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Antigenically sequence variable M proteins of the major bacterial pathogen Streptococcus pyogenes (Strep A) are responsible for recruiting human C4b-binding protein (C4BP) to the bacterial surface, which enables Strep A to evade destruction by the immune system. The most sequence divergent portion of M proteins, the hypervariable region (HVR), is responsible for binding C4BP. Structural evidence points to the conservation of two C4BP-binding sequence patterns (M2 and M22) in the HVR of numerous M proteins, with this conservation applicable to vaccine immunogen design. These two patterns, however, only partially explain C4BP binding by Strep A. Here, we identified several M proteins that lack these patterns but still bind C4BP and determined the structures of two, M68 and M87 HVRs, in complex with a C4BP fragment. Mutagenesis of these M proteins led to the identification of amino acids that are crucial for C4BP binding, enabling formulation of new C4BP-binding patterns. Mutagenesis was also carried out on M2 and M22 proteins to refine or generate experimentally grounded C4BP-binding patterns. The M22 pattern was the most prevalent among M proteins, followed by the M87 and M2 patterns, while the M68 pattern was rare. These patterns, except for M68, were also evident in numerous M-like Enn proteins. Binding of C4BP via these patterns to Enn proteins was verified. We conclude that C4BP-binding patterns occur frequently in Strep A strains of differing M types, being present in their M or Enn proteins, or frequently both, providing further impetus for their use as vaccine immunogens., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

An important element of antibody-guided vaccine design is the use of neutralizing or opsonic monoclonal antibodies to define protective epitopes in their native three-dimensional conformation. Here, we demonstrate a multimodal mass spectrometry-based strategy for in-depth characterization of antigen-antibody complexes to enable the identification of protective epitopes using the cytolytic exotoxin Streptolysin O (SLO) from Streptococcus pyogenes as a showcase. We first discovered a monoclonal antibody with an undisclosed sequence capable of neutralizing SLO-mediated cytolysis. The amino acid sequence of both the antibody light and the heavy chain was determined using mass-spectrometry-based de novo sequencing, followed by chemical cross-linking mass spectrometry to generate distance constraints between the antibody fragment antigen-binding region and SLO. Subsequent integrative computational modeling revealed a discontinuous epitope located in domain 3 of SLO that was experimentally validated by hydrogen-deuterium exchange mass spectrometry and reverse engineering of the targeted epitope. The results show that the antibody inhibits SLO-mediated cytolysis by binding to a discontinuous epitope in domain 3, likely preventing oligomerization and subsequent secondary structure transitions critical for pore-formation. The epitope is highly conserved across >98% of the characterized S. pyogenes isolates, making it an attractive target for antibody-based therapy and vaccine design against severe streptococcal infections.

Background: Streptococcus pyogenes (GAS) is a human bacterial pathogen that generates various mild to severe diseases. Worldwide, there are approximately 700 million cases of GAS infections per year. In some strains of GAS, the surface-resident M-protein, plasminogen-binding group A streptococcal M-protein (PAM), binds directly to human host plasminogen (hPg), where it is activated to plasmin through a mechanism involving a Pg/bacterial streptokinase (SK) complex as well as endogenous activators. Binding to Pg and its activation are dictated by selected sequences within the human host Pg protein, making it difficult to generate animal models to study this pathogen., Objectives: To develop a murine model for studying GAS infection by minimally modifying mouse Pg to enhance the affinity to bacterial PAM and sensitivity to GAS-derived SK., Methods: We used a targeting vector that contained a mouse albumin-promoter and mouse/human hybrid plasminogen cDNA targeted to the Rosa26 locus. Characterization of the mouse strain consisted of both gross and histological techniques and determination of the effects of the modified Pg protein through surface plasmon resonance measurements, Pg activation analyses, and mouse survival post-GAS infection., Results: We generated a mouse line expressing a chimeric Pg protein consisting of 2 amino acid substitutions in the heavy chain of Pg and a complete replacement of the mouse Pg light chain with the human Pg light chain., Conclusion: This protein demonstrated an enhanced affinity for bacterial PAM and sensitivity to activation by the Pg-SK complex, making the murine host susceptible to the pathogenic effects of GAS., Competing Interests: Declaration of competing interests All authors declare no conflict of interest., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Streptococcus pyogenes (group A Streptococcus ) is a clinically important microbial pathogen that requires iron in order to proliferate. During infections, S. pyogenes uses the surface displayed Shr receptor to capture human hemoglobin (Hb) and acquires its iron-laden heme molecules. Through a poorly understood mechanism, Shr engages Hb via two structurally unique N-terminal Hb-interacting domains (HID1 and HID2) which facilitate heme transfer to proximal NEAr Transporter (NEAT) domains. Based on the results of X-ray crystallography, small angle X-ray scattering, NMR spectroscopy, native mass spectrometry, and heme transfer experiments, we propose that Shr utilizes a "cap and release" mechanism to gather heme from Hb. In the mechanism, Shr uses the HID1 and HID2 modules to preferentially recognize only heme-loaded forms of Hb by contacting the edges of its protoporphyrin rings. Heme transfer is enabled by significant receptor dynamics within the Shr-Hb complex which function to transiently uncap HID1 from the heme bound to Hb's β subunit, enabling the gated release of its relatively weakly bound heme molecule and subsequent capture by Shr's NEAT domains. These dynamics may maximize the efficiency of heme scavenging by S. pyogenes , enabling it to preferentially recognize and remove heme from only heme-loaded forms of Hb that contain iron.

Alternate sigma factors play a major role in the survival of pathogenic bacteria such as Streptococcus pyogenes in adverse environment conditions. Stress induced sigma factors mediate gene expression under conditions of pathogenesis, dormancy and unusual environmental cues. In the present work, ComX, an alternate sigma factor from S. pyogenes has been characterized. The structures of ComX, RpoB β subunit and RpoC β' subunit of RNA polymerase have been predicted using comparative and homology modelling respectively and validated. Attempts have been made to study RpoB-RpoC-ComX complex interactions with Double Strand (DS) and Single Strand (SS) promoter regions. Stability of these complexes and the promoter melting mechanism have been analysed using Molecular Dynamic (MD) simulations. This study suggests that ComX, although identifies promoter analogous to the alternate sigma factor SigH of M. tuberculosis , follows a distinctive promoter flip out mechanism.Communicated by Ramaswamy H. Sarma.

The Cas9 endonuclease is an essential component of the CRISPR-Cas-based genome editing tools. The attainment of high specificity and efficiency of Cas9 during targetted DNA cleavage is the main problem that limits the clinical application of the CRISPR-Cas9 system. A deep understanding of the Cas9 mechanism and its structural-functional relationships is required to develop strategies for precise gene editing. Here, we present the first attempt to describe the solution structure of Cas9 from S. pyogenes using hydrogen-deuterium exchange mass spectrometry (HDX-MS) coupled to molecular dynamics simulations. HDX data revealed multiple protein regions with deuterium uptake levels varying from low to high. By analysing the difference in relative deuterium uptake by apoCas9 and its complex with sgRNA, we identified peptides involved in the complex formation and possible changes in the protein conformation. The REC3 domain was shown to undergo the most prominent conformational change upon enzyme-RNA interactions. Detection of the HDX in two forms of the enzyme provided detailed information about changes in the Cas9 structure induced by sgRNA binding and quantified the extent of the changes. The study demonstrates the practical utility of HDX-MS for the elucidation of mechanistic aspects of Cas9 functioning.

