ABSTRACT
BACKGROUND: Streptococcus suis is an important zoonotic pathogen. Biofilm formation largely explains the difficulty in preventing and controlling S. suis. However, little is known about the molecular mechanism of S. suis biofilm formation. RESULTS: In this study, transcriptomic and metabolomic analyses of S. suis in biofilm and planktonic states were performed to identify key genes and metabolites involved in biofilm formation. A total of 789 differential genes and 365 differential metabolites were identified. By integrating transcriptomics and metabolomics, five main metabolic pathways were identified, including amino acid pathway, nucleotide metabolism pathway, carbon metabolism pathway, vitamin and cofactor metabolism pathway, and aminoacyl-tRNA biosynthesis metabolic pathway. CONCLUSIONS: These results provide new insights for exploring the molecular mechanism of S. suis biofilm formation.
Subject(s)
Biofilms , Streptococcus suis , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biofilms/growth & development , Gene Expression Profiling , Gene Expression Regulation, Bacterial , Metabolic Networks and Pathways/genetics , Metabolome , Metabolomics , Multiomics , Streptococcus suis/genetics , Streptococcus suis/metabolism , TranscriptomeABSTRACT
The subtilisin-like protease-1 (SspA-1) plays an important role in the pathogenesis of a highly virulent strain of Streptococcus suis 2. However, the mechanism of SspA-1-triggered excessive inflammatory response is still unknown. In this study, we demonstrated that activation of type I IFN signaling is required for SspA-1-induced excessive proinflammatory cytokine production. Further experiments showed that the TLR2 endosomal pathway mediates SspA-1-induced type I IFN signaling and the inflammatory response. Finally, we mapped the major signaling components of the related pathway and found that the TIR adaptor proteins Mal, TRAM, and MyD88 and the downstream activation of IRF1 and IRF7 were involved in this pathway. These results explain the molecular mechanism by which SspA-1 triggers an excessive inflammatory response and reveal a novel effect of type I IFN in S. suis 2 infection, possibly providing further insights into the pathogenesis of this highly virulent S. suis 2 strain.
Subject(s)
Cytokines , Endosomes , Interferon Type I , Signal Transduction , Streptococcus suis , Toll-Like Receptor 2 , Streptococcus suis/immunology , Streptococcus suis/pathogenicity , Streptococcus suis/metabolism , Interferon Type I/metabolism , Toll-Like Receptor 2/metabolism , Cytokines/metabolism , Animals , Endosomes/metabolism , Mice , Streptococcal Infections/immunology , Streptococcal Infections/microbiology , Streptococcal Infections/metabolism , Bacterial Proteins/metabolism , Type IV Secretion Systems/metabolism , Type IV Secretion Systems/genetics , Humans , Myeloid Differentiation Factor 88/metabolism , Myeloid Differentiation Factor 88/genetics , Mice, Inbred C57BLABSTRACT
Streptococcus suis (S. suis) is a significant zoonotic microorganism that causes a severe illness in both pigs and humans and is characterized by severe meningitis and septicemia. Suilysin (SLY), which is secreted by S. suis, plays a crucial role as a virulence factor in the disease. To date, the interaction between SLY and host cells is not fully understood. In this study, we identified the interacting proteins between SLY and human brain microvascular endothelial cells (HBMECs) using the TurboID-mediated proximity labeling method. 251 unique proteins were identified in TurboID-SLY treated group, of which six plasma membrane proteins including ARF6, GRK6, EPB41L5, DSC1, TJP2, and PNN were identified. We found that the proteins capable of interacting with SLY are ARF6 and PNN. Subsequent investigations revealed that ARF6 substantially increased the invasive ability of S. suis in HBMECs. Furthermore, ARF6 promoted SLY-induced the activation of p38 MAPK signaling pathway in HBMECs. Moreover, ARF6 promoted the apoptosis in HBMECs through the activation of p38 MAPK signaling pathway induced by SLY. Finally, we confirmed that ARF6 could increase the virulence of SLY in C57BL/6 mice. These findings offer valuable insights that contribute to a deeper understanding of the pathogenic mechanism of SLY.
