Search Results (29)

Listeria monocytogenes (LM) is a lethal foodborne intracellular pathogen. Internalins A and B (inlA and inlB) are critical virulence factors that promote LM’s adhesion and invasion into host cells. InlA is covalently anchored to the cell wall by LM SortaseA (SrtA), while inlB is anchored to the cell wall via non-covalent bonds. Therefore, inhibiting SrtA and inlB is expected to suppress LM’s adhesion and invasion of host cells, enabling the prevention and control of infections. This study demonstrated that Luteolin inhibited the activity of purified LM SrtA protein in vitro. Interactive mechanism analysis indicated that Luteolin generates interaction with the critical active sites of SrtA, which may affect its binding to its natural substrates, thereby reducing the anchoring of inlA on the cell wall and achieving the inhibition of bacterial adhesion and invasion. In addition, Luteolin binds to the groove at the binding interface between inlB and its host receptor. The key residues in inlB that interact with the host receptor form weak interactions (Hydrogen bonds and van der Waals interactions) with Luteolin, this binding may inhibit their binding, suppressing LM’s adhesion and invasion of host cells. At the tested concentrations, Luteolin did not affect the growth of LM, but remarkably reduced the mortality and alleviated the infection symptoms of LM-infected Galleria mellonella. These results provide additional theoretical evidence for the application of Luteolin in the prevention and control of LM infections, which is expected to accelerate its application progress. Full article

Streptococcus pneumoniae (S. pneumoniae) Sortase A (SrtA) anchors virulence proteins to the surface of the cell wall by recognizing and cleaving the LPXTG motif. These toxins help bacteria adhere to and colonize host cells, promote biofilm formation, and trigger host inflammatory responses. Therefore, SrtA is an ideal target for the development of new preparations for S. pneumoniae. In this study, we found that phloretin (pht) and phlorizin (phz) exhibited excellent affinities for SrtA based on virtual screening experiments. We analyzed the interactive mechanism between pht, phz, and alnusone (aln, a reported S. pneumoniae SrtA inhibitor) and SrtA based on molecular dynamics simulation experiments. The results showed that these inhibitors bound to the active pocket of SrtA, and the root mean square deviation (RMSD) and distance analyses showed that these compounds and SrtA maintained stable configuration and binding during the assay. The binding free energy analysis showed that both electrostatic forces (ele), van der Waals forces (vdw), and hydrogen bonds (Hbonds) promoted the binding between pht, phz, and SrtA; however, for the binding of aln and SrtA, the vdw force was much stronger than ele, and Hbonds were not found. The binding free energy decomposition showed that HIS141, ILE143, and PHE119 contributed more energy to promote pht and SrtA binding; ARG215, ASP188, and LEU210 contributed more energy to promote phz and SrtA binding; and HIS141, ASP209, and ARG215 contributed more energy to promote aln and SrtA binding. Finally, the transpeptidase activity of SrtA decreased significantly when treated with different concentrations of pht, phz, or aln, which inhibited S. pneumoniae biofilm formation and adhesion to A549 cells without affecting normal bacterial growth. These results suggest that pht, phtz, and aln are potential materials for the development of novel inhibitors against S. pneumoniae infection. Full article

Sortase A (SrtA) of Staphylococcus aureus has long been shown to be a relevant molecular target for antibacterial development. Moreover, the designed SrtA inhibitors act via the antivirulence mechanism, potentially causing less evolutional pressure and reduced antimicrobial resistance. However, no marketed drugs or even drug candidates have been reported until recently, despite numerous efforts in the field. SrtA has been shown to be a tough target for rational structure-based drug design (SBDD), which hampers the regular development of small-molecule inhibitors using the available arsenal of drug discovery tools. Recently, several oligopeptides resembling the sorting sequence LPxTG (Leu-Pro-Any-Thr-Gly) of the native substrates of SrtA were reported to be active in the micromolar range. Despite the good experimental design of those works, their molecular modeling parts are still not convincing enough to be used as a basis for a rational modification of peptidic inhibitors. In this work, we propose to use the ensemble docking approach, in which the relevant SrtA conformations are extracted from the molecular dynamics simulation of the LPRDA (Leu-Pro-Arg-Asp-Ala)-SrtA complex, to effectively represent the most significant and diverse target conformations. The developed protocol is shown to describe the known experimental data well and then is applied to a series of new peptidomimetic molecules resembling the active oligopeptide structures reported previously in order to prioritize structures from this work for further synthesis and activity testing. The proposed approach is compared to existing alternatives, and further directions for its development are outlined. Full article

