Bacterial resistance has become a global concern for public health agencies. Various resistance mechanisms found in Staphylococcus aureus strains grant this bacterium resistance to a wide range of antibiotics, contributing to the rise in human mortality worldwide. Resistance mediated by efflux pumps is one of the most prevalent mechanisms in multi-resistant bacteria, which has aroused the interest of several researchers in the search for possible efflux pump inhibitors. In view of the aforementioned considerations, it is important that new strategies, such as the synthesis of chalcones, be made available as a viable strategy in antimicrobial therapy. In this study, the synthesized chalcone (2E)-1-(3'-aminophenyl)-3-(4-dimethylaminophenyl)-prop-2-en-1-one was tested for its antibacterial activity, focusing on antibiotic modification and the inhibition of the MepA efflux pump present in S. aureus strain K2068. The broth microdilution method, using microdilution plates, was employed in microbiological tests to determine the minimum inhibitory concentration of the chalcone, antibiotics, and ethidium bromide. The results show that while the chalcone did not exhibit direct antibacterial activity, it synergistically enhanced the effects of ciprofloxacin and ethidium bromide, as evidenced by the reduction in MICs. In addition, computer simulations of molecular docking demonstrate that the tested chalcone acts on the same binding site as the efflux pump inhibitor chlorpromazine, interacting with essentially the same residues. These data suggest that the chalcone may act as a MepA inhibitor.
- Klíčová slova
- Staphylococcus aureus, Antibacterial, Efflux pumps, Molecular docking,
- MeSH
- antibakteriální látky * farmakologie chemie chemická syntéza MeSH
- bakteriální proteiny * metabolismus genetika chemie antagonisté a inhibitory MeSH
- chalkon * farmakologie chemie MeSH
- chalkonoidy * farmakologie chemie MeSH
- lidé MeSH
- membránové transportní proteiny * metabolismus genetika chemie MeSH
- mikrobiální testy citlivosti MeSH
- proteiny spojené s mnohočetnou rezistencí k lékům * metabolismus genetika chemie MeSH
- simulace molekulového dockingu MeSH
- Staphylococcus aureus * účinky léků genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antibakteriální látky * MeSH
- bakteriální proteiny * MeSH
- chalkon * MeSH
- chalkonoidy * MeSH
- membránové transportní proteiny * MeSH
- proteiny spojené s mnohočetnou rezistencí k lékům * MeSH
Finding effective antibiotics against multi-resistant strains of bacteria has been a challenging race. Linker-Evolved-Group-Optimized-Lipophosphonoxins (LEGO-LPPOs) are small modular synthetic antibacterial compounds targeting the cytoplasmic membrane. Here we focused on understanding the reasons for the variable efficacy of selected LEGO-LPPOs (LEGO-1, LEGO-2, LEGO-3, and LEGO-4) differing in hydrophobic and linker module structure and length. LEGO-1-4 permeabilized cytoplasmic membrane of Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, and Escherichia coli, LEGO-1 with the longest linker module being the most effective. Gram-positive bacteria were more sensitive to LEGO-LPPO action compared to Gram-negatives, which was manifested as a delayed membrane permeabilization, higher minimal inhibitory concentration and lower amount of LEGO-LPPO bound to the cells. Outer membrane permeability measurements and time-kill assay showed that presence of the intact outer membrane brought about reduced susceptibility of Gram-negatives. Using liposome leakage and in silico simulations, we showed that membranes with major content of phosphatidylethanolamine were more prone to LEGO-LPPO permeabilization. The proposed mechanism stems from an electrostatic repulsion between highly positively charged LEGO-1 molecules and positively charged amino groups of phosphatidylethanolamine which destabilizes the membrane. Collectively, these data suggest that LEGO-LPPO membrane activity is enhanced by presence of phosphatidylethanolamine but hindered by presence of intact outer membrane.
