Katy Vaillancourt

The cell envelope subtilisin-like proteinase is a virulence determinant for Streptococcus suis

BackgroundStreptococcus suis is a major swine pathogen and zoonotic agent that mainly causes septicemia, meningitis, and endocarditis. It has recently been suggested that proteinases produced by S. suis (serotype 2) are potential virulence determinants. In the present study, we screened a S. suis mutant library created by the insertion of Tn917 transposon in order to isolate a mutant deficient in a cell surface proteinase. We characterized the gene and assessed the proteinase for its potential as a virulence factor.ResultsTwo mutants (G6G and M3G) possessing a single Tn917 insertion were isolated. The affected gene coded for a protein (SSU0757) that shared a high degree of identity with Streptococccus thermophilus PrtS (95.9%) and, to a lesser extent, with Streptococcus agalactiae CspA (49.5%), which are cell surface serine proteinases. The SSU0757 protein had a calculated molecular mass of 169.6 kDa and contained the catalytic triad characteristic of subtilisin family proteinases: motif I (Asp200), motif II (His239), and motif III (Ser568). SSU0757 also had the Gram-positive cell wall anchoring motif (Leu-Pro-X-Thr-Gly) at the carboxy-terminus, which was followed by a hydrophobic domain. All the S. suis isolates tested, which belonged to different serotypes, possessed the gene encoding the SSU0757 protein. The two mutants devoid of subtilisin-like proteinase activity had longer generation times and were more susceptible to killing by whole blood than the wild-type parent strain P1/7. The virulence of the G6G and M3G mutants was compared to the wild-type strain in the CD1 mouse model. Significant differences in mortality rates were noted between the P1/7 group and the M3G and G6G groups (p < 0.001).ConclusionIn summary, we identified a gene coding for a cell surface subtilisin-like serine proteinase that is widely distributed in S. suis. Evidences were brought for the involvement of this proteinase in S. suis virulence.

Characterization of a galactokinase-positive recombinant strain of Streptococcus thermophilus.

ABSTRACT The lactic acid bacterium Streptococcus thermophilus is widely used by the dairy industry for its ability to transform lactose, the primary sugar found in milk, into lactic acid. Unlike the phylogenetically related species Streptococcus salivarius, S. thermophilus is unable to metabolize and grow on galactose and thus releases substantial amounts of this hexose into the external medium during growth on lactose. This metabolic property may result from the inability of S. thermophilus to synthesize galactokinase, an enzyme of the Leloir pathway that phosphorylates intracellular galactose to generate galactose-1-phosphate. In this work, we report the complementation of Gal− strain S. thermophilus SMQ-301 with S. salivarius galK, the gene that codes for galactokinase, and the characterization of recombinant strain SMQ-301K01. The recombinant strain, which was obtained by transformation of strain SMQ-301 with pTRKL2TK, a plasmid bearing S. salivarius galK, grew on galactose with a generation time of 55 min, which was almost double the generation time on lactose. Data confirmed that (i) the ability of SMQ-301K01 to grow on galactose resulted from the expression of S. salivarius galK and (ii) transcription of the plasmid-borne galK gene did not require GalR, a transcriptional regulator of the gal and lac operons, and did not interfere with the transcription of these operons. Unexpectedly, recombinant strain SMQ-301K01 still expelled galactose during growth on lactose, but only when the amount of the disaccharide in the medium exceeded 0.05%. Thus, unlike S. salivarius, the ability to metabolize galactose was not sufficient for S. thermophilus to simultaneously metabolize the glucose and galactose moieties of lactose. Nevertheless, during growth in milk and under time-temperature conditions that simulated those used to produce mozzarella cheese, the recombinant Gal+ strain grew and produced acid more rapidly than the Gal− wild-type strain.

Purification and characterization of the subtilisin-like protease of Streptococcus suis that contributes to its virulence.

