Identification of potential antivirulence agents by substitution-oriented screening for inhibitors of Streptococcus pyogenes sortase A.

Antimicrobial resistance resulting in ineffective treatment of infectious diseases is an increasing global problem, particularly in infections with pathogenic bacteria. In some bacteria, such as Streptococcus pyogenes, the pathogenicity is strongly linked to the attachment of virulence factors. Their attachment to the cellular membrane is a transpeptidation reaction, catalyzed by sortase enzymes. As such, sortases pose an interesting target for the development of new antivirulence strategies that could yield novel antimicrobial drugs. Using the substitution-oriented fragment screening (SOS) approach, we discovered a potent and specific inhibitor (C10) of sortase A from S. pyogenes. The inhibitor C10 showed high specificity towards S. pyogenes sortase A, with an IC50 value of 10 µM and a Kd of 60 µM. We envision that this inhibitor could be employed as a starting point for further exploration of sortase's potential as therapeutic target for antimicrobial drug development.

Identifying hQC Inhibitors of Alzheimer's Disease by Effective Customized Pharmacophore-Based Virtual Screening, Molecular Dynamic Simulation, and Binding Free Energy Analysis.

Human glutaminyl cyclase (hQC) appeared as a promising new target with its inhibitors attracted much attention for the treatment of Alzheimer's disease (AD) in recent years. But so far, only a few compounds have been reported as hQC inhibitors. To find novel and potent hQC inhibitors, a high-specificity ZBG (zinc-binding groups)-based pharmacophore model comprising customized ZBG feature was first generated using HipHop algorithm in Discovery Studio software for screening out hQC inhibitors from the SPECS database. After purification by docking studies and drug-like ADMET properties filters, four potential hit compounds were retrieved. Subsequently, these hit compounds were subjected to 30-ns molecular dynamic (MD) simulations to explore their binding modes at the active side of hQC. MD simulations demonstrated that these hit compounds formed a chelating interaction with the zinc ion, which was consistent with the finding that the electrostatic interaction was the major driving force for binding to hQC confirmed with MMPBSA energy decomposition. Higher binding affinities of these compounds were also verified by the binding free energy calculations comparing with the references. Thus, these identified compounds might be potential hQC candidates and could be used for further investigation.

Enhanced phytoremediation of uranium-contaminated soils by arbuscular mycorrhiza and rhizobium.

Uranium phytoextraction is a promising technology, however, facing difficult that limited plant biomass due to nutrient deficiency in the contaminated sites. The aim of this study is to evaluate the potential of a symbiotic associations of a legume Sesbania rostrata, rhizobia and arbuscular mycorrhiza fungi (AMF) for reclamation of uranium contaminated soils. Results showed AMF and rhizobia had a mutual beneficial relations in the triple symbiosis, which significantly increased plant biomass and uranium accumulation in S. rostrata plant. The highest uranium removal rates was observed in plant-AMF-rhizobia treated soils, in which 50.5-73.2% had been extracted, whereas 7.2-23.3% had been extracted in plant-treated soil. Also, the S. rostrata phytochelatin synthase (PCS) genes expression were increased in AMF and rhizobia plants compared with the plants. Meantime, content of malic acid, succinic acid and citric acid were elevated in S. rostrata root exudates of AMF and rhizobia inoculated plants. The facts suggest that the mutual interactions in the triple symbiosis help to improve phytoremediation efficiency of uranium by S. rostrata.

Whole Proteome Profiling of N-Myristoyltransferase Activity and Inhibition Using Sortase A.

