Payen et al. Veterinary Research (2023) 54:1 https://doi.org/10.1186/s13567-022-01128-8 RESEARCH ARTICLE Open Access Laminin‑binding protein of Streptococcus suis serotype 2 influences zinc acquisition and cytokine responses Servane Payen1, Jesús Aranda Rrodriguez2, Mariela Segura1 and Marcelo Gottschalk1*    Abstract Streptococcus suis serotype 2 is an important bacterial pathogen of swine, responsible for substantial economic losses to the swine industry worldwide. The knowledge on the pathogenesis of the infection caused by S. suis is still poorly known. It has been previously described that S. suis possesses at least one lipoprotein with double laminin and zinc (Zn)-binding properties, which was described in the literature as either laminin-binding protein (Lmb, as in the current study), lipoprotein 103, CDS 0330 or AdcAII. In the present study, the role of the Lmb in the pathogenesis of the infec- tion caused by S. suis serotype 2 was dissected. Using isogenic mutants, results showed that Lmb does not play an important role in the laminin-binding activity of S. suis, even when clearly exposed at the bacterial surface. In addition, the presence of this lipoprotein does not influence bacterial adhesion to and invasion of porcine respiratory epithelial and brain endothelial cells and it does not increase the susceptibility of S. suis to phagocytosis. On the other hand, the Lmb was shown to play an important role as cytokine activator when tested in vitro with dendritic cells. Finally, this lipoprotein plays a critical role in Zn acquisition from the host environment allowing bacteria to grow in vivo. The significant lower virulence of the Lmb defective mutant may be related to a combination of a lower bacterial survival due to the incapacity to acquire Zn from their surrounding milieu and a reduced cytokine activation. Keywords: Streptococcus suis, lipoprotein, laminin-binding protein, zinc metabolism, virulence, cell adhesion and invasion, cytokines Introduction factors and the pathogenesis of the infection remains Streptococcus suis is one of the most important swine incomplete [1]. A battery of surface proteins has been pathogens responsible for important economic losses, proposed as being important for virulence, although causing sudden death, meningitis, and arthritis among most of them have only been partially confirmed [2]. other clinical manifestations [1]. Moreover, S. suis is a Bacterial attachment to host cells by mucosal patho- zoonotic agent responsible for meningitis and septic gens like S. suis is frequently associated to the first steps shock in humans [1]. Although the knowledge on the of the pathogenesis, leading to colonization and infection pathogenesis of the infection caused by this pathogen has [3, 4]. Bacterial adhesion and invasion of cells would be improved in recent years, our understanding on virulence critical to allow successful infection of the host. Indeed, bacteria have evolved with several adhesins that spe- *Correspondence: marcelo.gottschalk@umontreal.ca cifically recognize and attach to host cell surface com- 1 ponents, including extracellular matrix proteins, such Research Group On Infectious Diseases in Production Animals (GREMIP) and Swine and Poultry Infectious Diseases Research Center (CRIPA), as fibronectin, collagens, and laminin [2, 3]. Several Department of Pathology and Microbiology, Faculty of Veterinary Medicine, laminin-binding proteins (Lmbs) expressed on the bacte- University of Montreal, Saint‑Hyacinthe, QC J2S 2M2, Canada rial surface have been described for different pathogenic Full list of author information is available at the end of the article © The Author(s) 2022. 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The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Payen et al. Veterinary Research (2023) 54:1 Page 2 of 13 streptococci. Lmb was first identified in Streptococcus the CDS 0330 previously described [10, 18, 19]; yet again, agalactiae (or group B Streptococcus) [5], and homologs no mention of a potential laminin-binding ability of such of this lipoprotein were reported in other streptococci, protein was discussed [19]. such as Streptococcus pyogenes or Group A Streptococcus In the present study, we further dissected the role of (named as Lbp or Lsp) [6, 7] and Streptococcus pneumo- the Lmb/AdcAII/lipoprotein 103/CDS 0330 as both, niae (called AdcAII, see below) [8]. Most studies demon- laminin-binding and Zn-uptake protein in the pathogen- strated that recombinant Lmbs can bind purified laminin esis of the infection caused by S. suis serotype 2. which is a large (900 kDa), highly glycosylated multid- omain protein found in all human tissues [5, 7, 9–11]. In Materials and methods addition, in S. agalactiae, a mutant lacking Lmb showed Bacterial strains and growth conditions decreased adherence to human laminin and reduced The strains and plasmids used in this study are listed in invasion of human brain microvascular endothelial cells Table 1. The classical S. suis serotype 2 virulent European [12]. Lsp-negative S. pyogenes mutants were shown to reference P1/7 strain (wild type) was used throughout be defective in adhesion and invasion of epithelial cells this study including for construction of the isogenic defi- in vitro and they were also highly attenuated in a murine cient mutants [18]. S. suis strains were cultured in Todd subcutaneous ulcer model [7, 13]. Hewitt broth (THB; Becton Dickinson, Mississauga, ON, Although many studies mostly focused on its function Canada) as previously described [20]. Escherichia coli as an adhesin, Lmbs (or homologous proteins) may be strains, and different plasmids used in this study are also also involved