuni activation of Nod2, this may reflect differences between cell lines used in these assays, and/or differences in reporter sensitivity. Nod activation is an important aspect of innate immunity against C. jejuni [40], [41]; however, the mechanism by which C. jejuni PG fragments may reach the cytosol to activate Nod1 and/or Nod2 has not been elucidated. C. jejuni can be internalized into intestinal epithelial cells, surviving within an intracellular membrane-bound compartment known as the C. jejuni-containing vacuole (CCV) [45]. An oligopeptide transporter expressed in the early endosome has been implicated in the transport of Nod ligands from the endosome to the cytoplasm in HEK293T cells [46]; a similar system could allow cytoplasmic delivery of PG fragments from C. jejuni in the CCV. Another possibility is that during growth in the extracellular environment C. jejuni may shed part of its PG, as shown for other bacteria [44], [47], [48]; released Nod1 and Nod2-stimulatory molecules could then be transported to the cytoplasm by oligopeptide transporters or taken up by phagocytosis or clathrin-dependent endocytosis and transported to the cytosol to activate Nods [44], [49]–[51]. Bacterial outer membrane vesicles may also play a role in delivering C. jejuni Nod ligands to the cytoplasm [52]. Production of IL-8 and other proinflammatory mediators by intestinal epithelial cells infected with C. jejuni is thought to be key to the development of diarrhea and clearance of infection. Zilbauer et al. (2007) suggested that C. jejuni Nod1 activation is the primary signaling event required for IL-8 expression in Caco-2 intestinal epithelial cells. However, other work indicates that C. jejuni-induced IL-8 secretion can be triggered by other pathways in addition to the activation of Nod1, such as through Toll-like receptors (TLRs) and a pathway independent of Nods and TLRs that has yet to be identified [40], [53]–[58]. Thus while the increase in IL-8 secretion in response to Δpgp1 may be due to the modest increase in hNod1 activation observed in our luciferase assays, it cannot be ruled out that deletion of pgp1 leads to a change in another, as-yet unidentified factor stimulating IL-8 expression. Future work is planned to address these hypotheses. Our colonization data are in agreement with the longstanding hypothesis that the corkscrew morphology of C. jejuni is critical for burrowing into the mucus layer; however, the impact of shape and PG on colonization may be multifactorial. For instance, motility is a key factor in colonization [59]–[64], thus the Δpgp1 colonization defect may in part be due to its decreased motility. However, a ΔcarB mutant with a similar motility defect was not deficient for chick colonization (Figure S1). When the strongly colonizing C. jejuni strain 305/94 was re-isolated from chickens, it displayed a more pronounced motility defect than Δpgp1 despite having normal flagella, and had lost its helical shape, exhibiting a similar straight morphology as Δpgp1 [65]. It is intriguing to hypothesize that not only shape, but also the underlying structure of the PG may play an important role in colonization. We have found that a straight phenotype can arise from various changes in muropeptides based on preliminary analysis of other straight mutants identified in our laboratory (E. Frirdich, J. Vermeulen, and E. C. Gaynor, unpublished). PG changes or changes resulting from the loss of pgp1 could cause as-yet uncharacterized alterations to the cell surface affecting the ability of C. jejuni to survive in the chicken cecum, or affect host cell interactions and stimulation of innate immune receptors. Our understanding of the chicken innate immune system and how it responds to C. jejuni is in its infancy. The chicken genome encodes Nod1 but not Nod2, although chickens possess an ortholog of the NLRP3/NALP3 Nod-like receptor that is similar to Nod2 and also binds MDP [66], [67]. NLRP3 may substitute for Nod2 in chickens. Further insight into mechanisms by which C. jejuni survives commensally in the chicken cecum requires additional studies of these and other innate immune system factors. Identification and characterization of pgp1 provides a critical first step in understanding how shape and PG modifications impact C. jejuni pathogenesis. However, the shape program itself in C. jejuni is likely to be somewhat complicated. For instance, to