Salmonella enterica Serovar Enteritidis Genes Induced during Oviduct ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2008, p. 6616–6622 0099-2240/08/$08.00⫹0 doi:10.1128/AEM.01087-08 Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Vol. 74, No. 21

Salmonella enterica Serovar Enteritidis Genes Induced during Oviduct Colonization and Egg Contamination in Laying Hens䌤 I. Gantois, R. Ducatelle, F. Pasmans, F. Haesebrouck, and F. Van Immerseel* Department of Pathology, Bacteriology and Avian Diseases, Research Group Veterinary Public Health and Zoonoses, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium Received 15 May 2008/Accepted 29 August 2008

Salmonella enterica serovar Enteritidis is the predominant serovar associated with salmonellosis worldwide, which is in part due to its ability to contaminate the internal contents of the hen’s egg. It has been shown that S. enterica serovar Enteritidis has an unusual tropism for the avian reproductive tract and an ability to persist in the oviduct and ovary. Factors allowing S. enterica serovar Enteritidis strains to contaminate eggs could be a specific interaction with the oviduct tissue, leading to persisting oviduct colonization. In vivo expression technology, a promoter-trap strategy, was used to identify genes expressed during oviduct colonization and egg contamination with S. enterica serovar Enteritidis. A total of 25 clones with in vivo-induced promoters were isolated from the oviduct tissue and from laid eggs. Among the 25 clones, 7 were isolated from both the oviducts and the eggs. DNA sequencing of the cloned promoters revealed that genes involved in amino acid and nucleic acid metabolism, motility, cell wall integrity, and stress responses were highly expressed in the reproductive tract tissues of laying hens. Salmonella enterica serovar Enteritidis is the leading cause of food-borne salmonellosis, and this is due at least in part to its unusual tropism for the hen’s reproductive tract and the production of contaminated eggs (4, 37). Very little is known about the way eggs become contaminated or what mechanisms have evolved to enable the S. enterica serovar Enteritidis to do this. Two possible routes of egg contamination with Salmonella are known. The horizontal transmission route implies Salmonella penetration through the eggshell, after the eggs are covered by the shell (14, 42). In contrast, the vertical transmission route refers to direct contamination of the egg content before oviposition, as a result of Salmonella infection of the reproductive organs (48, 49). It is believed that the most important route of egg contamination is via infected reproductive tissues, both the oviduct and the ovary (18, 33). Salmonella bacteria have been found on the mucosal surface and within epithelial cells, lining the oviduct in naturally and experimentally infected hens (12, 26). Some virulence factors have been identified as playing key roles in the infection pathway resulting in egg contamination, namely, type 1 fimbriae and capsular-like lipopolysaccharide (13, 22, 44, 52, 53). It was also shown that variants of Salmonella enterica serovar Enteritidis able to grow to high cell density are more efficient in contaminating eggs (23). A phenotype microarray analysis along with whole-genome DNA-DNA hybridizations were used to find a correlation between the genotype and the phenotype of S. enterica serovar Enteritidis strains that vary in terms of their abilities to contaminate eggs (45). As detected by the phenotype microarray analysis, a number of compounds (vitamins, amino acids, and fatty acids) were identified that may stimulate bacterial

growth by the activation of alternative metabolic pathways and in this way contribute to high cell density and thus enhanced egg contamination (45). A small set of single nucleotide polymorphisms was linked to the phenotypic divergence between two related S. enterica serovar Enteritidis strains varying in terms of egg contamination (http://ncbi.nlm.nih.gov/genomes /static/Salmonella_SNPS.html) (21). Once Salmonella bacteria are inside the forming egg in the oviduct, other bacterial survival strategies become important. Indeed, the contamination of forming eggs in the reproductive tract is not correlated with egg contamination, suggesting killing of the Salmonella bacteria in the egg albumen at 41°C (34). Furthermore, Salmonella bacteria that are located in the albumens of laid eggs are capable of migrating to and penetrating the vitelline membrane and thus gaining access to the yolk. Salmonella bacteria thus need mechanisms to survive in the egg white containing antimicrobial peptides, such as lysozyme and ovotransferrin, and a high pH. Studies identifying bacterial factors needed to survive within eggs are scarce. In a previous study using a genomic DNA library, it was shown that YafD, a putative DNA repair enzyme, and XthA, exonuclease III, were important for survival in egg albumen (39). Moreover, in a transposon mutant library approach, genes involved in amino acid and nucleic acid metabolism and cell wall integrity were indicated as important for S. enterica serovar Enteritidis to survive in egg albumen (9). Once in the yolk, and in the absence of antibodies from previous exposure to Salmonella, the bacteria gain access to an environment that is rich in nutrients and that lacks inhibiting conditions and/or compounds such as lysozyme, iron-binding ovotransferrin, and an alkaline pH (10). To more specifically identify S. enterica serovar Enteritidis genes potentially involved in oviduct colonization, a genomewide screen was performed using in vivo expression technology (IVET), a promoter-trap method specifically designed to examine the induction of bacterial genes in a specific biological niche.

