Streptococcal Protein Research Articles

A Newly Developed Anti-L1CAM Monoclonal Antibody Targets Small Cell Lung Carcinoma Cells.

Few effective treatments are available for small cell lung cancer (SCLC), indicating the need to explore new therapeutic options. Here, we focus on an antibody-drug conjugate (ADC) targeting the L1 cell adhesion molecule (L1CAM). Several publicly available databases reveal that (1) L1CAM is expressed at higher levels in SCLC cell lines and tissues than in those of lung adenocarcinoma and (2) the expression levels of L1CAM are slightly higher in SCLC tissues than in adjacent normal tissues. We conducted a series of in vitro experiments using an anti-L1CAM monoclonal antibody (termed HSL175, developed in-house) and the recombinant protein DT3C, which consists of diphtheria toxin lacking the receptor-binding domain but containing the C1, C2, and C3 domains of streptococcal protein G. Our HSL175-DT3C conjugates theoretically kill cells only when the conjugates are internalized by the target (L1CAM-positive) cells through antigen-antibody interaction. The conjugates (an ADC analog) were effective against two SCLC-N (NEUROD1 dominant) cell lines, Lu-135 and STC-1, resulting in decreased viability. In addition, L1CAM silencing rendered the two cell lines resistant to HSL175-DT3C conjugates. These findings suggest that an ADC consisting of a humanized monoclonal antibody based on HSL175 and a potent anticancer drug would be effective against SCLC-N cells.

Deciphering GB1's Single Mutational Landscape: Insights from MuMi Analysis.

Mutational changes that affect the binding of the C2 fragment of Streptococcal protein G (GB1) to the Fc domain of human IgG (IgG-Fc) have been extensively studied using deep mutational scanning (DMS), and the binding affinity of all single mutations has been measured experimentally in the literature. To investigate the underlying molecular basis, we perform in silico mutational scanning for all possible single mutations, along with 2 μs-long molecular dynamics (WT-MD) of the wild-type (WT) GB1 in both unbound and IgG-Fc bound forms. We compute the hydrogen bonds between GB1 and IgG-Fc in WT-MD to identify the dominant hydrogen bonds for binding, which we then assess in conformations produced by Mutation and Minimization (MuMi) to explain the fitness landscape of GB1 and IgG-Fc binding. Furthermore, we analyze MuMi and WT-MD to investigate the dynamics of binding, focusing on the relative solvent accessibility of residues and the probability of residues being located at the binding interface. With these analyses, we explain the interactions between GB1 and IgG-Fc and display the structural features of binding. In sum, our findings highlight the potential of MuMi as a reliable and computationally efficient tool for predicting protein fitness landscapes, offering significant advantages over traditional methods. The methodologies and results presented in this study pave the way for improved predictive accuracy in protein stability and interaction studies, which are crucial for advancements in drug design and synthetic biology.

The Streptococcus phage protein paratox is an intrinsically disordered protein.

The bacteriophage protein paratox (Prx) blocks quorum sensing in its streptococcal host by directly binding the signal receptor and transcription factor ComR. This reduces the ability of Streptococcus to uptake environmental DNA and protects phage DNA from damage by recombination. Past work characterizing the Prx:ComR molecular interaction revealed that paratox adopts a well-ordered globular fold when bound to ComR. However, solution-state biophysical measurements suggested that Prx may be conformationally dynamic. To address this discrepancy, we investigated the stability and dynamic properties of Prx in solution using circular dichroism, nuclear magnetic resonance, and several fluorescence-based protein folding assays. Our work shows that under dilute buffer conditions Prx is intrinsically disordered. We also show that the addition of kosmotropic salts or protein stabilizing osmolytes induces Prx folding. However, the solute stabilized fold is different from the conformation Prx adopts when it is bound to ComR. Furthermore, we have characterized Prx folding thermodynamics and folding kinetics through steady-state fluorescence and stopped flow kinetic measurements. Our results show that Prx is a highly dynamic protein in dilute solution, folding and refolding within the 10 ms timescale. Overall, our results demonstrate that the streptococcal phage protein Prx is an intrinsically disordered protein in a two-state equilibrium with a solute-stabilized folded form. Furthermore, the solute-stabilized fold is likely the predominant form of Prx in a solute-crowded bacterial cell. Finally, our work suggests that Prx binds and inhibits ComR, and thus quorum sensing in Streptococcus, by a combination of conformational selection and induced-fit binding mechanisms.

