Comprehensive virulence profiling and evolutionary analysis of specificity determinants in Staphylococcus aureus two-component systems.

In the Staphylococcus aureus genome, a set of highly conserved two-component systems (TCSs) composed of histidine kinases (HKs) and their cognate response regulators (RRs) sense and respond to environmental stimuli, which drive the adaptation of the bacteria. This study investigates the complex interplay between TCSs in S. aureus USA300, a predominant methicillin-resistant S. aureus strain, revealing shared and unique virulence regulatory pathways and genetic variations mediating signal specificity within TCSs. Using TCS-related mutants from the Nebraska Transposon Mutant Library, we analyzed the effects of inactivated TCS HKs and RRs on the production of various virulence factors, in vitro infection abilities, and adhesion assays. We found that the TCSs' influence on virulence determinants was not associated with their phylogenetic relationship, indicating divergent functional evolution. Using the co-crystallized structure of the DesK-DesR from Bacillus subtilis and the modeled structures of the four NarL TCSs in S. aureus, we identified interacting residues, revealing specificity determinants and conservation within the same TCS, even from different strain backgrounds. The interacting residues were highly conserved within strains but varied between species due to selection pressures and the coevolution of cognate pairs. This study unveils the complex interplay and divergent functional evolution of TCSs, highlighting their potential for future experimental exploration of phosphotransfer between cognate and non-cognate recombinant HK and RRs.IMPORTANCEGiven the widespread conservation of two-component systems (TCSs) in bacteria and their pivotal role in regulating metabolic and virulence pathways, they present a compelling target for anti-microbial agents, especially in the face of rising multi-drug-resistant infections. Harnessing TCSs therapeutically necessitates a profound understanding of their evolutionary trajectory in signal transduction, as this underlies their unique or shared virulence regulatory pathways. Such insights are critical for effectively targeting TCS components, ensuring an optimized impact on bacterial virulence, and mitigating the risk of resistance emergence via the evolution of alternative pathways. Our research offers an in-depth exploration of virulence determinants controlled by TCSs in S. aureus, shedding light on the evolving specificity determinants that orchestrate interactions between their cognate pairs.

Characterization of the broad-spectrum antibacterial activity of bacteriocin-like inhibitory substance-producing probiotics isolated from fermented foods.

Antimicrobial peptides, such as bacteriocin, produced by probiotics have become a promising novel class of therapeutic agents for treating infectious diseases. Selected lactic acid bacteria (LAB) isolated from fermented foods with probiotic potential were evaluated for various tests, including exopolysaccharide production, antibiotic susceptibility, acid and bile tolerance, antibacterial activity, and cell adhesion and cytotoxicity to gastric cell lines. Six selected LAB strains maintained their high viability under gastrointestinal conditions, produced high exopolysaccharides, showed no or less cytotoxicity, and adhered successfully to gastric cells. Furthermore, three strains, Weissella confusa CYLB30, Lactiplantibacillus plantarum CYLB47, and Limosilactobacillus fermentum CYLB55, demonstrated a strong antibacterial effect against drug-resistant Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enterica serovar Choleraesuis, Enterococcus faecium, and Staphylococcus aureus. Whole genome sequencing was performed on these three strains using the Nanopore platform; then, the results showed that all three strains did not harbor genes related to toxins, superantigens, and acquired antimicrobial resistance, in their genome. The bacteriocin gene cluster was found in CYLB47 genome, but not in CYLB30 and CYLB55 genomes. In SDS-PAGE, the extract of CYLB30 and CYLB47 bacteriocin-like inhibitory substance (BLIS) yielded a single band with a size of less than 10 kDa. These BLIS inhibited the growth and biofilm formation of drug-resistant P. aeruginosa and methicillin-resistant S. aureus (MRSA), causing membrane disruption and inhibiting adhesion ability to human skin HaCaT cells. Moreover, CYLB30 and CYLB47 BLIS rescued the larvae after being infected with P. aeruginosa and MRSA infections. In conclusion, CYLB30 and CYLB47 BLIS may be potential alternative treatment for multidrug-resistant bacteria infections.