* Corresponding author. Mailing address: Department of Pathology, Bacteriology and Avian Diseases, Research Group Veterinary Public Health and Zoonoses, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium. Phone: 32 9 2647748. Fax: 32 9 264 7741. E-mail: [email protected]. 䌤 Published ahead of print on 5 September 2008. 6616

S. ENTERICA GENE EXPRESSION IN OVIDUCTS OF LAYING HENS

VOL. 74, 2008 MATERIALS AND METHODS

Bacterial strains and growth conditions. The bacterial strain used in this study was an isogenic streptomycin-resistant variant (59) of the S. enterica serovar Enteritidis 147 strain, originally isolated from egg content. The virulence of this strain has been tested in laying hens and a high capability to colonize the reproductive tract and internal organs has been demonstrated (43). The ⌬purA deletion mutant was constructed using the one-step inactivation method with PCR products described by Datsenko and Wanner (11). Bacteria were cultured aerobically at 37°C in Luria-Bertani (LB) broth (Sigma, St. Louis, MO). Antibiotics, when required, were included at the following concentrations: for streptomycin (Certa, Braine l’Alleud, Belgium), 100 ␮g/ml; and for ampicillin (Sigma), 50 ␮g/ml. The ⌬purA deletion mutant was grown in LB broth supplemented with 1.35% adenine (Sigma) and 0.337% thiamine (Sigma). This is called AdB1 hereafter. MacConkey agar (Oxoid, Basingstoke, Hampshire, England) supplemented with 1% filter-sterilized lactose (Merck, Darmstadt, Germany) was used to monitor lacZY gene expression. Plasmid. pIVET1 is a derivate of the suicide vector pGP704 (25). This plasmid contains a promoterless synthetic operon of purA coupled to lacZY, preceded by a restriction enzyme site (BglII). This plasmid also has a mobilization site (mob), which results in wide bacterial host transfer through conjugation. The selectable marker for the pIVET1 is an ampicillin resistance gene (bla). Construction of IVET fusion pool. A Sau3AI (New England Biolabs, Ipswich, MA) partial restriction digestion of genomic DNA of S. enterica serovar Enteritidis was performed. Sau3AI is a four-base recognition cutter allowing one to easily carry out a partial digestion generating a complete library of overlapping genomic DNA fragments. This is followed by size fractionation on a 0.8% agarose gel to obtain random fragments of 1 to 4 kb. The DNA fragments were extracted using the Zymoclean gel DNA recovery kit (Zymo Research, Orange, CA). Cloning of Sau3AI S. enterica serovar Enteritidis 147 chromosomal fragments into the BglII (New England Biolabs) site 5⬘ to the purA gene resulted in the construction of transcriptional fusions, by which properly positioned S. enterica serovar Enteritidis promoters drive the transcription of wild-type copies of purA and lacZY. The plasmids were transferred to Escherichia coli DH5␣␭pir by electroporation to obtain a pool of purA-lacZY fusion constructs (after selection for ampicillin resistance). The resulting plasmid pool contained more than 42,000 clones, which is sufficient to state with 99% certainty that with every promoter situated on the S. enterica serovar Enteritidis chromosome a fusion construct was made (8). After pooling Escherichia coli pIVET1 transformants, the pool was grown to the late logarithmic phase in 500 ml of LB supplemented with ampicillin at 37°C at 225 rpm. Subsequently, plasmids were extracted with the plasmid midi kit (Qiagen, Venlo, The Netherlands) and retransformed in the conjugative strain Escherichia coli SM10 ␭pir. The pool of transformants was grown again as mentioned above. The fusion construct library was then mobilized into the S. enterica serovar Enteritidis 147 ⌬purA strain by conjugation using Escherichia coli SM10 ␭pir as a donor strain. Integration in the chromosome resulted in a single crossover and therefore did not lead to disruption of the wild-type locus on the chromosome. The single crossover generated a duplication of S. enterica serovar Enteritidis DNA in which the native chromosomal promoter drove the purA-lacZY fusion, whereas the cloned promoter drove the expression of the wild-type copy of the gene. Integration of the recombinant plasmids was assessed by growth on MacConkey lactose agar plates supplemented with ampicillin, streptomycin, and AdB1. Approximately 42,000 recombinant fusion strains were scraped off the agar plates and pooled. S. enterica serovar Enteritidis ⌬purA mutant in vivo behavior in laying hens. A preliminary experiment was carried out to study the behavior of the S. enterica serovar Enteritidis ⌬purA mutant in laying hens. Two groups of 35 ISA Brown Salmonella-free laying hens were intravenously inoculated with 1 ml of 2 ⫻ 108 CFU/ml S. enterica serovar Enteritidis 147 wild-type and the S. enterica serovar Enteritidis 147 ⌬purA strain, respectively. The animals were euthanized after different time intervals (12 h, 24 h, 48 h, 3 days, 4 days, 7 days, and 10 days); and samples of the oviducts and spleens were taken. During the period of infection, eggs were collected. Bacteriological analysis of the oviducts, spleens, and eggs was performed as outlined below. IVET selection in laying hens. (i) Animals. Thirty ISA Brown Salmonella-free laying hens were housed in HEPA-filtered rooms from the age of 18 weeks until the end of the experiment. At the day of arrival at the animal facility, it was confirmed that the animals were Salmonella free using bacteriological analysis of cloacal swabs and an enzyme-linked immunosorbent assay for the detection of anti-Salmonella antibodies in serum (15). The animals had free access to drinking water and were fed ad libitum, and they received a lighting scheme of 16 h of light and 8 h of darkness.

