Insight into the role of Streptococcus suis zinc metalloprotease C from the new serotype causing meningitis in piglets.

Streptococcus suis (S. suis) is an important gram-positive pathogen and an emerging zoonotic pathogen that causes meningitis in swine and humans. Although several virulence factors have been characterized in S. suis, the underlying mechanisms of pathogenesis are not fully understood. In this study, we identified Zinc metalloproteinase C (ZmpC) probably as a critical virulence factor widely distributed in S. suis strains. ZmpC was identified as a critical facilitator in the development of bacterial meningitis, as evidenced by the detection of increased expression of TNF-α, IL-8, and matrix metalloprotease 9 (MMP-9). Subcellular localization analysis further revealed that ZmpC was localized to the cell wall surface and gelatin zymography analysis showed that ZmpC could cleave human MMP-9. Mice challenge demonstrated that ZmpC provided protection against S. suis CZ130302 (serotype Chz) and ZY05719 (serotype 2) infection. In conclusion, these results reveal that ZmpC plays an important role in promoting CZ130302 to cause mouse meningitis and may be a potential candidate for a S. suis CZ130302 vaccine.

Quantitative proteomic analysis reveals that serine/threonine kinase is involved in Streptococcus suis virulence and adaption to stress conditions.

The eukaryotic-type serine/threonine kinase of Streptococcus suis serotype 2 (SS2) performs critical roles in bacterial pathogenesis. In this study, isobaric tags for relative and absolute quantification (iTRAQ) MS/MS were used to analyze the protein profiles of wild type strain SS2-1 and its isogenic STK deletion mutant (Δstk). A total of 281 significant differential proteins, including 147 up-regulated and 134 down-regulated proteins, were found in Δstk. Moreover, 69 virulence factors (VFs) among these 281 proteins were predicted by the Virulence Factor Database (VFDB), including 38 downregulated and 31 up-regulated proteins in Δstk, among which 15 down regulated VFs were known VFs of SS2. Among the down-regulated proteins, high temperature requirement A (HtrA), glutamine synthase (GlnA), ferrichrome ABC transporter substrate-binding protein FepB, and Zinc-binding protein AdcA are known to be involved in bacterial survival and/or nutrient and energy acquisition under adverse host conditions. Overall, our results indicate that STK regulates the expression of proteins involved in virulence of SS2 and its adaption to stress environments.

Application of a dissolved oxygen control strategy to increase the expression of Streptococcus suis glutamate dehydrogenase in Escherichia coli.

The accumulation of acetate in Escherichia coli inhibits cell growth and desired protein synthesis, and cell density and protein expression are increased by reduction of acetate excretion. Dissolved oxygen (DO) is an important parameter for acetate synthesis, and the accumulation of acetate is inversely correlated to DO level. In this study, the effect of DO levels on glutamate dehydrogenase (GDH) expression was investigated, and then different DO control strategies were tested for effects on GDH expression. DO control strategy IV (50% 0-9 h, 30% 9-18 h) provided the highest cell density (15.43 g/L) and GDH concentration (3.42 g/L), values 1.59- and 1.99-times higher than those achieved at 10% DO. The accumulation of acetate was 2.24 g/L with DO control strategy IV, a decrease of 40.74% relative to that achieved for growth at 10% DO. Additionally, under DO control strategy IV, there was lower expression of PoxB, a key enzyme for acetate synthesis, at both the transcriptional and translational level. At the same time, higher transcription and protein expression levels were observed for a glyoxylate shunt gene (aceA), an acetate uptake gene (acs), gluconeogensis and anaplerotic pathways genes (pckA, ppsA, ppc, and sfcA), and a TCA cycle gene (gltA). The flux of acetate with DO strategy IV was 8.4%, a decrease of 62.33% compared with the flux at 10% DO. This decrease represents both lower flux for acetate synthesis and increased flux of reused acetate.

CrfP, a fratricide protein, contributes to natural transformation in Streptococcus suis.

