Lipoprotein signal peptidase of Streptococcus suis serotype 2

Astrid de Greeff1,2, Andrea Hamilton4, Iain C. Sutcliffe4, Herma Buys2, Loek van Alphen1,3 and Hilde E. Smith2

1 Department of Medical Microbiology, University of Amsterdam, 1100 DD Amsterdam, The Netherlands
2 Division of Infectious Diseases and Food Chain Quality, Cluster of Endemic Diseases, Institute of Animal Science and Health, 8200 AB Lelystad, The Netherlands
3 Laboratory for Vaccine Research, RIVM, National Institute of Public Health and the Environment, 3720 BA Bilthoven, The Netherlands
4 Institute of Pharmacy, Chemistry and Biomedical Sciences, University of Sunderland, UK

Correspondence
Astrid de Greeff
a.degreeff{at}id.wag-ur.nl


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
This paper reports the complete coding sequence for a proliprotein signal peptidase (SP-ase) of Streptococcus suis, Lsp. This is believed to be the first SP-ase described for S. suis. SP-ase II is involved in the removal of the signal peptide from glyceride-modified prolipoproteins. By using in vitro transcription/translation systems, it was shown that the lsp gene was transcribed in vitro. Functionality of Lsp in Escherichia coli was demonstrated by using an in vitro globomycin resistance assay, to show that expression of Lsp in E. coli increased the globomycin resistance. An isogenic mutant of S. suis serotype 2 unable to produce Lsp was constructed and shown to process lipoproteins incorrectly, including an S. suis homologue of the pneumococcal PsaA lipoprotein. Five piglets were inoculated with a mixture of both strains in an experimental infection, to determine the virulence of the mutant strain relative to that of the wild-type strain in a competitive challenge experiment. The data showed that both strains were equally virulent, indicating that the knockout mutant of lsp is not attenuated in vivo.


The GenBank accession number for the sequence of lsp reported in this paper is AF303229.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Streptococcus suis is a Gram-positive organism that is an important cause of meningitis, arthritis and septicaemia in piglets. Piglets often do not survive the disease (Clifton-Hadley, 1983; Vecht et al., 1985). Sows are symptomless carriers of S. suis on their tonsils and pass the bacteria on to their piglets, which are sensitive to the infection. Occasionally, humans get infected by S. suis (Arends & Zanen, 1988). Control of the disease is hampered by the lack of effective vaccines and sufficient, sensitive diagnostic tools. PCR assays have been developed for the detection of serotypes 1, 2, 7 and 9 (Smith et al., 1999b; Wisselink et al., 1999, 2002). For other serotypes no sensitive diagnostics are available yet. For development of vaccines and diagnostics, more insight into the process of pathogenesis will be very helpful. Virulence factors of S. suis are attractive vaccine candidates and can potentially be used as tools for detection of virulent serotypes of S. suis. Recently, an in vivo expression system was used to identify environmentally regulated promoters under different selection conditions (Smith et al., 2001). With this system, 36 environmentally regulated genes were identified.

One of these genes, ivs-23/iri-24 (Smith et al., 2001) showed similarity in the database to the regulatory genes cpsY of Streptococcus agalactiae (Koskiniemi et al., 1998) and oxyR of Escherichia coli (Demple, 1999). In S. agalactiae, a putative regulatory function on capsule expression was attributed to cpsY (Koskiniemi et al., 1998). Because, in S. suis, ivs-23/iri-24 is not linked to the capsule operon (Smith et al., 1999a), we here determined the gene sequences flanking ivs-23/iri-24 in S. suis. In this paper we describe a putative operon expressing the transcriptional regulator and show that the operon contains a prolipoprotein signal peptidase. This type II signal peptidase of S. suis (lsp) was cloned and characterized. Functionality of Lsp was demonstrated in E. coli. An insertional knockout mutant of lsp was constructed and the role of the prolipoprotein signal peptidase in the pathogenesis of S. suis was studied by comparing wild-type and mutant strains in an experimental infection in piglets. The data show that the lsp mutant is not attenuated in vivo.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and culture conditions.
The bacterial strains and plasmids used in this study are listed in Table 1. S. suis strains were grown in Todd–Hewitt broth (code CM 189; Oxoid) and plated on Columbia blood base agar plates (code CM 331; Oxoid), containing 6 % (v/v) horse blood. E. coli strains were grown in Luria broth (Miller, 1972) and plated on Luria broth containing 1·5 % (w/v) agar. If required, antibiotics were added in the following concentrations: chloramphenicol (Roche), 3·4 µg ml-1 for S. suis and 10 µg ml-1 for E. coli; and ampicillin (Roche), 100 µg ml-1 for E. coli.