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(ii) Experimental design. At the age of 21 weeks, all birds were intravenously inoculated with 1 ml of a 2 ⫻ 108-CFU solution of the fusion library transformants of the S. enterica serovar Enteritidis ⌬purA mutant. At different time points (7 days, 14 days, and 21 days postinfection [p.i.]), 10 birds were euthanized and samples of the oviducts were taken. Throughout the period of infection, eggs were collected. (iii) Bacteriological analysis of oviduct and eggs. Samples of oviduct were weighed, sliced into very small pieces, and subsequently homogenized with a stomacher in 10 times the volume of buffered peptone water (Oxoid). After the oviduct homogenates were plated, the recovered bacterial cells were pooled and used as an inoculum for a second round of selection (same experimental setup as for the first infection round). This was done to enrich the desired colonies in vivo. After isolation from the oviduct tissue, the transcriptional activity was monitored in vitro by growing the bacteria on MacConkey lactose agar with ampicillin, streptomycin, and AdB1 and analyzing their ability to utilize lactose as a carbon source. This allowed the detection of bacterial strains containing promoters expressed in vivo in the reproductive tract tissues (purA expression) and not in vitro. Fusion strains containing promoters induced in vitro showed red colonies (high-level lacZY expression) on MacConkey agar, whereas fusion strains carrying promoters inactive in vitro (low-level lacZY expression) displayed white colonies on MacConkey medium. Because we were interested in genes that were specifically induced in vivo and not in vitro, the bacteria with low-level lacZY expression were picked up for sequencing. Upon collection, the shell was decontaminated with 70% ethanol. Then the eggs were broken aseptically and the contents were pooled in a sterile plastic bag. The contents were homogenized with a stomacher for 3 min. Forty milliliters of buffered peptone water was added to the content of each egg, and this mixture was incubated for 48 h at 37°C. Further enrichment in tetrathionate brilliant green broth (1/10) (Merck) was done overnight at 37°C. A loopful of broth was plated on MacConkey lactose agar. The white bacteria were picked up for sequencing. The initial pool consisted of approximately 15% colonies with high-level lacZY expression (red), 16% colonies with intermediate-level lacZY expression (pink), and 69% colonies with low-level lacZY expression (white) colonies. After two rounds of selection in oviduct tissue, 92% of the bacteria recovered from the oviduct had high-level lacZY expression, 3% had intermediate-level lacZY expression, and 5% had low-level lacZY expression. This phenomenon is called a “red shift” and was a clear indication that there was a selection for the transcriptionally active fusions in the oviduct. We focused on the 5% colonies with low-level lacZY expression, because the cloned promoters were active in vivo in the oviduct tissue but not in vitro on culture plates. Approximately 300 white colonies recovered from the oviduct and contaminated eggs were picked up for sequence analysis. Sequencing IVET fusions. The sequences that were cloned at the 5⬘ site of the purA-lacZY fusion were identified by a modification of the PCR-based method of Kwon and Ricke (36). Genomic DNA of the selected IVET fusion strains of interest was isolated and completely digested with NlaIII (New England Biolabs), which resulted in a 4-bp overhang of CATG. This restriction enzyme was chosen because it recognizes a restriction site in the purA gene and it digests the genomic DNA frequently enough to yield average sizes of DNA fragments that were in the range to be sequenced. The digested DNA was ligated to a Y-linker using T4 DNA ligase (New England Biolabs). The Y-linker was synthesized using the following two linker sequences: for linker 1, 5⬘ TTT CTG CTC GAA TTC AAG CTT CTA ACG ATG TAC GGG GAC ACA TG 3⬘; and for linker 2, 5⬘ TGT CCC CGT ACA TCG TTA GAA CTA CTC GTA CCA TCC ACA T 3⬘. Linker 2 was 5⬘ phosphorylated using T4 polynucleotide kinase (New England Biolabs), whereafter linker 1 was added. The mixture was heated to 95°C and slowly cooled down to form the Y-linker. The Y-linker thus was designed to have 3⬘ overhang sequence complementary to the sticky end generated by NlaIII and a region of noncomplementary sequence on the opposite strand of the 5⬘ end. After ligation of the Y-linker to the digested chromosomal DNA, a PCR was performed with a primer binding the purA sequence and a primer binding the Y-linker. The sequence between the purA gene and the Y-linker was thus amplified and the resulting PCR product was sequenced using the primer binding to purA. Identification of the sequence of the cloned bacterial promoter was done by BLAST analysis using the genome sequences of S. enterica serovars Enteritidis, Typhi, and Typhimurium (http://www.ncbi.nih.gov; http://www.sanger.ac.uk /Projects/Microbes/).

