1 Biosafety Research Institute and College of Veterinary Medicine, Chonbuk National University, Jeonju 561-756, Korea
2 Halim Inc., 13-14 Euryang-Ri, Samgi-Myeon, Iksan 570-883, Korea
Correspondence
Joon-Seok Chae
jschae{at}chonbuk.ac.kr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AY956822AY956839.
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INTRODUCTION |
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Several recent investigations involving comparative studies between SG and other Salmonella serotypes have disclosed subtle differences in the cellular mechanisms that may be responsible for specificity or adaptability of SG to an avian host (Barrow et al., 1994; Pascopella et al., 1995
; Wallis et al., 1999
; Wilson et al., 2000
; Kaiser et al., 2000
; Paulin et al., 2002
; Wigley et al., 2002
; Chadfield et al., 2003
). For instance, unlike several serotypes of Salmonella, SG is incapable of establishing an infection in mice via the oral route (Pascopella et al., 1995
). In contrast to other Salmonella serotypes, very little is known about the genetic basis of SG virulence and the molecular mechanisms involved in systemic infection and development of FT. An 85 kb plasmid was reported to influence virulence in SG (Barrow et al., 1987
), and some of the plasmid-associated virulence genes were identified (Rychlik et al., 1998
). However, an SG strain cured of this virulence-associated plasmid showed only partial attenuation (Barrow et al., 1987
). Moreover, classical SG strains lacking a large virulence-associated plasmid may also occur (Patel et al., 2004
). Therefore, a large plasmid may not be sufficient or necessarily required, but a plethora of other unidentified chromosomal genes may be responsible for the full virulence of SG (Barrow & Lovell, 1989
). Interestingly, recent work has revealed that the Salmonella pathogenicity island-2 type III secretion system (SPI-2 TTSS), and not SPI-1, is essential for virulence in SG (Jones et al., 2001
). The above studies have disclosed some important aspects of SG pathogenicity and generated indirect evidence that the virulence mechanisms of SG may be different from those of other widely studied Salmonella serotypes such as Salmonella typhimurium or Salmonella typhi. Nevertheless, our knowledge regarding the precise role of virulence factors and their implication in establishment and/or maintenance of SG infection is relatively scarce. Therefore, it is important to elucidate the molecular basis of SG virulence in relation to early hostpathogen interactions that may influence the outcome of SG infection in chickens.
Signature-tagged mutagenesis (STM) has emerged as a powerful tool for identification of in vivo-essential genes and new targets for the development of rationally attenuated genetically defined live vaccines or potential antibiotic agents (Hensel et al., 1995; Handfield & Levesque, 1999
; Lehoux & Levesque, 2000
). STM is based on negative selection in which individual mutants are identified within a pool of bacteria. Mutants are generated by chromosomal insertion of a mini-transposon containing a specific DNA tag that uniquely labels each mutant within a library. Using this system, a large number of mutants can be analysed in parallel to identify in vivo-essential and infection-related genes (Lehoux & Levesque, 2000
; Lehoux et al., 2001
). The original STM technique (Hensel et al., 1995
) required radiolabelling and hybridization analysis for identification of in vivo-attenuated mutants and was later modified by a relatively simpler and non-hazardous PCR-based technique (Lehoux et al., 1999
).
In the present study, we employed PCR-based STM for screening and identification of in vivo-essential and infection-related genes of SG in a chicken model (chickens are the only natural host for this bacterium). Screening of 1152 mini-Tn5 mutants led to the identification of 20 potentially attenuated mutants that were found to have insertions in 18 unique loci. All 20 STM-identified SG mutants had competitiveness defects confirmed by in vitro and in vivo competition assays. In order to investigate the future possible applications of these mutants as novel live vaccine candidates, each potentially attenuated mutant was tested individually in a challenge experiment to verify and determine the degree of attenuation caused by the transposon mutation.
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METHODS |
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Generation of 12 mini-Tn5 SG mutant libraries.