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Table 1. Bacterial strains and plasmids

 
In vitro transcription/translation assay.
This assay was performed using the Prokaryotic DNA-Directed Translation Kit (Amersham Life Science) according to the manufacturer's instructions. TRAN35S-LABEL (containing 70 % L-[35S]methionine) (43·5 GBq mmol-1; ICN Biomedicals) was used to label de novo-synthesized proteins. After the in vitro transcription/translation, SDS-PAGE was performed with the NuPAGE system according to the manufacturer's instructions. Proteins were separated on a 4–12 % gradient gel in NuPAGE MES buffer. The gel was dried under vacuum and the signal was detected using a phosphor-imager (STORM, Molecular Dynamics).

Globomycin resistance assay.
Stationary-phase E. coli cells were diluted 1 : 50 in Luria broth containing appropriate antibiotics and grown at 37 °C for 8 h. Ten microlitres of exponential-phase E. coli culture was used to inoculate 100 µl Luria broth containing various concentrations of globomycin (a generous gift of Dr Shunichi Miyakoshi, Sankyo Co. Ltd, Tokyo, Japan). The globomycin concentrations tested were 0, 10, 20, 40, 80, 160 and 320 µg ml-1. The cells were allowed to grow for 16 h, and the OD630 was measured in a spectrophotometer.

DNA manipulations and sequence analysis.
Routine DNA manipulations 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 use of an ABI Prism dye terminator cycle sequencing ready reaction kit (Applied Biosystems). Sequencing data were assembled and analysed using the Lasergene program (DNASTAR). The BLAST software tool (Altschul et al., 1997) was used to search for protein sequences homologous to the deduced amino acid sequences in the GenBank/EMBL databases using the National Center for Biotechnology Information server (http://www.ncbi.nlm.nih.gov/BLAST/), typically using the tBlastN option with sequence filtering switched off and the maximum Expect value set at 0·001. Profile scanning for significant matches to database motifs was performed using the on-line ISREC ProfileScan Server (http://hits.isb-sib.ch/cgi-bin/hits_motifscan).

Southern blotting and hybridization.
Chromosomal DNA was isolated as described by Sambrook et al. (1989). After digestion with restriction enzymes, DNA fragments were separated on 0·8 % (w/v) agarose gels and transferred to GeneScreen Plus hybridization transfer membrane (NEN Life Science Products) as described by Sambrook et al. (1989). DNA probes for the lsp and chloramphenicol resistance genes were labelled with [{alpha}-32P]dCTP (111 GBq mmol-1; Amersham Life Science) by use of a random-primed DNA labelling kit (Boehringer). The DNA on the blots was pre-hybridized at 65 °C for at least 30 min and subsequently hybridized at 65 °C for 16 h with the appropriate DNA probes in a buffer containing 0·5 M sodium phosphate, pH 7·2, 1 mM EDTA and 7 % SDS. After hybridization, the membranes were washed twice with a buffer containing 40 mM sodium phosphate, pH 7·2, 1 mM EDTA and 5 % SDS at 65 °C for 30 min and twice with a buffer containing 40 mM sodium phosphate, pH 7·2, 1 mM EDTA and 1 % SDS at 65 °C for 30 min. The signal was detected using a phosphor-imager (Storm, Molecular Dynamics).

Construction of a lsp knockout mutant
To construct the mutant strain 10{Delta}Lsp, the pathogenic strain 10 (Vecht et al., 1989, 1992) of S. suis serotype 2 was electrotransformed (Smith et al., 1995) with the plasmid pLSP-3 (Fig. 1). In this plasmid the lsp gene was inactivated by the insertion of a resistance cassette, consisting of the chloramphenicol resistance gene from pC194 (Horinouchi & Weisblum, 1982b), preceded by the promoter region of the mrp gene (Smith et al., 1992). To create pLSP-3, pLSP2 (Fig. 1) was digested with StuI, and ligated to the 900 bp SmaI–EcoRV fragment from pUK21-Cm containing the chloramphenicol resistance cassette preceded by the promoter region of the mrp gene. The ligation mixture was transformated to E. coli XL-1blue cells, and ampicillin- and chloramphenicol-resistant colonies were selected on Luria broth containing 1·5 % (w/v) agar, 100 µg ampicillin ml-1 and 10 µg chloramphenicol ml-1. After electrotransformation of strain 10 with pLSP-3, chloramphenicol-resistant colonies were selected on Columbia blood base agar plates containing 3·4 µg chloramphenicol ml-1. Southern blotting and hybridization experiments were used to screen for double-crossover integration events (data not shown).