Streptococcus suis (S. suis) is an important zoonotic pathogen that causes septicaemia, meningitis and streptococcal toxic shock-like syndrome in its host, and recent studies have shown that S. suis could be competent for natural genetic transformation. Transformation is an important mechanism for the horizontal transfer of DNA, but some elements that affect the transformation process need to be further explored. Upon entering the competent state, Streptococcus species stimulate the transcription of competence-related genes that are responsible for exogenous DNA binding, uptake and processing. In this study, we performed conserved promoter motif and qRT-PCR analyses and identified CrfP as a novel murein hydrolase that is widespread in S. suis and stimulated with a peptide pheromone in the competent state through a process controlled by ComX. A bioinformatics analysis revealed that CrfP consists of a CHAP hydrolase domain and two bacterial Src homology 3-binding (SH3b) domains. Further characterization showed that CrfP could be exported to extracellular bacterial cells and lytic S. suis strains of different serotypes, and this finding was verified by TEM and a turbidity assay. To investigate the potential effect of CrfP in vivo, a gene-deletion mutant (ΔcrfP) was constructed. Instead of stopping the natural transformation process, the inactivation of CrfP clearly reduced the effective transformation rate. Overall, these findings provide evidence showing that CrfP is important for S. suis serovar 2 competence.

Caveolae/rafts protect human cerebral microvascular endothelial cells from Streptococcus suis serotype 2 α-enolase-mediated injury.

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that causes meningitis. The ubiquitously expressed 40S ribosome protein SA (RPSA) is a multifunctional protein involved in the pathogenesis of multiple pathogens, especially those causing meningitis. However, the role of RPSA in SS2-induced meningitis is not clear. In this study, immunofluorescence staining revealed that SS2 infection promoted the intracellular transfer of RPSA to the surface of human cerebral microvascular endothelial cells (HCMECs). Moreover, SS2 infection promoted the accumulation of caveolin 1 (CAV1) and the formation of membrane bulges where RPSA enveloped CAV1 on the cell surface. SS2 infection also caused dynamic changes in the localization of RPSA and CAV1 on the cell surface which could be eliminated by disruption of caveolae/rafts by addition of methyl-ß-cyclodextrin (MßCD). Co-immunoprecipitation analysis demonstrated that α-enolase (ENO), a key virulence factor of SS2, interacted with RPSA, and promoted the interaction between RPSA and CAV1. Immunofluorescence staining, western blotting and flow cytometry analyses showed that damaged caveolae/rafts significantly enhanced ENO adhesion to HCMECs, promoted the "destruction" of RPSA by ENO, and enhanced the toxic effect of ENO on HCMECs. Importantly, these effects could be relieved upon the addition of cholesterol. We conclude that caveolae/rafts weaken the toxic effect of SS2 ENO on RPSA-mediated events in HCMECs. Our study has led to better understanding of the roles of RPSA and caveolae/rafts upon SS2 infection, and a new pathological role for RPSA in infection.

Role of Bacterial and Host DNases on Host-Pathogen Interaction during Streptococcus suis Meningitis.

Streptococcus suis is a zoonotic agent causing meningitis in pigs and humans. Neutrophils, as the first line of defense against S. suis infections, release neutrophil extracellular traps (NETs) to entrap pathogens. In this study, we investigated the role of the secreted nuclease A of S. suis (SsnA) as a NET-evasion factor in vivo and in vitro. Piglets were intranasally infected with S. suis strain 10 or an isogenic ssnA mutant. DNase and NET-formation were analyzed in cerebrospinal fluid (CSF) and brain tissue. Animals infected with S. suis strain 10 or S. suis 10ΔssnA showed the presence of NETs in CSF and developed similar clinical signs. Therefore, SsnA does not seem to be a crucial virulence factor that contributes to the development of meningitis in pigs. Importantly, DNase activity was detectable in the CSF of both infection groups, indicating that host nucleases, in contrast to bacterial nuclease SsnA, may play a major role during the onset of meningitis. The effect of DNase 1 on neutrophil functions was further analyzed in a 3D-cell culture model of the porcine blood-CSF barrier. We found that DNase 1 partially contributes to enhanced killing of S. suis by neutrophils, especially when plasma is present. In summary, host nucleases may partially contribute to efficient innate immune response in the CSF.

Identification and characterization of a Streptococcus suis immunogenic ornithine carbamoytransferase involved in bacterial adherence.