According to the PCR-based STM working scheme (Lehoux et al., 1999), 12 uniquely tagged pUT mini-Tn5Km2 plasmids were transferred from E. coli S17-
pir (donor strain) to SGNalr (recipient strain) by conjugation. For generation of a single library of 96 mutants in a 96-well microtitre dish, independent mating experiments were performed using 12 uniquely tagged pUT mini-Tn5Km2. Briefly, 400 µl of each donor strain was mixed individually with 400 µl of mid-exponential-phase SGNalr (recipient strain). The mixture of bacteria (50 µl) was immobilized on 0·45 µm pore-size filters (Millipore) placed on non-selective M9 agar (Sigma) plates with no carbon source and incubated at 37 °C for 16 h. Transconjugants were recovered in 10 ml phosphate-buffered saline (PBS) and a 100 µl aliquot was plated on LB agar containing Na and Km. All the potential exconjugants were analysed for exclusive mini-Tn5Km2 insertion by confirmation of ampicillin sensitivity. Eight exconjugants (carrying a unique tag) from each mating experiment were arrayed in a designated well of a 96-well microtitre dish to form 8 pools of 12 uniquely tagged exconjugants (8x12=96). To minimize the number of sibling transconjugants, 12 such independent mating experiments were carried out to generate 12 libraries, each consisting of 96 clones (12x96=1152 mutants). In order to ensure that all the mutants had a single transposon insertion at a unique position, Southern blot analysis of EcoRI-digested genomic DNAs from 36 representative STM mutant strains was performed. Genomic DNA (1 µg) from each mutant was digested with EcoRI and separated by agarose gel electrophoresis. The DNA was transferred to a nylon membrane by using a vacuum blotter (model 782, Bio-Rad) according to the instructions provided by the manufacturer. The probe in the Southern hybridization was produced by PCR using the DIG labelling system (Roche) with primers pUTKanaR1 (5'-GCG GCC TCG AGC AAG ACG TTT-3') and Tag1 (5'-GTA CCG CGC TTA AAC GTT CAG-3'), spanning a
500 bp region from the kanamycin-resistance gene. The membrane was hybridized overnight at 42 °C in Dig-Easy Hyb (Roche), washed at 65 °C and developed by a colorimetric method as described in the Dig labelling and detection protocol (Roche).
In vivo screening of the mutant library in a chicken model.
The library of 96 mutants, containing 8 pools of 12 mutants each, was grown in LB broth supplemented with Na and Km in a 96-well microtitre dish at 37 °C for 16 h. The aliquots of 12 SG mutants from each pool were mixed and a sample was removed for PCR analysis (the input pool). A second aliquot from the same pool was washed twice, resuspended in PBS and used to infect 1-day-old White Leghorn broiler chicks (straight run). Chicks (n=4 per group) were infected orally with 0·1 ml of the bacterial suspension containing either 105,
106 or
107 c.f.u. (for preliminary experiments using chicks pretreated with 0·5 ml of 1 % sodium bicarbonate), or
107 c.f.u. (for screening the libraries in chicks without pretreatment with sodium bicarbonate). Four days post-infection (p.i.), bacteria were recovered from spleens of at least three chicks by plating homogenized tissue suspension on LB agar supplemented with Na and Km. An aliquot of 104 colonies, recovered after in vivo selection, was pooled (the output pool) and processed for the extraction of genomic DNA using a DNeasy Tissue kit (Qiagen) according to the instructions provided by the manufacturer. PCR identification of the mutants was performed as described by Lehoux et al. (1999)
, with the following modifications: the cycling conditions used were initial denaturation at 94 °C for 5 min followed by 30 three-step cycles of denaturation at 94 °C for 1 min, annealing at 70 °C for 1 min, extension at 72 °C for 1 min and a final extension cycle at 72 °C for 7 min. PCR amplicons present in the input pools were compared with the PCR amplicons present in the output pools.
In vivo and in vitro competition assays.
For an in vivo (chicken) competition assay, mutant and wild-type (WT) parent strains were grown separately for 16 h in LB broth. Then, equal amounts (2·5x107 each in 100 µl PBS) of each mutant and WT bacteria were mixed, and administered orally to 1-day-old broiler chicks (n=4 per group). The ratio of mutant to WT bacteria in the inoculum was verified by viable counts after plating serial dilutions of the inoculum in parallel on LB with Na (to measure total c.f.u.) and LB with Na and Km (to determine mutant c.f.u.). Four days p.i., spleens were collected from at least three chicks and individually homogenized. To determine the proportion of mutant to WT, dilutions of spleen homogenates were plated on LB with Na as well as LB with Na and Km. The competitive index (CI) was calculated as the ratio of mutant (c.f.u. at day 4/c.f.u. at day 1) divided by the ratio of WT (c.f.u. at day 4/c.f.u. at day 1). Then the mean in vivo CI from at least three chicks was calculated.