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Fig. 1. Schematic representation of pLSP-2 and pLSP-3. A 3 kb ClaI–DraI fragment that hybridized with probe ivs-23/iri-24 was cloned into pGEM7Zf(+) digested with ClaI and SmaI, yielding pLSP-2. To create pLSP-3, the 900 bp SmaI–EcoRV fragment of pUK21-Cm, containing the chloramphenicol resistance gene from pC194 (Horinouchi & Weisblum, 1982b), preceded by the promoter region of the mrp gene (Smith et al., 1992), was inserted in the StuI site of lsp.

 
Phenotypic characterization of the lsp knockout mutant.
S. suis strains S10 and 10{Delta}Lsp were grown in the presence and absence of [U-14C]palmitate (Amersham Bioscience) to allow radiolabelling of lipoproteins. Overnight cultures of S. suis S10 and 10{Delta}Lsp were diluted 1 : 20 into 5 ml pre-warmed Todd–Hewitt broth in triplicate. They were incubated at 37 °C for 1–2 h (early exponential phase), before the addition of either [14C]palmitate (25 µCi [925 kBq] in 137 µl ethanol) or ethanol (137 µl) as controls. Control broths were used to monitor growth (OD595) until late-exponential phase. Cells from the cultures containing radiolabel were harvested by microcentrifugation (13 000 g for 5 min), resuspended in 1 ml sterile phosphate-buffered saline and recentrifuged. This was repeated three times and the washed cells were then prepared for SDS-PAGE analysis as described previously (Hamilton et al., 2000). After drying, the gels were exposed to Kodak BIOMAX MR2 film (Anachem) at -80 °C for about 14 days. Autoradiograms were developed using Kodak GBX developer and fixer (Sigma) according to the manufacturer's instructions.

Western blotting following SDS-PAGE was performed as described previously (Hamilton et al., 2000) using a rabbit polyclonal antiserum raised against the PsaA putative lipoprotein of Streptococcus pneumoniae (kindly supplied by Dr Jacquelyn Sampson, National Center for Infectious Diseases, Atlanta, GA, USA). This antiserum cross-reacts with PsaA and other lipoproteins in the streptococcal LraI family (Harrington et al., 2000). BLAST searches of the draft S. suis genome, which has been made available by the Sanger Centre (http://www.sanger.ac.uk/Projects/S_suis/) suggested the presence of a PsaA homologue (LraI family member) in S. suis and that this sequence includes a signal peptide matching the consensus for proven Gram-positive bacterial lipoproteins (Sutcliffe & Harrington, 2002).

Experimental infections.
Germfree piglets, crossbreeds of Great Yorkshire and Dutch Landrace, were obtained from sows by caesarean sections. The surgery was performed in sterile flexible film isolators. Piglets were allotted to groups of 5, and were housed in sterile stainless steel incubators. Housing conditions and feeding regimens were as described before (Vecht et al., 1989, 1992). Six-day-old piglets were inoculated intranasally with about 107 c.f.u. of Bordetella bronchiseptica 92932, to predispose them to infection with S. suis. Two days later the piglets were inoculated intranasally with 106 c.f.u. of S. suis strain 10 plus 106 c.f.u. of S. suis strain 10{Delta}Lsp. To determine differences in virulence between wild-type and mutant strains, LD50 values should be determined. To do this, large numbers of piglets are required, which for ethical reasons is not acceptable. To circumvent this problem, we performed co-colonization studies. To monitor for the presence of S. suis and B. bronchiseptica and to check for absence of contaminants, swabs taken from the nasopharynx and the faeces were cultured three times a week. The swabs were plated directly onto Columbia agar containing 6 % horse blood, or grown for 48 h in Todd–Hewitt broth and subsequently again plated onto Columbia agar containing 6 % horse blood. Piglets were monitored twice a day for clinical signs and symptoms, such as fever, nervous signs and lameness. Blood samples from each piglet were collected three times a week. Leukocytes were counted with a cell counter. The piglets were killed when specific signs of an S. suis infection were observed, such as arthritis or meningitis, or when they became mortally ill. The other piglets were killed 2 weeks after inoculation with S. suis. All piglets were examined for pathological changes. Tissue specimens from heart, lung, liver, kidney, spleen and tonsil, and from the organs specifically involved in an S. suis infection (central nervous system, serosae and joints), were sliced with a scalpel or a tissuemiser. Tissue slices from each organ or site were resuspended in 2–25 ml Todd–Hewitt broth containing 15 % (v/v) glycerol, depending on the size of the tissue slice. The suspension was centrifuged at 3000 r.p.m. for 5 min. The supernatant was collected and serial dilutions were plated on Columbia agar containing 6 % horse blood, as well as on Columbia agar plates containing 6 % horse blood and 3·4 µg chloramphenicol ml-1 to quantitate the number of wild-type and mutant bacteria present. The number of mutant strain 10{Delta}Lsp cells was determined by counting the number of c.f.u. on the appropriate serial dilution on the selective plates; the number of wild-type strain 10 cells was determined by counting the number of c.f.u. on the appropriate serial dilution on the Columbia agar blood plates, from which the number of c.f.u. counted on the selective plates was subtracted. When both wild-type and mutant bacteria were found in tissues, the ratio of wild-type and mutant strain was determined more precisely, by toothpicking about 100 individual colonies onto both Columbia blood base agar plates and Columbia blood base agar plates containing 3·4 µg chloramphenicol ml-1.