in zinc (Zn) uptake. Indeed, crystal struc- listed in Table 1. For in vitro cell culture assays, bacteria ture analysis of Lmb homologs from different strepto- were prepared as previously described [21, 22] and resus- cocci revealed that the two globular domains form a pended in cell culture medium. When needed, antibiot- pocket to facilitate a tetrahedral geometry for Zn-binding ics (Sigma-Aldrich, Oakville, ON, Canada) were added [8, 11, 14]. Zinc is crucial in any normal metabolic con- to the media at the following concentrations: for S. suis, dition, but during infection this element becomes par- spectinomycin (Spc) at 100 μg/mL; for E. coli, kanamycin ticularly critical for bacterial pathogens [15, 16]. Indeed, (Km) and spectinomycin at 50 μg/mL and ampicillin (Ap) both host and pathogen compete for the same essential at 100 μg/mL. metals, which are at very low concentration in infec- tion sites [17]. In several pathogenic streptococci, dele- DNA manipulations tion of one or more Zn-acquisition lipoproteins results Genomic DNA was extracted from the S. suis wild-type in lower growth under Zn-restricted conditions as well strain using InstaGene Matrix solution (BioRad Labora- as decreased virulence, adhesion, and biofilm formation, tories, Hercules, CA, USA). Mini preparations of recom- underlining the importance of zinc metabolism during binant plasmids were carried out using the QIAprep Spin colonization and infection. Miniprep Kit (Qiagen, Valencia, CA, USA). Restriction In S. suis, an homologue of Lmb has been found in enzymes and DNA-modifying enzymes (Fisher Scientific, different strains [10] and a Lmb defective mutant was Ottawa, ON, Canada) were used according to the manu- shown to be avirulent in a mouse model of infection facturer’s recommendations. Oligonucleotide primers [18]. In addition, lipoproteins involved in Zn-acquisition (Table 2) were obtained from Integrated DNA Technolo- have also been described and reported to be crucial for S. gies (Coralville, IA, USA) and PCRs carried out with the suis virulence [19]. However, it seems to be some confu- iProof proofreading DNA polymerase (BioRad Labora- sion in the literature concerning the proteins involved in tories, Mississauga, ON, Canada) or the Taq DNA poly- both laminin adhesion and Zn uptake. Originally, a criti- merase (Qiagen). Amplification products were purified cal role as virulence factor of the so-called Zn-binding using the QIAquick PCR Purification Kit (Qiagen) and lipoprotein 103 was reported, without any mention of sequenced using an ABI 310 Automated DNA Sequencer a possible role as a laminin-binding protein [18]. Later, and ABI PRISM Dye Terminator Cycle Sequencing Kit Zhang et al. described a laminin-binding protein of S. (Applied Biosystems, Carlsbad, CA, USA). suis (called CDS 0330) [10], without mentioning that this protein is the same Zn-binding lipoprotein protein 103 Construction of the laminin‑binding defective/ previously described by Aranda et al. [18]. Interestingly, non‑encapsulated double mutant both research groups independently reported this pro- Precise in-frame deletion of cpsG gene from strain P1/7 tein as being protective using a mouse model of infection Δlmb [18] was constructed using splicing-by-over- [10, 18]. More recently, Zheng et al. reported that S. suis lap-extension PCRs as previously described [27, 28]. possesses two Zn-binding proteins, AdcA and AdcAII. Overlapping PCR product were cloned into pCR2.1 (Inv- Indeed, AdcAII corresponds to both lipoprotein 103 and itrogen, Burlington, ON, Canada), extracted with EcoRI, Payen et al. Veterinary Research (2023) 54:1 Page 3 of 13 Table 1 List of strains and plasmids used in this study Strain or plasmid Characteristics References Streptococcus suis P1/7 Virulent serotype 2 ST1 strain isolated from a case of pig meningitis in the United Kingdom [23] 5P1/7Δlmb Isogenic mutant derived from P1/7; in frame deletion of lmb gene [18] P1/7 Δlmb/ΔcpsG Isogenic mutant derived from P1/7; in frame deletion of lmb and cpsG gene This study P1/7 ΔcpsF Isogenic mutant derived from P1/7; in frame deletion of cpsF [21] P1/7 comp Δlmb Mutant Δlmb complemented with pMX1-lmb complementation vector This study Escherichia coli TOP10 F− mrcA Δ(mrr-hsdRMS-mcrBC) φ80 lacZΔM15 ΔlacX74 recA1 araD139 Δ(araleu) 7697 galU galK rpsL ­(StrR) Invitrogen endA1 nupG MC1061 Host for pMX1 derivatives [24] Plasmids pCR2.1 Apr, ­Kmr, pUC ori, lacZΔM15 Invitrogen pSET4s Spcr, pUC ori, thermosensitive pG + host3 ori, lacZΔM15 [25] pMX1 Replication functions of pSSU1, MCS pUC19 lacZ SpR, malX promoter of S. suis, derivative of pSET2 [25, 26] p4ΔcpsG pSET-4 s carrying the construct for lmb allelic replacement This study pMX1-lmb pMX1 carrying intact lmb gene This study Table 2 List of oligonucleotide primers used in this study This plasmid was introduced into E. coli MC1061 for Name Sequence (5′ – 3′) Construct verification of the sequence and then into the deletion mutant derived from S. suis P1/7 to construct lmb-com- cpsG-ID1 CCA​GCA​AAG​TAT​GGT​GGT​T TCG​ p4ΔcpsG plemented mutants. cpsG-ID2 CCA​CGC​CAG​ATT​CAA​TGA​GC p4ΔcpsG cpsG-ID3 GGG​T TC​GAT​AAA​GAT​AAG​CG p4ΔcpsG Bacterial surface hydrophobicity assay cspG-ID4 GCG​AAT​T TG​GAG​T TA​CGA​AAGC​ p4ΔcpsG Relative surface hydrophobicity of the S. suis wild-type, cpsG-ID5 CGA​T TC​AAA​TCC​ACG​GAA​AC p4ΔcpsG Lmb and Lmb/non-encapsulated mutant strains was cpsG-ID6 GCT​C TT​GGC​TAA​TAG​C TC​G p4ΔcpsG determined by measuring adsorption to n-hexadecane as pMX1-lmb-F CCG​CCA​TGG​ACA​GAT​GGG​GTT​TGA​TGC​AAC​ pMX1-lmb previously described [30]. pMX1-lmb-R CGC​GAA​T TC​GGA​CAA​GGC​AAT​AAT​CAA​GAC​ pMX1-lmb Microtiter plate laminin‑binding assay described [31]. Briefly, Maxisorp™ flat-bottom microtiter Laminin binding assay was performed as previously recloned into the thermosensitive E. coli–S. suis shuttle plasmid pSET4s, and digested with the same enzyme, 96-well plates (Nunc, VWR, Mississauga, ON, Canada) giving rise to the knockout vector p4ΔcpsG. Electropo- were coated with 5 μg/mL of laminin from human pla- ration of wild-type strain P1/7 procedures to obtain the centa from Sigma Chemical Co. (St. Louis, MO, USA) in mutant were previously described [25]. Allelic replace- 0.1 M carbonate coating buffer (pH 9.6) and were incu- ment was confirmed by PCR and DNA sequencing bated overnight at 4 °C. The plates were washed with analyses. Amplification products were purified with the PBS containing 0.05% (v/v) Tween 20 (PBST, pH 7.3), QIAgen PCR Purification Kit (Qiagen) and sequenced as and 200 μL of 3% (w/v) non-fat dry milk in PBST was described above. added to each well to prevent non-specific bacterial bind- ing. After 1 h at 37 °C, the wells were washed with PBST. Complementation of the laminin‑binding defective mutant Formaldehyde-killed bacterial suspensions of wild-type The pMX1 vector was used for the generation of recom- or mutant strains were added (concentration equivalent binant plasmids for complementation analysis (Table 1). to ­108 CFU/mL), and the plates were incubated for 2 h at This vector is a derivative of the E. coli- S. suis shuttle 37 °C. All unbound bacteria were subsequently removed cloning vector pSET2 [29] and possesses the S. suis malX by washing the wells with PBST. S. suis serotype 2-spe- promoter for transgene expression in S. suis. The entire cific rabbit antiserum diluted 1/5000 in PBST prepared lmb gene was amplified from genomic DNA of S. suis as previously described [31] was added to each well. This P1/7 strain and cloned into pMX1 via EcoRI and NcoI antiserum equally recognized both encapsulated and sites, generating complementation vector pMX1-lmb. non-encapsulated serotype 2 S. suis as previously shown Payen et al. Veterinary Research (2023) 54:1 Page 4 of 13 [31]. The plates were incubated for 1 h at 37 °C and wells collected from the caudal vein of surviving mice 12 h, were then washed, followed by the addition of horserad- 24 h and 48 h post-infection and plated as previously ish peroxidase-labelled anti-rabbit IgG (Jackson Immu- described [36]. noresearch Laboratories Inc., West Grove, PA, USA) (diluted 1/8000 in PBST). The plates were incubated for Measurement of plasma (systemic) pro‑inflammatory 1 h at 37 °C with the secondary antibody. After washing, mediators 3,3′,5,5′-tetramethylbenzidine (Zymed, San Francisco, In addition, 8 mice per group were intraperitoneally CA, USA) was used as the enzyme substrate according mock-infected (THB) or infected with 1 × ­107 CFU to the manufacturer’s instructions. The reactions were of wild-type or Δlmb mutant strains and blood sam- stopped by adding 25 μL of ­H2SO4 (1 N) and were read at ples were collected 12 h post-infection by intracardiac 450 nm. Uncoated wells served as background controls. puncture following euthanasia and anti-coagulated with Casein-coated wells served as a control for non-specific EDTA (Sigma-Aldrich) as previously described [36, 37]. adhesion of S. suis to protein-coated wells. Each experi- Plasma supernatants were collected following centrifuga- ment was repeated at least 3 times. tion at 10 000 × g for 10 min at 4 °C and stored at −80 °C. The 12 h post-infection time point was selected to obtain Bacterial adhesion and invasion assays using porcine maximal pro-inflammatory mediator production in the tracheal epithelial and brain microvascular endothelial absence of significant mouse mortality as determined cells previously [36]. Plasmatic concentrations of interleu- The neonatal porcine tracheal epithelial cell line (NPTr) kin (IL)-6 and C-X-C motif chemokine ligand (CXCL) 1 and the porcine brain microvascular endothelial cell line were quantified by sandwich. ELISA using pair-matched (PBMEC) were used and cultured until confluence as antibodies from R&D Systems (Minneapolis, MN, USA) previously described [32, 33]. Cells were infected with according to the manufacturer’s recommendations. 1 × ­106 CFU/well (multiplicity of infection (MOI) = 10) of the different S. suis strains and incubated for 2 h at 37 °C Phagocytosis assay in 5% C ­ O2. The adhesion assay, which quantifies total J774A.1 murine macrophages (ATCC TIB-67; Rock- cell-associated bacteria (surface-adherent and intracel- ville, MD, USA) were maintained in Dulbecco’s Modi- lular bacteria), and invasion assay (using the antibiotic fied Eagle’s Medium (Gibco, Burlington, ON, Canada) protection assay) were performed as previously described supplemented with 10% fetal bovine serum (Gibco) and [32, 33]. grown at 37 °C with 5% C ­ O2. Confluent cell cultures were scraped, seeded at 1 × ­105 cells/mL, and incubated for 3 h S. suis virulence mouse model of systemic infection at 37 °C with 5% C­ O2 to allow cell adhesion. Cells were A C57BL/6 J mouse model of infection was used. These infected by adding 1 × ­107 CFU/mL of bacterial suspen- studies were carried out in strict accordance with the sion in complete culture medium (MOI = 100), incubated recommendations of and approved by the University of for 2 h at 37 °C with 5% ­CO2, and phagocytosis assays Montreal Animal Welfare Committee guidelines and performed as previously described using the antibiotic policies, including euthanasia to minimize animal suffer- protection assay [21]. ing by the use of humane endpoints, applied throughout this study when animals were seriously affected (mortal- S. suis activation of marrow‑derived dendritic cells (bmDC) ity