CRISPR-Cas systems are RNA-guided nucleases that provide adaptive immune protection in bacteria and archaea against intruding genomic materials. Cas9, a type-II CRISPR effector protein, is widely used for gene editing applications since a single guide RNA can direct Cas9 to cleave specific genomic targets. The conformational changes associated with RNA/DNA binding are being modulated to develop Cas9 variants with reduced off-target cleavage. Previously, we showed that proline substitutions in the arginine-rich bridge helix (BH) of Streptococcus pyogenes Cas9 (SpyCas9-L64P-K65P, SpyCas9 2Pro ) improve target DNA cleavage selectivity. In this study, we establish that kinetic analysis of the cleavage of supercoiled plasmid substrates provides a facile means to analyze the use of two parallel routes for DNA linearization by SpyCas9: (i) nicking by HNH followed by RuvC cleavage (the TS (target strand) pathway) and (ii) nicking by RuvC followed by HNH cleavage (the NTS (nontarget strand) pathway). BH substitutions and DNA mismatches alter the individual rate constants, resulting in changes in the relative use of the two pathways and the production of nicked and linear species within a given pathway. The results reveal coordinated actions between HNH and RuvC to linearize DNA, which is modulated by the integrity of the BH and the position of the mismatch in the substrate, with each condition producing distinct conformational energy landscapes as observed by molecular dynamics simulations. Overall, our results indicate that BH interactions with RNA/DNA enable target DNA discrimination through the differential use of the parallel sequential pathways driven by HNH/RuvC coordination.

Mechanisms of disulfide bond formation in the human pathogen Streptococcus pyogenes are currently unknown. To date, no disulfide bond-forming thiol-disulfide oxidoreductase (TDOR) has been described and at least one disulfide bonded protein is known in S. pyogenes. This protein is the superantigen SpeA, which contains 3 cysteine residues (Cys 87, Cys90, and Cys98) and has a disulfide bond formed between Cys87 and Cys98. In this study, candidate TDORs were identified from the genome sequence of S. pyogenes MGAS8232. Using mutational and biochemical approaches, one of the candidate proteins, SpyM18_2037 (named here SdbA), was shown to be the catalyst that introduces the disulfide bond in SpeA. SpeA in the culture supernatant remained reduced when sdbA was inactivated and restored to the oxidized state when a functional copy of sdbA was returned to the sdbA -knockout mutant. SdbA has a typical C 46 XXC 49 active site motif commonly found in TDORs. Site-directed mutagenesis experiments showed that the cysteines in the CXXC motif were required for the disulfide bond in SpeA to form. Interactions between SdbA and SpeA were examined using cysteine variant proteins. The results showed that SdbA C49A formed a mixed disulfide with SpeA C87A , suggesting that the N-terminal Cys46 of SdbA and the C-terminal Cys98 of SpeA participated in the initial reaction. SpeA oxidized by SdbA displayed biological activities suggesting that SpeA was properly folded following oxidation by SdbA. In conclusion, formation of the disulfide bond in SpeA is catalyzed by SdbA and the findings represent the first report of disulfide bond formation in S. pyogenes. IMPORTANCE Here, we reported the first example of disulfide bond formation in Streptococcus pyogenes. The results showed that a thiol-disulfide oxidoreductase, named SdbA, is responsible for introducing the disulfide bond in the superantigen SpeA. The cysteine residues in the CXXC motif of SdbA are needed for catalyzing the disulfide bond in SpeA. The disulfide bond in SpeA and neighboring amino acids form a disulfide loop that is conserved among many superantigens, including those from Staphylococcus aureus. SpeA and staphylococcal enterotoxins lacking the disulfide bond are biologically inactive. Thus, the discovery of the enzyme that catalyzes the disulfide bond in SpeA is important for understanding the biochemistry of SpeA production and presents a target for mitigating the virulence of S. pyogenes.

Streptococcus pyogenes causes a wide range of human infections. Currently, antibiotics are the main treatment for S. pyogenes infection, but serious anti-microbial resistance requires alternative treatment options. To develop a novel strategy for treatment, we physicochemically characterized SPs0871, a putative maltose/maltodextrin-binding protein that is thought to have important roles in the pathogenesis of invasive streptococci. We obtained a variable domain of heavy chain of heavy-chain antibody, the smallest unit of an antibody, which specifically binds to SPs0871. Although the VHH completely inhibited the binding of maltodextrins to SPs0871, the inhibition did not lead to growth suppression of the bacteria. Our results provide important insights for development of VHH as an anti-streptococcal therapeutic., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Streptococcus pneumoniae , S. pyogenes (Group A Streptococcus ; GAS) and S. agalactiae (Group B Streptococcus ; GBS) are major aetiological agents of diseases in humans. The cellular membrane, a crucial site in host-pathogen interactions, is poorly characterized in streptococci. Moreover, little is known about whether or how environmental conditions influence their lipid compositions. Using normal phase liquid chromatography coupled with electrospray ionization MS, we characterized the phospholipids and glycolipids of S. pneumoniae , GAS and GBS in routine undefined laboratory medium, streptococcal defined medium and, in order to mimic the host environment, defined medium supplemented with human serum. In human serum-supplemented medium, all three streptococcal species synthesize phosphatidylcholine (PC), a zwitterionic phospholipid commonly found in eukaryotes but relatively rare in bacteria. We previously reported that S. pneumoniae utilizes the glycerophosphocholine (GPC) biosynthetic pathway to synthesize PC. Through substrate tracing experiments, we confirm that GAS and GBS scavenge lysoPC, a major metabolite in human serum, thereby using an abbreviated GPC pathway for PC biosynthesis. Furthermore, we found that plasmanyl-PC is uniquely present in the GBS membrane during growth with human serum, suggesting GBS possesses unusual membrane biochemical or biophysical properties. In summary, we report cellular lipid remodelling by the major pathogenic streptococci in response to metabolites present in human serum.

The Streptococcus pyogenes Cas9 protein (SpCas9), a component of CRISPR-based immune system in microbes, has become commonly utilized for genome editing. This nuclease forms a ribonucleoprotein (RNP) complex with guide RNA (gRNA) which induces Cas9 structural changes and triggers its cleavage activity. Here, electronic circular dichroism (ECD) spectroscopy was used to confirm the RNP formation and to determine its individual components. The ECD spectra had characteristic features differentiating Cas9 and gRNA, the former showed a negative/positive profile with maxima located at 221, 209 and 196 nm, while the latter revealed positive/negative/positive/negative pattern with bands observed at 266, 242, 222 and 209 nm, respectively. For the first time, the experimental ECD spectrum of the gRNA:Cas9 RNP complex is presented. It exhibits a bisignate positive/negative ECD couplet with maxima at 273 and 235 nm, and it differs significantly from individual spectrum of each RNP components. Additionally, the Cas9 protein and RNP complex retained biological activity after ECD measurements and they were able to bind and cleave DNA in vitro. Hence, we conclude that ECD spectroscopy can be considered as a quick and non-destructive method of monitoring conformational changes of the Cas9 protein as a result of Cas9 and gRNA interaction, and identification of the gRNA:Cas9 RNP complex.