N-myristoylation is the covalent addition of a 14-carbon saturated fatty acid (myristate) to the N-terminal glycine of specific protein substrates by N-myristoyltransferase (NMT) and plays an important role in protein regulation by controlling localization, stability, and interactions. We developed a novel method for whole-proteome profiling of free N-terminal glycines through labeling with S. Aureus sortase A (SrtA) and used it for assessment of target engagement by an NMT inhibitor. Analysis of the SrtA-labeling pattern with an engineered biotinylated depsipeptide SrtA substrate (Biotin-ALPET-Haa, Haa = 2-hydroxyacetamide) enabled whole proteome identification and quantification of de novo generated N-terminal Gly proteins in response to NMT inhibition by nanoLC-MS/MS proteomics, and was confirmed for specific substrates across multiple cell lines by gel-based analyses and ELISA. To achieve optimal signal over background noise we introduce a novel and generally applicable improvement to the biotin/avidin affinity enrichment step by chemically dimethylating commercial NeutrAvidin resin and combining this with two-step LysC on-bead/trypsin off-bead digestion, effectively eliminating avidin-derived tryptic peptides and enhancing identification of enriched peptides. We also report SrtA substrate specificity in whole-cell lysates for the first time, confirming SrtA promiscuity beyond its recognized preference for N-terminal glycine, and its usefulness as a tool for unbiased labeling of N-terminal glycine-containing proteins. Our new methodology is complementary to metabolic tagging strategies, providing the first approach for whole proteome gain-of signal readout for NMT inhibition in complex samples which are not amenable to metabolic tagging.

Selective Enrichment and Quantification of N-Terminal Glycine Peptides via Sortase A Mediated Ligation.

The identification and quantification of low-abundant proteins are always impeded by high-abundant proteins in proteomic analysis because of the extreme complexity of peptide mixtures and wide dynamic range of protein abundances. Here, we developed a novel approach to enrich and quantify N-terminal glycine peptides through sortase A mediated ligation. This strategy was based on the formation of a covalent bond between the sortase A recognition motif LPXTG and a N-terminal glycine residue. Also, the quantification was achieved by introducing isotopically labeled threonine in the motif LPXTG. In this strategy, both the enrichment of N-terminal glycine peptides and the stable isotope labeling were achieved in a single step. We applied this approach for the proteome analysis of MCF-7 cell line. It was demonstrated a significant reduction in sample complexity via highly selective and efficient enrichment of N-terminal glycine peptides, thereby detecting lots of less abundant proteins and enhancing proteome coverage. In comparison to the untreated sample, an increase of 34% of proteins was additionally identified. Furthermore, 97% of proteins were successfully quantified with high accuracy. In summary, this quantitative N-terminal glycine peptides enrichment strategy is expected for high-throughput qualitative and quantitative proteomic analysis as a complementary approach to conventional shotgun proteomics.

Isovitexin, a Potential Candidate Inhibitor of Sortase A of Staphylococcus aureus USA300.

Staphylococcus aureus (S. aureus) causes a broad variety of diseases. The spread of multidrugresistant S. aureus highlights the need to develop new ways to combat S. aureus infections. Sortase A (SrtA) can anchor proteins containing LPXTG binding motifs to the bacteria surface and plays a key role in S. aureus infections, making it a promising antivirulence target. In the present study, we used aSrtA activity inhibition assay to discover that isovitexin, a Chinese herbal product, can inhibit SrtA activity with an IC50 of 28.98 µg/ml. Using a fibrinogenbinding assay and a biofilm formation assay, we indirectly proved the SrtA inhibitory activity of isovitexin. Additionally, isovitexin treatment decreased the amount of staphylococcal protein A (SpA) on the surface of the cells. These data suggest that isovitexin has the potential to be an anti-infective drug against S. aureus via the inhibition of sortase activity.

Biofilm inhibition mechanism from extract of Hymenocallis littoralis leaves.

