Dermonecrosis caused by a spitting cobra snakebite results from toxin potentiation and is prevented by the repurposed drug varespladib.

Snakebite envenoming is a neglected tropical disease that causes substantial mortality and morbidity globally. The venom of African spitting cobras often causes permanent injury via tissue-destructive dermonecrosis at the bite site, which is ineffectively treated by current antivenoms. To address this therapeutic gap, we identified the etiological venom toxins in Naja nigricollis venom responsible for causing local dermonecrosis. While cytotoxic three-finger toxins were primarily responsible for causing spitting cobra cytotoxicity in cultured keratinocytes, their potentiation by phospholipases A2 toxins was essential to cause dermonecrosis in vivo. This evidence of probable toxin synergism suggests that a single toxin-family inhibiting drug could prevent local envenoming. We show that local injection with the repurposed phospholipase A2-inhibiting drug varespladib significantly prevents local tissue damage caused by several spitting cobra venoms in murine models of envenoming. Our findings therefore provide a therapeutic strategy that may effectively prevent life-changing morbidity caused by snakebite in rural Africa.

African polyvalent antivenom can maintain pharmacological stability and ability to neutralise murine venom lethality for decades post-expiry: evidence for increasing antivenom shelf life to aid in alleviating chronic shortages.

INTRODUCTION: Antivenom is a lifesaving medicine for treating snakebite envenoming, yet there has been a crisis in antivenom supply for many decades. Despite this, substantial quantities of antivenom stocks expire before use. This study has investigated whether expired antivenoms retain preclinical quality and efficacy, with the rationale that they could be used in emergency situations when in-date antivenom is unavailable. METHODS: Using WHO guidelines and industry test requirements, we examined the in vitro stability and murine in vivo efficacy of eight batches of the sub-Saharan African antivenom, South African Institute for Medical Research polyvalent, that had expired at various times over a period of 30 years. RESULTS: We demonstrate modest declines in immunochemical stability, with antivenoms older than 25 years having high levels of turbidity. In vitro preclinical analysis demonstrated all expired antivenoms retained immunological recognition of venom antigens and the ability to inhibit key toxin families. All expired antivenoms retained comparable in vivo preclinical efficacy in preventing the lethal effects of envenoming in mice versus three regionally and medically important venoms. CONCLUSIONS: This study provides strong rationale for stakeholders, including manufacturers, regulators and health authorities, to explore the use of expired antivenom more broadly, to aid in alleviating critical shortages in antivenom supply in the short term and the extension of antivenom shelf life in the longer term.

Virus-like particles displaying conserved toxin epitopes stimulate polyspecific, murine antibody responses capable of snake venom recognition.

Antivenom is currently the first-choice treatment for snakebite envenoming. However, only a low proportion of antivenom immunoglobulins are specific to venom toxins, resulting in poor dose efficacy and potency. We sought to investigate whether linear venom epitopes displayed on virus like particles can stimulate an antibody response capable of recognising venom toxins from diverse medically important species. Bioinformatically-designed epitopes, corresponding to predicted conserved regions of group I phospholipase A2 and three finger toxins, were engineered for display on the surface of hepatitis B core antigen virus like particles and used to immunise female CD1 mice over a 14 weeks. Antibody responses to all venom epitope virus like particles were detectable by ELISA by the end of the immunisation period, although total antibody and epitope specific antibody titres were variable against the different epitope immunogens. Immunoblots using pooled sera demonstrated recognition of various venom components in a diverse panel of six elapid venoms, representing three continents and four genera. Insufficient antibody yields precluded a thorough assessment of the neutralising ability of the generated antibodies, however we were able to test polyclonal anti-PLA2 IgG from three animals against the PLA2 activity of Naja nigricollis venom, all of which showed no neutralising ability. This study demonstrates proof-of-principle that virus like particles engineered to display conserved toxin linear epitopes can elicit specific antibody responses in mice which are able to recognise a geographically broad range of elapid venoms.