Subject(s)
ADP-Ribosylation Factor 6 , ADP-Ribosylation Factors , Apoptosis , Endothelial Cells , Hemolysin Proteins , Streptococcus suis , Streptococcus suis/pathogenicity , Streptococcus suis/metabolism , Humans , Animals , Apoptosis/drug effects , Mice , ADP-Ribosylation Factors/metabolism , ADP-Ribosylation Factors/genetics , Endothelial Cells/metabolism , Endothelial Cells/drug effects , Endothelial Cells/microbiology , Hemolysin Proteins/metabolism , Hemolysin Proteins/pharmacology , p38 Mitogen-Activated Protein Kinases/metabolism , Streptococcal Infections/microbiology , Streptococcal Infections/metabolism , Virulence , Brain/metabolismABSTRACT
Streptococcus suis is an emerging zoonotic pathogen that can cause invasive disease commonly associated with meningitis in pigs and humans. To cause meningitis, S. suis must cross the blood-brain barrier (BBB) comprising blood vessels that vascularize the central nervous system (CNS). The BBB is highly selective due to interactions with other cell types in the brain and the composition of the extracellular matrix (ECM). Purified streptococcal surface enolase, an essential enzyme participating in glycolysis, can bind human plasminogen (Plg) and plasmin (Pln). Plg has been proposed to increase bacterial traversal across the BBB via conversion to Pln, a protease which cleaves host proteins in the ECM and monocyte chemoattractant protein 1 (MCP1) to disrupt tight junctions. The essentiality of enolase has made it challenging to unequivocally demonstrate its role in binding Plg/Pln on the bacterial surface and confirm its predicted role in facilitating translocation of the BBB. Here, we report on the CRISPR/Cas9 engineering of S. suis enolase mutants eno261, eno252/253/255, eno252/261, and eno434/435 possessing amino acid substitutions at in silico predicted binding sites for Plg. As expected, amino acid substitutions in the predicted Plg binding sites reduced Plg and Pln binding to S. suis but did not affect bacterial growth in vitro compared to the wild-type strain. The binding of Plg to wild-type S. suis enhanced translocation across the human cerebral microvascular endothelial cell line hCMEC/D3 but not for the eno mutant strains tested. To our knowledge, this is the first study where predicted Plg-binding sites of enolase have been mutated to show altered Plg and Pln binding to the surface of S. suis and attenuation of translocation across an endothelial cell monolayer in vitro.
Subject(s)
Meningitis , Streptococcus suis , Animals , Humans , Swine , Plasminogen/metabolism , Blood-Brain Barrier , Streptococcus suis/genetics , Streptococcus suis/metabolism , Bacterial Translocation , Fibrinolysin/metabolism , Binding Sites , Phosphopyruvate Hydratase/chemistryABSTRACT
Streptococcus suis (S. suis), a significant zoonotic bacterial pathogen impacting swine and human, is associated with severe systemic diseases such as streptococcal toxic shock-like syndrome, meningitis, septicaemia, and abrupt fatality. The multifaceted roles of complement components C5a and C3a extend to orchestrating inflammatory cells recruitment, oxidative burst induction, and cytokines release. Despite the pivotal role of subtilisin-like serine proteases in S. suis pathogenicity, their involvement in immune evasion remains underexplored. In the present study, we identify two cell wall-anchored subtilisin-like serine proteases in S. suis, SspA-1 and SspA-2, as binding partners for C3a and C5a. Through Co-Immunoprecipitation, Enzyme-Linked Immunosorbent and Far-Western Blotting Assays, we validate their interactions with the aforementioned components. However, SspA-1 and SspA-2 have no cleavage activity against complement C3a and C5a performed by Cleavage assay. Chemotaxis assays reveal that recombinant SspA-1 and SspA-2 effectively attenuate monocyte chemotaxis towards C3a and C5a. Notably, the ΔsspA-1, ΔsspA-1, and ΔsspA-1/2 mutant strains exhibit compromised survival in blood, and resistance of opsonophagocytosis, alongside impaired survival in blood and in vivo colonization compared to the parental strain SC-19. Critical insights from the murine and Galleria mellonella larva infection models further underscore the significance of sspA-1 in altering mortality rates. Collectively, our findings indicate that SspA-1 and SspA-2 are novel binding proteins for C3a and C5a, thereby shedding light on their pivotal roles in S. suis immune evasion and the pathogenesis.