- MeSH
- antibakteriální látky * farmakologie chemie MeSH
- buněčná membrána metabolismus MeSH
- Escherichia coli metabolismus účinky léků MeSH
- fosfatidylethanolaminy * chemie metabolismus MeSH
- mikrobiální testy citlivosti * MeSH
- permeabilita buněčné membrány účinky léků MeSH
- Staphylococcus aureus účinky léků metabolismus MeSH
- vnější bakteriální membrána metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antibakteriální látky * MeSH
- fosfatidylethanolaminy * MeSH
- phosphatidylethanolamine MeSH Prohlížeč
The pathogenic fungus Aspergillus fumigatus utilizes a cyclic ferrioxamine E (FOXE) siderophore to acquire iron from the host. Biomimetic FOXE analogues were labeled with gallium-68 for molecular imaging with PET. [68Ga]Ga(III)-FOXE analogues were internalized in A. fumigatus cells via Sit1. Uptake of [68Ga]Ga(III)-FOX 2-5, the most structurally alike analogue to FOXE, was high by both A. fumigatus and bacterial Staphylococcus aureus. However, altering the ring size provoked species-specific uptake between these two microbes: ring size shortening by one methylene unit (FOX 2-4) increased uptake by A. fumigatus compared to that by S. aureus, whereas lengthening the ring (FOX 2-6 and 3-5) had the opposite effect. These results were consistent both in vitro and in vivo, including PET imaging in infection models. Overall, this study provided valuable structural insights into the specificity of siderophore uptake and, for the first time, opened up ways for selective targeting and imaging of microbial pathogens by siderophore derivatization.
- MeSH
- Aspergillus fumigatus * metabolismus chemie MeSH
- aspergilóza * diagnostické zobrazování mikrobiologie MeSH
- biomimetické materiály chemie metabolismus MeSH
- cyklické peptidy MeSH
- deferoxamin chemie MeSH
- druhová specificita MeSH
- myši MeSH
- pozitronová emisní tomografie * metody MeSH
- radioizotopy galia * chemie MeSH
- siderofory * chemie metabolismus MeSH
- Staphylococcus aureus * metabolismus MeSH
- železité sloučeniny chemie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- cyklické peptidy MeSH
- deferoxamin MeSH
- ferrioxamine E MeSH Prohlížeč
- Gallium-68 MeSH Prohlížeč
- radioizotopy galia * MeSH
- siderofory * MeSH
- železité sloučeniny MeSH
Bovine mastitis produced by Staphylococcus aureus (S. aureus) causes major problems in milk production due to the staphylococcal enterotoxins produced by this bacterium. These enterotoxins are stable and cannot be eradicated easily by common hygienic procedures once they are formed in dairy products. Here, magnetic microrobots (MagRobots) are developed based on paramagnetic hybrid microstructures loaded with IgG from rabbit serum that can bind and isolate S. aureus from milk in a concentration of 3.42 104 CFU g-1 (allowable minimum level established by the United States Food and Drug Administration, FDA). Protein A, which is present on the cell wall of S. aureus, selectively binds IgG from rabbit serum and loads the bacteria onto the surface of the MagRobots. The selective isolation of S. aureus is confirmed using a mixed suspension of S. aureus and Escherichia coli (E. coli). Moreover, this fuel-free system based on magnetic robots does not affect the natural milk microbiota or add any toxic compound resulting from fuel catalysis. This system can be used to isolate and transport efficiently S. aureus and discriminate it from nontarget bacteria for subsequent identification. Finally, this system can be scaled up for industrial use in food production.
