Inactivation of the gene encoding zinc-binding lipoprotein 103 impairs the infectivity of Streptococcus suis

Abstract

The Streptococcus suis 103 gene product is an immunogenic and protective lipoprotein that is a component of an ATP-binding cassette transporter implicated in zinc uptake. Belonging to the same transcriptional unit and downstream of the 103 gene is a gene that encodes a homologue of the pneumococcal histidine triad (Pht) protein Pht309. In an intraperitoneal mouse model the virulence of a mutant lacking the 103 gene was more than 50 times lower than that of the wild-type (WT) parent strain, S. suis serotype 2 strain P1/7. In addition, the immunogenicity of this mutant was dramatically decreased. In striking contrast, the virulence and immunogenicity of a P1/7 mutant lacking the Pht309 gene were similar to those of the parent strain. These results demonstrate that the 103 lipoprotein is strongly involved in S. suis virulence and support the hypothesis that this lipoprotein might be an excellent candidate for vaccines aiming to achieve broad protection against streptococci.

Streptococcus suis is a gram-positive bacterium that causes important economic losses to the swine industry worldwide (1). This microorganism is also an emerging zoonotic agent, having been responsible in recent years for many human outbreaks in Southeast Asia (2). Serotype 2 of S. suis is the most frequently associated with disease in both pigs and humans; septicemia and meningitis are the main clinical manifestations of infections produced by this bacterial species (1).

The ATP-binding cassette (ABC) transporters play a key role in maintenance of bacterial fitness and are important for bacterial pathogenesis (3). Several ABC transporters related to divalent-cation uptake, such as ZnuACB and MtsABC, have been shown to be involved in the virulence of gram-negative and gram-positive bacteria, respectively (4,5). Since these proteins are located on the bacterial surface, their ability to confer protection against bacterial infections has been evaluated in a number of bacterial species. Vaccination of mice with the zinc-binding lipoprotein encoded by the SsuiDRAFT0103 gene (from this point on designated as 103) of S. suis serotype 2 strain 89/1591 conferred a high degree of protection against challenge with this pathogen (6). This lipoprotein is a member of an ABC transporter regulated by AdcR, a transcriptional regulatory factor of the MarR protein family (7). In the highly virulent S. suis serotype 2 strain P1/7 the gene homologous to the 103 gene is SSU0308. This gene is cotranscribed with SSU0309, which encodes a protein homologous to the pneumococcal histidine triad (Pht) proteins (8). The Pht proteins have been found only in pathogenic streptococcal species and are involved in the virulence of these organisms (9).

In this work we deleted the genes encoding the 103 and Pht309 proteins in the virulent P1/7 strain of S. suis in order to study the virulence and immunogenicity of the resulting S. suis mutants. The genes (Table I) were inactivated by precise in-frame deletion to avoid polar effects in the expression of downstream genes. Briefly, upstream and downstream regions of SSU0308 and SSU0309 (including partially coding regions) were amplified by overlap-extension polymerase chain reaction (PCR) with the use of the oligonucleotide primers listed in Table II and genomic DNA from S. suis P1/7 as the template. The purified PCR fragments were cloned into the EcoRI site of the thermosensitive suicide vector pSET4s (10). Recombinant plasmids were used to electroporate the S. suis P1/7. Electrotransformation and allelic replacement were performed as described elsewhere (8,11). Gene replacement in candidate clones was confirmed by PCR and sequencing of the region in the resulting mutants.

Table I.

Nomenclature of the genes inactivated in Streptococcus suis serotype 2 strain P1/7 in this work and their homologues in S. suis serotype 2 strain 89/1591

P1/7a	89/1591b	Putative function (and nomenclature used in this work)
SSU0308	SsuiDRAFT0103	ATP-binding cassette (ABC) transporter component; zinc-binding lipoprotein (103)
SSU0309	SsuiDRAFT1195	Pneumococcal histidine triad (Pht) protein (Pht309)

^aOpen reading frame (ORF) name (http://www.sanger.ac.uk/Projects/S_suis/).

^bORF name (http://www.ncbi.nlm.nih.gov).

Table II.