Subject(s)
Streptococcal Infections , Streptococcus suis , Animals , Humans , Swine , Mice , Immune Evasion , Complement C3a , Streptococcus suis/metabolism , Cytokines , Subtilisins/metabolism , Streptococcal Infections/microbiologyABSTRACT
IMPORTANCE: Phase variation allows a single strain to produce phenotypic diverse subpopulations. Phase-variable restriction modification (RM) systems are systems that allow for such phase variation via epigenetic regulation of gene expression levels. The phase-variable RM system SsuCC20p was found in multiple streptococcal species and was acquired by an emerging zoonotic lineage of Streptococcus suis. We show that the phase variability of SsuCC20p is dependent on a recombinase encoded within the SsuCC20p locus. We characterized the genome methylation profiles of the different phases of SsuCC20p and demonstrated the consequential impact on the transcriptome and virulence in a zebrafish infection model. Acquiring mobile genetic elements containing epigenetic regulatory systems, like phase-variable RM systems, enables bacterial pathogens to produce diverse phenotypic subpopulations that are better adapted to specific (host) environments encountered during infection.
Subject(s)
Streptococcal Infections , Streptococcus suis , Animals , Streptococcus suis/genetics , Streptococcus suis/metabolism , Epigenesis, Genetic , DNA Restriction-Modification Enzymes/genetics , Zebrafish/microbiology , Virulence , Larva/microbiology , Epigenome , Transcriptome , Streptococcal Infections/microbiology , Bacterial Proteins/genetics , Bacterial Proteins/metabolismABSTRACT
Emodin was extracted from Rheum officinale Baill by ultrasound-assisted extraction (UAE), and ethanol was chosen as the suitable solvent through SEM and molecular dynamic simulation. Under the optimum conditions (power 541 W, time 23 min, liquid to material ratio 13:1 mL/g, ethanol concentration 83 %) predicted by RSM, the yield of emodin was 2.18 ± 0.11 mg/g. Moreover, ultrasound power and time displayed the significant effects on the extraction process. Extracting dynamics analysis indicated that the extraction process of emodin by UAE conformed to Fick's second diffusion law. The results of antibacterial experiments suggested that emodin can damage cell membrane and inhibit the expression of cps2A, sao, mrp, epf, neu and the hemolytic activity of S. suis. Biolayer interferometry and FT-IR multi-peak fitting assays demonstrated that emodin induced a secondary conformational shift in CcpA. Molecular docking and molecular dynamics confirmed that emodin bound to CcpA through hydrogen bonding (ALA248, GLU249, GLY129 and ASN196) and π-π T-shaped interaction (TYR225 and TYR130), and the mutation of amino acid residues affected the affinity of CcpA to emodin. Therefore, emodin inhibited the sugar utilization of S. suis through binding to CcpA, and CcpA may be a potential target to inhibit the growth of S. suis.
Subject(s)
Emodin , Rheum , Streptococcus suis , Emodin/pharmacology , Emodin/chemistry , Rheum/chemistry , Streptococcus suis/genetics , Streptococcus suis/metabolism , Molecular Docking Simulation , Spectroscopy, Fourier Transform Infrared , Anti-Bacterial Agents/pharmacology , Ethanol/metabolismABSTRACT
Streptococcus suis is a zoonotic Gram-positive bacterium that causes invasive infections such as sepsis and meningitis, threatening public health worldwide. For successful establishment of infection, the bacterium should subvert the innate effectors of immune defence, including the cathelicidin family of host-defence peptides that combat pathogenic bacteria by directly disrupting cell membranes and coordinating immune responses. Here, our study shows that an extracellular endopeptidase O (PepO) of S. suis contributes to assisting the bacterium to resist cathelicidin-mediated killing, as the deletion of the pepO gene makes S. suis more sensitive to the human cathelicidin LL-37, as well as its mouse equivalent, mCRAMP. This protease targets and cleaves both LL-37 and mCRAMP, degrading them into shorter peptides with only a few amino acids, thereby abrogating their ability to kill S. suis. By cleaving LL-37 and mCRAMP, PepO impairs their chemotactic properties for neutrophil migration and undermines their anti-apoptosis activity, which is required for prolonging neutrophil lifespan. Also, PepO inhibits the ability of LL-37 and mCRAMP to promote lysosome development in macrophages. Moreover, the loss of PepO attenuates organ injury and decreases bacterial burdens in a murine model of S. suis bacteraemia. Taken together, these data provide novel insights into the role of the intrinsic proteolytic characteristics of PepO in S. suis-host interaction. Our findings demonstrate that S. suis utilizes the PepO protease to cleave cathelicidins, which is an immunosuppressive strategy adopted by this bacterium to facilitate pathogenesis.