Exploring the Utility of Recombinant Snake Venom Serine Protease Toxins as Immunogens for Generating Experimental Snakebite Antivenoms.

Snakebite is a neglected tropical disease that causes high rates of global mortality and morbidity. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Despite polyclonal antibody-based antivenoms being the mainstay life-saving therapy for snakebite, they are associated with limited cross-snake species efficacy, as there is often extensive toxin variation between snake venoms, including those used as immunogens for antivenom production. This restricts the therapeutic utility of any antivenom to certain geographical regions. In this study, we explored the feasibility of using recombinantly expressed toxins as immunogens to stimulate focused, pathology-specific, antibodies in order to broadly counteract specific toxins associated with snakebite envenoming. Three snake venom serine proteases (SVSP) toxins, sourced from geographically diverse and medically important viper snake venoms, were successfully expressed in HEK293F mammalian cells and used for murine immunisation. Analyses of the resulting antibody responses revealed that ancrod and RVV-V stimulated the strongest immune responses, and that experimental antivenoms directed against these recombinant SVSP toxins, and a mixture of the three different immunogens, extensively recognised and exhibited immunological binding towards a variety of native snake venoms. While the experimental antivenoms showed some reduction in abnormal clotting parameters stimulated by the toxin immunogens and crude venom, specifically reducing the depletion of fibrinogen levels and prolongation of prothrombin times, fibrinogen degradation experiments revealed that they broadly protected against venom- and toxin-induced fibrinogenolytic functional activities. Overall, our findings further strengthen the case for the use of recombinant venom toxins as supplemental immunogens to stimulate focused and desirable antibody responses capable of neutralising venom-induced pathological effects, and therefore potentially circumventing some of the limitations associated with current snakebite therapies.

Physiological constraints dictate toxin spatial heterogeneity in snake venom glands.

BACKGROUND: Venoms are ecological innovations that have evolved numerous times, on each occasion accompanied by the co-evolution of specialised morphological and behavioural characters for venom production and delivery. The close evolutionary interdependence between these characters is exemplified by animals that control the composition of their secreted venom. This ability depends in part on the production of different toxins in different locations of the venom gland, which was recently documented in venomous snakes. Here, we test the hypothesis that the distinct spatial distributions of toxins in snake venom glands are an adaptation that enables the secretion of venoms with distinct ecological functions. RESULTS: We show that the main defensive and predatory peptide toxins are produced in distinct regions of the venom glands of the black-necked spitting cobra (Naja nigricollis), but these distributions likely reflect developmental effects. Indeed, we detected no significant differences in venom collected via defensive 'spitting' or predatory 'biting' events from the same specimens representing multiple lineages of spitting cobra. We also found the same spatial distribution of toxins in a non-spitting cobra and show that heterogeneous toxin distribution is a feature shared with a viper with primarily predatory venom. CONCLUSIONS: Our findings suggest that heterogeneous distributions of toxins are not an adaptation to controlling venom composition in snakes. Instead, it likely reflects physiological constraints on toxin production by the venom glands, opening avenues for future research on the mechanisms of functional differentiation of populations of protein-secreting cells within adaptive contexts.

Investigating Viral Inoculation and Recovery from Medical Masks.

The SARS-CoV-2 pandemic from 2019 onwards has significantly increased the usage of surgical style medical masks, both in healthcare and public settings. It is important to study the contamination of and viral transfer from such masks. However, accepted standard test methods such as ISO 18184 have prescribed inoculation methods which may not be fully representative of the type of viral insult experienced in the clinic or community. In addition to studying a conventional mask, the performance of a mask featuring an antimicrobial photosensitiser was also studied.

Anticoagulant Activity of Naja nigricollis Venom Is Mediated by Phospholipase A2 Toxins and Inhibited by Varespladib.