BACKGROUND: Streptococcus suis (SS) is a major swine pathogen and a serious zoonotic pathogen causing septicemia and meningitis in piglets and humans. Using an immunoproteomic approach, we previously brought evidence that ornithine carbamoytransferase (OCT) may represent a vaccine candidate to protect against S. suis biofilm-related and acute infections. METHOD: In this study, the gene encoding OCT was cloned into the expression vector pET-28a and the recombinant protein was expressed in Escherichia coli BL21. The immunogenicity and protective efficacy of the SS OCT was further investigated in a mouse model. RESULTS: The protein was found to be expressed in vivo and elicited high antibody titers following SS infections in mice. An animal challenge experiment with SS showed that 62.5% of mice immunized with the OCT protein were protected. Using an in vitro competitive adherence inhibition assay of adherence, evidence was obtained that OCT could significantly reduce the number of SS cells adhered to porcine kidney PK-15 cells. The bacterial levels recovered in mice of the OCT immunized group were significantly decreased in some organs, compared with the control group. CONCLUSION: In summary, our results suggest that the recombinant SS OCT protein, which is involved in bacterial adherence, may efficiently stimulate an immune response conferring protection against SS infections. It may therefore be considered as a potential vaccine candidate, although further studies are necessary to evaluate their use in swine.

Improvement of Streptococcus suis glutamate dehydrogenase expression in Escherichia coli through genetic modification of acetate synthesis pathway.

Escherichia coli generates acetate as an undesirable by-product that has several negative effects on protein expression, and the reduction of acetate accumulation by modifying genes of acetate synthesis pathway can improve the expression of recombinant proteins. In the present study, the effect of phosphotransacetylase (pta) or/and acetate kinase (ackA) deletion on glutamate dehydrogenase (GDH) expression was investigated. The results indicated that the disruptions of pta or/and ackA decreased the acetate accumulation and synthesis of per gram cell, and increased cell density, and GDH expression and synthesis of per gram cell. The pta gene was more important for acetate formation than the ackA gene. Using the strain with deletions of pta-ackA (SSGPA) for GDH expression, acetate accumulation (2·61 g l-1 ) and acetate synthesis of per gram cell (0·229 g g-1 ) were lowest, decreasing by 28·29 and 41·43% compared with those of the parental strain (SSG) respectively. The flux of acetate synthesis (6·6%) was decreased by 72·15% compared with that of SSG, and the highest cell density (11·38 g l-1 ), GDH expression (2·78 mg ml-1 ), and GDH formation of per gram cell (0·2442 mg mg-1 ) were obtained, which were 1·22-, 1·43- and 1·17-times higher than the parental strain respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Significance and Impact of the Study: Acetate is the key undesirable by-product in Escherichia coli cultivation, and both biomass and production of desired products are increased by the reduction of acetate accumulation. In the present study, the strains with deletions of pta or/and ackA were constructed to reduce the acetate accumulation and improve the GDH expression, and the highest expression level of GDH was obtained using the strain with lesion in pta-ackA that was 1·17-times higher than that of the parental strain. The construction strategy of recombinant E. coli for decreasing the acetate excretion can be used for high expression level of other desired products.

The cysteine protease ApdS from Streptococcus suis promotes evasion of innate immune defenses by cleaving the antimicrobial peptide cathelicidin LL-37.

Streptococcus suis is a globally distributed zoonotic pathogen associated with meningitis and septicemia in humans, posing a serious threat to public health. To successfully invade and disseminate within its host, this bacterium must overcome the innate immune system. The antimicrobial peptide LL-37 impedes invading pathogens by directly perforating bacterial membranes and stimulating the immune function of neutrophils, which are the major effector cells against S. suis However, little is known about the biological relationship between S. suis and LL-37 and how this bacterium adapts to and evades LL-37-mediated immune responses. In this study by using an array of approaches, including enzyme, chemotaxis, cytokine assays, quantitative RT-PCR, and CD spectroscopy, we found that the cysteine protease ApdS from S. suis cleaves LL-37 and thereby plays a key role in the interaction between S. suis and human neutrophils. S. suis infection stimulated LL-37 production in human neutrophils, and S. suis exposure to LL-37 up-regulated ApdS protease expression in the bacterium. We observed that ApdS targets and rapidly cleaves LL-37, impairing its bactericidal activity against S. suis We attributed this effect to the decreased helical content of the secondary structure in the truncated peptide. Moreover, ApdS rescued S. suis from killing by human neutrophils and neutrophil extracellular traps because LL-37 truncation attenuated neutrophil chemotaxis and inhibited the formation of extracellular traps and the production of reactive oxygen species. Altogether, our findings reveal an immunosuppressive strategy of S. suis whereby the bacterium blunts the innate host defenses via ApdS protease-mediated LL-37 cleavage.