Subject(s)
Cathelicidins , Streptococcus suis , Animals , Humans , Mice , Cathelicidins/metabolism , Cathelicidins/pharmacology , Antimicrobial Cationic Peptides/metabolism , Immune Evasion , Streptococcus suis/genetics , Streptococcus suis/metabolism , Metalloendopeptidases , Bacteria/metabolismABSTRACT
BACKGROUND: Streptococcus suis serotype 2 (S. suis 2) is an important zoonotic pathogen. Orphan response regulator CovR plays crucial regulative functions in the survivability and pathogenicity of S. suis 2. However, research on the CovR in S. suis 2 is limited. RESULTS: In this study, the regulative functions of CovR in the survivability and pathogenicity were investigated in S. suis 2 isolated from a diseased pig. The deletion of CovR significantly weakened the survivability and pathogenicity of S. suis 2. Compared with the wild-type strain, ΔcovR showed slower growth rates and thinner capsular polysaccharides. Moreover, ΔcovR showed reduced adhesion and invasion to Hep-2 cells as well as anti-phagocytosis and anti-killing ability to 3D4 cells and anti-serum killing ability. In addition, the deletion of CovR significantly reduced the colonisation ability of S. suis 2 in mice. The survival rate of mice infected with ΔcovR was increased by 16.7% compared with that of mice infected with S. suis 2. Further, the deletion of CovR led to dramatic changes in metabolism-related pathways in S. suis 2, five of those, including fructose and mannose metabolism, glycerolipid metabolism, ABC transporters, amino sugar and nucleotide sugar metabolism and phosphotransferase system, were significantly down-regulated. CONCLUSIONS: Based on the results, CovR plays positive regulative functions in the survivability and pathogenicity of S. suis 2 SC19 strain isolated from a pig.
Subject(s)
Streptococcal Infections , Streptococcus suis , Swine Diseases , Animals , Mice , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Serogroup , Streptococcal Infections/veterinary , Streptococcus suis/metabolism , Swine , Virulence , Virulence Factors/metabolismABSTRACT
Streptococcus suis is an encapsulated bacterium causing severe diseases in swine. Here, we compared the protective properties of the capsular polysaccharide (CPS) of different S. suis serotypes by using serotype-switched mutants in a mouse model of infection. CPS structure influenced bacterial survival in mice, antibody binding, and antibody-mediated bacterial killing. The CPS of serotypes 3, 4 and 14 allowed more antibody binding and bacterial elimination than the CPS of serotypes 2, 7 and 9. Results suggest that the different CPS structures of S. suis provide varying levels of protection by influencing antigen availability and elimination by the host immune system.
Subject(s)
Streptococcal Infections , Streptococcus suis , Animals , Mice , Swine , Polysaccharides, Bacterial , Streptococcus suis/metabolism , Bacterial Capsules , Serogroup , Antibodies , Streptococcal Infections/microbiology , Antibodies, BacterialABSTRACT
Streptococcus suis (S. suis) regulates biofilm formation through LuxS/AI-2 quorum sensing system, increasing drug resistance and exacerbating infection. The anti-hyperglycaemic agent metformin has anti-bacterial and anti-biofilm activities. This study aimed to investigate the anti-biofilm and anti-quorum sensing activity of metformin in S. suis. We first determined the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of metformin on S. suis. The results indicated that metformin showed no obvious inhibitory or bactericidal effect. Crystal violet staining showed that metformin significantly inhibited the formation of S. suis biofilm at sub-MIC concentration, which was also confirmed by scanning electron microscopy. Then, we quantified the AI-2 signal molecules in S. suis, and the results showed that metformin had a significant inhibitory effect on the production of AI-2 signal in S. suis. Inhibition of enzyme activity and molecular docking experiments showed that metformin has a significant binding activity to LuxS protein. In addition, qRT-PCR results showed that metformin significantly down-regulated the expression of AI-2 synthesis-related genes luxS and pfs, and adhesion-related genes luxS, pfs, gapdh, sly, fbps, and ef. Western blotting also showed that metformin significantly reduced the expression of LuxS protein. Our study suggests that metformin seems to be a suitable candidate for the inhibition of S. suis LuxS/AI-2 QS system and prevention of biofilm formation, which provided a new idea for the prevention and control of S. suis.
