Mupirocin induced expression of genes encoding efflux pumps NorA and MepA as well as a yellow fluorescent protein (YFP) fluorescence reporter of NorA. Mupirocin exposure also produced reduced ...susceptibility to pump substrates ciprofloxacin and chlorhexidine, a change that was dependent on intact
and
, respectively.
Staphylococcus aureus Tet38 efflux pump has multiple functions, including conferring resistance to tetracycline and other compounds and enabling internalization and survival within epithelial cells. ...In this study, we evaluated the effects of sodium and potassium on
expression. These monovalent cations are known to play a role in transport by the related S. aureus TetK and B. subtilis TetL transporters.
transcription decreased with increasing sodium concentrations by means of direct repression by the salt stress-dependent KdpD/E regulator.
transcription increased 20-fold and tetracycline minimum inhibitory concentration (MIC) increased 4-fold in a Δ
mutant. KdpE bound specifically to the
promoter. Under extreme salt stress, the survival of S. aureus with intact
was reduced compared to that of a Δ
mutant. To study the effect of sodium on Tet38 function, we generated constructs overexpressing
and
and introduced them into Escherichia coli TO114, which is deficient in major sodium transporters. Tet38 tetracycline efflux was directly demonstrated in a fluorescence assay, and tetracycline efflux of both Tet38 and TetK was abolished by the protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP). In contrast, NaCl inhibited efflux by Tet38 but not TetK, whereas KCl inhibited efflux by TetK but not Tet38. Cell-associated Na increased with heterologous overexpression of Tet38. These data indicate that S. aureus Tet38 is a tetracycline efflux pump regulated by the KdpD/E regulator. Under salt stress, S. aureus adjusted its survival in part by reducing the expression of
through KdpD/E. The mechanisms by which Tet38 is detrimental to salt tolerance in S. aureus and inhibited by sodium remain to be determined.
This study shows that S. aureus Tet38 is a tetracycline efflux pump regulated by KdpD/E regulator. These findings are the first direct demonstration of Tet38-mediated tetracycline efflux, which had previously been inferred from its ability to confer tetracycline resistance. Under salt stress, S. aureus adjusts its survival in part by reducing the expression of
through KdpD/E. We demonstrated the differences in the respective functions of S. aureus Tet38 and other tetracycline efflux transporters (S. aureus TetK, B. subtilis TetL) regarding their transport of tetracycline and Na
/K
. Notably, sodium selectively reduced tetracycline efflux by Tet38, and potassium selectively reduced tetracycline efflux by TetK. The multiple functions of Tet38 emphasize its importance in bacterial adaptation to and survival in diverse environments.
The intra-articular administration of tranexamic acid (TXA) has been shown to be effective in reducing blood loss in unicompartmental knee arthroplasty and anterior cruciate reconstruction. The ...effects on human articular cartilage, however, remains unknown. Our aim, in this study, was to investigate any detrimental effect of TXA on chondrocytes, and to establish if there was a safe dose for its use in clinical practice. The hypothesis was that TXA would cause a dose-dependent damage to human articular cartilage.
The cellular morphology, adhesion, metabolic activity, and viability of human chondrocytes when increasing the concentration (0 mg/ml to 40 mg/ml) and length of exposure to TXA (0 to 12 hours) were analyzed in a 2D model. This was then repeated, excluding cellular adhesion, in a 3D model and confirmed in viable samples of articular cartilage.
Increasing concentrations above 20 mg/ml resulted in atypical morphology, reduced cellular adhesion and metabolic activity associated with increased chondrocyte death. However, the cell matrix was not affected by the concentration of TXA or the length of exposure, and offered cellular protection for concentrations below 20 mg/ml.
These results show that when in vitro chondrocytes are exposed to higher concentrations of TXA, such as that expected following recommended intra-articular administration, cytotoxicity is observed. This effect is dose-dependent, such that a tissue concentration of 10 mg/ml to 20 mg/ml could be expected to be safe. Cite this article: Bone Joint J 2018;100-B:404-12.
TetR21 controls the expression of Tet38 and LmrS efflux pumps. A
mutant, QT21, exhibited a 4-fold increase in the transcription level of
overexpressor showed increases of 4-fold and 2-fold, ...respectively, in the MICs of chloramphenicol and erythromycin, while the MICs of
mutant QT18 and
mutant QT1821 remained similar to those of parental strain RN6390. TetR21 does not bind to the promoter of
, suggesting indirect regulation of
.
