Departments of Bacteriology1 and Immunology, Pathology and Epidemiology2, Institute for Animal Science and Health, PO Box 65, 8200 AB Lelystad, The Netherlands
Author for correspondence: Hilde E. Smith. Tel: +31 320 238270. Fax: +31 320 238153. e-mail: h.e.smith{at}id.wag-ur.nl
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ABSTRACT |
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Keywords: Streptococcus suis, in vivo expression, iron-restriction-induced, virulence
Abbreviations: IVET, in vivo expression technology
The GenBank accession numbers for the sequences determined in this work are AF302190AF302207 (iri genes) and AF303226303247 (ivs genes).
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INTRODUCTION |
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To meet these shortages it is necessary to identify the genes that are involved in the pathogenic process. At present, however, only a limited number of Strep. suis genes are known (Smith et al., 1992 , 1993
, 1999
; Serhir et al., 1997
; Segers et al., 1998
; and GenBank accession nos AF106927, Z95920 and A57222) and of these, only a few are putatively involved in virulence (Smith et al., 1992
, 1993
, 1999
; Jacobs et al., 1994
; Gottschalk et al., 1995
; Segers et al., 1998
). Previously, putative virulence factors have been identified after growth of bacteria in standard laboratory media. However, it is known that many important virulence factors are environmentally regulated and are induced at specific stages of the infection process (Mahan et al., 1993
). Recently, several approaches have been reported that allow the identification of genes that are specifically expressed in the host. Examples are signature-tagged mutagenesis (STM) and in vivo expression technology (IVET; Mahan et al., 1993
, 1995
; Camilli & Mekalanos, 1995
; Hensel et al., 1995
; Mei et al., 1997
; Young & Miller, 1997
; Chiang & Mekalanos, 1998
; Coulter et al., 1998
; Lowe et al., 1998
; Polissi et al., 1998
; Camacho et al., 1999
; Darwin & Miller, 1999
; Edelstein et al., 1999
; Fuller et al., 1999
; Zhao et al., 1999
). In addition, important virulence proteins could also be identified by the selection of genes specifically expressed under conditions mimicking in vivo conditions, for example by growth under iron-restricted conditions (Litwin & Calderwood, 1993
; Martinez et al., 1990
).
The aim of the present work was to identify virulence genes of Strep. suis by the selection of environmentally regulated genes by the use of iron-restricted conditions in vitro and by experimental infection of piglets. For this purpose, chromosomal DNA fragments of Strep. suis were cloned in a plasmid in front of a promoterless erythromycin resistance gene. Subsequently, the library was used for the selection of bacteria in which erythromycin resistance was induced under iron-restricted conditions. In addition, we selected for erythromycin-resistant bacteria after infection of piglets with the library and treatment of the piglets with erythromycin. We used pigs instead of mice for these experiments as we recently showed that virulence of Strep. suis is different in these two animal species (Vecht et al., 1997 ).
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METHODS |
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DNA techniques.
Routine DNA manipulations and PCR reactions were performed as described by Sambrook et al. (1989 ). DNA sequences were determined on a 373A DNA Sequencing System (Applied Biosystems). Samples were prepared by using the ABI/PRISM Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems). Custom-made sequencing primers were purchased from Life Technologies. Sequencing data were assembled and analysed using the McMollyTetra software package. The BLAST program was used to search for protein sequences similar to the deduced amino acid sequences.
PCR reaction mixtures (50 µl) contained 10 mM Tris/HCl pH 8·3, 1·5 mM MgCl2, 50 mM KCl, 0·2 mM of each of the four deoxynucleotide triphosphates, 1 µM of each of the primers and 1 U of AmpliTaq Gold DNA Polymerase (Perkin Elmer-Applied Biosystems). DNA amplification was carried out in a Perkin Elmer 9600 thermal cycler and the program consisted of incubation for 10 min at 95 °C and 30 cycles of 1 min at 95 °C, 2 min at 56 °C and 2 min at 72 °C.
Assessment of erythromycin levels in treated piglets.