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. Full article

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. Full article

Staphylococcus aureus (S. aureus), commonly known as “superbugs”, is a highly pathogenic bacterium that poses a serious threat to human health. There is an urgent need to replace traditional antibiotics with novel drugs to combat S. aureus. Sortase A (SrtA) is a crucial transpeptidase involved in the adhesion process of S. aureus. The reduction in virulence and prevention of S. aureus infections have made it a significant target for antimicrobial drugs. In this study, we combined virtual screening with experimental validation to identify potential drug candidates from a drug library. Three hits, referred to as Naldemedine, Telmisartan, and Azilsartan, were identified based on docking binding energy and the ratio of occupied functional sites of SrtA. The stability analysis manifests that Naldemedine and Telmisartan have a higher binding affinity to the hydrophobic pockets. Specifically, Telmisartan forms stable hydrogen bonds with SrtA, resulting in the highest binding energy. Our experiments prove that the efficiency of adhesion and invasion by S. aureus can be decreased without significantly affecting bacterial growth. Our work identifies Telmisartan as the most promising candidate for inhibiting SrtA, which can help combat S. aureus infection. Full article

Bacterial surface proteins assembled into amyloids contribute to biofilm formation and host immune evasion. Streptococcus sanguinis, a pioneer colonizer of teeth commonly involved in cardiovascular infections, expresses about thirty-three proteins anchored to the cell wall by sortase A. Here, we characterized the production of amyloid in S. sanguinis strains differing in biofilm and immune evasion phenotypes and investigated the role of sortase A in amyloidogenesis. Amyloid was identified in biofilms formed by nine strains, using Congo red (CR) staining and cross-polarized light microscopy. Additionally, EGCG, an amyloid inhibitor, impaired biofilm maturation in a strain-specific fashion. The amounts of amyloid-like components quantified in culture fluids of nine strains using thioflavin T and fluorimetry negatively correlated with bacterial binding to complement-activating proteins (SAP, C1q), C3b deposition and rates of opsonophagocytosis in PMNs, implying amyloid production in immune evasion. The deletion of the sortase A gene (srtA) in strain SK36 compromised amyloid production and sucrose-independent biofilm maturation. The srtA mutant further showed increased susceptibility to C3b deposition and altered interactions with PMNs as well as reduced persistence in human blood. These findings highlight the contribution of amyloids to biofilm formation and host immune evasion in S. sanguinis strains, further indicating the participation of sortase A substrates in amyloidogenesis. Full article

Sortase A (SrtA) of Staphylococcus aureus is a well-defined molecular target to combat the virulence of these clinically important bacteria. However up to now no efficient drugs or even clinical candidates are known, hence the search for such drugs is still relevant and necessary. SrtA is a complex target, so many straight-forward techniques for modeling using the structure-based drug design (SBDD) fail to produce the results they used to bring for other, simpler, targets. In this work we conduct theoretical studies of the binding/activity of Leu-Pro-Arg-Asp-Ala (LPRDA) polypeptide, which was recently shown to possess antivirulence activity against S. aureus. Our investigation was aimed at establishing a framework for the estimation of the key interactions and subsequent modification of LPRDA, targeted at non-peptide molecules, with better drug-like properties than the original polypeptide. Firstly, the available PDB structures are critically analyzed and the criteria to evaluate the quality of the ligand–SrtA complex geometry are proposed. Secondly, the docking protocol was investigated to establish its applicability to the LPRDA–SrtA complex prediction. Thirdly, the molecular dynamics studies were carried out to refine the geometries and estimate the stability of the complexes, predicted by docking. The main finding is that the previously reported partially chaotic movement of the β6/β7 and β7/β8 loops of SrtA (being the intrinsically disordered parts related to the SrtA binding site) is exaggerated when SrtA is complexed with LPRDA, which in turn reveals all the signs of the flexible and structurally disordered molecule. As a result, a wealth of plausible LPRDA–SrtA complex conformations are hard to distinguish using simple modeling means, such as docking. The use of more elaborate modeling approaches may help to model the system reliably but at the cost of computational efficiency. Full article