Electrochemical immunosensor based on antibody-oriented probe for the detection of AFB1 in rice bran oil

Aflatoxin B1 is one of the most toxic mycotoxins, posing a serious threat to human health. There is an urgent need to develop sensitive, rapid and selective ways for the detection of AFB1. Herein, an electrochemical immunosensor was fabricated using the antibody-oriented probe (gold nanoparticle-Streptococcal protein A-anti-AFB1 monoclonal antibody, AuNP-SPA-mAb) to analyze AFB1 in rice bran oil. AuNP-SPA-mAb probe was constructed by the introduction of streptococcal protein A to ensure the controlled immobilization of antibody. AFB1 competed with antigen loaded on the gold electrode for binding AuNP-SPA-mAb in the presence of the target AFB1, inhibiting the capture of horse radish peroxidase (HRP)-labeled second antibody. Subsequently, HRP could facilitate the oxidization of hydroquinone in the presence of hydrogen peroxide, resulting in the production of an electrochemical signal. Under optimal conditions, this immunosensor displayed a linear range of 10–8000 pg/mL and a limit of detection of 2.2 pg/mL. The fabricated immunosensor was further utilized to verify the potential application by detecting samples of rice bran oil. Recovery rates varied from 88.0% to 107.9%. This immunosensor provides a highly sensitive and rapid approach for the determination of such mycotoxins.

Quantification of Adaptive Immune Responses Against Protein-Binding Interfaces in the Streptococcal M1 Protein

Bacterial or viral antigens can contain subdominant protein regions that elicit weak antibody responses upon vaccination or infection although there is accumulating evidence that antibody responses against subdominant regions can enhance the protective immune response. One proposed mechanism for subdominant protein regions is the binding of host proteins that prevent antibody production against epitopes hidden within the protein binding interfaces. Here, we used affinity purification combined with quantitative mass spectrometry (AP-MS) to examine the level of competition between antigen-specific antibodies and host–pathogen protein interaction networks using the M1 protein from Streptococcus pyogenes as a model system. As most humans have circulating antibodies against the M1 protein, we first used AP-MS to show that the M1 protein interspecies protein network formed with human plasma proteins is largely conserved in naïve mice. Immunizing mice with the M1 protein generated a time-dependent increase of anti-M1 antibodies. AP-MS analysis comparing the composition of the M1-plasma protein network from naïve and immunized mice showed significant enrichment of 292 IgG peptides associated with 56 IgG chains in the immune mice. Despite the significant increase of bound IgGs, the levels of interacting plasma proteins were not significantly reduced in the immune mice. The results indicate that the antigen-specific polyclonal IgG against the M1 protein primarily targets epitopes outside the other plasma protein binding interfaces. In conclusion, this study demonstrates that AP-MS is a promising strategy to determine the relationship between antigen-specific antibodies and host-pathogen interaction networks that could be used to define subdominant protein regions of relevance for vaccine development.