ETHNOPHARMACOLOGICAL RELEVANCE: Hymenocallis littoralis (Jacq.) Salisb. has been referred as beach spider lily and commonly known for its rich phytochemical diversity. Phytochemicals such as alkaloids, volatile constituents, phenols, flavonoids, flavonols extracted from different parts of these plants like bulbs, flowers, leaf, stem and root had been used in folk medicines from ancient times because of their excellent antimicrobial and antioxidant properties. The leaf and bulb extract of H. littoralis plant was traditionally used for wound healing. Alkaloids extracted from bulb of this plant possess anti-viral, anti-neoplastic and cytotoxic properties. However, these phytochemicals have also shown antibiofilm activity, which is considered as one of the important factor accountable for the drug resistance in microorganisms. Thus, the investigation of medicinal properties of H. littoralis could be useful to control biofilm producing pathogens. AIM OF THE STUDY: Explore antimicrobial, antibiofilm and antioxidant potentials of H. littoralis against pathogenic microorganisms using experimental and computational biology approach. MATERIALS AND METHODS: Phytochemical extraction from dried powder of H. littoralis leaves was done by solvent extraction using methanol. Antimicrobial and antibiofilm activities of leaves extract were carried out using agar well diffusion method, growth curve, minimum inhibitory concentration (MIC) and Scanning Electron Microscopy (SEM). Liquid Chromatography and Mass Spectroscopy (LCMS) technique was used for the identification of phytochemicals. Molecular docking studies of antibiofilm agents with adhesin proteins were performed using Autodock 4.2. Antioxidant activity of extract was carried out by FRAP assay. The noxious effect of extract was investigated by histological studies on rat skin. RESULTS: The preliminary phytochemical analysis of methanolic leaves extract revealed the presence of alkaloids, flavonoids, terpenoid, glycosides, terpene, terpenoids and phenolics. The various phytochemicals such as Apigenin 7-(4'', 6'' diacetylalloside)-4'- alloside, Catechin 7-O- apiofuranoside, Emodic acid, Epicatechin 3-O- ß-D-glucopyranoside, 4 - Methylesculetin, Methylisoeugenol, Quercetin 5,7,3',4'-tetramethyl ether 3-rutinoside, 4 - Methylumbelliferyl ß-D- glucuronide were extracted, characterized and recognized from the leaves extract of H. littoralis. The identification of these phytochemicals was performed using LC-MS. The antimicrobial property of H. littoralis leaf extract was investigated against different pathogenic microorganisms. Out of these tested microorganisms, promising antibiofilm and antimicrobial activities were confirmed against S. aureus NCIM 2654 and C. albicans NCIM 3466 by using growth curve and SEM analysis. MIC of this leaf extract was identified as 45 µg/ml and 70 µg/ml for S. aureus NCIM 2654 and C. albicans NCIM 3466 respectively. The leaves extract also showed good antioxidant activity due to presence of phenols and flavonoids. Molecular docking of these identified antibiofilm components interacts with the active site residues of adhesin proteins, Sortase A and Als3 from S. aureus and C. albicans respectively. Histological studies of extracted phytochemicals revealed non-noxious effects on rat skin. CONCLUSION: Thus, the present study revealed that the leaves extract of H. littoralis contains various phytochemicals having good extent of antimicrobial, antibiofilm and antioxidant properties. The in-vitro and in-silico results would be useful to design new lead compounds against biofilm producing pathogenic microorganisms.

Role of D-aminoacyl-tRNA deacylase beyond chiral proofreading as a cellular defense against glycine mischarging by AlaRS.

Strict L-chiral rejection through Gly-cisPro motif during chiral proofreading underlies the inability of D-aminoacyl-tRNA deacylase (DTD) to discriminate between D-amino acids and achiral glycine. The consequent Gly-tRNAGly 'misediting paradox' is resolved by EF-Tu in the cell. Here, we show that DTD's active site architecture can efficiently edit mischarged Gly-tRNAAla species four orders of magnitude more efficiently than even AlaRS, the only ubiquitous cellular checkpoint known for clearing the error. Also, DTD knockout in AlaRS editing-defective background causes pronounced toxicity in Escherichia coli even at low-glycine levels which is alleviated by alanine supplementation. We further demonstrate that DTD positively selects the universally invariant tRNAAla-specific G3•U70. Moreover, DTD's activity on non-cognate Gly-tRNAAla is conserved across all bacteria and eukaryotes, suggesting DTD's key cellular role as a glycine deacylator. Our study thus reveals a hitherto unknown function of DTD in cracking the universal mechanistic dilemma encountered by AlaRS, and its physiological importance.

Streptococcus suis sortase A is Ca2+ independent and is inhibited by acteoside, isoquercitrin and baicalin.