Streptococcus suis is a major swine pathogen that is responsible for severe infections such as meningitis, endocarditis, and septicemia. S. suis is also recognized as a zoonotic agent and expresses several virulence factors. The recently identified subtilisin-like protease (SspA) of S. suis plays an important role in the pathogenicity of this bacterium in animal models. The objective of the present study was to clone, purify, and characterize the SspA of serotype 2 S. suis P1/7. The SSU0757 gene encoding SspA was amplified and a 4798-bp DNA fragment was obtained. It was cloned into the expression plasmid pBAD/HisB and then inserted into Escherichia coli to overproduce the protein. The recombinant protease was purified by chromatography procedures and showed a molecular weight of 170 kDa by SDS-PAGE. Its activity was optimal at pH 7 and at temperatures ranging from 25°C to 37°C. It had a high specificity for the chromogenic substrate succinyl-Ala-Ala-Pro-Phe-pNa while specific inhibitors of serine proteases inhibited its activity. In addition to degrading gelatin, the protease hydrolyzed the Aα chain of fibrinogen, which prevented fibrin formation by thrombin. The recombinant subtilisin-like protease also showed toxicity towards brain microvascular endothelial cells. Lastly, sera from pigs infected with S. suis reacted with the recombinant SspA, indicating that it is produced during infections. In conclusion, the SspA of S. suis shared similarities with subtilisin-like proteases produced by other pathogenic streptococci and may contribute to the pathogenic process of S. suis infections.

The Doubly Phosphorylated Form of HPr, HPr(Ser-P)(His∼P), Is Abundant in Exponentially Growing Cells of Streptococcus thermophilus and Phosphorylates the Lactose Transporter LacS as Efficiently as HPr(His∼P)

ABSTRACT In Streptococcus thermophilus, lactose is taken up by LacS, a transporter that comprises a membrane translocator domain and a hydrophilic regulatory domain homologous to the IIA proteins and protein domains of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The IIA domain of LacS (IIALacS) possesses a histidine residue that can be phosphorylated by HPr(His∼P), a protein component of the PTS. However, determination of the cellular levels of the different forms of HPr, namely, HPr, HPr(His∼P), HPr(Ser-P), and HPr(Ser-P)(His∼P), in exponentially lactose-growing cells revealed that the doubly phosphorylated form of HPr represented 75% and 25% of the total HPr in S. thermophilus ATCC 19258 and S. thermophilus SMQ-301, respectively. Experiments conducted with [32P]PEP and purified recombinant S. thermophilus ATCC 19258 proteins (EI, HPr, and IIALacS) showed that IIALacS was reversibly phosphorylated by HPr(Ser-P)(His∼P) at a rate similar to that measured with HPr(His∼P). Sequence analysis of the IIALacS protein domains from several S. thermophilus strains indicated that they can be divided into two groups on the basis of their amino acid sequences. The amino acid sequence of IIALacS from group I, to which strain 19258 belongs, differed from that of group II at 11 to 12 positions. To ascertain whether IIALacS from group II could also be phosphorylated by HPr(His∼P) and HPr(Ser-P)(His∼P), in vitro phosphorylation experiments were conducted with purified proteins from Streptococcus salivarius ATCC 25975, which possesses a IIALacS very similar to group II S. thermophilus IIALacS. The results indicated that S. salivarius IIALacS was phosphorylated by HPr(Ser-P)(His∼P) at a higher rate than that observed with HPr(His∼P). Our results suggest that the reversible phosphorylation of IIALacS in S. thermophilus is accomplished by HPr(Ser-P)(His∼P) as well as by HPr(His∼P).

Role of galK and galM in Galactose Metabolism by Streptococcus thermophilus

ABSTRACT Streptococcus thermophilus is unable to metabolize the galactose moiety of lactose. In this paper, we show that a transformant of S. thermophilus SMQ-301 expressing Streptococcus salivarius galK and galM was able to grow on galactose and expelled at least twofold less galactose into the medium during growth on lactose.

Phosphorylation of Streptococcus salivarius lactose permease (LacS) by HPr(His ~ P) and HPr(Ser-P)(His ~ P) and effects on growth.