Cryo-EM analysis of S. aureus TarL, a polymerase in wall teichoic acid biogenesis central to virulence and antibiotic resistance.

Wall teichoic acid (WTA), a covalent adduct of Gram-positive bacterial cell wall peptidoglycan, contributes directly to virulence and antibiotic resistance in pathogenic species. Polymerization of the Staphylococcus aureus WTA ribitol-phosphate chain is catalyzed by TarL, a member of the largely uncharacterized TagF-like family of membrane-associated enzymes. We report the cryo-electron microscopy structure of TarL, showing a tetramer that forms an extensive membrane-binding platform of monotopic helices. TarL is composed of an amino-terminal immunoglobulin-like domain and a carboxyl-terminal glycosyltransferase-B domain for ribitol-phosphate polymerization. The active site of the latter is complexed to donor substrate cytidine diphosphate-ribitol, providing mechanistic insights into the catalyzed phosphotransfer reaction. Furthermore, the active site is surrounded by electropositive residues that serve to retain the lipid-linked acceptor for polymerization. Our data advance general insight into the architecture and membrane association of the still poorly characterized monotopic membrane protein class and present molecular details of ribitol-phosphate polymerization that may aid in the design of new antimicrobials.

Novel antimicrobial peptides identified in legume plant, Medicago truncatula.

One of the major issues in healthcare today is antibiotic resistance. Antimicrobial peptides (AMPs), a subclass of host defense peptides, have been suggested as a viable solution for the multidrug resistance problem. Legume plants express more than 700 nodule-specific cysteine-rich (NCR) peptides. Three NCR peptides (NCR094, NCR888, and NCR992) were predicted to have antimicrobial activity using in silico AMP prediction programs. This study focused on investigating the roles of the NCRs in antimicrobial activity and antibiofilm activity, followed by in vitro toxicity profiling. Different variants were synthesized, i.e., mutated and truncated derivatives. The effect on the growth of Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus (MRSA) was monitored post-treatment, and survived cells were counted using an in vitro and ex vivo killing assay. The antibiofilm assay was conducted using subinhibitory concentrations of the NCRs and monitoring K. pneumoniae biomass, followed by crystal violet staining. The cytotoxicity profile was evaluated using erythrocyte hemolysis and leukemia (K562) cell line toxicity assays. Out of the NCRs, NCR094 and NCR992 displayed mainly in vitro and ex vivo bactericidal activity on K. pneumoniae. NCR094 wild type (WT) and NCR992 eradicated K. pneumoniae at different potency; NCR094 and NCR992 killed K. pneumoniae completely at 25 and 50 µM, respectively. However, both peptides in the wild type showed negligible bactericidal effect on MRSA in vitro and ex vivo. NCR094 and its derivatives relatively retained the antimicrobial activity on K. pneumoniae in vitro and ex vivo. NCR992 WT lost its antimicrobial activity on K. pneumoniae ex vivo, yet the different truncated and mutated variants retained some of the antimicrobial role ex vivo. All the different variants of NCR094 had no effect on MRSA in vitro and ex vivo. Similarly, NCR992's variants had a negligible bactericidal role on MRSA in vitro, yet the truncated variants had a significantly high bactericidal effect on MRSA ex vivo. NCR094.3 (cystine replacement variant) and NCR992.1 displayed significant antibiofilm activity more than 90%. NCR992.3 and NCR992.2 displayed more than 50% of antibiofilm activity. All the NCR094 forms had no toxicity, except NCR094.1 (49.38%, SD ± 3.46) and all NCR992 forms (63%-93%), which were above the cutoff (20%). Only NCR992.2 showed low toxicity on K562 (24.8%, SD ± 3.40), yet above the 20% cutoff. This study provided preliminary antimicrobial and safety data for the potential use of these peptides for therapeutical applications.IMPORTANCEThe discovery of new antibiotics is urgently needed, given the global expansion of antibiotic-resistant bacteria and the rising mortality rate. One of the initial lines of defense against microbial infections is antimicrobial peptides (AMPs). Plants can express hundreds of such AMPs as defensins and defensin-like peptides. The nodule-specific cysteine-rich (NCR) peptides are a class of defensin-like peptides that have evolved in rhizobial-legume symbioses. This study screened the antimicrobial activity of a subset of NCR sequences using online computational AMP prediction algorithms. Two novel NCRs, NCR094 and NCR992, with different variants were identified to exhibit antimicrobial activity with various potency on two problematic pathogens, K. pneumoniae and MRSA, using in vitro and ex vivo killing assays. Yet, one variant, NCR094.3, had no toxicity toward human cells and displayed antibiofilm activity, which make it a promising lead for antimicrobial drug development.