Sortase A (SrtA) has long been recognized as an ideal drug target for therapeutic agents against Gram-positive pathogens. However, the SrtA of Streptococcus suis (Ss-SrtA), an important zoonotic agent, has not been studied. In this study, the enzymatic properties of Ss-SrtA were investigated, and inhibition of Ss-SrtA by natural products was evaluated. Ss-SrtA was expressed and purified. The purified recombinant Ss-SrtA had maximal activity at pH 6.0-7.5, 45°C, and showed a Km of 6.7 µM for the hydrolysis of substrate abz-LPATG-dnp. Different from Staphylococcus aureus SrtA (Sa-SrtA) which is stimulated by Ca2+, Ss-SrtA was observed to be Ca2+ independent. Structural analysis showed that salt bridges formed between K111 and D180 in Ss-SrtA replaced the function of Ca2+ in Sa-SrtA to stabilize the substrate-binding cleft. Site-directed mutagenesis identified H126, C192 and R200 as the key residues of Ss-SrtA active site. To discover potential inhibitors, the percent inhibition of sortase activity by natural products was measured. Among these selected natural products, acteoside, isoquercitrin and baicalin were discovered as novel SrtA inhibitors, with IC50 values of 36.3 ± 1.3 µM, 100.0 ± 1.3 µM and 85.4 ± 1.5 µM, respectively. The inhibitory effects of these three natural products were further confirmed on endogenous Sa-SrtA. Using a previously established S. aureus model with a fluorescent-labeled Sa-SrtA substrate, acteoside, isoquercitrin, and baicalin showed 86%, 28% and 45% inhibition on endogenous Sa-SrtA activity, respectively. Overall, these findings shed new light on enzymatic properties, Ca2+-independent catalytic mechanism and potential inhibitors of Ss-SrtA.

Spatholobus suberectus Dunn. constituents inhibit sortase A and Staphylococcus aureus cell clumping to fibrinogen.

Sortases are a family of Gram-positive transpeptidases responsible for anchoring surface protein virulence factors to the peptidoglycan cell wall layer. In Staphylococcus aureus (S. aureus), deletion of sortase isoform results in a significant reduction in virulence and infection potential. Twenty flavonoids were isolated from the stem of the folk medicinal plant Spatholobus suberectus Dunn. These compounds were tested against S. aureus-derived sortase A (SrtA), a key transpeptidase for bacterial virulence. Among these active flavonoids, 7-hydroxy-6-methoxy-flavanone (3) and formononetin (10) were identified as compounds with promising SrtA inhibitory activity. These compounds also exhibited inhibitory activity against S. aureus cell clumping to fibrinogen. The suppression of cell-clumping activity indicates the potential of these compounds in the treatment of S. aureus infections via the inhibition of SrtA.

Identification of potential glutaminyl cyclase inhibitors from lead-like libraries by in silico and in vitro fragment-based screening.

A glutaminyl cyclase (QC) fragment library was in silico selected by disconnection of the structure of known QC inhibitors and by lead-like 2D virtual screening of the same set. The resulting fragment library (204 compounds) was acquired from commercial suppliers and pre-screened by differential scanning fluorimetry followed by functional in vitro assays. In this way, 10 fragment hits were identified ([Formula: see text]5 % hit rate, best inhibitory activity: 16 [Formula: see text]). The in vitro hits were then docked to the active site of QC, and the best scoring compounds were analyzed for binding interactions. Two fragments bound to different regions in a complementary manner, and thus, linking those fragments offered a rational strategy to generate novel QC inhibitors. Based on the structure of the virtual linked fragment, a 77-membered QC target focused library was selected from vendor databases and docked to the active site of QC. A PubChem search confirmed that the best scoring analogues are novel, potential QC inhibitors.

A Mutation in the Catalytic Subunit of the Glycosylphosphatidylinositol Transamidase Disrupts Growth, Fertility, and Stomata Formation.