Macrocyclization via an Arginine-Tyrosine Crosslink Broadens the Reaction Scope of Radical S-Adenosylmethionine Enzymes.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are an ascendant class of natural products with diverse structures and functions. Recently, we identified a wide array of RiPP gene clusters that are regulated by quorum sensing and encode one or more radical S-adenosylmethionine (RaS) enzymes, a diverse protein superfamily capable of catalyzing chemically difficult transformations. In this work, we characterize a novel reaction catalyzed by one such subfamily of RaS enzymes during RiPP biosynthesis: installation of a macrocyclic carbon-carbon bond that links the unactivated δ-carbon of an arginine side chain to the ortho-position of a tyrosine-phenol. Moreover, we show that this transformation is, unusually for RiPP biogenesis, largely insensitive to perturbations of the leader portion of the precursor peptide. This reaction expands the already impressive scope of RaS enzymes and contributes a unique macrocyclization motif to the growing body of RiPP architectures.

PmtA functions as a ferrous iron and cobalt efflux pump in Streptococcus suis.

Transition metals are nutrients essential for life. However, an excess of metals can be toxic to cells, and host-imposed metal toxicity is an important mechanism for controlling bacterial infection. Accordingly, bacteria have evolved metal efflux systems to maintain metal homeostasis. Here, we established that PmtA functions as a ferrous iron [Fe(II)] and cobalt [Co(II)] efflux pump in Streptococcus suis, an emerging zoonotic pathogen responsible for severe infections in both humans and pigs. pmtA expression is induced by Fe(II), Co(II), and nickel [Ni(II)], whereas PmtA protects S. suis against Fe(II) and ferric iron [Fe(III)]-induced bactericidal effect, as well as Co(II) and zinc [Zn(II)]-induced bacteriostatic effect. In the presence of elevated concentrations of Fe(II) and Co(II), ΔpmtA accumulates high levels of intracellular iron and cobalt, respectively. ΔpmtA is also more sensitive to streptonigrin, a Fe(II)-activated antibiotic. Furthermore, growth defects of ΔpmtA under Fe(II) or Co(II) excess conditions can be alleviated by manganese [Mn(II)] supplementation. Finally, PmtA plays a role in tolerance to H2O2-induced oxidative stress, yet is not involved in the virulence of S. suis in mice. Together, these data demonstrate that S. suis PmtA acts as a Fe(II) and Co(II) efflux pump, and contributes to oxidative stress resistance.

pdh modulate virulence through reducing stress tolerance and biofilm formation of Streptococcus suis serotype 2.

Streptococcus suis serotype 2 (S. suis 2) is a zoonotic pathogen. It causes meningitis, arthritis, pneumonia and sepsis in pigs, leading to extremely high mortality, which seriously affects public health and the development of the pig industry. Pyruvate dehydrogenase (PDH) is an important sugar metabolism enzyme that is widely present in microorganisms, mammals and higher plants. It catalyzes the irreversible oxidative decarboxylation of pyruvate to acetyl-CoA and reduces NAD+ to NADH. In this study, we found that the virulence of the S. suis ZY05719 sequence type 7 pdh deletion strain (Δpdh) was significantly lower than the wild-type strain (WT) in the mouse infection model. The distribution of viable bacteria in the blood and organs of mice infected with the Δpdh was significantly lower than those infected with WT. Bacterial survival rates were reduced in response to temperature stress, salt stress and oxidative stress. Additionally, compared to WT, the ability to adhere to and invade PK15 cells, biofilm formation and stress resistance of Δpdh were significantly reduced. Moreover, real-time PCR results showed that pdh deletion reduced the expression of multiple adhesion-related genes. However, there was no significant difference in the correlation biological analysis between the complemented strain (CΔpdh) and WT. Moreover, the survival rate of Δpdh in RAW264.7 macrophages was significantly lower than that of the WT strain. This study shows that PDH is involved in the pathogenesis of S. suis 2 and reduction in virulence of Δpdh may be related to the decreased ability to resist stress of the strain.

Aliphatic Ether Bond Formation Expands the Scope of Radical SAM Enzymes in Natural Product Biosynthesis.

The biosynthetic pathways of microbial natural products provide a rich source of novel enzyme-catalyzed transformations. Using a new bioinformatic search strategy, we recently identified an abundance of gene clusters for ribosomally synthesized and post-translationally modified peptides (RiPPs) that contain at least one radical S-adenosylmethionine (RaS) metalloenzyme and are regulated by quorum sensing. In the present study, we characterize a RaS enzyme from one such RiPP gene cluster and find that it installs an aliphatic ether cross-link at an unactivated carbon center, linking the oxygen of a Thr side chain to the α-carbon of a Gln residue. This reaction marks the first ether cross-link installed by a RaS enzyme. Additionally, it leads to a new heterocyclization motif and underlines the utility of our bioinformatics approach in finding new families of RiPP modifications.

