A single-chain fab derived drug conjugate for HER2 specific delivery.

Despite the development of antibody-drug conjugates, the fragment Fab-based drug conjugates offer some unique capabilities in terms of safety, clearance, penetration and others. Current methods for preparing Fab drug conjugates are limited by the availability and stability of Fab proteins, leaving reports on this rare. Here, we found that a single-chain scaffold of Fab enables stabilization of the paired structure and supports high-yield expression in bacteria cytoplasm. Furthermore, we conjugated anti-neoplastic agent SN38 to the C-terminus by sortase A ligation and generated a homogenous Fab conjugate with the drug-to-Fab ratio of 1. The resulting anti-HER2 Fab-SN38 conjugate demonstrated potent and antigen-dependent cell-killing ability with the aid of its special cathepsin-triggered cyclization-promoted release mechanism. In vivo, Fab-SN38 can prevent growths of HER2-positive tumors in athymic mice and be well tolerated to the treatment at 7 mg/kg per dose. Anti-tumor activity, high dose tolerance and penetration advantage observed in this study would merit Fab conjugate investigation in target chemotherapy.

Antibiofilm mechanism of 2R,3R-dihydromyricetin by targeting sortase A and its application against Staphylococcus aureus adhesion on eggshell.

Biofilm formation of Staphylococcus aureus in food processing environments raises significant safety concerns, necessitating the development of new antibiofilm approaches for controlling S. aureus contamination. This study aimed to elucidate the antibiofilm mechanism of 2R,3R-dihydromyricetin (DMY), a natural flavonoid, against S. aureus and evaluate its efficacy in reducing bacterial adhesion to eggshell. The results revealed that DMY was a potent inhibitor of S. aureus sortase A (SrtA) with an IC50 of 73.43 µM, preventing bacterial adhesion to fibrinogen and subsequent biofilm formation. Fluorescence quenching assay and surface plasmon resonance analysis confirmed that DMY could directly bind to S. aureus SrtA. Notably, circular dichroism spectra demonstrated a conformational change in SrtA from α-helical to ß-sheet structure upon DMY binding. Molecular dynamics simulation suggested that DMY bound to the catalytic pocket of S. aureus SrtA via hydrophobic interactions and hydrogen bonds. Furthermore, fluorescence microscopic observations further revealed that DMY attenuated the biofilm-related phenotype of SrtA by decreasing the anchoring of S. aureus protein A (SpA) onto cell wall. Importantly, pretreatment with 125 µg/mL DMY significantly reduced 1.14-1.75 log CFU/cm2 of S. aureus adhered on eggshells. Overall, these findings highlight how specific targeting of SrtA by DMY inhibits the attachment stages of biofilm development in S. aureus, making it a promising candidate for a novel disinfectant against this pathogen in the food industry.

Structural studies and functional validation of Eugenol and Ferulic acid against Enterococcus faecalis Sortase A.

Enterococcus faecalis (E. faecalis) is commonly occurring pathogen                                                          associated with nosocomial infections. Infections are difficult to treat because of their multidrug-resistant (MDR) nature and their tendency to form biofilms. Therefore, it is essential to find alternative medicinal approaches of treatment. In this regard, targeting an important  protein for drug development can be an alternative approach. Sortase A (SrtA) is an important enzyme involved in anchoring cell surface-exposed proteins to the cell envelope. SrtA is present in Gram-positive bacteria which catalyses the attachment of several virulence factors and other proteins to the cell membrane. It is involved in bacterial pathogenesis, therefore, it's a promising drug target for the development of anti-microbial drugs targeting cell adhesion, evasion, and biofilm development. To identify SrtA potential inhibitors, we have purified E. faecalis Sortase A (EfSrtAΔN59).  Structural studies along with molecular docking of protein with selected ligand molecules were done and confirmed by MD simulation experiments. We have also performed functional validation of these compounds on bacterial growth, anti-biofilm assays and inhibition assay of selected ligands were also done against E. faecalis individually and in synergistic combinations.  Results indicated that both Eugenol and Ferulic acid bind to EfSrtAΔN59 with significant interactions and show promising results.

Maple compounds prevent biofilm formation in Listeria monocytogenes via sortase inhibition.