The coding region for the sortase A (SrtA) of Staphylococcus aureus was fused at the N-terminus of LfcinB. The SrtA-LfcinB fusion protein in E. coli C43(DE3) was expressed with the expected sizes of 21 kDa and 38 kDa by pET21b-SrtA-LfcinB and pET32-1SrtA-LfcinB constructs, respectively. Increased levels of the TrxA-His-SrtA-SrtA-LfcinB fusion protein were detected by the pET32-3SrtA-LfcinB construct having three expression cassettes. LfcinB is released from the expressed SrtA-LfcinB protein by SrtA self-cleavage which is induced in the presence of Ca²⁺. The antibacterial activity was detected after SrtA-mediated cleavage of LfcinB. Furthermore, to reduce the antimicrobial peptide toxicity to the E. coli host, the human interferon-γ (hIFN-γ) sequences were mutated into a negatively charged mIFc2 protein (7 kDa), which was co-expressed with LfcinB in an insoluble form. The yield of LfcinB was elevated while changing the gene order of LfcinB and mIFc2 (pET21b-fLfcinB-bmIFc2). Furthermore, increased levels of LfcinB were detected using the pET21b-(fLfcinB-bmIFc2)₂ construct. To increase the dissolution rate of inclusion bodies, inclusion bodies treated with different temperatures and pH and resuspended in different volumes of 50 mM Tris-HCl were assayed. Our results reveal that heat-treated LfcinB/mIFc2 inclusion bodies at 90 °C, pH 10, and 16X resuspended volumes have the best resolubilization rate. This work suggests that the mIFc2 co-expression system shows higher efficiency for LfcinB production than the SrtA fusion system. The expressed LfcinB from the mIFc2 co-expression system exhibits excellent broad-spectrum antibacterial activities against thirteen Gram-negative and ten Gram-positive bacteria species with a range of minimum inhibitory concentrations (MIC) between 37–150 ug/mL. Full article

Sortase A (SrtA) of Staphylococcus aureus has been identified as a promising target to a new type of antivirulent drugs, and therefore, the design of lead molecules with a low nanomolar range of activity and suitable drug-like properties is important. In this work, we aimed at identifying new fragment-sized starting points to design new noncovalent S. aureus SrtA inhibitors by making use of the dedicated molecular motif, 5-arylpyrrolidine-2-carboxylate, which has been previously shown to be significant for covalent binding SrtA inhibitors. To this end, an in silico approach combining QSAR and molecular docking studies was used. The known SrtA inhibitors from the ChEMBL database with diverse scaffolds were first employed to derive descriptors and interpret their significance and correlation to activity. Then, the classification and regression QSAR models were built, which were used for rough ranking of the virtual library of the synthetically feasible compounds containing the dedicated motif. Additionally, the virtual library compounds were docked into the “activated” model of SrtA (PDB:2KID). The consensus ranking of the virtual library resulted in the most promising structures, which will be subject to further synthesis and experimental testing in order to establish new fragment-like molecules for further development into antivirulent drugs. Full article

Growth of the lactic acid bacterium Streptococcus thermophilus in milk depends on its capacity to hydrolyze proteins of this medium through its surface proteolytic activity. Thus, strains exhibiting the cell envelope proteinase (CEP) PrtS are able to grow in milk at high cellular density. Due to its LPNTG motif, which is possibly the substrate of the sortase A (SrtA), PrtS is anchored to the cell wall in most S. thermophilus strains. Conversely, a soluble extracellular PrtS activity has been reported in the strain 4F44. It corresponds, in fact, to a certain proportion of PrtS that is not anchored to the cell wall but rather is released in the growth medium. The main difference between PrtS of strain 4F44 (PrtS_4F44) and other PrtS concerns the absence of a 32-residue imperfect duplication in the prodomain of the CEP, postulated as being required for the maturation and correct subsequent anchoring of PrtS. In fact, both mature (without the prodomain at the N-terminal extremity) and immature (with the prodomain) forms are found in the soluble PrtS_4F44 form along with an intact LPNTG at their C-terminal extremity. Investigations we present in this work show that (i) the imperfect duplication is not implied in PrtS maturation; (ii) the maturase PrtM is irrelevant in PrtS maturation which is probably automaturated; and (iii) SrtA allows for the PrtS anchoring in S. thermophilus but the SrtA of strain 4F44 (SrtA_4F44) displays an altered activity. Full article