Gram-positive bacteria colonize mucosal tissues, withstanding large mechanical perturbations such as coughing, which generate shear forces that exceed the ability of non-covalent bonds to remain attached. To overcome these challenges, the pathogen Streptococcus pyogenes utilizes the protein Cpa, a pilus tip-end adhesin equipped with a Cys-Gln thioester bond. The reactivity of this bond towards host surface ligands enables covalent anchoring; however, colonization also requires cell migration and spreading over surfaces. The molecular mechanisms underlying these seemingly incompatible requirements remain unknown. Here we demonstrate a magnetic tweezers force spectroscopy assay that resolves the dynamics of the Cpa thioester bond under force. When folded at forces <6 pN, the Cpa thioester bond reacts reversibly with amine ligands, which are common in inflammation sites; however, mechanical unfolding and exposure to forces >6 pN block thioester reformation. We hypothesize that this folding-coupled reactivity switch (termed a smart covalent bond) could allow the adhesin to undergo binding and unbinding to surface ligands under low force and remain covalently attached under mechanical stress.

HupZ is an expected heme degrading enzyme in the heme acquisition and utilization pathway in Group A Streptococcus. The isolated HupZ protein containing a C-terminal V5-His 6 tag exhibits a weak heme degradation activity. Here, we revisited and characterized the HupZ-V5-His 6 protein via biochemical, mutagenesis, protein quaternary structure, UV-vis, EPR, and resonance Raman spectroscopies. The results show that the ferric heme-protein complex did not display an expected ferric EPR signal and that heme binding to HupZ triggered the formation of higher oligomeric states. We found that heme binding to HupZ was an O 2 -dependent process. The single histidine residue in the HupZ sequence, His111, did not bind to the ferric heme, nor was it involved with the weak heme-degradation activity. Our results do not favor the heme oxygenase assignment because of the slow binding of heme and the newly discovered association of the weak heme degradation activity with the His 6 -tag. Altogether, the data suggest that the protein binds heme by its His 6 -tag, resulting in a heme-induced higher-order oligomeric structure and heme stacking. This work emphasizes the importance of considering exogenous tags when interpreting experimental observations during the study of heme utilization proteins.

Currently, either highly multiplexed genetic manipulations can be delivered to mammalian cells all at once or extensive engineering of gene regulatory sequences can be used to conditionally activate a few manipulations. Here, we provide proof of principle for a new system enabling multiple genetic manipulations to be executed as a preprogrammed cascade of events. The system leverages the programmability of the S. pyogenes Cas9 and is based on flexible arrangements of individual modules of activity. The basic module consists of an inactive single-guide RNA (sgRNA)-like component that is converted to an active state through the effects of another sgRNA. Modules can be arranged to bring about an algorithmic program of sequential genetic manipulations without the need for engineering cell-type-specific promoters or gene regulatory sequences. With the expanding diversity of available tools that use spCas9, this sgRNA-based system provides multiple levels of interfacing with mammalian cell biology., Competing Interests: Declaration of Interests The following competing interests are declared: R.C., A.R.T., H.P., M.S.M., M.R., and B.J.M. as shareholders in Cellgorithmics; R.C., B.J.M., H.P., and M.S.M. as cofounders of Cellgorithmics; and R.C., H.P., M.S.M., and B.J.M. as inventors on patent application no. PCT/US2018/052211., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Streptococcus pyogenes (Group A streptococcus; GAS) is an important human pathogen responsible for mild to severe, life-threatening infections. GAS expresses a wide range of virulence factors, including the M family proteins. The M proteins allow the bacteria to evade parts of the human immune defenses by triggering the formation of a dense coat of plasma proteins surrounding the bacteria, including IgGs. However, the molecular level details of the M1-IgG interaction have remained unclear. Here, we characterized the structure and dynamics of this interaction interface in human plasma on the surface of live bacteria using integrative structural biology, combining cross-linking mass spectrometry and molecular dynamics (MD) simulations. We show that the primary interaction is formed between the S-domain of M1 and the conserved IgG Fc-domain. In addition, we show evidence for a so far uncharacterized interaction between the A-domain and the IgG Fc-domain. Both these interactions mimic the protein G-IgG interface of group C and G streptococcus. These findings underline a conserved scavenging mechanism used by GAS surface proteins that block the IgG-receptor (FcγR) to inhibit phagocytic killing. We additionally show that we can capture Fab-bound IgGs in a complex background and identify XLs between the constant region of the Fab-domain and certain regions of the M1 protein engaged in the Fab-mediated binding. Our results elucidate the M1-IgG interaction network involved in inhibition of phagocytosis and reveal important M1 peptides that can be further investigated as future vaccine targets., Competing Interests: The authors have declared that no competing interests exist.

The pathogenicity of group A Streptococcus (GAS) is mediated by direct bacterial invasivity and toxin-associated damage. Among the extracellular products, the exotoxin streptolysin O (SLO) is produced by almost all GAS strains. SLO is a pore forming toxin (PFT) hemolitically active and extremely toxic in vivo . Recent evidence suggests that human serum albumin (HSA), the most abundant protein in plasma, is a player in the innate immunity "orchestra." We previously demonstrated that HSA acts as a physiological buffer, partially neutralizing Clostridioides difficile toxins that reach the bloodstream after being produced in the colon. Here, we report the in vitro and ex vivo capability of HSA to neutralize the cytotoxic and hemolytic effects of SLO. HSA binds SLO with high affinity at a non-conventional site located in domain II, which was previously reported to interact also with C. difficile toxins. HSA:SLO recognition protects HEp-2 and A549 cells from cytotoxic effects and cell membrane permeabilization induced by SLO. Moreover, HSA inhibits the SLO-dependent hemolytic effect in red blood cells isolated from healthy human donors. The recognition of SLO by HSA may have a significant protective role in human serum and sustains the emerging hypothesis that HSA is an important constituent of the innate immunity system., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Vita, De Simone, Leboffe, Montagnani, Mariotti, Di Bella, Luzzati, Gori, Ascenzi and di Masi.)

Sortases are a group of enzymes displayed on the cell-wall of Gram-positive bacteria. They are responsible for the attachment of virulence factors onto the peptidoglycan in a transpeptidation reaction through recognition of a pentapeptide substrate. Most housekeeping sortases recognize one specific pentapeptide motif; however, Streptococcus pyogenes sortase A (SpSrtA WT) recognizes LPETG, LPETA and LPKLG motifs. Here, we examined SpSrtA's flexible substrate specificity by investigating the role of the β7/β8 loop in determining substrate specificity. We exchanged the β7/β8 loop in SpSrtA with corresponding β7/β8 loops from Staphylococcus aureus (SaSrtA WT) and Bacillus anthracis (BaSrtA WT). While the BaSrtA-derived variant showed no enzymatic activity toward either LPETG or LPETA substrates, the activity of the SaSrtA-derived mutant toward the LPETA substrate was completely abolished. Instead, the mutant had an improved activity toward LPETG, the preferred substrate of SaSrtA WT., (© 2020 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals, Inc.)