Mycosynthesis of selenium nanoparticles using Penicillium tardochrysogenum as a therapeutic agent and their combination with infrared irradiation against Ehrlich carcinoma.

Over the past years, the assessment of myco-fabricated selenium nanoparticles (SeNPs) properties, is still in its infancy. Herein, we have highly stable myco-synthesized SeNPs using molecularly identified soil-isolated fungus; Penicillium tardochrysogenum OR059437; (PeSeNPs) were clarified via TEM, EDX, UV-Vis spectrophotometer, FTIR and zeta potential. The therapeutic efficacy profile will be determined, these crystalline PeSeNPs were examined for antioxidant, antimicrobial, MIC, and anticancer potentials, indicating that, PeSeNPs have antioxidant activity of (IC50, 109.11 µg/mL) using DPPH free radical scavenging assay. Also, PeSeNPs possess antimicrobial potential against Penicillium italicum RCMB 001,018 (1) IMI 193,019, Methicillin-Resistant Staphylococcus aureus (MRSA) ATCC 4330 and Porphyromonas gingivalis RCMB 022,001 (1) EMCC 1699; with I.Z. diameters and MIC; 16 ± 0.5 mm and MIC 500 µg/ml, 11.9 ± 0.6 mm, 500 µg/ml and 15.9±0.6 mm, 1000 µg/ml, respectively. Additionally, TEM micrographs were taken for P. italicum treated with PeSeNPs, demonstrating the destruction of hyphal membrane and internal organelles integrity, pores formation, and cell death. PeSeNP alone in vivo and combined with a near-infrared physiotherapy lamp with an energy intensity of 140 mW/cm2 showed a strong therapeutic effect against cancer cells. Thus, PeSeNPs represent anticancer agents and a suitable photothermal option for treating different kinds of cancer cells with lower toxicity and higher efficiency than normal cells. The combination therapy showed a very large and significant reduction in tumor volume, the tumor cells showed large necrosis, shrank, and disappeared. There was also improvement in liver ultrastructure, liver enzymes, and histology, as well as renal function, urea, and creatinine.

Precursor-directed biosynthesis and biological activity of tripropeptin Cpip, a new tripropeptin C analog containing pipecolic acid.

Tripropeptin C, a non-ribosomal cyclic lipopeptide containing three proline residues, exhibits excellent efficacy in the mouse-methicillin-resistant Staphylococcus aureus septicemia model. Since tripropeptins contain L-prolyl-D-proline and, as a result, are known to form a hairpin structure in proteins, it was of interest to determine whether this substructure contributes to their antibacterial activity. In this study, prolines in tripropeptin C were replaced with pipecolic acid(s) using precursor-directed biosynthesis. Only a new tripropeptin analog, tripropeptin Cpip, which has one L-pipecolic acid in place of L-proline, was isolated. The in vitro antimicrobial activity of the new analog was approximately two to four times weaker activity against Gram-positive bacteria, including drug-resistant species, compared with that of tripropeptin C. These results suggest that the L-prolyl-D-proline substructure plays an important role in the observed potency of tripropeptins.

Prostaglandin E2 accumulation is closely associated with S. aureus-infected bovine endometritis.