All animal experiments were approved by the ethical committee of the Institute for Animal Science and Health in accordance with the Dutch law on animal experiments.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
lsp is located in a putative operon
One of the clones selected from a genomic library of S. suis serotype 2 was induced both in vivo and under iron-restricted conditions. This clone, ivs-23/iri-24, showed similarity in the database to the 5' part of cpsY of Streptococcus agalactiae, and to oxyR of E. coli (Smith et al., 2001). A 32P-labelled probe of ivs-23/iri-24 was used to identify the flanking regions from chromosomal DNA of S. suis strain 10. A 3 kb ClaI–DraI fragment was detected and cloned into pGEM7Zf(+), yielding pLSP-2 (Fig. 1). The complete sequence of the insert was determined. Examination of the sequence revealed the presence of three complete and partially overlapping ORFs, and two incomplete ORFs (Fig. 1). The two incomplete ORFs are located at the 5' and 3' ends of the sequence and showed similarity in the database to genes encoding a regulatory protein for C-P lyase of Streptomyces griseus and to a gene encoding PHNA protein of E. coli respectively. 5' of the first complete ORF, a putative -35 and -10 promoter sequence was found. Upstream of each start codon putative RBSs were found. 3' of the third complete ORF, putative -35 and -10 promoter sequences were found. Together these data suggest that the three ORFs are transcribed as an operon and that the incomplete ORFs at the 3' and 5' end of the sequence do not belong to this operon.

The first ORF of the putative operon encoded a 304 amino acid protein that was highly similar (219/300 [73 %] amino acid identity) to Streptococcus pneumoniae SP0927, a putative transcriptional regulator of the LysR family, along with similarities to other regulators such as CpsY and OxyR (as described above). Sequence analysis confirmed that this translated sequence matched the database motifs for both the LysR family helix–turn–helix (Pfam PF00126) and substrate binding (Pfam PF03466) domains. The second ORF of the putative operon encoded a 154 amino acid protein that was highly similar (91/152 [59 %] amino acid identity) to Streptococcus agalactiae SAG1366 and to Streptococcus pneumoniae SP0928 (78/144 [54 %] amino acid identity), both of which are annotated as putative prolipoprotein signal peptidases (Lsp; type II signal peptidase). Sequence analysis confirmed that this translated sequence matched the database motif for the signal peptidase II family (Pfam PF01252). Finally, the last ORF of the putative operon encoded a 297 amino acid protein that was highly similar (254/297 [85 %] amino acid identity) to Streptococcus pneumoniae SP0929, a putative ribosomal pseudouridine synthase. Sequence analysis confirmed that this translated sequence matched the database motifs for both an S4 domain (Pfam PF01479), which probably mediates binding to RNA, and the RluD pseudouridine synthase family (Pfam PF00849; PDOC00869). Pseudouridine synthases modify RNA base composition by converting uracil bases to pseudouridine (Conrad et al., 1998). Inspection of the published microbial genomes and the publicly available unpublished genomes for Streptococcus gordonii and Streptococcus mitis indicated that this putative three-ORF operon is well conserved within the streptococcal genomes (e.g. the SP0927–SP0929 locus: Tettelin et al., 2001). Moreover, chromosomal loci encoding putative Lsp and putative pseudouridine synthase enzymes are apparently uniformly present in the genomes of Gram-positive bacteria (e.g. LL0997 and LL0998 in the genome of Lactococcus lactis: Bolotin et al., 2001) although this gene arrangement is not conserved in other taxa such as the Proteobacteria. The upstream presence of the putative LysR family member is not conserved in the non-streptococcal Gram-positive bacterial genomes and a variety of genes may be found preceding lsp (e.g. L. lactis LL0996 encodes a putative lumazine synthase). In this respect it is noted that in all the available streptococcal genomes there is sequence overlap between the ORFs encoding the putative Lsp and the pseudouridine synthase, whereas the overlap between lysR and lsp is not fully conserved.