Oligonucleotide primers used in this work

Oligonucleotide	Sequence	Mutant constructed
103-ID1	5′-GCTTGCTCGGCTCAGAAG	UA5004 (Δ103)
103-ID2	5′-ACAGCTTCCTGACCAACC	UA5004 (Δ103)
103-ID3	5′-AGTGATCCTAAGAATGCG	UA5004 (Δ103)
103-ID4	5′-TGGATCTGCTTCAAGAGG	UA5004 (Δ103)
103-ID5a	5′-CGAATTCAGTCACCTCATTTTACCC	UA5004 (Δ103)
103-ID6	5′-GCAGTGTAAGAGAAGGCTCTTGACAGATGAACCTTG	UA5004 (Δ103)
103-ID7	5′-CAAGGTTCATCTGTCAAGAGCCTTCTCT TACACTGC	UA5004 (Δ103)
103-ID8a	5′-CGAATTCTCTACACCTGTCGAACTC	UA5004 (Δ103)
Pht309-ID1	5′-AGGGATGAGTCTCTGTAG	UA5005 (ΔPht309)
Pht309-ID2	5′-AGCAGGAATTTGACGTCC	UA5005 (ΔPht309)
Pht309-ID3	5′-ACCTTACACATGGTTGAG	UA5005 (ΔPht309)
Pht309-ID4	5′-CCATCGTCTGAAACTGGG	UA5005 (ΔPht309)
Pht309-ID5a	5′-CGAATTCAATGAAGTCCAGGATGGC	UA5005 (ΔPht309)
Pht309-ID6	5′-GTAGTCCAAGCAGATGGATTGGGAGTGACTTGCTTG	UA5005 (ΔPht309)
Pht309-ID7	5′-CAAGCAAGTCACTCCCAATCCATCTGCTTGGACTAC	UA5005 (ΔPht309)
Pht309-ID8a	5′-CGAATTCTGCTCCTGTTTCAGTTGG	UA5005 (ΔPht309)

^aOligonucleotides that include an EcoRI restriction site (underlined).

The contributions of the 103 and Pht309 proteins to S. suis infectivity were evaluated with the use of S. suis wild-type (WT) parent strain (P1/7), UA5004 (Δ103), and UA5005 (ΔPht309) in a mouse virulence assay. All the animal experiments were approved by the Animal Ethics Committee of the Universitat Autònoma de Barcelona. Four groups of 3 female 8-week-old BALB/cAnNHsd mice obtained from Harlan Iberica (Barcelona, Spain) were used for each bacterial strain. The concentration of inoculated bacteria was calculated according to previous studies (6). Briefly, the mice were injected intraperitoneally with 0.1 mL of serial 10-fold dilutions [approximately 10⁸ to 10⁵ colony-forming units (CFU) per animal] of bacterial suspensions at the exponential phase (optical density 600:0.6) in Todd–Hewitt broth (Difco, Sparks, Maryland, USA) supplemented with 2% yeast extract (Difco) (THY) and further supplemented with 10% inactivated bovine serum (Invitrogen, Carlsbad, California, USA). The concentration of the original inocula was determined by plate enumeration. The number of animals that survived 1 wk after inoculation was recorded, and the median lethal dose (LD₅₀) was calculated as previously described (12). The data were analyzed by means of chi-squared tests. A P-value of less than 0.05 was considered significant.

Bacterial colonization of the liver, spleen, and brain of the infected animals was evaluated as follows. Briefly, surviving mice of all groups were sacrificed at day 7 after inoculation, and small pieces of these organs were trimmed, placed in 500 μL of phosphate-buffered saline, and homogenized. The homogenized organ samples were then enriched by inoculation of 300 μL into THY broth, incubation at 37°C, and subsequent dilution and plating onto sheep blood agar. The presence of S. suis in isolated bacterial colonies was confirmed by PCR with the use of primers 103ID-1 and 103ID-4.

To obtain serum for Western blot assays, 2 female 8-week-old BALB/cAnNHsd mice were used for each bacterial strain. The animals were inoculated intraperitoneally with a sublethal dose of the WT (P1/7), UA5004 (Δ103), or UA5005 (ΔPht309) strain of S. suis. Two additional mice inoculated with THY broth served as negative controls. At days 15 and 30 after inoculation 1 mouse from each group was anesthetized with ketamine/xylazine (90 mg/kg and 10 mg/kg, respectively). Blood was collected by cardiac puncture, incubated at 37°C for 2 h to facilitate clot formation, and then centrifuged at 300 × g for 10 min. Serum was recovered and stored at −20°C till the time of assay. After blood collection, the mice were euthanized. The titers of antibodies generated against WT, UA5004, and UA5005 were evaluated by Western blot analysis as described previously (13), with 10 μg of the highly immunogenic cell-surface-associated proteins obtained from the UA5002 (ΔadcR Δfur) S. suis strain as described before (8) used as the antigen.

The data obtained from the virulence assay (Figure 1) showed that inactivation of the S. suis gene encoding the 103 lipoprotein increased the LD₅₀ for the BALB/cAnNHsd mice from 4.4 × 10⁶ CFU per mouse (LD₅₀ of WT) to > 2.3 × 10⁸ CFU per mouse (more than 50 times; P < 0.01). The in vitro growth rates of the mutant strains and the parent strain did not differ significantly (data not shown). In striking contrast, inactivation of the gene encoding Pht309 did not significantly affect S. suis virulence in the mice: the LD₅₀ was 6.4 × 10⁶ CFU per mouse, less than 1.5 times different from the LD₅₀ of the parent strain (P > 0.05).

Figure 1.

