ABSTRACT
Salmonella enterica serovar Enteritidis has developed the potential to contaminate table eggs internally, by colonization of the chicken reproductive tract and internalization in the forming egg. The serotype Enteritidis has developed mechanisms to colonize the chicken oviduct more successfully than other serotypes. Until now, the strategies exploited by Salmonella Enteritidis to do so have remained largely unknown. For that reason, a microarray-based transposon library screen was used to identify genes that are essential for the persistence of Salmonella Enteritidis inside primary chicken oviduct gland cells in vitro and inside the reproductive tract in vivo. A total of 81 genes with a potential role in persistence in both the oviduct cells and the oviduct tissue were identified. Major groups of importance include the Salmonella pathogenicity islands 1 and 2, genes involved in stress responses, cell wall, and lipopolysaccharide structure, and the region-of-difference genomic islands 9, 21, and 40.
INTRODUCTION
Chicken eggs and egg-derived products were traced back as the primary food vehicle responsible for a Salmonella enterica serovar Enteritidis pandemic that started in the late 1980s (1, 2). It is still incompletely understood why this particular Salmonella serotype is more successful than other serotypes in colonizing the chicken egg. Indeed, many other serotypes can be present in the environment of the laying hen, but Salmonella Enteritidis is the main serotype associated with eggs (3). Intensive cleaning and disinfection of the eggshell surface failed to eliminate the serotype from eggs, indicating that it must often be present in the internal egg content (2). Most likely, the bacteria are incorporated in the egg during its formation in the chicken reproductive tract (4–7). Reproductive tract colonization capacities of different serotypes have been compared in various studies and show that Salmonella Enteritidis is superior in reproductive organ colonization compared to other serotypes, with the exception of Salmonella Typhimurium (8–10).
A previously performed in vivo expression technology (IVET) screening identified genes involved in amino acid and nucleic acid metabolism, motility, cell wall integrity, and stress responses as being expressed by Salmonella Enteritidis inside the reproductive tract (11). A role for type 1 fimbriae (12), lipopolysaccharide (LPS) structure (13), and stress responses (10) in reproductive tract colonization has already been confirmed through the use of defined deletion mutants. Gene knockout strains can provide important information on the relevance of single genes in the pathogenesis of egg infections, but a whole-genome approach that identifies all mutated genes that are important in this process is hitherto lacking. In the present study, we identified genes that are required for the persistence of Salmonella Enteritidis inside the reproductive tract by using a genome-wide microarray-based transposon library that selects mutations that are causing decreased colonization of the oviduct. Our results confirm those found by other studies as genes encoding cell wall components and proteins involved in stress responses were identified. In addition, we demonstrate a potential role for the Salmonella pathogenicity islands (SPIs) and phage-associated genes, as well as unique regions present in Salmonella Enteritidis but not in Salmonella Typhimurium, the so-called regions of difference (ROD).
MATERIALS AND METHODS
Bacterial strains, growth conditions, and construction of the transposon library.For the construction of the transposon library, an isogenic streptomycin resistant variant of the Salmonella Enteritidis 147 strain (147str) was used. The library was made according to the method previously described (14–16). In a first step, the pJA1 plasmid was transferred to the Escherichia coli SM10 λpir strain and maintained under ampicillin (100 μg/ml; Sigma-Aldrich, St. Louis, MO) selective pressure. The pJA1 suicide vector contains IS10 inverted repeats flanking a kanamycin resistance cassette with an adjacent, outward-directed T7 transcriptional promoter. In addition, the plasmid harbors a mini-Tn10 transposase under the control of an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible LacIq/Ptac promoter and the RP4 mob region for conjugation. A standard overnight conjugation reaction, in the presence of IPTG (Sigma-Aldrich) but without antibiotics, was performed between E. coli SM10 λpir and Salmonella Enteritidis 147str. Under influence of IPTG, the transposable element, containing a kanamycin resistance gene and the T7 promoter, was excised from the pJA1 plasmid and integrated randomly into the genome of Salmonella Enteritidis 147str. The next day, all colonies were scraped from the plate into 10 mM MgSO4 (Sigma-Aldrich), and 10-fold dilutions were streaked onto Luria-Bertani (LB) agar plates containing 200 μg/ml streptomycin (Sigma-Aldrich) and 30 μg/ml kanamycin (Sigma-Aldrich) to select for successfully transposed Salmonella Enteritidis 147str mutants. About 50,000 mutants were collected and stored in 15% glycerol (Sigma-Aldrich) solution at −80°C.