Background: antibiotic resistance encourages the development of new therapies, or the discovery of novel antibacterial agents. Previous research revealed that Myrmecodia pendans (Sarang Semut) contain potential antibacterial agents. However, specific proteins inhibited by them have not yet been identified as either proteins targeted by antibiotics or proteins that have a role in the quorum-sensing system. This study aims to investigate and predict the action mode of antibacterial compounds with specific proteins by following the molecular docking approach. Methods: butein (1), biflavonoid (2), 3″-methoxyepicatechin-3-O-epicatechin (3), 2-dodecyl-4-hydroxylbenzaldehyde (4), 2-dodecyl-4-hydroxylbenzaldehyde (5), pomolic acid (6), betulin (7), and sitosterol-(6′-O-tridecanoil)-3-O-β-D-glucopyranoside (8) from M. pendans act as the ligand. Antibiotics or substrates in each protein were used as a positive control. To screen the bioactivity of compounds, ligands were analyzed by Prediction of Activity Spectra for Substances (PASS) program. They were docked with 12 proteins by AutoDock Vina in the PyRx 0.8 software application. Those proteins are penicillin-binding protein (PBP), MurB, Sortase A (SrtA), deoxyribonucleic acid (DNA) gyrase, ribonucleic acid (RNA) polymerase, ribosomal protein, Cytolysin M (ClyM), FsrB, gelatinase binding-activating pheromone (GBAP), and PgrX retrieved from UniProt. The docking results were analyzed by the ProteinsPlus and Discovery Studio software applications. Results: most compounds have Pa value over 0.5 against proteins in the cell wall. In nearly all proteins, biflavonoid (2) has the strongest binding affinity. However, compound 2 binds only three residues, so that 2 is the non-competitive inhibitor. Conclusion: compound 2 can be a lead compound for an antibacterial agent in each pathway. Full article

Staphylococcus aureus is still one of the leading causes of both hospital- and community-acquired infections. Due to the very high percentage of drug-resistant strains, the participation of drug-tolerant biofilms in pathological changes, and thus the limited number of effective antibiotics, there is an urgent need to search for alternative methods of prevention or treatment for S. aureus infections. In the present study, biochemically characterized (HPLC/UPLC–QTOF–MS) acetonic, ethanolic, and water extracts from fruits and bark of Viburnum opulus L. were tested in vitro as diet additives that potentially prevent staphylococcal infections. The impacts of V. opulus extracts on sortase A (SrtA) activity (Fluorimetric Assay), staphylococcal protein A (SpA) expression (FITC-labelled specific antibodies), the lipid composition of bacterial cell membranes (LC-MS/MS, GC/MS), and biofilm formation (LIVE/DEAD BacLight) were assessed. The cytotoxicity of V. opulus extracts to the human fibroblast line HFF-1 was also tested (MTT reduction). V. opulus extracts strongly inhibited SrtA activity and SpA expression, caused modifications of S. aureus cell membrane, limited biofilm formation by staphylococci, and were non-cytotoxic. Therefore, they have pro-health potential. Nevertheless, their usefulness as diet supplements that are beneficial for the prevention of staphylococcal infections should be confirmed in animal models in the future. Full article

Rapid spread of antibiotic resistance throughout the kingdom bacteria is inevitably bringing humanity towards the “post-antibiotic” era. The emergence of so-called “superbugs”—pathogen strains that develop resistance to multiple conventional antibiotics—is urging researchers around the globe to work on the development or perfecting of alternative means of tackling the pathogenic bacteria infections. Although various conceptually different approaches are being considered, each comes with its advantages and drawbacks. While drug-resistant pathogens are undoubtedly represented by both Gram(+) and Gram(−) bacteria, possible target spectrum across the proposed alternative approaches of tackling them is variable. Numerous anti-virulence strategies aimed at reducing the pathogenicity of target bacteria rather than eliminating them are being considered among such alternative approaches. Sortase A (SrtA) is a membrane-associated cysteine protease that catalyzes a cell wall sorting reaction by which surface proteins, including virulence factors, are anchored to the bacterial cell wall of Gram(+) bacteria. Although SrtA inhibition seems perspective among the Gram-positive pathogen-targeted antivirulence strategies, it still remains less popular than other alternatives. A decrease in virulence due to inactivation of SrtA activity has been extensively studied in Staphylococcus aureus, but it has also been demonstrated in other Gram(+) species. In this manuscript, results of past studies on the discovery of novel SrtA inhibitory compounds and evaluation of their potency were summarized and commented on. Here, we discussed the rationale behind the inhibition of SrtA, raised some concerns on the comparability of the results from different studies, and touched upon the possible resistance mechanisms as a response to implementation of such therapy in practice. The goal of this article is to encourage further studies of SrtA inhibitory compounds. Full article