Sequence analysis of Lsp of S. suis
A hydrophobicity plot (Kyte–Doolittle) and various tools for the prediction of membrane-spanning domains revealed the presence of four hydrophobic regions (data not shown), suggesting a similar membrane localization for S. suis Lsp as described for other Lsp enzymes (Witke & Götz, 1995; Prágai et al., 1997; Tjalsma et al., 1999b). The protein sequence of S. suis Lsp was compared to the sequences of other characterized signal peptidase II enzymes, notably that of Bacillus subtilis, wherein five conserved domains forming a potential active site have recently been identified (Zhao & Wu, 1992; Sankaran & Wu, 1994a; Witke & Götz, 1995; Prágai et al., 1997; Tjalsma et al., 1999b). This analysis confirmed that the strictly conserved N-terminal Domain I aspartic acid (D14 in the B. subtilis sequence) which is necessary for enzyme stability and function (Sankaran & Wu, 1994b; Tjalsma et al., 1999b) is conserved in the Lsp sequence. The other five critical residues identified by Tjalsma et al. (1999b) are located in the two motifs NXXD (Domain III) and FNXAD (Domain V), wherein the two aspartates are proposed to form a catalytic dyad in the protease active site. All five of the critical residues identified in these two motifs are fully conserved in the S. suis Lsp sequence.

Lsp is expressed in vitro
Expression of Lsp was determined by using an E. coli-based in vitro transcription/translation system on pLSP-2. pGEM7Zf(+) was used as a negative control. Three proteins were expressed that corresponded very well to the predicted sizes of the S. suis LysR family protein, the putative pseudouridine synthase and Lsp (respectively 34·7, 32·8 and 17·5 kDa) (data not shown). The negative control pGEM7Zf(+) expressed the {alpha}-LacZ fragment as well as {beta}-lactamase (Amp) (data not shown). These data clearly showed that all three genes were expressed very efficiently from pLSP-2.

Globomycin resistance
To test whether Lsp was functional in E. coli, we used a globomycin sensitivity assay. Globomycin is a cyclic peptide antibiotic that specifically inhibits the processing of prolipoprotein to mature lipoprotein (Inukai et al., 1978). Gram-negative organisms, like E. coli, are especially sensitive to this antibiotic due to inhibition of the murein lipoprotein processing. Overexpression of cloned signal peptidase genes from both Gram-negative and Gram-positive bacteria was shown to cause globomycin resistance in E. coli (Tokunaga et al., 1983; Zhao & Wu, 1992; Witke & Götz, 1995). This globomycin resistance is generally used to demonstrate functionality of lipoprotein signal peptidases. pLSP-2 was used for the expression of Lsp. In pLSP-3, a chloramphenicol resistance cassette was inserted into the lsp gene to inactivate the gene. This construct was used as a negative control. As a second negative control, pGEM7Zf(+) without insert was included in the assay. E. coli cultures containing either one of the plasmids were grown overnight in media containing various concentrations of globomycin. After 16 h of growth the OD630 of the cultures was measured. Fig. 2 shows that E. coli harbouring pGEM7Zf(+) or pLSP-3 did not grow in the presence of a concentration of globomycin higher than 20–40 µg ml-1. In contrast, E. coli harbouring pLSP-2 could grow in the presence of a concentration of globomycin of at least 320 µg ml-1 (Fig. 2), although the maximum optical density measured for this strain was lower than for the other strains. This indicates that the expression of Lsp and/or either of the other proteins encoded on pLSP-2 slightly inhibited the growth of E. coli. The increase of globomycin resistance due to expression of Lsp demonstrates that lsp indeed encodes a signal peptidase II that is functional in E. coli.