Isolation of oviduct tubular gland cells.Primary tubular gland cells of the chicken reproductive tract were isolated according to the protocol of Jung-Testas et al. (17), with minor modifications. A daily subcutaneous injection of 1 mg of estradiol-benzoate (Sigma-Aldrich) dissolved in sesame oil was given to 7-day-old chicks for 10 consecutive days. Three weeks later, the chicks received a second round of injection for 7 days with the same amount of estradiol-benzoate. The next day, the chicks were euthanized with an intravenous embutramid injection (T61; Intervet, Belgium). Part of the oviduct, ranging from the beginning of the magnum to the end of the isthmus, was aseptically removed, opened longitudinally, and rinsed three times in Hanks balanced salt solution (HBSS; Gibco/Invitrogen). The oviduct segment was cut into small pieces and allowed to dissociate for 30 min at 37°C in Dulbecco minimal essential medium (DMEM; Gibco/Invitrogen) containing collagenase (1 mg/ml; Sigma-Aldrich) supplemented with 50 μg/ml penicillin-streptomycin (Gibco/Invitrogen). The tissue suspension was centrifuged for 5 min at 335 × g at 37°C, the supernatant was removed, and the tissue was further treated with trypsin (0.25% trypsin plus 3 mM EDTA in DMEM) for 5 min under agitation at 37°C. Fetal calf serum (FCS; Gibco/Invitrogen), was added to neutralize the trypsin, and the cells were centrifuged at 335 × g for 5 min at 37°C. Next, cells were treated with lysis buffer (0.87% NH4Cl and 0.1% NaHCO3 in HBSS) to eliminate red blood cells. The resulting cell suspension was filtered, using a 70-μm-pore-size cell strainer, followed by centrifugation at 335 × g. The cells were then washed twice with DMEM containing 10% FCS. The cells were seeded at 106 cells/ml in cell culture flasks in DMEM supplemented with 15% FCS, insulin (0.12 IU/ml; Sigma-Aldrich), fibronectin (1 μg/ml; Sigma-Aldrich), β-estradiol (50 nM; Sigma-Aldrich), penicillin-streptomycin (50 μg/ml), and amphotericin B (Fungizone; 250 ng/ml; Gibco/Invitrogen). Flasks were placed in a cell incubator at 37°C with 5% CO2 for 2 h. During this period fibroblasts attached to the cell culture flask, while the oviduct cells remained in suspension. Nonadhering oviduct cells were removed and seeded in 24-well tissue culture plates at 106 cells/ml. At 2 days postisolation, the wells were evaluated for confluent growth and used for in vitro experiments. The experimental protocol was approved by the ethical committee of the Faculty of Veterinary Medicine, Ghent University.
Transposon library selection on oviduct cells in vitro and oviduct loops in vivo. (i) Identification of Salmonella Enteritidis genes important for persistence inside primary tubular gland cells of the oviduct in vitro.The Salmonella Enteritidis transposon library (initial library) was grown for 7 h at 37°C in LB medium (Sigma-Aldrich) with agitation in the presence of streptomycin (200 μg/ml) and kanamycin (30 μg/ml). The bacterial suspension was centrifuged, resuspended in cell medium without antibiotics, amphotericin B, and FCS, and added to the oviduct tubular gland cells (see above) at a concentration of 107 CFU/ml (multiplicity of infection of 10). The plates were centrifuged for 10 min at 524 × g to obtain optimal contact of the bacteria with the cell layer. The cells were incubated for 1 h at 37°C and rinsed three times with HBSS, and then cell medium containing gentamicin (100 μg/ml; Gibco/Invitrogen) was added. After 1 h, the gentamicin concentration was lowered to 30 μg/ml, and the cells were incubated for another 14 h. The plates were rinsed three times with HBSS, and the cells were lysed using 1% Triton X-100 (Sigma-Aldrich). The plates were placed on an MTS 2/4 digital microtiter plate shaker (IKA, Staufen, Germany) for 10 min at maximum speed. Afterward, HBSS was added, and the intracellular bacteria were collected. Harvested intracellular bacteria (output library) were grown in LB medium with streptomycin and kanamycin for 7 h and then used for a second round of invasion. In all, three subsequent enrichment passages were performed, and the experiment was repeated in five independent replicates.