Staphylococcus aureus and Streptococcus pyogenes are significant human pathogens, causing infections at multiple body sites, including across the skin. Both are organisms that cause human diseases and secrete superantigens, including toxic shock syndrome toxin-1 (TSST-1), staphylococcal enterotoxins (SEs), and streptococcal pyrogenic exotoxins (SPEs). On the skin, human keratinocytes represent the first cell type to encounter these superantigens. We employed transcriptome sequencing (RNA-seq) to evaluate the human primary keratinocyte response to both TSST-1 and staphylococcal enterotoxin B (SEB) in triplicate analyses. Both superantigens caused large numbers of genes to be up- and downregulated. The genes that exhibited 2-fold differential gene expression compared to vehicle-treated cells, whether up- or downregulated, totaled 5,773 for TSST-1 and 4,320 for SEB. Of these, 4,482 were significantly upregulated by exposure of keratinocytes to TSST-1, whereas 1,291 were downregulated. For SEB, expression levels of 3,785 genes were upregulated, whereas those of 535 were downregulated. There was the expected high overlap in both upregulation (3,412 genes) and downregulation (400 genes). Significantly upregulated genes included those associated with chemokine production, with the possibility of stimulation of inflammation. We also tested an immortalized human keratinocyte line, from a different donor, for chemokine response to four superantigens. TSST-1 and SEB caused production of interleukin-8 (IL-8), MIP-3α, and IL-33. SPEA and SPEC were evaluated for stimulation of expression of IL-8 as a representative chemokine; both stimulated production of IL-8. IMPORTANCE Staphylococcus aureus and Streptococcus pyogenes are common human pathogens, causing infections that include the skin. Both pathogens produce a family of secreted toxins called superantigens, which have been shown to be important in human diseases. The first cell types encountered by superantigens on skin are keratinocytes. Our studies demonstrated, that the human keratinocyte pathway, among other pathways, responds to superantigens with production of chemokines, setting off inflammation. This inflammatory response may be harmful, facilitating opening of the skin barrier., (Copyright © 2020 Schlievert et al.)

CRISPR/Cas9 is a programmable genome editing tool widely used for biological applications and engineered Cas9s have increased discrimination against off-target cleavage compared with wild-type Streptococcus pyogenes (SpCas9) in vivo. To understand the basis for improved discrimination against off-target DNA containing important mismatches at the distal end of the guide RNA, we performed kinetic analyses on the high-fidelity (Cas9-HF1) and hyper-accurate (HypaCas9) engineered Cas9 variants. We show that DNA cleavage is impaired by more than 100- fold for the high-fidelity variants. The high-fidelity variants improve discrimination by slowing the observed rate of cleavage without increasing the rate of DNA rewinding and release. The kinetic partitioning favors release rather than cleavage of a bound off-target substrate only because the cleavage rate is so low. Further improvement in discrimination may require engineering increased rates of dissociation of off-target DNA.

The CRISPR/Cas9 nucleases have been widely applied for genome editing in various organisms. Cas9 nucleases complexed with a guide RNA (Cas9-gRNA) find their targets by scanning and interrogating the genomic DNA for sequences complementary to the gRNA. Recognition of the DNA target sequence requires a short protospacer adjacent motif (PAM) located outside this sequence. Given that the efficiency of target location may depend on the strength of interactions that promote target recognition, here we sought to compare affinities of different Cas9 nucleases for their cognate PAM sequences. To this end, we measured affinities of Cas9 nucleases from Streptococcus pyogenes , Staphylococcus aureus , and Francisella novicida complexed with guide RNAs (gRNAs) (SpCas9-gRNA, SaCas9-gRNA, and FnCas9-gRNA, respectively) and of three engineered SpCas9-gRNA variants with altered PAM specificities for short, PAM-containing DNA probes. We used a "beacon" assay that measures the relative affinities of DNA probes by determining their ability to competitively affect the rate of Cas9-gRNA binding to fluorescently labeled target DNA derivatives called "Cas9 beacons." We observed significant differences in the affinities for cognate PAM sequences among the studied Cas9 enzymes. The relative affinities of SpCas9-gRNA and its engineered variants for canonical and suboptimal PAMs correlated with previous findings on the efficiency of these PAM sequences in genome editing. These findings suggest that high affinity of a Cas9 nuclease for its cognate PAM promotes higher genome-editing efficiency., (© 2020 Mekler et al.)

Crosslinking-mass spectrometry (XL-MS) serves to identify interaction sites between proteins. Numerous search engines for crosslink identification exist, but lack of ground truth samples containing known crosslinks has precluded their systematic validation. Here we report on XL-MS data arising from measuring synthetic peptide libraries that provide the unique benefit of knowing which identified crosslinks are true and which are false. The data are analysed with the most frequently used search engines and the results filtered to an estimated false discovery rate of 5%. We find that the actual false crosslink identification rates range from 2.4 to 32%, depending on the analysis strategy employed. Furthermore, the use of MS-cleavable crosslinkers does not reduce the false discovery rate compared to non-cleavable crosslinkers. We anticipate that the datasets acquired during this research will further drive optimisation and development of XL-MS search engines, thereby advancing our understanding of vital biological interactions.

Background: Atomic Force Microscopy (AFM) is an experimental technique to study structure-function relationship of biomolecules. AFM provides images of biomolecules at nanometer resolution. High-speed AFM experiments produce a series of images following dynamics of biomolecules. To further understand biomolecular functions, information on three-dimensional (3D) structures is beneficial., Method: We aim to recover 3D information from an AFM image by computational modeling. The AFM image includes only low-resolution representation of a molecule; therefore we represent the structures by a coarse grained model (Gaussian mixture model). Using Monte-Carlo sampling, candidate models are generated to increase similarity between AFM images simulated from the models and target AFM image., Results: The algorithm was tested on two proteins to model their conformational transitions. Using a simulated AFM image as reference, the algorithm can produce a low-resolution 3D model of the target molecule. Effect of molecular orientations captured in AFM images on the 3D modeling performance was also examined and it is shown that similar accuracy can be obtained for many orientations., Conclusions: The proposed algorithm can generate 3D low-resolution protein models, from which conformational transitions observed in AFM images can be interpreted in more detail., General Significance: High-speed AFM experiments allow us to directly observe biomolecules in action, which provides insights on biomolecular function through dynamics. However, as only partial structural information can be obtained from AFM data, this new AFM based hybrid modeling method would be useful to retrieve 3D information of the entire biomolecule., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Fmoc solid-phase peptide synthesis (SPPS) is the most common approach used to synthesize natural and unnatural peptides. However, the synthesis of sequences longer than 30-60 amino acids is associated with a drastic reduction in peptide quality. Thus, large and complex peptides are normally synthesized as fragments, which are then conjugated together. Here, we describe the synthesis of a large, branched peptide, a multi-epitope vaccine candidate against Group A Streptococcus, with the help of microwave-assisted Fmoc-SPPS, thiol-maleimide conjugation, and copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) "click" reaction.

Gram-positive bacteria use their adhesive pili to attach to host cells during early stages of a bacterial infection. These extracellular hair-like appendages experience mechanical stresses of hundreds of picoNewtons; however, the presence of an internal isopeptide bond prevents the pilus protein from unfolding. Here, we describe a method to interfere with nascent pili proteins through a peptide that mimics one of the β-strands of the molecule. By using AFM-based force spectroscopy, we study the isopeptide bond formation and the effect of the peptide in the elasticity of the pilus protein. This method could be used to afford a new strategy for mechanically targeted antibiotics by simply blocking the folding of the bacterial pilus protein.