The Pss exopolysaccharide (EPS) enhances the ability of the foodborne pathogen Listeria monocytogenes to colonize and persist on surfaces of fresh fruits and vegetables. Eradicating listeria within EPS-rich biofilms is challenging due to their increased tolerance to disinfectants, desiccation, and other stressors. Recently, we discovered that extracts of maple wood, including maple sap, are a potent source of antibiofilm agents. Maple lignans, such as nortrachelogenin-8'-O-ß-D-glucopyranoside and lariciresinol, were found to inhibit the formation of, and promote the dispersion of pre-formed L. monocytogenes EPS biofilms. However, the mechanism remained unknown. Here, we report that these lignans do not affect Pss EPS synthesis or degradation. Instead, they promote EPS detachment, likely by interfering with an unidentified lectin that keeps EPS attached to the cell surfaces. Furthermore, the maple lignans inhibit the activity of L. monocytogenes sortase A (SrtA) in vitro. SrtA is a transpeptidase that covalently anchors surface proteins, including the Pss-specific lectin, to the cell wall peptidoglycan. Consistent with this, deletion of the srtA gene results in Pss EPS detachment from listerial cells. We also identified several additional maple compounds, including epicatechin gallate, isoscopoletin, scopoletin, and abscisic acid, which inhibit L. monocytogenes SrtA activity in vitro and prevent biofilm formation. Molecular modelling indicates that, despite their structural diversity, these compounds preferentially bind to the SrtA active site. Since maple products are abundant and safe for consumption, our finding that they prevent biofilm formation in L. monocytogenes offers a viable source for protecting fresh produce from this foodborne pathogen.

OVEREXPRESSION, PURIFICATION AND BIOCHEMICAL STUDIES OF SORTASE A FROM Enterococcus faecalis (Ef) AND ITS INHIBITION STUIDES WITH ALOENIN.

Sortase A (SrtA) is a bacterial transpeptidase that garnishes the bacterial surface by adding various virulent factors or proteins by cleaving the LPXTG-specific motif between T and G amino acids. These virulence factors assist in the attachment of host cells, which are essential for bacterial virulence. Enterococcus species are among the multidrug-resistant bacteria that cause nosocomial infections; they have drawn a lot of attention recently. SrtA from E. faecalis (Ef) plays a critical role in pathogenesis, making it a suitable target for the development of antibacterial agents. Since SrtA is not involved in bacterial growth and is present on the surface of bacteria, the probability of antibiotic resistance developing is minimal. In this work, we have done cloning, expression and purification of Ef-SrtA using IMAC (Immobilised Metal Affinity Chromatography) followed by Gel filtration chromatography. Purified Ef-SrtA showed maximum activity at pH-8 and temperature between 45-55°C. The fluorescent assay for kinetic studies of Ef-SrtA showed Vmax 3.852 µM.min-1 and kcat 7.7 × 10-2s-1 for the hydrolysis of substrate using Abz-LPETG-K(Dnp)-NH2. We have selected fifteen aloe vera extracted compounds and performed virtual screening and docking experiments to identify potential inhibitors against Ef-SrtA. Among fifteen molecules, Aloenin-a which was bound to the active site with a binding energy of -6.1 kcal/mol, interacted with the active site residues, Arg139, Pro105, Leu39, Ala46, and Cys126. Aloenin-ashowed a significant inhibitory effect against Ef-SrtA, with an IC50 value of 20.68 µM. Aloenin-a inhibits biofilm formation at concentrations of 20-250 µg/mL. The fibrinogen assay showed adherence to fibrinogen was reduced in the presence of Aloenin-a for E. faecalis. The results demonstrated that aloe vera extracts containing Aloenin-a can be a significant antagonist of Ef-SrtA.

QSAR Studies on a Class of Benzofuranene Cyanide Derivatives as Potential Inhibitors Targeting Staphylococcus aureus Sortase A.

BACKGROUND: Staphylococcus aureus is a widely distributed and highly pathogenic zoonotic bacterium. Sortase A represents a crucial target for the research and development of novel antibacterial drugs. OBJECTIVE: This study aims to establish quantitative structure-activity relationship models based on the chemical structures of a class of benzofuranene cyanide derivatives. The models will be used to screen new antibacterial agents and predict the properties of these molecules. METHOD: The compounds were randomly divided into a training set and a test set. A large number of descriptors were calculated using the software, and then the appropriate descriptors were selected to build the models through the heuristic method and the gene expression programming algorithm. RESULTS: In the heuristic method, the determination coefficient, determination coefficient of cross-validation, F-test, and mean squared error values were 0.530, 0.395, 9.006, and 0.047, respectively. In the gene expression programming algorithm, the determination coefficient and the mean squared error values in the training set were 0.937 and 0.008, respectively, while in the test set, they were 0.849 and 0.035. The results showed that the minimum bond order of a C atom and the relative number of benzene rings had a significant positive contribution to the activity of compounds. CONCLUSION: In this study, two quantitative structure-activity relationship models were successfully established to predict the inhibitory activity of a series of compounds targeting Staphylococcus aureus Sortase A, providing insights for further development of novel anti-Staphylococcus aureus drugs.