Bites from elapid snakes typically result in neurotoxic symptoms in snakebite victims. Neurotoxins are, therefore, often the focus of research relating to understanding the pathogenesis of elapid bites. However, recent evidence suggests that some elapid snake venoms contain anticoagulant toxins which may help neurotoxic components spread more rapidly. This study examines the effects of venom from the West African black-necked spitting cobra (Naja nigricollis) on blood coagulation and identifies potential coagulopathic toxins. An integrated RPLC-MS methodology, coupled with nanofractionation, was first used to separate venom components, followed by MS, proteomics and coagulopathic bioassays. Coagulation assays were performed on both crude and nanofractionated N. nigricollis venom toxins as well as PLA2s and 3FTx purified from the venom. Assays were then repeated with the addition of either the phospholipase A2 inhibitor varespladib or the snake venom metalloproteinase inhibitor marimastat to assess whether either toxin inhibitor is capable of neutralizing coagulopathic venom activity. Subsequent proteomic analysis was performed on nanofractionated bioactive venom toxins using tryptic digestion followed by nanoLC-MS/MS measurements, which were then identified using Swiss-Prot and species-specific database searches. Varespladib, but not marimastat, was found to significantly reduce the anticoagulant activity of N. nigricollis venom and MS and proteomics analyses confirmed that the anticoagulant venom components mostly consisted of PLA2 proteins. We, therefore, conclude that PLA2s are the most likely candidates responsible for anticoagulant effects stimulated by N. nigricollis venom.

Preclinical validation of a repurposed metal chelator as an early-intervention therapeutic for hemotoxic snakebite.

Snakebite envenoming causes 138,000 deaths annually, and ~400,000 victims are left with permanent disabilities. Envenoming by saw-scaled vipers (Viperidae: Echis) leads to systemic hemorrhage and coagulopathy and represents a major cause of snakebite mortality and morbidity in Africa and Asia. The only specific treatment for snakebite, antivenom, has poor specificity and low affordability and must be administered in clinical settings because of its intravenous delivery and high rates of adverse reactions. This requirement results in major treatment delays in resource-poor regions and substantially affects patient outcomes after envenoming. Here, we investigated the value of metal ion chelators as prehospital therapeutics for snakebite. Among the tested chelators, dimercaprol (British anti-Lewisite) and its derivative 2,3-dimercapto-1-propanesulfonic acid (DMPS) were found to potently antagonize the activity of Zn2+-dependent snake venom metalloproteinases in vitro. Moreover, DMPS prolonged or conferred complete survival in murine preclinical models of envenoming against a variety of saw-scaled viper venoms. DMPS also considerably extended survival in a "challenge and treat" model, where drug administration was delayed after venom injection and the oral administration of this chelator provided partial protection against envenoming. Last, the potential clinical scenario of early oral DMPS therapy combined with a delayed, intravenous dose of conventional antivenom provided prolonged protection against the lethal effects of envenoming in vivo. Our findings demonstrate that the safe and affordable repurposed metal chelator DMPS can effectively neutralize saw-scaled viper venoms in vitro and in vivo and highlight the promise of this drug as an early, prehospital, therapeutic intervention for hemotoxic snakebite envenoming.

Isolation and purification of recombinant immunoglobulin light chain variable domains from the periplasmic space of Escherichia coli.