Intramolecular isopeptide bonds, formed autocatalytically between Lys and Asn/Asp side chains, are widely present in the immunoglobulin-like domains of Gram-positive bacterial adhesins, including Group A Streptococcus, and confer considerable mechanical and chemical stability. These properties make them attractive for applications in biotechnology. Here, we detail the practical considerations that are involved in engineering isopeptide bonds into Ig-like proteins, including the choice of a site where bond-forming residues could be introduced and the appropriate methodology for mutagenesis. We specify how to determine whether an isopeptide bond has formed, what strategies can be adopted to overcome problems, and how to monitor the stability of the engineered protein.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complex is an RNA-guided DNA-nuclease that is part of the bacterial adaptive immune system. CRISPR/Cas9 RNP has been adapted for targeted genome editing within cells and whole organisms with new applications vastly outpacing detection and quantification of gene-editing reagents. Detection of the CRISPR/Cas9 RNP within biological samples is critical for assessing gene-editing reagent delivery efficiency, retention, persistence, and distribution within living organisms. Conventional detection methods are effective, yet the expense and lack of scalability for antibody-based affinity reagents limit these techniques for clinical and/or field settings. This necessitates the development of low cost, scalable CRISPR/Cas9 RNP affinity reagents as alternatives or augments to antibodies. Herein, we report the development of the Streptococcus pyogenes anti-CRISPR/Cas9 protein, AcrIIA4, as a novel affinity reagent. An engineered cysteine linker enables covalent immobilization of AcrIIA4 onto glassy carbon electrodes functionalized via aryl diazonium chemistry for detection of CRISPR/Cas9 RNP by electrochemical, fluorescent, and colorimetric methods. Electrochemical measurements achieve a detection of 280 pM RNP in reaction buffer and 8 nM RNP in biologically representative conditions. Our results demonstrate the ability of anti-CRISPR proteins to serve as robust, specific, flexible, and economical recognition elements in biosensing/quantification devices for CRISPR/Cas9 RNP., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The human-adapted pathogen group A Streptococcus (GAS) utilizes wounds as portals of entry into host tissue, wherein surface adhesins interact with the extracellular matrix, enabling bacterial colonization. The streptococcal collagen-like protein 1 (Scl1) is a major adhesin of GAS that selectively binds to two fibronectin type III (FnIII) repeats within cellular fibronectin, specifically the alternatively spliced extra domains A and B, and the FnIII repeats within tenascin-C. Binding to FnIII repeats was mediated through conserved structural determinants present within the Scl1 globular domain and facilitated GAS adherence and biofilm formation. Isoforms of cellular fibronectin that contain extra domains A and B, as well as tenascin-C, are present for several days in the wound extracellular matrix. Scl1-FnIII binding is therefore an example of GAS adaptation to the host's wound environment. Similarly, cellular fibronectin isoforms and tenascin-C are present in the tumor microenvironment. Consistent with this, FnIII repeats mediate GAS attachment to and enhancement of biofilm formation on matrices deposited by cancer-associated fibroblasts and osteosarcoma cells. These data collectively support the premise for utilization of the Scl1-FnIII interaction as a novel method of anti-neoplastic targeting in the tumor microenvironment., (© 2019 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.)

The group A streptococci are associated with a group of diseases affecting the heart, brain, and joints that are collectively referred to as acute rheumatic fever. The streptococcal immune-mediated sequelae, including acute rheumatic fever, are due to antibody and cellular immune responses that target antigens in the heart and brain as well as the group A streptococcal cross-reactive antigens as reviewed in this article. The pathogenesis of acute rheumatic fever, rheumatic heart disease, Sydenham chorea, and other autoimmune sequelae is related to autoantibodies that are characteristic of autoimmune diseases and result from the immune responses against group A streptococcal infection by the host. The sharing of host and streptococcal epitopes leads to molecular mimicry between the streptococcal and host antigens that are recognized by the autoantibodies during the host response. This article elaborates on the discoveries that led to a better understanding of the pathogenesis of disease and provides an overview of the history and the most current thought about the immune responses against the host and streptococcal cross-reactive antigens in group A streptococcal sequelae.

Group A Streptococcus (GAS) ( Streptococcus pyogenes ) is an important human pathogen associated with significant global morbidity and mortality for which there is no safe and efficacious vaccine. The T antigen, a protein that polymerizes to form the backbone of the GAS pilus structure, is a potential vaccine candidate. Previous surveys of the tee gene, which encodes the T antigen, have identified 21 different tee types and subtypes such that any T antigen-based vaccine must be multivalent and carefully designed to provide broad strain coverage. In this study, the crystal structures of three two-domain T antigens (T3.2, T13, and T18.1) were determined and found to have remarkable structural similarity to the previously reported T1 antigen, despite moderate overall sequence similarity. This has enabled reliable modeling of all major two-domain T antigens to reveal that T antigen sequence variation is distributed along the full length of the protein and shields a highly conserved core. Immunoassays performed with sera from immunized animals and commercial T-typing sera identified a significant cross-reactive antibody response between T18.1, T18.2, T3.2, and T13. The existence of shared epitopes between T antigens, combined with the remarkably conserved structure and high level of surface sequence divergence, has important implications for the design of multivalent T antigen-based vaccines., (Copyright © 2019 American Society for Microbiology.)

The SpyCatcher-SpyTag system was developed seven years ago as a method for protein ligation. It is based on a modified domain from a Streptococcus pyogenes surface protein (SpyCatcher), which recognizes a cognate 13-amino-acid peptide (SpyTag). Upon recognition, the two form a covalent isopeptide bond between the side chains of a lysine in SpyCatcher and an aspartate in SpyTag. This technology has been used, among other applications, to create covalently stabilized multi-protein complexes, for modular vaccine production, and to label proteins (e.g., for microscopy). The SpyTag system is versatile as the tag is a short, unfolded peptide that can be genetically fused to exposed positions in target proteins; similarly, SpyCatcher can be fused to reporter proteins such as GFP, and to epitope or purification tags. Additionally, an orthogonal system called SnoopTag-SnoopCatcher has been developed from an S. pneumoniae pilin that can be combined with SpyCatcher-SpyTag to produce protein fusions with multiple components. Furthermore, tripartite applications have been produced from both systems allowing the fusion of two peptides by a separate, catalytically active protein unit, SpyLigase or SnoopLigase. Here, we review the current state of the SpyCatcher-SpyTag and related technologies, with a particular emphasis on their use in vaccine development and in determining outer membrane protein localization and topology of surface proteins in bacteria.

Objective: Intravenous immune globulin (IVIG), pooled from human blood, is a polyspecific antibody preparation that inhibits the super-antigenic proteins associated with streptococcal and staphylococcal toxic shock, and the Shiga toxin. In addition to this toxin-neutralising activity, IVIG contains other pathogen-reactive antibodies that may confer additional therapeutic benefits. We sought to determine if pathogen-reactive antibodies that promote opsonophagocytosis of different organisms can be sequentially affinity-purified from one IVIG preparation., Results: Antibodies that recognise cell wall antigens of Streptococcus pyogenes, Staphylococcus aureus, and vancomycin-resistant enterococcus (VRE) were sequentially affinity-purified from a single preparation of commercial IVIG and opsonophagocytic activity was assessed using a flow cytometry assay of neutrophil uptake. Non-specific IgG-binding proteins were removed from the S. aureus preparations using an immobilised Fc fragment column, produced using IVIG cleaved with the Immunoglobulin G-degrading enzyme of S. pyogenes (IdeS). Affinity-purified anti-S. aureus and anti-VRE immunoglobulin promoted significantly higher levels of opsonophagocytic uptake by human neutrophils than IVIG when identical total antibody concentrations were compared, confirming activity previously shown for affinity-purified anti-S. pyogenes immunoglobulin. The opsonophagocytic activities of anti-S. pyogenes, anti-S. aureus, and anti-VRE antibodies that were sequentially purified from a single IVIG preparation were undiminished compared to antibodies purified from previously unused IVIG.