We previously identified the protein Tet38 as a chromosomally encoded efflux pump of Staphylococcus aureus that confers resistance to tetracycline and certain unsaturated fatty acids. Tet38 also ...contributes to mouse skin colonization. In this study, we discovered a novel regulator of tet38, named tetracycline regulator 21 (TetR21), that bound specifically to the tet38 promoter and repressed pump expression. A ΔtetR21 mutant showed a 5-fold increase in tet38 transcripts and an 8-fold increase in resistance to tetracycline and fatty acids. The global regulator MgrA bound to the tetR21 promoter and indirectly repressed the expression of tet38. To further assess the full role of Tet38 in S. aureus adaptability, we tested its effect on host cell invasion using A549 (lung) and HMEC-1 (heart) cell lines. We used S. aureus RN6390, its Δtet38, ΔtetR21, and ΔmgrA mutants, and a Δtet38 ΔtetR21 double mutant. After 2 h of contact, the Δtet38 mutant was internalized in 6-fold-lower numbers than RN6390 in A549 and HMEC-1 cells, and the ΔtetR21 mutant was internalized in 2-fold-higher numbers than RN6390. A slight increase of 1.5-fold in internalization was found for the ΔmgrA mutant. The growth patterns of RN6390 and the ΔmgrA and ΔtetR21 mutants within A549 cells were similar, while no growth was observed for the Δtet38 mutant. These data indicate that the Tet38 efflux pump is regulated by TetR21 and contributes to the ability of S. aureus to internalize and replicate within epithelial cells.
Endogenous transporters protect
against antibiotics and also contribute to bacterial defense from environmental toxins. We evaluated the effect of overexpression of four efflux pumps, NorA, NorB, ...NorC, and Tet38, on
survival following exposure to pyocyanin (PYO) of
, using a well diffusion assay. We measured the PYO-created inhibition zone and found that only an overexpression of NorA reduced
susceptibility to pyocyanin killing. The MIC
of the NorA overexpressor increased threefold compared to that of wild-type RN6390 and was reduced 2.5-fold with reserpine, suggesting that increased NorA efflux caused PYO resistance. The PYO-created inhibition zone of a
mutant was consistently larger than that of a plasmid-borne NorA overexpressor. PYO also produced a modest increase in
expression (1.8-fold at 0.25 µg/mL PYO) that gradually decreased with increasing PYO concentrations. Well diffusion assays carried out using
showed that
mutant was less susceptible to killing by PYO-deficient mutants PA14
and PA14
than to killing by PA14. NorA overexpression led to reduced killing by all tested
. We evaluated the NorA-PYO interaction using a collection of 22 clinical isolates from adult and pediatric cystic fibrosis (CF) patients, which included both
(CF-SA) and
(CF-PA). We found that when isolated alone, CF-PA and CF-SA expressed varying levels of PYO and
transcripts, but all four CF-PA/CF-SA pairs isolated concurrently from CF patients produced a low level of PYO and low
transcript levels, respectively, suggesting a partial adaptation of the two bacteria in circumstances of persistent co-colonization.
Hooper et al assessed the evidence for correlation between aboveground and belowground diversity and concluded that a variety of mechanisms could lead to positive, negative, or no ...relationship--depending on the strength and type of interactions among species.
Abstract Pathological changes occur in areas of CNS tissue remote from inflammatory lesions in multiple sclerosis (MS) and its animal model experimental allergic encephalomyelitis (EAE). To determine ...if oxidative stress is a significant contributor to this non-inflammatory pathology, cortex tissues from mice with clinical signs of EAE were examined for evidence of inflammation and oxidative stress. Histology and gene expression analysis showed little evidence of immune/inflammatory cell invasion but reductions in natural antioxidant levels and increased protein oxidation that paralleled disease severity. Two-dimensional oxyblots and mass-spectrometry-based protein fingerprinting identified glutamine synthetase (GS) as a particular target of oxidation. Oxidation of GS was associated with reductions in enzyme activity and increased glutamate/glutamine levels. The possibility that this may cause neurodegeneration through glutamate excitotoxicity is supported by evidence of increasing cortical Ca2+ levels in cortex extracts from animals with greater disease severity. These findings indicate that oxidative stress occurs in brain areas that are not actively undergoing inflammation in EAE and that this can lead to a neurodegenerative process due to the susceptibility of GS to oxidative inactivation.
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