Subject(s)
Streptococcus suis , Streptococcus suis/metabolism , Molecular Docking Simulation , Homoserine/metabolism , Bacterial Proteins/metabolism , Quorum Sensing , Biofilms , Carbon-Sulfur Lyases/genetics , Carbon-Sulfur Lyases/metabolism , Carbon-Sulfur Lyases/pharmacology , Lactones/metabolismABSTRACT
Due to the large amount of antibiotics used for human therapy, agriculture, and even aquaculture, the emergence of multidrug-resistant Streptococcus suis (S. suis) led to serious public health threats. Antibiotic-assisted strategies have emerged as a promising approach to alleviate this crisis. Here, the polyphenolic compound gallic acid was found to enhance sulfonamides against multidrug-resistant S. suis. Mechanistic analysis revealed that gallic acid effectively disrupts the integrity and function of the cytoplasmic membrane by dissipating the proton motive force of bacteria. Moreover, we found that gallic acid regulates the expression of dihydrofolate reductase, which in turn inhibits tetrahydrofolate synthesis. As a result of polypharmacology, gallic acid can fully restore sulfadiazine sodium activity in the animal infection model without any drug resistances. Our findings provide an insightful view into the threats of antibiotic resistance. It could become a promising strategy to resolve this crisis.
Subject(s)
Streptococcus suis , Animals , Humans , Streptococcus suis/genetics , Streptococcus suis/metabolism , Microbial Sensitivity Tests , Anti-Bacterial Agents/metabolism , Sulfanilamide/metabolism , Sulfanilamide/pharmacology , Cell MembraneABSTRACT
Bacterial morphology is largely determined by the spatial and temporal regulation of peptidoglycan (PG) biosynthesis. Ovococci possess a unique pattern of PG synthesis different from the well studied Bacillus, and the mechanism of the coordination of PG synthesis remains poorly understood. Several regulatory proteins have been identified to be involved in the regulation of ovococcal morphogenesis, among which DivIVA is an important one to regulate PG synthesis in streptococci, while its mechanism is largely unknown. Here, the zoonotic pathogen Streptococcus suis was used to investigate the regulation of DivIVA on PG synthesis. Fluorescent d-amino acid probing and 3D-structured illumination microscopy found that DivIVA deletion caused abortive peripheral PG synthesis, resulting in a decreased aspect ratio. The phosphorylation-depleted mutant (DivIVA3A) cells displayed a longer nascent PG and became longer, whereas the phosphorylation-mimicking mutant (DivIVA3E) cells showed a shorter nascent PG and became shorter, suggesting that DivIVA phosphorylation is involved in regulating peripheral PG synthesis. Several DivIVA-interacting proteins were identified, and the interaction was confirmed between DivIVA and MltG, a cell wall hydrolase essential for cell elongation. DivIVA did not affect the PG hydrolysis activity of MltG, while the phosphorylation state of DivIVA affected its interaction with MltG. MltG was mislocalized in the ΔdivIVA and DivIVA3E cells, and both ΔmltG and DivIVA3E cells formed significantly rounder cells, indicating an important role of DivIVA phosphorylation in regulating PG synthesis through MltG. These findings highlight the regulatory mechanism of PG synthesis and morphogenesis of ovococci. IMPORTANCE The peptidoglycan (PG) biosynthesis pathway provides a rich source of novel antimicrobial drug targets. However, bacterial PG synthesis and its regulation is a very complex process involving dozens of proteins. Moreover, unlike the well studied Bacillus, ovococci undergo unusual PG synthesis with unique mechanisms of coordination. DivIVA is an important regulator of PG synthesis in ovococci, while its exact role in regulating PG synthesis remains poorly understood. In this study, we determined the role of DivIVA in regulating lateral PG synthesis of Streptococcus suis and identified a critical interacting partner, MltG, in which DivIVA influenced the subcellular localizations of MltG through its phosphorylation. Our study characterizes the detailed role of DivIVA in regulating bacterial PG synthesis, which is very helpful for understanding the process of PG synthesis in streptococci.