GPI-anchored proteins (GPI-APs) are essential for plant growth and development; knockout mutations in enzymes responsible for anchor biosynthesis or attachment are gametophyte or embryo lethal. In a genetic screen targeted to identify genes regulating stomata formation, we discovered a missense mutation in the Arabidopsis (Arabidopsis thaliana) homolog of GPI8/PIG-K, a Cys protease that transfers an assembled GPI anchor to proteins. The Arabidopsis genome has a single copy of AtGPI8, and the atgpi8-1 mutation reduces the efficiency of this enzyme, leading to reduced accumulation of GPI-anchored proteins. While the atgpi8-1 mutation strongly disrupts plant growth, it is not lethal. Phenotypic analysis of atgpi8-1 mutants suggests that GPI-APs are important for root and shoot growth, stomata formation, apical dominance, transition to flowering, and male gametophyte viability. In addition, atgpi8-1 mutants accumulate higher levels of callose and have reduced plasmodesmata permeability. Genetic interactions of atgpi8-1 with mutations in ERECTA family (ERf) genes suggest the existence of a GPI-AP in a branch of the ERf signaling pathway that regulates stomata formation. Activation of the ERf signal transduction cascade by constitutively active YODA rescues stomata clustering in atgpi8-1, indicating that a GPI-AP functions upstream of the MAP kinase cascade. TOO MANY MOUTHS (TMM) is a receptor-like protein that is able to form heterodimers with ERfs. Our analysis demonstrates that tmm-1 is epistatic to atgpi8-1, indicating that either TMM is a GPI-AP or there is another GPI-AP regulating stomata development whose function is dependent upon TMM.

Elongation Factor Tu Prevents Misediting of Gly-tRNA(Gly) Caused by the Design Behind the Chiral Proofreading Site of D-Aminoacyl-tRNA Deacylase.

D-aminoacyl-tRNA deacylase (DTD) removes D-amino acids mischarged on tRNAs and is thus implicated in enforcing homochirality in proteins. Previously, we proposed that selective capture of D-aminoacyl-tRNA by DTD's invariant, cross-subunit Gly-cisPro motif forms the mechanistic basis for its enantioselectivity. We now show, using nuclear magnetic resonance (NMR) spectroscopy-based binding studies followed by biochemical assays with both bacterial and eukaryotic systems, that DTD effectively misedits Gly-tRNAGly. High-resolution crystal structure reveals that the architecture of DTD's chiral proofreading site is completely porous to achiral glycine. Hence, L-chiral rejection is the only design principle on which DTD functions, unlike other chiral-specific enzymes such as D-amino acid oxidases, which are specific for D-enantiomers. Competition assays with elongation factor thermo unstable (EF-Tu) and DTD demonstrate that EF-Tu precludes Gly-tRNAGly misediting at normal cellular concentrations. However, even slightly higher DTD levels overcome this protection conferred by EF-Tu, thus resulting in significant depletion of Gly-tRNAGly. Our in vitro observations are substantiated by cell-based studies in Escherichia coli that show that overexpression of DTD causes cellular toxicity, which is largely rescued upon glycine supplementation. Furthermore, we provide direct evidence that DTD is an RNA-based catalyst, since it uses only the terminal 2'-OH of tRNA for catalysis without the involvement of protein side chains. The study therefore provides a unique paradigm of enzyme action for substrate selection/specificity by DTD, and thus explains the underlying cause of DTD's activity on Gly-tRNAGly. It also gives a molecular and functional basis for the necessity and the observed tight regulation of DTD levels, thereby preventing cellular toxicity due to misediting.

Chinese herb medicine against Sortase A catalyzed transformations, a key role in gram-positive bacterial infection progress.

Many Gram-positive bacteria can anchor their surface proteins to the cell wall peptidoglycan covalently by a common mechanism with Sortase A (SrtA), thus escaping from the host's identification of immune cells. SrtA can complete this anchoring process by cleaving LPXTG motif conserved among these surface proteins and thus these proteins anchor on the cell wall. Moreover, those SrtA mutants lose this capability to anchor these relative proteins, with these bacteria no longer infectious. Therefore, SrtA inhibitors can be promising anti-infective agents to cure bacterial infections. Chinese herb medicines (CHMs) (chosen from Science Citation Index) have exhibited inhibition on SrtA of Gram-positive pathogens irreversibly or reversibly. In general, CHMs are likely to have important long-term impact as new antibacterial compounds and sought after by academia and the pharmaceutical industry. This review mainly focuses on SrtA inhibitors from CHMs and the potential inhibiting mechanism related to chemical structures of compounds in CHMs.