For the in vitro competition assay, 2·5x104 c.f.u. of mutant and WT parent were co-inoculated in 5 ml LB. Cultures were grown at 37 °C for 16 h with shaking (200 r.p.m.) and the input and output ratios of mutant and WT were determined by selective plating as described above. For each mutant strain, the mean in vitro CI from two such experiments was recorded.
Cloning and sequence analysis of disrupted genes from attenuated mutants.
Chromosomal DNA was isolated from transposon mutants and digested with one of the several restriction enzymes (EcoRI, SalI, PstI or KpnI) that cut on either end of the transposon (leaving the kanamycin-resistance gene intact). As described above, Southern hybridization analysis using a portion of the kanamycin-resistance gene from mini-Tn5Km2 as a probe was used to identify the fragments of suitable size for cloning. Digested fragments were ligated into pUC19 and transformed into E. coli TOP10. Transformants were selected on LB agar plates containing Km. Plasmid from each of these strains, containing both transposon and flanking DNA, was purified using Wizard Plus SV minipreps (Promega) and sequenced with the primers reading in from the plasmid polylinker (M13F and M13R; Genotech) and primer Tn5Km2, 5'-TGC AAT GTA ACA TCA GAG-3' reading out from the kanamycin-resistance gene. Sequencing was performed (by Genotech, Korea) with an ABI Prism 377 DNA sequencer. DNA sequences flanking the transposon insertion were assembled. Subsequently, amino acid sequences were deduced and analysed by searching the BLAST and FASTA network service at the National Center for Biotechnology Information GenBank database. For the analysis of genomic organization of mutants and complete ORFs of the disrupted genes, similarity searches were performed with the completed S. typhimurium LT2 genome (http://www.ncbi.nlm.nih.gov/genomes).
Confirmation of virulence attenuation of individual STM mutants.
The virulence attenuation of STM mutants was assessed by determining the 50 % lethal dose (LD50) for each mutant. Groups of 1-day-old broiler chicks (n=5 per group) were orally inoculated with doses of 106, 107, 108, 109 or 1010 c.f.u. in a volume of 0·2 ml. Bacteria for challenge were grown to mid-exponential phase (approx. 6 h) at 37 °C with shaking (200 r.p.m.) in LB broth supplemented with Na and Km. The number of c.f.u. in the inoculum was verified by viable counts after plating a diluted aliquot of inoculum on to LB agar plates. After 7 days, cumulative mortality data were used to estimate the LD50 values (Reed & Muench, 1938). Internal organs (liver, spleen, heart, lung and caeca) from each chick that died during the experiment were collected and processed for isolation of mutants by plating on LB agar containing Km and Na. The isolated colonies of mutants were confirmed by PCR using primers specific for the designated tag of the mutants, as described above. Each group of chicks was monitored daily for observable clinical signs of FT such as drowsiness/lethargy, drowsiness with ruffled feathers and whitish diarrhoea. At the end of each experiment, all the surviving chicks were killed and internal organs (spleen, heart and liver) were processed for isolation of the respective mutants. The results of isolation were recorded as positive or negative.
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RESULTS AND DISCUSSION |
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Screening of tagged mutants in a chicken infection model
Previous STM screens have revealed that all the mutants in a pool used to infect a single animal must be present in a sufficient numbers to be able to establish an infection simultaneously. In practice, this means that large doses must be used in infection studies. Since the SGNalr strain is highly virulent for 1-day-old chickens (Shah et al., 2005b), preliminary experiments were performed to standardize the infection dose and period, and to test the reproducibility of the STM screen. Chicks were orally administered with 0·5 ml 1 % (w/v) sodium bicarbonate followed by infection with a pool of 12 mutants containing either
105 or
106 c.f.u. Four days p.i., only 78 mutants were present in the output pool recovered from spleen homogenates. When a dose of
107 c.f.u. was administered, 11 out of 12 mutants could be recovered from the resulting output pool, while one mutant was consistently missing from all chicks (data not shown). For further in vivo screening of the mutant library, a dose of
107 c.f.u. was used without any pretreatment with sodium bicarbonate. This dose is approximately 102 times greater than the LD50 (2·7x105) of the SG WT strain. Since the infection inocula in this study were administered orally without pretreatment with sodium bicarbonate, the situation may be considered equivalent to natural infection. Increase in the duration of infection period beyond 4 days resulted in death of one or more chicks, indicating that the dose of
107 c.f.u. was sufficient for establishing an infection in which the organisms could overcome a gastric barrier to successfully attach and invade the intestinal epithelium. Therefore, further investigations to improve the reproducibility of our screen by increasing either the dose or infection period were not undertaken. Additionally, in order to avoid selection of spurious mutants, we performed two rounds of screening as described by Darwin & Miller (1999)
. In the first round, all 96 pools, each containing 12 uniquely tagged mutants, were screened in 1-day-old chicks. The mutants that did not generate PCR amplicons in the output pools as compared to the input pools were reassembled into a new 96-well microtitre plate. In the second round, these reassembled mutants were screened in duplicate and the mutants with reproducible absence of PCR amplicons were identified. This led to the identification of 20 putatively attenuated mutants that were retained for further analysis.