Subject(s)
Streptococcus suis , Streptococcus suis/genetics , Streptococcus suis/metabolism , Peptidoglycan/metabolism , Hydrolases/metabolism , Cell Wall/metabolism , Phosphorylation , Bacteria/metabolismABSTRACT
Synthesis of capsular polysaccharide (CPS), an important virulence factor of pathogenic bacteria, is modulated by the CpsBCD phosphoregulatory system in Streptococcus. Serine/threonine kinases (STKs, e.g. Stk1) can also regulate CPS synthesis, but the underlying mechanisms are unclear. Here, we identify a protein (CcpS) that is phosphorylated by Stk1 and modulates the activity of phosphatase CpsB in Streptococcus suis, thus linking Stk1 to CPS synthesis. The crystal structure of CcpS shows an intrinsically disordered region at its N-terminus, including two threonine residues that are phosphorylated by Stk1. The activity of phosphatase CpsB is inhibited when bound to non-phosphorylated CcpS. Thus, CcpS modulates the activity of phosphatase CpsB thereby altering CpsD phosphorylation, which in turn modulates the expression of the Wzx-Wzy pathway and thus CPS production.
Subject(s)
Streptococcus suis , Phosphorylation , Streptococcus suis/metabolism , Polysaccharides, Bacterial/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Phosphoric Monoester Hydrolases/metabolism , Bacterial Capsules/metabolismABSTRACT
Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that can infect humans in contact with infected pigs or their byproducts. It can employ different types of genes to defend against oxidative stress and ensure its survival. The thioredoxin (Trx) system is a key antioxidant system that contributes adversity adaptation and pathogenicity. SS2 has been shown to encode putative thioredoxin genes, but the biological roles, coding sequence, and underlying mechanisms remains uncharacterized. Here, we demonstrated that SSU05_0237-ORF, from a clinical SS2 strain, ZJ081101, encodes a protein of 104 amino acids with a canonical CGPC active motif and an identity 70-85% similar to the thioredoxin A (TrxA) in other microorganisms. Recombinant TrxA efficiently catalyzed the thiol-disulfide oxidoreduction of insulin. The deletion of TrxA led to a significantly slow growth and markedly compromised tolerance of the pathogen to temperature stress, as well as impaired adhesion ability to pig intestinal epithelial cells (IPEC-J2). However, it was not involved in H2O2 and paraquat-induced oxidative stress. Compared with the wild-type strain, the ΔTrxA strain was more susceptible to killing by macrophages through increasing NO production. Treatment with TrxA mutant strain also significantly attenuated cytotoxic effects on RAW 264.7 cells by inhibiting inflammatory response and apoptosis. Knockdown of pentraxin 3 in RAW 264.7 cells was more vulnerable to phagocytic activity, and TrxA promoted SS2 survival in phagocytic cells depending on pentraxin 3 activity compared with the wild-type strain. Moreover, a co-inoculation experiment in mice revealed that TrxA mutant strain is far more easily cleared from the body than the wild type strain in the period from 8-24 h, and exhibits significantly attenuated oxidative stress and liver injury. In summary, we reveal the important role of TrxA in the pathogenesis of SS2.
Subject(s)
Macrophages , Streptococcal Infections , Streptococcus suis , Animals , Humans , Mice , Bacterial Proteins/metabolism , Hydrogen Peroxide/pharmacology , Hydrogen Peroxide/metabolism , Macrophages/metabolism , Macrophages/microbiology , Serogroup , Streptococcus suis/metabolism , Streptococcus suis/pathogenicity , Swine , Thioredoxins/genetics , Thioredoxins/metabolism , Thioredoxins/pharmacology , VirulenceABSTRACT
Streptococcus suis (SS) is a zoonotic pathogen that affects the health of humans and the development of the pig industry. The SS Cba protein is a collagen adhesin, and a few of its homologs are related to the enhancement of bacterial adhesion. We compared the phenotypes of SS9-P10, SS9-P10 cba knockout strains and its complementary strains in vitro and in vivo and found that knocking out the cba gene did not affect the growth characteristics of the strain, but it significantly reduced the ability of SS to form biofilms, adhesion to host cells, phagocytic resistance to macrophages and attenuated virulence in a mouse infection model. These results indicated that Cba was a virulence related factor of SS9. In addition, Mice immunized with the Cba protein had higher mortality and more serious organ lesions after challenge, and the same was observed in passive immunization experiments. This phenomenon is similar to the antibody-dependent enhancement of infection by bacteria such as Acinetobacter baumannii and Streptococcus pneumoniae. To our knowledge, this is the first demonstration of antibody-dependent enhancement of SS, and these observations highlight the complexity of antibody-based therapy for SS infection.