was not an endpoint measurement). Thirty 6-week- The femur and tibia from C57BL/6 J mice (Jackson old female C57BL/6 J (Jackson Research Laboratories, Research Laboratories) were used to generate bmDCs, Bar Harbor, ME, USA) were used for these experiments as previously described [21]. Briefly, hematopoietic (15 mice per group). Early stationary phase bacteria were bone marrow stem cells were cultured in complete cul- washed twice in phosphate-buffered saline, pH 7.4, and ture medium (RPMI-1640 supplemented with 5% heat- resuspended in THB [34–36]. Bacterial cultures were inactivated fetal bovine serum, 10 mM HEPES, 2 mM appropriately diluted and plated on THB agar (THA) L-glutamine, and 50 µM 2-mercaptoethanol (Gibco, to accurately determine bacterial concentrations. Mice Burlington, ON, Canada) and complemented with 20% were inoculated with 1 × ­107 CFU via the intraperito- granulocyte-macrophages colony-stimulating factor from neal route and health and behavior monitored at least mouse-transfected Ag8653 cells [38]. Cell purity was thrice daily until 72 h post-infection and twice thereafter confirmed to be at least 90% CD11c + by flow cytometry until the end of the experiment (12 days post-infection) as previously described [21]. Albeit this culture system for the development of clinical signs of sepsis, such as cannot completely rule out the presence of other innate depression, swollen eyes, rough hair coat, prostration, cells such as macrophages, it represents an enriched and lethargy. For bacteremia studies, blood samples were source of bmDCs [39]. Payen et al. Veterinary Research (2023) 54:1 Page 5 of 13 All experiments were performed in the absence of mutant in which the cpsG gene, encoding a glycosyltrans- endotoxin (lipopolysaccharide) contamination and under ferase, was deleted from the P1/7 Δlmb strain was con- non-toxic conditions (data not shown), the latter being structed and compared to the wild-type P1/7, P1/7 Δlmb CytoTox ­96® Non-Radioactive Cytotoxicity Assay (Pro- evaluated by the lactate dehydrogenase release with the and P1/7 ΔcpsF. Surface hydrophobicity of P1/7 Δlmb was low (less than 6%) and comparable to that of wild- mega, Madison, WI, USA). Since the P1/7Δlmb mutant type P1/7 (Additional file 1). Meanwhile, and as expected, strain did not grow in the RPMI-1640 complete medium, deletion of the cpsG gene significantly increased surface heat-killed S. suis suspensions (prepared as previously hydrophobicity, with similar values to those obtained described [40]) of all tested strains were used for bmDC with P1/7 ΔcpsF non-encapsulated mutant (Additional stimulation at a concentration equivalent to 2 × ­109 CFU/ file 1). These results indicate that the Lmb does not play mL. Cells were resuspended at 1 × ­106 cells/mL in com- any role in the surface hydrophobicity of the strain tested. plete medium and stimulated with the different heat- killed S. suis strains. Supernatants were collected at 4 h, Lack of lipoprotein Lmb does not affect S. suis‑binding 6 h, 8 h, 12 h and 16 h following stimulation with heat-kill to laminin S. suis, incubation times at which secreted cytokine lev- Although previous studies revealed that purified Lmb els were maximal in the absence of cytotoxicity (data not of S. suis could bind human laminin [31], the ability of shown) [20, 21]. Mock-infected cells served as negative a Δlmb mutant to bind immobilized human placental controls. Secreted levels of tumor necrosis factor (TNF), laminin was not previously evaluated. Binding capacity interleukin (IL)-6, C-C motif chemokine ligand (CCL) 3, to laminin of the Δlmb mutant was comparable to that of and C-X-C motif chemokine ligand (CXCL) 1 were quan- the wild-type strain (Figure 1). It was also confirmed that tified by sandwich ELISA using pair-matched antibodies a non-encapsulated mutant strain is able to bind laminin from R&D Systems (Minneapolis, MN, USA) according more efficiently than the wild-type strain, as previously to the manufacturer’s recommendations. described [31]. Interestingly, adhesion to laminin of a double mutant Δlmb/non-encapsulated (Δlmb/ΔcpsG) Bacterial growth analysis was similar to that observed with the single non-encap- Bacterial growth experiments were performed in micro- sulated ΔcpsF mutant (Figure 1). Taken, together, these tubes (500-μL culture volume). A bacterial overnight results show that Lmb would not be critical for the culture grown (1 × ­104 CFU/mL) in THB was used to inoculate THB, plasma naturally poor in zinc, plasma with various ­ZnSO4 concentrations (from 0 to 50 μM) and plasma with ­ZnSO4 and the addition of the appropri- ate concentration of the chelating agent Tetrakis-(2-Pyri- dylmethyl) ethylenediamine (TPEN, Sigma Aldrich). Growth was followed during 24 h of incubation at 37 °C. The total numbers of CFU/mL were evaluated at different incubation times. Statistical analyses Normality of data was verified using the Shapiro–Wilk test. Accordingly, parametric (unpaired t-test) or non- parametric tests (Mann-Whitney rank sum test), where appropriate, were performed to evaluate statistical dif- ferences between groups. Log-rank test was used to compare survival rates between wild-type-infected mice and those infected with mutant strains. Each in vitro test was repeated in at least three independent experiments. p < 0.05 was considered as statistically significant. Figure 1 The Δlmb mutant is not impaired in its capacity of adhesion to laminin. Adhesion to laminin of the wild-ype P1/7 strain Results (dark blue), Δlmb (light blue), ΔcpsF (dark brown) and Δlmb/ΔcpsG Surface hydrophobicity of the strains included in this study (light brown) mutant strains as evaluated by ELISA. Data