Competition assays for in vitro and in vivo growth of mutants
Since STM identifies mutants that are outcompeted by multiple other strains during a mixed infection, the attenuation of each STM mutant was confirmed and quantified by an in vivo competition assay. Each mutant was mixed with an equal number of c.f.u. of the WT parent strain and used to infect four chicks via the oral route. Four days p.i., bacteria were recovered from the spleens and an in vivo competition index (CI) was calculated (see Methods). An in vivo CI of 0·1 and 0·000001, respectively, indicated that the mutant grew at least ten times and 106 times less rapidly in vivo as compared to the WT parent, and mutants with these in vivo CI values were referred to as significantly or highly attenuated (Table 1). In vitro CIs for all the attenuated mutants were also determined to identify whether the mutation led to a general growth defect or an in vivo-specific reduction in competitive fitness. The results revealed that although most of the mutants did have an in vitro growth defect (low CI), this was much less marked than the in vivo growth disadvantage (Table 1
). Moreover, for the majority of mutants, the in vitro CI was markedly higher than the in vivo CI, indicating a specific effect on virulence of these mutants (Table 1
). Interestingly, the gcpA mutant (SGC-1) had low CI values both in vitro and in vivo, while the ssaP mutant (SGB-10) seemed unusually more competitive in vitro than the WT strain.
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In a previously published study, an SPI-1-defective mutant of SG carrying a defined mutation in the spaS gene did not show attenuation of virulence in 3-week-old Rhode Island Red chickens, indicating that the SPI-1 TTSS was not required for virulence of SG (Jones et al., 2001). In contrast, our screening of five SPI-1 mutants in 1-day-old White Leghorn chickens revealed that SPI-1 has an important role in the pathogenesis of SG infection. The disparity in these data is likely to be due to the differences in the age and breed of chicken used. Significant differences have been shown to exist in the resistance to SG infection in inbred lines of chickens (Bumstead & Barrow, 1993
). The White Leghorn breed of chicken has been shown to be relatively more resistant to infection with host-adapted Salmonella than heavy breeds like Rhode Island Red, New Hampshire, or crosses between the two (Hutt & Crawford, 1960
). Therefore, while it can be said that SPI-1 has a role in SG pathogenesis, the age and genetic makeup of the chicken may also contribute to this effect. Further studies comparing the effects of defined mutations in SPI-1 TTSS during early as well as later phases of the infection in various breeds and age groups of chicken may help to determine the precise role of SPI-1 in SG pathogenesis.
There is also a great deal of variation in available data regarding the role of SPI-1 in virulence of S. typhimurium in chickens. Following experimental oral inoculation of 1-day-old White Leghorn chicks, strains of S. typhimurium with mutations in invA, invB and invC were found to be attenuated (Porter & Curtiss, 1997). However, an SPI-1 sipC mutant of S. typhimurium did not show a consistent defect in colonization in 3-week-old Light Sussex chicks (Turner et al., 1998
). In contrast, mutations in several SPI-1 genes did not produce colonization-defective phenotypes of S. typhimurium when tested in 1-day-old Light Sussex chicks (Morgan et al., 2004
). Thus, it is possible that different genetic factors are required by SG (a chicken-host-adapted serotype) and S. typhimurium (non-host-adapted serotype) in order to initiate an infection in a chicken model.
SPI-2 TTSS.