Subject(s)
Streptococcal Infections , Streptococcus suis , Humans , Animals , Mice , Swine , Virulence Factors/genetics , Virulence Factors/metabolism , Streptococcus suis/metabolism , Serogroup , Antibody-Dependent Enhancement , Virulence/genetics , Streptococcal Infections/microbiologyABSTRACT
Bacterial outer membrane vesicles (OMVs) are considered a promising vaccine platform for their high built-in adjuvanticity and ability to efficiently induce immune responses. OMVs can be engineered with heterologous antigens based on genetic engineering strategies. However, several critical issues should still be validated, including optimal exposure to the OMV surface, increased production of foreign antigens, nontoxicity, and induction of powerful immune protection. In this study, engineered OMVs with the lipoprotein transport machinery (Lpp) were designed to present SaoA antigen as a vaccine platform against Streptococcus suis. The results suggest that Lpp-SaoA fusions can be delivered on the OMV surface and do not have significant toxicity. Moreover, they can be engineered as lipoprotein and significantly accumulated in OMVs at high levels, thus accounting for nearly 10% of total OMV proteins. Immunization with OMVs containing Lpp-SaoA fusion antigen induced strong specific antibody responses and high levels of cytokines, as well as a balanced Th1/Th2 immune response. Furthermore, the decorated OMV vaccination significantly enhanced microbial clearance in a mouse infection model. It was found that antiserum against lipidated OMVs significantly promoted the opsonophagocytic uptake of S. suis in RAW246.7 macrophages. Lastly, OMVs engineered with Lpp-SaoA induced 100% protection against a challenge with 8× the 50% lethal dose (LD50) of S. suis serotype 2 and 80% protection against a challenge with 16× the LD50 in mice. Altogether, the results of this study provide a promising versatile strategy for the engineering of OMVs and suggest that Lpp-based OMVs may be a universal adjuvant-free vaccine platform for important pathogens. IMPORTANCE Bacterial outer membrane vesicles (OMVs) have become a promising vaccine platform due to their excellent built-in adjuvanticity properties. However, the location and amount of the expression of the heterologous antigen in the OMVs delivered by the genetic engineering strategies should be optimized. In this study, we exploited the lipoprotein transport pathway to engineer OMVs with heterologous antigen. Not only did lapidated heterologous antigen accumulate in the engineered OMV compartment at high levels, but also it was engineered to be delivered on the OMV surface, thus leading to the optimal activation of antigen-specific B cells and T cells. Immunization with engineered OMVs induced a strong antigen-specific antibodies in mice and conferred 100% protection against S. suis challenge. In general, the data of this study provide a versatile strategy for the engineering of OMVs and suggest that OMVs engineered with lipidated heterologous antigens may be a vaccine platform for significant pathogens.
Subject(s)
Streptococcus suis , Vaccines , Animals , Mice , Streptococcus suis/genetics , Streptococcus suis/metabolism , Antigens, Heterophile , Bacterial Outer Membrane Proteins/metabolism , Bacterial Outer Membrane/metabolism , Lipoproteins/genetics , Antibodies, Bacterial , Bacterial Vaccines/geneticsABSTRACT
The cpdB gene is pro-virulent in avian pathogenic Escherichia coli and in Salmonella enterica, where it encodes a periplasmic protein named CpdB. It is structurally related to cell wall-anchored proteins, CdnP and SntA, encoded by the also pro-virulent cdnP and sntA genes of Streptococcus agalactiae and Streptococcus suis, respectively. CdnP and SntA effects are due to extrabacterial hydrolysis of cyclic-di-AMP, and to complement action interference. The mechanism of CpdB pro-virulence is unknown, although the protein from non-pathogenic E. coli hydrolyzes cyclic dinucleotides. Considering that the pro-virulence of streptococcal CpdB-like proteins is mediated by c-di-AMP hydrolysis, S. enterica CpdB activity was tested as a phosphohydrolase of 3'-nucleotides, 2',3'-cyclic mononucleotides, linear and cyclic dinucleotides, and cyclic tetra- and hexanucleotides. The results help to understand cpdB pro-virulence in S. enterica and are compared with E. coli CpdB and S. suis SntA, including the activity of the latter on cyclic-tetra- and hexanucleotides reported here for the first time. On the other hand, since CpdB-like proteins are relevant to host-pathogen interactions, the presence of cpdB-like genes was probed in eubacterial taxa by TblastN analysis. The non-homogeneous genomic distribution revealed taxa with cpdB-like genes present or absent, identifying eubacteria and plasmids where they can be relevant.
