Synthesis and X-ray crystal structure of a bridging trispiran ligand

The document attached has been archived with permission from the publisher.Spiro[3,3]heptane-2,6-dispiro-4,5-diazafluorene (4) has been synthesized in three steps from 1,10-phenanthroline. It crystallizes in the orthorhombic space group P212121 and has the two 4,5-diazafluorene metal binding domains mutually orthogonal.Peter J. Steel and Christopher J. Sumb

Protein surface functionalisation as a general strategy for facilitating biomimetic mineralisation of ZIF-8

The durability of enzymes in harsh conditions can be enhanced by encapsulation within metal-organic frameworks (MOFs) via a process called biomimetic mineralisation. Herein we show that the surface charge and chemistry of a protein determines its ability to seed MOF growth. We demonstrate that chemical modification of amino acids on the protein surface is an effective method for systematically controlling biomimetic mineralisation by zeolitic imidazolate framework-8 (ZIF-8). Reaction of surface lysine residues with succinic (or acetic) anhydride facilitates biomimetic mineralisation by increasing the surface negative charge, whereas reaction of surface carboxylate moieties with ethylenediamine affords a more positively charged protein and hinders the process. Moreover, computational studies confirm that the surface electrostatic potential of a protein is a good indicator of its ability to induce biomimetic mineralisation. This study highlights the important role played by protein surface chemistry in encapsulation and outlines a general method for facilitating the biomimetic mineralisation of proteins

Pre-organisation or a hydrogen bonding mismatch; silver(I) diamide ligand coordination polymers versus discrete metallo-macrocyclic assemblies

The investigation of novel motifs to selectively complex anions is an area of considerable importance due to the significant environmental, biological and medicinal roles of anions. The synthesis of discrete metallo-macrocyclic compounds or coordination polymers displaying anion-binding pockets can generate specific anion receptors from relatively simple components. Here, we examine the self-assembly of a series of flexible diamide compounds L1–L5 with silver(I) metal salts. A new diamide ligand, 2,6-[N,N0-bis(di-(pyridin-2-yl)methyl)pyridine]-2,6-dicarboxamide (L5), with two chelating di-2- pyridylmethyl donor groups, was also prepared. Compounds L1–L3, lacking the pre-organising effect of a central 2,6-pyridine dicarboxamide core, form 1D coordination polymers {[Ag(L1)(CH3CN)](PF6)}n (6), {[Ag(L2)](NO3)•(H2O)]}n (7) and {[AgNO3(L3)]•(CH3OH)]}n (9) which in turn form 2D and 3D hydrogen-bonded networks through orthogonal hydrogen bonding. In one instance, L2 gives rise to a dinuclear metallo-macrocycle in the solid state, [Ag2(CF3CO2)2(L2)2][Ag2(m2- CF3CO2)2(L2)2] (8). Both diamide ligands L4 and L5 form dinuclear metallo-macrocycles, [Ag2(NO2)2(L4)2] (10) and [Ag2(L5)2](NO3)2•2CH3OH•2H2O (11), in solution and in the solid state. Where possible, all compounds were investigated in solution and their solid-state structures were determined using X-ray crystallography. This enabled the effect of competing supramolecular synthons, covalent MZL bonding and hydrogen bonding, to be examined by comparing the solution and solid-state behaviour of each metal–ligand combination.Maisara Abdul-Kadir, Philip R. Clements, Lyall R. Hanton, Courtney A. Hollis and Christopher J. Sumb

Incompetence of Neutrophils to Invasive Group A streptococcus Is Attributed to Induction of Plural Virulence Factors by Dysfunction of a Regulator