Streptococcus pyogenes (group A Streptococcus, GAS) has caused a wide variety of human diseases. Its multifunctional surface dehydrogenase (SDH) is crucial for GAS life cycle. Furthermore, GAS infection into human pharyngeal cells has been previously shown to be mediated by the interaction between SDH and host urokinase-type plasminogen activator receptor (uPAR). However, the structural information of SDH remains to be elucidated and there are few detailed studies to characterize its interaction with uPAR. In-depth research on these issues will provide potential targets and strategies for combating GAS. Here, we prepared recombinant SDH tetramer in Escherichia coli BL21 (DE3) cells. After purification and crystallization, we determined its crystal structure at 1.74 Å. The unique characteristics might be potentially explored as drug targets or vaccine immunogen. We subsequently performed gel filtration chromatography, native-polyacrylamide gel electrophoresis (PAGE) and in vitro pull-down analyses. The results showed that their interaction was too weak to form stable complexes and the role of uPAR involved in GAS infection needs further demonstration. Altogether the current work provides the first view of SDH and deepens the knowledge of GAS infection., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The native octameric structure of streptococcal enolase from Streptococcus pyogenes increasingly dissociates as amino acid residues are removed one by one from the carboxy-terminus. These truncations gradually convert native octameric enolase into monomers and oligomers. In this work, we investigated how these truncations influence the interaction between Streptococcal enolase and canine plasminogen. We used dual polarization interferometry (DPI), localized surface plasmon resonance (LSPR), and sedimentation velocity analytical ultracentrifugation (AUC) to study the interaction. The DPI was our first technique, was performed on all the truncations and used one exclusive kind of chip. The LSRP was used to show that the DPI results were not dependent on the type of chip used. The AUC was required to show that our surface results were not the result of selecting a minority population in any given sample; the majority of the protein was responsible for the binding phenomenon we observed. By comparing results from these techniques we identified one detail that is essential for streptococcal enolase to bind plasminogen: In our hands the individual monomers bind plasminogen; dimers, trimers, tetramers may or may not bind, the fully intact, native, octamer does not bind plasminogen. We also evaluated the contribution to the equilibrium constant made by surface binding as well as in solution. On a surface, the association coefficient is about twice that in solution. The difference is probably not significant. Finally, the fully octameric form of the protein that does not contain a hexa-his N-terminal peptide does not bind to a silicon oxynitride surface, does not bind to an Au-nanoparticle surface, does not bind to a surface coated with Ni-NTA nor does it bind to a surface coated with DPgn. The likelihood is great that the enolase species on the surface of Streptococcus pyogenes is an x-mer of the native octamer., Competing Interests: The authors have declared that no competing interests exist.

Group A Streptococcus (GAS) infections are responsible for significant morbidity and mortality worldwide. The outlook for an effective global vaccine is reduced because of significant antigenic variation among GAS strains worldwide. Other challenges in GAS therapy include the lack of common access to antibiotics in developing countries, as well as allergy to and treatment failures with penicillin and increasing erythromycin resistance in the industrialized world. At the portal of entry, GAS binds to newly deposited extracellular matrix, which is rich in cellular fibronectin isoforms with extra domain A (EDA, also termed EIIIA) via the surface adhesin, the streptococcal collagen-like protein 1 (Scl1). Recombinant Scl1 constructs, derived from diverse GAS strains, bind the EDA loop segment situated between the C and C' β-strands. Despite the sequence diversity in Scl1 proteins, multiple sequence alignments and secondary structure predictions of Scl1 variants, as well as crystallography and homology modeling studies, point to a conserved mechanism of Scl1-EDA binding. We propose that targeting this interaction may prevent the progression of infection. A synthetic cyclic peptide, derived from the EDA C-C' loop, binds to recombinant Scl1 with a micromolar dissociation constant. This review highlights the current concept of EDA binding to Scl1 and provides incentives to exploit this binding to treat GAS infections and wound colonization., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Since its discovery, the CRISPR-Cas9 system has been in the center of attention for its promising applications in genome editing. However, in order to apply this system successfully in genetic engineering, all aspects of its molecular mechanism have to be well understood. One of the best ways to investigate the intricacies of the molecular mechanism is single-molecule studies as they allow real-time observation of the kinetic processes and provide high spatiotemporal resolution. This chapter describes single-molecule fluorescence resonance energy transfer experiments carried out to investigate the Cas9 protein from Streptococcus pyogenes, providing information on the effects of target truncation, Cas9-induced double-stranded DNA dynamics, and target search., (© 2019 Elsevier Inc. All rights reserved.)

In order to proliferate and mount an infection, many bacterial pathogens need to acquire iron from their host. The most abundant iron source in the body is the oxygen transporter hemoglobin (Hb). Streptococcus pyogenes , a potentially lethal human pathogen, uses the Shr protein to capture Hb on the cell surface. Shr is an important virulence factor, yet the mechanism by which it captures Hb and acquires its heme is not well-understood. Here, we show using NMR and biochemical methods that Shr binds Hb using two related modules that were previously defined as domains of unknown function (DUF1533). These hemoglobin-interacting domains (HIDs), called HID1 and HID2, are autonomously folded and independently bind Hb. The 1.5 Å resolution crystal structure of HID2 revealed that it is a structurally unique Hb-binding domain. Mutagenesis studies revealed a conserved tyrosine in both HIDs that is essential for Hb binding. Our biochemical studies indicate that HID2 binds Hb with higher affinity than HID1 and that the Hb tetramer is engaged by two Shr receptors. NMR studies reveal the presence of a third autonomously folded domain between HID2 and a heme-binding NEAT1 domain, suggesting that this linker domain may position NEAT1 near Hb for heme capture., (© 2018 Macdonald et al.)