SPI-2 encodes a second TTSS which secretes effector proteins across the membrane of the Salmonella-containing vacuole and enables the persistence of Salmonella inside host cells by modulating vesicular trafficking (Hensel, 2000). Our analysis of SG genes identified by STM revealed six potentially attenuated mutants with an insertion mutation in the ssaP (SGB-10), ssaG (SGC-2), ssaV (SGC-9 and SGA-3), sseF (SGH-1) and ssrA (SGD-8) genes (Table 1
, Fig. 1b
). Most of the SPI-2 mutants were highly attenuated, as evidenced by greatly lowered in vivo CI numbers (Table 1
). The degree of attenuation of the above mutants was confirmed in a challenge experiment, which showed 102105-fold increase in LD50 when compared with the WT parent. Moreover, ssaG, ssaV (two mutants) and ssrA mutants did not cause any mortality or clinical symptoms of FT (Table 2
), indicating that SPI-2 has a major role in the virulence of SG infection. Our results are in corroboration with the previous study, in which the SPI-2 mutant of SG carrying mutations in the ssaU gene was found to be highly attenuated in virulence in 3-week-old chickens (Jones et al., 2001
). Interestingly, a previous analysis of ssaG, ssaV and ssrA defective mutants of S. typhimurium did not reveal colonization defects in 1-day-old chicks (Morgan et al., 2004
). However, we found that ssaG, ssrA and both ssaV mutants of SG were highly attenuated in virulence in 1-day-old chicks. These results further support our assumption that different genetic factors may be required by S. typhimurium and SG for infection in a chicken host.
SPI-10.
Mutant SGE-3 had an insertion mutation in sefD (a component of the sef operon that encodes a major adhesion subunit of SEF14 fimbriae); this gene showed 100 % homology to sefD of S. enteritidis and S. typhi (Table 1). sefD is a component of SPI-10 (33 kb), which carries phage 46 and the sefAR chaperone-usher fimbrial operon in S. typhi (Parkhill et al., 2001
). Interestingly, SEF14 fimbriae are found only in serogroup D Salmonella and are known to elicit a strong protective immune response against S. enteritidis infection in mice (Ogunniyi et al., 1994
). In this study, the sefD mutant of SG was highly attenuated in vivo (chicken CI, <0·000002), with >103-fold increase in LD50 as compared to the WT parent (Table 2
). The transposon mutation in sefD is unlikely to be polar because sefD is a terminal gene of an operon (Fig. 1c
) and its functional expression is known to be required for virulence in a mouse model as well as for the uptake or survival of S. enteritidis in macrophages (Edwards et al., 2000
). Therefore, our results are confirmatory and provide the first evidence for the role of sefD in the virulence of SG in chickens.
SPI-13.
Three STM mutants had insertion mutations in the genes showing 100 % homology to the following putative ORFs of S. typhimurium LT2: STM3118 (SGD-3), STM3120 (SGG-1) and STM3121 (SGA-10) (Table 1). These ORFs are clustered at cs 67.5 of the S. typhimurium LT2 genome (Fig. 1d
). A close inspection of these genes showed that at least 18 ORFs are clustered at cs 67.5, representing a region adjacent to tRNA pheV (McClelland et al., 2001
). The G+C content of this region is 48·1 mol%, which is considerably lower than the mean G+C content (52 mol%) of the S. typhimurium genome, indicating characteristic features that represent an SPI, and we designate this region SPI-13. These SPI-13 mutants showed a 102105-fold increase in LD50 as compared to the WT parent. The in vivo CIs of these mutants were also significantly lowered. Moreover, oral challenge of chicks with SGD-3 (gacD) and SGA-10 (gtrB) mutants did not produce any symptoms of FT or mortality, indicating their important role in the pathogenesis of SG infection in chickens.
We also investigated whether the above ORFs or their homologues have been documented to play any role in the virulence of other Salmonella serotypes. The deduced amino acid sequences of disrupted genes in mutants SGD-3, SGG-1 and SGA-10 were 100 % homologous to S. enteritidis genes cat-2 (accession no. AAK97550), citE (AAK97548) and stmR (AAK97547), respectively. The importance of cat-2 for the ability of S. enteritidis to invade and/or survive in chicken macrophages was recently documented (Zhao et al., 2002), but the role of other genes of SPI-13 in the virulence of S. enterica is not yet known. In order to get an insight into the possible differences between the genomes of human- and avian-adapted Salmonella serotypes, complete ORFs of STM3118, STM3120 and STM3121 were compared with the complete genome sequences of the following Salmonella serotypes: S. Paratyphi A (CP000026), S. typhi TY2 (AE014613) and S. typhi CT18 (AL513382). No homologues to the above three ORFs were found in any of these Salmonella serotypes.
