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
The role of oxidants and proinflammatory cytokines in the pathogenesis of pneumonia caused by Staphylococcus aureus (S. aureus) has been demonstrated. The present study aims to investigate the protective effect of ethyl acetate extract (EtOAc) obtained from Usnea longissima (UL) against acute oxidative and inflammatory lung damage due to S. aureus infection in rats. Albino Wistar-type male rats were divided into three groups: Healthy (HG), S. aureus inoculated (SaG), and S. aureus inoculated + ULEtOAc administered (SUL). SaG (n = 6) and SUL (n = 6) group rats' left nostrils (excluding HG) were inoculated with 0.1 ml bacterial mixture. After 24 hours, ULEtOAc (50 mg/kg) was administered orally to the SUL group, and the same volume of normal saline was administered orally to the HG (n = 6) and SaG groups. This procedure was performed once a day for seven days. Levels of oxidant and antioxidant parameters such as malondialdehyde (MDA) and total glutathione (tGSH), as well as pro-inflammatory cytokine levels such as nuclear factor-kappa B (NF-κB), tumor necrosis factor-alpha (TNF-α), interleukin-one beta (IL-1β), were measured in removed lung tissues. Tissues were also examined histopathologically. Biochemical results showed that ULEtOAc significantly suppressed the increase of MDA, NF-κB, TNF-α, and IL-1β levels and the decrease of tGSH caused by S. aureus in lung tissue. S. aureus inoculation caused severe mononuclear cell infiltration in interstitial areas, severe lymphoid hyperplasia in bronchial-associated lymphoid tissue and severe alveolar edema, histopathologically. Treatment with ULEtOAc had an attenuating effect on these histopathological findings. Experimental results from this study suggest that ULEtOAc may be beneficial in treating S. aureus-induced oxidative and inflammatory lung damage.
- MeSH
- cytokiny MeSH
- glutathion metabolismus farmakologie MeSH
- krysa rodu rattus MeSH
- NF-kappa B metabolismus MeSH
- oxidační stres MeSH
- plíce metabolismus patologie MeSH
- pneumonie * farmakoterapie patologie MeSH
- potkani Wistar MeSH
- stafylokokové infekce * farmakoterapie patologie MeSH
- Staphylococcus aureus metabolismus MeSH
- TNF-alfa metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články 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
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.
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.
- 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
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.
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
Intoxication by staphylococcal enterotoxins (SE) is among the most common causes of food-poisoning outbreaks resulting from the consumption of raw milk or products made thereof. The aim of our study was to analyze the thermal stability of SE and evaluate the inactivation of SE types A, B, and C (SEA, SEB, SEC) by autoclaving at 100°C, 110°C, and 121°C. Milk samples were inoculated with 38 Staphylococcus aureus strains that possessed the ability to produce SEA, SEB, or SEC and incubated at 37°C for 24 h. This incubation was followed by heat treatment at 100°C, 110°C, or 121°C for 3 min. Samples were analyzed by Staph. aureus plate count method on Baird-Parker agar and specifically for the presence of SE. An enzyme-linked immunofluorescent assay (ELFA) on a MiniVIDAS analyzer (bioMérieux, Marcy l'Étoile, France) was used to detect SE, which were determined semi-quantitatively based on test values. The obtained results were analyzed by means of nonparametric statistical methods. All samples (100%; 38/38) were SE-positive before heat treatment, and the positivity rates decreased after heat treatment at 100°C, 110°C, and 121°C to 36.8% (14/38), 34.2% (13/38), and 31.6% (12/38), respectively. The rates of positive samples differed between SEA, SEB, and SEC producers: SEA was detected in the highest amounts both before and after heat treatment. The amount of SE (expressed as test values) decreased significantly after heat treatment. Comparing amounts of SE in positive and negative samples before and after heat treatment, we can conclude that the success of SE inactivation depends on the amount present before heat treatment. The highest amount of SE and the highest rate of SE-positive samples after all heat treatments were found in samples with strains producing SEA. For SEB and SEC, lower amounts of enterotoxin were present and were inactivated at 100°C. Although temperatures of 100°C, 110°C, and 121°C may inactivate SE in milk, the key measures in prevention of staphylococcal enterotoxicosis are avoiding initial contamination of milk by Staph. aureus, promoting consumption of heat-treated milk, and preventing disruption of the cold chain during milk production and processing.
- MeSH
- chlazení MeSH
- enterotoxiny analýza chemie MeSH
- mastitida skotu mikrobiologie MeSH
- mléko chemie mikrobiologie MeSH
- potravinářská mikrobiologie metody MeSH
- skot MeSH
- stabilita léku MeSH
- stafylokokové infekce veterinární MeSH
- Staphylococcus aureus metabolismus MeSH
- vysoká teplota * MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