Background/aim: We aimed to evaluate the efficacy of a rapid antigen test in detecting group A beta-hemolytic streptococci (GAS) in throat samples in comparison with the culture method and to compare the efficiency of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and traditional methods in identifying GAS in cultures. Materials and methods: A total of 3668 throat samples from patients with a prediagnosis of tonsillopharyngitis were assessed by the QuickVue+Strep A antigen test and culture. For GAS identification from cultures, bacitracin sensitivity, PYR, and latex agglutination tests and MALDI-TOF MS were used. Results: A total of 567 (15.5%) and 536 (14.6%) of the samples were positive for GAS culture and rapid antigen testing, respectively. The sensitivity and specificity of the rapid antigen test compared to culture was 89.07% and 99%, respectively, while positive and negative predictive values were 94.22% and 98.02%. Traditional methods were in full concordance with MALDI-TOF-MS for all 567 isolates. In all densities of growth in culture, the time to diagnosis with MALDI-TOF MS was significantly lower than with traditional identification tests. Conclusion: This study shows that both the rapid antigen testing of samples and bacterial identification with MALDI-TOF MS contribute much to the rapid diagnosis of GAS tonsillopharyngitis., (CC-BY 4.0)

Group A Streptococcus (GAS) is a human-specific pathogen responsible for a wide range of diseases, ranging from superficial to life-threatening invasive infections, including endometritis, and autoimmune sequelae. GAS strains express a vast repertoire of virulence factors that varies depending on the strain genotype, and many adhesin proteins that enable GAS to adhere to host cells are restricted to some genotypes. GAS emm28 is the third most prevalent genotype in invasive infections in France and is associated with gyneco-obstetrical infections. emm28 strains harbor R28, a cell wall-anchored surface protein that has previously been reported to promote adhesion to cervical epithelial cells. Here, using cellular and biochemical approaches, we sought to determine whether R28 supports adhesion also to other cells and to characterize its cognate receptor. We show that through its N-terminal domain, R28 Nt , R28 promotes bacterial adhesion to both endometrial-epithelial and endometrial-stromal cells. R28 Nt was further subdivided into two domains, and we found that both are involved in cell binding. R28 Nt and both subdomains interacted directly with the laminin-binding α3β1, α6β1, and α6β4 integrins; interestingly, these bindings events did not require divalent cations. R28 is the first GAS adhesin reported to bind directly to integrins that are expressed in most epithelial cells. Finally, R28 Nt also promoted binding to keratinocytes and pulmonary epithelial cells, suggesting that it may be involved in supporting the prevalence in invasive infections of the emm28 genotype., (© 2018 Weckel et al.)

Bacteria anchor to their host cells through their adhesive pili, which must resist the large mechanical stresses induced by the host as it attempts to dislodge the pathogens. The pili of gram-positive bacteria are constructed as a single polypeptide made of hundreds of pilin repeats, which contain intramolecular isopeptide bonds strategically located in the structure to prevent their unfolding under force, protecting the pilus from degradation by extant proteases and oxygen radicals. Here, we demonstrate the design of a short peptide that blocks the formation of the isopeptide bond present in the pilin Spy0128 from the human pathogen Streptococcus pyogenes , resulting in mechanically labile pilin domains. We use a combination of protein engineering and atomic-force microscopy force spectroscopy to demonstrate that the peptide blocks the formation of the native isopeptide bond and compromises the mechanics of the domain. While an intact Spy0128 is inextensible at any force, peptide-modified Spy0128 pilins readily unfold at very low forces, marking the abrogation of the intramolecular isopeptide bond as well as the absence of a stable pilin fold. We propose that isopeptide-blocking peptides could be further developed as a type of highly specific antiadhesive antibiotics to treat gram-positive pathogens., Competing Interests: The authors declare no conflict of interest.

Cleavage of collagen by collagenases such as matrix metalloproteinase 1 (MMP-1) is a key step in development, tissue remodeling, and tumor proliferation. The abundant heterotrimeric type I collagen composed of two α1(I) chains and one α2(I) chain is efficiently cleaved by MMP-1 at a unique site in the triple helix, a process which may be initiated by local unfolding within the peptide chains. Atypical homotrimers of the α1(I) chain, found in embryonic and cancer tissues, are very resistant to MMP cleavage. To investigate MMP-1 cleavage, recombinant homotrimers were constructed with sequences from the MMP cleavage regions of human collagen chains inserted into a host bacterial collagen protein system. All triple-helical constructs were cleaved by MMP-1, with α2(I) homotrimers cleaved efficiently at a rate similar to that seen for α1(II) and α1(III) homotrimers, while α1(I) homotrimers were cleaved at a much slower rate. The introduction of destabilizing Gly to Ser mutations within the human collagenase susceptible region of the α2(I) chain did not interfere with MMP-1 cleavage. Molecular dynamics simulations indicated a greater degree of transient hydrogen bond breaking in α2(I) homotrimers compared with α1(I) homotrimers at the MMP-1 cleavage site, and showed an extensive disruption of hydrogen bonding in the presence of a Gly to Ser mutation, consistent with chymotrypsin digestion results. This study indicates that α2(I) homotrimers are susceptible to MMP-1, proves that the presence of an α1(I) chain is not a requirement for α2(I) cleavage, and supports the importance of local unfolding of α2(I) in collagenase cleavage., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Endogenous nucleic acids and their receptors may be involved in the initiation of systemic autoimmune diseases including rheumatoid arthritis (RA). As the role of the DNA sensing Toll-like receptor (TLR) 9 in RA is unclear, we aimed to investigate its involvement in the pathogenesis of autoimmune arthritis using three different experimental models of RA. The data obtained revealed involvement of TLR9 in the T cell-dependent phase of inflammatory arthritis. In rats with pristane-induced arthritis (PIA), TLR9 inhibition before disease onset reduced arthritis significantly and almost completely abolished bone erosion. Accordingly, serum levels of IL-6, α-1-acid-glycoprotein and rheumatoid factor were reduced. Moreover, in TLR9 -/- mice, streptococcal cell wall (SCW)-induced arthritis was reduced in the T cell-dependent phase, whereas T cell-independent serum-transfer arthritis was not affected. Remarkably, while TLR7 expression did not change during in vitro osteoclastogenesis, TLR9 expression was higher in precursor cells than in mature osteoclasts and partial inhibition of osteoclastogenesis was achieved only by the TLR9 antagonist. These results demonstrate a pivotal role for TLR9 in the T cell-dependent phases of inflammatory arthritis and additionally suggest some role during osteoclastogenesis. Hence, endogenous DNA seems to be crucially involved in the pathophysiology of inflammatory autoimmune arthritis., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Keratinized epidermis constitutes a powerful barrier of the mucosa and skin, effectively preventing bacterial invasion, unless it is wounded and no longer protective. Wound healing involves deposition of distinct extracellular matrix (ECM) proteins enriched in cellular fibronectin (cFn) isoforms containing extra domain A (EDA). The streptococcal collagen-like protein 1 (Scl1) is a surface adhesin of group A Streptococcus (GAS), which contains an N-terminal variable (V) domain and a C-terminally located collagen-like domain. During wound infection, Scl1 selectively binds EDA/cFn isoforms and laminin, as well as low-density lipoprotein (LDL), through its V domain. The trimeric V domain has a six-helical bundle fold composed of three pairs of anti-parallel α-helices interconnected by hypervariable loops, but the roles of these structures in EDA/cFn binding are unclear. Here, using recombinant Scl (rScl) constructs to investigate structure-function determinants of the Scl1-EDA/cFn interaction, we found that full-length rScl1, containing both the globular V and the collagen domains, is necessary for EDA/cFn binding. We established that the surface-exposed loops, interconnecting conserved α-helices, guide recognition and binding of Scl1-V to EDA and binding to laminin and LDL. Moreover, electrostatic surface potential models of the Scl1-V domains pointed to a conserved, negatively charged pocket, surrounded by positively charged and neutral regions, as a determining factor for the binding. In light of these findings, we propose an updated model of EDA/cFn recognition by the Scl1 adhesin from GAS, representing a significant step in understanding the Scl1-ECM interactions within the wound microenvironment that underlie GAS pathogenesis., (© 2018 McNitt et al.)