RESULTS AND DISCUSSION IVET selection in laying hens. The wild-type strain was able to reach colonization rates of up to 105 CFU/g in the oviduct, while for all time points, only very low numbers (1 to 10

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APPL. ENVIRON. MICROBIOL.

TABLE 1. Isolation of the S. enterica serovar Enteritidis ⌬purA mutant and the corresponding wild-type strain from spleens and oviducts of intravenously inoculated laying hens Spleen Strain

Time p.i. No.

Wild type

purA mutant

12 h 24 h 36 h 2 days 3 days 4 days 7 days 10 days 12 h 24 h 36 h 2 days 3 days 4 days 7 days 10 days

a

5/5 5/5 5/5 3/3 3/3 3/3 3/3 3/4 5/5 5/5 5/5 2/3 2/3 3/3 2/3 4/4

CFU/g

Oviduct b

4.4 ⫾ 0.5 3.6 ⫾ 0.5 4.0 ⫾ 0.7 3.8 ⫾ 0.2 3.4 ⫾ 0.6 3.6 ⫾ 0.3 3.7 ⫾ 0.4 2.7 ⫾ 0.7 4.2 ⫾ 0.3 4.0 ⫾ 0.6 4.0 ⫾ 0.1 3.8 ⫾ 0.8 2.8 ⫾ 1.6 4.2 ⫾ 0.1 2.8 ⫾ 1.5 2.9 ⫾ 0.5

No.

a

3/5 3/5 5/5 3/3 3/3 2/3 3/3 3/4 0/5 0/5 0/5 0/3 0/3 0/3 0/3 0/4

CFU/g

b

3.1 ⫾ 1.4 3.3 ⫾ 1.2 5.9 ⫾ 1.1 4.4 ⫾ 0.7 4.7 ⫾ 1.6 3.0 ⫾ 1.1 4.9 ⫾ 0.9 3.7 ⫾ 1.2 0.8 ⫾ 0.4 0.6 ⫾ 0.5 0.8 ⫾ 0.4 0.7 ⫾ 0.5 0.6 ⫾ 0.5 0.7 ⫾ 0.5 0.3 ⫾ 0.5 0.3 ⫾ 0.6

a Number of tissue samples positive after direct plating/total number of tissue samples. b Mean number (log10) of Salmonella detected per gram of tissue ⫾ standard deviation (samples positive after enrichment were presumed to contain 10 CFU/g).

CFU/g) of the ⌬purA mutant strain colonized the oviduct. The ⌬purA strain colonized the oviduct approximately 104 times less than the wild-type strain. In addition, all the analyzed eggs were negative for the ⌬purA mutant strain. These data indicate that purA expression is required for the persistence of S. enterica serovar Enteritidis in the reproductive tract during infection of the laying hen, and these findings allowed us to use the pIVET1 system. Table 1 shows the number of CFU isolated from the oviducts and spleens of chickens infected with the wild-type strain and the ⌬purA strain. The number of CFU in the spleen is also shown. These data indicate that the purA selection took place only in the oviduct and not in the spleen. The IVET selection resulted in the identification of 25 different genes, and the variability between different birds was relatively low. The fusion strains carrying the promoters asnS, purA, hflK, stcD, lrp, uspBA, and yrfI were isolated from both oviducts and eggs from different animals. There is a possibility that bacteria recovered were actually recovered from the shell. However, since seven out of eight fusion strains were also isolated from the oviduct, it is strongly believed that these strains were recovered from the egg shell membranes or the internal egg contents instead of the egg shell. Additionally, several fusion strains were isolated from more than one egg. Table 2 presents the egg production rate and the number of positive eggs laid during the infection. An overall egg production rate of 41% was obtained and 13.37% of the eggs were positive. The different genes being induced in oviduct tissue on day 7, day 14, and day 21 p.i. and in eggs are listed in Table 3, and their functions are summarized in Table 4. We have categorized the genes according to their functions. Seven categories were used: metabolic genes, motility genes, genes involved in cell membrane and cell wall integrity, regulatory genes, stress genes, virulence plasmid genes, and genes with an unknown function. The fact that different genes are induced at