represent the Previous studies have demonstrated that deletion of optical density (O.D.)450 mean ± SEM from at least three independent either cpsG or cpsF biosynthesis genes from S. suis results experiments. *Indicates a significant difference (p < 0.05). in a non-encapsulated phenotype [30, 41]. An isogenic Payen et al. Veterinary Research (2023) 54:1 Page 6 of 13 interaction between S. suis and laminin, even in the the Δlmb/ΔcpsG double mutant adhered and invaded the absence of the capsular polysaccharide (CPS). two cell types similarly to the non-encapsulated ΔcpsF mutant. These results indicate that the Lmb does not play Lack of lipoprotein Lmb does not impair the adhesion a critical role in the adhesion/invasion of the epithelial to and invasion of epithelial and endothelial swine cells and endothelial cells tested, even in the absence of the The role of the Lmb protein in the adhesion/invasion to CPS. NPTr and PBMEC cells was evaluated using the gener- ated mutant strains. For both cell types, and as expected Presence of the lipoprotein Lmb is required for full [42, 43], the non-encapsulated mutant strain significantly virulence of S. suis adhered and invaded cells more efficiently than the wild- To confirm the role of Lmb in S. suis virulence and type strain (Figures 2A–D). However, the Δlmb mutant development of clinical disease, a well-characterized similarly adhered and (weakly) invaded NPTr (Fig- C56BL/6 mouse model of infection was used [36, 44]. ures 2A, B) and PBMEC (Figures 2C, D) cells. Moreover, Wild-type strain-infected mice rapidly developed Figure 2 The Δlmb mutant is not impaired in its capacity of adhesion to and invasion of epithelial and endothelial cells. Adhesion to and invasion of NPTr (A, C) and PBMEC (B, D) of wild-type P1/7 strain (dark blue), Δlmb (light blue), ΔcpsF (dark brown) and Δlmb/ΔcpsG (light brown) mutant strains after 2 h of incubation. *Indicates a significant difference (p < 0.05). Each bar represents the mean bacterial concentration (CFU/ mL)+SEM from at least three independent experiments. Payen et al. Veterinary Research (2023) 54:1 Page 7 of 13 clinical signs of systemic disease characteristic of septic Blood bacterial burden was also evaluated at the shock with 60% of mice succumbing to infection within early infection times of 12 h, 24 h and 48 h (Figure 3B). 48 h (Figure 3A). On the other hand, only 20% of mice Twelve hours following infection, mice infected with infected with the Δlmb mutant succumbed to disease the wild-type strain presented elevated blood bacte- (Figure 3A). These mice developed transient signs of rial burdens averaging 1 × ­107 CFU/mL (Figure 3B). infection such as rough coat hair following inoculation Moreover, infection with the wild-type strain resulted of bacteria and rapidly recovered and presented a nor- in elevated bacterial burdens after 24 h and 48 h infec- mal behavior. These results confirm that the Lmb lipo- tion. On the other hand, mice infected with the Δlmb protein is crucial for the virulence of S. suis serotype 2 mutant show a significant decrease in blood bacte- strain P1/7. rial burden at 12, 24 h and 48 h post-infection. This Figure 3 Presence of the lipoprotein Lmb is important for S. suis systemic virulence and blood persistence following intraperitoneal inoculation. Survival (A) and blood bacterial burden at 12, 24 and 48 h post-infection (B) of C57BL/6 mice following intraperitoneal inoculation of the S. suis virulent wild-type P1/7 strain (dark blue) and Δlmb mutant strain (light blue). Data represent survival curves (A) (n = 15) or geometric mean (B) (n = survived mice at each time point). *(p < 0.05) indicates a significant difference between survival or blood bacterial burden of mice infected either with the wild-type or the Δlmb mutant strain. Payen et al. Veterinary Research (2023) 54:1 Page 8 of 13 Figure 4 Absence of the lipoprotein Lmb significantly decreases the cytokine levels in mouse plasma from infected animals. Plasma levels of IL-6 (A) and CXCL1 (B) in mice 12 h following intraperitoneal inoculation of the S. suis serotype 2 wild-type strain P1/7 (dark blue) or Δlmb mutant strain (light blue). Data represent mean ± SEM (n = 8). *p < 0.05 indicates a significant difference between plasma levels of mice infected with the mutant strain when compared to the wild-type P1/7 strain. difference would have been more evident at 48 h after The lipoprotein Lmb partially regulates bmDC release infection considering that almost 60% of mice infected of cytokines with the wild-type P1/7 strain were already dead and The role of the Lmb lipoprotein in cytokine release was the viable counts were performed with survived (less evaluated using bmDCs as an innate immune cell model, affected) mice only. These data indicate that the Δlmb given that dendritic cells play a critical role during S. mutant strain presents less survival capacity within the suis pathogenesis and that their inflammatory response bloodstream of infected mice. In addition, the inflammatory response of animals infected with the P1/7 and Δlmb mutant strain was evaluated. IL-6 and CXCL1 concentration were meas- ured after 12 h of infection. The concentrations of these plasma mediators were significantly lower for the Δlmb mutant strain when compared to P1/7 wild- type strain (Figure 4). Lmb does not promote S. suis resistance to phagocytosis by murine macrophages One of the hypotheses that may explain that the Δlmb mutant survives less in the bloodstream of infected mice is an increased susceptibility to phagocytosis. In vitro phagocytosis studies with murine macrophages showed that the wild-type strain was less phagocy- Figure 5 The lipoprotein Lmb is not