(ii) Identification of Salmonella Enteritidis genes important for colonization of the oviduct in vivo.For the identification of genes involved in oviduct colonization in vivo, an intraoviductal inoculation approach was chosen. The experimental protocol for this procedure was approved by the ethical committee of the Faculty of Veterinary Medicine, Ghent University. Three 21-week-old commercial laying hens were premedicated intramuscularly with buprenorphine hydrochloride at 0.05 mg/kg (Temgesic; Schering-Plough, Kenilworth, NJ) and atropine at 0.05 mg/kg. Anesthesia was induced by the administration of isoflurane (Schering-Plough) through a face mask. After intubation with a 3.0-mm uncuffed tracheal tube (Hudson RCI, Temecula, CA), a continuous oxygen flow of 1.5 to 2.0 liters/min was administered carrying 1.5 to 3% isoflurane. The birds were covered with a sterile surgical blanket and defeathered on the abdominal surface. After disinfection of the incision area with a povidone iodine solution (Braunol; B. Braunol Medical, Prague, Czech Republic), the abdomen was opened through a midline incision and the oviduct segments were carefully exposed. A 7-h-old culture of the Salmonella Enteritidis transposon library was centrifuged and diluted in HBSS until 107 CFU/ml were obtained. The oviducts were inoculated with 1 ml of the bacterial suspension at the isthmus-magnum transition zone using a 27-gauge needle. After inoculation, the oviduct was reintroduced into the abdomen, and the abdominal wall was sutured. After recovery from anesthesia, the birds were placed in separate cages on wood shavings. The animals had unrestricted access to drinking water and feed and, 2 days after infection, the hens were euthanized by intravenous injection of embutramid (T61; Intervet). The magnum and isthmus were aseptically removed and opened longitudinally. Oviducts were rinsed three times in HBSS supplemented with 100 μg/ml gentamicin to kill extracellular bacteria. Tubular gland cells were isolated according to the protocol of Jung-Testas et al. (17) as previously described but with an additional 50 μg of gentamicin/ml in all enzyme solutions and without penicillin and streptomycin until the cells were lysed with 1% Triton X-100 for 10 min.
DNA isolation and RNA transcription of transposon library.In order to perform microarray hybridization, genomic DNA (gDNA) from the initial library and from the in vitro and in vivo selections were purified using the phenol-chloroform extraction method. Next, 10 μg was digested with HinP1I (New England BioLabs, Ipswich, MA), and 2 μg was ligated to the Y-linkers described by Tavazoie and Church (18). Using a PCR with a transposon and linker-specific primer (14), the transposon containing fragments were amplified. Subsequently, 1 μg of PCR product was used in an in vitro transcription reaction from the T7 promoter in the transposon adjacent region. The MEGAscript T7 transcription kit (Ambion, Austin, TX) was used for this purpose according to the manufacturer's protocol, except that all reaction volumes and reagents were doubled. RNA was purified by lithium chloride precipitation as described within the MEGAscript kit manual.