One-week-old specific-pathogen-free (SPF) piglets were treated orally with erythromycin stearate [Abbott; 20 or 40 mg (kg body wt)-1] or intramuscularly with erythromycin [Erythrocin 200, Sanofi Santé; 20 or 40 mg (kg body wt)-1]. Blood samples were collected 3, 6 or 24 h after the administration of the antibiotics to determine erythromycin levels. To do this, 1 ml of blood sample was mixed for 30 min on a rotator at 40 r.p.m. with 4 ml 0·1 M sodium acetate buffer pH 4 and 4 ml acetonitrile. The sample was centrifuged for 10 min at 1500 g and the aqueous phase was collected. The pH of the sample was adjusted to a value of 9 by the addition of 1 ml 0·5 M potassium phosphate buffer pH 8 and 0·25 ml 10 M KOH. Subsequently, the sample was mixed with 5 ml chloroform on a rotator for 15 min at 40 r.p.m. Three millilitres of the lower organic phase was collected and evaporated to dryness with a gentle stream of nitrogen at 40 °C. The remaining residue was dissolved in 1 ml 0·1 M phosphate buffer pH 8 and used to determine the erythromycin concentration. On MuellerHinton agar plates containing Micrococcus luteus (ATCC 9341), 14 mm holes were punched. Duplicate 0·25 ml volumes of sample extracts and of standards (erythromycin stock solution with a concentration range from 0·01 to 0·08 µg ml-1) were added to the holes. The plates were incubated for 1618 h at 30 °C, the inhibition zones were measured and the erythromycin concentrations were calculated.
Experimental infections.
Gnotobiotic Great Yorkshire and Dutch Landrace cross piglets were obtained from sows by Caesarean section. The surgery was performed in sterile flexible film isolators. The piglets were allotted to groups, each consisting of four animals, and were housed in sterile stainless steel incubators. Housing conditions and feeding regimens were as described previously (Vecht et al., 1989 , 1992
). One-week-old piglets were inoculated intravenously with Strep. suis strain 10(pIVS-E), strain 10(pIVS-PE) or strain 10(pIVS-RE) as described previously (Vecht et al., 1989
, 1992
; see Table 3
). Two hours after infection the pigs were injected intramuscularly with erythromycin for the first time and thereafter received erythromycin twice a day, once intramuscularly [Erythrocin; 40 mg (kg body wt)-1] and once orally [erythromycin stearate; 40 mg (kg body wt)-1]. Piglets were monitored twice a day for clinical signs of disease, such as fever, symptoms of meningitis and encephalitis, and lameness. Blood samples were collected three times a week from each pig. Leucocyte concentrations were determined using a cell counter (Contraves). To monitor infection with Strep. suis and to check for absence of contaminants, we collected swabs of the nasopharynx and of faeces daily. The swabs were plated directly onto Columbia agar containing 6% (v/v) horse blood. After the piglets had been killed, they were examined for gross pathological changes. Tissue specimens were collected from the central nervous system, serosae (peritoneum, pericardium), joints, lungs, heart and tonsils. The tissues were homogenized in the presence of ToddHewitt medium using an Ultra-Turrax tissuemizer (Omni International) and frozen at -80 °C in the presence of 15% (v/v) glycerol.
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RESULTS |
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Selection of promoters induced under iron-restricted conditions
We first selected for gene sequences that were specifically induced on agar plates under iron-restricted conditions. For this purpose, about 96000 c.f.u. Strep. suis were plated under iron-limiting conditions on agar plates containing deferoxamine mesylate and erythromycin. The 1500 colonies that grew on these plates were inoculated onto plates containing erythromycin, deferoxamine mesylate and FeSO4. Twenty-four clones showed reduced growth in the presence of FeSO4. The inserts of the 24 selected iri clones were amplified by PCR using primers complementary to the 5' ends of the erythromycin and spectinomycin resistance genes and the nucleotide sequences of these fragments were determined. The sequence data showed that the 24 clones contained 18 unique sequences (Table 2). The 18 sequences were analysed for similarity to known genes by comparison with the sequences in the GenBank/EMBL and SWISS-PROT databases. One sequence, iri-31, was identical to cps2A, a previously identified Strep. suis gene putatively involved in the regulation of capsule expression (Smith et al., 1999
). Fourteen iri sequences were similar to sequences of known, non-Strep. suis, genes. Three of these sequences (iri-2, iri-1, 6 and 22, and iri-34) were similar to sequences of environmentally regulated genes previously selected by applying the IVET to Vibrio cholerae (Camilli & Mekalanos, 1995
), Staphylococcus aureus (Lowe et al., 1998
) and Pseudomonas aeruginosa (Wang et al., 1996
), respectively. One, contained in iri-1, 6 and 22, was similar to the agrA gene of Staph. aureus, a key locus involved in the regulation of numerous virulence proteins. Three iri sequences had no significant similarity to any sequences in the databases (Table 2
).