- Klíčová slova
- bacteria, collective behavior, infections, micromachines,
- MeSH
- enterotoxiny metabolismus MeSH
- Escherichia coli MeSH
- imunoglobulin G MeSH
- králíci MeSH
- magnetické jevy MeSH
- mléko MeSH
- skot MeSH
- stafylokokové infekce * MeSH
- Staphylococcus aureus * metabolismus MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- skot MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- enterotoxiny MeSH
- imunoglobulin G MeSH
Toxin-antitoxin (TA) systems are small genetic elements which encode toxin proteins that interfere with vital cellular functions. PepA1 and PepG1 toxin proteins, known also as SprA1 and SprG1, are type I TA. In Staphylococcus aureus (S. aureus), their expression without the antitoxin counterparts (SprA1AS and SprF1), is lethal to the pathogen. Molecular Dynamics (MD) simulation was performed for PepA1 and PepG1 to understand their dynamic state, conformational changes, and their toxicity. The protein structures were constructed and used for MD simulation and the conformational changes, stability, flexibility, fluctuations, hydrophobicity, and role of their dynamic state on function prediction were studied extensively by GROMACS MD simulation analysis tools. In silico study indicated that the PepA1 and PepG1 proteins change their structural conformation from an open to closed state where PepA1 conformational changes were faster (10 ns) than PepG1 (20 ns) while PepG1 exerted more stability and flexibility than PepA1. According to SASA values, PepG1 is more hydrophobic than the PepA1 and forms fewer hydrogen bonds than PepA1. The in vivo study with PepA1 and PepG1 proteins provided evidence that both the conformation changes between the open and closed states and the amino acid sequence are crucial for peptide toxicity.
- Klíčová slova
- Cloning, Molecular dynamics simulation, PepA1, PepG1, Staphylococcus aureus, Toxin-antitoxin system,
- MeSH
- antitoxiny * metabolismus MeSH
- bakteriální proteiny genetika metabolismus MeSH
- hydrofobní a hydrofilní interakce MeSH
- lidé MeSH
- peptidy metabolismus MeSH
- stafylokokové infekce * genetika MeSH
- Staphylococcus aureus metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antitoxiny * MeSH
- bakteriální proteiny MeSH
- peptidy MeSH
Bacterial resistance is a natural process carried out by bacteria, which has been considered a public health problem in recent decades. This process can be triggered through the efflux mechanism, which has been extensively studied, mainly related to the use of natural products to inhibit this mechanism. To carry out the present study, the minimum inhibitory concentration (MIC) tests of the compound limonene were performed, through the microdilution methodology in sterile 96-well plates. Tests were also carried out with the association of the compound with ethidium bromide and ciprofloxacin, in addition to the ethidium bromide fluorimetry, and later the molecular docking. From the tests performed, it was possible to observe that the compound limonene presented significant results when associated with ethidium bromide and the antibiotic used. Through the fluorescence emission, it was observed that when associated with the compound limonene, a greater ethidium bromide fluorescence was emitted. Finally, when analyzing the in silico study, it demonstrated that limonene can efficiently fit into the MepA structure. In this way, it is possible to show that limonene can contribute to cases of bacterial resistance through an efflux pump, so that it is necessary to carry out more studies to prove its effects against bacteria carrying an efflux pump and assess the toxicity of the compound.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální proteiny metabolismus MeSH
- limonen MeSH
- mikrobiální testy citlivosti MeSH
- proteiny spojené s mnohočetnou rezistencí k lékům * MeSH
- simulace molekulového dockingu MeSH
- Staphylococcus aureus * metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antibakteriální látky MeSH
- bakteriální proteiny MeSH
- limonen MeSH
- proteiny spojené s mnohočetnou rezistencí k lékům * MeSH
Bacterial cell wall peptidoglycan is essential, maintaining both cellular integrity and morphology, in the face of internal turgor pressure. Peptidoglycan synthesis is important, as it is targeted by cell wall antibiotics, including methicillin and vancomycin. Here, we have used the major human pathogen Staphylococcus aureus to elucidate both the cell wall dynamic processes essential for growth (life) and the bactericidal effects of cell wall antibiotics (death) based on the principle of coordinated peptidoglycan synthesis and hydrolysis. The death of S. aureus due to depletion of the essential, two-component and positive regulatory system for peptidoglycan hydrolase activity (WalKR) is prevented by addition of otherwise bactericidal cell wall antibiotics, resulting in stasis. In contrast, cell wall antibiotics kill via the activity of peptidoglycan hydrolases in the absence of concomitant synthesis. Both methicillin and vancomycin treatment lead to the appearance of perforating holes throughout the cell wall due to peptidoglycan hydrolases. Methicillin alone also results in plasmolysis and misshapen septa with the involvement of the major peptidoglycan hydrolase Atl, a process that is inhibited by vancomycin. The bactericidal effect of vancomycin involves the peptidoglycan hydrolase SagB. In the presence of cell wall antibiotics, the inhibition of peptidoglycan hydrolase activity using the inhibitor complestatin results in reduced killing, while, conversely, the deregulation of hydrolase activity via loss of wall teichoic acids increases the death rate. For S. aureus, the independent regulation of cell wall synthesis and hydrolysis can lead to cell growth, death, or stasis, with implications for the development of new control regimes for this important pathogen.