Microarray procedure.RNA recovered from the initial and selected transposon libraries was reverse transcribed into cDNA and subsequently Cy5 labeled using random hexamer primers and a Klenow fragment. cDNA was hybridized on a whole-genome Salmonella Typhimurium/Enteritidis SALSA cDNA microarray, carrying 5,877 coding sequences, together with Cy3-labeled Salmonella Enteritidis genomic reference DNA (19). Two microarray chips were hybridized for each biological replicate. Slides were scanned on an Axon 4000A scanner (Axon Instruments, Foster City, CA). Spots showing a reference signal lower than the background plus two standard deviations or obvious blemishes were excluded from subsequent analysis. Local background was subtracted from spot signals, and cyanin fluorescence ratios were calculated using GenePix version 1.4 software (Agilent, Santa Clara, CA). To compensate for unequal dye incorporation or any effect of the amount of template, data centering was performed by bringing the median natural logarithm of the ratios for each group of spots printed by the same pin to zero. The data that passed the quality controls were analyzed using GeneSpring version GX7.3 software (Agilent). Signal values of the output library were normalized against those of the initial library and used to identify mutants for which the gene value had at least a 2-fold decrease (fold difference of <0.5) after the selection procedure compared to the initial library grown in LB medium. The significance of the centered data, at P ≤ 0.001 for the in vitro tests and P ≤ 0.05 for the in vivo tests, was determined using a parametric-based statistical test, adjusting the individual P value with the Benjamini-Hochberg false discovery rate multiple test correction. The microarray protocols are described in detail at http://www.ifr.ac.uk/safety/microarrays/protocols.html. Since the microarray is mainly annotated for Salmonella Typhimurium, gene sequences were used in a BLAST search to look for their Salmonella Enteritidis (SEN) homologues.
RESULTS AND DISCUSSION
To obtain information about the oviduct colonization mechanisms of Salmonella Enteritidis, a genome-wide microarray-based transposon library screening was performed. The idea behind this technique is that mutants harboring transposon insertions in genes important for oviduct colonization are less able to persist or multiply inside oviduct cells and therefore, following oviduct-cell passage, decrease in number compared to mutants in which genes that are not essential for oviduct colonization are inactivated.
The transposon library screening was carried out in vitro using isolated primary tubular gland cells of the chicken oviduct and in vivo using isolated oviduct tubular gland cells after infection of 21-week-old commercial laying hens. Using both methods, we identified 241 (in vitro) and 323 (in vivo) genes as being important for oviduct colonization, respectively. Of these, 81 genes were found to be common to both screens (Table 1) and thus considered to be the most important for colonization and persistence within the chicken reproductive tract. Consequently, the following discussion focuses on these genes.
Genes important for persistence inside the reproductive tract, identified during both in vitro and in vivo screening
Pathogenicity islands.As shown in Table 1 a number of genes belonging to the Salmonella pathogenicity islands (SPI) 1, 2, and 3 were identified as important for the persistence of Salmonella Enteritidis inside the oviduct cells in the chicken reproductive tract. SPI-1 encodes a type 3 secretion system and associated effector proteins that facilitate the uptake of the bacteria by nonphagocytic host cells. The importance of SPI-1 genes in the invasion and survival of Salmonella Enteritidis in chicken oviduct epithelial cells was previously demonstrated by Li et al. (20). These authors showed that mutations in the effector proteins sipB, sipA, sopB, and sopE2 led to a decrease in the invasion of oviduct cells, while those in sipB and sipA were also less able to survive inside these cells. Intracellular survival/proliferation is a function mainly attributed to SPI-2 (19, 21). A Salmonella Enteritidis mutant in ssrA, encoding the key transcriptional regulator of SPI-2, has a reduced potential to colonize the reproductive tract, and this is probably due to a diminished systemic spread (22). In the present study we show that in addition to systemic spread to the reproductive tract, SPI-2 might also be involved in the persistence of Salmonella Enteritidis inside the reproductive tract in oviduct epithelial cells. In addition to SPI-1 and SPI-2, one gene, marT, belonging to SPI-3, was shown to be important for oviduct colonization. Tukel et al. (23) showed that marT is involved in the transcriptional activation of misL, a fibronectin binding protein increasing the invasiveness into epithelial cells.