TABLE 2. Isolation of fusion library transformants of the S. enterica serovar Enteritidis ⌬purA mutant from eggs of intravenously inoculated laying hens No. eggs laid/no. positive eggs at weeka:

Time p.i. (no. of days)

1 (n ⫽ 30)b

2 (n ⫽ 18)b

3 (n ⫽ 9)b

1 2 3 4 5 6 7

20/1 (2) 9/2 (1) 8/0 6/0 7/2 7/3 7/1

5/2 8/2 8/1 10/2 10/3 9/0 10/0

5/1 4/0 4/0 5/0 4/0 5/0 6/1

a Numbers of animals that died after the infection, if any, are shown in parentheses. b The number of birds at the indicated week is shown in parentheses.

several time points after infection may suggest that there is some shift over time in gene expression which can be due to changes in the egg-forming environment. However, it is also possible that the IVET screen failed to identify these genes at all time points. Metabolic genes. Among the bacterial genes that were expressed in oviducts and eggs, 14 out of 25 genes were related to the bacterial metabolism, of which 50% were involved in amino acid metabolism (asnS, folC, lysS, glyA, leuS, valS, and serS). The upregulation of genes related to amino acid metabolism could indicate that the requirement for amino acids is high. purA, tmk, nkdD, dnaX, and rpoZ are genes encoding

TABLE 3. List of genes induced in the oviduct at days 7, 14, and 21 p.i. and in contaminated eggs during the time of infection Gene(s) induced at indicated day Gene group 7

Metabolic

14

21

folC lysC asnS glyA leuS valS purA

Motility Cell membrane and cell wall integrity

gpmA flgG tatA

purA tmk ndkD dnaX rpoZ gpmA

asnS (1)

serS aceA purA

purA (3)

uspBA yrfI

hflK (4) peg-yohN (1) murA (1) lrp (3) uspBA (3) yrfI (5)

hflK peg-yohN Regulator Stress Virulence plasmid Unknown functions

Gene(s) induced in eggsa

uspBA

lrp uspBA yrfI repB, tap, repA STY3802

STY3802 ygfA

a The number of eggs that was contaminated with the fusion strain carrying the respective promoter is shown in parentheses.


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TABLE 4. Functions and/or features of the genes induced during oviduct colonization and egg contamination Gene group

Metabolic

Motility Cell membrane and cell wall integrity

Gene

folC lysC asnS glyA leuS valS serS aceA purA tmk ndkD dnaX rpoZ gpmA flgG tatA

hflK peg-yohN

murA Regulator

lrp

Stress

uspBA

yrfI Virulence plasmid

repB, tap, repA

Unknown function

STY3802 ygfA

Gene function and/or feature

Folylpolyglutamate synthase Lysine tRNA synthetase Asparagines tRNA synthetase Serine hydroxymethyltransferase Leucyl tRNA synthetase Valyl tRNA synthetase Seryl tRNA synthetase Isocitrate lyase Adenylosuccinate synthetase Thymidylate kinase Nucleoside diphosphate kinase D DNA polymerase III subunits gamma and tau RNA polymerase omega subunit Phosphoglycerate mutase 1 Flagellar basal body rod protein Twin-arginine translocation system; transports folded proteins across the bacterial plasma membrane Component of modulator for Fts protease Putative outer membrane lipoprotein, similar to Escherichia coli putative type 1 fimbrial protein/putative periplasmic protein UDP-N-acetylglucosamine 1carboxyvinyl transferase; cell wall formation Putative leucine response regulator; regulator of spv genes and conjugal transfer of virulence plasmid Universal stress protein B, involved in stationary-phase resistance to ethanol, universal stress protein A; required for resistance to DNA-damaging agents Heat shock protein 33, redox regulated chaperone Located on the PT4 plasmid, analogue to the virulence plasmid pSLT; encodes a replication initiation protein Putative GntR family regulatory protein Conserved hypothetical protein, similar to Escherichia coli putative ligase