Immunoglobulin light chain amyloidosis is the most common form of systemic amyloidosis. However, very little is known about the underlying mechanisms that initiate and modulate the associated protein aggregation and deposition. Model systems have been established to investigate these disease-associated processes. One of these systems comprises two 114 amino acid light-chain variable domains of the kappa 4 IgG family, SMA and LEN. Despite high sequence identity (93%), SMA is amyloidogenic in vivo, but LEN adopts a stable dimer, displaying amyloidogenic properties only under destabilising conditions in vitro. We present here a refined and reproducible periplasmic expression and purification protocol for SMA and LEN that improves on existing methods and provides high yields of pure protein (10-50mg/L), particularly suitable for structural studies that demand highly concentrated and purified proteins. We confirm that recombinant SMA and LEN proteins have structure and dimerization capabilities consistent with the native proteins and employ fluorescence to probe internalization and cellular localization within cardiomyocytes. We propose periplasmic expression and simplified chromatographic steps outlined here as an optimized method for production of these and other variable light chain domains to investigate the underlying mechanisms of light chain amyloidosis. We show that SMA and LEN can be internalised within cardiomyocytes and were observed to localise to the perinuclear area, assessed by confocal microscopy as a possible mechanism for underlying cytotoxicity and pathogenesis associated with amyloidosis.

Analysis of snake venom metalloproteinases from Myanmar Russell's viper transcriptome.

Snake venom metalloproteinases (SVMPs) are the key enzymes in Russell's viper (RV) venom which target all important components of haemostasis, such as clotting factors, platelets, endothelial cells and basement membrane. The structural diversity of SVMPs contributes to the broad spectrum of biological activities. The aim of the study was to investigate the SVMP transcript profile to gain better insights into the characteristic clinical manifestations of the Myanmar Russell's viper (MRV) bites that distinguish it from the RVs of other habitats. Next generation sequencing (RNA-Seq) of mRNA from MRV venom glands (2 males and 1 female) was performed on an Illumina HiSeq2000 platform and then de novo assembled using Trinity software. A total of 59 SVMP contigs were annotated through a Blastn search against the serpent nucleotide database from NCBI. Among them, disintegrins were the most abundant transcripts (75%) followed by the P-III class SVMPs (25%). The P-II SVMPs were scarce (0.002%), while no P-I SVMPs were detectable in the transcriptome. For detailed structural analysis, contigs were conceptually translated and compared with amino acid sequences from other RVs and other vipers using Clustal Omega. The RTS-disintegrin (jerdostatin homolog) was the most abundant among transcripts corresponding to 5 disintegrin isoforms. From 10 isoforms of SVMPs, RVV-X, and Vipera lebetina apoptosis-inducing protease (VLAIP) homolog, hereby termed Daboia siamensis AIP (DSAIP), were found to be highly expressed. Venom protein analysis using SDS-PAGE followed by mass spectrometry revealed that the disintegrin was scarce, while the latter two SVMPs were abundant. These two proteins can contribute to severe clinical manifestations caused by MRV envenomation.

Heparin binding preference and structures in the fibroblast growth factor family parallel their evolutionary diversification.

The interaction of a large number of extracellular proteins with heparan sulfate (HS) regulates their transport and effector functions, but the degree of molecular specificity underlying protein-polysaccharide binding is still debated. The 15 paracrine fibroblast growth factors (FGFs) are one of the paradigms for this interaction. Here, we measure the binding preferences of six FGFs (FGF3, FGF4, FGF6, FGF10, FGF17, FGF20) for a library of modified heparins, representing structures in HS, and model glycosaminoglycans, using differential scanning fluorimetry. This is complemented by the identification of the lysine residues in the primary and secondary binding sites of the FGFs by a selective labelling approach. Pooling these data with previous sets provides good coverage of the FGF phylogenetic tree, deduced from amino acid sequence alignment. This demonstrates that the selectivity of the FGFs for binding structures in sulfated polysaccharides and the pattern of secondary binding sites on the surface of FGFs follow the phylogenetic relationship of the FGFs, and so are likely to be the result of the natural selection pressures that led to the expansion of the FGF family in the course of the evolution of more complex animal body plans.

Snake Venom Metalloproteinases and Their Peptide Inhibitors from Myanmar Russell's Viper Venom.