Structures of two bacterial resistance factors mediating tRNA-dependent aminoacylation of phosphatidylglycerol with lysine or alanine.

The cytoplasmic membrane is probably the most important physical barrier between microbes and the surrounding habitat. Aminoacylation of the polar head group of the phospholipid phosphatidylglycerol (PG) catalyzed by Ala-tRNA(Ala)-dependent alanyl-phosphatidylglycerol synthase (A-PGS) or by Lys-tRNA(Lys)-dependent lysyl-phosphatidylglycerol synthase (L-PGS) enables bacteria to cope with cationic peptides that are harmful to the integrity of the cell membrane. Accordingly, these synthases also have been designated as multiple peptide resistance factors (MprF). They consist of a separable C-terminal catalytic domain and an N-terminal transmembrane flippase domain. Here we present the X-ray crystallographic structure of the catalytic domain of A-PGS from the opportunistic human pathogen Pseudomonas aeruginosa. In parallel, the structure of the related lysyl-phosphatidylglycerol-specific L-PGS domain from Bacillus licheniformis in complex with the substrate analog L-lysine amide is presented. Both proteins reveal a continuous tunnel that allows the hydrophobic lipid substrate PG and the polar aminoacyl-tRNA substrate to access the catalytic site from opposite directions. Substrate recognition of A-PGS versus L-PGS was investigated using misacylated tRNA variants. The structural work presented here in combination with biochemical experiments using artificial tRNA or artificial lipid substrates reveals the tRNA acceptor stem, the aminoacyl moiety, and the polar head group of PG as the main determinants for substrate recognition. A mutagenesis approach yielded the complementary amino acid determinants of tRNA interaction. These results have broad implications for the design of L-PGS and A-PGS inhibitors that could render microbial pathogens more susceptible to antimicrobial compounds.

Streptococcus mutans sortase A inhibitory metabolites from the flowers of Sophora japonica.

A new maltol derivative (2) along with three known maltol derivative (1) and flavonol glycosides (3 and 4) were isolated from the dried flowers of Sophora japonica. Based upon the results of combined spectroscopic methods, the structure of new compound (2) was determined to be maltol-3-O-(4'-O-cis-p-coumaroyl-6'-O-(3-hydroxy-3-methylglutaroyl))-ß-glucopyranoside, an isomer of 1. These compounds strongly inhibited the action of sortase A (SrtA) from Streptococcus mutans, a primary etiologic agent of human dental caries. The onset and magnitude of inhibition of the saliva-induced aggregation in S. mutans treated with compound 2 (4×IC50) were comparable to the behavior of untreated srtA-deletion mutant.

Virtual screening of LPXTG competitive SrtA inhibitors targeting signal transduction mechanism in Bacillus anthracis: a combined experimental and theoretical study.

Members of the sortase enzyme super family decorate the surfaces of Bacillus anthracis cell wall with proteins that play key roles in microbial pathogenesis and its biofilm formation. Bacillus anthracis Sortase-A (Ba-SrtA) is a potential target for new therapeutics as it is required for B. anthracis survival and replication within macrophages. An understanding of the binding site pocket and substrate recognition mechanism by SrtA enzymes may serve to be beneficial in the rational development of sortase inhibitors. Here, the LPXTG signal peptide-based competitive inhibitors are screened against the Ba-SrtA and compounds with reasonable inhibition, specificity, and mechanisms of inactivation of SrtA have been covered. The screened compounds are experimentally validated against the phylogenetically similar Gram-positive pathogen B. cereus. In situ microscopic visualizations suggest that these screened compounds showed the microbial and biofilm inhibitory activity against B. cereus. It facilitates the further development of these molecules into useful anti-infective agents to treat infections caused by B. anthracis and other Gram-positive pathogens. These results provide insight into basic design principles for generating new clinically relevant lead molecules. It also provides an alternative strategy where a screened ligand molecule can be used in combination to battle increasingly against the Gram-positive pathogens.