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Fig. 2. Globomycin resistance assay. OD630 of E. coli cultures harbouring different plasmids was measured after 16 h of growth. Black bars, pLSP-2; white bars, pGEM7Zf(+); hatched bars, pLSP-3. Bars represent the means of triplicate experiments; error bars indicate the standard error of the mean (SEM).

 
Role of Lsp in pathogenesis of S. suis serotype 2
To test the role of Lsp in the pathogenesis of S. suis, an isogenic knockout mutant of Lsp was constructed in strain 10, yielding strain 10{Delta}Lsp. Strain 10{Delta}Lsp was constructed by electroporation of pLSP-3, in which the lsp gene was inactivated by insertion of a chloramphenicol resistance cassette, into S. suis (Fig. 1) (Smith et al., 1995). Southern blotting and hybridization experiments were used to discriminate isolates in which double-crossover events had occurred from those in which single-crossover events had occurred (data not shown). To characterize the mutant phenotype, the strain 10 parent and strain 10{Delta}Lsp were grown in the presence of radiolabelled palmitic acid, and radiolabelled lipoproteins were visualized after SDS-PAGE and autoradiography (Fig. 3). The signal peptide processing of at least four radiolabelled proteins was affected in strain 10{Delta}Lsp, as indicated by a shift of the radiolabelled protein band to a higher molecular mass, which is consistent with the accumulation of the prolipoprotein form (Fig. 3, lane 2). However, the appearance of doublets (prolipoprotein and putative ‘mature-like’ forms) suggested that some alternative path of lipoprotein processing was also active in the case of some lipoproteins. To verify the mutant phenotype further, Western blot analysis was performed using a polyclonal antibody to the pneumococcal PsaA lipoprotein. Consistent with the bioinformatic detection of a pneumococcal PsaA homologue (LraI family lipoprotein) in S. suis (see Methods), a cross-reacting band of about 35 kDa was detected in strain 10, whereas this band was shifted to a position consistent with the accumulation of the prolipoprotein in strain 10{Delta}Lsp (Fig. 4). Cumulatively, these data confirmed that lipoprotein processing is disrupted in strain 10{Delta}Lsp.



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Fig. 3. Radiolabelling of S. suis lipoproteins with [U-14C]palmitic acid. S. suis strains S10 and 10{Delta}Lsp were grown in the presence of [U-14C]palmitate and the labelled proteins were analysed by SDS-PAGE and autoradiography. Lane 1, S. suis strain S10; lane 2, S. suis strain 10{Delta}Lsp. Positions of the molecular mass standards in kDa are marked on the left. Some of the radiolalled bands shifted to higher molecular masses (consistent with the accumulation of prolipoproteins with uncleaved signal peptides) are indicated on the right ({bullet}).

 


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Fig. 4. Western blot analysis of the putative S. suis homologue of pneumococcal PsaA. Cell extracts of S. suis strains S10 and 10{Delta}Lsp were analysed by SDS-PAGE and Western blotting using a polyclonal anti-PsaA antibody. Lane 1, Bio-Rad prestained standards with molecular masses in kDa marked on the left. Lane 2, S. suis strain S10. Lane 3, S. suis strain 10{Delta}Lsp. The shifting to a higher molecular mass of the cross-reacting band in lane 3 is consistent with the accumulation of a prolipoprotein with an uncleaved signal peptide.

 
Because we were unable to determine LD50 values for the mutant in pigs for ethical reasons, it was decided to do a competitive co-colonization experiment with the wild-type strain. The virulence of strain 10{Delta}Lsp was compared to that of the wild-type in an experimental infection in piglets. The mutant strain 10{Delta}Lsp was mixed with wild-type bacteria in competition challenge experiments to determine the relative attenuation of the mutant strain. Using in vitro conditions, the growth rates of wild-type and mutant strain in Todd–Hewitt medium were found to be essentially identical (data not shown). Wild-type and mutant bacteria were inoculated at an actual ratio of 4 : 1 (1·5x106 c.f.u. of strain 10 and 4x105 c.f.u. of strain 10{Delta}Lsp), in five piglets (nos 1, 26, 42, 44 and 49). During the experiment, piglets that developed specific S. suis symptoms (meningitis, arthritis or mortal illness) were killed. Piglets that did not develop these symptoms were killed at the end of the experiment. Three out of five piglets developed central nervous signs (nos 26, 42 and 44); one of them also developed a severe pericarditis (no. 44). Piglet no. 49 developed a severe arthritis in one leg. Piglet no. 1 survived until the end of the experiment, but at the post-mortem section a peritonitis and probably a starting meningitis was observed. As shown in Fig. 5(a), wild-type and mutant bacteria were re-isolated from tonsils in a ratio of about 14 : 1 (wild-type : mutant), while the input ratio was 4 : 1. This indicated that the mutant bacteria colonized the tonsil less efficiently than the wild-type bacteria. Fig. 5(b) shows that no large differences were found in the ratio of re-isolated wild-type bacteria and mutant bacteria, nor in the number of organs that were colonized by wild-type or mutant bacteria. Taken together, these data indicate that, although the mutant seemed to colonize the tonsil slightly less efficient that the wild-type strain, the mutant strain 10{Delta}Lsp is as virulent as the wild-type strain.