Regions of difference.Genes belonging to regions of difference (ROD) 9, 21, and 40 were identified in our study to be involved in chicken reproductive tract colonization. RODs are genomic islands that were shown to be present in the genome of Salmonella Enteritidis but not in Salmonella Typhimurium (24).
ROD9 is a truncated form of SPI-19 found in Salmonella Gallinarum, where it encodes a type 6 secretion system (T6SS) indispensable for efficient colonization of the gut, spleen, and liver in young chickens (25). SPI-19 is necessary for survival of Salmonella Gallinarum inside avian macrophages, without contributing to cytotoxicity and cell death (26). Since SPI-19 of Salmonella Enteritidis lost many of the T6SS essential components, it was suggested that this system is not functional within this serotype (27). Transfer of the complete Salmonella Gallinarum SPI-19 cluster to Salmonella Enteritidis resulted in increased ileum, liver, and spleen colonization 1 day postinfection despite a colonization defect after 3 days (25). Other researchers hypothesized that some genes of ROD9 might be retained in Salmonella Enteritidis to enhance its colonization potential, since the SEN1001 gene of the Salmonella Enteritidis ROD9 cluster appeared to be essential for the colonization of mice and survival inside murine macrophages (28). Finally, a Salmonella Enteritidis ROD9 deletion mutant was not defective in reproductive tract colonization (29).
ROD21 is present in Salmonella Enteritidis, Gallinarum, and Dublin but absent in Salmonella Typhimurium (24, 30). The cluster contains a Toll/interleukin-1 receptor domain containing protein SEN1975 or TlpA that might suppress NF-κB induction while it promotes host cell apoptosis (28, 31, 32). A tlpA mutant was defective in intracellular survival inside human THP1 macrophages, and mice infected with the tlpA mutant survived the challenge better compared to the wild type (32). ROD21 also comprises two putative type IV pilin proteins (SEN1976 and SEN1978), which might be linked to several functions, including motility, adhesion, and biofilm and bacterial aggregate formation, as well as invasion (28, 31). Also, two conjugational transfer proteins (SEN1979 and SEN1980) and a histone-like nucleoid-structuring regulator (SEN1993) are located on ROD21, of which the latter was shown to be a homologue of the uropathogenic E. coli hnsT gene, which promotes the expression of virulence genes (31). Nevertheless, a role for ROD21 in reproductive tract colonization could not be demonstrated since a ROD21 deletion mutant had the same colonization levels in this organ as the wild type after oral inoculation (29).
The ROD40 locus encodes a type I restriction/modification system, which degrades foreign DNA (24), but the role of ROD40 in reproductive tract colonization still remains to be investigated.
Phage-associated genes.Five Salmonella Enteritidis genes belonging to ϕSE10, ϕSE12, ϕSE12A, or ϕSE14 phages have been identified during our screening to be important for oviduct colonization. Shah et al. (33) showed that the SEN1393 gene of ϕSE14 is involved in the invasion of Salmonella Enteritidis inside Caco-2 and LMH chicken liver cells. In addition, this mutant was attenuated for survival inside chicken macrophages and showed a significantly reduced growth in egg albumen compared to the wild type. In contrast a ϕSE14 deletion strain colonized the liver and spleen of BALB/c mice, as well as the wild-type strain, 2 days postinfection (34).
Virulence plasmid.Some serotypes of Salmonella, including Enteritidis, harbor serotype-specific plasmids, encoding genes necessary for replication (repA and repA2) and conjugation (the often-degraded tra operon) and carrying virulence genes such as the Salmonella plasmid virulence (spv) operon and the pef and fae fimbrial operons (35, 36). Our screening suggests that repA2, traA, and the hypothetical protein (pSENV_079) are important for Salmonella Enteritidis colonization of the chicken reproductive tract.