Russell's viper bites are potentially fatal from severe bleeding, renal failure and capillary leakage. Snake venom metalloproteinases (SVMPs) are attributed to these effects. In addition to specific antivenom therapy, endogenous inhibitors from snakes are of interest in studies of new treatment modalities for neutralization of the effect of toxins. Two major snake venom metalloproteinases (SVMPs): RVV-X and Daborhagin were purified from Myanmar Russell's viper venom using a new purification strategy. Using the Next Generation Sequencing (NGS) approach to explore the Myanmar RV venom gland transcriptome, mRNAs of novel tripeptide SVMP inhibitors (SVMPIs) were discovered. Two novel endogenous tripeptides, pERW and pEKW were identified and isolated from the crude venom. Both purified SVMPs showed caseinolytic activity. Additionally, RVV-X displayed specific proteolytic activity towards gelatin and Daborhagin showed potent fibrinogenolytic activity. These activities were inhibited by metal chelators. Notably, the synthetic peptide inhibitors, pERW and pEKW, completely inhibit the gelatinolytic and fibrinogenolytic activities of respective SVMPs at 5 mM concentration. These complete inhibitory effects suggest that these tripeptides deserve further study for development of a therapeutic candidate for Russell's viper envenomation.

Bacterial sensing underlies artificial sweetener-induced growth of gut Lactobacillus.

Disruption in stable establishment of commensal gut microbiota by early weaning is an important factor in susceptibility of young animals to enteric disorders. The artificial sweetener SUCRAM [consisting of neohesperidin dihydrochalcone (NHDC) and saccharin] included in piglets' feed reduces incidence of enteric disease. Pyrosequencing of pig caecal 16S rRNA gene amplicons identified 25 major families encompassing seven bacterial classes with Bacteroidia, Clostridia and Bacilli dominating the microbiota. There were significant shifts in microbial composition in pigs maintained on a diet containing SUCRAM, establishing SUCRAM as a major influence driving bacterial community dynamics. The most notable change was a significant increase of Lactobacillaceae population abundance, almost entirely due to a single phylotype, designated Lactobacillus 4228. The sweetener-induced increase in Lactobacillaceae was observed in two different breeds of pigs signifying a general effect. We isolated Lactobacillus 4228, sequenced its genome and found it to be related to Lactobacillus amylovorus. In vitro analyses of Lactobacillus 4228 growth characteristics showed that presence of NHDC significantly reduces the lag phase of growth and enhances expression of specific sugar transporters, independently of NHDC metabolism. This study suggests that sensing of NHDC by a bacterial plasma membrane receptor underlies sweetener-induced growth of a health promoting gut bacterium.

Oxidative Stress Alters the Morphology and Toxicity of Aortic Medial Amyloid.

The aggregation and fibril deposition of amyloid proteins have been implicated in a range of neurodegenerative and vascular diseases, and yet the underlying molecular mechanisms are poorly understood. Here, we use a combination of cell-based assays, biophysical analysis, and atomic force microscopy to investigate the potential involvement of oxidative stress in aortic medial amyloid (AMA) pathogenesis and deposition. We show that medin, the main constituent of AMA, can induce an environment rich in oxidative species, increasing superoxide and reducing bioavailable nitric oxide in human cells. We investigate the role that this oxidative environment may play in altering the aggregation process of medin and identify potential posttranslational modification sites where site-specific modification and interaction can be unambiguously demonstrated. In an oxidizing environment, medin is nitrated at tyrosine and tryptophan residues, with resultant effects on morphology that lead to longer fibrils with increased toxicity. This provides further motivation to investigate the role of oxidative stress in AMA pathogenicity.

Pharmacokinetics of Meropenem for Use in Bacterial Keratitis.

