Identification and characterization of the cell division protein MapZ from Streptococcus suis.

Streptococcus suis, an emerging zoonotic pathogen, causes invasive diseases in pigs, including sepsis, meningitis, endocarditis, pneumonia, and arthritis. Importantly, similar pathologies are reported in human S. suis infections. In previous work, the locus SSU0375 of S. suis strain P1.7 had been identified as a conditionally essential gene by intrathecal experimental infection of pigs with a transposon library of S. suis. This study aimed to identify the function of the corresponding gene product. Bioinformatics analysis and homology modeling revealed sequence and structural homologies with the Streptococcus pneumoniae mid-cell-anchored protein Z (MapZ) that is involved in cell division in different bacterial species. Indeed, depletion of this locus in S. suis strain 10 revealed a growth defect as compared to the wild type. Electron microscopy analysis of the corresponding mutant demonstrated morphological growth defects as compared to the wild-type strain, including an irregular cell shape and size as well as mispositioned division septa. Light microscopy and subsequent quantitative image analysis confirmed these morphological alterations. In the genetic rescue strain, the wild-type phenotype was completely restored. In summary, we proposed that SSU0375 or the corresponding locus in strain 10 encode for a S. suis MapZ homolog that guides septum positioning as evidenced for other members of the Streptococci family.

Pdh is involved in the cell division and Normal septation of Streptococcus suis.

Streptococcus suis (S. suis) is an important zoonotic pathogen that causes major economic losses in the pig industry worldwide. The S. suis cell division process is an integral part of its growth and reproduction, which is controlled by a complex regulatory network. Pyruvate dehydrogenase (PDH), which catalyzes the oxidative decarboxylation of pyruvate to form acetyl-CoA, while reducing NAD + to NADH, plays an important role in energy metabolism. Recently, we reported that pdh regulates virulence by reducing stress tolerance and biofilm formation in S. suis serotype 2. In this study, we found that deletion of the pdh gene in S. suis resulted in abnormal cell chains, plump morphology and abnormal localization of the Z rings, indicating that the knockout mutant is impaired in its ability to divide. In addition, the interaction between FtsZ and PDH in vitro was confirmed by ELISA, and qRT-PCR analysis revealed that the deletion of the pdh gene results in differential expression of the division-related genes ftsZ, ftsK, ftsl, zapA, divIC, pbp1a, rodA, mreD, and sepF. These results indicate that pdh is involved in the normal formation of Z rings and cell morphology during S. suis cell division.

Co-regulation of CodY and (p)ppGpp synthetases on morphology and pathogenesis of Streptococcus suis.

CodY and (p)ppGpp synthetases are two important global regulators of bacteria. In some pathogens, such as Listeria monocytogenes, the GTP pool links these two regulatory systems, and introducing a codY mutant into the ΔrelA strain restored the pathogenicity of the attenuated ΔrelA mutant. In previous studies, we identified the (p)ppGpp synthetases (RelA and RelQ) and CodY of Streptococcus suis. To understand the interrelationships between these two regulators in S. suis, a ΔrelAΔrelQΔcodY mutant was constructed, and its growth, morphology, and pathogenicity were evaluated. Compared with ΔrelAΔrelQ, ΔcodY, its growth was very slow, but its chain length was partly restored to the wild-type length and its capsule became thick and rough. The adherence, invasion ability, and resistance to whole-blood killing in vitro of ΔrelAΔrelQΔcodY and its lethality and colonization ability in mice were clearly reduced, which differs from the effects of these mutations in L. monocytogenes. An analysis of gene expression showed that CodY interacted with the relA promoter in a GTP-independent manner to positively regulate the expression of relA. The introduction of a codY mutant into the ΔrelAΔrelQ strain further reduced the expression of virulence factors, which suggests a novel interaction between the (p)ppGpp synthetases and CodY. This study extends our understanding of the relationship between the (p)ppGpp-mediated stringent response and the regulation of CodY in S. suis.

Streptococcus suis DivIVA Protein Is a Substrate of Ser/Thr Kinase STK and Involved in Cell Division Regulation.

Streptococcus suis serotype 2 is an important swine pathogen and an emerging zoonotic agent that causes severe infections. Recent studies have reported a eukaryotic-like Ser/Thr protein kinase (STK) gene and characterized its role in the growth and virulence of different S. suis 2 strains. In the present study, phosphoproteomic analysis was adopted to identify substrates of the STK protein. Seven proteins that were annotated to participate in different cell processes were identified as potential substrates, which suggests the pleiotropic effects of stk on S. suis 2 by targeting multiple pathways. Among them, a protein characterized as cell division initiation protein (DivIVA) was further investigated. In vitro analysis demonstrated that the recombinant STK protein directly phosphorylates threonine at amino acid position 199 (Thr-199) of DivIVA. This effect could be completely abolished by the T199A mutation. To determine the specific role of DivIVA in growth and division, a divIVA mutant was constructed. The ΔdivIVA strain exhibited impaired growth and division, including lower viability, enlarged cell mass, asymmetrical division caused by aberrant septum, and extremely weak pathogenicity in a mouse infection model. Collectively, our results reveal that STK regulates the cell growth and virulence of S. suis 2 by targeting substrates that are involved in different biological pathways. The inactivation of DivIVA leads to severe defects in cell division and strongly attenuates pathogenicity, thereby indicating its potential as a molecular drug target against S. suis.

(p)ppGpp synthetases regulate the pathogenesis of zoonotic Streptococcus suis.