involved in S. suis tosed than the non-encapsulated ΔcpsF mutant, as phagocytosis resistance to J774A.1 murine macrophages. previously described [30] (Figure 5). The Δlmb mutant Internalization of the P1/7 wild-type strain (dark blue), Δlmb (light was internalized similarly to the wild-type P1/7 strain, blue), ΔcpsF (dark brown) and Δlmb/ΔcpsG (light brown) mutant strains after 2 h of incubation. * (p < 0.05) indicate a significant whereas the Δlmb/ΔcpsG double mutant was phagocy- difference between the wild-type and ΔcpsF mutant and a significant tosed similarly to the non-encapsulated ΔcpsF mutant. difference between the Δlmb and Δlmb/ΔcpsG mutants. Each bar These results indicate that the lipoprotein Lmb does represents the mean bacterial concentration (CFU/mL) + SEM from at not play any role in the resistance to the phagocytosis. least three independent experiments. Payen et al. Veterinary Research (2023) 54:1 Page 9 of 13 to S. suis has been well-characterized [20, 21]. bmDCs (a poor medium that mimic in vivo conditions), growth were activated for up to 16 h with heat-killed bacteria. of the Δlmb mutant strain was seriously reduced when For all experiments and at all incubation times, con- compared to the wild-type strain (Figure 7B). The com- trol mock infected cells presented negligible cytokine plemented Δlmb mutant completely restored the growth values < 300 pg/mL (not shown). The Δlmb and Δlmb/ to the levels of the wild-type strain. To investigate if the cpsG mutant strains induced significant lower levels loss of Lmb was associated with a reduced ability to grow of IL-6 and TNF (Figures 6A, C) and, to a lesser extent, under conditions of zinc starvation, Z ­ nSO4 was added CXCL1 (Figure 6B), when compared to wild-type P1/7 to the plasma at concentrations of 25 and 50 µM (Fig- and non-encapsulated ΔcpsF mutant strains, respec- ure 7C). Growth of Δlmb mutant was partially and totally tively (Figure 6). The complemented Δlmb:: pMX1-lmb restored with 25 µM and 50 µM ­ZnSO4, respectively, strain restored the capacity of cytokine induction of the indicating a dose-effect of zinc concentration (Figure 7C). Δlmb mutant to values similar to those of the wild-type Finally, the chelating agent N,N,N’,N’-Tetrakis-(2-pyridyl- strain (Figure 6). On the other hand, the presence of Lmb methyl)ethylenediamine (TPEN), a cell permeable high- did not affect the induction of CCL3 (Figure 6D). These affinity ­Zn2+ chelator used to reduce concentration of results indicate that the Lmb lipoprotein is one of the S. zinc in zinc homeostasis studies (15, 49, 51) was added suis components responsible for cytokine release. to the plasma supplemented with Z ­ nSO4 (Figure 7C) [13, 45, 46]. Addition of 20 μM TPEN in plasma supple- The lipoprotein Lmb is essential for S. suis growth mented with 50 µM Z ­ nSO4 impaired the growth of the under zinc starvation conditions Δlmb mutant strain (Figure 7D). Considering the limited role observed for the Lmb as laminin-binding protein, the second characteristic Discussion already described in the literature for this lipoprotein The pathogenesis of the infection caused by S. suis is (Zn uptake) was evaluated. First, the wild-type P1/7 only partially known and the definition of critical viru- and Δlmb strains cultured in rich media (THB) pre- lence factors is controversial [2]. During the first steps sented similar good growth rate, as evaluated by bacterial of the infection, the interaction of microorganisms with counts (Figure 7A). In contrast, when cultured in plasma extracellular matrix proteins can promote bacterial Figure 6 Presence of the lipoprotein Lmb modulates S. suis induced dendritic cell (DC) cytokine production. Pro-inflammatory cytokine production by bmDCs following activation with heat killed bacteria of the wild-type P1/7 strain (dark blue), Δlmb (light blue), Δlmb:: pMX1-lmb (grey), ΔcpsF (dark brown) and Δlmb/ΔcpsG (light brown) mutant strains. Production of IL-6 (A), CXCL1 (B), TNF (C) and CCL3 (D). Data represent the mean+SEM of at least three independent experiments. *Indicates a significant difference (p < 0.05). Payen et al. Veterinary Research (2023) 54:1 Page 10 of 13 Figure 7 Growth of Δlmb mutant strain under different conditions. Growth of the wild-type P1/7 strain (dark blue), Δlmb (light blue) and Δlmb::pMX1-lmb (grey, plasma only) in THB (A), plasma (B), plasma supplemented with 25 µM ZnSO4 (pink dash) or 50 µM ZnSO4 (pink dot) (C) and plasma supplemented with 50 µM ZnSO4 (pink dot) added with 20 µM TPEN (green). *(p < 0.05) indicate a significant difference between the wild-type and Δlmb mutant in plasma, between Δlmb mutant with and without supplementation of ZnSO4 in plasma or between Δlmb mutant with supplementation of ZnSO4 and with supplementation of ZnSO4 with addition of TPEN. Each point represents mean bacterial concentration (CFU/mL) + / − SEM of at least three different independent experiments. colonization and invasion [47]. It has been shown that Based on the previous observation that some S. suis S. suis is able to interact with several of these proteins, strains are able to bind laminin [31], Zhang et al. showed including laminin [31]. On the other hand, in order for that S. suis possesses a lmb gene which is homologous bacterial pathogens to survive inside their hosts, they to those of S. pyogenes, S. agalactiae and S. pneumoniae need to efficiently acquire and use the available nutri- and the cloned and purified protein clearly adhered to ents from their surrounding medium. One of these com- laminin in vitro. However, it is not clear if this lipoprotein ponents playing an important role in the host-pathogen is mainly responsible for the binding of S. suis