S. aureus isolated from bacterial bovine endometritis is common in epidemiological reports, but is often ignored as a subclinical pathogenic microorganism. In a previous study, we showed that live S. aureus (LSA) and heat killed S. aureus (HK-SA) induce different inflammatory responses in bovine endometrial tissue, and possibly being associated with the accumulation of prostaglandin E2 (PGE2). Thus, in this study, we varied PGE2 concentrations using inhibitors or agonists in HK-SA-treated bovine endometrial tissues. The results demonstrated that PGE2 has a positive relationship with IL-6, TNF-α, and damage-associated molecular patterns (DAMPs; e.g., HMGB-1 and HABP-1) expression and tissues damage, and is regulated by the EP4-p38 MAPK pathway. We concluded that lipoproteins of S. aureus are associated with PGE2 generation. To further explore the relationship between LSA and PGE2 accumulation, we used the S. aureus strain SA113 lipoprotein knockout (SA113Δlpl) to infect bovine endometrial epithelial cells (BECs). LSA decreased PGE2, cAMP, EP4, IL-6, IL-8, cAMP secretion, and the MAPK and PKA signaling pathways when infected with SA113Δlpl, as compared with SA113-infected groups. Moreover, the adhesion and invasion of BECs were similarly downregulated when lipoproteins in S. aureus were knocked out. The results of this study show that PGE2 is involved in both HK-SA- and LSA-induced inflammatory responses in the bovine endometrium. We suggest that S. aureus infection is associated with bovine endometritis, and although HK-SA and LSA induce different inflammatory responses, the strategy of decreasing PGE2 accumulation is helpful in reducing the inflammation stage caused by S. aureus.

Synergistic effect of secondary metabolites isolated from Pestalotiopsis sp. FKR-0115 in overcoming ß-lactam resistance in MRSA.

Six aromatic secondary metabolites, pestalone (1), emodin (2), phomopsilactone (3), pestalachlorides B (4), C (5), and D (6), were isolated from Pestalotiopsis sp. FKR-0115, a filamentous fungus collected from white moulds growing on dead branches in Minami Daito Island. The efficacy of these secondary metabolites against methicillin-resistant Staphylococcus aureus (MRSA) with and without meropenem (ß-lactam antibiotic) was evaluated using the paper disc method and broth microdilution method. The chemical structures of the isolated compounds (1-6) were characterised using spectroscopic methods, including nuclear magnetic resonance and mass spectrometry. All six isolated compounds exhibited synergistic activity with meropenem against MRSA. Among the six secondary metabolites, pestalone (1) overcame bacterial resistance in MRSA to the greatest extent.

Engraulisin: A novel marine derived cell penetrating peptide with activity against drug resistant bacteria.

Cell penetrating peptides (CPP) with their intrinsic ability to penetrate plasma membranes facilitate intracellular uptake of various macromolecules. Although a substantial number of CPPs have been reported over the last three decades, the number is still inadequate when compared to the theoretically feasible peptides with similar physicochemical composition. Marine organisms, due to their hostile environment, are an immense source of several high-valued therapeutically relevant peptides. Various marine derived antibacterial, antimycotic and anticancer peptides have demonstrated improved activity in comparison to peptides of terrestrial origin. While a significant number of marine bioactive peptides exist, cell penetrating peptides from marine organisms remain unravelled. In this study, we report Engraulisin from Engraulis japonicus, a computationally derived novel cell penetrating peptide of marine origin. Engraulisin manifest successful uptake in mammalian cells at 5 µM concentration with negligible cytotoxicity observed through MTT assay. Analysis of its cellular uptake mechanism revealed significant inhibition at 4 °C suggesting endocytosis as the major route of cellular entry. Interestingly, the novel peptide also demonstrated selective antimicrobial activity against Methicillin-resistant Staphylococcus aureus (MRSA). Additionally, molecular dynamics simulation with POPC and POPG bilayer system unveiled significance of positively charged residues in forming a stable membrane interaction. Engraulisin represents a novel marine-derived cell penetrating peptide which can be explored for cellular delivery of pharmaceutically relevant molecules.

Quercetin targets SarA of methicillin-resistant Staphylococcus aureus to mitigate biofilm formation.