Group A streptococcal collagen-like protein 1 restricts tumor growth in murine pancreatic adenocarcinoma and inhibits cancer-promoting neutrophil extracellular traps.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer associated with an immunosuppressive environment. Neutrophil extracellular traps (NETs) were initially described in the context of infection but have more recently been implicated in contributing to the tolerogenic immune response in PDAC. Thus, NETs are an attractive target for new therapeutic strategies. Group A Streptococcus (GAS) has developed defensive strategies to inhibit NETs. In the present work, we propose utilizing intra-tumoral GAS injection to stimulate anti-tumor activity by inhibiting cancer-promoting NETs. Mice harboring Panc02 or KPC subcutaneous tumors injected with three different M-type GAS strains. Tumors and spleens were harvested at the endpoint of the experiments to assess bacterial colonization and systemic spread, while sera were analyzed for humoral responses toward the streptococcal antigens, especially the M1 and Scl1 proteins. Role of the streptococcal collagen-like protein 1 (Scl1) in anti-PDAC activity was assessed in vivo after intratumoral injection with M1 GAS wild-type, an isogenic mutant strain devoid of Scl1, or a complemented mutant strain with restored scl1 expression. In addition, recombinant Scl1 proteins were tested for NET inhibition using in vitro and ex vivo assays assessing NET production and myeloperoxidase activity. Injection of three different M-type GAS strains reduced subcutaneous pancreatic tumor volume compared to control in two different murine PDAC models. Limitation of tumor growth was dependent on Scl1, as isogenic mutant strain devoid of Scl1 did not reduce tumor size. We further show that Scl1 plays a role in localizing GAS to the tumor site, thereby limiting the systemic spread of bacteria and off-target effects. While mice did elicit a humoral immune response to GAS antigens, tested sera were weakly immunogenic toward Scl1 antigen following intra-tumoral treatment with Scl1-expressing GAS. M1 GAS inhibited NET formation when co-cultured with neutrophils while Scl1-devoid mutant strain did not. Recombinant Scl1 protein inhibited NETs ex vivo in a dose-dependent manner by suppressing myeloperoxidase activity. Altogether, we demonstrate that intra-tumoral GAS injections reduce PDAC growth, which is facilitated by Scl1, in part through inhibition of cancer promoting NETs. This work offers a novel strategy by which NETs can be targeted through Scl1 protein and potentiates its use as a cancer therapeutic.

Abstract 1982 Targeting Sialic Acids with Streptococcal Adhesin Proteins

Abstract 1982 Targeting Sialic Acids with Streptococcal Adhesin Proteins

Streptococcal pyrogenic exotoxin B is a superantigen that induces murine splenocyte proliferation and secretion of IL-2 and IFN-γ ex vivo.

Streptococcus pyogenes is a significant human pathogen, producing a range of virulence factors, including streptococcal pyrogenic exotoxin B (SpeB) that is associated with foodborne outbreaks. It was only known that this cysteine protease mediates cleavage of transmembrane proteins to permit bacterial penetration and is found in 25% of clinical isolates from streptococcal toxic shock syndrome patients with extreme inflammation. Its interaction with host and streptococcal proteins has been well characterized, but doubt remains about whether it constitutes a superantigen. In this study, for the first time it is shown that SpeB acts as a superantigen, similarly to other known superantigens such as staphylococcal enterotoxin A or streptococcal pyrogenic exotoxin type C, by inducing proliferation of murine splenocytes and cytokine secretion, primarily of interleukin-2 (IL-2), as shown by cytometric bead array analysis. IL-2 secretion was confirmed by enzyme-linked immunosorbent assay (ELISA) as well as secretion of interferon-γ. ELISA showed a dose-dependent relationship between SpeB concentration in splenocyte cells and IL-2 secretion levels, and it was shown that SpeB retains activity in milk pasteurized for 30 min at 63°C.

Molecular characterization of the interaction between human IgG and the M-related proteins from Streptococcus pyogenes

Group A Streptococcal M-related proteins (Mrp) are dimeric α-helical-coiled-coil cell membrane-bound surface proteins. During infection, Mrp recruit the fragment crystallisable region of human immunoglobulin G (IgG-Fc) via their A-repeat regions to the bacterial surface, conferring upon the bacteria enhanced phagocytosis resistance and augmented growth in human blood. However, Mrps show a high degree of sequence diversity, and it is currently not known whether this diversity affects the Mrp-IgG interaction. Herein, we report that diverse Mrps all bind human IgG subclasses with nanomolar affinity, with differences in affinity which ranged from 3.7-11.1 nM for mixed IgG. Using surface plasmon resonance, we confirmed Mrps display preferential IgG-subclass binding. All Mrps were found to have a significantly weaker affinity for IgG3 (p<0.05) compared to all other IgG-subclasses. Furthermore, plasma pulldown assays analysed via western blotting revealed that all Mrp were able to bind IgG in the presence of other serum proteins at both 25°C and 37°C. Finally, we report for the first time that dimeric Mrps bind to IgG with a 1:1 stoichiometry, enhancing our understanding of this important host-pathogen interaction.