PURPOSE: To investigate the toxicity and corneal pharmacokinetics of meropenem as a potential antimicrobial for bacterial keratitis. METHODS: Corneal epithelial cell and keratocyte toxicity was investigated using methyl thiazolyl tetrazolium (MTT) and LIVE/DEAD assays. The penetration of meropenem through the human cornea was measured using an artificial anterior chamber. In one group of corneas, the epithelial and endothelial layers were removed and in a second group these layers were left intact. We applied 50 µL (10 mg/mL) meropenem to the corneal surface and collected samples in the anterior chamber from 45 minutes up to 24 hours. Meropenem concentrations were estimated with a bioassay and HPLC. RESULTS: Meropenem had significantly higher cellular metabolic activity (MTT assay) at both 5 mg/mL and 2.5 mg/mL compared with moxifloxacin (P = 0.029 and P = 0.018, respectively), with 96% cell viability (LIVE/DEAD assay). The measured values for meropenem concentrations in corneal and aqueous samples were significantly higher using a bioassay than with HPLC (P = 0.004). For both intact and denuded corneas, the concentrations in the anterior chamber increased from 0.48 µg/mL (SD 0.89) and 0.89 µg/mL (SD 0.81) to 6.35 µg/mL (SD 0.81) and 13.48 µg/mL (SD 14.82) using HPLC, and from 0.68 µg/mL (SD 1.50) and 1.31 µg/mL (SD 1.55) to 47.03 µg/mL (SD 5.51) and 43.69 µg/mL (SD 27.22) measured with a bioassay. CONCLUSIONS: Meropenem has very low toxicity in vitro. It has good corneal penetration, achieving anterior chamber concentrations above MIC90 for bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, streptococci, coagulase-negative staphylococci, and the Enterobacteriaceae.

Diadenosine 5', 5'''-P(1),P(4)-tetraphosphate (Ap4A) is synthesized in response to DNA damage and inhibits the initiation of DNA replication.

The level of intracellular diadenosine 5', 5'''-P(1),P(4)-tetraphosphate (Ap4A) increases several fold in mammalian cells treated with non-cytotoxic doses of interstrand DNA-crosslinking agents such as mitomycin C. It is also increased in cells lacking DNA repair proteins including XRCC1, PARP1, APTX and FANCG, while >50-fold increases (up to around 25 µM) are achieved in repair mutants exposed to mitomycin C. Part of this induced Ap4A is converted into novel derivatives, identified as mono- and di-ADP-ribosylated Ap4A. Gene knockout experiments suggest that DNA ligase III is primarily responsible for the synthesis of damage-induced Ap4A and that PARP1 and PARP2 can both catalyze its ADP-ribosylation. Degradative proteins such as aprataxin may also contribute to the increase. Using a cell-free replication system, Ap4A was found to cause a marked inhibition of the initiation of DNA replicons, while elongation was unaffected. Maximum inhibition of 70-80% was achieved with 20 µM Ap4A. Ap3A, Ap5A, Gp4G and ADP-ribosylated Ap4A were without effect. It is proposed that Ap4A acts as an important inducible ligand in the DNA damage response to prevent the replication of damaged DNA.

HaloTag is an effective expression and solubilisation fusion partner for a range of fibroblast growth factors.

The production of recombinant proteins such as the fibroblast growth factors (FGFs) is the key to establishing their function in cell communication. The production of recombinant FGFs in E. coli is limited, however, due to expression and solubility problems. HaloTag has been used as a fusion protein to introduce a genetically-encoded means for chemical conjugation of probes. We have expressed 11 FGF proteins with an N-terminal HaloTag, followed by a tobacco etch virus (TEV) protease cleavage site to allow release of the FGF protein. These were purified by heparin-affinity chromatography, and in some instances by further ion-exchange chromatography. It was found that HaloTag did not adversely affect the expression of FGF1 and FGF10, both of which expressed well as soluble proteins. The N-terminal HaloTag fusion was found to enhance the expression and yield of FGF2, FGF3 and FGF7. Moreover, whereas FGF6, FGF8, FGF16, FGF17, FGF20 and FGF22 were only expressed as insoluble proteins, their N-terminal HaloTag fusion counterparts (Halo-FGFs) were soluble, and could be successfully purified. However, cleavage of Halo-FGF6, -FGF8 and -FGF22 with TEV resulted in aggregation of the FGF protein. Measurement of phosphorylation of p42/44 mitogen-activated protein kinase and of cell growth demonstrated that the HaloTag fusion proteins were biologically active. Thus, HaloTag provides a means to enhance the expression of soluble recombinant proteins, in addition to providing a chemical genetics route for covalent tagging of proteins.