enzymes playing a role in nucleic acid biosynthesis. In a transposon bank approach, identifying genes involved in egg albumen resistance at 37°C, egg albumen-susceptible mutants frequently had insertions in genes encoding proteins involved in amino acid and nucleic acid metabolism (9). The magnum compartment of the oviduct produces egg albumen, and Lu et al. (39) discovered that egg albumen has nuclease activity. Therefore, it can be hypothesized that the expression of genes responsible for amino acid and nucleic acid biosynthesis is required for survival in the hostile environment of the reproductive tract. glyA encodes a serine hydroxymethyltransferase and it was suggested that upregulation of glyA was an integral

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response to signals eliciting curli fimbria formation (7). Curli promote binding to a variety of host proteins, such as fibronectin, and increase bacterial internalization in eukaryotic cells (20, 51). Although the expression of Salmonella curli was shown to correlate with a high frequency of growth in eggs (10), their role in the colonization of the reproductive tract has not been demonstrated yet. Two genes involved in carbohydrate degradation and energy metabolism were identified. gpmA encodes a phosphoglycerate mutase and is involved in glycolysis. aceA was also identified in this screen and encodes isocitrate lyase. Isocitrate lyase is required for acetic acid utilization via the glyoxylate shunt. Phenotype microarray data revealed that strains varying in their ability to contaminate eggs show drastic differences in amino acid and nucleic acid metabolism (45). Recently, a set of small single nucleotide polymorphisms were identified that differ in strains that vary in terms of egg contamination (http://ncbi .nlm.nih.gov/genomes/static/Salmonella_SNPS.html) (21). Evolutionary variations were observed for folD, lysR and lysC, leuO and leuC, and purF and aceB. This means that a single amino acid change in these genes could impact the transcriptional activations of folC, lysS, leuS, purA, and aceA, respectively. The fact that these genes within several operons were detected by two different methods strengthens the association between the expression of individual genes and evolutionary trends that contribute to reproductive tract colonization and egg contamination. Motility genes. Only one gene involved in motility was identified. flgG encodes a component of the flagellar basal body rod. It is believed that motility afforded by flagella mediates the initial interaction between bacterium and host cell. S. enterica serovar Enteritidis mutant strains unable to express flagella were less able to attach to explants in vitro and were less pathogenic to chicks in vivo (1, 54). Studies in our lab showed a reduced invasion in primary tubular gland cells of the oviduct and reduced survival in egg albumen at chicken body temperature (42°C) in comparison with what was seen for the wild-type S. enterica serovar Enteritidis strain (data not shown). The topic of motility is controversial, however, since nonflagellated S. enterica serovar Pullorum is very efficient at colonizing the reproductive tract and contaminating eggs, which suggests that flagella are not required for egg contamination (50). Genes involved in cell membrane and cell wall integrity. The IVET approach allowed the identification of four genes that are involved in cell membrane and cell wall structure and integrity: tatA, hflK, stcD, and murA. It is believed that cell wall structural and functional integrity is essential for S. enterica serovar Enteritidis to survive the stress of exposure to egg albumen. Possibly, the upregulation of genes important for cell wall integrity is essential for the bacteria to counter the antimicrobial activities of ovotransferrin, lysozyme, and other yetunidentified antimicrobial components present in the oviduct and eggs. tatA is the first gene of the tatABC operon, and it encodes the twin-arginine translocation system. The Tat pathway is involved in the transport of folded proteins across bacterial plasma membranes (55). Previous work has demonstrated that strains with mutations in genes encoding the Tat pathway components have defective cell walls, reflected in an increased sensitivity to hydrophobic drugs and lysozyme (60). It was