Biointerface engineering through amalgamation of gene technology and site-specific growth factor conjugation for efficient osteodifferentiation.

The development of bone implants through bioinspired immobilization of growth factors remains a key issue in the generation of biological interfaces, especially in enhancing osteodifferentiation ability. In this study, we developed a strategy for surface functionalization of poly(lactide-glycolide) (PLGA) and hydroxyapatite (HA) composite substrates through site-specific conjugation of bone morphogenetic protein 2 containing 3,4-hydroxyphenalyalanine (DOPA-BMP2) mediated by tyrosinase and sortase A (SrtA). Firstly, the growth factor BMP2-LPETG containing LPETG motif was successfully expressed in Escherichia coli through recombinant DNA technology. The excellent binding affinity of binding growth factor (DOPA-BMP2) was achieved by converting the tyrosine residue (Y) of YKYKY-GGG peptide into DOPA (X) by tyrosinase, which bound to the substrates. Then its GGG motif was specifically bound to the end of BMP2-LPETG mediated by SrtA. Therefore, the generated bioactive DOPA-BMP2/PLGA/HA substrates significantly promoted the osteogenic differentiation of MC3T3-E1 cells. Thanks to this microbial-assisted engineering approach, our work presents a facile and highly site-specific strategy to engineer biomimetic materials for orthopedics and dentistry by effectively delivering growth factors, peptides, and other biomacromolecules.

Biocatalytic Method for Producing an Affinity Resin for the Isolation of Immunoglobulins.

Affinity chromatography is a widely used technique for antibody isolation. This article presents the successful synthesis of a novel affinity resin with a mutant form of protein A (BsrtA) immobilized on it as a ligand. The key aspect of the described process is the biocatalytic immobilization of the ligand onto the matrix using the sortase A enzyme. Moreover, we used a matrix with primary amino groups without modification, which greatly simplifies the synthesis process. The resulting resin shows a high dynamic binding capacity (up to 50 mg IgG per 1 mL of sorbent). It also demonstrates high tolerance to 0.1 M NaOH treatment and maintains its effectiveness even after 100 binding, elution, and sanitization cycles.

An approach for the intracellular delivery of IgG via enzymatic ligation with a cell-permeable attenuated cationic amphiphilic lytic peptide.

Achieving effective intracellular delivery of therapeutic molecules such as antibodies (IgG) is a challenge in biomedical research and pharmaceutical development. Conjugation of IgG with a cell-penetrating peptide is a rational approach. Here, not only the efficacy of the conjugates in internalizing into cells, but also the physicochemical property of the conjugates allowing their solubilized states in solution without forming aggregates are critical. In this study, we have shown that the first requirement can be addressed using a cell-permeable attenuated cationic amphiphilic lytic (CP-ACAL) peptide, L17ER4. The second requirement can be addressed by ligation of IgG to L17ER4 using sortase A, where the use of a linker of appropriate chain length is also important. For evaluation, the intracellular delivery efficacy was studied using conjugate structures with different orientations and conjugation modes of L17ER4 in ligation to a model protein, green fluorescent protein fused to a nuclear localization signal (NLS-EGFP). The effect of tetraarginine positioning in the L17ER4 sequence was also investigated. Following these studies, an optimized peptide sequence containing L17ER4 was ligated to an anti-green fluorescent protein (GFP) IgG bearing a sortase A recognition sequence. Treatment of the cells with the conjugate of anti-GFP IgG and L17ER4 resulted in a high efficiency of cytosolic translocation of the conjugate and the binding to the target protein in the cell without significant aggregate formation. The feasibility of the d-form of L17ER4 as a CP-ACAL was also confirmed.

Halenaquinol Blocks Staphylococcal Protein A Anchoring on Cell Wall Surface by Inhibiting Sortase A in Staphylococcus aureus.