- Klíčová slova
- antibiotics, cell wall, methicillin, peptidoglycan, vancomycin,
- MeSH
- antibakteriální látky farmakologie MeSH
- antiinfekční látky metabolismus farmakologie MeSH
- bakteriální proteiny metabolismus MeSH
- buněčná stěna metabolismus fyziologie MeSH
- homeostáza MeSH
- kyseliny teichoové metabolismus MeSH
- methicilin farmakologie MeSH
- N-acetylmuramoyl-L-alaninamidasa metabolismus MeSH
- peptidoglykan metabolismus MeSH
- stafylokokové infekce mikrobiologie MeSH
- Staphylococcus aureus růst a vývoj metabolismus MeSH
- vankomycin farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antibakteriální látky MeSH
- antiinfekční látky MeSH
- bakteriální proteiny MeSH
- kyseliny teichoové MeSH
- methicilin MeSH
- N-acetylmuramoyl-L-alaninamidasa MeSH
- peptidoglykan MeSH
- vankomycin MeSH
The stringent response is characterized by the synthesis of the messenger molecules pppGpp, ppGpp or pGpp (here collectively designated (pp)pGpp). The phenotypic consequences resulting from (pp)pGpp accumulation vary among species and can be mediated by different underlying mechanisms. Most genome-wide analyses have been performed under stress conditions, which often mask the immediate effects of (pp)pGpp-mediated regulatory circuits. In Staphylococcus aureus, (pp)pGpp can be synthesized via the RelA-SpoT-homolog, RelSau upon amino acid limitation or via one of the two small (pp)pGpp synthetases RelP or RelQ upon cell wall stress. We used RNA-Seq to compare the global effects in response to induction of the synthetase of rel-Syn (coding for the enzymatic region of RelSau) or relQ without the need to apply additional stress conditions. Induction of rel-Syn resulted in changes in the nucleotide pool similar to induction of the stringent response via the tRNA synthetase inhibitor mupirocin: a reduction in the GTP pool, an increase in the ATP pool and synthesis of pppGpp, ppGpp and pGpp. Induction of all three enzymes resulted in similar changes in the transcriptome. However, RelQ was less active than Rel-Syn and RelP, indicating strong restriction of its (pp)pGpp-synthesis activity in vivo. (pp)pGpp induction resulted in the downregulation of many genes involved in protein and RNA/DNA metabolism. Many of the (pp)pGpp upregulated genes are part of the GTP sensitive CodY regulon and thus likely regulated through lowering of the GTP pool. New CodY independent transcriptional changes were detected including genes involved in the SOS response, iron storage (e.g. ftnA, dps), oxidative stress response (e.g., perR, katA, sodA) and the psmα1-4 and psmß1-2 operons coding for cytotoxic, phenol soluble modulins (PSMs). Analyses of the ftnA, dps and psm genes in different regulatory mutants revealed that their (pp)pGpp-dependent regulation can occur independent of the regulators PerR, Fur, SarA or CodY. Moreover, psm expression is uncoupled from expression of the quorum sensing system Agr, the main known psm activator. The expression of central genes of the oxidative stress response protects the bacteria from anticipated ROS stress derived from PSMs or exogenous sources. Thus, we identified a new link between the stringent response and oxidative stress in S. aureus that is likely crucial for survival upon phagocytosis.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- fyziologický stres * MeSH
- ligasy genetika metabolismus MeSH
- regulace genové exprese u bakterií * MeSH
- Staphylococcus aureus genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bakteriální proteiny MeSH
- guanosine 3',5'-polyphosphate synthetases MeSH Prohlížeč
- ligasy MeSH
Here, we describe a fluorescent assay developed to study competitive binding of the glycopeptide antibiotics to live bacteria cells. This assay demonstrated that the mechanism of action of the lipoglycopeptide antibiotics strongly depends on the hydrophobicity of the substitutes, with the best antibacterial activity of the glycopeptide antibiotics equally sharing properties of binding to D-Ala-D-Ala residues of the nascent peptidoglycan and to the membrane.