The oral bacterium Streptococcus salivarius takes up lactose via a transporter called LacS that shares 95% identity with the LacS from Streptococcus thermophilus, a phylogenetically closely related organism. S. thermophilus releases galactose into the medium during growth on lactose. Expulsion of galactose is mediated via LacS and stimulated by phosphorylation of the transporter by HPr(His approximately P), a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS). Unlike S. thermophilus, S. salivarius grew on lactose without expelling galactose and took up galactose and lactose concomitantly when it is grown in a medium containing both sugars. Analysis of the C-terminal end of S. salivarius LacS revealed a IIA-like domain (IIA(LacS)) almost identical to the IIA domain of S. thermophilus LacS. Experiments performed with purified proteins showed that S. salivarius IIA(LacS) was reversibly phosphorylated on a histidine residue at position 552 not only by HPr(His approximately P) but also by HPr(Ser-P)(His approximately P), a doubly phosphorylated form of HPr present in large amounts in rapidly growing S. salivarius cells. Two other major S. salivarius PTS proteins, IIAB(L)(Man) and IIAB(H)(Man), were unable to phosphorylate IIA(LacS). The effect of LacS phosphorylation on growth was studied with strain G71, an S. salivarius enzyme I-negative mutant that cannot synthesize HPr(His approximately P) or HPr(Ser-P)(His approximately P). These results indicated that (i) the wild-type and mutant strains had identical generation times on lactose, (ii) neither strain expelled galactose during growth on lactose, (iii) both strains metabolized lactose and galactose concomitantly when grown in a medium containing both sugars, and (iv) the growth of the mutant was slightly reduced on galactose.

Suicin 3908, a New Lantibiotic Produced by a Strain of Streptococcus suis Serotype 2 Isolated from a Healthy Carrier Pig

While Streptococcus suis serotype 2 is known to cause severe infections in pigs, it can also be isolated from the tonsils of healthy animals that do not develop infections. We hypothesized that S. suis strains in healthy carrier pigs may have the ability to produce bacteriocins, which may contribute to preventing infections by pathogenic S. suis strains. Two of ten S. suis serotype 2 strains isolated from healthy carrier pigs exhibited antibacterial activity against pathogenic S. suis isolates. The bacteriocin produced by S. suis 3908 was purified to homogeneity using a three-step procedure: ammonium sulfate precipitation, cationic exchange HPLC, and reversed-phase HPLC. The bacteriocin, called suicin 3908, had a low molecular mass; was resistant to heat, pH, and protease treatments; and possessed membrane permeabilization activity. Additive effects were obtained when suicin 3908 was used in combination with penicillin G or amoxicillin. The amino acid sequence of suicin 3908 suggested that it is lantibiotic-related and made it possible to identify a bacteriocin locus in the genome of S. suis D12. The putative gene cluster involved in suicin production by S. suis 3908 was amplified by PCR, and the sequence analysis revealed the presence of nine open reading frames (ORFs), including the structural gene and those required for the modification of amino acids, export, regulation, and immunity. Suicin 3908, which is encoded by the suiA gene, exhibited approximately 50% identity with bovicin HJ50 (Streptococcus bovis), thermophilin 1277 (Streptococcus thermophilus), and macedovicin (Streptococcus macedonicus). Given that S. suis 3908 cannot cause infections in animal models, that it is susceptible to conventional antibiotics, and that it produces a bacteriocin with antibacterial activity against all pathogenic S. suis strains tested, it could potentially be used to prevent infections and to reduce antibiotic use by the swine industry.