Sortase A (SrtA) is a cysteine transpeptidase that binds to the periplasmic membrane and plays a crucial role in attaching surface proteins, including staphylococcal protein A (SpA), to the peptidoglycan cell wall. Six pentacyclic polyketides (1-6) were isolated from the marine sponge Xestospongia sp., and their structures were elucidated using spectroscopic techniques and by comparing them to previously reported data. Among them, halenaquinol (2) was found to be the most potent SrtA inhibitor, with an IC50 of 13.94 µM (4.66 µg/mL). Semi-quantitative reverse transcription PCR data suggest that halenaquinol does not inhibit the transcription of srtA and spA, while Western blot analysis and immunofluorescence microscopy images suggest that it blocks the cell wall surface anchoring of SpA by inhibiting the activity of SrtA. The onset and magnitude of the inhibition of SpA anchoring on the cell wall surface in S. aureus that has been treated with halenaquinol at a value 8× that of the IC50 of SrtA are comparable to those for an srtA-deletion mutant. These findings contribute to the understanding of the mechanism by which marine-derived pentacyclic polyketides inhibit SrtA, highlighting their potential as anti-infective agents targeting S. aureus virulence.

Unraveling the efficacy of verbascoside in thwarting MRSA pathogenicity by targeting sortase A.

In the fight against hospital-acquired infections, the challenge posed by methicillin-resistant Staphylococcus aureus (MRSA) necessitates the development of novel treatment methods. This study focused on undermining the virulence of S. aureus, especially by targeting surface proteins crucial for bacterial adherence and evasion of the immune system. A primary aspect of our approach involves inhibiting sortase A (SrtA), a vital enzyme for attaching microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) to the bacterial cell wall, thereby reducing the pathogenicity of S. aureus. Verbascoside, a phenylethanoid glycoside, was found to be an effective SrtA inhibitor in our research. Advanced fluorescence quenching and molecular docking studies revealed a specific interaction between verbascoside and SrtA, pinpointing the critical active sites involved in this interaction. This molecular interaction significantly impedes the SrtA-mediated attachment of MSCRAMMs, resulting in a substantial reduction in bacterial adhesion, invasion, and biofilm formation. The effectiveness of verbascoside has also been demonstrated in vivo, as shown by its considerable protective effects on pneumonia and Galleria mellonella (wax moth) infection models. These findings underscore the potential of verbascoside as a promising component in new antivirulence therapies for S. aureus infections. By targeting crucial virulence factors such as SrtA, agents such as verbascoside constitute a strategic and potent approach for tackling antibiotic resistance worldwide. KEY POINTS: • Verbascoside inhibits SrtA, reducing S. aureus adhesion and biofilm formation. • In vivo studies demonstrated the efficacy of verbascoside against S. aureus infections. • Targeting virulence factors such as SrtA offers new avenues against antibiotic resistance.

Fraxetin Targeting to Sortase A Decreases the Pathogenicity of Streptococcus agalactiae to Nile Tilapia.

Sortase A (SrtA) is responsible for anchoring surface proteins to the cell wall, and has been identified as a promising target developing anti-infective drugs of Gram-positive bacteria. The aim of the study was to identify inhibitors of Streptococcus agalactiae (S. agalactiae) SrtA from natural compounds to overcome the spread of antibiotic resistance in aquaculture. Here, we found that the MIC of fraxetin against S. agalactiae was higher than 256 µg/mL, indicating that fraxetin had no anti- S. agalactiae activity. But fraxetin could dose-dependently decrease the activity of SrtA in vitro at concentrations ranging between 4-32 µg/mL by a fluorescence resonance energy transfer (FRET) assay. Moreover, the inhibition of SrtA by fraxetin decreased the anchoring of surface proteins with the LPXTG motif to the cell wall by detecting the immunofluorescence change of serine-rich repeat protein 1 (Srr1) on the bacterial cell surface. The results of fibronectin binding and cell adhesion assays indicated that fraxetin could significantly decrease the adhesion ability of S. agalactiae in a dose-dependent manner. The results were further proven by immunofluorescence staining. Animal challenge results showed that treatment with fraxetin could reduce the mortality of tilapia infected with S. agalactiae to 46.67%, indicating that fraxetin could provide a significant amount of protection to tilapia by inactivating SrtA. Taken together, these findings provided a novel inhibitor of S. agalactiae SrtA and a promising candidate for treating S. agalactiae infections in aquaculture.

The Complete Assessment of Small Molecule and Peptidomimetic Inhibitors of Sortase A Towards Antivirulence Treatment.