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To test this hypothesis we inoculated pigs either with Strep. suis strain 10(pIVS-PE) or with strain 10(pIVS-E). In pIVS-PE the promoter of the mrp gene of Strep. suis (Smith et al., 1992 ), which is highly expressed in vivo as well as in vitro, drives expression of the erythromycin resistance gene. The control plasmid, pIVS-E, does not contain a promoter in front of the erythromycin resistance gene. The strains were inoculated intravenously or intranasally. All pigs infected with strain 10(pIVS-PE) showed specific clinical signs of Strep. suis infection (Table 3
) and, except for one, all pigs died in the course of the experiment. Moreover, high numbers of bacteria were isolated from the central nervous system, the serosae and from the joints. In contrast, none of the pigs inoculated with strain 10(pIVS-E) showed specific clinical signs of disease and all survived the infection until the end of the experiment. Moreover, bacteria were not isolated from the central nervous system, the serosae or from the joints of these animals. These data clearly demonstrated that in-vivo-expressed sequences could be selected from pigs using the applied antibiotic treatment regimen.
Selection of gene sequences expressed in vivo in piglets
Piglets were inoculated intravenously with different doses (5x1055x108 c.f.u.) of the Strep. suis library (Table 3) and treated with erythromycin as described above. Specific signs of disease developed in all animals 38 d after infection (Table 3
). High numbers of bacteria were recovered from tissues (central nervous system, joints, serosae, lung, liver, spleen, heart and kidney) of the individual piglets. Analysis of the recovered bacteria showed that only a limited number of different clones were present in each of the bacterial samples isolated from the diseased pigs. For example, 30 randomly selected clones from the joints of one pig all possessed identical DNA inserts as assessed by PCR and DNA sequence analysis (results not shown). In addition, at 80% of the 62 sample sites analysed, four randomly selected clones were all identical. However, from different tissues of a single animal, different clones could be isolated. On the other hand, identical clones could be isolated from different as well as from corresponding tissues of different animals. These findings indicated that a limited number of clones had been selected in vivo and were greatly enriched in the affected tissues. The observed selection was not tissue-specific. Finally, none of the selected clones failed to grow on agar plates that contained 1 µg erythromycin ml-1.
Two hundred and forty-five clones were analysed by PCR and partial sequence analysis. Among these, 22 unique ivs clones were found. The 22 sequences were analysed for similarity to sequences of known genes by comparison with the GenBank/EMBL and SWISS-PROT databases (Table 4). The sequences of two genes showed similarity to genes encoding putative virulence factors: ivs-21, 26 and 30 which was identical to epf, a previously identified Strep. suis gene putatively involved in virulence (Smith et al., 1993
, 1996
); and ivs-31, which was similar to the fibronectin-binding protein of Streptococcus gordonii. Moreover, the sequences of two ivs genes (ivs-25 and ivs-6, 7, 13 and 14) were homologous to two environmentally regulated ivi genes, previously identified using IVET selection in other bacterial species (Camilli & Mekalanos, 1995
; Lowe et al., 1998
). Four ivs sequences (ivs-25; ivs-23 and 24; ivs-2, 4 and 28; and ivs-6, 7, 13 and 14) were also found when the library was selected using iron-restricted conditions. The remainder of the sequences showed similarity to sequences of known, non-Strep. suis genes, including two showing similarity to mobile elements and five showing similarity to genes of unknown function.