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shown that the mislocation of two cell wall amidases, AmiA and AmiC, was responsible for the cell envelope defect (30). In addition, using microarrays, the global effect of a tat null mutation in Escherichia coli was investigated. The Tat system proved to be essential for the export of phospholipases, iron uptake, anaerobic respiration, osmotic stress defense, motility, and biofilm formation (31). The conserved nature of the structure and the functions of the Tat system in Escherichia coli and S. enterica serovar Typhimurium (47) assumes that the tat expression in the reproductive tract is necessary for many aspects of the survival and virulence of the bacteria, including growth in iron-limiting conditions and resistance against antimicrobial components, such as lysozyme. Fusions of the promoter of hflK to purA-lacZY were isolated from both the oviduct and eggs. hflK is involved in the quality control of the plasma membrane. In Escherichia coli, HflK and HflC, two periplasmic membrane proteins, form a complex with FtsH, an ATP-dependent protease that is embedded in the plasma membrane, and modulate its proteolytic activity (58). FtsH may serve to maintain quality control of some cytoplasmic and membrane proteins. It was shown that when the FtsH-HflKC complex recognizes a proper substrate, HflKC dissociates from FtsH to activate its proteolytic functions, and therefore it was proposed that HflKC acts as a proteolytic inhibitor of FtsH (35). It was shown that FtsH is involved in the degradation of the heat shock transcription factor ␴32, a key element in the regulation of the Escherichia coli heat shock response (62). Lower levels of FtsH protease activity would influence heat shock transcription factor ␴32 and subsequently DnaK and ultimately FliC production. Since HflKC inhibits FtsH protease activity, it may be possible that the sequestration of FtsH by HflKC can induce the heat shock response and thereby activate bacterial motility. Fusions containing two genes, a gene belonging to the peg operon and yohN, were both isolated from oviduct tissue as from a contaminated egg postlay. The peg genes are so far restricted to S. enterica serovar Enteritidis, S. enterica serovar Gallinarum, and S. enterica serovar Paratyphi (61). The peg fimbrial proteins of S. enterica serovar Enteritidis show 58 to 64% identity with their predicted functional equivalents in the S. enterica serotype Typhimurium LT2 stc cluster (61). The stc operon is a putative fimbrial operon. Although little is known on this fimbrial operon, it was shown that the stc operon of S. enterica serotype Typhimurium is required for intestinal persistence in mice (63). Interestingly, expression of the stc operon in S. enterica serotype. Typhimurium does not assemble into fimbrial filaments but rather appears to coat the bacterial surface (28). The exact role of these stc genes remains to be determined, but they can play a role in increasing the resistance of the bacterial cell wall to antimicrobial peptides in egg white. yohN encodes a putative periplasmic protein of unknown function. Finally, the fusion of the promoter of murA to purA-lacZY was recovered from one infected egg postlay. murA is involved in peptidoglycan synthesis. The cationic peptide lysozyme, present in the egg albumen, catalyzes the hydrolysis of 1,4beta-linkages between N-acetylmuramic acid and N-acetyl-Dglucosamine residues in the peptidoglycan in order to kill the bacteria (27). Therefore, the expression of murA may be trig-


gered as a response to the disruption of the peptidoglycan cell wall layer. Regulatory genes. We have isolated the fusion of the promoter of lrp with purA-lacZY from multiple oviducts and eggs. The leucine-responsive regulatory protein (Lrp) is a DNA binding protein that acts as a global regulator to influence transcription (5, 6, 29, 40). Lrp is known to regulate many fimbrial genes, and its contributions have been studied in detail for Escherichia coli pap and fim fimbrial operons. The pef (plasmid-encoded fimbria) genes in Salmonella encode Pef pili and are regulated by a mechanism that is strikingly similar to that found for the Escherichia coli pap systems (46). Lrp directly influences the phase-variable expression of the pef operon. The on-off pef switch is controlled by DNA methylation of 5⬘-GATC-3⬘ sites in the pef regulatory DNA by Dam. To obtain access to these sites, Dam needs to compete with Lrp (46). There is currently little evidence for the role of Pef fimbriae in virulence and persistence in the chicken, but they were shown to mediate adhesion to mouse intestinal epithelium and the pefC mutant strain was less virulent in mice (2). In a recent study, it was demonstrated that a knockout mutation in lrp inhibited fim transcription in S. enterica serotype Typhimurium (41). Lrp is a positive regulator of the expression of type 1 fimbriae in Salmonella through direct interaction with the fimZ promoter region. There is mounting evidence that S. enterica serovar Enteritidis adheres to oviduct epithelial tissues in vitro via type 1 fimbriae (12). These findings could thus implicate lrp as a gene affecting Salmonella oviduct colonization and, as a consequence, the contamination of eggs. Stress genes. Two stress-related genes were identified: uspBA and yrfI. Clones carrying two adjacent genes uspB and uspA were recovered four times independently from the oviduct at 1 and 3 weeks p.i. In addition, uspBA fusions were found in three contaminated eggs. uspBA encodes the universal stress proteins A and B. The expression of the UspA stress protein is affected by metabolic, temperature, and oxidative stresses (38). Inactivation of the uspA gene in S. enterica serotype Typhimurium leads to decreased survival in stress conditions (38). Most likely, the unfavorable environment of the reproductive tract triggers the expression of the uspA gene. It should also be noted that UspA is also implicated in membrane function during stationary phase because it is regulated, in part, by FadR, the global regulator of fatty acid synthesis and degradation (16). UspB is required for stationary-phase resistance to ethanol (17). Bacteria exposed to lethal concentrations of ethanol lyse, presumably because of the effects on the lipids of the inner and outer membranes. It is speculated that UspB may play a role in sensing or mediating alterations in membrane composition during stationary phase. Thus, both universal stress proteins may be involved in the alterations of the membrane composition during stationary phase and are possibly required for resistance against the antimicrobial peptides in the oviduct and eggs. Another gene found in our IVET screen is yrfI, which encodes the redox-regulated chaperone Hsp33. The fusion strain harboring the yrfI promoter was isolated from three oviducts and five eggs. The YrfI heat shock protein is under heat shock control at the transcriptional level, while it is under oxidative stress control at the posttranslational level (32). Upon stress exposure, it binds to a large number of cellular proteins and