(p)ppGpp-mediated stringent response is one of the main adaption mechanism in bacteria, and the ability to adapt to environment is linked to the pathogenesis of bacterial pathogens. In the zoonotic pathogen Streptococcus suis, there are two (p)ppGpp synthetases, RelA and RelQ. To investigate the regulatory functions of (p)ppGpp/(p)ppGpp synthetases on the pathogenesis of S. suis, the phenotypes of the [(p)ppGpp(0)] mutant ΔrelAΔrelQ and its parental strain were compared. Light and electron microscopy observation showed that the mutant strain had a longer chain-length than its parental strain. Disruption of relA and relQ led to decreased adhesive and invasive ability to HEp-2 cells, and increased sensitivity to the blood killing and phagocytosis. Mouse infection experiments showed that the mutant strain was attenuated and easier to be cleaned up in vivo. Quantitative reverse transcription PCR (qRT-PCR) analysis indicated that the expressions of virulence related genes involving in morphology and virulence were down-regulated in the mutant strain. Our study demonstrated that the (p)ppGpp synthetases or (p)ppGpp can regulate the pathogenesis of this important zoonotic pathogen.

Polar invasion and translocation of Neisseria meningitidis and Streptococcus suis in a novel human model of the blood-cerebrospinal fluid barrier.

Acute bacterial meningitis is a life-threatening disease in humans. Discussed as entry sites for pathogens into the brain are the blood-brain and the blood-cerebrospinal fluid barrier (BCSFB). Although human brain microvascular endothelial cells (HBMEC) constitute a well established human in vitro model for the blood-brain barrier, until now no reliable human system presenting the BCSFB has been developed. Here, we describe for the first time a functional human BCSFB model based on human choroid plexus papilloma cells (HIBCPP), which display typical hallmarks of a BCSFB as the expression of junctional proteins and formation of tight junctions, a high electrical resistance and minimal levels of macromolecular flux when grown on transwell filters. Importantly, when challenged with the zoonotic pathogen Streptococcus suis or the human pathogenic bacterium Neisseria meningitidis the HIBCPP show polar bacterial invasion only from the physiologically relevant basolateral side. Meningococcal invasion is attenuated by the presence of a capsule and translocated N. meningitidis form microcolonies on the apical side of HIBCPP opposite of sites of entry. As a functionally relevant human model of the BCSFB the HIBCPP offer a wide range of options for analysis of disease-related mechanisms at the choroid plexus epithelium, especially involving human pathogens.

[Biological profiles of recombinant Suilysin].

OBJECTIVE: To provide insights into the role of Suilysin in the pathogenesis of Streptococcus suis by analysing the biological profiles of recombinant Suilysin (rSLY). METHODS: sly gene of S. suis strain SC22 was cloned into pET30a(+) vector and overexpressed in E. coli Rosseta, and rSLY was purified with Ni2+ affinity chromatography, anion exchange chromatography and gel filtration. The hemolytic unit of rSLY was measured with human red blood cells. The cellular toxicity of rSLY to human peripheral white blood cell and cells derived from fetal heart (HEH), liver (HEL), kidney (HEK), lung (HPL) was detected by lactate dehydrogenase (LDH) release assay. The blocking effects of water-soluble cholesterol, serum and specific antibody on the hemolytic activity of rSLY were detected. The amount of proinflammatory cytokines in the serum of mice stimulated by rSLY was measured by Luminex-100. RESULTS: The hemolytic unit of rSLY was 0.125 nmol/L, but the cellular toxicity of 1 nmol/L rSLY to human peripheral white blood cell, HEH, HEL, HEK and HPF was 20%-25%. The hemolytic activity of rSLY was blocked completely by equal molar of water-soluble cholesterol. Human serum containing 2.8 mmol/L-5.7 mmol/L of cholesterol in physical condition, could merely blocked 1 nmol/L of rSLY to lyse erythrocytes. The human serum with the addition of rabbit anti-rSLY IgG to a final concentration of 15 mg/ml decreased the hemolysis by 10 nmol/L rSLY from 77% down to 5% successfully, but hemolysis remained 60% when tested with 100 nmol/L rSLY. Although IL-1 beta and TNF-alpha didn't display any observable change, the level of interleukin -6 (IL-6) and KC were increased continually in C57BL/6 mice injected intraperitoneumly with rSLY whereas the IL-6 and KC level of mice in the control group only increased slightly and quickly decreased. CONCLUSION: The data suggest that rSLY could not only lyse cells but also induce strong inflammatory response and damage immune cells recruited to the sites of inflammation. This finding implies that serum could provide light protection against rSLY, and specific antibody show a concentration-dependent protection.

Determination of the cell adhesion specificity of Streptococcus suis with the complete set of monodeoxy analogues of globotriose.

Streptococcus suis causes meningitis and other serious infections in pigs and humans, and binds to host cell globotriosylceramide. In order to determine the essential hydroxyls involved in binding, the complete set of monodeoxy derivatives of the receptor trisaccharide Gal alpha1-Gal beta1-4Glc were tested as inhibitors of bacterial hemagglutination. Removal of the 4''-, 6'', 2' or 3'-hydroxyls abolished inhibitory activity, which indicated that they were critically involved in binding. The same results were obtained using synthetic lipid-linked monodeoxy derivatives of the trisaccharides in a thin-layer overlay assay. The P(N) and P(O) subtypes of the S. suis adhesin showed similar binding patterns. The hydroxyls of the glucose moiety were not critical for binding, although the adhesin binds better to the trisaccharide Gal alpha1-4Gal beta1-4Glc than the disaccharide Gal alpha1-4Gal.