the best of our knowledge, Pgp1 is the only PG modifying enzyme to date where overexpression causes cell straightening, indicating that the precise dose of Pgp1 is important for maintenance of morphology. One interpretation of this finding is that the proper ratio of monomeric tripeptides to dipeptides, which is disrupted in both the Δpgp1 mutant and pgp1-overexpressing strains, may be required for proper shape determination. It is also possible that excess Pgp1 could, as suggested for loss of Pgp1 above, indirectly affect other PG hydrolases by disrupting the stoichiometry of a putative PG biosynthetic/modification complex. Additional complexities in the shape program are evidenced by reports of straight Campylobacter strains in the literature for which the morphological change has not yet been attributed to mutations in specific gene product (s). For instance, some C. jejuni flagellar mutants appeared to have a straight morphology [68]–[70]; however, other mutants with lesions in the same flagellar genes remained helical ([71], [72]; E. Frirdich and E. C. Gaynor, unpublished). Two reports also cited that passage through the chicken gut or chick embryos resulted in straight C. jejuni isolates [65], [73]. Conversely, laboratory passage has led to the isolation of poorly colonizing rod-shaped C. jejuni and C. coli strains [74], [75]. In the 11168-GS (straight) and 11168-O (helical) strains of the 2004 study, sequence and expression levels of pgp1 were found to be identical (E. Frirdich and E. C. Gaynor, unpublished). Collectively, this suggests not only that genes other than pgp1 affecting morphology remain to be discovered, but also that mechanisms such as phase variation and/or epigenetics may be involved. The pgp1 homolog in H. pylori (named csd4) was identified in a screen for cell shape mutants and is described by Sycuro et al. [22]. Morphology, PG profile, and enzymatic activity indicate conserved functions for the gene products in both organisms. While some enzymes and their effects on morphology are conserved between these related helical bacteria, there are also likely to be differences in the overall PG remodeling and shape determining programs, particularly given their very different wild-type muropeptide profiles. The Δpgp1 mutant will be a valuable tool to continue to study the effects of the loss of C. jejuni helical shape on its biology and pathogenesis. So far, pgp1 has been found to be important for all aspects of the C. jejuni life cycle. As this study represents the first identification of a gene involved in C. jejuni helical shape and of a role for PG in shape determination, it will also provide the basis for work characterizing additional enzymes involved in C. jejuni PG biosynthesis and shape determination. These studies have now been made easier by the availability of PG structural data for the wild-type C. jejuni strain 81-176 published as part of this work. Future detailed biochemical and structural studies on Pgp1 will also provide interesting insight into the function of this key protein in C. jejuni physiology.
Animal experiments were carried out in strict accordance with the University of Michigan Committee on Use and Care of Animals (UCUCA). Animal infection and euthanasia protocols were approved by the University of Michigan UCUCA and assigned approval number 10462. Oral gavage was carried out under humane guidelines using an approved protocol judged not to cause distress or harm to the animals. Euthanasia was carried out under humane guidelines using a lethal dose of isofluorane. All animal use procedures are in compliance with University guidelines, State and Federal regulations, and the standards of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The University of Michigan Animal Welfare Assurance Number on file with the NIH Office of Laboratory Animal Welfare (OLAW) is A3114-01, and the University is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC, Intl.). Bacterial strains and plasmids used in this study and their construction are described in Text S1. C. jejuni strains were grown at 38°C in Mueller-Hinton (MH; Oxoid) broth or 8. 5% (w/v) agar supplemented with vancomycin (10 µg/mL) and trimethoprim (5 µg/mL) (unless otherwise indicated) under microaerobic/capnophilic conditions (6% O2,12% CO2) in a Sanyo tri-gas incubator for plates or using the Oxoid CampyGen system for broth cultures. Growth media were supplemented with chloramphenicol (Cm; 20 µg/mL) or kanamycin (Km; 50 µg/mL), where appropriate. E. coli strains used for plasmid construction were grown at 37°C in Luria-Bertani (LB; Sigma) broth or 7. 