This review covers the most recent advances in the development of inhibitors for the bacterial enzyme sortase A (SrtA). Sortase A (SrtA) is a critical virulence factor, present ubiquitously in Gram-positive bacteria of which many are pathogenic. Sortases are key enzymes regulating bacterial adherence to host cells, by anchoring extracellular matrix-binding proteins to the bacterial outer cell wall. By targeting virulence factors, effective treatment can be achieved, without inducing antibiotic resistance to the treatment. This is a potentially more sustainable, long-term approach to treating bacterial infections, including ones that display multiple resistance to current therapeutics. There are many promising approaches available for SrtA inhibition, some of which have the potential to advance into further clinical development, with peptidomimetic and in vivo active small molecules being among the most promising. There are currently no approved drugs on the market targeting SrtA, despite its promise, adding to the relevance of this review article, as it extends to the pharmaceutical industry additionally to academic researchers.

Chondrocyte-Targeted Delivery System of Sortase A-Engineered Extracellular Vesicles Silencing MMP13 for Osteoarthritis Therapy.

Targeted drug delivery and the reduction of off-target effects are crucial for the promising clinical application of nucleic acid drugs. To address this challenge, a new approach for treating osteoarthritis (OA) that accurately delivers antisense oligonucleotides (ASO) targeting matrix metalloproteinase-13 (ASO-MMP13) to chondrocytes, is developed. Small extracellular vesicles (exos) are ligated with chondrocyte affinity peptide (CAP) using Sortase A and subsequently incubated with cholesterol-modified ASO-MMP13 to construct a chondrocyte-targeted drug delivery exo (CAP-exoASO). Compared with exos without CAP (ExoASO), CAP-exoASOs attenuate IL-1ß-induced chondrocyte damage and prolong the retention time of ASO-MMP13 in the joint without distribution in major organs following intra-articular injection. Notably, CAP-exoASOs decrease MMP13 expression (P < 0.001) and upregulate COL2A1 expression (P = 0.006), resulting in reorganization of the cartilage matrix and alleviation of progression in the OA model. Furthermore, the Osteoarthritis Research Society International (OARSI) score of articular cartilage tissues treated with CAP-exoASO is comparable with that of healthy rats (P = 0.148). A mechanistic study demonstrates that CAP-exoASO may reduce inflammation by suppressing the IL-17 and TNF signaling pathways. Based on the targeted delivery effect, CAP-exoASOs successfully accomplish cartilage repair and have considerable potential for development as a promising therapeutic modality for satisfactory OA therapy.

Sortase-mediated labeling: Expanding frontiers in site-specific protein functionalization opens new research avenues.

New applications for biomolecules demand novel approaches for their synthesis and modification. Traditional methods for modifying proteins and cells using non-specific labeling chemistry are insufficiently precise to rigorously interrogate the mechanistic biological and physiological questions at the forefront of biomedical science. Site-specific catalytic modification of proteins promises to meet these challenges. Here, we describe recent applications of the enzyme sortase A in facilitating precise biomolecule labeling. We focus on describing new chemistries to broaden the scope of sortase-mediated labeling (sortagging), the development of new probes for imaging via enzymatic labeling, and the modulation of biological systems using probes and reactions mediated by sortase.

Synthesis, Antimicrobial Activities, and Molecular Modeling Studies of Agents for the Sortase A Enzyme.

Sortase A (SrtA) is an attractive target for developing new anti-infective drugs that aim to interfere with essential virulence mechanisms, such as adhesion to host cells and biofilm formation. Herein, twenty hydroxy, nitro, bromo, fluoro, and methoxy substituted chalcone compounds were synthesized, antimicrobial activities and molecular modeling strategies against the SrtA enzyme were investigated. The most active compounds were found to be T2, T4, and T19 against Streptococcus mutans (S. mutans) with MIC values of 1.93, 3.8, 3.94 µg/mL, and docking scores of -6.46, -6.63, -6.73 kcal/mol, respectively. Also, these three active compounds showed better activity than the chlorohexidine (CHX) (MIC value: 4.88 µg/mL, docking score: -6.29 kcal/mol) in both in vitro and in silico. Structural stability and binding free energy analysis of S.mutans SrtA with active compounds were measured by molecular dynamic (MD) simulations throughout 100 nanoseconds (ns) time. It was observed that the stability of the critical interactions between these compounds and the target enzyme was preserved. To prove further, in vivo biological evaluation studies could be conducted for the most promising precursor compounds T2, T4, and T19, and it might open new avenues to the discovery of more potent SrtA inhibitors.

Affinity Resins for the Isolation of Immunoglobulins G Obtained Using Biocatalytic Technology.