LPS composition and fimbriae.A previously performed IVET screening already showed that genes involved in cell membrane and cell wall integrity were highly expressed during colonization of the reproductive tract by Salmonella Enteritidis (11). Seven of the genes identified in our study are involved in the composition of the bacterial membrane. Of the cell wall structural proteins, seven are involved in LPS biosynthesis (rfaB, rfbI, rfbN rfbP, rfbS, rfbX, and rfbV). The importance of LPS biosynthesis in oviduct colonization was already demonstrated by Coward et al. (13). These authors showed that both the wzy (exhibiting only one O-antigen repeat) and the febE (defective in the production of very long O-antigen repeats with more than 100 subunits) deletion strains are less efficient in the colonization of the ovary and oviduct than a wild-type strain. Also, one gene of SPI-17 was found to be involved in the persistence of Salmonella Enteritidis inside the reproductive tract in our study. SPI-17 of Salmonella Enteritidis is a degenerate prophage harboring the gtrA and gtrB genes, which are involved in LPS O-antigen modification in Shigella flexneri (24, 37, 38). Additionally, the UDP-glucose 6-dehydrogenase gene (ugd, udg, pagA, or pmrE), shown to modify the lipid A component of LPS (39), was identified in our study as involved in oviduct colonization. Finally, a gene of the peptidoglycan recycling system (ampD) and an envelope lipoprotein-encoding gene (envF) were identified, but their role in reproductive tract colonization is not clear.
Although type 1 (SEF21) fimbriae are necessary for adhesion to the isthmus secretions (40), we demonstrate here a potential role for the peg fimbrial operon (pegABCD) in oviduct colonization since a pegC mutant was identified during the transposon library screening. This operon is only found in Salmonella Enteritidis, Gallinarum, and Paratyphi B. Transcription of this operon was induced during reproductive tract colonization (11), and a Salmonella Enteritidis pegD mutant had a decreased egg white survival capacity compared to the wild type (33).
DNA biosynthesis/repair and stress responses.The importance of nucleic acid biosynthesis and stress responses for the colonization of the reproductive tract by Salmonella Enteritidis was already shown by Gantois et al. (11). These researchers demonstrated that the expression of many genes of the nucleic acid biosynthesis pathways was upregulated in the oviduct. In addition, a deletion of the purA gene, involved in the pyrimidine metabolism, resulted in a reduced colonization potential of the chicken reproductive tract compared to spleen colonization (11). In the present study, the pyrimidine metabolism pathway (pyrD) and the O6-methylguanine-DNA-transferase gene (ogt), necessary for the repair of DNA alkylation damage, were identified. The ogt gene is regulated by the alternative sigma factor σS (RpoS) and is involved in the survival of bacteria under starvation or stress conditions (41), which can be encountered in the reproductive tract.
One of these stress conditions encountered during colonization of the reproductive tract is the high chicken body temperature of 42°C. Consequently, it is not surprising that in our study two heat-inducible genes (hslV and htrA) were identified. HslV is the proteolytic subunit of the HslVU complex, whereas HtrA is a periplasmic serine protease. Both genes are involved in the elimination of unfolded and damaged proteins important for bacteria facing unfavorable conditions. Finally, yciG of the yciGFE-katN operon is transcribed as a polycistronic messenger and harbors a putative RpoS-dependent promoter upstream of yciG, directing the transcription of genes essential for the general stress response (42).
Conclusion.In order to identify genes that are necessary for the persistence of Salmonella Enteritidis inside the chicken reproductive tract, a genome-wide transposon screening was performed. We can hypothesize that Salmonella Enteritidis invades and survives within the chicken oviduct epithelial cells by use of its pathogenicity islands. To further cope with the antimicrobial factors present inside these cells or the reproductive tract, the bacterium might alter its membrane composition, enhance its DNA repair strategies and activate some stress management strategies. Finally, our study also suggests a potential role for the RODs 9, 21, and 40.
ACKNOWLEDGMENTS
This research was supported by the Institute of Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen grant 81040). A.T. and N.S. were supported by a Core Strategic Grant from the Biotechnology and Biology Sciences Research Council to the Gut Health Food Safety program of the Institute of Food Research.
FOOTNOTES
- Received 8 September 2014.
- Accepted 30 September 2014.
- Accepted manuscript posted online 3 October 2014.
- Copyright © 2014, American Society for Microbiology. All Rights Reserved.