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DISCUSSION |
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We used a promoter trap to identify environmentally regulated Strep. suis genes expressed under specific conditions, i.e. during iron restriction or during experimental infection. This system differs from the antibiotic-based IVET system described for Salmonella typhimurium (Mahan et al., 1995 ) in two ways. One is that the lacZ reporter gene fusion is omitted in our vector constructions because inclusion of the lacZ gene resulted in structural instability of the vector. The other difference is that we used a plasmid system rather than a chromosomal integration system. We used a plasmid system because the low transformation efficiency of Strep. suis (Smith et al., 1995
) might prevent the generation of a complete gene library using a chromosomal integration system. From the data obtained, it is evident that we selected for a number of inducible and environmentally regulated sequences. Interestingly, four iri genes were identical to four ivs genes. Because most bacteria require iron for their growth and because there is a limited amount of free iron available within the host (Payne, 1993
), it might be expected that the expression of some ivs genes is regulated by iron. With the in vivo selection system we did not observe tissue-specific colonization: clones isolated from one piglet were also isolated from other piglets from corresponding as well as from different tissues. Furthermore, it was striking, and different to the observations made with IVET systems, that only a limited number of clones could be selected. This might be due to the mechanisms involved in the molecular pathogenesis of Strep. suis infections in pigs. Alternatively, a limited number of clones might be found because essentially all selection occurred in the initial period when the bacteria were still in the bloodstream. Further experiments will be required to discriminate between these possibilities. In addition, we were not able to demonstrate that we selected for gene sequences that are exclusively expressed in vivo. This could be explained either by the absence of promoter sequences exclusively expressed in vivo among the 22 identified ivs genes, and/or by the inability of this plasmid-based system to identify such sequences due to gene dose effects.
A number of interesting genes were selected. Two ivs genes showed similarity to genes encoding putative virulence factors. ivs-21, 26 and 30 were shown to be identical to the epf gene of Strep. suis (Smith et al., 1993 ), which is found in virulent strains of Strep. suis serotypes 1 and 2 (Stockhofe-Zurwieden et al., 1996
; Vecht et al., 1991
, 1992
). ivs-31 showed similarity to the fibronectin/fibrinogen-binding protein of Strep. gordonii (GenBank accession no. X65164) and group A streptococci (Courtney et al., 1994
). In streptococci, fibronectin/fibrinogen-binding proteins play an important role in adhesion to host cells and are considered to be important virulence factors. The selection of these two ivs genes clearly demonstrated the selectivity of the system and might be indicative of the relevance of the other ivs genes in the pathogenesis of Strep. suis infections in pigs. The performance of the system was further demonstrated by the observation that two ivs genes, ivs-25 and ivs-6, 7, 13 and 14, showed similarity to environmentally regulated genes previously identified using an IVET selection system in other bacterial species.
ivs-25 showed significant similarity to the sapR gene of Streptococcus mutans (GenBank accession no. P72485) and Lactobacillus sake Lb706 (Axelsson & Holck, 1995 ) as well as to the agrA gene of Staph. aureus (Projan & Novick, 1997
), both of which encode response regulator proteins of bacterial two-component signal transduction systems, thereby mediating the response to an environmental signal (Projan & Novick, 1997
). Use of an IVET selection system for Staph. aureus in mice selected the region preceding the agrA gene, suggesting induction of agrA expression under in vivo conditions (Lowe et al., 1998
). Moreover, in Staph. aureus the agr locus was shown to play an important role in altering the expression of a considerable number of virulence factors in response to cell density (Projan & Novick, 1997
). In future experiments the putative role of ivs-25 in the expression of virulence factors in Strep. suis will be analysed further.
Clones ivs-6, 7, 13 and 14 showed similarity to a gene, iviVI, previously identified by IVET selection in V. cholerae (Camilli & Mekalanos, 1995 ). The function of iviVI is unknown. However, the genes showed similarity to members of the ATP-binding cassette family of transporters. The sequenced portion of ivs-6, 7, 13 and 14 included an N-terminal ATP-binding Walker A box motif, which is highly conserved in this transporter family.