Multi-eGO: Model Improvements toward the Study of Complex Self-Assembly Processes.

Structure-based models have been instrumental in simulating protein folding and suggesting hypotheses about the mechanisms involved. Nowadays, at least for fast-folding proteins, folding can be simulated in explicit solvent using classical molecular dynamics. However, other self-assembly processes, such as protein aggregation, are still far from being accessible. Recently, we proposed that a hybrid multistate structure-based model, multi-eGO, could help to bridge the gap toward the simulation of out-of-equilibrium, concentration-dependent self-assembly processes. Here, we further improve the model and show how multi-eGO can effectively and accurately learn the conformational ensemble of the amyloid β42 intrinsically disordered peptide, reproduce the well-established folding mechanism of the B1 immunoglobulin-binding domain of streptococcal protein G, and reproduce the aggregation as a function of the concentration of the transthyretin 105-115 amyloidogenic peptide. We envision that by learning from the dynamics of a few minima, multi-eGO can become a platform for simulating processes inaccessible to other simulation techniques.

Recombinant streptococcal protein G-modified metal-organic framework ZIF-8 for the highly selective purification of immunoglobulin G from human serum

A novel solid phase extractant His-rSPG@ZIF-8 was prepared by covalently coupling recombinant streptococcal protein G (His-rSPG) with ZIF-8. The His-rSPG@ZIF-8 composite was characterized by Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy (Raman), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Due to the specific binding between the immunoglobulin binding region of His-rSPG and the Fc region of immunoglobulin G (IgG), the His-rSPG@ZIF-8 composite demonstrated exceptional selectivity in adsorbing IgG. In Britton-Robinson buffer (BR buffer) with a salt concentration of 500 mmol L−1 (0.04 mol L−1, pH 8.0), the His-rSPG@ZIF-8 composite exhibited a remarkable adsorption efficiency of 99.8 % for 0.05 mg of the composite on 200 μL of IgG solution (100 μg mL−1). The adsorption behavior of the His-rSPG@ZIF-8 composite aligns with the Langmuir adsorption model, and the theoretical maximum adsorption capacity is 1428.6 mg g−1. The adsorbed IgG molecules were successfully eluted using a SDS solution (0.5 %, m/m), resulting in a recovery rate of 91.2 %. Indeed, the His-rSPG@ZIF-8 composite was successfully utilized for the isolation and purification of IgG from human serum samples. The obtained IgG exhibited high purity, as confirmed by SDS-PAGE analysis. Additionally, LC-MS/MS analysis was employed to identify the human serum proteins following the adsorption and elution process using the His-rSPG@ZIF-8 composite material. The results revealed that the recovered solution contained an impressive content of immunoglobulin, accounting for 62.4 % of the total protein content. Furthermore, this process also led to the significant enrichment of low abundance proteins such as Serpin B4 and Cofilin-1. Consequently, the His-rSPG@ZIF-8 composite holds great promise for applications such as IgG purification and immunoassays. At the same time, it expands the application of metal-organic frameworks in the field of proteomics.

Dairy phages escape CRISPR defence of Streptococcus thermophilus via the anti-CRISPR AcrIIA3

Bacterial community collapse due to phage infection is a major risk in cheese making processes. As virulent phages are ubiquitous and diverse in milk fermentation factories, the use of phage-resistant lactic acid bacteria (LAB) is essential to obtain high-quality fermented dairy products. The LAB species Streptococcus thermophilus contains two type II-A CRISPR-Cas systems (CRISPR1 and CRISPR3) that can effectively protect against phage infection. However, virulent streptococcal phages carrying anti-CRISPR proteins (ACR) that block the activity of CRISPR-Cas systems have emerged in yogurt and cheese environments. For example, phages carrying AcrIIA5 can impede both CRISPR1 and CRISPR3 systems, while AcrIIA6 stops only CRISPR1. Here, we explore the activity and diversity of a third streptococcal phage anti-CRISPR protein, namely AcrIIA3. We were able to demonstrate that AcrIIA3 is efficiently active against the CRISPR3-Cas system of S. thermophilus. We used AlphaFold2 to infer the structure of AcrIIA3 and we predicted that this new family of functional ACR in virulent streptococcal phages has a new α-helical fold, with no previously identified structural homologs. Because ACR proteins are being explored as modulators in genome editing applications, we also tested AcrIIA3 against SpCas9. We found that AcrIIA3 could block SpCas9 in bacteria but not in human cells. Understanding the diversity and functioning of anti-defence mechanisms will be of importance in the design of long-term stable starter cultures.