Endoglycosidase S (EndoS) is a bacterial endo-β-N-acetylglucosaminidase that specifically catalyzes the hydrolysis of the β-1,4 linkage between the first two N-acetylglucosamine residues of the biantennary complex-type N-linked glycans of IgG Fc regions. It is used for the chemoenzymatic synthesis of homogeneously glycosylated antibodies with improved therapeutic properties, but the molecular basis for its substrate specificity is unknown. Here, we report the crystal structure of the full-length EndoS in complex with its oligosaccharide G2 product. The glycoside hydrolase domain contains two well-defined asymmetric grooves that accommodate the complex-type N-linked glycan antennae near the active site. Several loops shape the glycan binding site, thereby governing the strict substrate specificity of EndoS. Comparing the arrangement of these loops within EndoS and related endoglycosidases, reveals distinct-binding site architectures that correlate with the respective glycan specificities, providing a basis for the bioengineering of endoglycosidases to tailor the chemoenzymatic synthesis of monoclonal antibodies.

Zinc is an essential metal in bacteria. One important bacterial zinc transporter is AdcA, and most bacteria possess AdcA homologs that are single-domain small proteins due to better efficiency of protein biogenesis. However, a double-domain AdcA with two zinc-binding sites is significantly overrepresented in Streptococcus species, many of which are major human pathogens. Using molecular simulation and experimental validations of AdcA from Streptococcus pyogenes , we found here that the two AdcA domains sequentially stabilize the structure upon zinc binding, indicating an organization required for both increased zinc affinity and transfer speed. This structural organization appears to endow Streptococcus species with distinct advantages in zinc-depleted environments, which would not be achieved by each single AdcA domain alone. This enhanced zinc transport mechanism sheds light on the significance of the evolution of the AdcA domain fusion, provides new insights into double-domain transporter proteins with two binding sites for the same ion, and indicates a potential target of antimicrobial drugs against pathogenic Streptococcus species., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Generally, the high diversity of protein properties necessitates the development of unique nanoparticle bio-conjugation methods, optimized for each different protein. Here we describe a universal bio-conjugation approach which makes use of a new recombinant fusion protein combining two distinct domains. The N-terminal part is Glutathione S-Transferase (GST) from Schistosoma japonicum, for which we identify and characterize the remarkable ability to bind gold nanoparticles (GNPs) by forming gold-sulfur bonds (Au-S). The C-terminal part of this multi-domain construct is the SpyCatcher from Streptococcus pyogenes, which provides the ability to capture recombinant proteins encoding a SpyTag. Here we show that SpyCatcher can be immobilized covalently on GNPs through GST without the loss of its full functionality. We then show that GST-SpyCatcher activated particles are able to covalently bind a SpyTag modified protein by simple mixing, through the spontaneous formation of an unusual isopeptide bond.

Streptococcus pyogenes is a major global health burden causing a wide variety of diseases. Because a vaccine against this bacterium is still lacking, vaccine candidates or antimicrobial therapies are urgently needed. Here we use an invasive and clinically relevant streptococcal M1 serotype to characterize the bacterial proteome in-depth. An elaborate fractionation technique is employed to separate the different cell fractions, followed by shotgun mass-spectrometry analysis, allowing us to confirm the expression of nearly two-thirds (1022) of the 1690 open reading frames predicted for the streptococcal M1 reference proteome. In contrast with other studies, we present the entire isolated membrane proteome, which opens up a whole new source for drug targets. We show both the unique and most prevalent proteins for each cellular fraction and analyze the presence of predicted cell-wall-anchored proteins and lipoproteins. With our approach, we also identify a variety of novel proteins whose presence has not been reported in previous proteome studies. Proteins of interest, potential virulence factors, and drug or vaccine targets are discussed for each cellular fraction. Overall, the results of this work represent the so-far widest proteomic approach to characterize the protein composition and localization in S. pyogenes.

B-lymphocytes have the ability to repair their plasma membranes following injury, such as by bacterial cholesterol-dependent cytolysins. The repair process includes the removal of the pore from the inflicted region of the plasma membrane via lipid raft-mediated internalization. Lipid rafts are critical for B cell receptor (BCR) activation. Cholesterol-dependent pore forming bacterial toxins provide a useful tool for examining the role of lipid rafts in B cell activation and the underlying cellular mechanisms. This method serves as a great alternative of known cholesterol disruption reagents such as filipin, nystatin, and methyl-β-cyclodextrin. Here, we describe a method of damaging primary murine B cell plasma membranes with the Streptococcus pyogenes cytolysin, Streptolysin O (SLO), and monitoring levels of damage, repair and BCR activation.

In many Lactobacillales species ( i.e. lactic acid bacteria), peptidoglycan is decorated by polyrhamnose polysaccharides that are critical for cell envelope integrity and cell shape and also represent key antigenic determinants. Despite the biological importance of these polysaccharides, their biosynthetic pathways have received limited attention. The important human pathogen, Streptococcus pyogenes , synthesizes a key antigenic surface polymer, the Lancefield group A carbohydrate (GAC). GAC is covalently attached to peptidoglycan and consists of a polyrhamnose polymer, with N -acetylglucosamine (GlcNAc) side chains, which is an essential virulence determinant. The molecular details of the mechanism of polyrhamnose modification with GlcNAc are currently unknown. In this report, using molecular genetics, analytical chemistry, and mass spectrometry analysis, we demonstrated that GAC biosynthesis requires two distinct undecaprenol-linked GlcNAc-lipid intermediates: GlcNAc-pyrophosphoryl-undecaprenol (GlcNAc-P-P-Und) produced by the GlcNAc-phosphate transferase GacO and GlcNAc-phosphate-undecaprenol (GlcNAc-P-Und) produced by the glycosyltransferase GacI. Further investigations revealed that the GAC polyrhamnose backbone is assembled on GlcNAc-P-P-Und. Our results also suggested that a GT-C glycosyltransferase, GacL, transfers GlcNAc from GlcNAc-P-Und to polyrhamnose. Moreover, GacJ, a small membrane-associated protein, formed a complex with GacI and significantly stimulated its catalytic activity. Of note, we observed that GacI homologs perform a similar function in Streptococcus agalactiae and Enterococcus faecalis In conclusion, the elucidation of GAC biosynthesis in S. pyogenes reported here enhances our understanding of how other Gram-positive bacteria produce essential components of their cell wall., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Programmable colloidal assembly enables the creation of mesoscale materials in a bottom-up manner. Although DNA oligonucleotides have been used extensively as the programmable units in this paradigm, proteins, which exhibit more diverse modes of association and function, have not been widely used to direct colloidal assembly. Here we use protein-protein interactions to drive controlled aggregation of polystyrene microparticles, either through reversible coiled-coil interactions or through intermolecular isopeptide linkages. The sizes of the resulting aggregates are tunable and can be controlled by the concentration of immobilized surface proteins. Moreover, particles coated with different protein pairs undergo orthogonal assembly. We demonstrate that aggregates formed by association of coiled-coil proteins, in contrast to those linked by isopeptide bonds, are dispersed by treatment with chemical denaturants or soluble competing proteins. Finally, we show that protein-protein interactions can be used to assemble complex core-shell aggregates. This work illustrates a versatile strategy for engineering colloidal systems for use in materials science and biotechnology.