- MeSH
- antibakteriální látky metabolismus MeSH
- barvení a značení MeSH
- buněčná stěna mikrobiologie MeSH
- Enterococcus faecium metabolismus MeSH
- enterokoky rezistentní vůči vankomycinu metabolismus MeSH
- fluorescence MeSH
- glykopeptidy metabolismus MeSH
- lipoglykopeptidy chemie metabolismus MeSH
- mikrobiální testy citlivosti MeSH
- peptidoglykan metabolismus MeSH
- rhodaminy chemie MeSH
- Staphylococcus aureus metabolismus MeSH
- teikoplanin analogy a deriváty chemie metabolismus MeSH
- vankomycin chemie metabolismus MeSH
- vazba proteinů fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antibakteriální látky MeSH
- dalbavancin MeSH Prohlížeč
- glykopeptidy MeSH
- lipoglykopeptidy MeSH
- oritavancin MeSH Prohlížeč
- peptidoglykan MeSH
- rhodamine isothiocyanate MeSH Prohlížeč
- rhodaminy MeSH
- teikoplanin MeSH
- vankomycin MeSH
The presented study deals with the observation of properties of methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) in the toxic arsenic environment and influence of arsenic on antioxidant capacity. Two forms of arsenic (As(III), As(V)) with different concentrations were used for induction of the oxidative stress in tested strains. Microbiological methods showed that the growth inhibition of MSSA was higher than that of MRSA in presence of both arsenic ions. As(III) showed 24% and 33% higher anti-microbial effects than As(V) against MSSA and MRSA respectively. A similar result was found also in the experiment of reduction of biofilm-formation. By using spectrophotometry, it was revealed that As(III) induced higher antioxidant production in both bacterial cultures. Methicillin-susceptible S. aureus produced an app. 50 mg equivalent of gallic acid (GAE/1 mg of protein) and MRSA produced an app. 15 mg of GAE/1 mg of protein. The productions of metallothionein in MSSA and MRSA were decreased up to 62.41% and 55.84% respectively in presence of As ions. Reduction of As(III) and As(V) concentrations leads to a decrease in antioxidant production and increased the formation of metallothionein. All of these changes in the results were found to be significant statistically. Taken together, these experiments proved that in comparison with MSSA, MRSA is less susceptible not only to the antimicrobial effects of antibiotics but also against effects caused by metalloids, as arsenic. Thus, it can be stated that MRSA abounds with complex defensive mechanisms, which may in the future constitute significant problem in the efficiency of antibiotics alternatives as metal ions or nanoparticles.
- Klíčová slova
- Antibiotic resistance, Growth inhibition, Metals, Microbes, Oxidative stress,
- MeSH
- antibakteriální látky MeSH
- antioxidancia metabolismus MeSH
- arsen farmakologie MeSH
- kyselina gallová metabolismus MeSH
- methicilin rezistentní Staphylococcus aureus účinky léků metabolismus MeSH
- mikrobiální testy citlivosti MeSH
- oxidační stres * MeSH
- Staphylococcus aureus účinky léků metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antibakteriální látky MeSH
- antioxidancia MeSH
- arsen MeSH
- kyselina gallová MeSH