Streptococcus suis contains multiple phase-variable methyltransferases that show a discrete lineage distribution.

Streptococcus suis is a major pathogen of swine, responsible for a number of chronic and acute infections, and is also emerging as a major zoonotic pathogen, particularly in South-East Asia. Our study of a diverse population of S. suis shows that this organism contains both Type I and Type III phase-variable methyltransferases. In all previous examples, phase-variation of methyltransferases results in genome wide methylation differences, and results in differential regulation of multiple genes, a system known as the phasevarion (phase-variable regulon). We hypothesized that each variant in the Type I and Type III systems encoded a methyltransferase with a unique specificity, and could therefore control a distinct phasevarion, either by recombination-driven shuffling between different specificities (Type I) or by biphasic on-off switching via simple sequence repeats (Type III). Here, we present the identification of the target specificities for each Type III allelic variant from S. suis using single-molecule, real-time methylome analysis. We demonstrate phase-variation is occurring in both Type I and Type III methyltransferases, and show a distinct association between methyltransferase type and presence, and population clades. In addition, we show that the phase-variable Type I methyltransferase was likely acquired at the origin of a highly virulent zoonotic sub-population.

Characterization of the zinc metalloprotease of Streptococcus suis serotype 2.

Streptococcus suis is a swine pathogen and zoonotic agent responsible for meningitis and septic shock. Although several putative virulence factors have been described, the initial steps of the S. suis pathogenesis remain poorly understood. While controversial results have been reported for a S. suis serotype 2 zinc metalloprotease (Zmp) regarding its IgA protease activity, recent phylogenetic analyses suggested that this protein is homologous to the ZmpC of Streptococcus pneumoniae, which is not an IgA protease. Based on the previously described functions of metalloproteases (including IgA protease and ZmpC), different experiments were carried out to study the activities of that of S. suis serotype 2. First, results showed that S. suis, as well as the recombinant Zmp, were unable to cleave human IgA1, confirming lack of IgA protease activity. Similarly, S. suis was unable to cleave P-selectin glycoprotein ligand-1 and to activate matrix metalloprotease 9, at least under the conditions tested. However, S. suis was able to partially cleave mucin 16 and syndecan-1 ectodomains. Experiments carried out with an isogenic Δzmp mutant showed that the Zmp protein was partially involved in such activities. The absence of a functional Zmp protein did not affect the ability of S. suis to adhere to porcine bronchial epithelial cells in vitro, or to colonize the upper respiratory tract of pigs in vivo. Taken together, our results show that S. suis serotype 2 Zmp is not a critical virulence factor and highlight the importance of independently confirming results on S. suis virulence by different teams.

Streptococcus suis synthesizes deoxyadenosine and adenosine by 5'-nucleotidase to dampen host immune responses.

Streptococcus suis is a major porcine bacterial pathogen and emerging zoonotic agent. S. suis 5'-nucleotidase is able to convert adenosine monophosphate to adenosine, resulting in inhibiting neutrophil functions in vitro and it is an important virulence factor. Here, we show that S. suis 5'-nucleotidase not only enables producing 2'-deoxyadenosine from 2'-deoxyadenosine monophosphate by the enzymatic assay and reversed-phase high performance liquid chromatography (RP-HPLC) analysis in vitro, but also synthesizes both 2'-deoxyadenosine and adenosine in mouse blood in vivo by RP-HPLC and liquid chromatography with tandem mass spectrometry analyses. Cellular cytotoxicity assay and Western blot analysis indicated that the production of 2'-deoxyadenosine by 5'-nucleotidase triggered the death of mouse macrophages RAW 264.7 in a caspase-3-dependent way. The in vivo infection experiment showed that 2'-deoxyadenosine synthesized by 5'-nucleotidase caused monocytopenia in mouse blood. The in vivo transcriptome analysis in mouse blood showed the inhibitory effect of 5'-nucleotidase on neutrophil functions and immune responses probably mediated through the generation of adenosine. Taken together, these findings indicate that S. suis synthesizes 2'-deoxyadenosine and adenosine by 5'-nucleotidase to dampen host immune responses, which represents a new mechanism of S. suis pathogenesis.