Glycosylated yellow laccases of the basidiomycete Stropharia aeruginosa.

Here we describe the identification, purification and characterisation of glycosylated yellow laccase proteins from the basidiomycete fungus Stropharia aeruginosa. Biochemical characterisation of two yellow laccases, Yel1p and Yel3p, show that they are both secreted, monomeric, N-glycosylated proteins of molecular weight around 55kDa with substrate specificities typical of laccases, but lacking the absorption band at 612nm typical of the blue laccase proteins. Low coverage, high throughput 454 transcriptome sequencing in combination with inverse-PCR was used to identify cDNA sequences. One of the cDNA sequences has been assigned to the Yel1p protein on the basis of identity between the translated protein sequence and the peptide data from the purified protein, and the full length gene sequence has been obtained. Biochemical properties, substrate specificities and protein sequence data have been used to discuss the unusual spectroscopic properties of S. aeruginosa proteins in the context of recent theories about the differences between yellow and blue laccases.

Expression and purification of the aortic amyloid polypeptide medin.

The 50-amino acid protein medin is the main fibrillar component of human aortic medial amyloid (AMA), the most common form of localised amyloid which affects 97% of Caucasians over the age of 50. Structural models for several amyloid assemblies, including the Alzheimer's amyloid-ß peptides, have been defined from solid-state nuclear magnetic resonance (SSNMR) measurements on (13)C- and (15)N-labelled protein fibrils. SSNMR-derived structural information on fibrillar medin is scant, however, because studies to date have been restricted to limited measurements on site-specifically labelled protein prepared by solid-phase synthesis. Here we report a procedure for the expression of a SUMO-medin fusion protein in Escherichia coli and IMAC purification yielding pure, uniformly (13)C,(15)N-labelled medin in quantities required for SSNMR analysis. Thioflavin T fluorescence and dynamic light scattering measurements and transmission electron microscopy analysis confirm that recombinant medin assembles into amyloid-like fibrils over a 48-h period. The first (13)C and (15)N SSNMR spectra obtained for uniformly-labelled fibrils indicate that medin adopts a predominantly ß-sheet conformation with some unstructured elements, and provide the basis for further, more detailed structural investigations.

SalB inactivation modulates culture supernatant exoproteins and affects autolysis and viability in Enterococcus faecalis OG1RF.

The culture supernatant fraction of an Enterococcus faecalis gelE mutant of strain OG1RF contained elevated levels of the secreted antigen SalB. Using differential fluorescence gel electrophoresis (DIGE) the salB mutant was shown to possess a unique complement of exoproteins. Differentially abundant exoproteins were identified using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Stress-related proteins including DnaK, Dps family protein, SOD, and NADH peroxidase were present in greater quantity in the OG1RF salB mutant culture supernatant. Moreover, several proteins involved in cell wall synthesis and cell division, including d-Ala-d-Lac ligase and EzrA, were present in reduced quantity in OG1RF salB relative to the parent strain. The salB mutant displayed reduced viability and anomalous cell division, and these phenotypes were exacerbated in a gelE salB double mutant. An epistatic relationship between gelE and salB was not identified with respect to increased autolysis and cell morphological changes observed in the salB mutant. SalB was purified as a six-histidine-tagged protein to investigate peptidoglycan hydrolytic activity; however, activity was not evident. High-pressure liquid chromatography (HPLC) analysis of reduced muropeptides from peptidoglycan digested with mutanolysin revealed that the salB mutant and OG1RF were indistinguishable.