Arginyltransferase ATE1 catalyzes midchain arginylation of proteins at side chain carboxylates in vivo.

Arginylation is an emerging posttranslational modification mediated by Arg-tRNA-protein-transferase (ATE1). It is believed that ATE1 links Arg solely to the N terminus of proteins, requiring prior proteolysis or action by Met-aminopeptidases to expose the arginylated site. Here, we tested the possibility of Arg linkage to midchain sites within intact protein targets and found that many proteins in vivo are modified on the side chains of Asp and Glu by unconventional chemistry that targets the carboxy rather than the amino groups at the target sites. Such arginylation appears to be functionally regulated, and it can be directly mediated by ATE1, in addition to the more conventional ATE1-mediated linkage of Arg to the N-terminal alpha amino group. This midchain arginylation implies an unconventional mechanism of ATE1 action that likely facilitates its major biological role.

Sortase A inhibitory metabolites from the roots of Pulsatilla koreana.

Three new lignans (3, 4, and 6) along with eight known lignans and phenyl propanoids were isolated from the dried roots of Pulsatilla koreana, an oriental folk medicine. Based upon the results of combined spectroscopic and chemical methods, the structures of new compounds were determined to be lignan glycosides. Included among the known compounds are three compounds (5, 7, and 8) isolated first time from this plant as well as two compounds (2 and 11) previously reported only as synthetic derivatives. These compounds significantly inhibited the action of Sortase A from Streptococcus mutans OMZ65, an isolate from human oral cavity.

Role of the LytSR two-component regulatory system in adaptation to cationic antimicrobial peptides in Staphylococcus aureus.

Many host defense cationic antimicrobial peptides (HDPs) perturb the staphylococcal cell membrane (CM) and alter transmembrane potential (ΔΨ) as key parts of their lethal mechanism. Thus, a sense-response system for detecting and mediating adaptive responses to such stresses could impact organism survival; the Staphylococcus aureus LytSR two-component regulatory system (TCRS) may serve as such a ΔΨ sensor. One well-known target of this system is the lrgAB operon, which, along with the related cidABC operon, has been shown to be a regulator in the control of programmed cell death and lysis. We used an isogenic set of S. aureus strains: (i) UAMS-1, (ii) its isogenic ΔlytS and ΔlrgAB mutants, and (iii) plasmid-complemented ΔlytSR and ΔlrgAB mutants. The ΔlytS strain displayed significantly increased in vitro susceptibilities to all HDPs tested (neutrophil-derived human neutrophil peptide 1 [hNP-1], platelet-derived thrombin-induced platelet microbicidal proteins [tPMPs], and the tPMP-mimetic peptide RP-1), as well as to calcium-daptomycin (DAP), a cationic antimicrobial peptide (CAP). In contrast, the ΔlrgAB strain exhibited no significant changes in susceptibilities to these cationic peptides, indicating that although lytSR positively regulates transcription of lrgAB, increased HDP/CAP susceptibilities in the ΔlytS mutant were lrgAB independent. Further, parental UAMS-1 (but not the ΔlytS mutant) became more resistant to hNP-1 and DAP following pretreatment with carbonyl cyanide m-chlorophenylhydrazone (CCCP) (a CM-depolarizing agent). Of note, lytSR-dependent survival against CAP/HDP killing was not associated with changes in either surface positive charge, expression of mprF and dlt, or CM fluidity. The ΔlytS strain (but not the ΔlrgAB mutant) displayed a significant reduction in target tissue survival in an endocarditis model during DAP treatment. Collectively, these results suggest that the lytSR TCRS plays an important role in adaptive responses of S. aureus to CM-perturbing HDPs/CAPs, likely by functioning as a sense-response system for detecting subtle changes in ΔΨ.