Autoimmune streptococcal glomerulonephritis: the problem of nephritogenicity of &lt;i&gt;Streptococcus pyogenes&lt;/i&gt;

Acute post-streptococcal glomerulonephritis usually occurs as a complication after a streptococcal infection due to untimely or inadequate antibiotic therapy. The etiology of post-streptococcal glomerulonephritis has been studied rather comprehensively. Today, both clinicians and microbiologists do not deny the dominant role of Streptococcus pyogenes (streptococcus attributed to serological group A, GAS). Usually, emergence of acute post-streptococcal glomerulonephritis (APSGN) is associated with the so-called GAS-related "nephritogenicity" often judged by appearance and accumulation of antibodies to the antigens and extracellular products of streptococcal cells in patient blood. This interpretation is quite loose and most likely evidence about a link to the bacterial strain, rather than its nephritogenicity. Many studies refer and still attribute a leading role of "nephritogenic" factors to various streptococcal antigens and related biologically active products. Streptococcal nephritogenic factors include cross-reacting antigens, streptokinase, cysteine proteinase, endostreptosin a GAS cell membrane protein as well as plasmin-tropic enzyme glyceraldehyde-3-phosphate dehydrogenase. Nephritogenicity of all such streptococcal products is suspected to result from the fact that they are found in renal biopsies like specific patient blood serum antibodies. Regarding a term of nephritogenicity, it has been evidenced that it cannot be attributed to any specific streptococcal cell product. This review attempted to analyze a number of bacterial products as starting factors triggering this process. APSGN can be reproduced experimentally in rabbits by intravenous administration of a heat-killed Streptococcus pyogenes culture. In our experiments, strains of serotypes 1, 4, 12, 15, 22 were used. They produced M-proteins and had the ability to bind human and rabbit immunoglobulin G by interacting with the Fc part of the IgG molecule. In numerous series of experiments, evidence was obtained regarding the initiating role of GAS IgGFc-receptor proteins in developing APSGN. Recent studies confirmed the role of streptococcal IgGFc-binding proteins in the initiation of glomerulonephritis after animals were inoculated with temperature-killed IgGFc-positive GAS. This approach excluded a large group of bacterial extracellular agents from the list of APSGN-initiating candidates. An unconventional view on the pathogenesis of GAS-infection-coupled complications may allow approaching their prevention or new treatment strategies.

Generation of a CD70-Specific Fusion Nanobody with IgG Recruiting Capacity for Tumor Killing.

Due to its competitive advantages such as small size, high stability, easy production, and good tissue penetration compared with monoclonal antibodies (mAb), nanobodies (Nbs) were considered the next generation of therapeutics. However, the absence of Fc fragments and Fc-triggered immune effectors limits their clinical applications. In order to overcome these limitations, we develop a novel approach by attaching an IgG binding domain (IgBD) to Nbs for recruiting endogenous IgG and recovering the immune effectors for tumor killing. We linked a Streptococcal Protein G-derived IgBD, termed C3Fab, at the C-terminus of a CD70-specific Nb 3B6 to construct an endogenous IgG recruitment antibody (termed EIR). The recombinant Nb3B6-C3Fab was expressed in E. coli BL21 (DE3) and purified by nickel affinity chromatography. We further evaluated the binding, recruitment of IgG, and the serum half-life of Nb3B6-C3Fab. The tumor-killing effects on CD70 positive cells mediated by antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity were also detected. We successfully constructed a IgBD fused Nb3B6-C3Fab with high affinity for CD70 and mouse IgG (mIgG). Nb3B6-C3Fab can specifically bind to CD70 positive tumor cells and recruit mIgG on the cell surface. Ligating of Nb3B6 with C3Fab increased its serum half-life in mice almost 39-fold from 0.96 h to 37.67 h. Moreover, we demonstrated remarkable cytotoxicity of Nb3B6-C3Fab to CD70 positive tumor cells via C3Fab by immune effector cells. Our study demonstrates that IgBD fusion endows Nbs with the ability for endogenous IgG recruitment andhalf-life promotion. Linking IgBD to Nbs is an effective strategy to recovering immune effectors for tumor killing.