5% agar (w/v) and supplemented with ampicillin (100 µg/mL), Cm (15 µg/mL), or Km (25 µg/mL), as necessary. RNA was extracted from log phase broth bacteria (OD 0. 3), and cDNA was generated from the RNA, as described previously [74], [76]. The expression ratio of pgp1 was determined relative to the C. jejuni gyrA gene encoding DNA gyrase subunit A. The primer and TaqMan probe sequences used for qPCR are described in Text S1. Each qPCR reaction mixture consisted of 25 ng of cDNA, 950 nM of each primer, 250 nM TaqMan probe, and 10 µl 2× TaqMan Gene Expression Master Mix (Applied Biosystems) in a total volume of 20 µl. Duplicate reactions for the gene of interest and the housekeeping gene were run in a Stratagene Mx3000P real-time PCR system for 2 min at 60°C, 10 min at 95°C, and then 40 cycles of 15 s at 95°C and 1 min at 60°C. The quantitative PCR cycle threshold (CT) results were analyzed by the comparative CT method (ΔΔCT method). The C. jejni 81-176: : solo (KmR) and C. jejuni 81-176: : picard (CmR) Tn libraries (Tn library construction is described in Text S1) and were screened on CFW, as described previously [10]. Transmission electron microscopy (TEM) was carried out on 18 h broth cultures. Samples were fixed in a final concentration of 2. 5% (v/v) of gluteraldehyde for 2–3 h on ice. Cells were then harvested, resuspended in an equal volume of H2O, and stored at 4°C. For imaging, 2 µL of bacteria was spotted onto parafilm to which 4 µL of 0. 5% uranyl acetate was added for 1 min. A formavar-carbon film on 300 mesh copper grid (Canemco, Lakefield, Quebec, Canada) was added to the bacteria-uranyl acetate spot for 2 min. The grid was then removed, dried, washed ten times in sterile water, dried again and visualized on a Hitachi H7600 TEM equipped with a side mount AMT Advantage (1 mega-pixel) CCD camera (Hamamatsu ORCA) at the UBC Bioimaging facility (The University of British Columbia, Vancouver, BC, Canada). Phenotypic assays were carried out with strains grown in shaking MH-TV broth or biphasic cultures grown for 18 h. CFW fluorescence was assayed as described previously [10]. For motility, cultures were diluted to an OD600 of 0. 2 in MH-TV and 2 µl was point inoculated into MH-TV plates containing 0. 4% agar. Plates were incubated for 20 h and the halo diameter was measured. Biofilm formation was assayed using crystal violet as described previously [11] with the exception that the absorbance was measured at 570 nm. C. jejuni strains were passaged once from frozen stocks and then passaged to 20–25 MH plates and grown for 20 h to obtain log-phase bacteria at a final OD of 200–600. Cells were collected into cold MH broth by scraping, harvested by centrifugation at 8 000× g for 15 min and then resuspended in 6 mL ice cold H2O. Cells were lysed by dropwise addition to 6 mL 8% SDS boiling under reflux. PG was purified from the cell lysate, digested with the muramidase cellosyl (kindly provided by Hoechst, Frankfurt, Germany), and the resulting muropeptides were reduced with sodium borohydride and separated by HPLC as described [77]. Muropeptide fractions were collected, concentrated in a SpeedVac, acidified by 1% trifluoroacetic acid, and analysed by offline electrospray mass spectrometry on a Finnigan LTQ-FT mass spectrometer (ThermoElectron, Bremen, Germany) at the Newcastle University Pinnacle facility as described [25]. Muropeptide structures were assigned based on (i) comparison with retention times of known muropeptides from H. pylori, Caulobacter crescentus and E. coli and (ii) the obtained MS data (Table S3) and MS/MS fragmentation patterns (not shown). The C. jejuni 81-176 pgp1 gene was cloned for expression in E. coli without its signal peptide (amino acids 16–464 of the protein) in frame with the thioredoxin- and His-tag of the pET32a vector to give plasmid pEF46. A detailed description of the cloning of the expression construct, and the expression and purification protocol is included in Text S1. For enzyme assays, the purified protein was dialysed against 0. 05 M Tris-Cl, pH 7. 5 containing 0. 01 M ZnCl2,0. 3 M NaCl and 20% glycerol. Purified Δpgp1 PG (1. 0 mg/ml) was incubated with Trx-His6-Pgp1 (5 mM) in 0. 