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Fig. 5. Efficacy of colonization of wild-type and mutant bacteria on various organs of infected pigs. (a) Colonization of wild-type strain 10 and mutant strain 10{Delta}Lsp on the tonsils: {blacklozenge}, piglet no. 1; {square}, piglet no. 26; {blacktriangleup}, tonsil piglet no. 42; {triangleup}, tonsil piglet no. 44; {blacksquare}, tonsil piglet no. 49. (b) Colonization of the specific organs (CNS, central nervous system): {blacktriangleup}, CNS piglet no. 1; {blacksquare}, joints piglet no. 1; {bullet}, peritoneum piglet no. 1; {square}, spleen piglet no. 26; {triangleup}, CNS piglet no. 26; _, kidney piglet no. 42; {blacklozenge}, CNS piglet no. 42; x, joints piglet no. 42; +, CNS piglet no. 44; , joints piglet no. 44; {circ}, pericardium piglet no. 44; {triangledown}, spleen piglet no. 49; {blacktriangledown}, heart piglet no. 49; {lozenge}, joint piglet no. 49. Each symbol represents the numbers of wild-type or mutant bacteria isolated from one particular organ, from one pig.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this paper we describe the identification and characterization of Lsp, a lipoprotein signal peptidase of S. suis serotype 2. The lsp gene was found to be part of a putative operon containing three overlapping genes. These genes were preceded by a putative promoter sequence. Downstream of the third gene was another putative promoter sequence. These findings suggest that this putative operon contains only the three overlapping genes. Besides lsp, this putative operon contained a gene encoding a protein that is predicted to belong to the LysR family of transcriptional regulators and a gene encoding a putative pseudouridine synthase. This chromosomal locus was noted to be conserved in other streptococcal genomes, whilst the association of the lsp gene with that for a putative pseudouridine synthase is conserved in Gram-positive bacterial genomes generally. The LysR family transcriptional regulator was identified with an IVET system as being environmentally regulated (Smith et al., 2001). Two different conditions were used to select environmentally regulated promoters: iron-restricted conditions and in vivo conditions. With both selections, the regulator gene was found. It is generally accepted that environmentally regulated genes, especially in vivo-induced genes, are potential virulence factors (Mahan et al., 1993). For this reason we studied the role of the predicted Lsp enzyme encoded by the gene adjacent to the regulator in the pathogenesis of S. suis serotype 2.

Lsp is involved in the removal of the signal peptide from diacylglyceride-modified prolipoproteins (Sankaran & Wu, 1994a, b). In Gram-negative organisms several enzymes involved in the lipid-modification of prolipoproteins, including Lsp, are essential for normal growth, cell division and viability (Sankaran & Wu, 1994a, b; Gan et al., 1993; Gupta et al., 1993; Williams et al., 1989; Yamagata et al., 1982). In contrast, lsp of the Gram-positive organism B. subtilis is not necessary for growth and viability, even though the PrsA lipoprotein is an essential maturase enzyme (Tjalsma et al., 1999a). Moreover, in S. pneumoniae, one of the enzymes involved in lipid-modification, prolipoprotein diacylglyceryl transferase (encoded by lgt), is not essential for cell growth in vitro, but is essential for viability during infection (Petit et al., 2001). In Gram-positive organisms several roles have been attributed to lipoproteins such as participation in antibiotic resistance, ABC transporter systems, adhesion, protein export and extracytoplasmic folding, and sensory systems (Sutcliffe & Russell, 1995; Sutcliffe & Harrington, 2002). The abundance of putative lipoproteins in typical bacterial genomes (Sutcliffe & Harrington, 2002) suggests that this class of proteins is of considerable physiological significance. In streptococci other than S. suis, several lipoproteins have been shown to be involved in virulence (Burnette-Curley et al., 1995; Berry & Paton, 1996; Kitten et al., 2000; Brown et al., 2001; Marra et al., 2002). Moreover, mutants disrupted in lsp have been shown to be attenuated in signature-tagged mutagenesis studies of Staphylococcus aureus (Mei et al., 1997; Coulter et al., 1998).