Modular Set of Reagents in Lateral Flow Immunoassay: Application for Antibiotic Neomycin Detection in Honey

A scheme of modular competitive immunochromatography with an analyte-independent test strip and changeable specific immunoreactants has been proposed. Native (detected) and biotinylated antigens interact with specific antibodies during their preincubation in solution, that is, without the immobilization of reagents. After this, the detectable complexes on the test strip are formed by the use of streptavidin (which binds biotin with high affinity), anti-species antibodies, and immunoglobulin-binding streptococcal protein G. The technique was successfully applied for the detection of neomycin in honey. The visual and instrumental detection limits were 0.3 and 0.014 mg/kg, respectively, and the degree of neomycin revealed in honey samples varied from 85% to 113%. The efficiency of the modular technique with the use of the same test strip for different analytes was confirmed for streptomycin detection. The proposed approach excludes the necessity of finding the condition of immobilization for each new specific immunoreactant and transferring the assay to other analytes by a simple choice of concentrations for preincubated specific antibodies and the hapten-biotin conjugate.

Junctional adhesion molecule 3 is a potential therapeutic target for small cell lung carcinoma

There are few effective therapies for small cell lung carcinoma (SCLC); thus, we need to develop novel and efficacious treatments. We hypothesized that an antibody–drug conjugate (ADC) could be a promising option for SCLC. Several publicly available databases were used to demonstrate the extent to which junctional adhesion molecule 3 (JAM3) mRNA was expressed in SCLC and lung adenocarcinoma cell lines and tissues. Three SCLC cell lines, Lu-135, SBC-5, and Lu-134 A, were selected and examined for JAM3 protein expression by flow cytometry. Finally, we examined the response of the three SCLC cell lines to a conjugate between an anti-JAM3 monoclonal antibody HSL156 (developed in-house) and the recombinant protein DT3C, which consists of diphtheria toxin lacking the receptor-binding domain but containing the C1, C2, and C3 domains of streptococcal protein G. In silico analyses revealed that JAM3 mRNA was expressed higher in SCLC cell lines and tissues than in those of lung adenocarcinoma. As expected, all the three SCLC cell lines examined were positive for JAM3 at the mRNA and protein levels. Consequently, control SCLC cells, but not JAM3-silenced ones, were highly sensitive to HSL156-DT3C conjugates, resulting in dose- and time-dependent decreased viability. Finally, silencing JAM3 alone suppressed the growth of all SCLC cell lines examined. Taken together, these findings suggest that an ADC targeting JAM3 could represent a new approach to treating SCLC patients.

StaR Is a Positive Regulator of Topoisomerase I Activity Involved in Supercoiling Maintenance in Streptococcus pneumoniae

The DNA topoisomerases gyrase and topoisomerase I as well as the nucleoid-associated protein HU maintain supercoiling levels in Streptococcus pneumoniae, a main human pathogen. Here, we characterized, for the first time, a topoisomerase I regulator protein (StaR). In the presence of sub-inhibitory novobiocin concentrations, which inhibit gyrase activity, higher doubling times were observed in a strain lacking staR, and in two strains in which StaR was over-expressed either under the control of the ZnSO4-inducible PZn promoter (strain ΔstaRPZnstaR) or of the maltose-inducible PMal promoter (strain ΔstaRpLS1ROMstaR). These results suggest that StaR has a direct role in novobiocin susceptibility and that the StaR level needs to be maintained within a narrow range. Treatment of ΔstaRPZnstaR with inhibitory novobiocin concentrations resulted in a change of the negative DNA supercoiling density (σ) in vivo, which was higher in the absence of StaR (σ = -0.049) than when StaR was overproduced (σ = -0.045). We have located this protein in the nucleoid by using super-resolution confocal microscopy. Through in vitro activity assays, we demonstrated that StaR stimulates TopoI relaxation activity, while it has no effect on gyrase activity. Interaction between TopoI and StaR was detected both in vitro and in vivo by co-immunoprecipitation. No alteration of the transcriptome was associated with StaR amount variation. The results suggest that StaR is a new streptococcal nucleoid-associated protein that activates topoisomerase I activity by direct protein-protein interaction.