IMPORTANCE: Anti-biofilm is an important strategy against Staphylococcus aureus chronic infection. SarA is a positive regulator of biofilm formation in S. aureus. In this study, we identified the SarA inhibitor quercetin using computer simulation screening. Previous studies have shown that quercetin inhibits biofilm; however, the underlying mechanism remains unknown. This study revealed the inhibitory effect of quercetin on the SarA protein. We also isolated the SarA protein and confirmed its interaction with quercetin in vitro. Besides, the inhibitory effect of quercetin on the transcription and translation levels of the SarA protein was also determined. The effects of quercetin on S. aureus biofilm inhibition and biofilm components were consistent with the changes in the transcription level of biofilm-related genes regulated by SarA. In summary, our study revealed the mechanism by which quercetin affects biofilm formation by inhibiting the transcriptional regulator SarA of S. aureus.

Endogenous blocking of TLR2 along with TNF-α and IL-1ß ameliorates the severity of the S. aureus arthritis via modulating STAT3/SOCS3 expressions in tissue resident macrophages.

In vivo studies identifying a role of TLR2 in septic arthritis models are lacking. TNF-α played as the most important proinflammatory cytokine, and connected directly to the pathogenesis of bacterial arthritis. IL-1ß is another central mediator cytokine in arthritis. It is therefore reasonable to question the role of neutralization of endogenous TNF-α and IL-1ß along with TLR2 and associated downstream signaling as crucial mediators in the S. aureus -induced inflammatory arthritis. In reaction to an injury or a pathogen encounter, innate immune cells serve as the initial line of defense. TLR2 mediated entry of S. aureus into macrophage cells initiates an array of inflammatory cascades. After macrophage cell gets activated at the site inflammation, they generate elevated number of cytokines which includes TNF-α, IL-1ß. This cytokines signals through STAT1/STAT3 mediated pathways. Thus, aim of this study was to discover how This bone damage could be altered by altering the STAT/STAT3/SOCS3 ratio by blocking TLR2, a particular S. aureus binding site, in conjunction with the use of IL-1 and TNF- antibodies for neutralizing endogenous IL-1ß and TNF-α. Additionally, the role of local macrophages in therapy of arthritis was investigated in synovial and Splenic tissue. To comprehend the inflammatory milieu within the system, ROS and other antioxidant enzymes, along with the expression of mTOR in macrophage cells, were also taken into consideration. The detrimental impact of bacterial burden on synovial joints was reduced by simultaneously inhibiting TLR2, TNF-α, and IL-1ß. Lowered IFN-γ decreases its sensitivity to STAT1 and lowered IL-6 reduces STAT3 expressions. Whereas, elevated IL-10 enhances SOSC3 expression, which thereby able to limits STAT1/STAT3 inter-conversion. As a result, NF-κB activity was downregulated.

Prenylated isoflavonoids from Fabaceae against the NorA efflux pump in Staphylococcus aureus.

Overexpression of NorA efflux pumps plays a pivotal role in the multidrug-resistance mechanism in S. aureus. Here, we investigated the activities of prenylated isoflavonoids, present in the legume plant family (Fabaceae), as natural efflux pump inhibitors (EPIs) in fluoroquinolone-resistant S. aureus. We found that four prenylated isoflavonoids, namely neobavaisoflavone, glabrene, glyceollin I, and glyceollin III, showed efflux pump inhibition in the norA overexpressing S. aureus. At sub-inhibitory concentrations, neobavaisoflavone (6.25 µg/mL, 19 µM) and glabrene (12.5 µg/mL, 39 µM), showed up to 6 times more Eth accumulation in norA overexpressing S. aureus than in the control. In addition, these two compounds boosted the MIC of fluoroquinolones up to eightfold. No fluoroquinolone potentiation was observed with these isoflavonoids in the norA knockout strain, indicating NorA as the main target of these potential EPIs. In comparison to the reported NorA EPI reserpine, neobavaisoflavone showed similar potentiation of fluoroquinolone activity at 10 µM, higher Eth accumulation, and less cytotoxicity. Neobavaisoflavone and glabrene did not exhibit membrane permeabilization effects or cytotoxicity on Caco-2 cells. In conclusion, our findings suggest that the prenylated isoflavonoids neobavaisoflavone and glabrene are promising phytochemicals that could be developed as antimicrobials and resistance-modifying agents to treat fluoroquinolone-resistant S. aureus strains.