Group A streptococcus (GAS) causes variety of diseases ranging from common pharyngitis to life-threatening severe invasive diseases, including necrotizing fasciitis and streptococcal toxic shock-like syndrome. The characteristic of invasive GAS infections has been thought to attribute to genetic changes in bacteria, however, no clear evidence has shown due to lack of an intriguingly study using serotype-matched isolates from clinical severe invasive GAS infections. In addition, rare outbreaks of invasive infections and their distinctive pathology in which infectious foci without neutrophil infiltration hypothesized us invasive GAS could evade host defense, especially neutrophil functions. Herein we report that a panel of serotype-matched GAS, which were clinically isolated from severe invasive but not from non-invaive infections, could abrogate functions of human polymorphnuclear neutrophils (PMN) in at least two independent ways; due to inducing necrosis to PMN by enhanced production of a pore-forming toxin streptolysin O (SLO) and due to impairment of PMN migration via digesting interleukin-8, a PMN attracting chemokine, by increased production of a serine protease ScpC. Expression of genes was upregulated by a loss of repressive function with the mutation of csrS gene in the all emm49 severe invasive GAS isolates. The csrS mutants from clinical severe invasive GAS isolates exhibited high mortality and disseminated infection with paucity of neutrophils, a characteristic pathology seen in human invasive GAS infection, in a mouse model. However, GAS which lack either SLO or ScpC exhibit much less mortality than the csrS-mutated parent invasive GAS isolate to the infected mice. These results suggest that the abilities of GAS to abrogate PMN functions can determine the onset and severity of invasive GAS infection

A Combination of Independent Transcriptional Regulators Shapes Bacterial Virulence Gene Expression during Infection

Transcriptional regulatory networks are fundamental to how microbes alter gene expression in response to environmental stimuli, thereby playing a critical role in bacterial pathogenesis. However, understanding how bacterial transcriptional regulatory networks function during host-pathogen interaction is limited. Recent studies in group A Streptococcus (GAS) suggested that the transcriptional regulator catabolite control protein A (CcpA) influences many of the same genes as the control of virulence (CovRS) two-component gene regulatory system. To provide new information about the CcpA and CovRS networks, we compared the CcpA and CovR transcriptomes in a serotype M1 GAS strain. The transcript levels of several of the same genes encoding virulence factors and proteins involved in basic metabolic processes were affected in both ΔccpA and ΔcovR isogenic mutant strains. Recombinant CcpA and CovR bound with high-affinity to the promoter regions of several co-regulated genes, including those encoding proteins involved in carbohydrate and amino acid metabolism. Compared to the wild-type parental strain, ΔccpA and ΔcovRΔccpA isogenic mutant strains were significantly less virulent in a mouse myositis model. Inactivation of CcpA and CovR alone and in combination led to significant alterations in the transcript levels of several key GAS virulence factor encoding genes during infection. Importantly, the transcript level alterations in the ΔccpA and ΔcovRΔccpA isogenic mutant strains observed during infection were distinct from those occurring during growth in laboratory medium. These data provide new knowledge regarding the molecular mechanisms by which pathogenic bacteria respond to environmental signals to regulate virulence factor production and basic metabolic processes during infection

Allelic replacement of the streptococcal cysteine protease SpeB in a Δsrv mutant background restores biofilm formation

<p>Abstract</p> <p>Background</p> <p>Group A <it>Streptococcus </it>(GAS) is a Gram-positive human pathogen that is capable of causing a wide spectrum of human disease. Thus, the organism has evolved to colonize a number of physiologically distinct host sites. One such mechanism to aid colonization is the formation of a biofilm. We have recently shown that inactivation of the streptococcal regulator of virulence (Srv), results in a mutant strain exhibiting a significant reduction in biofilm formation. Unlike the parental strain (MGAS5005), the streptococcal cysteine protease (SpeB) is constitutively produced by the <it>srv </it>mutant (MGAS5005Δ<it>srv</it>) suggesting Srv contributes to the control of SpeB production. Given that SpeB is a potent protease, we hypothesized that the biofilm deficient phenotype of the <it>srv </it>mutant was due to the constitutive production of SpeB. In support of this hypothesis, we have previously demonstrated that treating cultures with E64, a commercially available chemical inhibitor of cysteine proteases, restored the ability of MGAS5005Δ<it>srv </it>to form biofilms. Still, it was unclear if the loss of biofilm formation by MGAS5005Δ<it>srv </it>was due only to the constitutive production of SpeB or to other changes inherent in the <it>srv </it>mutant strain. To address this question, we constructed a Δ<it>srv</it>Δ<it>speB </it>double mutant through allelic replacement (MGAS5005Δ<it>srv</it>Δ<it>speB</it>) and tested its ability to form biofilms <it>in vitro</it>.</p> <p>Findings</p> <p>Allelic replacement of <it>speB </it>in the <it>srv </it>mutant background restored the ability of this strain to form biofilms under static and continuous flow conditions. Furthermore, addition of purified SpeB to actively growing wild-type cultures significantly inhibited biofilm formation.</p> <p>Conclusions</p> <p>The constitutive production of SpeB by the <it>srv </it>mutant strain is responsible for the significant reduction of biofilm formation previously observed. The double mutant supports a model by which Srv contributes to biofilm formation and/or dispersal through regulation of <it>speB</it>/SpeB.</p