Survival curve for mice (6 per group) inoculated with the highly virulent Streptococcus suis serotype 2 strain P1/7 (WT, ) or mutant strains of P1/7 in which the genes encoding the 103 and Pht309 proteins had been deleted: UA5004 (Δ103, ●) and UA5005 (ΔPht309, ). All the mice inoculated with, < 10⁷ colony-forming units per animal survived until the end of the experiment and are not represented. The asterisk represents a significant difference (P < 0.01) relative to the WT parent strain.

When the mice surviving 1 wk after inoculation were sacrificed, S. suis was isolated from at least 1 of the analyzed organs of all the animals inoculated with either the virulent parent strain P1/7 or the ΔPht309 mutant, without significant differences (data not shown). However, S. suis could not be detected in the organs of any of the mice injected with the Δ103 mutant. In addition, whereas the titers of antibodies generated against sublethal doses of P1/7 and the ΔPht309 mutant increased at similar levels with time, this was not the case for the animals inoculated with the Δ103 mutant (Figure 2), despite the fact that the concentration of the inoculum of the Δ103 mutant (10⁷ CFU per animal) was 100 times greater than the concentrations of the inocula of P1/7 and the ΔPht309 mutant (10⁵ CFU per animal). In fact, the immunogenic response detected against the Δ103 mutant was similar to that obtained in the negative controls.

Figure 2.

Western blot analysis of serum recovered from mice inoculated with a sublethal dose of S. suis P1/7, UA5004, or UA5005 or with Todd–Hewitt broth supplemented with 2% yeast extract (negative control, C−) on days 15 and 30 after inoculation. Cell-surface-associated proteins obtained from the S. suis UA5002 strain were used as the antigen. The sizes of the major proteins are indicated.

The ABC 103 component is a putative zinc-binding streptococcal lipoprotein regulated by AdcR (a transcriptional regulatory factor implicated in zinc homeostasis) that confers protection against S. suis in mice (6,8). The ABC transporters are widespread among living organisms, and there is increasing evidence that they play either a direct or an indirect role in the virulence of bacteria (14). For instance, mutations in the psaA gene of S. pneumoniae, which encodes a lipoprotein component of an ABC transporter involved in cation uptake, caused deficiencies in virulence and adherence (15). In S. pneumoniae, PsaA does not appear to act directly as an adhesin; rather, psaA mutations indirectly affect the adherence process through the disruption of Mn²⁺ transport (15). Thus, we speculate that the reduced virulence of the Δ103 mutant may result from its impaired capacity to traverse host barriers, and we propose an indirect mechanism in which deficient Zn²⁺ uptake affects the ability of the strain to adhere to host tissues.

The Pht309 gene is cotranscribed with the 103 gene and encodes a protein homologous to the Pht family (8). The Pht proteins are involved in the interaction between the host cell and the pathogen, and it has been hypothesized that they have been selected to become sensitive to AdcR regulation in order to exploit zinc as a compartment- specific regulatory cue (16). In S. pneumoniae the Pht family has been described as comprising 4 members (PhtA, PhtB, PhtD, and PhtE), which extensively share sequence identity (9). Recently it has been reported that none of the single or double mutants of genes encoding Pht proteins in S. pneumoniae are significantly attenuated compared with the WT parent (17). Nevertheless, the mutant lacking all 4 proteins was completely avirulent (17). Similarly, it has been reported that S. suis presents at least 2 Pht proteins encoded by genes SSU0309 (Pht309) and SSU1103 (Pht 1103) (8), and our results show that the S. suis mutant lacking the gene for Pht309 was not impaired in virulence when compared with the parent strain. Therefore, we believe that the observed results could be explained by a functionally redundant role for Pht proteins (17).

Previous studies showed that the S. suis lipoprotein 103 is not only immunogenic but also confers a high level of protection against S. suis challenge in the mouse model (6). Furthermore, the presence of Pht proteins (which also belong to the S. suis adcR regulon) at the S. suis cell surface has been demonstrated (8), suggesting that these proteins might be suitable as vaccines against streptococcal infections (9,18). Taking into account that an adcR-mutant strain overexpresses genes encoding immunogenic proteins, and considering that genes encoding the 103 and Pht309 proteins were demonstrated to be cotranscribed and under the regulation of AdcR (8), we analyzed the immunogenic capacities of S. suis mutants lacking these genes. Although the immunogenicity of the ΔPht309 mutant was similar to that of the parent strain, the Δ103 mutant was less immunogenic than the other 2 strains. This could be due to an impairment in virulence of the Δ103 mutant, probably reflecting a decreased ability to escape innate immune clearance mechanisms and, consequently, a reduction in persistence of the mutant. The data obtained in this work support the hypothesis that lipoprotein 103, whose encoding gene is present in practically all streptococci (6), may have important implications for the development of vaccines against streptococcal pathogens.

In conclusion, the findings of this study clearly show the dramatic impact of mutagenesis of the 103 gene on S. suis virulence and strongly demonstrate that its encoded protein plays a very important role in the pathogenesis of streptococcal disease.