IgM cleavage by Streptococcus suis reduces IgM bound to the bacterial surface and is a novel complement evasion mechanism.

Streptococcus suis (S. suis) causes meningitis, arthritis and endocarditis in piglets. The aim of this study was to characterize the IgM degrading enzyme of S. suis (IdeSsuis) and to investigate the role of IgM cleavage in evasion of the classical complement pathway and pathogenesis. Targeted mutagenesis of a cysteine in the putative active center of IdeSsuis abrogated IgM cleavage completely. In contrast to wt rIdeSsuis, point mutated rIdeSsuis_C195S did not reduce complement-mediated hemolysis indicating that complement inhibition by rIdeSsuis depends on the IgM proteolytic activity. A S. suis mutant expressing IdeSsuis_C195S did not reduce IgM labeling, whereas the wt and complemented mutant showed less IgM F(ab')2 and IgM Fc antigen on the surface. IgM cleavage increased survival of S. suis in porcine blood ex vivo and mediated complement evasion as demonstrated by blood survival and C3 deposition assays including the comparative addition of rIdeSsuis and rIdeSsuis_C195S. However, experimental infection of piglets disclosed no significant differences in virulence between S. suis wt and isogenic mutants without IgM cleavage activity. This work revealed for the first time in vivo labeling of S. suis with IgM in the cerebrospinal fluid of piglets with meningitis. In conclusion, this study classifies IdeSsuis as a cysteine protease and emphasizes the role of IgM cleavage for bacterial survival in porcine blood and complement evasion though IgM cleavage is not crucial for the pathogenesis of serotype 2 meningitis.

Dipeptidylpeptidase IV of Streptococcus suis degrades the porcine antimicrobial peptide PR-39 and neutralizes its biological properties.

Streptococcus suis is a major swine pathogen causing pathologies such as meningitis, sepsis, endocarditis, and arthritis. Several surface-bound and secreted proteases produced by S. suis have been identified and proposed as virulence factors. PR-39 is a proline/arginine-rich antimicrobial peptide produced by porcine leucocytes. In addition to play a role in innate immunity, this peptide possesses immunomodulatory properties. In this study, we hypothesized that proteases produced by S. suis inactivate PR-39. Most strains of S. suis tested were relatively resistant to PR-39, with minimal inhibitory concentration (MIC) values ≥ 200 µg/ml. The proteolytic cleavage of PR-39 by recombinant subtilisin-like protease and dipeptidylpeptidase IV (DPPIV) of S. suis was assessed by SDS-PAGE. While PR-39 was not cleaved by the subtilisin-like protease, it was time-dependently degraded by DPPIV. Whole cells of S. suis also degraded PR-39. When S. suis was grown in a culture medium supplemented with recombinant DPPIV, its susceptibility to PR-39 was decreased. Activation of brain microvascular endothelial cells with PR-39 resulted in an increased secretion of the chemokine interleukin-8 (IL-8) thus confirming the immunomodulatory activity of this porcine antimicrobial peptide. However, a pre-treatment of PR-39 with DPPIV completely neutralized the increased IL-8 secretion. In this study, we showed that DPPIV produced by S. suis can degrade PR-39 and neutralize its antibacterial and immunomodulatory properties. This may allow survival of S. suis in the central nervous system by resisting to killing by this antimicrobial peptide and delaying the recruitment of phagocytic cells such as neutrophils to the site of infection.

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.

Mechanistic Investigations of Lysine-Tryptophan Cross-Link Formation Catalyzed by Streptococcal Radical S-Adenosylmethionine Enzymes.

Streptide is a ribosomally synthesized and post-translationally modified peptide with a unique cyclization motif consisting of an intramolecular lysine-tryptophan cross-link. Three radical S-adenosylmethionine enzymes, StrB, AgaB, and SuiB from different species of Streptococcus, have been shown to install this modification onto their respective precursor peptides in a leader-dependent fashion. Herein, we conduct detailed investigations to differentiate among several plausible mechanistic proposals, specifically addressing radical versus electrophilic addition to the indole during cross-link formation, the role of substrate side chains in binding in the enzyme active site, and the identity of the catalytic base in the reaction cycle. Our results are consistent with a radical electrophilic aromatic substitution mechanism for the key carbon-carbon bond-forming step. They also elaborate on other mechanistic features that underpin this unique and synthetically challenging post-translational modification.