SPI-14.
Two mutants had insertion mutations in the SG genes that were 100 % homologous to the following putative ORFs of S. typhimurium LT2: STM0855 (SGA-8) and STM0859 (SGC-8). Both mutants showed a 105-fold increase in LD50 when compared with the WT parent, and a significantly lowered in vivo CI (Tables 1 and 2). Analysis of the sequences flanking the transposon insertions showed that the above two ORFs are clustered at cs 19 of the S. typhimurium LT2 genome (Fig. 1e
). At least six putative ORFs are clustered in this region, which is not flanked by tRNA. The G+C content of this region is 41 mol%, which is significantly lower than the mean G+C content of the S. typhimurium genome, indicating that the region constitutes another SPI, which we have designated SPI-14. Further analysis of the deduced protein sequences of all six ORFs (STM0854 to STM0859) of SPI-14 showed that the first five genes were transcribed in the same direction and the terminal ORF (STM0859) was in the opposite orientation (Fig. 1e
). The insertion mutation in ORF0855 of the SGA-8 mutant may have a polar effect on products of genes downstream of the operon. Nucleotide sequence analysis revealed that STM0854 and STM0855 have an overlapping region of 80 bp while the ORFs STM0855/STM0856 and STM0856/STM0857 are separated by a very short spacer region of 13 bp and 9 bp, respectively. Such overlapping frames and short spacer regions are characteristics of a polycistronic message and this warrants further detailed characterization of these genes. To date, the genes clustered on SPI-14 have not been documented to play a role in the virulence of S. enterica. In future, it will be interesting to fully characterize all the components of SPI-14.
Similar to SPI-13, no sequence homologues for SPI-14 ORFs were found in the complete genome sequences of primarily human-adapted Salmonella serotypes (S. typhi TY2, S. typhi CT18 and S. Paratyphi A). It is possible that both SPI-13 and SPI-14 have been acquired by SG and S. typhimurium via horizontal transfer from a phage or plasmid of unknown origin. However, none of the genes of SPI-13 and SPI-14 have yet been reported to be implicated in the virulence of S. typhimurium. Thus, it is possible that the functional expression of these genes may be conserved in SG. SG has several important characteristics, including (i) a non-motile serotype; (ii) membership of Salmonella serogroup D; and (iii) an avian host adaptation. These characteristics differentiate the SG serotype from all motile Salmonella serotypes. Therefore, identification of genes in both SPI-13 and SPI-14, and their relevance to SG virulence as shown in this study, is important. It will be of interest to perform detailed functional characterization of the SPI-13 and SPI-14 region, which may be useful in disclosing some of the important aspects of pathogenesis, host restriction and possibly the evolutionary origin of SG.
Non-SPI-encoded virulence-associated genes.
We identified one SG mutant (SGA-1) with a transposon insertion in the envZ gene (Fig. 1f). This mutant showed a high degree of attenuation in vivo and a 105-fold increase in LD50 when compared with the WT parent, indicating that envZ has an important role in establishment and maintenance of SG infection in chickens. Our results are in corroboration with earlier studies, in which an envZ mutant of S. typhimurium was reported to have severely attenuated virulence in mice (Chatfield et al., 1991
; Lee et al., 2000
). However, no mutant with an insertion mutation in the envZ gene was reported when over 1054 STM mutant strains of S. typhimurium were screened in 1-day-old chicks (Morgan et al., 2004
). Further investigations using defined envZ mutants of S. typhimurium in a chicken model may reveal the differences in the genetic mechanisms governing the pathogenic processes of SG and S. typhimurium in different animal hosts.