Identification and characterization of a new cell surface protein possessing factor H-binding activity in the swine pathogen and zoonotic agent Streptococcus suis

Streptococcus suis is a major swine pathogen and an emerging zoonotic agent. The ability of pathogenic bacteria to bind the complement regulator factor H on their cell surface may allow them to avoid complement attack and phagocytosis. The aim of this study was to characterize a new cell surface protein possessing factor H-binding activity in S. suis serotype 2. The capacity of S. suis to bind the complement regulator factor H on its surface was demonstrated by ELISA. Using a factor I-cofactor assay, it was found that the functional activity of factor H bound to S. suis was kept. Since the product of gene SSU0186 in S. suis P1/7 shared similarity with a Streptococcus pneumoniae protein (named PspC) possessing factor H-binding activity, it was proposed as a putative factor H receptor in S. suis. SSU0186 has a 1686 bp open reading frame encoding a 561 amino acid protein containing the Gram-positive cell wall anchoring motif (LPXTG) at the carboxy-terminal, an amino-terminal signal sequence, an α-helix domain, a proline-rich region and a G5 domain. The SSU0186 gene was cloned in Escherichia coli and the purified recombinant factor H-binding protein showed a molecular mass of 95 kDa, as determined by SDS-PAGE. The protein possessed the functional property of binding factor H. Sera from S. suis-infected pigs reacted with the recombinant factor H receptor, suggesting that it is produced during the course of infections. In conclusion, we identified a novel S. suis cell surface protein that binds the complement factor H. This cell surface protein may help S. suis to resist complement attack and phagocytosis and contribute to pathogenesis.

Characterization of DNase activity and gene in Streptococcus suis and evidence for a role as virulence factor

BackgroundThe Gram-positive bacterium Streptococcus suis serotype 2 is an important swine pathogen and emerging zoonotic agent. Multilocus sequence typing allowed dividing S. suis serotype 2 into sequence types (STs). The three major STs of S. suis serotype 2 from North America are 1 (most virulent), 25 (intermediate virulence) and 28 (less virulent). Although the presence of DNase activity in S. suis has been previously reported, little data is available. The aim of this study was to investigate DNase activity in S. suis according to STs, to characterize the activity and gene, and to provide evidence for a potential role in virulence.ResultsWe showed that ST1 and ST28 strains exhibited DNase activity that was absent in ST25 strains. The lack of activity in ST25 isolates was associated with a 14-bp deletion resulting in a shifted reading frame and a premature stop codon. The DNase of S. suis P1/7 (ST1) was cell-associated and active on linear DNA. A DNase-deficient mutant of S. suis P1/7 was found to be less virulent in an amoeba model. Stimulation of macrophages with the DNase mutant showed a decreased secretion of pro-inflammatory cytokines and matrix metalloproteinase-9 compared to the parental strain.ConclusionsThis study further expands our knowledge of S. suis DNase and its potential role in virulence.

The HPr(Ser) kinase of Streptococcus salivarius: a hexameric bifunctional enzyme controlled by glycolytic intermediates and inorganic phosphate

Phosphorylation of HPr, the small phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system, on Ser46 by the HPr(Ser) kinase (HPrK/P) is a vital step in catabolite repression in Gram-positive bacteria. Streptococcus salivarius HPrK/P is reported to be a multimeric protein not regulated by metabolic intermediates. We re-evaluated the molecular mass of S. salivarius HPrK/P using sedimentation equilibrium ultracentrifugation, demonstrated that S. salivarius HPrK/P dephosphorylated HPr(Ser-P) and further characterised the effect of fructose 1,6-bisphosphate and other metabolic intermediates on enzyme activities. The molecular mass of S. salivarius HPrK/P was 201305 Da, suggesting that streptococcal HPrK/P was a hexameric protein. Fructose 1,6-bisphosphate poorly activated streptococcal HPrK/P but protected kinase activity against inhibition by inorganic phosphate and inhibited dephosphorylation of HPr(Ser-P). Phosphoenolpyruvate and 2-phosphoglycerate, but not fructose 1-P, fructose 6-P, and ribulose 1,5-bisphosphate, also protected kinase activity against inhibition by inorganic phosphate. Thus, unlike previous reports, we show that fructose 1,6-bisphosphate and other key glycolytic intermediates played a pivotal role as a modulator of streptococcal HPrK/P activities.