02 M NaH2PO4, pH 4. 8,0. 005 M ZnCl2, and 0. 1 M NaCl for 4 h at 37°C on a Thermomixer at 750 rpm. A control sample received no enzyme and another enzyme sample contained 0. 01 M EDTA and no ZnCl2. The samples were incubated with 10 µg of cellosyl (Hoechst, Frankfurt am Main, Germany) for 1 h, boiled for 10 min and centrifuged at room temperature for 15 min at 16 000 g. The muropeptides present in the supernatant were reduced with sodium borohydride and analyzed by HPLC, as described [24]. Chick colonization was performed as described previously [61], [76], with an infective dose of 104 CFU. Chicken experiments were carried out under protocol #10462 approved by the University of Michigan Committee on Care and Use of Animals (UCUCA). The human epithelial cell lines T84, Caco-2 and INT407 and the murine RAW 264. 7 and human Thp-1 macrophage cell lines were used for C. jejuni infections. Carboxymethylcellulose (CMC) was added to the tissue culture media to increase viscosity. A detailed description of the tissue culture infections is included in Text S1. Luciferase asssays were performed as previously described [46]. Briefly, HEK293T cells were transfected overnight with 75 ng of NF-κB luciferase reporter plasmid (Igκ-luc, Invitrogen) and either human Nod1 (hNod1,3 ng), mouse Nod1 (mNod1,0. 1 ng), or human Nod2 (hNod2,0. 1 ng). The empty vector (pcDNA3. 1, Invitrogen) was used to balance the transfected DNA concentration. At the same time, either C. jejuni 81-176 or Δpgp1 PG muropeptides at 0. 1 µg/mL were added, and the NF-κB-dependent luciferase activation was then measured following 18–24 h of co-incubation. Positive controls were TriDAP (tripeptide l-Ala-γ-d-Glu-meso-DAP, 5 µg/ml), FK565 (a synthetic immunostimulant tetrapeptide, heptanoyl-d-glutamyl-meso-DAP-d-alanine, 5 ug/ml), and MDP (muramyldipeptide, 10 µg/ml) for hNod1, mNod1, and Nod2 assays, respectively. Data are representative of three independent experiments. The INT407 human epithelial cell line was seeded at approximately 1×105 cells/ml in MEM supplemented with 10% FBS into 24-well tissue culture plates and allowed to grow for 20–24 h prior to infection. The cells were washed three times with MEM and either left uninfected or infected with either C. jejuni wild-type strain 81-176 or Δpgp1 at an O. D. 600 of 0. 002/mL taken from an 18 h shaking broth culture. Supernatants were collected after 8 h and 24 h, centrifuged for 10 min to pellet residual cells and bacteria, and frozen at −80°C until assayed. The concentration of IL-8 present in the supernatants was measured by the human IL-8 ELISA kit (Invitrogen, Camarillo, CA).
Bacterial cell shape is dictated by the composition of the cell envelope component peptidoglycan. Some important pathogens have a characteristic helical corkscrew morphology that may help them burrow into mucus overlaying cells to initiate colonization and pathogenicity. One example is Campylobacter jejuni, the leading cause of bacterial-induced diarrheal disease in the developed world. Direct evidence supporting the hypothesis that C. jejuni shape is related to its pathogenicity traits has not previously been provided. We identified a gene encoding a peptidase modifying peptidoglycan that is essential for maintaining the C. jejuni corkscrew shape. We can now connect a C. jejuni gene with morphology and peptidoglycan biosynthesis. Loss of this gene was also found to affect pathogenic attributes such as chicken colonization, biofilms, motility, and activation of host inflammatory mediators. In addition, this is the first study to thoroughly characterize C. jejuni peptidoglycan structure and to identify a gene involved in peptidoglycan maintenance. Our findings highlight an emerging theme in bacterial pathogenesis research: the connection between bacterial cell biology and pathogenesis. Finally, our characterization of C. jejuni cell shape and peptidoglycan provides a starting point for further work in this area in C. jejuni and other bacteria with curved and helical morphologies.
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infectious diseases
bacterial diseases
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genetics and genomics
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microbiology
glycobiology
bacterial pathogens
Peptidoglycan-Modifying Enzyme Pgp1 Is Required for Helical Cell Shape and Pathogenicity Traits in Campylobacter jejuni