Subject(s)
Escherichia coli Proteins , Salmonella enterica , Streptococcus suis , Escherichia coli/metabolism , Salmonella enterica/metabolism , Streptococcus suis/metabolism , Virulence , Cyclic AMP , Genomics , Escherichia coli Proteins/metabolism , 2',3'-Cyclic-Nucleotide Phosphodiesterases/geneticsABSTRACT
Streptococcus suis is a zoonotic pathogen that continuously threatens animal husbandry and public health worldwide. Studies have shown that S. suis can cause persistent infection by forming biofilms. In this study, a model of S. suis biofilm-related infection was successfully constructed for the first time by simulating the natural infection of S. suis, and biofilm of S. suis in vivo was successfully observed in the lung tissue of infected pigs by a variety of detection methods. Subsequently, selective capture of transcribed sequences (SCOTS) was used to identify genes expressed by S. suis in vivo biofilms. Sixty-nine genes were captured in in vivo biofilms formed by S. suis for the first time by SCOTS; they were mainly involved in metabolism, cell replication, and division, transport, signal transduction, cell wall, etc. Genes related to S. suis in vitro biofilm formation were also identified by SCOTS and RNA sequencing. Approximately half of the genes captured by SCOTS in the in vivo and in vitro biofilms were found to be different. In summary, our study provides powerful clues for future exploration of the mechanisms of S. suis biofilm formation. IMPORTANCE Streptococcus suis is considered an important zoonotic pathogen, and persistent infection caused by biofilm is currently considered to be the reason why S. suis is difficult to control in swine. However, to date, a model of the biofilm of S. suis in vivo has not been successfully constructed. Here, we successfully detected biofilms of S. suis in vivo in lung tissues of piglets infected with S. suis. Selective capture of transcribed sequences and the transcriptome were used to obtain gene profiles of S. suis in vivo and in vitro biofilms, and the results showed large differences between them. Such data are of importance for future experimental studies exploring the mechanism of biofilm formation by S. suis in vivo.
Subject(s)
Streptococcus suis , Transcriptome , Animals , Swine , Streptococcus suis/genetics , Streptococcus suis/metabolism , Persistent Infection , Biofilms , LungABSTRACT
Streptococcus suis LuxS/AI-2 quorum sensing system regulates biofilm formation, resulting in increased pathogenicity and drug resistance, and diminished efficacy of antibiotic treatment. The remaining peony seed cake after oil extraction is rich in monoterpenoid glycosides, which can inhibit the formation of bacterial biofilm. In this study, we investigated the effect of seven major monocomponents (suffruticosol A, suffruticosol B, suffruticosol C, paeonifloin, albiflorin, trans-ε-viniferin, gnetin H) of peony seed meal on minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of S. suis. The results showed that the MICs of the seven single components were all greater than 200 µg/mL, with no significant bacteriostatic and bactericidal advantages. Crystal violet staining and scanning electron microscope observation showed that the seven single components had a certain inhibitory effect on the biofilm formation ability of S. suis at sub-MIC concentration. Among them, the ability of paeoniflorin to inhibit biofilm was significantly higher than that of the other six single components. AI-2 signaling molecules were detected by bioreporter strain Vibrio harvey BB170. The detection results of AI-2 signal molecules found that at 1/2 MIC concentration, paeoniflorin significantly inhibited the production of S. suis AI-2 signal, and the inhibitory effect was better than that of the other six single components. In addition, molecular docking analysis revealed that paeoniflorin had a significant binding activity with LuxS protein compared with the other six single components. The present study provides evidence that paeoniflorin plays a key role in the regulation of the inhibition of S. suis LuxS/AI-2 system and biofilm formation in peony seed meal.