Genomic Distribution of Pro-Virulent cpdB-like Genes in Eubacteria and Comparison of the Enzyme Specificity of CpdB-like Proteins from Salmonella enterica, Escherichia coli and Streptococcus suis

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.

How Sugars Protect Dry Protein Structure.

Extremotolerant organisms and industry exploit sugars as desiccation protectants, with trehalose being widely used by both. How sugars, in general, and the hydrolytically stable sugar trehalose, in particular, protect proteins is poorly understood, which hinders the rational design of new excipients and implementation of novel formulations for preserving lifesaving protein drugs and industrial enzymes. We employed liquid-observed vapor exchange nuclear magnetic resonance (LOVE NMR), differential scanning calorimetry (DSC), and thermal gravimetric analysis (TGA) to show how trehalose and other sugars protect two model proteins: the B1 domain of streptococcal protein G (GB1) and truncated barley chymotrypsin inhibitor 2 (CI2). Residues with intramolecular H-bonds are most protected. The LOVE NMR and DSC data indicate that vitrification may be protective. Combining LOVE NMR and TGA data shows that water retention is not important. Our data suggest that sugars protect protein structure as they dry by strengthening intraprotein H-bonds and water replacement and that trehalose is the stress-tolerance sugar of choice because of its covalent stability.

Identification, genome sequencing, and virulence variation analysis of MCRV-NH, an epidemic reovirus strain in cultured mud crab, Scylla paramamosain

Mud Crab Reovirus (MCRV) is a major pathogen of cultured mud crab Scylla paramamosain. Early studies showed that the mortality rate caused by MCRV was high. However, current studies show that MCRV has been widely present in cultured mud crab ponds, and the mortality rate has dramatically decreased, suggesting that it has become more infectious but less virulent. In this study, an epidemic MCRV strain named MCRV-NH was isolated from a representative farming areas of mud crabs to reveal the intrinsic molecular mechanism of the virulence variation of MCRV. Electron microscopy results showed that MCRV-NH was similar to the early reported MCRV or Scylla serrata reovirus (SsRV) in terms of morphology and structure. Viral infection experiments showed that no mud crab was dead during the infection period of 11 days even though MCRV-NH could rapidly proliferate in mud crabs at 21–23 °C, demonstrating that the mortality rate caused by MCRV-NH significantly decreased. Sequencing results of the MCRV-NH genome showed that eleven nucleotides were deleted in the sequencing region, and its nucleotide sequence identities with those of MCRV and SsRV were 99.43% and 99.33%, and the numbers of corresponding nucleotide mutations were 150 and 126, respectively. Evolutionary analysis showed that MCRV-NH belonged to the same species as MCRV and SsRV, and MCRV-NH should be a variant. In comparison with the open reading frames (ORFs) of MCRV and SsRV, the ORFs of MCRV-NH had no nucleotide deletions, but the numbers of nucleotide mutations were 100 and 123, and the numbers of amino acid mutations were 32 and 35, respectively. Eight mutated amino acids compared with the two reoviruses were located at the predicted active sites of the viral proteins. Among them, three mutations resulted in the generation of new phosphorylation sites, and two mutations led to the loss of the original phosphorylation sites. Meanwhile, a heptapeptide repeat sequence in VP9 disappeared, and a streptococcal histidine triad protein in VP6 and a Big-1 (bacterial Ig-like domain 1) domain in VP11 were generated due to key amino acid mutations in these viral proteins, which are involved in the infection and replication of MCRV. These changes in the active sites of the viral proteins may be responsible for the MCRV virulence variation. This study provided essential evidence to support the conclusion that MCRV-NH was a variant of MCRV with weaker pathogenicity and revealed the underlying possible molecular mechanism of the virulence variation of MCRV-NH.