to laminin. interplay is zinc (Zn) [48]. Zn-binding lipoproteins have Results from the current study using a Lmb-defective also been described in S. suis as being critically important mutant indicate that the Lmb does not play a critical role for bacterial fitness and survival during the infection [19]. in the bacterial binding to this extracellular matrix pro- Interestingly, one of these S. suis laminin- and Zn-bind- tein. Although Lmb was reported as a surface protein ing proteins is a single lipoprotein with a double func- [10], it was previously described that the presence of the tion previously identified as lipoprotein 103, Lmb CDS CPS may prevent S. suis to attach to laminin by hinder- 0330 or AdcAII [18, 19]. This protein (or genes coding ing surface proteins [31]. Hence, a double mutant with- for it) presents high homology with that of other strep- out Lmb and CPS was constructed and tested. The ΔcpsF tococci [10], in which this double function has also been non-encapsulated mutant adhered significantly more to described [5, 7, 13, 46]. In the case of S. suis, it is not clear laminin when compared to the wild-type strain confirm- whether the laminin-binding or the Zn-binding capaci- ing previous results. However, the double Δlmb/ΔcpsG ties of this protein (or both) plays an important role dur- mutant adhered similarly to the non-encapsulated ing the infection. In the present study, the more precise mutant, indicating that even in the absence of CPS the role of this lipoprotein (called Lmb in the current study surface expressed Lmb does not seem to play an impor- for simplicity purposes) in the pathogenesis of the infec- tant role in S. suis adhesion to the laminin. The presence tion has been studied. of additional proteins able to bind laminin that have been Payen et al. Veterinary Research (2023) 54:1 Page 11 of 13 described as being present in S. suis serotype 2 may com- (ST) 1 as wild-type reference strain, whereas Zhang et al. pensate the absence of Lmb [49, 50]. [19] used a ST7 highly virulent strain from China. Previ- The role of the Lmb in the adhesion/invasion of host ous studies have also reported some differences within S. cells seems to vary among different streptococci. For suis serotype 2 strains from different background in the example, the presence of the Lmb seems to be important pathogenesis of the infection [22, 55]. It may be hypoth- for S. agalactiae colonization of the epithelium and sub- esized that regulation of Zn may vary depending on the sequent translocation into the bloodstream, having a tro- virulence/phenotype of the strain. pism for the central nervous system [51]. The Lbp from S. The lower virulence of the Δlmb mutant can be pneumoniae has also been reported to play an important explained by a lower survival (lower bacteremia) in vivo role in the adhesion of the pathogen to human micro- as shown in the current study. This would be directly vascular endothelial cells [52] and S. pyogenes Δlmb (or related to the in-vivo Zn-restricted conditions rather to Lsp, or Lbp) mutants show a decrease adhesion only [6] a higher susceptibility of the mutant to bacterial killing. or a decrease in adhesion and invasion [7] to host cells. Indeed, no difference in the phagocytosis rate between Results of the current study showed that the Lmb does the Δlmb mutant and the wild-type strains could be not play any major role in S. suis adhesion to or invasion observed. In addition, induction of production of large of swine respiratory epithelial and brain microvascular amounts of pro-inflammatory mediators leading to endothelial cells, which correlates with the fact that this exacerbated inflammation is a hallmark of S. suis infec- protein is not critical for laminin binding. As mentioned, tions and is responsible, at least in part, for host death other proteins playing similar overlapped functions may [56]. Although the current study clearly showed a lower compensate and allow S. suis to adhere to this extracel- cytokine concentration in mice infected with the Δlmb lular matrix protein and host cells. mutant, this effect might be the simple consequence of There are several studies on the role of a Lmb (or lower bacteremia. However, in addition to this, in vitro homologous proteins) as Zn-binding proteins of strepto- results showed that the presence of the Lmb is also cocci [5–8], with the exception of that of S. pneumoniae important as cytokine activator. Similarly, the laminin- which plays an important role as a Zn-binding lipopro- binding protein from S. pneumoniae (Lbp) was reported tein but it does not bind laminin [53]. As mentioned, to activate human brain microvascular-endothelial cells two Zn-binding proteins have been described in S. suis: [52], whereas that of S. agalactiae was not involved in AdcA and AdcAII [19]. Although both lipoproteins may IL-8 release when using the same cells [12]. As lipopro- play a role in Zn-binding, Zhang et al. reported that teins have been clearly shown to be responsible for the AdcAII expression is significantly more up-regulated cytokine activation of cells by S. suis [57, 58], it can also than AdcA, but only the absence of both lipoproteins be hypothesized that the lower cytokine production affected survival under Zn-restricted conditions and in vivo by the Δlmb mutant is a double combination of virulence in a mouse model of infection [19]. Results of lower bacteremia and reduced activation due to the the current study confirm only partially such observa- absence of this lipoprotein. tions. Indeed, the Δlmb mutant (which corresponds to In summary, results from the current study showed the ΔAdcAII previously described) could not grow under that Lmb does