The 1.7 Å crystal structure of the C5a peptidase from Streptococcus agalactiae (ScpB) reveals an active site competent for catalysis.

A 1.7 Å structure is presented for an active form of the virulence factor ScpB, the C5a peptidase from Streptococcus agalactiae. The previously reported structure of the ScpB active site mutant exhibited a large separation (~20 Å) between the catalytic His and Ser residues. Significant differences are observed in the catalytic domain between the current and mutant ScpB structures resulting with a high RMSDCα (4.6 Å). The fold of the active form of ScpB is nearly identical to ScpA (RMSDCα 0.2 Å), the C5a-peptidase from Streptococcus pyogenes. Both ScpA and ScpB have comparable activity against human C5a, indicating neither enzyme require host proteins for C5a-ase activity. These studies are a first step in resolving reported differences in the specificities of these enzymes.

The protease associated (PA) domain in ScpA from Streptococcus pyogenes plays a role in substrate recruitment.

Annually, over 18 million disease cases and half a million deaths worldwide are estimated to be caused by Group A Streptococcus. ScpA (or C5a peptidase) is a well characterised member of the cell enveleope protease family, which possess a S8 subtilisin-like catalytic domain and a shared multi-domain architecture. ScpA cleaves complement factors C5a and C3a, impairing the function of these critical anaphylatoxins and disrupts complement-mediated innate immunity. Although the high resolution structure of ScpA is known, the details of how it recognises its substrate are only just emerging. Previous studies have identified a distant exosite on the 2nd fibronectin domain that plays an important role in recruitment via an interaction with the substrate core. Here, using a combination of solution NMR spectroscopy, mutagenesis with functional assays and computational approaches we identify a second exosite within the protease-associated (PA) domain. We propose a model in which the PA domain assists optimal delivery of the substrate's C terminus to the active site for cleavage.

Crystal Structure of Staphopain C from Staphylococcus aureus.

Staphylococcus aureus is a common opportunistic pathogen of humans and livestock that causes a wide variety of infections. The success of S. aureus as a pathogen depends on the production of an array of virulence factors including cysteine proteases (staphopains)-major secreted proteases of certain strains of the bacterium. Here, we report the three-dimensional structure of staphopain C (ScpA2) of S. aureus, which shows the typical papain-like fold and uncovers a detailed molecular description of the active site. Because the protein is involved in the pathogenesis of a chicken disease, our work provides the foundation for inhibitor design and potential antimicrobial strategies against this pathogen.

Systematic single-cell pathway analysis to characterize early T cell activation.

Pathway analysis is a key analytical stage in the interpretation of omics data, providing a powerful method for detecting alterations in cellular processes. We recently developed a sensitive and distribution-free statistical framework for multisample distribution testing, which we implement here in the open-source R package single-cell pathway analysis (SCPA). We demonstrate the effectiveness of SCPA over commonly used methods, generate a scRNA-seq T cell dataset, and characterize pathway activity over early cellular activation. This reveals regulatory pathways in T cells, including an intrinsic type I interferon system regulating T cell survival and a reliance on arachidonic acid metabolism throughout T cell activation. A systems-level characterization of pathway activity in T cells across multiple tissues also identifies alpha-defensin expression as a hallmark of bone-marrow-derived T cells. Overall, this work provides a widely applicable tool for single-cell pathway analysis and highlights regulatory mechanisms of T cells.

A joint-ParB interface promotes Smc DNA recruitment.

Chromosomes readily unlink and segregate to daughter cells during cell division, highlighting a remarkable ability of cells to organize long DNA molecules. SMC complexes promote DNA organization by loop extrusion. In most bacteria, chromosome folding initiates at dedicated start sites marked by the ParB/parS partition complexes. Whether SMC complexes recognize a specific DNA structure in the partition complex or a protein component is unclear. By replacing genes in Bacillus subtilis with orthologous sequences from Streptococcus pneumoniae, we show that the three subunits of the bacterial Smc complex together with the ParB protein form a functional module that can organize and segregate foreign chromosomes. Using chimeric proteins and chemical cross-linking, we find that ParB directly binds the Smc subunit. We map an interface to the Smc joint and the ParB CTP-binding domain. Structure prediction indicates how the ParB clamp presents DNA to the Smc complex, presumably to initiate DNA loop extrusion.

Skin Microbiota and Clinical Associations in Netherton Syndrome.