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prevents their irreversible aggregation. Both the uspBA and yrfI genes indicate a protective response against protein or cellular damage in the hostile environment of the oviduct and the egg. Virulence plasmid genes. Two weeks after infection, two independent oviducts were colonized with bacteria with lowlevel lacZY expression containing fusions of the promoters of repA, repB, and tap to purA-lacZY. These genes are located on the S. enterica serovar Enteritidis virulence plasmid and are responsible for plasmid replication and control the low copy number of the plasmid (56). The exact role of the virulence plasmid in pathogenesis is unclear, but evidence exists that spv (Salmonella plasmid virulence) genes enable S. enterica serotype Typhimurium to infect the internal organs by increasing the rate of bacterial replication within the host cells (24). Therefore, it can be speculated that the virulence plasmid may play a role in the multiplication of Salmonella in oviduct cells. Furthermore, the virulence plasmid also expresses the plasmidencoded fimbriae (Pef). As already mentioned earlier, Baümler et al. (3) showed that Pef mediates adhesion to the small intestine of mice. In addition, in vivo assays have shown that the pef genes are expressed by both S. enterica serovar Enteritidis and S. enterica serotype Typhimurium, as orally infected chickens develop specific antibodies (19). Interestingly, virulence plasmids have been found in only a few serovars, particularly those showing host adaptation. These plasmids are 50 to 90 kb in size and have been called serovar-specific plasmids (57). It is believed that virulence plasmid acquisition may have expanded the host range of Salmonella, and Baümler et al. (3) suggested that the acquisition of different fimbrial operons may have enabled Salmonella to adapt to different situations of colonization. Therefore, it may be speculated that the S. enterica serovar Enteritidis-specific virulence plasmid may enable this serovar to adapt to the specific environment of the reproductive tracts of laying hens. Genes with unknown function. Gene STY3802, with an unknown function and encoding a putative GntR family regulatory protein, was identified in two different oviducts at weeks 2 and 3 p.i. In addition, one cloned fragment contained the ygfA gene, which encodes a putative uncharacterized protein. Conclusion. Although it is clear that IVET cannot identify all genes expressed in the oviduct, this study resulted in the identification of 25 genes that are induced in S. enterica serovar Enteritidis during colonization of the reproductive tract and within eggs. The data suggest that the oviduct and eggs are stressful environments for Salmonella bacteria. The bacteria appear to react by stress-induced protective and reparative responses, protein and nucleic acid synthesis, cell wall component synthesis, and expression changes in fimbrial and flagellar operons. Identification of S. enterica serovar Enteritidis genes necessary for oviduct colonization can improve our understanding of the molecular mechanism behind the ability of S. enterica serovar Enteritidis to colonize the reproductive tracts of laying hens, potentially leading to the development of control products, such as vaccines. Further research is needed to unravel the exact roles of these genes in oviduct colonization and egg contamination.

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ACKNOWLEDGMENTS We thank D. Heithoff for his invaluable suggestions on the IVET work. We acknowledge Lohmann Animal Health GmbH & Co. for funding this research project. F. Van Immerseel is supported by a postdoctoral research grant of the Research Foundation—Flanders (FWO) and the Research Fund of Ghent University. REFERENCES 1. Allen-Vercoe, E., and M. J. Woodward. 1999. The role of flagella, but not fimbriae, in the adherence of Salmonella enterica serotype Enteritidis to chick gut explant. J. Med. Microbiol. 48:771–780. 2. Ba ümler, A. J., R. M. Tsolis, F. A. Bowe, J. G. Kusters, S. Hoffmann, and F. Heffron. 1996. The pef fimbrial operon of Salmonella Typhimurium mediates adhesion to murine small intestine and is necessary for fluid accumulation in the infant mouse. Infect. Immun. 64:61–68. 3. Ba ümler, A. J., A. J. Gilde, R. M. Tsolis, A. W. M. van der Velden, B. M. M. Ahmer, and F. Heffron. 1997. 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