not play an important role in the Zn-restricted conditions (plasma) and the addition of laminin-binding activity of S. suis serotype 2. In addi- Zn restored this defect. In addition, the complemented- tion, the presence of this lipoprotein does not influence mutant was able to grow at similar levels than the wild- bacterial adhesion to and invasion of porcine respira- type strain. In addition, and also different from the study tory epithelial and brain endothelial cells and it does of Zhang et al. [19], in the current work, the Δlmb mutant not increase the susceptibility of S. suis serotype 2 to presented a markedly lower virulence than the wild-type phagocytosis. On the other hand, this lipoprotein was strain, confirming previous results reported by Aranda shown to play an important role as cytokine activator et al. [18], who used mice from a different background when tested in vitro with dendritic cells. Finally, the (BALB/c). The critical role of ΔAdcAII has also been Lmb plays a critical role in Zn acquisition from the host observed in S. pneumoniae (16), whereas both ΔAdcA environment allowing bacteria to grow in vivo. The sig- and ΔAdcAII presented less virulence than a wild-type nificant lower virulence of the Lmb defective mutant strain of Group A Streptococcus [54]. In our hands, the may be related to a combination of a lower bacterial presence of ΔAdcAII is critical for Zn uptake and viru- survival due to the incapacity to acquire and use Zn lence in S. suis serotype 2. Differences observed among from their surrounding milieu and a reduced cytokine the S. suis studies might be explained by the background activation in the absence of this lipoprotein. Since the of the S. suis strains used: the current study and that of gene coding for this protein is also present in other Aranda et al. [18] used the virulent P1/7 sequence type serotypes of S. suis (data from GeneBank, not shown), Payen et al. Veterinary Research (2023) 54:1 Page 12 of 13 it would be interesting to confirm if the role of the Lmb Received: 13 September 2022 Accepted: 8 November 2022 described in the current study applies also to other pathogenic serotypes of S. suis. References Abbreviations 1. Gottschalk M, Xu J, Calzas C, Segura M (2010) Streptococcus suis: a new Ap: ampicillin; CFU: colony-forming unit; CCL: C-C motif chemokine ligand; emerging or an old neglected zoonotic pathogen? Future Microbiol CXCL: C-X-C motif chemokine ligand; DC: dendritic cell; ELISA: enzyme-linked 5:371–391 immunosorbent assay; Ig: immunoglobulin; IL: interleukin; Km: kanamycin; LB: 2. Segura M, Fittipaldi N, Calzas C, Gottschalk M (2017) Critical Streptococ- Luria-Bertani; Lmb: laminin binding protein; Nptr: Neonatal Porcine Tracheal cus suis virulence factors: are they all really critical? Trends Microbiol Epithelial cell; PBMEC: Porcine Brain Microvascular Endothelial cell; PCR: poly- 25:585–599 merase chain reaction; ST: sequence type; THA: THB agar; THB: Todd Hewitt 3. Segura M, Calzas C, Grenier D, Gottschalk M (2016) Initial steps of the broth; TNF: tumor necrosis factor; Zn: zinc; TPEN: Tetrakis-(2-Pyridylmethyl) pathogenesis of the infection caused by Streptococcus suis: fighting ethylenediamine. against nonspecific defenses. FEBS Lett 590:3772–3799 4. Jiang Q, Zhou X, Cheng L, Li M (2019) The adhesion and invasion mecha- nisms of Streptococci. Curr Issues Mol Biol 32:521–560 Supplementary Information 5. Spellerberg B, Rozdzinski E, Martin S, Weber-Heynemann J, Schnitzler N, The online version contains supplementary material available at https://​doi.​ Lutticken R, Podbielski A (1999) Lmb, a protein with similarities to the org/​10.​1186/​s13567-​022-​01128-8. LraI adhesin family, mediates attachment of Streptococcus agalactiae to human laminin. 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Loisel E, Jacquamet L, Serre L, Bauvois C, Ferrer JL, Vernet T, Di Guilmi Acknowledgements AM, Durmort C (2008) AdcAII, a new pneumococcal Zn-binding protein The authors would like to thank S. Lacouture for technical help and Dr N. Fit- homologous with ABC transporters: biochemical and structural analysis. J tipaldi for constructive suggestions with mutant constructions. Mol Biol 381:594–606 9. Kikkawa Y, Sanzen N, Sekiguchi K (1998) Isolation and charac- Authors’ contributions terization of laminin-10/11 secreted by human lung carcinoma cells: Conception of the work: SP, JAR, MS, MG; laboratory techniques: SP, JAR; acqui- laminin-10/11 mediates cell adhesion through integrin α3β1. J Biol Chem sition, analysis and interpretation of data: SP, MS, MG; preparation of the manu- 273:15854–15859 script: SP, JAR, MS, MG. All authors read and approved the final manuscript. 10. Zhang YM, Shao ZQ, Wang J, Wang L, Li X, Wang C, Tang J, Pan X (2014) Prevalent distribution and conservation of Streptococcus suis Lmb protein Funding and its protective capacity against the Chinese highly virulent strain This study was funded by the Natural Sciences and Engineering Research infection. Microbiol Res 169:395–401 Council of Canada (NSERC) to MG (grant #-2022–03730) and to MS (Grant # 11. Linke C, Caradoc-Davies TT, Young PG, Proft T, Baker EN (2009) The 2021–03020). laminin-binding protein Lbp from Streptococcus pyogenes is a zinc recep- tor. J Bacteriol 191:5814–5823 12. Tenenbaum T, Spellerberg B, Adam R, Vogel M, Kim KS, Schroten H (2007) Declarations Streptococcus agalactiae invasion of human brain microvascular endothe- lial cells is promoted by the laminin-binding protein Lmb. Microbes Infect Ethics approval and consent to participate 9:714–720 All experiments involving animals were conducted in accordance with the 13. 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