Comparative Chemical Profiling and Antimicrobial/Anticancer Evaluation of Extracts from Farmed versus Wild Agelas oroides and Sarcotragus foetidus Sponges.

Marine sponges are highly efficient in removing organic pollutants and their cultivation, adjacent to fish farms, is increasingly considered as a strategy for improving seawater quality. Moreover, these invertebrates produce a plethora of bioactive metabolites, which could translate into an extra profit for the aquaculture sector. Here, we investigated the chemical profile and bioactivity of two Mediterranean species (i.e., Agelas oroides and Sarcotragus foetidus) and we assessed whether cultivated sponges differed substantially from their wild counterparts. Metabolomic analysis of crude sponge extracts revealed species-specific chemical patterns, with A. oroides and S. foetidus dominated by alkaloids and lipids, respectively. More importantly, farmed and wild explants of each species demonstrated similar chemical fingerprints, with the majority of the metabolites showing modest differences on a sponge mass-normalized basis. Furthermore, farmed sponge extracts presented similar or slightly lower antibacterial activity against methicillin-resistant Staphylococcus aureus, compared to the extracts resulting from wild sponges. Anticancer assays against human colorectal carcinoma cells (HCT-116) revealed marginally active extracts from both wild and farmed S. foetidus populations. Our study highlights that, besides mitigating organic pollution in fish aquaculture, sponge farming can serve as a valuable resource of biomolecules, with promising potential in pharmaceutical and biomedical applications.

Naphthyl-Substituted Indole and Pyrrole Carboxylic Acids as Effective Antibiotic Potentiators-Inhibitors of Bacterial Cystathionine γ-Lyase.

Over the past decades, the problem of bacterial resistance to most antibiotics has become a serious threat to patients' survival. Nevertheless, antibiotics of a novel class have not been approved since the 1980s. The development of antibiotic potentiators is an appealing alternative to the challenging process of searching for new antimicrobials. Production of H2S-one of the leading defense mechanisms crucial for bacterial survival-can be influenced by the inhibition of relevant enzymes: bacterial cystathionine γ-lyase (bCSE), bacterial cystathionine ß-synthase (bCBS), or 3-mercaptopyruvate sulfurtransferase (MST). The first one makes the main contribution to H2S generation. Herein, we present data on the synthesis, in silico analyses, and enzymatic and microbiological assays of novel bCSE inhibitors. Combined molecular docking and molecular dynamics analyses revealed a novel binding mode of these ligands to bCSE. Lead compound 2a manifested strong potentiating activity when applied in combination with some commonly used antibiotics against multidrug-resistant Acinetobacter baumannii, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus. The compound was found to have favorable in vitro absorption, distribution, metabolism, excretion, and toxicity parameters. The high effectiveness and safety of compound 2a makes it a promising candidate for enhancing the activity of antibiotics against high-priority pathogens.

Comparative Antibacterial and Efflux Pump Inhibitory Activity of Isolated Nerolidol, Farnesol, and α-Bisabolol Sesquiterpenes and Their Liposomal Nanoformulations.

The efflux systems are considered important mechanisms of bacterial resistance due to their ability to extrude various antibiotics. Several naturally occurring compounds, such as sesquiterpenes, have demonstrated antibacterial activity and the ability to inhibit efflux pumps in resistant strains. Therefore, the objective of this research was to analyze the antibacterial and inhibitory activity of the efflux systems NorA, Tet(K), MsrA, and MepA by sesquiterpenes nerolidol, farnesol, and α-bisabolol, used either individually or in liposomal nanoformulation, against multi-resistant Staphylococcus aureus strains. The methodology consisted of in vitro testing of the ability of sesquiterpenes to reduce the Minimum Inhibitory Concentration (MIC) and enhance the action of antibiotics and ethidium bromide (EtBr) in broth microdilution assays. The following strains were used: S. aureus 1199B carrying the NorA efflux pump, resistant to norfloxacin; IS-58 strain carrying Tet(K), resistant to tetracyclines; RN4220 carrying MsrA, conferring resistance to erythromycin. For the EtBr fluorescence measurement test, K2068 carrying MepA was used. It was observed the individual sesquiterpenes exhibited better antibacterial activity as well as efflux pump inhibition. Farnesol showed the lowest MIC of 16.5 µg/mL against the S. aureus RN4220 strain. Isolated nerolidol stood out for reducing the MIC of EtBr to 5 µg/mL in the 1199B strain, yielding better results than the positive control CCCP, indicating strong evidence of NorA inhibition. The liposome formulations did not show promising results, except for liposome/farnesol, which reduced the MIC of EtBr against 1199B and RN4220. Further research is needed to evaluate the mechanisms of action involved in the inhibition of resistance mechanisms by the tested compounds.