Proteomic analysis at the sites of clinical infection with invasive Streptococcus pyogenes

Invasive Streptococcus pyogenes infections are rare, with often-unexplained severity. Prompt diagnosis is desirable, as deaths can occur rapidly following onset and there is an increased, but preventable, risk to contacts. Here, proteomic analyses of clinical samples from invasive human S. pyogenes infections were undertaken to determine if novel diagnostic targets could be detected, and to augment our understanding of disease pathogenesis. Fluid samples from 17 patients with confirmed invasive S. pyogenes infection (empyema, septic arthritis, necrotising fasciitis) were analysed by proteomics for streptococcal and human proteins; 16/17 samples had detectable S. pyogenes DNA. Nineteen unique S. pyogenes proteins were identified in just 6/17 samples, and 15 of these were found in a single pleural fluid sample including streptococcal inhibitor of complement, trigger factor, and phosphoglycerate kinase. In contrast, 469 human proteins were detected in patient fluids, 177 (38%) of which could be identified as neutrophil proteins, including alpha enolase and lactotransferrin which, together, were found in all 17 samples. Our data suggest that streptococcal proteins are difficult to detect in infected fluid samples. A vast array of human proteins associated with leukocyte activity are, however, present in samples that deserve further evaluation as potential biomarkers of infection

Control of structure topology and spatial distribution of biomacromolecules in Protein@ZIF-8 biocomposites

The protective capacity and applications of biomimetically mineralized biomacromolecule zeolitic imidazolate framework (ZIF) composites are likely dependent on the localization of the biomolecule and the topology of the mineralized ZIF coating. Herein, we identify reaction conditions to reliably yield the porous ZIF-8 sodalite topology (high ZIF-8 precursor concentrations; high 2-methylimidazole:Zn²⁺ ratios) in preference to other more dense phases. Furthermore, protocols to universally prepare biocomposites with a range of biomacromolecules are canvassed. Through the use of fluorophore-tagged proteins and confocal laser scanning microscopy (CLSM), we further establish the positioning of biomolecules within ZIF-8 crystals. CLSM reveals subsurface localization with fluorescein-tagged bovine serum albumin (BSA) or full encapsulation with rhodamine Btagged BSA. These observations allowed us to demonstrate that core−shell ZIF-8 growth strategies afford complete encapsulation with varying thicknesses of potentially active biocomposite or protective ZIF-8. The demonstrated control over ZIF topology (enabling mass transport) and biomacromolecule localization is critical for applications of MOF biocomposites in catalysis.Weibin Liang, Raffaele Ricco, Natasha K. Maddigan, Robert P. Dickinson, Huoshu Xu, Qiaowei Li, Christopher J. Sumby, Stephen G. Bell, Paolo Falcaro and Christian J. Doona

A Naturally Occurring Mutation in ropB Suppresses SpeB Expression and Reduces M1T1 Group A Streptococcal Systemic Virulence

Epidemiological studies of group A streptococcus (GAS) have noted an inverse relationship between SpeB expression and invasive disease. However, the role of SpeB in the course of infection is still unclear. In this study we utilize a SpeB-negative M1T1 clinical isolate, 5628, with a naturally occurring mutation in the gene encoding the regulator RopB, to elucidate the role of RopB and SpeB in systemic virulence. Allelic exchange mutagenesis was used to replace the mutated ropB allele in 5628 with the intact allele from the well characterized isolate 5448. The inverse allelic exchange was also performed to replace the intact ropB in 5448 with the mutated allele from 5628. An intact ropB was found to be essential for SpeB expression. While the ropB mutation was shown to have no effect on hemolysis of RBC's, extracellular DNase activity or survival in the presence of neutrophils, strains with the mutated ropB allele were less virulent in murine systemic models of infection. An isogenic SpeB knockout strain containing an intact RopB showed similarly reduced virulence. Microarray analysis found genes of the SpeB operon to be the primary target of RopB regulation. These data show that an intact RopB and efficient SpeB production are necessary for systemic infection with GAS