Netherton syndrome (NS) is a rare, life-threatening syndrome caused by serine protease inhibitor Kazal-type 5 gene (SPINK 5) mutations, resulting in skin barrier defect, bacterial skin infections, and allergic sensitization in early childhood. Recent data on adult patients with NS suggest that the presence of Staphylococcus aureus further promotes barrier disruption and skin inflammation. We analyzed the skin microbiota by shotgun sequencing in 12 patients with NS from eight Finnish families with healthy family controls as the reference and correlated the findings with allergen-specific IgE prevalence, immune cell phenotype, and infection history of the patients. Compared with healthy family controls, skin microbiome diversity and normal skin site variability were measurably decreased in patients with NS. No correlation was found between allergic sensitization and skin microbiota as such, but low circulating CD57+ and/or CD8+ T cells significantly correlated with lower microbial diversity and less abundance of S. aureus (P < 0.05). S. aureus was the most prevalent species in patients with NS but also Streptococcus agalactiae was abundant in four patients. The genomic DNA relative abundance of S. aureus secreted virulence peptides and proteases PSMα, staphopain A, and staphopain B were increased in most of the samples of patients with NS, and their abundance was significantly (P < 0.05) associated with recurrent childhood skin infections, confirming the clinical relevance of S. aureus dominance in the NS skin microbiome.

Coronary Sinus Ostial Obstruction in Single-Ventricle Congenital Heart Disease: Two Patients With Different Outcomes.

Coronary sinus ostial obstruction is an exceedingly rare anomaly that is particularly important to diagnose in patients with single-ventricle heart disease before surgical palliation. We present 2 cases, an infant and an adult, diagnosed with coronary sinus ostial obstruction, with different clinical outcomes due to timing of diagnosis. (Level of Difficulty: Intermediate.).

Gradual opening of Smc arms in prokaryotic condensin.

Multi-subunit SMC ATPases control chromosome superstructure apparently by catalyzing a DNA-loop-extrusion reaction. SMC proteins harbor an ABC-type ATPase "head" and a "hinge" dimerization domain connected by a coiled coil "arm." Two arms in a SMC dimer can co-align, thereby forming a rod-shaped particle. Upon ATP binding, SMC heads engage, and arms are thought to separate. Here, we study the shape of Bacillus subtilis Smc-ScpAB by electron-spin resonance spectroscopy. Arm separation is readily detected proximal to the heads in the absence of ligands, and separation near the hinge largely depends on ATP and DNA. Artificial blockage of arm opening eliminates DNA stimulation of ATP hydrolysis but does not prevent basal ATPase activity. We report an arm contact as being important for controlling the transformations. Point mutations at this arm interface eliminated Smc function. We propose that partially open, intermediary conformations provide directionality to SMC DNA translocation by (un)binding suitable DNA substrates.

DIY enzyme labelled fluorescence alcohol (ELFA) standard production protocol to quantify single-cell phosphatase activity (SCPA) of microplankton.

Extracellular enzyme activities (EEA) are crucial components of microbial food web interactions and biogeochemical cycles in aquatic ecosystems. They also represent relevant biological traits in the ecophysiology of phytoplankton and other components of microbial plankton. To assess species-specific and (sub-)population-level characteristics of phytoplankton EEA at the single-cell level and close-to-in-situ conditions solely the enzyme labelled fluorescence (ELF)-based substrates have been used, because they become fluorescent and precipitate around the enzyme activity location upon enzymatic cleavage. However, the enzyme-labelled fluorescence alcohol (ELFA) standard is no longer commercially available, hence standard curves cannot be run anymore and single-cell phosphatase activity (SCPA) is no longer quantifiable. Therefore, we introduce a simple protocol for an ELFA standard do it yourself (DIY) production to enable quantifying microplankton SCPA again. This protocol is based on fluorescence measurements easily available to environmental enzyme activity laboratories, and it circumvents any need for chemical synthesis equipment and knowledge. The method is based on a controlled reaction of the ELF-phosphate (ELFP) substrate with commercially available alkaline phosphatase, which efficiently turns all the substrate into ELFA product. The ELFA product was dried out and dissolved again in dimethyl sulfoxide (DMSO) for storage. The ELFA concentration of that standard-to-be ELFA solution in DMSO was determined by linear regression between a low concentration dilution series of ELFA solution measured fluorimetrically and parallel measurements of a series of phosphatase-catalysed reactions at an overlapping ELFP concentration range. Finally, the fluorescence- and concentration-stable ELFA solution in DMSO with a known concentration constitutes the ELFA standard that is necessary to quantify bulk (fluorimeter) and single-cell (microscope and flow cytometer) phosphatase activity in microplankton.

Improvement of sabinene tolerance of Escherichia coli using adaptive laboratory evolution and omics technologies.