Increased Activity of ß-Lactam Antibiotics in Combination with Carvacrol against MRSA Bacteremia and Catheter-Associated Biofilm Infections.

ß-Lactam antibiotics are the mainstay for the treatment of staphylococcal infections, but their utility is greatly limited by the emergence and rapid dissemination of methicillin-resistant Staphylococcus aureus (MRSA). Herein, we evaluated the ability of the plant-derived monoterpene carvacrol to act as an antibiotic adjuvant, revitalizing the anti-MRSA activity of ß-lactam antibiotics. Increased susceptibility of MRSA to ß-lactam antibiotics and significant synergistic activities were observed with carvacrol-based combinations. Carvacrol significantly inhibited MRSA biofilms and reduced the production of exopolysaccharide, polysaccharide intercellular adhesin, and extracellular DNA and showed synergistic biofilm inhibition in combination with ß-lactams. Transcriptome analysis revealed profound downregulation in the expression of genes involved in two-component systems and S. aureus infection. Mechanistic studies indicate that carvacrol inhibits the expression of staphylococcal accessory regulator sarA and interferes with SarA-mecA promoter binding that decreases mecA-mediated ß-lactam resistance. Consistently, the in vivo experiment also supported that carvacrol restored MRSA sensitivity to ß-lactam antibiotic treatments in both murine models of bacteremia and biofilm-associated infection. Our results indicated that carvacrol has a potential role as a combinatorial partner with ß-lactam antibiotics to address MRSA infections.

A Review on Five and Six-Membered Heterocyclic Compounds Targeting the Penicillin-Binding Protein 2 (PBP2A) of Methicillin-Resistant Staphylococcus aureus (MRSA).

Staphylococcus aureus is a common human pathogen. Methicillin-resistant Staphylococcus aureus (MRSA) infections pose significant and challenging therapeutic difficulties. MRSA often acquires the non-native gene PBP2a, which results in reduced susceptibility to ß-lactam antibiotics, thus conferring resistance. PBP2a has a lower affinity for methicillin, allowing bacteria to maintain peptidoglycan biosynthesis, a core component of the bacterial cell wall. Consequently, even in the presence of methicillin or other antibiotics, bacteria can develop resistance. Due to genes responsible for resistance, S. aureus becomes MRSA. The fundamental premise of this resistance mechanism is well-understood. Given the therapeutic concerns posed by resistant microorganisms, there is a legitimate demand for novel antibiotics. This review primarily focuses on PBP2a scaffolds and the various screening approaches used to identify PBP2a inhibitors. The following classes of compounds and their biological activities are discussed: Penicillin, Cephalosporins, Pyrazole-Benzimidazole-based derivatives, Oxadiazole-containing derivatives, non-ß-lactam allosteric inhibitors, 4-(3H)-Quinazolinones, Pyrrolylated chalcone, Bis-2-Oxoazetidinyl macrocycles (ß-lactam antibiotics with 1,3-Bridges), Macrocycle-embedded ß-lactams as novel inhibitors, Pyridine-Coupled Pyrimidinones, novel Naphthalimide corbelled aminothiazoximes, non-covalent inhibitors, Investigational-ß-lactam antibiotics, Carbapenem, novel Benzoxazole derivatives, Pyrazolylpyridine analogues, and other miscellaneous classes of scaffolds for PBP2a. Additionally, we discuss the penicillin-binding protein, a crucial target in the MRSA cell wall. Various aspects of PBP2a, bacterial cell walls, peptidoglycans, different crystal structures of PBP2a, synthetic routes for PBP2a inhibitors, and future perspectives on MRSA inhibitors are also explored.