Four ivs genes were identical to four iri genes. The first gene, ivs-23 and 24, which is identical to iri-24, showed similarity to cpsY of Streptococcus agalactiae (Koskiniemi et al., 1998 ) and to oxyR of various organisms (Demple, 1999
). CpsY of Strep. agalactiae is involved in the regulation of capsule expression and environmental induction of expression of the cpsY gene has been suggested by Koskiniemi et al. (1998
). In Strep. suis, ivs-23 and 24 and iri-24 are not linked to the capsular locus (Smith et al., 1999
). The oxyR gene is the central regulator of oxidative stress response in E. coli (Demple, 1999
) and approximately 10 genes are under the control of the OxyR protein. The second gene, ivs-2, 4 and 28, which is identical to iri-10 and 20, showed similarity to the yoaE gene of E. coli (GenBank accession no. P76262), a putative ABC transporter protein. The third and the fourth genes, ivs-25 and ivs-6, 7, 13 and 14 were identical to iri-1, 6 and 22 and iri-2, respectively. These genes also showed similarity to ivi genes selected using IVET in other bacterial species.
Based on data recently presented by Niven et al. (1999 ), selection of iri genes of Strep. suis might not be expected. These authors suggested that Strep. suis does not require iron for growth. However, in their studies these authors used media with iron concentrations reduced using EDDA. Therefore, the different conditions used in vitro may explain the different results obtained.
Two of the Strep. suis ivs genes, ivs-1 and ivs-8, showed similarity to transposon sequences. Moreover, one Strep. suis ivs gene, ivs-2, 4 and 28, had a G+C content that was considerably higher than that of the rest of the selected genes. The relevance of these ivs genes in the pathogenesis of Strep. suis infection in pigs needs to be investigated further. However, it is striking that in Sal. typhimurium several of the ivi clones that are required for full virulence have been found to be associated with mobile elements. Their atypical base composition and codon usage has led to the suggestion that they have been acquired from other bacterial species by horizontal transfer (Conner et al., 1998 ).
Importantly, our screen identified five ivs genes that showed similarity to sequences encoding proteins of unknown function. It seems unlikely that these genes are standard housekeeping or metabolic genes. Therefore, strains of Strep. suis carrying mutations in each of these genes are currently being constructed and the effect of these mutations on bacterial virulence will be examined.
Besides the four ivs/iri genes, a considerable number of other iri genes have been selected in this study by plating the library under iron-restricted conditions. Interestingly, one of the selected iri genes, iri-31, is identical to the cps2A gene of Strep. suis. This gene was previously isolated as a part of the capsular locus of Strep. suis serotype 2 (Smith et al., 1999 ) and was implicated in the regulation of capsular polysaccharide biosynthesis (Kolkman et al., 1997
; Smith et al., 1999
). Moreover, because the size of the capsule of Strep. suis is greater after growth in vivo than after growth in vitro (Quessy et al., 1994
), regulated expression of cps2A might be expected. Another iri gene, iri-7, showed similarity to the rpgG gene of Strep. mutans. This gene was shown to be required for the biosynthesis of rhamnose-glucose polysaccharide (Yamashita et al., 1999
). Because rhamnose is part of the polysaccharide capsule in Strep. suis serotype 2 (Elliott & Tai, 1978
), a role of the iri-7 gene in capsule biosynthesis can be proposed. iri-34 showed similarity to the np16 gene, previously identified using IVET selection in P. aeruginosa and suspected to encode threonine dehydratase activity (Wang et al., 1996
). Together with the observation that four iri genes could be selected by the in vivo approach, these data suggest that a number of the iri genes may encode important virulence factors for Strep. suis.
The promoter selection system described in this paper has been successfully used for the identification of many environmentally regulated genes potentially involved in the pathogenesis of Strep. suis infections in piglets. To further investigate the role of these genes, strains of Strep. suis carrying mutations in each of these genes need to be constructed and their effect on virulence needs to be examined in piglets.
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ACKNOWLEDGEMENTS |
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Received 13 June 2000;
revised 12 November 2000;
accepted 20 November 2000.