BACKGROUND: Biosynthesis of sabinene, a bicyclic monoterpene, has been accomplished in engineered microorganisms by introducing heterologous pathways and using renewable sugar as a carbon source. However, the efficiency and titers of this method are limited by the low host tolerance to sabinene (in both eukaryotes and prokaryotes). RESULTS: In this study, Escherichia coli BL21(DE3) was selected as the strain for adaptive laboratory evolution. The strain was evolved by serial passaging in the medium supplemented with gradually increasing concentration of sabinene, and the evolved strain XYF(DE3), which exhibited significant tolerance to sabinene, was obtained. Then, XYF(DE3) was used as the host for sabinene production and an 8.43-fold higher sabinene production was achieved compared with the parental BL21(DE3), reaching 191.76 mg/L. Whole genomes resequencing suggested the XYF(DE3) strain is a hypermutator. A comparative analysis of transcriptomes of XYF(DE3) and BL21(DE3) was carried out to reveal the mechanism underlying the improvement of sabinene tolerance, and 734 up-regulated genes and 857 down-regulated genes were identified. We further tested the roles of the identified genes in sabinene tolerance via reverse engineering. The results demonstrated that overexpressions of ybcK gene of the DLP12 family, the inner membrane protein gene ygiZ, and the methylmalonyl-CoA mutase gene scpA could increase sabinene tolerance of BL21(DE3) by 127.7%, 71.1%, and 75.4%, respectively. Furthermore, scanning electron microscopy was applied to monitor cell morphology. Under sabinene stress, the parental BL21(DE3) showed increased cell length, whereas XYF(DE3) showed normal cell morphology. In addition, overexpression of ybcK, ygiZ or scpA could partially rescue cell morphology under sabinene stress and overexpression of ygiZ or scpA could increase sabinene production in BL21(DE3). CONCLUSIONS: This study not only obtained a sabinene-tolerant strain for microbial production of sabinene but also revealed potential regulatory mechanisms that are important for sabinene tolerance. In addition, for the first time, ybcK, ygiZ, and scpA were identified to be important for terpene tolerance in E. coli BL21(DE3).

Evidence for binary Smc complexes lacking kite subunits in archaea.

SMC complexes play a central role in chromosome organization in all domains of life. The bacterial Smc-ScpAB complex is a three-subunit complex composed of Smc, ScpA and ScpB. ScpA bridges the two ATPase domains of the Smc homodimer, while ScpB, which belongs to the kite family of proteins, interacts with ScpA. The three subunits are known to be equally important for the function of Smc-ScpAB in bacteria. From crystallographic and biochemical studies, evidence is provided that six archaeal ScpA proteins are unable to interact with the only putative ScpB found in these species. Structure-based sequence alignment reveals that these archaeal ScpAs lack the ScpB-binding segment that is commonly present in the middle of bacterial ScpA sequences, which is thus responsible for their inability to interact with ScpB. ScpA proteins lacking the ScpB-binding segment are found to prevail in archaea. Moreover, two archaeal ScpA proteins with a longer middle region also failed to bind their putative ScpB partner. Furthermore, all or most species belonging to five out of 14 euryarchaeotal orders contain Smc and ScpA but not a detectable ScpB homologue. These data support the notion that archaeal Smc-based complexes generally function as a two-subunit complex composed of only Smc and ScpA.

Group A Streptococcus Cell Wall Oligosaccharide-Streptococcal C5a Peptidase Conjugates as Effective Antibacterial Vaccines.

Group A streptococcus (GAS) is one of the common Gram-positive pathogenic bacteria accounting for a variety of infectious diseases. Currently, there is no commercial vaccine for GAS. To develop efficient GAS vaccines, synthetic tri-, hexa-, and nonasaccharides of a conserved group A carbohydrate (GAC) were conjugated with an inactive mutant of group A streptococcal C5a peptidase (ScpA), ScpA193, to create bivalent conjugate vaccines, which were compared with the corresponding CRM197 and TT conjugates. Systematic evaluations of these semisynthetic conjugates demonstrated that they could induce robust and comparable T-cell-dependent immune responses in mice. It was further disclosed that antibodies provoked by the ScpA193 conjugates, especially that of hexa- and nonasaccharides, could recognize and bind to GAS cells and mediate GAS opsonophagocytosis in vitro. In vivo evaluations of the hexa- and nonasaccharide-ScpA193 conjugates using a mouse model revealed that immunizing mice with especially the latter conjugate could effectively protect the animals from GAS challenges and GAS-induced pulmonary damage and significantly increase animal survival. Further in vitro studies suggested that the two ScpA193 conjugates could function through activating CD4+ T cells and promoting helper T cells (Th) to differentiate into antigen-specific Th1 and Th2 cells. In conclusion, the nonasaccharide-ScpA193 conjugate was identified as a particularly promising GAS vaccine candidate that is worthy of further investigation and development.