Echinacoside, a promising sortase A inhibitor, combined with vancomycin against murine models of MRSA-induced pneumonia.

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogenic bacterium responsible for a range of severe infections, such as skin infections, bacteremia, and pneumonia. Due to its antibiotic-resistant nature, current research focuses on targeting its virulence factors. Sortase A (SrtA) is a transpeptidase that anchors surface proteins to the bacterial cell wall and is involved in adhesion and invasion to host cells. Through fluorescence resonance energy transfer (FRET), we identified echinacoside (ECH), a natural polyphenol, as a potential SrtA inhibitor with an IC50 of 38.42 µM in vitro. It was demonstrated that ECH inhibited SrtA-mediated S. aureus fibrinogen binding, surface protein A anchoring, and biofilm formation. The fluorescence quenching assay determined the binding mode of ECH to SrtA and calculated the KA-binding constant of 3.09 × 105 L/mol, demonstrating the direct interaction between the two molecules. Molecular dynamics simulations revealed that ECH-SrtA interactions occurred primarily at the binding sites of A92G, A104G, V168A, G192A, and R197A. Importantly, the combination of ECH and vancomycin offered protection against murine models of MRSA-induced pneumonia. Therefore, ECH may serve as a potential antivirulence agent against S. aureus infections, either alone or in combination with vancomycin.

Staphylococcus aureus adapts to the immunometabolite itaconic acid by inducing acid and oxidative stress responses including S-bacillithiolations and S-itaconations.

Staphylococcus aureus is a major pathogen, which has to defend against reactive oxygen and electrophilic species encountered during infections. Activated macrophages produce the immunometabolite itaconate as potent electrophile and antimicrobial upon pathogen infection. In this work, we used transcriptomics, metabolomics and shotgun redox proteomics to investigate the specific stress responses, metabolic changes and redox modifications caused by sublethal concentrations of itaconic acid in S. aureus. In the RNA-seq transcriptome, itaconic acid caused the induction of the GlnR, KdpDE, CidR, SigB, GraRS, PerR, CtsR and HrcA regulons and the urease-encoding operon, revealing an acid and oxidative stress response and impaired proteostasis. Neutralization using external urea as ammonium source improved the growth and decreased the expression of the glutamine synthetase-controlling GlnR regulon, indicating that S. aureus experienced ammonium starvation upon itaconic acid stress. In the extracellular metabolome, the amounts of acetate and formate were decreased, while secretion of pyruvate and the neutral product acetoin were strongly enhanced to avoid intracellular acidification. Exposure to itaconic acid affected the amino acid uptake and metabolism as revealed by the strong intracellular accumulation of lysine, threonine, histidine, aspartate, alanine, valine, leucine, isoleucine, cysteine and methionine. In the proteome, itaconic acid caused widespread S-bacillithiolation and S-itaconation of redox-sensitive antioxidant and metabolic enzymes, ribosomal proteins and translation factors in S. aureus, supporting its oxidative and electrophilic mode of action in S. aureus. In phenotype analyses, the catalase KatA, the low molecular weight thiol bacillithiol and the urease provided protection against itaconic acid-induced oxidative and acid stress in S. aureus. Altogether, our results revealed that under physiological infection conditions, such as in the acidic phagolysome, itaconic acid is a highly effective antimicrobial against multi-resistant S. aureus isolates, which acts as weak acid causing an acid, oxidative and electrophilic stress response, leading to S-bacillithiolation and itaconation.

SarS and Rot are necessary for the repression of lukED and lukSF-PV in Staphylococcus aureus.

IMPORTANCE: The leukocidins play an important role in disarming the host immune system and promoting infection. While both SarS and Rot have been established as repressors of leukocidins, the importance of each repressor in infection is unclear. Here, we demonstrate that repression by SarS and Rot is not additive and show that in addition to upregulating expression of each other, they are also able to bind concurrently to the leukocidin promoters. These findings suggest that both repressors are necessary for maximal repression of lukED and lukSF-PV and illuminate another complex relationship among Staphylococcus aureus virulence regulators.