Affinity chromatography resins that are obtained by conjugation of matrices with proteins of bacterial origin, like protein A, are frequently used for the purification of numerous therapeutic monoclonal antibodies. This article presents the development of a biocatalytic method for the production of novel affinity resins with an immobilized mutant form of protein A via sortase A mediated reaction. The conditions for activation of the agarose Seplife 6FF matrix, selection of different types of linkers with free amino groups and conditions for immobilization of recombinant protein A on the surface of the activated matrix were studied. Finally, the basic operational properties, like dynamic binding capacity (DBC), temperature dependance of DBC and stability during the cleaning-in-place process of the affinity resin with the Gly-Gly-EDA-Gly-Gly linker, were assessed using recombinant hyperchimeric monoclonal antibodies. The main characteristics show comparable results with the widely used commercial samples.

SpSrtA-Catalyzed Isopeptide Ligation on Lysine Residues.

Sortase-mediated ligation (SML) is widely used for protein bioconjugation. However, the sortase used in this strategy typically recognizes only the N-terminal oligoglycine, which is absent in most natural proteins. To broaden the spectrum of substrates compatible with SML, we focus on a novel sortase, sortase A from Streptococcus pneumoniae (SpSrtA), known for its expanded substrate specificity (N-terminal glycine, alanine, and serine). We present the first evidence showing that the reported SpSrtA mutant (SpSrtA*) can modify lysine residues in itself and other proteins. The modification sites of SpSrtA* were identified through LC-MS/MS analysis. Moreover, we discovered an optimal lysine-containing peptide tag by fusing it onto sfGFP, resulting in a labeling efficiency of 57%. Inspired by this, we applied the method to modify proteins on microorganism surfaces up to 13.5-fold. To enhance labeling efficiency, we fused the SpSrtA* onto a surface protein and achieved a 2.64-fold improvement. We further developed a high-throughput yeast display screening method for the directed evolution of SpSrtA*, achieving a 10-fold improvement in the labeling efficiency of this surface protein. Our study provides a novel strategy for modifying the lysine residues that will be a powerful addition to the protein bioconjugation toolbox.

Localization of the Remnant of a Cell Wall Sorting Signal and Its Interaction with a Sensor Kinase.

Sortases are highly conserved enzymes with endopeptidase and transpeptidase activities in Gram-positive bacteria. Sortase A cleaves within an LPXTG-motif and covalently crosslinks cell wall proteins to become anchored to the peptidoglycan of the cell wall. We showed that a peptide cleaved by sortase A from the C-terminus (C-pep) of the LPXTG-adhesin SspA intercalates in the cell membrane. Nested in the membrane, this C-pep docks with the intramembrane sensor histidine kinase, SraS, to activate the response regulator, SraR. SraR signals that the C-pep has been cleaved as an indicator of the fidelity of sortase A processing. SraSR also signals that key LPXTG-proteins in concert with lipoteichoic acid engage the mucin, MUC5B, which elicits a different transcriptional response than the binding of other salivary constituents. To visualize the C-pep intercalating in the cell membrane in vivo, we used Structured Illumination Microscopy (SIM). And to show that the C-pep complexes with SraS, we used bimolecular fluorescence experiments. The C-pep and SraS were each expressed with one or the other half of yellow fluorescence protein (YFP). Reconstitution of the complete YFP signal indicated that the C-pep and SraS interacted at molecular distances within the cell membrane in vivo. Using these imaging protocols, we learned that the C-pep functions as a signaling molecule within the cell membrane of the streptococcal cell.

New Cyclopiane Diterpenes and Polyketide Derivatives from Marine Sediment-Derived Fungus Penicillium antarcticum KMM 4670 and Their Biological Activities.

Two new cyclopiane diterpenes and a new cladosporin precursor, together with four known related compounds, were isolated from the marine sediment-derived fungus Penicillium antarcticum KMM 4670, which was re-identified based on phylogenetic inference from ITS, BenA, CaM, and RPB2 gene regions. The absolute stereostructures of the isolated cyclopianes were determined using modified Mosher's method and quantum chemical calculations of the ECD spectra. The isolation from the natural source of two biosynthetic precursors of cladosporin from a natural source has been reported for the first time. The antimicrobial activities of the isolated compounds against Staphylococcus aureus, Escherichia coli, and Candida albicans as well as the inhibition of staphylococcal sortase A activity were investigated. Moreover, the cytotoxicity of the compounds to mammalian cardiomyocytes H9c2 was studied. As a result, new cyclopiane diterpene 13-epi-conidiogenone F was found to be a sortase A inhibitor and a promising anti-staphylococcal agent.