Availability of tanning salons in Ontario relative to indoor tanning policy (2001-2017).

Ultraviolet (UV) radiation from indoor tanning equipment is a known cause of skin cancer; however, little is known about how the availability of indoor tanning salons has been impacted by indoor tanning legislation, including Ontario's Skin Cancer Prevention Act: Tanning Beds (SCPA). Tanning salon listings were obtained from the 2001 to 2017 editions of InfoCanada's Ontario Business to Business Sales and Marketing directories. Using descriptive statistics and regression analysis, we assessed the number of tanning salons before and after: 1) the 2006 International Agency for Research on Cancer (IARC) report on indoor tanning and skin cancer; 2) the 2009 World Health Organization (WHO) reclassification of artificial UV radiation as carcinogenic; and 3) the passing and enactment of Ontario's SCPA in 2013 and 2014, respectively. There were fewer tanning salon listings in the years after vs. before the IARC report, the WHO reclassification, and the passing and enactment of the SCPA. The number of tanning salons in Ontario, Canada has been declining since 2006, which may reflect a decline in indoor tanning bed use. Key public health policy instruments, including legislation and public education, appear to be associated with this trend, suggesting they may contribute to deterring indoor tanning.

Study on Quasi-Static Uniaxial Compression Properties and Constitutive Equation of Spherical Cell Porous Aluminum-Polyurethane Composites.

Quasi-static uniaxial compression properties and the constitutive equation of spherical cell porous aluminum-polyurethane composites (SCPA-PU composites) were investigated in this paper. The effects of relative density on the densification strain, plateau stress and energy absorption properties of the SCPA-PU composites were analyzed. It is found that the stress-strain curves of SCPA-PU composites consist of three stages: The linear elastic part, longer plastic plateau segment and densification region. The results also demonstrate that both the plateau stress and the densification strain energy of the SCPA-PU composites can be improved by increasing the relative density of the spherical cell porous aluminum (SCPA), while the densification strain of the SCPA-PU composites shows little dependence on the relative density of the SCPA. Furthermore, the applicability of three representative phenomenological models to the constitutive equations of SCPA-PU composites are verified and compared based on the experimental results. The error analysis result indicates that the Avalle model is the best model to characterize the uniaxial compression constitutive equation of SCPA-PU composites.

Structure of Full-Length SMC and Rearrangements Required for Chromosome Organization.

Multi-subunit SMC complexes control chromosome superstructure and promote chromosome disjunction, conceivably by actively translocating along DNA double helices. SMC subunits comprise an ABC ATPase "head" and a "hinge" dimerization domain connected by a 49 nm coiled-coil "arm." The heads undergo ATP-dependent engagement and disengagement to drive SMC action on the chromosome. Here, we elucidate the architecture of prokaryotic Smc dimers by high-throughput cysteine cross-linking and crystallography. Co-alignment of the Smc arms tightly closes the interarm space and misaligns the Smc head domains at the end of the rod by close apposition of their ABC signature motifs. Sandwiching of ATP molecules between Smc heads requires them to substantially tilt and translate relative to each other, thereby opening up the Smc arms. We show that this mechanochemical gating reaction regulates chromosome targeting and propose a mechanism for DNA translocation based on the merging of DNA loops upon closure of Smc arms.

Overall Shapes of the SMC-ScpAB Complex Are Determined by Balance between Constraint and Relaxation of Its Structural Parts.

The SMC-ScpAB complex plays a crucial role in chromosome organization and segregation in many bacteria. It is composed of a V-shaped SMC dimer and an ScpAB subcomplex that bridges the two Structural Maintenance of Chromosomes (SMC) head domains. Despite its functional significance, the mechanistic details of SMC-ScpAB remain obscure. Here we provide evidence that ATP-dependent head-head engagement induces a lever movement of the SMC neck region, which might help to separate juxtaposed coiled-coil arms. Binding of the ScpA N-terminal domain (NTD) to the SMC neck region is negatively regulated by the ScpB C-terminal domain. Mutations in the ScpA NTD compromise this regulation and profoundly affect the overall shape of the complex. The SMC hinge domain is structurally relaxed when free from coiled-coil juxtaposition. Taken together, we propose that the structural parts of SMC-ScpAB are subjected to the balance between constraint and relaxation, cooperating to modulate dynamic conformational changes of the whole complex.