The SGC-3 (gppA) mutant had an insertion mutation in ORF1026, encoding the hypothetical phage (Gifsy-2)-related protein in S. typhimurium LT2 (Fig. 1f). Gifsy-2 (a cryptic lambdoid phage) is present in the lysogenic state in S. typhimurium and plays a role in mouse virulence (Figueroa-Bossi & Bossi, 1999
). Interestingly, in SG, Gifsy-1 and Gifsy-2 carry sopE, which encodes an effector protein translocated by the SPI-1 TTSS (Mirold et al., 2001
). The gppA mutant was highly attenuated in vivo and had a 105-fold increase in LD50. Moreover, no mortality or clinical symptoms of FT were recorded in chicks challenged with this mutant. Such phage-related loci encoding virulence properties have been reported to transfer between Salmonella of the same or different serovars, providing the ability for an organism to gain diverse virulence traits and to adapt to different animal hosts (Figueroa-Bossi et al., 2001
). It will be interesting to further disclose the role of these prophages in virulence as well as in evolution and host restriction of SG.
The transposon insertion in mutant SGC-1 (gcpA) had 100 % homology to the ORF STM3820, encoding cytochrome c peroxidase (CCP) in S. typhimurium LT2 (Fig. 1f). CCP is an antioxidant that catalyses degradation of hydrogen peroxide and protects bacteria from oxidative stress (oxidative burst and generation of nitric oxide) during infection (Turner et al., 2005
). Disruption of the gene encoding CCP has been recently reported to cause an in vivo attenuation of Campylobacter jejuni in chicken intestine (Hendrixson & DiRita, 2004
). In this study, the gcpA mutant had low CI values both in vivo (0·023) and in vitro (0·06). However, in a challenge experiment the gcpA mutant showed a 105-fold increase in LD50 when compared with the WT parent, indicating that this mutant had a defect in virulence (Table 2
). Therefore, while it can be said that gcpA is indeed a virulence gene, this strain could not be classified as in vivo-attenuated in the strict sense on the basis of our criteria for the in vivo CI. These results however indicate that CCP has an important role in the pathogenesis of SG infection in chickens. Further mutational and biochemical analysis of gcpA gene may be helpful in elucidating the mechanisms by which SG evades the barriers of the immune system for its systemic spread in a chicken host.
Concluding remarks
The design of our infection study to confirm attenuation was solely based on in vivo competitive growth defects and mortality rates at dosage levels that would detect significant changes in the ability of a strain to cause a severe infection. In a challenge study, chicks inoculated with two SPI-2 mutants (ssaP, sseF) and one SPI-13 mutant (gtrA) showed mortality as well as clinical symptoms typical of FT. These mutant strains were reisolated from the internal organs of all challenged chicks. The increase in LD50 of these mutants (102-fold) was markedly lower than for other mutant strains (103105-fold). Nevertheless, these mutants were highly attenuated in an in vivo mixed competition assay, indicating that ssaP, sseF and gtrA contribute to virulence, but the LD50 test revealed that their level of attenuation was less marked than for other mutants. Our results are in corroboration with a previous study in which an sseF mutant of S. typhimurium failed to demonstrate an attenuation of virulence by LD50 test, but revealed a CI defect (Hensel et al., 1998). It is possible that the kinetics of disease progression may be slower in these mutants, as the CI values in this study were measured 3 days p.i. while the LD50 was determined 7 days p.i. Further studies involving inoculation of such mutants by various parenteral routes and determination of their growth curves in the reticuloendothelial system of chickens at various stages of infection may be useful to elucidate the specific roles of such genes. Moreover, most of the genes identified in this study are parts of large operons. Thus, it is possible that in some mutants the transposon insertion occurred in a gene not directly responsible for attenuation, the relevant gene being the next one in the operon. In such cases, further studies involving allelic replacement without polar effect may confirm the gene responsible for attenuation.
In conclusion, our study provides the results of the first large-scale screening of STM mutants of SG and identification of virulence genes in a chicken (natural host) infection model. Most of the genes identified were not previously known to play a role in the virulence of SG, suggesting numerous future avenues of study towards elucidation of the virulence mechanisms as well as the evolutionary origin and host restriction of SG. The identification of SPI-13 and SPI-14 is interesting, because these genes have not been previously reported to play any role in the virulence of S. enterica in general and SG in particular. A gene encoding CCP has been identified, which is required for virulence of SG in chickens and could be the target for the development of new antibiotics. Over half of the mutants analysed did not cause mortality during the challenge experiment, indicating a strong possibility that these mutant strains represent new targets for the development of a rationally attenuated live vaccine against FT.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 16 April 2005;
revised 25 August 2005;
accepted 27 September 2005.
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