Type II signal peptidase activity of Lsp was initially demonstrated using a globomycin assay. We showed that overexpression of Lsp in E. coli resulted in an increased resistance to globomycin. To study the role of Lsp in the pathogenesis of S. suis, an isogenic knockout mutant of lsp was constructed in a wild-type S. suis serotype 2 strain. To construct the mutant, we used a plasmid replicating in E. coli in which the lsp gene was inactivated by the insertion of a chloramphenicol resistance gene (pLSP-3). In the globomycin assay, E. coli containing pLSP-3 did not show an increased globomycin resistance, whereas the construct containing the intact lsp gene did. This strongly indicated that in pLSP-3, Lsp was non-functional. To confirm the phenotype of strain 10{Delta}Lsp, lipoproteins were radiolabelled by growth in the presence of radiolabelled [14C]palmitic acid. Comparison with the lipoprotein profile for strain 10 indicated that multiple radiolabelled bands accumulated in a higher molecular mass form, consistent with a failure to remove signal peptides from prolipoproteins (Fig. 3). This observation was confirmed by Western blot analysis, which demonstrated the accumulation in strain 10{Delta}Lsp of the prolipoprotein form of a putative LraI family member (PsaA homologue; Fig. 4).

The virulence of the S. suis lsp mutant was tested in an experimental infection model in piglets. Since we are unable to determine LD50 values for the mutant strain in pigs, it was decided to compare the virulence of strain 10{Delta}Lsp to the wild-type S. suis strain 10 in a competitive co-colonization assay in piglets. These kinds of co-colonization experiments have been successfully applied to determine the virulence of mutants of Actinobacillus pleuropneumoniae in piglets (Fuller et al., 2000). Moreover, we recently successfully used this procedure to determine the virulence of an isogenic knockout of a gene encoding a fibronectin- and fibrinogen-binding protein (de Greeff et al., 2002). The data clearly showed that the lsp mutant strain was capable of colonizing both the tonsil and the organs specific for an S. suis infection, as efficiently as the wild-type strain. This means that both strains are equally virulent, and that the knockout mutant of lsp is not attenuated in vivo. Thus this phenotype contrasts that of other Gram-pathogens defective in enzymes necessary for lipoprotein biosynthesis (Mei et al., 1997; Coulter et al., 1998; Petit et al., 2001). This could suggest that lipoproteins do not play a role in the pathogenicity of S. suis but may also indicate that lipoproteins can be processed via an alternative route, independently of Lsp. Our data (Fig. 4) indicate that the PsaA homologue at least is not processed by an alternative path but several other lipoproteins radiolabelled with palmitic acid may be (Fig. 4). Based on Southern blot experiments, we have no reason to assume that a second Lsp gene is present in S. suis, and BLAST searches of the draft S. suis genome indicate that there is only a single copy of Lsp (i.e. the sequence reported herein). Thus the processing of lipoproteins in strain 10{Delta}Lsp may be due to the presence of cryptic sites for other signal peptidases in some lipoprotein signal peptides. Alternatively, it may be that as yet undescribed pathway(s) for processing of lipoproteins exist in Gram-positive bacteria. This idea is supported by the observations of Tjalsma et al. (1999a), who showed that mature-like forms of the lipoprotein PrsA were still found in a knockout mutant of lsp in B. subtilis. Whilst these authors excluded the possibility of processing by type I signal peptidases taking over the function of Lsp, how the mature-like forms were alternatively processed could not be explained. Thus, based on our data, and the information available in the literature, we hypothesize that in S. suis alternative processing of lipoproteins can also take place.

In conclusion, we describe the cloning and characterization of the prolipoprotein signal peptidase of S. suis, Lsp. We also show that an isogenic mutant of lsp is not attenuated in vivo in piglets. Further research is necessary to determine whether lipoproteins can be alternatively processed in S. suis.


   ACKNOWLEDGEMENTS
 
We thank the people from the Animal and Laboratory Services and the people from Pathobiology of the ID-Lelystad for their assistance during the animal experiments.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 3 March 2003; accepted 5 March 2003.